Meningococcal antigens

Abstract

The invention provides proteins from Neisseria meningitidis (strains A & B), including amino acid sequences, the corresponding nucleotide sequences, expression data, and serological data. The proteins are useful antigens for vaccines, immunogenic compositions, and/or diagnostics.

Description

This application is a continuation-in-part of international patent application PCT/IB99/00103, filed Jan. 14, 1999, from which priority is claimed under 35 U.S.C. § 119.

This invention relates to antigens from the bacterium Neisseria meningitidis.

BACKGROUND

Neisseria meningitidis is a non-motile, gram negative diplococcus human pathogen. It colonises the pharynx, causing meningitis and, occasionally, septicaemia in the absence of meningitis. It is closely related to N. gonorrhoeae, although one feature that clearly differentiates meningococcus from gonococcus is the presence of a polysaccharide capsule that is present in all pathogenic meningococci.

N. meningitidis causes both endemic and epidemic disease. In the United States the attack rate is 0.6-1 per 100,000 persons per year, and it can be much greater during outbreaks (see Lieberman el al. (1996) Safety and Immunogenicity of a Serogroups A/C Neisseria meningitidis Oligosaccharide-Protein Conjugate Vaccine in Young Children. JAMA 275(19):1499-1503; Schuchat et al (1997) Bacterial Meningitis in the United States in 1995. N Engl J Med 337(14):970-976). In developing countries, endemic disease rates are much higher and during epidemics incidence rates can reach 500 cases per 100,000 persons per year. Mortality is extremely high, at 10-20% in the United States, and much higher in developing countries. Following the introduction of the conjugate vaccine against Haemophilus influenzae, N. meningitidis is the major cause of bacterial meningitis at all ages in the United States (Schuchat et al (1997) supra).

Based on the organism's capsular polysaccharide, 12 serogroups of N. meningitidis have been identified. Group A is the pathogen most often implicated in epidemic disease in sub-Saharan Africa. Serogroups B and C are responsible for the vast majority of cases in the United States and in most developed countries. Serogroups W135 and Y are responsible for the rest of the cases in the United States and developed countries. The meningococcal vaccine currently in use is a tetravalent polysaccharide vaccine composed of serogroups A, C, Y and W135. Although efficacious in adolescents and adults, it induces a poor immune response and short duration of protection, and cannot be used in infants [eg. Morbidity and Mortality weekly report, Vol. 46, No. RR-5 (1997)]. This is because polysaccharides are T-cell independent antigens that induce a weak immune response that cannot be boosted by repeated immunization. Following the success of the vaccination against H. influenzae, conjugate vaccines against serogroups A and C have been developed and are at the final stage of clinical testing (Zollinger W D “New and Improved Vaccines Against Meningococcal Disease” in: New Generation Vaccines, supra, pp. 469-488; Lieberman et al (1996) supra; Costantino et al (1992) Development and phase I clinical testing of a conjugate vaccine against meningococcus A and C. Vaccine 10:691-698).

Meningococcus B remains a problem, however. This serotype currently is responsible for approximately 50% of total meningitis in the United States, Europe, and South America. The polysaccharide approach cannot be used because the menB capsular polysaccharide is a polymer of α(2-8)-linked N-acetyl neuraminic acid that is also present in mammalian tissue. This results in tolerance to the antigen; indeed, if an immune response were elicited, it would be anti-self, and therefore undesirable. In order to avoid induction of autoimmunity and to induce a protective immune response, the capsular polysaccharide has, for instance, been chemically modified substituting the N-acetyl groups with N-propionyl groups, leaving the specific antigenicity unaltered (Romero & Outschoorn (1994) Current status of Meningococcal group B vaccine candidates: capsular or non-capsular? Clin Microbiol Rev 7(4):559-575).

Alternative approaches to menB vaccines have used complex mixtures of outer membrane proteins (OMPs), containing either the OMPs alone, or OMPs enriched in porins, or deleted of the class 4 OMPs that are believed to induce antibodies that block bactericidal activity. This approach produces vaccines that are not well characterized. They are able to protect against the homologous strain, but are not effective at large where there are many antigenic variants of the outer membrane proteins. To overcome the antigenic variability, multivalent vaccines containing up to nine different porins have been constructed (eg. Poolman J T (1992) Development of a meningococcal vaccine. Infect. Agents Dis. 4:13-28). Additional proteins to be used in outer membrane vaccines have been the opa and opc proteins, but none of these approaches have been able to overcome the antigenic variability (eg. Ala'Aldeen & Borriello (1996) The meningococcal transferrin-binding proteins 1 and 2 are both surface exposed and generate bactericidal antibodies capable of killing homologous and heterologous strains. Vaccine 14(1):49-53).

A certain amount of sequence data is available for meningococcal and gonococcal genes and proteins (eg. EP-A-0467714, WO96/29412), but this is by no means complete. The provision of further sequences could provide an opportunity to identify secreted or surface-exposed proteins that are presumed targets for the immune system and which are not antigenically variable. For instance, some of the identified proteins could be components of efficacious vaccines against meningococcus B, some could be components of vaccines against all meningococcal serotypes, and others could be components of vaccines against all pathogenic Neisseriae.

The Invention

The invention provides proteins comprising the N. meningitidis amino acid sequences disclosed in the examples.

It also provides proteins comprising sequences homologous (ie. having sequence identity) to the N. meningitidis amino acid sequences disclosed in the examples. Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more). These homologous proteins include mutants and allelic variants of the sequences disclosed in the examples. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between the proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.

The invention further provides proteins comprising fragments of the N. meningitidis amino acid sequences disclosed in the examples. The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more). Preferably the fragments comprise an epitope from the sequence.

The proteins of the invention can, of course, be prepared by various means (eg. recombinant expression, purification from cell culture, chemical synthesis etc.) and in various forms (eg. native, fusions etc.). They are preferably prepared in substantially pure form (ie. substantially free from other N. meningitidis or host cell proteins).

According to a further aspect, the invention provides antibodies which bind to these proteins. These may be polyclonal or monoclonal and may be produced by any suitable means.

According to a further aspect, the invention provides nucleic acid comprising the N. meningitidis nucleotide sequences disclosed in the examples. In addition, the invention provides nucleic acid comprising sequences homologous (ie. having sequence identity) to the N. meningitidis nucleotide sequences disclosed in the examples.

Furthermore, the invention provides nucleic acid which can hybridise to the N. meningitidis nucleic acid disclosed in the examples, preferably under “high stringency” conditions (eg. 65° C. in a 0.1×SSC, 0.5% SDS solution).

Nucleic acid comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides from the N. meningitidis sequences and, depending on the particular sequence, n is 10 or more (eg 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).

According to a further aspect, the invention provides nucleic acid encoding the proteins and protein fragments of the invention.

It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (eg. for antisense or probing purposes).

Nucleic acid according to the invention can, of course, be prepared in many ways (eg. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (eg. single stranded, double stranded, vectors, probes etc.).

In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.

According to a further aspect, the invention provides vectors comprising nucleotide sequences of the invention (eg. expression vectors) and host cells transformed with such vectors.

According to a further aspect, the invention provides compositions comprising protein, antibody, and/or nucleic acid according to the invention. These compositions may be suitable as vaccines, for instance, or as diagnostic reagents, or as immunogenic compositions.

The invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments (eg. as vaccines) or as diagnostic reagents. It also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of: (i) a medicament for treating or preventing infection due to Neisserial bacteria; (ii) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria; and/or (iii) a reagent which can raise antibodies against Neisserial bacteria Said Neisserial bacteria may be any species or strain (such as N. gonorrhoeae) but are preferably N. meningitidis, especially strain A, strain B or strain C.

The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of nucleic acid, protein, and/or antibody according to the invention.

According to further aspects, the invention provides various processes.

A process for producing proteins of the invention is provided, comprising the step of culturing a host cell according to the invention under conditions which induce protein expression.

A process for producing protein or nucleic acid of the invention is provided, wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.

A process for detecting polynucleotides of the invention is provided, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting said duplexes.

A process for detecting proteins of the invention is provided, comprising the steps of: (a) contacting an antibody according to the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.

Unlike the sequences disclosed in PCT/IB98/01665, the sequences disclosed in the present application are believed not to have any significant homologs in N. gonorrhoeae. Accordingly, the sequences of the present invention also find use in the preparation of reagents for distinguishing between N. meningitidis and N. gonorrhoeae.

A summary of standard techniques and procedures which may be employed in order to perform the invention (eg. to utilise the disclosed sequences for vaccination or diagnostic purposes) follows. This summary is not a limitation on the invention but, rather, gives examples that may be used, but are not required.

General

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature eg. Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and ii (D. N Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in this specification.

All publications, patents, and patent applications cited herein are incorporated in full by reference. In particular, the contents of UK patent applications 9800760.2, 9819015.0 and 9822143.5 are incorporated herein.

Definitions

A composition containing X is “substantially free of” Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.

The term “comprising” means “including” as well as “consisting” eg. a composition “comprising” X may consist exclusively of X or may include something additional to X, such as X+Y.

A “conserved” Neisseria amino acid fragment or protein is one that is present in a particular Neisserial protein in at least x % of Neisseria. The value of x may be 50% or more, e.g., 66%, 75%, 80%, 90%, 95% or even 100% (i.e. the amino acid is found in the protein in question in all Neisseria). In order to determine whether an animo acid is “conserved” in a particular Neisserial protein, it is necessary to compare that amino acid residue in the sequences of the protein in question from a plurality of different Neisseria (a reference population). The reference population may include a number of different Neisseria species or may include a single species. The reference population may include a number of different serogroups of a particular species or a single serogroup. A preferred reference population consists of the 5 most common Neisseria.

The term “heterologous” refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene. Another example is where a Neisserial sequence is heterologous to a mouse host cell. A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.

An “origin of replication” is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector. The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control. An origin of replication may be needed for a vector to replicate in a particular host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell. Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.

A “mutant” sequence is defined as DNA, RNA or amino acid sequence differing from but having sequence identity with the native or disclosed sequence. Depending on the particular sequence, the degree of sequence identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the Smith-Waterman algorithm as described above). As used herein, an “allelic variant” of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared. An allelic variant can also comprise an alteration in the 5′ or 3′ untranslated regions of the gene, such as in regulatory control regions (eg. see U.S. Pat. No. 5,753,235).

Expression Systems

The Neisserial nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, plants, bacteria, and yeast.

i. Mammalian Systems

Mammalian expression systems are known in the art. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al. (1989) “Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: A Laboratory Manual, 2nd ed.]

Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallotheionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.

The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989) Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range. Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J. 4:761] and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:6777] and from human cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237].

A DNA molecule may be expressed intracellularly in mammalian cells. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus triparite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.

Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3′ terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) “Termination and 3′ end processing of eukaryotic RNA. In Transcription and splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105]. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) “Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning: A Laboratory Manual].

Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs. Enhancers; introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO [Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].

The transformation procedure used depends upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg. Hep G2), and a number of other cell lines.

ii. Baculovirus Systems

The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art. Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine. The packaged recombinant-virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit). These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter “Summers and Smith”).

Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector). This construct may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements. Intermediate transplacement constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.

Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5′ to 3) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.

Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) “The Regulation of Baculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the gene encoding the p10 protein, Vlak et al., (1988), J. Gen. Virol. 69:765.

DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human α-interferon, Maeda et al., (1985), Nature 315:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci. USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also be used to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted. Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus—usually by co-transfection. The promoter and transcription termination sequence of the construct will usually comprise a 2-5 kb section of the baculovirus genome. Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art (See Summers and Smith supra; Ju et al. (1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. Miller et al., (1989), Bioessays 4:91 The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5′ and 3′ by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about 5%); thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses. An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished. The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles. These occlusion bodies, up to 15 μm in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope. Cells infected with recombinant viruses lack occlusion bodies. To distinguish recombinant virus from wild-type virus, the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies. “Current Protocols in Microbiology” Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990); Summers and Smith, supra; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25:225).

Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art. See, eg. Summers and Smith supra.

The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host. Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced. Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients. The product may be purified by such techniques as chromatography, eg. HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresis; density gradient centrifugation; solvent extraction, or the like. As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so as to provide a product which is at least substantially free of host debris, eg. proteins, lipids and polysaccharides.

In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.

iii. Plant Systems

There are many plant cell culture and whole plant genetic expression systems known in the art. Exemplary plant cellular genetic expression systems include those described in patents, such as: U.S. Pat. No. 5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat. No. 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30:3861-3863 (1991). Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al., Mol. Gen. Genet. 209:33-40 (1987); Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J. Biol. Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356 (1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992). A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R. L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology, Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52. References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038 (1990); Maas et al., EMBO J. 9:3447-3452 (1990); Benkel and Hickey, Proc. Natl. Acad. Sci 84:1337-1339 (1987)

Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants. The expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host. The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host. The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker, and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed. A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.

Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended. These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome. Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.

The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest. Usually, there will be only one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5′ untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence. Unique restriction enzyme sites at the 5′ and 3′ ends of the cassette allow for easy insertion into a pre-existing vector.

A heterologous coding sequence may be for any protein relating to the present invention. The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane. Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested. Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed. While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.

Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery. If so, site-directed mutagenesis of the “intron” region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41:95-105, 1985.

The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genet, 202:179-185, 1985. The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982. Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley. Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.

The vector may also be introduced into the plant cells by electroporation. (Fromm et al., Proc. Natl Acad. Sci. USA 82:5824, 1985). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.

All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.

Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.

In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant. Where the desired protein of the invention is secreted into the medium, it may be collected. Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues. The mixture may be suspended in a buffer solution to retrieve soluble proteins. Conventional protein isolation and purification methods will be then used to purify the recombinant protein. Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein.

iv. Bacterial Systems

Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 and EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981) “The cloning of interferon and other mistates.” In Interferon 3 (ed I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature 292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also provide useful promoter sequences.

In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).

In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al. (1975) Nature 254:34). The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ and of E. coli 16S rRNA [Steitz et al. (1979) “Genetic signals and nucleotide sequences in messenger RNA.” In Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger)]. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al. (1989) “Expression of cloned genes in Escherichia coli.” In Molecular Cloning: A Laboratory Manual].

A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO-A-0 219 237).

Fusion proteins provide an alternative to direct expression. Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5′ terminus of a foreign gene and expressed in bacteria The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al. (1984) Nature 309:810]. Fusion proteins can also be made with sequences from the lacZ [Jia et al. (1987) Gene 60:197], trpE (Allen et al. (1987) J. Biotechnol. 5:93; Makoff et al. (1989) J. Gen. Microbiol. 135:11], and Chey [EP-A-0 324 647] genes. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated [Miller et al. (1989) Bio/Technology 7:698].

Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [U.S. Pat. No. 4,336,336]. The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.

DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) [Masui et al. (1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212]. As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 244 042].

Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trip gene in E. coli as well as other biosynthetic genes.

Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as bacteria The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.

Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0 127 328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev. Microbiol. 32:469]. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.

Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol. 189:113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ. Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].

Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl₂ or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See eg. [Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978) “An improved method for transformation of Escherichia coli with ColE1-derived plasmids. In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H. W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol. Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol. 144:698; Harlander (1987) “Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infect Immun. 32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus].

v. Yeast Expression

Yeast expression systems are also known to one of ordinary skill in the art. A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site (the “TATA Box”) and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203). The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].

In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PHO5 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoff et al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol. Immunol. 96:119; Hollenberg et al. (1979) “The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids of Medical, Environmental and Commercial Importance (eds. K. N. Timmis and A. Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109;].

A DNA molecule may be expressed intracellularly in yeast. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5′ terminus of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See eg. EP-A-0 196 056. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated (eg. WO88/024066).

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP-A-0 012 873; JPO. 62,096,086) and the A-factor gene (U.S. Pat. No. 4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EP-A-0 060 057).

A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a “pre” signal sequence, and a “pro” region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos. 4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor. (eg. see WO 89/02463.)

Usually, transcription termination sequences recognized by yeast are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.

Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al. (1979) Gene 8:17-24], pCl/1 [Brake et al. (1984) Proc. Natl. Acad. Sci USA 81:4642-4646], and YRp17 [Stinchcomb et al. (1982) J. Mol. Biol. 158:157]. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See eg. Brake et al., supra.

Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol. 101:228-245]. An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector. See Orr-Weaver et al., supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750]. The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed. Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al. (1987) Microbiol, Rev. 51:351].

Alternatively, some of the above described components can be put together into transformation vectors. Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, inter alia, the following yeasts: Candida albicans [Kurtz, et al. (1986) Mol. Cell. Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. Basic Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302], Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichia guillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichia pastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet. 10:380471 Gaillardin, et al (1985) Curr. Genet. 10:49].

Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See eg. [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol. 25:141; Candida]; [Gleeson et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302; Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat. Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75;1929; Ito et al. (1983) J. Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse (1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].

Antibodies

As used herein, the term “antibody” refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen. “Antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies. Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishing/identifying Neisserial proteins.

Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods. In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). A dose of 50-200 μg/injection is typically sufficient. Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. One may alternatively generate antibodies by in vitro immunization using methods known in the adt, which for the purposes of this invention is considered equivalent to in vivo immunization. Polyclonal antisera is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25° C. for one hour, followed by incubating at 4° C. for 2-18 hours. The serum is recovered by centrifugation (eg. 1,000 g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits.

Monoclonal antibodies are prepared using the standard method of Kohler & Milstein [Nature (1975) 256:495-96], or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (eg. hypoxanthine, aminopterin, thymidine medium, “HAT”). The resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected MAb-secreting hybridomas are then cultured either in vitro (eg. in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).

If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly ³²P and ¹²⁵I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. “Specific binding partner” refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, ¹²⁵I may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with ¹²⁵I, or with an anti-biotin MAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.

Pharmaceutical Compositions

Pharmaceutical compositions can comprise either polypeptides, antibodies, or nucleic acid of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.

The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.

For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

Delivery Methods

Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Vaccines

Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).

Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with “pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents (“adjuvants”). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.

Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59™ are preferred.

As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamine (MTP-PE), etc.

The immunogenic compositions (eg. the immunising antigen/immunogen/polypeptide/protein/nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctors assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

The immunogenic compositions are conventionally administered parenterally, eg. by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg. WO98/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.

As an alternative to protein-based vaccines, DNA vaccination may be employed [eg. Robinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648; see later herein].

Gene Delivery Vehicles

Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.

The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picomavirus, poxvirus, or togavirus viral vector. See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.

Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol. 53:160) polytropic retrovirus eg. MCF and MCF-MLV (see Kelly (1983) J. Virol. 45:291), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.

Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.

These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S. Pat. No. 5,591,624). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see WO96/37626). It is preferable that the recombinant viral vector is a replication defective recombinant virus.

Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO95/30763 and WO92/05266), and can be used to create producer cell lines (also termed vector cell lines or “VCLs”) for the production of recombinant vector particles. Preferably, the packaging cell lines are made from human parent cells (eg. HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.

Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus. Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection (“ATCC”) in Rockville, Md. or isolated from known sources using commonly available techniques.

Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat. No. 5,219,740, U.S. Pat. No. 4,405,712, U.S. Pat. No. 4,861,719, U.S. Pat. No. 4,980,289, U.S. Pat. No. 4,777,127, U.S. Pat. No. 5,591,624. See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Rain (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.

Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and WO93/07283, WO93/06223, and WO93/07282. Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in WO94/12649, WO93/03769, WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum. Gene Ther. 3:147-154 may be employed. The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors. Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, WO93/09239. Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides. The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie. there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position. Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262. Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol. 61:3096). Another exemplary AAV vector is the Double-D ITR vector. Construction of the Double-D ITR vector is disclosed in U.S. Pat. No. 5,478,745. Still other vectors are those disclosed in Carter U.S. Pat. No. 4,797,368 and Muzyczka U.S. Pat. No. 5,139,941, Chartejee U.S. Pat. No. 5,474,935, and Kotin WO94/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470. Additional AAV gene therapy vectors are described in U.S. Pat. No. 5,354,678, U.S. Pat. No. 5,173,414, U.S. Pat. No. 5,139,941, and U.S. Pat. No. 5,252,479.

The gene therapy vectors of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Pat. No. 5,288,641 and EP0176170 (Roizman). Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in WO95/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in WO90/09441 and WO92/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.

Also contemplated are alpha virus gene therapy vectors that can be employed in this invention. Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat. Nos. 5,091,309, 5,217,879, and WO92/10578. More particularly, those alpha virus vectors described in U.S. Ser. No. 08/405,627, filed Mar. 15, 1995, WO94/21792, WO92/10578, WO95/07994, U.S. Pat. No. 5,091,309 and U.S. Pat. No. 5,217,879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Md. or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see U.S. Ser. No. 08/679,640).

DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. See WO95/07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.

Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol. Standardization 1:115; rhinovirus, for example ATCC VR-1110 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; in U.S. Pat. No. 4,603,112 and U.S. Pat. No. 4,769,330 and WO89/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in U.S. Pat. No. 5,166,057 and in Enami (1990) Proc Nail Acad Sci 87:3802-3805; Enami & Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NEJ Med 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol. 66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med 121:190.

Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see U.S. Ser. No. 08/366,787, filed Dec. 30, 1994 and Curiel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655, ionizing radiation as described in U.S. Pat. No. 5,206,152 and in WO92/11033, nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl Acad Sci 91:1581-1585.

Particle mediated gene transfer may be employed, for example see U.S. Ser. No. 60/023,867. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu & Wu (1987) J. Biol. Chem. 262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.

Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.

Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968. As described in U.S. S No. 60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin. Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters. Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Pat. No. 5,206,152 and WO92/11033

Exemplary liposome and polycationic gene delivery vehicles are those described in U.S. Pat. Nos. 5,422,120 and 4,762,915; in WO 95/13796; WO94/23697, and WO91/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.

A polynucleotide composition can comprises therapeutically effective amount of a gene therapy vehicle, as the term is defined above. For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

Delivery Methods

Once formulated, the polynucleotide compositions of the invention can be administered (1) directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) in vitro for expression of recombinant proteins. The subjects to be treated can be mammals or birds. Also, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in eg. WO93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.

Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.

Polynucleotide and Polypeptide Pharmaceutical Compositions

In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.

A. Polypeptides

One example are polypeptides which include, without limitation: asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin. Viral antigens, such as envelope proteins, can also be used. Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein of plasmodium falciparum known as RII.

B. Hormones, Vitamins, etc.

Other groups that can be included are, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.

C. Polyalkylenes, Polysaccharides, etc.

Also, polyalkylene glycol can be included with the desired polynucleotides/polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, or polysaccharides can be included. In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosan and poly(lactide-co-glycolide)

D. Lipids, and Liposomes

The desired polynucleotide/polypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom.

Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim. Biophys. Acta. 1097:1-17; Straubinger (1983) Meth. Enzymol. 101:512-527.

Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol. Chem. 265:10189-10192), in functional form.

Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner supra). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, eg. Szoka (1978) Proc. Nail. Acad. Sci. USA 75:41944198; WO90/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art See eg. Straubinger (1983) Meth. Immunol. 101:512-527; Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim. Biophys. Acia 443:629; Ostro (1977) Biochem. Biophys. Res. Commun. 76:836; Fraley (1979) Proc. Natl. Acad. Sci. USA 76:3348); Enoch & Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145; Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka & Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder (1982) Science 215:166.

E. Lipoproteins

In addition, lipoproteins can be included with the polynucleotide/polypeptide to be delivered. Examples of lipoproteins to be utilized include: chylomicrons, HDL, DL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL. These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors. Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.

Naturally occurring lipoproteins comprise a lipid and a protein portion. The protein portion are known as apoproteins. At the present, apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins, designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.

A lipoprotein can comprise more than one apoprotein. For example, naturally occurring chylomicrons comprises of A, B, C, and E, over time these lipoproteins lose A and acquire C and E apoproteins. VLDL comprises A, B, C, and E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, and E.

The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. Exp Med. Biol. 151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.

Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phospholipids. The composition of the lipids varies in naturally occurring lipoproteins. For example, chylomicrons comprise mainly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986). The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.

Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance. Such methods are described in Meth. Enzymol. (supra); Pitas (1980) J. Biochem. 255:5454-5460 and Mahey (1979) J. Clin. Invest 64:743-750. Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acia 30: 443. Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Mass., USA. Further description of lipoproteins can be found in Zuckermann et al. PCT/US97/14465.

F. Polycationic Agents

Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.

Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location. These agents have both in vitro, ex vivo, and in vivo applications. Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.

The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine. Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.

Organic polycationic agents include: spermine, spermidine, and purtrescine.

The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.

Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene. Lipofectin™, and lipofectAMINE™ are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.

Immunodiagnostic Assays

Neisserial antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Neisserial antibodies can be used to detect antigen levels). Immunoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics methods. Antibodies to Neisserial proteins within biological samples, including for example, blood or serum samples, can be detected. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the invention, in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc.) required for the conduct of the assay, as well as suitable set of assay instructions.

Nucleic Acid Hybridisation

“Hybridization” refers to the association of two nucleic acid sequences to one another by hydrogen bonding. Typically, one sequence will be fixed to a solid support and the other will be free in solution. Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization. See Sambrook et al. [supra] Volume 2, chapter 9, pages 9.47 to 9.57.

“Stringency” refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ. For example, the combination of temperature and salt concentration should be chosen that is approximately 120 to 200° C. below the calculated Tm of the hybrid under study. The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies. See Sambrook et al. at page 9.50.

Variables to consider when performing, for example, a Southern blot are (1) the complexity of the DNA being blotted and (2) the homology between the probe and the sequences being detected. The total amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to 1 g for a plasmid or phage digest to 10⁻⁹ to 10⁻⁸ g for a single copy gene in a highly complex eukaryotic genome. For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes can be used. For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with 1 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 10⁸ cpm/μg. For a single-copy mammalian gene a conservative approach would start with 10 μg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 10⁸ cpm/μg, resulting in an exposure time of 24 hours.

Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing. In many cases the probe is not 100% homologous to the fragment. Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and formamide content of the hybridization buffer. The effects of all of these factors can be approximated by a single equation:
Tm—81+16.6(log₁₀ Ci)+0.4[% (G+C)]−0.6(% formamide)-600/n−1.5(% mismatch).
where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth & Wahl (1984) Anal. Biochem. 138: 267-284).

In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered. The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust. As the temperature of the hybridization increases (ie. stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases. If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase. The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner. The stringency of the washes is also increased with decreasing salt concentrations.

In general, convenient hybridization temperatures in the presence of 50% formamide are 42° C. for a probe with is 95% to 100% homologous to the target fragment, 37° C. for 90% to 95% homology, and 32° C. for 85% to 90% homology. For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above. If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent. If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed. If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.

Nucleic Acid Probe Assays

Methods such as PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determine the presence of cDNA or mRNA. A probe is said to “hybridize” with a sequence of the invention if it can form a duplex or double stranded complex, which is stable enough to be detected.

The nucleic acid probes will hybridize to the Neisserial nucleotide sequences of the invention (including both sense and antisense strands). Though many different nucleotide sequences will encode the amino acid sequence, the native Neisserial sequence is preferred because it is the actual sequence present in cells. mRNA represents a coding sequence and so a probe should be complementary to the coding sequence; single-stranded cDNA is complementary to mRNA, and so a cDNA probe should be complementary to the non-coding sequence.

The probe sequence need not be identical to the Neisserial sequence (or its complement)—some variation in the sequence and length can lead to increased assay sensitivity if the nucleic acid probe can form a duplex with target nucleotides, which can be detected. Also, the nucleic acid probe can include additional nucleotides to stabilize the formed duplex. Additional Neisserial sequence may also be helpful as a label to detect the formed duplex. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of the probe, with the remainder of the probe sequence being complementary to a Neisserial sequence. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a Neisserial sequence in order to hybridize therewith and thereby form a duplex which can be detected.

The exact length and sequence of the probe will depend on the hybridization conditions, such as temperature, salt condition and the like. For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least 30 nucleotides, although it may be shorter than this. Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.

Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al. [J. Am. Chem. Soc. (1981) 103:3185], or according to Urdea et al. [Proc. Natl. Acad. Sci. USA (1983) 80: 7461], or using commercially available automated oligonucleotide synthesizers.

The chemical nature of the probe can be selected according to preference. For certain applications, DNA or RNA are appropriate. For other applications, modifications may be incorporated eg. backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc. [eg. see Agrawal & Iyer (1995) Curr Opin Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387]; analogues such as peptide nucleic acids may also be used [eg. see Corey (1997) TIBTECH 15:224-229; Buchardt et al. (1993) TIBTECH 11:384-386].

Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acids. The assay is described in: Mullis et al. [Meth. Enzymol. (1987) 155: 335-350]; U.S. Pat. Nos. 4,683,195 and 4,683,202. Two “primer” nucleotides hybridize with the target nucleic acids and are used to prime the reaction. The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site. Typically, such sequence will flank the desired Neisserial sequence.

A thermostable polymerase creases copies of target nucleic acids from the primers using the original target nucleic acids as a template. After a threshold amount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots. When using the Southern blot method, the labelled probe will hybridize to the Neisserial sequence (or its complement).

Also, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al [supra]. mRNA, or cDNA generated from mRNA using a polymerase enzyme, can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labelled with a radioactive moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-7 show biochemical data and sequence analysis pertaining to Examples 1, 2, 3, 7, 13, 16 and 19, respectively, with ORFs 40, 38, 44, 52, 114, 41 and 124. M1 and M2 are molecular weight markers. Arrows indicate the position of the main recombinant product or, in Western blots, the position of the main N. meningitidis immunoreactive band. TP indicates N. meningitidis total protein extract; OMV indicates N. meningitidis outer membrane vesicle preparation. In bactericidal assay results: a diamond (♦) shows preimmune data; a triangle (▴) shows GST control data; a circle (●) shows data with recombinant N. meningitidis protein. Computer analyses show a hydrophilicity plot (upper), an antigenic index plot (middle), and an AMPHI analysis (lower). The AMPHI program has been used to predict T-cell epitopes [Gao et al. (1989) J. Immunol. 143:3007; Roberts et al. (1996) AIDS Res Hum Retrovir 12:593; Quakyi et al. (1992) Scand J Immunol suppl. 11:9) and is available in the Protean package of DNASTAR, Inc. (1228 South Park Street, Madison, Wis. 53715 USA).

FIG. 8 shows an alignment comparison of amino acid sequences for ORF 40 for several strains of Neisseria. Dark shading indicates regions of homology, and gray shading indicates the conservation of amino acids with similar characteristics. The Figure demonstrates a high degree of conservation among the various strains, further confirming its utility as an antigen for both vaccines and diagnostics.

EXAMPLES

The examples describe nucleic acid sequences which have been identified in N. meningitidis, along with their putative translation products. Not all of the nucleic acid sequences are complete ie. they encode less than the full-length wild-type protein. It is believed at present that none of the DNA sequences described herein have significant homologs in N. gonorrhoeae.

The examples are generally in the following format:

• • a nucleotide sequence which has been identified in N. meningitidis (strain B)
  • the putative translation product of this sequence
  • a computer analysis of the translation product based on database comparisons
  • a corresponding gene and protein sequence identified in N. meningitidis (strain A)
  • a description of the characteristics of the proteins which indicates that they might be suitably antigenic
  • results of biochemical analysis (expression, purification, ELISA, FACS etc.)

The examples typically include details of sequence homology between species and strains. Proteins that are similar in sequence are generally similar in both structure and function, and the homology often indicates a common evolutionary origin. Comparison with sequences of proteins of known function is widely used as a guide for the assignment of putative protein function to a new sequence and has proved particularly useful in whole-genome analyses.

Sequence comparisons were performed at NCBI (http://www.ncbi.nlm.nih.gov) using the algorithms BLAST, BLAST2, BLASTn, BLASTp, tBLASTn, BLASTx, & tBLASTx [eg. see also Altschul et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25:2289-3402]. Searches were performed against the following databases: non-redundant GenBank+EMBL+DDBJ+PDB sequences and non-redundant GenBank CDS translations+PDB+SwissProt+SPupdate+PIR sequences.

Dots within nucleotide sequences (eg. position 288 in Example 12) represent nucleotides which have been arbitrarily introduced in order to maintain a reading frame. In the same way, double-underlined nucleotides were removed. Lower case letters (eg. position 589 in Example 12) represent ambiguities which arose during alignment of independent sequencing reactions (some of the nucleotide sequences in the examples are derived from combining the results of two or more experiments).

Nucleotide sequences were scanned in all six reading frames to predict the presence of hydrophobic domains using an algorithm based on the statistical studies of Esposti et al. [Critical evaluation of the hydropathy of membrane proteins (1990) Eur J Biochem 190:207-219]. These domains represent potential transmembrane regions or hydrophobic leader sequences.

Open reading frames were predicted from fragmented nucleotide sequences using the program ORFFINDER (NCBI).

Underlined amino acid sequences indicate possible transmembrane domains or leader sequences in the ORFs, as predicted by the PSORT algorithm (http://www.psort.nibb.ac.jp). Functional domains were also predicted using the MOTIFS program (GCG Wisconsin & PROSITE).

Various tests can be used to assess the in vivo immunogenicity of the proteins identified in the examples. For example, the proteins can be expressed recombinantly and used to screen patient sera by immunoblot A positive reaction between the protein and patient serum indicates that the patient has previously mounted an immune response to the protein in question ie. the protein is an immunogen. This method can also be used to identify immunodominant proteins.

The recombinant protein can also be conveniently used to prepare antibodies eg. in a mouse. These can be used for direct confirmation that a protein is located on the cell-surface. Labelled antibody (eg. fluorescent labelling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein.

In particular, the following methods (A) to (S) were used to express, purify and biochemically characterise the proteins of the invention:

A) Chromosomal DNA Preparation

N. meningitidis strain 2996 was grown to exponential phase in 100 ml of GC medium, harvested by centrifugation, and resuspended in 5 ml buffer (20% Sucrose, 50 mM Tris-HCl, 50 mM EDTA, pH8). After 10 minutes incubation on ice, the bacteria were lysed by adding 10 ml lysis solution (50 mM NaCl, 1% Na-Sarkosyl, 50 μg/ml Proteinase K), and the suspension was incubated at 37° C. for 2 hours. Two phenol extractions (equilibrated to pH 8) and one ChCl₃/isoamylalcohol (24:1) extraction were performed. DNA was precipitated by addition of 0.3M sodium acetate and 2 volumes ethanol, and was collected by centrifugation. The pellet was washed once with 70% ethanol and redissolved in 4 ml buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The DNA concentration was measured by reading the OD at 260 nm.

B) Oligonucleotide Design

Synthetic oligonucleotide primers were designed on the basis of the coding sequence of each ORF, using (a) the meningococcus B sequence when available, or (b) the gonococcus/meningococcus A sequence, adapted to the codon preference usage of meningococcus as necessary. Any predicted signal peptides were omitted, by deducing the 5′-end amplification primer sequence immediately downstream from the predicted leader sequence.

The 5′ primers included two restriction enzyme recognition sites (BamHI-NdeI, BamHI-NheI, or EcoRI-NheI, depending on the gene's own restriction pattern); the 3′ primers included a XhoI restriction site. This procedure was established in order to direct the cloning of each amplification product (corresponding to each ORF) into two different expression systems: pGEX-KG (using either BamHI-XhoI or EcoRI-XhoI), and pET21b+ (using either NdeI-XhoI or NheI-XhoI).

5′end primer tail:
CGCGGATCCCATATG   (BamHI-NdeI)
CGCGGATCCGCTAGC   (BamHI-NheI)
CCGGAATTCTAGCTAGC (EcoRI-NheI)
3′-end primer tail:
CCCGCTCGAG        (XhoI)

As well as containing the restriction enzyme recognition sequences, the primers included nucleotides which hybridised to the sequence to be amplified. The number of hybridizing nucleotides depended on the melting temperature of the whole primer, and was determined for each primer using the formulae:
T_m=4 (G+C)+2 (A+T)  (tail excluded)
T_m=64.9+0.41 (% GC)−600/N  (whole primer)

The average melting temperature of the selected oligos were 65-70° C. for the whole oligo and 50-55° C. for the hybridising region alone.

Table I shows the forward and reverse primers used for each amplification. Oligos were synthesized by a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2 ml NH₄OH, and deprotected by 5 hours incubation at 56° C. The oligos were precipitated by addition of 0.3M Na-Acetate and 2 volumes ethanol. The samples were then centrifuged and the pellets resuspended in either 100 μl or 1 ml of water. OD₂₆₀ was determined using a Perkin Elmer Lambda Bio spectrophotometer and the concentration was determined and adjusted to 2-10 pmol/μl.

TABLE I
PCR primers
ORF	Primer	Sequence	Restriction sites
ORF 38	Forward	CGCGGATCCCATATG-TCGCCGCAAAATTCCGA	BamHI-NdeI
		<SEQ ID 112>
	Reverse	CCCGCTCGAG-TTTTGCCGCGTTAAAAGC	XhoI
		<SEQ ID 113>
ORF 40	Forward	CGCGGATCCCATATG-ACCGTGAAGACCGCC	BamHI-NdeI
		<SEQ ID 114>
	Reverse	CCCGCTCGAG-CCACTGATAACCGACAGA	XhoI
		<SEQ ID 115>
ORF 41	Forward	CGCGGATCCCATATG-TATTTGAAACAGCTCCAAG	BamHI-NdeI
		<SEQ ID 116>
	Reverse	CCCGCTCGAG-TTCTGGGTGAATGTTA	XhoI
		<SEQ ID 117>
ORF 44	Forward	GCGGATCCCATATG-GGCACGGACAACCCC	BamHI-NdeI
		<SEQ ID 118>
	Reverse	CCCGCTCGAG-ACGTGGGGAACAGTCT	XhoI
		<SEQ ID 119>
ORF 51	Forward	GCGGATCCCATATG-AAAAATATTCAAGTAGTTGC	BamHI-NdeI
		<SEQ ID 120>
	Reverse	CCCGCTCGAG-AAGTTTGATTAAACCCG	XhoI
		<SEQ ID 121>
ORF 52	Forward	CGCGGATCCCATATG-TGCCAACCGCAATCCG	BamHI-NdeI
		<SEQ ID 122>
	Reverse	CCCGCTCGAG-TTTTTCCAGCTCCGGCA	XhoI
		<SEQ ID 123>
ORF 56	Forward	GCGGATCCCATATG-GTTATCGGAATATTACTCG	BamHI-NdeI
		<SEQ ID 124>
	Reverse	CCCGCTCGAG-GGCTGCAGAAGCTGG	XhoI
		<SEQ ID 125>
ORF 69	Forward	CGCGGATCCCATATG-CGGACGTGGTTGGTTTT	BamHI-NdeI
		<SEQ ID 126>
	Reverse	CCCGCTCGAG-ATATCTTCCGTTTTTTTCAC	XhoI
		<SEQ ID 127>
ORF 82	Forward	CGCGGATCCGCTAGC-GTAAATTTATTATTTTTAGAA	BamHI-NheI
		<SEQ ID 128>
	Reverse	CCCGCTCGAG-TCCAACTCATTGAAGTA	XhoI
		<SEQ ID 129>
ORF 114	Forward	CGCGGATCCCATATG-AATAAAGGTTTACATCGCAT	BamHI-NheI
		<SEQ ID 130>
	Reverse	CCCGCTCGAG-AATCGCTGCACCGGCT	XhoI
		<SEQ ID 131>
ORF 124	Forward	CGCGGATCCCATATG-ACTGCCTTTTCGACA	BamHI-NheI
		<SEQ ID 132>
	Reverse	CCCGCTCGAG-GCGTGAAGCGTCAGGA	XhoI
		<SEQ ID 133>

C) Amplification

The standard PCR protocol was as follows: 50-200 ng of genomic DNA were used as a template in the presence of 20-40 μM of each oligo, 400-8004M dNTs solution, 1×PCR buffer (including 1.5 mM MgCl₂), 2.5 units TaqI DNA polymerase (using Perkin-Elmer AmpliTaQ, GIBCO Platinum, Pwo DNA polymerase, or Tahara Shuzo Taq polymerase).

In some cases, PCR was optimised by the addition of 10 μl DMSO or 50 μl 2M betaine.

After a hot start (adding the polymerase during a preliminary 3 minute incubation of the whole mix at 95° C.), each sample underwent a double-step amplification: the first 5 cycles were performed using as the hybridization temperature the one of the oligos excluding the restriction enzymes tail, followed by 30 cycles performed according to the hybridization temperature of the whole length oligos. The cycles were followed by a final 10 minute extension step at 72° C.

The standard cycles were as follows:

	Denaturation	Hybridisation	Elongation
First 5 cycles	30 seconds	30 seconds	30-60 seconds
	95° C.	50-55° C.	72° C.
Last 30 cycles	30 seconds	30 seconds	30-60 seconds
	95° C.	65-70° C.	72° C.

The elongation time varied according to the length of the ORF to be amplified.

The amplifications were performed using either a 9600 or a 2400 Perkin Elmer GeneAmp PCR System. To check the results, 1/10 of the amplification volume was loaded onto a 1-1.5% agarose gel and the size of each amplified fragment compared with a DNA molecular weight marker.

The amplified DNA was either loaded directly on a 1% agarose gel or first precipitated with ethanol and resuspended in a suitable volume to be loaded on a 1% agarose gel. The DNA fragment corresponding to the right size band was then eluted and purified from gel, using the Qiagen Gel Extraction Kit, following the instructions of the manufacturer. The final volume of the DNA fragment was 30 μl or 500 of either water or 10 mM Tris, pH 8.5.

D) Digestion of PCR Fragments

The purified DNA corresponding to the amplified fragment was split into 2 aliquots and double-digested with:

• • NdeI/XhoI or NheI/XhoI for cloning into pET-21b+ and further expression of the protein as a C-terminus His-tag fusion
  • BamHI/XhoI or EcoRI/XhoI for cloning into pGEX-KG and further expression of the protein as N-terminus GST fusion.
  • EcoRI/PstI, EcoRI/SalI, SalI/PstI for cloning into pGex-His and further expression of the protein as N-terminus His-tag fusion

Each purified DNA fragment was incubated (37° C. for 3 hours to overnight) with 20 units of each restriction enzyme (New England Biolabs) in a either 30 or 40 μl final volume in the presence of the appropriate buffer. The digestion product was then purified using the QIAquick PCR purification kit, following the manufacturer's instructions, and eluted in a final volume of 30 or 50 μl of either water or 10 mM Tris-HCl, pH 8.5. The final DNA concentration was determined by 1% agarose gel electrophoresis in the presence of titrated molecular weight marker.

E) Digestion of the Cloning Vectors (pET22B, pGEX-KG, pTRC-His A, and pGex-His)

10 μg plasmid was double-digested with 50 units of each restriction enzyme in 200 μl reaction volume in the presence of appropriate buffer by overnight incubation at 37° C. After loading the whole digestion on a 1% agarose gel, the band corresponding to the digested vector was purified from the gel using the Qiagen QIAquick-Gel Extraction Kit and the DNA was eluted in 50 μl of 10 mM Tris-HCl, pH 8.5. The DNA concentration was evaluated by measuring OD₂₆₀ of the sample, and adjusted to 50 μg/μl. 1 μl of plasmid was used for each cloning procedure.

The vector pGEX-His is a modified pGEX-2T vector carrying a region encoding six histidine residues upstream to the thrombin cleavage site and containing the multiple cloning site of the vector pTRC99 (Pharmacia).

F) Cloning

The fragments corresponding to each ORF, previously digested and purified, were ligated in both pET22b and pGEX-KG. In a final volume of 20 μl, a molar ratio of 3:1 fragment/vector was ligated using 0.5 μl of NEB T4 DNA ligase (400 units/μl), in the presence of the buffer supplied by the manufacturer. The reaction was incubated at room temperature for 3 hours. In some experiments, ligation was performed using the Boehringer “Rapid Ligation Kit”, following the manufacturer's instructions.

In order to introduce the recombinant plasmid in a suitable strain, 100 μl E. coli DH5 competent cells were incubated with the ligase reaction solution for 40 minutes on ice, then at 37° C. for 3 minutes, then, after adding 800 μl LB broth, again at 37° C. for 20 minutes. The cells were then centrifuged at maximum speed in an Eppendorf microfuge and resuspended in approximately 200 μl of the supernatant. The suspension was then plated on LB ampicillin (100 mg/ml).

The screening of the recombinant clones was performed by growing 5 randomly-chosen colonies overnight at 37° C. in either 2 ml (pGEX or pTC clones) or 5 ml (pET clones) LB broth+100 μg/ml ampicillin. The cells were then pelletted and the DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions, to a final volume of 30 μl. 5 μl of each individual miniprep (approximately 1 g) were digested with either NdeI/XhoI or BamHI/XhoI and the whole digestion loaded onto a 1-1.5% agarose gel (depending on the expected insert size), in parallel with the molecular weight marker (1 Kb DNA Ladder, GIBCO). The screening of the positive clones was made on the base of the correct insert size.

G) Expression

Each ORF cloned into the expression vector was transformed into the strain suitable for expression of the recombinant protein product. 1 μl of each construct was used to transform 30 μl of E. coli BL21 (pGEX vector), E. coli TOP 10 (pTRC vector) or E. coli BL21-DE3 (pET vector), as described above. In the case of the pGEX-His vector, the same E. coli strain (W3110) was used for initial cloning and expression. Single recombinant colonies were inoculated into 2 ml LB+Amp (100 μg/ml), incubated at 37° C. overnight, then diluted 1:30 in 20 ml of LB+Amp (100 μg/ml) in 100 ml flasks, making sure that the OD₆₀₀ ranged between 0.1 and 0.15. The flasks were incubated at 30° C. into gyratory water bath shakers until OD indicated exponential growth suitable for induction of expression (0.4-0.8 OD for pET and pTRC vectors; 0.8-1 OD for pGEX and pGEX-His vectors). For the pET, pTRC and pGEX-His vectors, the protein expression was induced by addition of 1 mM IPTG, whereas in the case of pGEX system the final concentration of IPTG was 0.2 mM. After 3 hours incubation at 30° C., the final concentration of the sample was checked by OD. In order to check expression, 1 ml of each sample was removed, centrifuged in a microfuge, the pellet resuspended in PBS, and analysed by 12% SDS-PAGE with Coomassie Blue staining. The whole sample was centrifuged at 6000 g and the pellet resuspended in PBS for further use.

H) GST-Fusion Proteins Large-Scale Purification.

A single colony was grown overnight at 37° C. on LB+Amp agar plate. The bacteria were inoculated into 20 ml of LB+Amp liquid culture in a water bath shaker and grown overnight. Bacteria were diluted 1:30 into 600 ml of fresh medium and allowed to grow at the optimal temperature (20-37° C.) to OD₅₅₀ 0.8-1. Protein expression was induced with 0.2 mM IPTG followed by three hours incubation. The culture was centrifuged at 800 rpm at 4° C. The supernatant was discarded and the bacterial pellet was resuspended in 7.5 ml cold PBS. The cells were disrupted by sonication on ice for 30 sec at 40 W using a Branson sonifier B-15, frozen and thawed twice and centrifuged again. The supernatant was collected and mixed with 150 μl Glutatione-Sepharose 4B resin (Pharmacia) (previously washed with PBS) and incubated at room temperature for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4° C. The resin was washed twice with 10 ml cold PBS for 10 minutes, resuspended in 1 ml cold PBS, and loaded on a disposable column. The resin was washed twice with 2 ml cold PBS until the flow-through reached OD₂₈₀ of 0.02-0.06. The GST-fusion protein was eluted by addition of 70011 cold Glutathione elution buffer (10 mM reduced glutathione, 50 mM Tris-HCl) and fractions collected until the OD₂₈₀ was 0.1. 21 μl of each fraction were loaded on a 12% SDS gel using either Biorad SDS-PAGE Molecular weight standard broad range (M1) (200, 116.25, 97.4, 66.2, 45, 31, 21.5, 14.4, 6.5 kDa) or Amersham Rainbow Marker (M2) (220, 66, 46, 30, 21.5, 14.3 kDa) as standards. As the MW of GST is 26 kDa, this value must be added to the MW of each GST-fusion protein.

I) His-Fusion Solubility Analysis

To analyse the solubility of the His-fusion expression products, pellets of 3 ml cultures were resuspended in buffer M1 [500 μl PBS pH 7.2]. 25 μl lysozyme (10 mg/ml) was added and the bacteria were incubated for 15 min at 4° C. The pellets were sonicated for 30 sec at 40 W using a Branson sonifier B-15, frozen and thawed twice and then separated again into pellet and supernatant by a centrifugation step. The supernatant was collected and the pellet was resuspended in buffer M2 [8M urea, 0.5M NaCl, 20 mM imidazole and 0.1M NaH₂ PO₄] and incubated for 3 to 4 hours at 4° C. After centrifugation, the supernatant was collected and the pellet was resuspended in buffer M3 [6M guanidinium-HCl, 0.5M NaCl, 20 mM imidazole and 0.1M NaH₂PO₄] overnight at 4° C. The supernatants from all steps were analysed by SDS-PAGE.

J) His-Fusion Large-Scale Purification.

A single colony was grown overnight at 37° C. on a LB+Amp agar plate. The bacteria were inoculated into 20 ml of LB+Amp liquid culture and incubated overnight in a water bath shaker. Bacteria were diluted 1:30 into 600 ml fresh medium and allowed to grow at the optimal temperature (20-37° C.) to OD₅₅₀ 0.6-0.8. Protein expression was induced by addition of 1 mM IPTG and the culture further incubated for three hours. The culture was centrifuged at 8000 rpm at 4° C., the supernatant was discarded and the bacterial pellet was resuspended in 7.5 ml of either (i) cold buffer A (300 mM NaCl, 50 mM phosphate buffer, 10 mM imidazole, pH 8) for soluble proteins or (ii) buffer B (urea 8M, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 8.8) for insoluble proteins.

The cells were disrupted by sonication on ice for 30 sec at 40 W using a Branson sonifier B-15, frozen and thawed two times and centrifuged again.

For insoluble proteins, the supernatant was stored at −20° C., while the pellets were resuspended in 2 ml buffer C (6M guanidine hydrochloride, 100 mM phosphate buffer, 10 mM Tris-HCl, pH 7.5) and treated in a homogenizer for 10 cycles. The product was centrifuged at 13000 rpm for 40 minutes.

Supernatants were collected and mixed with 150 μl Ni²⁺-resin (Pharmacia) (previously washed with either buffer A or buffer B, as appropriate) and incubated at room temperature with gentle agitation for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4° C. The resin was washed twice with 10 ml buffer A or B for 10 minutes, resuspended in 1 ml buffer A or B and loaded on a disposable column. The resin was washed at either (i) 4° C. with 2 ml cold buffer A or (ii) room temperature with 2 ml buffer B, until the flow-through reached OD₂₈₀ of 0.02-0.06.

The resin was washed with either (i) 2 ml cold 20 mM imidazole buffer (300 mM NaCl, 50 mM phosphate buffer, 20 mM imidazole, pH 8) or (ii) buffer D (urea 8M, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 6.3) until the flow-through reached the O.D₂₈₀ of 0.02-0.06. The His-fusion protein was eluted by addition of 700 μl of either (i) cold elution buffer A (300 mM NaCl, 50 mM phosphate buffer, 250 mM imidazole, pH 8) or (ii) elution buffer B (urea 8M, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 4.5) and fractions collected until the O.D₂₈₀ was 0.1. 21 μl of each fraction were loaded on a 12% SDS gel.

K) His-Fusion Proteins Renaturation

10% glycerol was added to the denatured proteins. The proteins were then diluted to 20 μg/ml using dialysis buffer I (10% glycerol, 0.5M arginine, 50 mM phosphate buffer, 5 mM reduced glutathione, 0.5 mM oxidised glutathione, 2M urea, pH 8.8) and dialysed against the same buffer at 4° C. for 12-14 hours. The protein was further dialysed against dialysis buffer II (10% glycerol, 0.5M arginine, 50 mM phosphate buffer, 5 mM reduced glutathione, 0.5 mM oxidised glutathione, pH 8.8) for 12-14 hours at 4° C. Protein concentration was evaluated using the formula:
Protein (mg/ml)=(1.55×OD₂₈₀)−(0.76×OD₂₆₀)
L) His-Fusion Large-Scale Purification

500 ml of bacterial cultures were induced and the fusion proteins were obtained soluble in buffer M1, M2 or M3 using the procedure described above. The crude extract of the bacteria was loaded onto a Ni-NTA superflow column (Qiagen) equilibrated with buffer M1, M2 or M3 depending on the solubilization buffer of the fusion proteins. Unbound material was eluted by washing the column with the same buffer. The specific protein was eluted with the corresponding buffer containing 500 mM imidazole and dialysed against the corresponding buffer without imidazole. After each run the columns were sanitized by washing with at least two column volumes of 0.5 M sodium hydroxide and reequilibrated before the next use.

M) Mice Immunisations

20 μg of each purified protein were used to immunise mice intraperitoneally. In the case of ORF 44, CD1 mice were immunised with Al(OH)₃ as adjuvant on days 1, 21 and 42, and immune response was monitored in samples taken on day 56. For ORF 40, CD1 mice were immunised using Freund's adjuvant, rather than Al(OH)₃, and the same immunisation protocol was used, except that the immune response was measured on day 42, rather than 56. Similarly, for ORF 38, CD1 mice were immunised with Freund's adjuvant, but the immune response was measured on day 49.

N) ELISA Assay (Sera Analysis)

The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 7 ml of Mueller-Hinton Broth (Difco) containing 0.25% Glucose. Bacterial growth was monitored every 30 minutes by following OD₆₂₀. The bacteria were let to grow until the OD reached the value of 0.3-0.4. The culture was centrifuged for 10 minutes at 10000 rpm. The supernatant was discarded and bacteria were washed once with PBS, resuspended in PBS containing 0.025% formaldehyde, and incubated for 2 hours at room temperature and then overnight at 4° C. with stirring. 100 μl bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4° C. The wells were then washed three times with PBT washing buffer (0.1% Tween-20 in PBS). 200 μl of saturation buffer (2.7% Polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 200 μl of diluted sera (Dilution buffer: 1% BSA, 0.1% Tween-20, 0.1% NaN₃ in PBS) were added to each well and the plates incubated for 90 minutes at 37° C. Wells were washed three times with PBT. 100 μl of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37° C. Wells were washed three times with PBT buffer. 100 μl of substrate buffer for HRP (25 ml of citrate buffer pH5, 10 mg of O-phenildiamine and 10 μl of H₂O) were added to each well and the plates were left at room temperature for 20 minutes. 100 μl H₂SO₄ was added to each well and OD₄₉₀ was followed. The ELISA was considered positive when OD₄₉₀ was 2.5 times the respective pre-immune sera.

O) FACScan Bacteria Binding Assay Procedure.

The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 4 tubes containing 8 ml each Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD₆₂₀. The bacteria were let to grow until the OD reached the value of 0.35-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer (1% BSA, 0.4% NaN₃) and centrifuged for 5 minutes at 4000 rpm. Cells were resuspended in blocking buffer to reach OD₆₂₀ of 0.07. 100 μl bacterial cells were added to each well of a Costar 96 well plate. 100 μl of diluted (1:200) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4° C. Cells were centrifuged for 5 minutes at 4000 rpm, the supernatant aspirated and cells washed by addition of 200 μl/well of blocking buffer in each well. 100 μl of R-Phicoerytin conjugated F(ab)₂ goat anti-mouse, diluted 1:100, was added to each well and plates incubated for 1 hour at 4° C. Cells were spun down by centrifugation at 4000 rpm for 5 minutes and washed by addition of 200 μl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200 μl/well of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan setting were: FL1 on, FL2 and FL3 off; FSC-H threshold: 92; FSC PMT Voltage: E 02; SSC PMT: 474; Amp. Gains 7.1; FL-2 PMT: 539; compensation values: 0.

P) OMV Preparations

Bacteria were grown overnight on 5 GC plates, harvested with a loop and resuspended in 10 ml 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes and the bacteria disrupted by sonication for 10 minutes on ice (50% duty cycle, 50% output). Unbroken cells were removed by centrifugation at 5000 g for 10 minutes and the total cell envelope fraction recovered by centrifugation at 50000 g at 4° C. for 75 minutes. To extract cytoplasmic membrane proteins from the crude outer membranes, the whole fraction was resuspended in 2% sarkosyl (Sigma) and incubated at room temperature for 20 minutes. The suspension was centrifuged at 10000 g for 10 minutes to remove aggregates, and the supernatant further ultracentrifuged at 50000 g for 75 minutes to pellet the outer membranes. The outer membranes were resuspended in 10 mM Tris-HCl, pH8 and the protein concentration measured by the Bio-Rad Protein assay, using BSA as a standard.

Q) Whole Extracts Preparation

Bacteria were grown overnight on a GC plate, harvested with a loop and resuspended in 1 ml of 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes.

R) Western Blotting

Purified proteins (500 ng/lane), outer membrane vesicles (5 μg) and total cell extracts (25 μg) derived from MenB strain 2996 were loaded on 15% SDS-PAGE and transferred to a nitrocellulose membrane. The transfer was performed for 2 hours at 150 mA at 4° C., in transferring buffer (0.3% Tris base, 1.44% glycine, 20% methanol). The membrane was saturated by overnight incubation at 4° C. in saturation buffer (10% skimmed milk, 0.1% Triton X100 in PBS). The membrane was washed twice with washing buffer (3% skimmed milk, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37° C. with mice sera diluted 1:200 in washing buffer. The membrane was washed twice and incubated for 90 minutes with a 1:2000 dilution of horseradish peroxidase labelled anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.

S) Bactericidal Assay

MC58 strain was grown overnight at 37° C. on chocolate agar plates. 5-7 colonies were collected and used to inoculate 7 ml Mueller-Hinton broth. The suspension was incubated at 37° C. on a nutator and let to grow until OD₆₂₀ was 0.5-0.8. The culture was aliquoted into sterile 1.5 ml Eppendorf tubes and centrifuged for 20 minutes at maximum speed in a microfuge. The pellet was washed once in Gey's buffer (Gibco) and resuspended in the same buffer to an OD₆₂₀ of 0.5, diluted 1:20000 in Gey's buffer and stored at 25° C.

50 μl of Gey's buffer/1% BSA was added to each well of a 96-well tissue culture plate. 25 μl of diluted mice sera (1:100 in Gey's buffer/0.2% BSA) were added to each well and the plate incubated at 4° C. 25 μl of the previously described bacterial suspension were added to each well. 25 μl of either heat-inactivated (56° C. waterbath for 30 minutes) or normal baby rabbit complement were added to each well. Immediately after the addition of the baby rabbit complement, 22 μl of each sample/well were plated on Mueller-Hinton agar plates (time 0). The 96-well plate was incubated for 1 hour at 37° C. with rotation and then 22 μl of each sample/well were plated on Mueller-Hinton agar plates (time 1). After overnight incubation the colonies corresponding to time 0 and time 1 hour were counted.

Table II gives a summary of the cloning, expression and purification results.

TABLE II
Cloning, expression and purification
		His-fusion	GST-fusion
ORF	PCR/cloning	expression	expression	Purification
orf 38	+	+	+	His-fusion
orf 40	+	+	+	His-fusion
orf 41	+	n.d.	n.d.
orf 44	+	+	+	His-fusion
orf 51	+	n.d.	n.d.
orf 52	+	n.d.	+	GST-fusion
orf 56	+	n.d.	n.d.
orf 69	+	n.d.	n.d.
orf 82	+	n.d.	n.d.
orf 114	+	n.d.	+	GST-fusion
orf 124	+	n.d.	n.d.

Example 1

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1>:

1   ACACTGTTGT TTGCAACGGT TCAGGCAAGT GCTAACCAAT GAAGAGCAAG
51  AAGAAGATTT ATATTTAGAC CCCGTACAAC GCACTGTTGC CGTGTTGATA
101 GTCAATTCCG ATAAAGAAGG CACGGGAGAA AAAGAAAAAG TAGAAGAAAA
151 TTCAGATTGG GCAGTATATT TCAACGAGAA AGGAGTACTA ACAGCCAGAG
201 AAATCACCyT CAAAGCCGGC GACAACCTGA AAATCAAACA AAACGGCACA
251 AACTTCACCT ACTCGCTGAA AAALGACCTC ACAGATCTGA CCAGTGTTGG
301 AACTGAAAAA TTATCGTTTA GCGCAAACGG CAATAAAGTC AACATCACAA
351 GCGACACCAA AGGCTTGAAT TTTGCGAAAG AAACGGCTGG sACGAACGgC
401 GACACCACGG TTCATCTGAA CGGTATTGGT TCGACTTTGA CCGATACGCT
451 GCTGAATACC GGAGCGACCA CAAACGTAAC CAACGACAAC GTTACCGATG
501 ACGAGAAAAA ACGTGCGGCA AGCGTTAAAG ACGTATTAAA CGCTGGCTGG
551 AACATTAAAG GCGTTAAACC CGGTACAACA GCTTCCGATA ACGTTGATTT
601 CGTCCGCACT TACGACACAG TCGAGTTCTT GAGCGCAGAT ACGAAAACAA
651 CGACTGTTAA TGTGGAAAGC AAAGACAACG GCAAGAAAAC CGAAGTTAAA
701 ATCGGTGCGA AGACTTCTGT TATTAAAGAA AAAGAC...

This corresponds to the amino acid sequence <SEQ ID 2; ORF40>:

1   ..TLLFATVQAS ANQEEQEEDL YLDPVQRTVA VLIVNSDKEG TGEKEKVEEN
51  SDWAVYFNEK GVLTAREITX KAGDNLKIKQ NGTNFTYSLK KDLTDLTSVG
101 TEKLSFSANG NKVNITSDTK GLNFAKETAG TNGDTTVHLN GIGSTLTDTL
151 LNTGATTNVT NDNVTDDEKK RAASVKDVLN AGWNIKGVKP GTTASDNVDF
201 VRTYDTVEFL SADTKTITVN VESKDNGKKT EVKIGAXTSV IKEKD...

Further work revealed the complete DNA sequence <SEQ ID 3>:

1    ATGAACAAAA TATACCGCAT CATTTGGAAT AGTGCCCTCA ATGCCTGGGT
51   CGTCGTATCC GAGCTCACAC GCAACCACAC CAAACGCGCC TCCGCAACCG
101  TGAAGACCGC CGTATTGGCG ACACTGTTGT TTGCAACGGT TCAGGCAAGT
151  GCTAACAATG AAGAGCAAGA AGAAGATTTA TATTTAGACC CCGTACAACG
201  CACTGTTGCC GTGTTGATAG TCAATTCCGA TAAAGAAGGC ACGGGAGAAA
251  AAGAAAAAGT AGAAGAAAAT TCAGATTGGG CAGTATATTT CAACGAGAAA
301  GGAGTACTAA CAGCCAGAGA AATCACCCTC AAAGCCGGCG ACAACCTGAA
351  AATCAAACAA AACGGCACAA ACTTCACCTA CTCGCTGAAA AAAGACCTCA
401  CAGATCTGAC CAGTGTTGGA ACTGAAAAAT TATCGTTTAG CGCAAACGGC
451  AATAAAGTCA ACATCACAAG CGACACCAAA GGCTTGAATT TTGCGAAAGA
501  AACGGCTGGG ACGAACGGCG ACACCACGGT TCATCTGAAC GGTATTGGTT
551  CGACTTTGAC CGATACGCTG CTGAATACCG GAGCGACCAC AAACGTAACC
601  AACGACAACG TTACCGATGA CGAGAAAAAA CGTGCGGCAA GCGTTAAAGA
651  CGTATTAAAC GCTGGCTGGA ACATTAAAGG CGTTAAACCC GGTACAACAG
701  CTTCCGATAA CGTTGATTTC GTCCGCACTT ACGACACAGT CGAGTTCTTG
751  AGCGCAGATA CGAAAACAAC GACTGTTAAT GTGGAAAGCA AAGACAACGG
801  CAAGAAAACC GAAGTTAAAA TCGGTGCGAA GACTTCTGTT ATTAAAGAAA
851  AAGACGGTAA GTTGGTTACT GGTAAAGACA AAGGCGAGAA TGGTTCTTCT
901  ACAGACGAAG GCGAAGGCTT AGTGACTGCA AAAGAAGTGA TTGATGCAGT
951  AAACAAGGCT GGTTGGAGAA TGAAAACAAC AACCGCTAAT GGTCAAACAG
1001 GTCAAGCTGA CAAGTTTGAA ACCGTTACAT CAGGCACAAA TGTAACCTTT
1051 GCTAGTGGTA AAGGTACAAC TGCGACTGTA AGTAAAGATG ATCAAGGCAA
1101 CATCACTGTT ATGTATGATG TAAATGTCGG CGATGCCCTA AACGTCAATC
1151 AGCTGCAAAA CAGCGGTTGG AATTTGGATT CCAAAGCGGT TGCAGGTTCT
1201 TCGGGCAAAG TCATCAGCGG CAATGTTTCG CCGAGCAAGG GAAAGATGGA
1251 TGAAACCGTC AACATTAATG CCGGCAACAA CATCGAGATT ACCCGCAACG
1301 GTAAAAATAT CGACATCGCC ACTTCGATGA CCCCGCAGTT TTCCAGCGTT
1351 TCGCTCGGCG CGGGGGCGGA TGCGCCCACT TTGAGCGTGG ATGGGGACGC
1401 ATTGAATGTC GGCAGCAAGA AGGACAACAA ACCCGTCCGC ATTACCAATG
1451 TCGCCCCGGG CGTTAAAGAG GGGGATGTTA CAAACGTCGC ACAACTTAAA
1501 GGCGTGGCGC AAAACTTGAA CAACCGCATC GACAATGTGG ACGGCAACGC
1551 GCGTGCGGGC ATCGCCCAAG CGATTGCAAC CGCAGGTCTG GTTCAGGCGT
1601 ATTTGCCCGG CAAGAGTATG ATGGCGATCG GCGGCGGCAC TTATCGCGGC
1651 GAAGCCGGTT ACGCCATCGG CTACTCCAGT ATTTCCGACG GCGGAAATTG
1701 GATTATCAAA GGCACGGCTT CCGGCAATTC GCGCGGCCAT TTCGGTGCTT
1751 CCGCATCTGT CGGTTATCAG TGGTAA

This corresponds to the amino acid sequence <SEQ ID 4; ORF40-1>:

1   MNKIYRIIWN SALNAWVVVS ELTRNHTKRA SATVKTAVLA TLLFATVQAS
51  ANNEEQEEDL YLDFVQRTVA VLIVNSDKEG TGEKEKVEEN SDWAVYFNEK
101 GVLTAREITL KAGDNLKIKQ NGTNFTYSLK KDLTDLTSVG TEKLSFSMIG
151 NKVNITSDTK GLNFAKETAG TNGDTTVHLN GIGSTLTDTL LNTGATTNVT
201 NDNVTDDEKK RAASVKDVLN AGWNIKGVKP GTTASDNVDF VRTYDTVEFL
251 SADTKTTTVN VESKDNGKKT EVKIGAKTSV IKEKDGKLVT GKDKGENGSS
301 TDEGEGLVTA KEVIDAYNKA GWRMKTTTAN GQTGQADKFE TVTSGTNVTF
351 ASGKGTTATV SKDDQGNITV NYDVNVGDAL NVNQLQNSGW NLDSKAVAGS
401 SGKVISGNVS PSKGKMDETV NINAGNNIEI TRNGKNIDIA TSHTPQFSSV
451 SLGAGADAPT LSVDGDALNV GSKKDNKPVR ITNVAPGVKE GOVTNVAQLK
501 GVAQNLNNRI DNVDGNARAG ZAQAIATAGL VQAYLPGKSM MAIGGGTYRG
551 EAGYAIGYSS ISDGGNWIIK GTASGNSRGH FGASASVGYQ W*

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 5>:

1    ATGAACAAAA TATACCGCAT CATTTGGAAT AGTGCCCTCA ATGCCTGNGT
51   CGCCGTATCC GAGCTCACAC GCAACCACAC CAAACGCGCC TCCGCAACCG
101  TGAAGACCGC CGTATTGGCG ACACTGTTGT TTGCAACGGT TCAGGCGAAT
151  GCTACCGATG AAGATGAAGA AGAAGAGTTA GAATCCGTAC AACGCTCTGT
201  CGTAGGGAGC ATTCAAGCCA GTATGGAAGG CAGCGGCGAA TTGGAAACGA
251  TATCATTATC AATGACTAAC GACAGCAAGG AATTTGTAGA CCCATACATA
301  GTAGTTACCC TCAAAGCCGG CGACAACCTG AAAATCAAAC AAAACACCAA
351  TGAAAACACC AATGCCAGTA GCTTCACCTA CTCGCTGAAA AAAGACCTCA
401  CAGGCCTGAT CAATGTTGAN ACTGAAAAAT TATCGTTTGG CGCAAACGGC
451  AAGAAAGTCA ACATCATAAG CGACACCAAA GGCTTGAATT TCGCGAAAGA
501  AACGGCTGGG ACGAACGGCG ACACCACGGT TCATCTGAAC GGTATCGGTT
551  CGACTTTGAC CGATACGCTT GCGGGTTCTT CTGCTTCTCA CGTTGATGCG
601  GGTAACCNAA GTACACATTA CACTCGTGCA GCAAGTATTA AGGATGTGTT
651  GAATGCGGGT TGGAATATTA AGGGTGTTAA ANNNGGCTCA ACAACTGGTC
701  AATCAGAAAA TGTCGATTTC GTCCGCACTT ACGACACAGT CGAGTTCTTG
751  AGCGCAGATA CGNAAACAAC GACNGTTAAT GTGGAAAGCA AAGACAACGG
801  CAAGAGAACC GAAGTTAAAA TCGGTGCGAA GACTTCTGTT ATTAAAGAAA
851  AAGACGGTAA GTTGGTTACT GGTAAAGGCA AAGGCGAGAA TGGTTCTTCT
901  ACAGACGAAG GCGAAGGCTT AGTGACTGCA AAAGAAGTGA TTGATGCAGT
951  AAACAAGGCT GGTTGGAGAA TGAAAACAAC AACCGCTAAT GGTCAAACAG
1001 GTCAAGCTGA CAAGTTTGAA ACCGTTACAT CAGGCACAAA TGTAACCTTT
1051 GCTAGTGGTA AAGGTACAAC TGCGACTGTA AGTAAAGATG ATCAAGGCAA
1101 CATCACTGTT ATGTATGATG TAAATGTCGG CGATGCCCTA AACGTCAATC
1151 AGCTGCAAAA CAGCGGTTGG AATTTGGATT CCAAAGCGGT TGCAGGTTCT
1201 TCGGGCAAAG TCATCAGCGG CAATGTTTCG CCGAGCAAGG GAAAGATGGA
1251 TGAAACCGTC AACATTAATG CCGGCAACAA CATCGACATT AGCCGCAACG
1301 GTAAAAATAT CGACATCGCC ACTTCGATGG CGCCGCAGTT TTCCAGCGTT
1351 TCGCTCGGCG CGGGGGCAGA TGCGCCCACT TTAAGCGTGG ATGACGAGGG
1401 CGCGTTGAAT GTCGGCAGCA AGGATGCCAA CAAACCCGTC CGCATTACCA
1451 ATGTCGCCCC GGGCGTTAAA GANGGGGATG TTACAAACGT CNCACAACTT
1501 AAAGGCGTGG CGCAAAACTT GAACAACCGC ATCGACAATG TGGACGGCAA
1551 CGCGCGTGCN GGCATCGCCC AAGCGATTGC AACCGCAGGT CTGGTTCAGG
1601 CGTATCTGCC CGGCAAGAGT ATGATGGCGA TCGGCGGCGG CACTTATCGC
1651 GGCGAAGCCG GTTACGCCAT CGGCTACTCC AGTATTTCCG ACGGCGGAAA
1701 TTGGATTATC AAAGGCACGG CTTCCGGCAA TTCGCGCGGC CATTTCGGTG
1751 CTTCCGCATC TGTCGGTTAT CAGTGGTAA

This encodes a protein having amino acid sequence <SEQ ID 6; ORF40a>:

1   MNKIYRIIWN SALNPXVAVS ELTRNHTKRA SATVKTAVLA TLLFATVQAN
51  ATDEDEKEEL ESVQRSVVGS IQASMEGSGE LETISLSHTN DSKEFVDPYI
101 VVTLKAGDNL KIKONTHENT NASSFTYSLK KDLTGLINVX TEKLSFGANG
151 KKVNIISDTK GLNFAXETAG TNGDTTVHLN GIGSTLTDTL AGSSASHVDA
201 GNXSTHYTRA ASIKDVLNAG WNIKGVKXGS TTGQSENVDF VRTYDTVEFL
251 SADTXTTTVN VESKDNGKRT EVXIGAXTSV IKEKDGKLVT GKGKGENGSS
301 TDEGEGLVTA KEVIDAVNKA GWRMKTTTAN GQTGQADKFE TVTSGTNVTF
351 ASGKGTTATV SXDDQGNITV MYDVNVGDAL NVNQLONSGW NLDSKAVAGS
401 SGKVISGNVS PSKGKMDETV NINAGNNIEI SRNGKNIDIA TSMAPQFSSV
451 SLGAGADAPT LSVDDEGALN VGSKDANKPV RITNVAPGVK XGDVTNVXQL
501 KGVAQNLNNR IDNYOGNARA GIAQAIATAG LVQAYLPGKS NNAIGGGTYR
551 GEAGYAIGYS SISDGGNWII KGTASGNSRG HFGASASVGY QW

The originally-identified partial strain B sequence (ORF40) shows 65.7% identity over a 254 aa overlap with ORF40a:

          10        20        30
orf40.pep TLLFATVQASANQEEQEEDLYLDPVQRTVA
          |||||||||:|::|::||:|  : |||:|
orf40a    SALNAXVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL--ESVQRSV-
          20        30        40        50        60
          40        50        60        70        80
orf40.pep VLIVNSDKEGTGEKEKVEEN-SDWAVYFNEKGVLTAREITXKAGDNLKIKQN------GT
          |  :::: ||:|| | :  : :: :  | :  ::    :| |||||||||||      ::
orf40a    VGSIQASMEGSGELETISLSMTNDSKEFVDPYIV----VTLKAGDNLKIKQNTNENTNAS
          70        80        90       100       110       120
          90       100       110       120       130       140
orf40.pep NFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLNGIG
          :|||||||||| | :| ||||||:|||:|||| |||||||||||||||||||||||||||
orf40a    SFTYSLKKDLTGLINVXTEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLNGIG
          130       140       150       160       170       180
          150       160       170       180       190       200
orf40.pep STLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTA--SDNVDFV
          ||||||| :::|: :|   | :  :  ||||:|||||||||||||| |:|:  |:|||||
orf40a    STLTDTLAGSSAS-HVDAGNXST-HYTRAASIKDVLNAGWNIKGVKXGSTTGQSENVDFV
          190       200       210       220       230       240
          210       220       230       240
orf40.pep RTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKD
          ||||||||||||| |||||||||||||:||||||||||||||||
orf40a    RTYDTVEFLSADTXTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSST
          250       260       270       280       290       300

The complete strain B sequence (ORF44-1) and ORF40a show 83.7% identity in 601 aa overlap:

            10        20        30        40        50        60
orf40-1.pep MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL
            ||||||||||||||| |:|||||||||||||||||||||||||||||||:|::|::||:|
orf40a      MNKIYRIIWNSALNAXVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
            10        20        30        40        50        60
            70        80        90       100       110       119
orf40-1.pep YLDPVQRTVAVLIVNSDKEGTGEKEKVEEN-SDWAVYFNEKGVLTAREITLKAGDNLKIK
            : |||:| |  :::: ||:|| | :  : :: :  | :  ::    :|||||||||||
orf40a      --ESVQRSV-VGSIQASMEGSGELETISLSMTNDSKEFVDPYIV----VTLKAGDNLKIK
            70        80        90       100       110
            120             130       140       150       160       170
orf40-1.pep QN------GTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNG
            ||      :::||||||||| | :| ||||||:|||:|||| |||||||||||||||||
orf40a      QNTNENTNASSFTYSLKKDLTGLINVXTEKLSFGANGKKVNIISDTKGLNFAKETAGTNG
            120       130       140       150       160       170
            180       190       200       210       220       230
orf40-1.pep DTTVHLNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTT
            ||||||||||||||||| :::|: :|   | :  :  ||||:|||||||||||||| |:|
orf40a      DTTVHLNGIGSTLTDTLAGSSAS-HVDAGNXST-HYTRAASIKDVLNAGWNIKGVKXGST
            180       190       200       210       220       230
            240       250       260       270       280       290
orf40-1.pep A--SDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTG
            :  |:|||||||||||||||||| |||||||||||||:||||||||||||||||||||||
orf40a      TGQSENVDFVRTYDTVEFLSADTXTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTG
            240       250       260       270       280       290
            300       310       320       330       340       350
orf40-1.pep KDKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFA
            | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf40a      KGKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFA
            300       310       320       330       340       350
            360       370       360       390       400       410
orf40-1.pep SGKGTTATVSKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSP
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf40a      SGKGTTATVSKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSP
            360       370       360       390       400       410
            420       430       440       450       460       470
orf40-1.pep SKGKMDETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGD-ALNV
            |||||||||||||||||||:||||||||||||:|||||||||||||||||||| : ||||
orf40a      SKGKMDETVNINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNV
            420       430       440       450       460       470
            480       490       500       510       520       530
orf40-1.pep GSKKDNKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGL
            |||  |||||||||||||| |||||| |||||||||||||||||||||||||||||||||
orf40a      GSKDANKPVRITNVAPGVKXGDVTNVXQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGL
            480       490       500       510       520       530
            540       550       560       570       580       590
orf40-1.pep VQAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQ
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf40a      VQAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQ
            540       550       560       570       580       590
orf40-1.pep WX
            ||
orf40a      WX

Computer analysis of these amino acid sequences gave the following results:

Homology with Hsf Protein Encoded by the Type b Surface Fibrils Locus of H. influenzae (Accession Number U41852)

ORF40 and Hsf protein show 54% aa identity in 251 aa overlap:

Orf40 1 TLLFATVQASANQEEQEEDLYLDPVQRTVAVLVINSDXXXXXXXXXXXXNSDWAVYFNEK 60
      TLLFATVQA+A  E++E    LDPV RT  VL  +SD            NS+W +YF+ K
Hsf   41 TLLFATVQANATDEDEE----LDPVVRTAPVLSFHSDKEGTGEKEVTE-NSNWGIYFDNK 95
Orf40 61 GVLTAREITXKAGDNLKIKQN------GTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVN 114
      GVL A  IT KAGDNLKIKQN       ++FTYSLKKDLTDLTSV TEKLSF ANG+KV+
Hsf   96 GVLKAGAITLKAGDNLKIKQNTDESTNASSFTYSLKKDLTDLTSVATEKLSFGANGDKVD 155
Orf40 115 ITSDTKGLNFAKETAGTNGDTTVhLNGIGSTLTDTLLNTGAXXXXXXXXXXXXEKKRAAS 174
      ITSD  GL  AK      G+  VHLNG+ STL D + NTG             EK RAA+
Hsf   156 ITSDANGLKLAK-----TGNGNVHLNGLDSTLPDAVTNTGVLSSSSFTPNDV-EKTRAAT 209
Orf40 175 VKDVLNAGWNIKGVKPGTTASDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKI 234
      VKDVLNAGWNIKG K      ++VD V  Y+ VEF++ D  T  V ++K+NGK TEVK
Hsf   210 VKDVLNAGWNIKGAKTAGGNVESVDLVSAYNNVEFITGDKNTLDVVLTAKENGKTTEVKF 269
Orf40 235 GAKTSVIKEKD 245
      KTSVIKEKD
Hsf   270 TPKTSVIKEKD 280

ORF40a also shows homology to Hsf:

gi|1666683 (U41852) hsf gene product [Haemophilus influenzae] Length = 2353
Score = 153 (67.7 bits), Expect = 1.5−116, Sum P(11) = 1.5e−116
Identities = 33/36 (91%), Positives = 34/36 (94%)
Query:   16 VAVSELTRNHTKRASATVKTAVLATLLFATVQANAT 51
V VSELTR HTKRASATV+TAVLATLLFATVQNAT
Sbjct:   17 VVVSELTRTHTKRASATVETAVLATLLFATVQANAT 52
Score = 161 (71.2 bits), Expect = 1.5e−116, Sum P(11) 1.5e−116
Identities = 32/38 (84%), Positives = 36/38 (94%)
Query:  101 VTLKAGDNLKIKQNTNENTNASSFTYSLKKDLTGLINV 138
+TLAGDNLKIKQNT+E+TNASSFTYSLKKDLT L +V
Sbjct:  103 ITLKAGDNLKIKQNTDESTNASSFFYSLKKDLTDLTSV 140
Score = 110 (48.7 bits), Expect = 1.5e−116, Sum P(11) = 1.5e−116
Identities = 21/29 (72%), Positives = 25/29 (86%)
Query:  138 VTEKLSFGANGKKVNIISDTKGLNFAKET 166
V++KLS G NG KVNI SDTKGLNFAK++
Sbjct: 1439 VSDKLSLGTNGNKVNITSDTXGLNFAKDS 1467
Score = 85 (37.6 bits), Expect = 1.5e−116, Sum P(11) = 1.5e−116
Identities = 18/32 (56%), Positives = 20/32 (62%)
Query:  169 TNGDTTVHLNGIGSTLTDTLAGSSASHVDAGN 200
T  D  +HLNGI STLTDTL  S A+    GN
Sbjct: 1469 TGDDANIHLNGIASTLTDTLLNSGATTNLGGN 1500
Score = 92 (40.7 bits), Expect = 1.5e−116, Sum P(11) = 1.5e−116
Identities = 16/19 (84%), Positives = 19/19 (100%)
Query:  206 RAASIKOVLNAGWNIKGVK 224
RAAS+KDVLNAGWN++GVK
Sbjct: 1509 RAASVKDVLNAGWNVRGVK 1527
Score = 90 (39.8 bits), Expect = 1.5e−116, Sum P(11) = 1.5e−116
Identities = 17/28 (60%), Positives 20/28 (71%)
Query:  226 STTGQSENVDFVRTYDTVEFLSADTTTT 253
S   Q EN+DFV TYDTV+F+S D  TT
Sbjct: 1530 SANNQVENIDFVATYDTVDFVSGDKDTT 1557

Based on homology with Hsf, it was predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

ORF40-1 (61 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 1A shows the results of affinity purification of the His-fusion protein, and FIG. 1B shows the results of expression of the GST-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for FACS analysis (FIG. 1C), a bactericidal assay (FIG. 1D), and ELISA (positive result). These experiments confirm that ORF40-1 is a surface-exposed protein, and that it is a useful immunogen.

FIG. 1E shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF40-1.

Example 2

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 7>

1   ATGTFACGTt TGACTGCtTT AGCCGTATGC ACCGCCCTCG CTTTGGGCGC
51  GTGTT~GCCG CAAAATTCCG ACTCTGCCCC ACAAGCCAAA GaACAGGCGG
101 TTTCCGCCGC ACAAACCGAA GgCGCGTCCG TTACCGTCAA AACCGCGCGC
151 GGCGACGTTC AAATACCGCA AAACCCCGAA CGCATCGCCG TTTACGATTT
201 GGGTATGCTC GACACCTTGA GCAAACTGGG CGTGAAAACC GGTTTGTCCG
251 TCGATAAAAA CCGCCTGCCG TATTTAGAGG AATATTTCAA AACGACAAAA
301 CCTGCcGGCA CTTTGTTCGA GCCGGATTAC GAAACGCTCA ACGCTTACAA
351 ACCGCAGCTC ATCATCATCG GCAGCCGCGC CgCCAAGGCG TTTGACAAAT
401 TGAAcGAAAT CGCGCCGACC ATCGrmwTGA CCGCCGATAC CGCCAACCTC
451 AAAGAAAGTG CCAArGAGGC ATCGACGCTG GCGCAAATCT TC..

This corresponds to the amino acid sequence <SEQ ID 8; ORF38>:

1   MLRLTALAVC TALALGACSP QNSDSAPOAK EQAVSAAQTE GASVTVKTAR
51  GDVQIPQNPE RIAVYDLGHL DTLSKLGVKT GLSVDKNRLP YLEEYFKTTK
101 PAGTLFEPDY ETLNAYKPQL IIIGSRAAKA FDKLNEIAPT IXXTADTANL
151 KESAKEASTL AQIF..

Further work revealed the complete nucleotide sequence <SEQ ID 9>:

1   ATGTTACGTT TGACTGCTTT AGCCGTATGC ACCGCCCTCG CTTTGGGCGC
51  GTGTTCGCCG CAAAATTCCG ACTCTGCCCC ACAAGCCAAA GAACAGGCGG
101 TTTCCGCCGC ACAAACCGAA GGCGCGTCCG TTACCGTCAA AACCGCGCGC
151 GGCGACGTTC AAATACCGCA AAACCCCGAA CGCATCGCCG TTTACGATTT
201 GGGTATGCTC GACACCTTGA GCAAACTGGG CGTGAAAACC GGTTTGTCCG
251 TCGATAAAAA CCGCCTGCCG TATTTAGAGG AATATTTCAA AACGACAAAA
301 CCTGCCGGCA CTTTGTTCGA GCCGGATTAC GAAACGCTCA ACGCTTACAA
351 ACCGCAGCTC ATCATCATCG GCAGCCGCGC CGCCAAGGCG TTTGACAAAT
401 TGAACGAAAT CGCGCCGACC ATCGAAATGA CCGCCGATAC CGCCAACCTC
451 AAAGAAAGTG CCAAAGAGCG CATCGACGCG CTGGCGCAAA TCTTCGGCAA
501 ACAGGCGGAA GCCGACAAGC TGAAGGCGGA AATCGACGCG TCTTTTGAAG
551 CCGCGAAAAC TGCCGCACAA GGTAAGGGCA AAGGTTTGGT GATTTTGGTC
601 AACGGCGGCA AGATGTCGGC TTTCGGCCCG TCTTCACGCT TGGGCGGCTG
651 GCTGCACAAA GACATCGGCG TTCCCGCTGT CGATGAATCA ATTAAAGAAG
701 GCAGCCACGG TCAGCCTATC AGCTTTGAAT ACCTGAAAGA GAAAAATCCC
751 GACTGGCTGT TTGTCCTTGA CCGAAGCGCG GCCATCGGCG AAGAGGGTCA
801 GGCGGCGAAA GACGTGTTGG ATAATCCGCT GGTTGCCGAA ACAACCGCTT
851 GGAAAAAAGG ACAGGTCGTG TACCTCGTTC CTGAAACTTA TTTGGCAGCC
901 GGTGGCGCGC AAGAGCTGCT GAATGCAAGC AAACAGGTTG CCGACGCTTT
951 TAACGCGGCA AAATAA

This corresponds to the amino acid sequence <SEQ ID 10; ORF38-1>:

1   MLRLTALAVC TALALGACSP QNSDSAPQAK EQAVSAAQTE GASVTVKTAR
51  GDVQIPQNPE RIAVYDLQIL DTLSXLGVKT GLSVDKNRLP YLEEYFKTTK
101 PAGTLFEPDY ETLNAYKPQL IIIGSRAAKA FDKLNEIAPT IENTADTANL
151 KESAKERIDA LAQIFGKQAE ADKLKAEIDA SFEAAKTAAQ GKGKGLVILV
201 NGGKMSAFGP SSRLGGWLKK DIGVPAVDES IKEGSHGQPI SFEYLKEKNP
251 DWLFVLDRSA AIGEEGQAAK DVLDNPLVAE TTAWKKGQVV YLVPETYLAA
301 GGAQELLNAS KQVADAFNAA K*

Computer analysis of this amino acid sequence reveals a putative prokaryotic membrane lipoprotein lipid attachment site (underlined).

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 11>:

1   ATGTTACGTT TGACTGCTTT AGCCGTATGC ACCGCCCTCG CTTTGGGCGC
51  GTGTTCGCCG CAAAATTCCG ACTCTGCCCC ACAAGCCAAA GAACAGGCGG
101 TTTCCGCCGC ACAATCCGAA GGCGTGTCCG TTACCGTCAA AACGGCGCGC
151 GGCGATGTTC AAATACCGCA AAACCCCGAA CGTATCGCCG TTTACGATTT
201 GGGTATGCTC GACACCTTGA GCAAACTGGG CGTGAAAACC GGTTTGTCCG
251 TCGATAAAAA CCGCCTGCCG TATTTAGAGG AATATTTCAA AACGACAAAA
301 CCTGCCGGAA CTTTGTTCGA GCCGGATTAC GAAACGCTCA ACGCTTACAA
351 ACCGCAGCTC ATCATCATCG GCAGCCGCGC AGCCAAAGCG TTTGACAAAT
401 TGAACGAAAT CGCGCCGACC ATCGAAATGA CCGCCGATAC CGCCAACCTC
451 AAAGAAAGTG CCAAAGAGCG TATCGACGCG CTGGCGCAAA TCTTCGGCAA
501 AAAGGCGGAA GCCGACAAGC TGAAGGCGGA AATCGACGCG TCTTTTGAAG
551 CCGCGAAAAC TGCCGCGCAA GGCAAAGGCA AGGGTTTGGT GATTTTGGTC
601 AAcGGCGGCA AGATGTCCGC CTTCGGCCCG TCTTCACGAC TGGGCGGCTG
651 GCTGCACAAA GACATCGGCG TTCCCGCTGT TGACGAAGCC ATCAAAGAAG
701 GCAGCCACGG TCAGCCTATC AGCTTTGAAT ACCTGAAAGA GAAAAATCCC
751 GACTGGCTGT TTGTCCTTGA CCGCAGCGCG GCCATCGGCG AAAAGGGTCA
601 GGCGGCGAAA GACGTGTTGA ACAATCCGCT GGTTGCCGAA ACAACCGCTT
851 GGAAAAATGG ACAAGTCGTT TACCTTGTTC CTGAAACTTA TTTGGCAGCC
901 GGTGGCGCGC AAGAGCTACT GAATGCAAGC AAACAGGTTG CCGACGCTTT
951 TAACGCGGCA AAATAA

This encodes a protein having amino acid sequence <SEQ ID 12; ORF38a>:

1   MLRLTALAVC TALALGACSP QNSDSAPOAK EQAVSAAQSE GVSVTVKTAR
51  GDVQIPQNPE RIAVYDLGHL DTLSKLGVKT GLSVDKNRLP YLEEYFKTTK
101 PAGTLFEPDY ETLNAYKPQL IIIGSRAAKA FDKLNEIAPT IENTADTANL
151 KESAKERIDA LAOIFGKKAE ADKLKAEIDA SFEAAKTAAQ GKGKGLVILV
201 NGGKMSAFGP SSRLGGWLHK DIGVPAVDEA IKEGSHGQPI SFEYLKEKNP
251 DWLFVLDRSA AIGEEGQAAK DVLNNPLVAE TTAWKKGQVV YLVPETYLAA
301 GGAQELLNAS KQVAOAFWAA K*

The originally-identified partial strain B sequence (ORF38) shows 95.2% identity over a 165 aa overlap with ORF38a:

          10        20        30        40        50        60
orf38.pep MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQTEGASVTVKTARGDVQIPQNPE
          ||||||||||||||||||||||||||||||||||||||:||:||||||||||||||||||
orf38a    MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQSEGVSVTVKTARGDVQIPQNPE
          10        20        30        40        50        60
          70        80        90       100       110       120
orf38.pep RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL
          ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf38a    RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL
          70        80        90       100       110       120
          130       140       150       160
orf38.pep IIIGSRAAKAFDKLNEIAPTIXXTADTANLKESAKE-ASTLAQIF
          |||||||||||||||||||||  |||||||||||||  ::|||||
orf39a    IIIGSRAAKAFDKLNEIAPTIEMTADTANLKESAKERIDALAQIFGKKAEADKLKAEIDA
          130       140       150       160
orf38a    SFEAAKTAAQGKGKGLVILVNGGKMSAFGPSSRLGGWLHKDIGVPAVDEAIKEGSHGQPI
          190       200       210       220       230       240

The complete strain B sequence (ORF38-1) and ORF38a show 98.4% identity in 321 aa overlap:

orf38a.pep MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQSEGVSVTVKTARGDVQIPQNPE
           ||||||||||||||||||||||||||||||||||||||:||:||||||||||||||||||
orf38-1    MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQTEGASVTVKTARGDVQIPQNPE
orf38a.pep RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf38-1    RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL
orf38a.pep IIIGSRAAKAFDKLNEIAPTIEMTADTANLKESAKERIDALAQIFGKKAEADKLKAEIDA
           |||||||||||||||||||||||||||||||||||||||||||||||:||||||||||||
orf38-1    IIIGSRAAKAFDKLNEIAPTIEMTADTANLKESAKERIDALAQIFGKQAEADKLKAEIDA
orf38a.pep SFEAAKTAAQGKGKGLVILVNGGKMSAFGPSSRLGGWLHKDIGVPAVDEAIKEGSHGQPI
           |||||||||||||||||||||||||||||||||||||||||||||||||:||||||||||
orf38-1    SFEAAKTAAQGKGKGLVILVNGGKMSAFGPSSRLGGWLHKDIGVPAVDESIKEGSHGQPI
orf38a.pep SFEYLKEKNPDWLFVLDRSAAIGEEGQAAKDVLNNPLVAETTAWKKGQVVYLVPETYLAA
           |||||||||||||||||||||||||||||||||:||||||||||||||||||||||||||
orf38-1    SFEYLKEKNPDWLFVLDRSAAIGEEGQAAKDVLDNPLVAETTAWKKGQVVYLVPETYLAA
orf38a.pep GGAQELLNASKQVADAFNAAK
           |||||||||||||||||||||
orf38-1    GGAQELLNASKQVADAFNAAK

Computer analysis of these sequences revealed the following:

Homology with a Lipoprotein (lipo) of C. jejuni (Accession Number X82427)

ORF38 and lipo show 38% aa identity in 96 aa overlap:

Orf38: 40 EGASVTVKTARGDVQIPQNPERIAVYDLGMLDTLSKLGVKTGLS-VKDNRLPYLEEYFKT 98
       EG S  VK  + G+ + P+NP  ++ + DLG+LDT   L +   ++ V    LP   + FK
Lipo:  51 EGDSFLVKDSLGENKTPKNPSKVVILDLGILDTFDALKLNDKVAGVPAKNLPKYLQQFKN 110
Orf38: 99 TKPAGTLFEPDYETLNAYKPQLIIIGSRAAKAFDKL 134
       G + + D+E +NA KP LIII  R +K +DKL
Lipo:  111 KPSVGGVQQVDFEAINALKPDLIIISGRQSKFYDKL 146

Based on this analysis, it was predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

ORF38-1 (32 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 2A shows the results of affinity purification of the His-fusion protein, and FIG. 2B shows the results of expression of the GST-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for Western blot analysis (FIG. 2C) and FACS analysis (FIG. 2D). These experiments confirm that ORF38-1 is a surface-exposed protein, and that it is a useful immunogen.

FIG. 2E shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF38-1.

Example 3

The following N. meningitidis DNA sequence was identified <SEQ ID 13>:

1   ATGAAACTTC TGACCACCGC AATCCTGTCT TCCGCAATCG CGCTCAGCAG
51  TATGGCTGCC GCCGCTGGCA CGGACAACCC CACTGTTGCA AAAAAAACCG
101 TCAGCTACGT CTGCCAGCAA GGTAAAAAAG TCAAAGTAAC CTACGGCTTC
151 AACAAACAGG GTCTGACCAC ATACGCTTCC GCCGTCATCA ACGGCAAACG
201 CGTGCAAATG CCTGTCAATT TGGACAAATC CGACAATGTG GAAACATTCT
251 ACGGCAAAGA AGGCGGTTAT GTTTTGGGTA CCGGCGTGAT GGATGGCAAA
301 TCCTACCGCA AACAGCCCAT TATGATTACC GCACCTGACA ACCAAATCGT
351 CTTCAAAGAC TGTTCCCCAC GTTAA

This corresponds to the amino acid sequence <SEQ ID 14; ORF44>:

1   MKLLTTAILS SAIALSSMAA AAGTDWPTVA KKTVSYVCQQ GKKVKVTYGF
51  NKQGLTTYAS AVINGKRVQH PVNLDKSDNV ETFYGKEGGY VLGTGVMDGK
101 SYRKQPIHIT APDNQIVFKD CSPR*

Computer analysis of this amino acid sequence predicted the leader peptide shown underlined.

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 15>:

1   ATGAAACTTC TGACCACCGC AATCCTGTCT TCCGCAATCG CGCTCAGCAG
51  TATGGCTGCT GCTGCCGGCA CGAACAACCC CACCGTTGCC AAAAAAACCG
101 TCAGCTACGT CTGCCAGCAA GGTAAAAAAG TCAAAGTAAC CTACGGCTTT
151 AACAAACAGG GCCTGACCAC ATACGCTTCC GCCGTCATCA ACGGCAAACG
201 TGTGCAAATG CCTGTCAATT TGGACAAATC CGACAATGTG GAAACATTCT
251 ACGGCAAAGA AGGCGGTTAT GTTTTGGGTA CCGGCGTGAT GGATGGCAAA
301 TCCTATCGCA AACAGCCTAT TATGATTACC GCACCTGACA ACCAAATCGT
351 CTTCAAAGAC TGTTCCCCAC GTTAA

This encodes a protein having amino acid sequence <SEQ ID 16; ORF44a>:

1   MKLLTTAILS SAIALSSMAA AAGTNNPTVA KKTVSYVCQQ GKKVKVTYGF
51  NKQGLTTYAS AVINGKRVQM PVNLDKSDNV ETFYGKEGGY VLGTGVMDGK
101 SYRKQPIMIT APDNQIVFKD CSPR*

The strain B sequence (ORF44) shows 99.2% identity over a 124 aa overlap with ORF44a:

          10        20        30        40        50        60
orf44.pep MKLLTTAILSSAIALSSMAAAAGTDNPTVAKKTVSYVCQQGKKVKVTYGFNKQGLTTYAS
          ||||||||||||||||||||||||:|||||||||||||||||||||||||||||||||||
orf44a    MKLLTTAILSSAIALSSMAAAAGTNNPTVAKKTVSYVCQQGKKVKVTYGFNKQGLTTYAS
          10        20        30        40        50        60
70        80        90       100       110       120
orf44.pep AVINGKRVQMPVNLDKSDNVETFYGKEGGYVLGTGVMDGKSYRKQPIMITAPDNQIVFKD
          ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf44a    AVINGKRVQMPVNLDKSDNVETFYGKEGGYVLGTGVMDGKSYRKQPIMITAPDNQIVFKD
          70        80        90       100       110       120
orf44.pep CSPRX
          |||||
orf44a    CSPRX

Computer analysis gave the following results:

Homology with the LecA Adhesin of Eikenella corrodens (Accession Number D78153)

ORF44 and LecA protein show 45% aa identity in 91 aa overlap:

Orf44 33 TVSYVCQQGKKVKVTYGFNKQGLTTYASAVINGKRVQMPVNLDKSDNVETFYGKEGGYVL 92
      +V+YVCQQG+++ V Y FN  G+ T A   +N + +++P NL  SDNV+T +    GY L
LecA  135 SVAYVCQQGRRLNVNYRFNSAGVPTSAELRVNNRNLRLPYNLSASDNVDTVF-SANGYRL 193
Orf44 93 GTGVHDGKSYRKQPIHITAPDNQIVFKDCSP 123
      T  MD  +YR Q I+++AP+ Q+++KDCSP
LecA  194 TTNAMDSANYRSQDIIVSAPNGQNLYKDCSP 224

Based on homology with the adhesin, it was predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

ORF44-1 (11.2 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 3A shows the results of affinity purification of the His-fusion protein, and FIG. 3B shows the results of expression of the GST-fusion in E-coli. Purified His-fusion protein was used to immunise mice, whose sera were used for ELISA, which gave positive results, and for a bactericidal assay (FIG. 3C). These experiments confirm that ORF44-1 is a surface-exposed protein, and that it is a useful immunogen.

FIG. 3D shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF44-1.

Example 4

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 17>

1   GGCACCGAAT TCAAAACCAC CCTTTCCGGA GCCGACATAC AGGCAGGGGT
51  GGGTGAAAAA GCCCGAGCCG ATGCGAAAAT TATCCTAAAA GGCATCGTTA
101 ACCGCATCCA AACCGAAGAA AAGCTGGAAT CCAACTCGAC CGTATGGCAA
151 AAGCAGGCCG GAAGCGGCAG CACGGTTGAA ACGCTGAAGC TACCGAGCTT
201 TGAAGGGCCG GCACTGCCTA AGCTGACCGC TCCCGGCGGC TATATCGCCG
251 ACATCCCCAA AGGCAACCTC AAAACCGAAA TCGAAAAGCT GGCCAAACAG
301 CCCGAATATG CCTATCTGAA ACAGCTTCAG ACGGTCAAGG ACGTGAACTG
351 GAACCAAGTA CAGCTCGCTT ACGACAAATG GGACTATAAA CAGGAAGGCC
401 TAACCGGAGC CGGAGCCGCA ATTANCGCAC TGGCCGTTAC CGTGGTCACC
451 TCAGGCGCAG GAACCGGAGC CGTATTGGGA TTAANACGNG TGGCCGCCGC
501 CGCAACCGAT GCAGCATTT...

This corresponds to the amino acid sequence <SEQ ID 18; ORF49>:

1   GTEFKTTLSG ADIQAGVGEK ARADPKIILK GIVNRIQTEE KLESNSTVWQ
51  KQAGSGSTVE TLKLPSFEGP ALPKLTAPGG YIADIPKGNL KTEIEKLAKQ
101 PEYAYLKQLQ TVKDVNWNQV QLAYDKWDYK QEGLTCAGAA IXALAVTVVT
151 SGAGTGAVLG LXRVAAAATD AAF..

Further work revealed the complete nucleotide sequence <SEQ ID 19>:

1    ATGCAACTGC TGGCAGCCGA AGGCATTCAC CAACACCAAT TGAATGTTCA
51   GAAAAGTACC CGTTTCATCG GCATCAAAGT GGGTAAAAGC AATTACAGCA
101  AAAACGAGCT GAACGAAACC AAACTGCCCG TACGCGTTAT CGCCCAAACA
151  GCCAAAACCC GTTCCGGCTG GGATACCGTA CTCGAAGGCA CCGAATTCAA
201  AACCACCCTT TCCGGAGCCG ACATACAGGC AGGGGTGGGT GAAAAAGCCC
251  GAGCCGATGC GAAAATTATC CTAAAAGGCA TCGTTAACCG CATCCAAACC
301  GAAGAAAAGC TGGAATCCAA CTCGACCGTA TGGCAAAAGC AGGCCGGAAG
351  CGGCAGCACG GTTGAAACGC TGAAGCTACC GAGCTTTGAA GGGCCGGCAC
401  TGCCTAAGCT GACCGCTCCC GGCGGCTATA TCGCCGACAT CCCCAAAGGC
451  AACCTCAAAA CCGAAATCGA AAAGCTGGCC AAACAGCCCG AATATGCCTA
501  TCTGAAACAG CTTCAGACGG TCAAGGACGT GAACTGGAAC CAAGTACAGC
551  TCGCTTACGA CAAATGGGAC TATAAACAGG AAGGCCTAAC CGGAGCCGGA
601  GCCGCAATTA TCGCACTGGC CGTTACCGTG GTCACCTCAG GCGCAGGAAC
651  CGGAGCCGTA TTGGGATTAA ACGGTGCGGC CGCCGCCGCA ACCGATGCAG
701  CATTTGCCTC TTTGGCCAGC CAGGCTTCCG TATCGTFCAT CAACAACAAA
751  CGCAATATCG GTAACACCCT GAAAGAGCTG GGCAGAAGCA GCACGGTGAA
801  AAATCTGATG GTTGCCGTCG CTACCGCAGG CGTAGCCGAC AAAATCGGTG
851  CTTCGGCACT GAACAATGTC AGCGATAAGC AGTGGATCAA CAACCTGACC
901  GTCAACCTGG CCAATGCGGG CAGTGCCGCA CTGATTAATA CCGCTGTCAA
951  CGGCGGCAGC CTGAAAGACA ATCTGGAAGC GAATATCCTT GCGGCTTTGG
1001 TGAATACTGC GCATGGAGAG GCAGCAAGTA AAATCAAACA GTTGGATCAG
1051 CACTACATTG CCCATAAGAT TGCCCATGCC ATAGCGGGCT GTGCGGCAGC
1101 GGCGGCGAAT AAGGGCAAGT GTCAAGATGG TGCGATCGGT GCGGCGGTCG
1151 GTGAAATCCT TGGCGAAACC CTACTGGACG GCAGAGACCC TGGCAGCCTG
1201 AATGTGAAGG ACAGGGCAAA AATCATTGCT AAGGCGAAGC TGGCAGCAGG
1251 GGCGGTTGCG GCGTTGAGTA AGGGGGATGT GAGTACGGCG GCGAATGCGG
1301 CTGCTGTGGC GGTAGAGAAT AATTCTTTAA ATOATATACA GGATCGTTTG
1351 TTGAGTGGAA ATTATGCTTT ATGTATGAGT GCAGGAGGAG CAGAAAGCTT
1401 TTGTGAGTCT TATCGACCAC TGGGCTTGCC ACACTTTGTA AGTGTTTCAG
1451 GAGAAATGAA ATTACCTAAT AAATTCGGGA ATCGTATGGT TAATGGAAAA
1531 TTAATTATTA ACACTAGAAA TGGCAATGTA TATTTCTCTG TAGGTAAAAT
1551 ATGGAGTACT GTAAAATCAA CAAAATCAAA TATAAGTGGG GTATCTGTCG
1601 GTTGGGTTTT AAATGTTTCC CCTAATGATT ATTTAAAAGA AGCATTTATG
1651 AATGATTTCA GAAATAGTAA TCAAAATAAA GCCTATGCAG AAATGATTTC
1701 CCAGACTTTG GTAGGTGAGA GTGTTGGTGG TAGTCTTTGT CTGACAAGAG
1751 CCTGCTTTTC GGTAAGTTCA ACAATATCTA AATCTAAATC TCCTTTTAAA
1801 GATTCAAAAA TTATTGGGGA AATCGGTTTG GGAAGTGGTG TTGCTGCAGG
1851 AGTAGAAAAA ACAATATACA TAGGTAACAT AAAAGATATT GATAAATTTA
1901 TTAGTGCAAA CATAAAAAAA TAG

This corresponds to the amino acid sequence <SEQ ID 20; ORF49-1>:

1   MQLLAAEGIH QHQLNVQKST RFIGIKVGKS NYSKNELNET KLPVRVIAQT
51  AKTRSGWDTV LEGTEFKTTL SGADIQAGVG EKARADAKII LKGIVNRIQT
101 EEKLESNSTV WQKQAGSGST VETLKLPSFE GPALPKLTAP GGYIADIPKG
151 NLKTEIEKLA KQPEYAYLKQ LQTVKDVNWN QVQLAYDKWD YKQEGLTGAG
201 AAIIALAVTV VTSGAGTGAV LGLNGAAAAA TDAAFASLAS QASVSFINNK
251 GNIGNTLKEL GRSSTVKNLM VAVATAGVAD KIGASALNNV SDKQWINNLT
301 VNLANAGSAA LINTAVNGGS LKDNLEANIL AALVNTAHGE AASKIKQLDQ
351 HYIAHKIAHA IAGCAAAAAN KGKCQDGAIG AAVGEILGET LLDGRDPGSL
401 NVKDRAKIIA KAKLAAGAVA ALSKGDVSTA ANAAAVAVEN NSLNDIQDRL
451 LSGNYALCNS AGGAESFCES YRPLGLPHFV SVSGENKLPN KFGNRNVNGK
501 LIINTRNGNV YFSVGKIWST VKSTKSNISG VSVGWVLNVS PNDYLKEASM
551 NDFRNSNQNK AYAEMISQTL VGESVGGSLC LTRACFSVSS TISKSKSPFK
601 DSKIIGEIGL GSGVAAGVEK TIYIGNIKDI DKFISANIKK *

Computer analysis predicts a transmembrane domain and also indicates that ORF49 has no significant amino acid homology with known proteins. A corresponding ORF from N. meningitidis strain A was, however, identified:

ORF49 shows 86.1% identity over a 173 aa overlap with an ORF (ORF49a) from strain A of N. meningitidis:

          10        20        30
orf49.pep GTEFKTTLSGADIQAGVGEKARADAKIILK
          ||||||||:|||||||| ||||:|||||||
orf49a    SKNELNETKLPVRVVAQXAATRSGWDTVLEGTEFKTTLAGADIQAGVXEKARVDAKIILK
          40        50        60        70        80        90
          40        50        60        70        80        90
orf49.pep GIVNRIQTEEKLESNSTVWQKQAGSGSTVETLKLPSFEGPALPKLTAPGGYIADIPKGNL
          |||||||:|||||:|||||||||| |||:|||||||||:|: |||:||||||:|||||||
orf49a    GIVNRIQSEEKLETNSTVWQKQAGRGSTIETLKLPSFESPTPPKLSAPGGYIVDIPKGNL
          100       110       120       130       140       150
          100       110       120       130       140       150
orf49.pep KTEIEKLAKQPEYAYLKQLQTVKDVNWNQVQLAYDKWDYKQEGLTGAGAAIXALAVTVVT
          |||||||:||||||||||||::|::||||||||||:||||||||| ||||| ||||||||
orf49a    KTEIEKLSKQPEYAYLKQLQVAKNINWNQVQLAYDRWDYKQEGLTEAGAAIIALAVTVVT
          160       170       180       190       200       210
          160       170
orf49.pep SGAGTGAVLGLXRVAAAATDAAF
          |||||||||||  : ||||||||
orf49a    SGAGTGAVLGLNGAXAAATDAAFASLASQASVSFINNKGDVGKTLKELGRSSTVKNLVVA
          220       230       240       250       260       270

ORF49-1 and ORF49a show 83.2% identity in 457 aa overlap:

orf49a.pep XQLLAEEGIHKHELDVQKSRRFIGIKVGXSNYSKNELNETKLPVRVVAQXAATRSGWDTV
           |||| ||||:|:|:|||| |||||||| |||||||||||||||||:||:| ||||||||
orf49-1    MQLLAAEGIHQHQLNVQKSTRFIGIKVGKSNYSKNELNETKLPVRVIAQTAKTRSGWDTV
orf49a.pep LEGTEFKTTLAGADIQAGVXEKARVDAKIILKGIVNRIQSEEKLETNSTVWQKQAGRGST
           ||||||||||:|||||||| ||||:||||||||||||||:|||||:|||||||||| |||
orf49-1    LEGTEFKTTLSGADIQAGVGEKARADAXIILKGIVNRIQTEEKLESNSTVWQKQAGSGST
orf49a.pep IETLKLPSFESPTPPKLSAPGGYIVDIPKGNLKTEIEKLSKQPEYAYLKQLQVAKNINWN
           :|||||||||:|: |||:||||||:||||||||||||||:||||||||||||::|::|||
orf49-1    VETLKLPSFEGPALPKLTAPGGYIADIPKGNLKTEIEKLAKQPEYAYLKQLQTVKDVNWN
orf49a.pep QVQLAYDRWDYKQEGLTEAGAAIIALAVTVVTSGAGTGAVLGLNGAXAAATDAAFASLAS
           |||||||:||||||||| |||||||||||||||||||||||||||| |||||||||||||
orf49-1    QVQLAYDKWDYKQEGLTGAGAAIIALAVTVVTSGAGTGAVLGLNGAAAAATDAAFASLAS
orf49a.pep QASVSFINNKGDVGKTLKELGRSSTVKNLVVAAATAGVADKIGASALXNVSDKQWINNLT
           |||||||||||::|:||||||||||||||:||:|||||||||||||| ||||||||||||
orf49-1    QASVSFINNKGNIGNTLKELGRSSTVKNLMVAVATAGVADKIGASALNNVSDKQWINNLT
orf49a.pep VNLANAGSAALINTAVNGGSLKDXLEANILAALVNTAHGEAASKIKQLDQHYIVHKIAHA
           ||||||||||||||||||||||| |||||||||||||||||||||||||||||:||||||
orf49-1    VNLANAGSAALINTAVNGGSLKDNLEANILAALVNTAHGEAASKIKQLDQHYIAHKIAHA
orf49a.pep IAGCAAAAANKGKCQDGAIGAAVGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVS
           ||||||||||||||||||||||||||:||:| :|::| :|::|:| :|:| :||:||:|:
orf49-1    IAGCAAAAANKGKCQDGAIGAAVGEILGETLLDGRDPGSLNVKDRAKIIAKAKLAAGAVA
orf49a.pep GVVGGDVNAAANAAEVAVKNNQLSDXEGREFDNEMTACAKQNXPQLCRKNTVKKYQNVAD
           ::  |||::||||| |||:||:|:| : | :::::: |
orf49-1    ALSKGDVSTAANAAAVAVENNSLNDIQDRLLSGNYALCMSAGGAESFCESYRPLGLPHFV
orf49a.pep KRLAASIAICTDISRSTECRTIRKQHLIDSRSLHSSWEAGLIGKDDEWYKLFSKSYTQAD
orf49-1    SVSGEMKLPNKFGNRMVNGKLIINTRNGNVYFSVGKIWSTVKSTKSNISGVSVGWVLNVS

The complete length ORF49a nucleotide sequence <SEQ ID 21> is:

1    NTGCAACTGC TGGCAGAAGA AGGCATCCAC AAGCACGAGT TGGATGTCCA
51   AAAAAGCCGC CGCTTTATCG GCATCAAGGT AGGTNAGAGC AATTACAGTA
101  AAAACGAACT GAACGAAACC AAATTGCCTG TCCGCGTCGT CGCCCAAANT
151  GCAGCCACCC GTTCAGGCTG GGATACCGTG CTCGAAGGTA CCGAATTCAA
201  AACCACGCTG GCCGGTGCCG ACATTCAGGC AGGTGTANGC GAAAAAGCCC
251  GTGTCGATGC GAAAATTATC CTCAAAGGCA TTGTGAACCG TATCCAGTCG
301  GAAGAAAAAT TAGAAACCAA CTCAACCGTA TGGCAGAAAC AGGCCGGACG
351  CGGCAGCACT ATCGAAACGC TAAAACTGCC CAGCTTCGAA AGCCCTACTC
401  CGCCCAAATT GTCCGCACCC GGCGGNTATA TCGTCGACAT TCCGAAAGGC
451  AATCTGAAAA CCGAAATCGA AAAGCTGTCC AAACAGCCCG AGTATGCCTA
501  TCTGAAACAG CTCCAAGTAG CGAAAAACAT CAACTGGAAT CAGGTGCAGC
551  TTGCTTACGA CAGATGGGAC TACAAACAGG AGGGCTTAAC CGAAGCAGGT
601  GCGGCGATTA TCGCACTGGC CGTTACCGTG GTCACCTCAG GCGCAGGAAC
651  CGGAGCCGTA TTGGGATTAA ACGGTGCGNC CGCCGCCGCA ACCGATGCAG
701  CATTCGCCTC TTTGGCCAGC CAGGCTTCCG TATCGTTCAT CAACAACAAA
751  GGCGATGTCG GCAAAACCCT GAAAGAGCTG GGCAGAAGCA GCACGGTGAA
801  AAATCTGGTG GTTGCCGCCG CTACCGCAGG CGTAGCCGAC AAAATCGGCG
851  CTTCGGCACT GANCAATGTC AGCGATAAGC AGTGGATCAA CAACCTGACC
901  GTCAACCTAG CCAATGCGGG CAGTGCCGCA CTGATTAATA CCGCTGTCAA
951  CGGCGGCAGC CTGAAAGACA NTCTGGAAGC GAATATCCTT GCGGCTTTGG
1001 TCAATACCGC GCATGGAGAA GCAGCCAGTA AAATCAAACA GTTGGATCAG
1051 CACTACATAG TCCACAAGAT TGCCCATGCC ATAGCGGGCT GTGCGGCAGC
1101 GGCGGCGAAT AAGGGCAAGT GTCAGGATGG TGCGATAGGT GCGGCTGTGG
1151 GCGAGATAGT CGGGGAGGCT TTGACAAACG GCAAAAATCC TGACACTTTG
1201 ACAGCTAAAG AACGCGAACA GATTTTGGCA TACAGCAAAC TGGTTGCCGG
1251 TACGGTAAGC GGTGTGGTCG GCGGCGATGT AAATGCGGCG GCGAATGCGG
1301 CTGAGGTAGC GGTGAAAAAT AATCAGCTTA GCGACTAAGA GGGTAGAGAA
1351 TTTGATAACG AAATGACTGC ATGCGCCAAA CAGAATANTC CTCAACTGTG
1401 CAGAAAAAAT ACTGTAAAAA AGTATCAAAA TGTTGCTGAT AAAAGACTTG
1451 CTGCTTCGAT TGCAATATGT ACGGATATAT CCCGTAGTAC TGAATGTAGA
1501 ACAATCAGAA AACAACATTT GATCGATAGT AGAAGCCTTC ATTCATCTTG
1551 GGAAGCAGGT CTAATTGGTA AAGATGATGA ATGGTATAAA TTATTCAGCA
1601 AATCTTACAC CCAAGCAGAT TTGGCTTTAC AGTCTTATCA TTTGAATACT
1651 GCTGCTAAAT CTTGGCTTCA ATCGGGCAAT ACAAAGCCTT TATCCGAATG
1701 GATGTCCGAC CAAGGTTATA CACTTATTTC AGGAGTTAAT CCTAGATTCA
1751 TTCCAATACC AAGAGGGITF GTAAAACAAA ATACACCTAT TACTAATGTC
1801 AAATACCCGG AAGGCATCAG TTTCGATACA AACCTANAAA GACATCTGGC
1851 AAATGCTGAT GGTTTTAGTC AAGAACAGGG CATTAAAGGA GCCCATAACC
1901 GCACCAATNT TATGGCAGAA CTAAATTCAC GAGGAGGANG NGTAAAATCT
1951 GAAACCCANA CTGATATTGA AGGCATTACC CGAATTAAAT ATGAGATTCC
2001 TACACTAGAC AGGACAGGTA AACCTGATGG TGGATTTAAG GAAATTTCAA
2051 GTATAAAAAC TGTTTATAAT CCTAAAAANT TTTNNGATGA TAAAATACTT
2101 CAAATGGCTC AANATGCTGN TTCACAAGGA TATTCAAAAG CCTCTAAAAT
2151 TGCTCAAAAT GAAAGAACTA AATCAATATC GGAAAGAAAA AATGTCATTC
2201 AATTCTCAGA AACCTTTGAC GGAATCAAAT TTAGANNNTA TNTNGATGTA
2251 AATACAGGAA GAATTACAAA CATTCACCCA GAATAATTTA A

This encodes a protein having amino acid sequence <SEQ ID 22>:

1   XQLLAEEGIH KHELDVQKSR RFIGIKVGXS NYSKNELNET KLPVRVVAQX
51  AATRSGWDTV LEGTEFKTTL AGADIQAGVX EKARVOAKII LKGIVNRIQS
101 EEKLETNSTV WQKQAGRGST IETLKLPSFE SPTPPKLSAP GGYIVDIPKG
151 NLKTEIEKLS KQPEYAYLKQ LQVAKNINWN QVQLAYDRWD YKQEGLTEAG
201 AAIIALAVTV VTSGAGTGAV LGLNGAXAAA TORAFASLAS QASVSFINNK
251 GDVGKTLKEL GRSSTVKNLV VAAATAGVAD KIGASALXNV SDKQNINNLT
301 VNLANAGSAA LINTAVNGGS LKDXLEANIL AALVNTAHGE AASKIKQLDQ
351 HYIVHKIAHA IAGCAAAAAN KGKCQDGAIG AAVGEIVGEA LTNGKNPDTL
401 TAKEREQILA YSKLVAGTVS GVVGGDVNAA ANAAEVAVKN NQLSDXEGRE
451 FDWEHTACAK QNXPQLCRXN TVKKYQNVAD KRLAASIAIC TDISRSTECR
501 TIRKQHLIDS RSLHSSWEAG LIGKDDEWYK LFSKSYTQAD LALOSYHLNT
551 AAKSWLQSGN TKPLSEWNSD QGYTLISGVN PRFIPIPRGF VKQNTPITNV
601 KYPEGISFDT NLXRHLATAD GFSQEQGIKG AHNRTNXMAE LNSRGGXVKS
651 ETXTDIEGIT RIKYEIPTLD RTGKPDGGFK EISSIKTVYN FKXFKDDKIL
701 QMAQXAXSQG YSKASKIAQN ERTKSISERK NVIQFSETFD GIKFRXYXDV
751 NTGRITNIHP E

Based on the presence of a putative transmembrane domain, it is predicted that these proteins from N. meningitidis, and their epitopes, could be useful antigens for vaccines or diagnostics.

Example 5

The following partial DNA sequence was identified in N. meningitidis SEQ ID 23>

1   ..CGGATCGTTG TAGGTTTGCG GATTTCTTGC GCCGTAGTCA CCGTAGTCCC
51  AAGTATAACC CAAGGCTTTG TCTTCGCCTT TCATTCCGAT AAGGGATATG
101 ACGCTTTGGT CGGTATAGCC GTCTTGGGAA CCTTTGTCCA CCCAACGCAT
151 ATCTGCCTGC GGATTCTCAT TGCCGCTTCT TGGCTGCTGA TTTTTCTGCC
201 TTCGCGTTTT TCAACTTCGC GCTTGAGGGC TTCGGCATAT TTGTCGGCCA
251 ACGCCATTTC TTTCGGATGC AGCTGCCTAT TGTTCCAATC TACATTCGCA
301 CCCACCACAG CACCACCACT ACCACCAGTT GCATAG

This corresponds to the amino acid sequence <SEQ ID 24; ORF50>:

1   ..RIVVGLRISC AVVTVVPSIT QGFVFAFHSD KGYDALVGIA VLGTFVHPTH
51  ICLRILIAAS WLLIFLPSRF STSRLRASAY LSANAISFGC SCLLFQSTFA
101 PTTAPPLPPV A*

Computer analysis predicts two transmembrane domains and also indicates that ORF50 has no significant amino acid homology with known proteins.

Based on the presence of a putative transmembrane domain, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 6

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 25>

1    ..AAGTTTGACT TTACCTGGTT TATTCCGGCG GTAATCAAAT ACCGCCGGTT
51   GTTTTTTGAA GTATTGGTGG TGTCGGTGGT GTTGCAGCTG TTTGCGCTGA
101  TTACGCCTCT GTTTTTCCAA GTGGTGATGG ACAAGGTGCT GGTACATCGG
151  GGATTCTCTA CTTTGGATGT GGTGTCGGTG GCTTTGTTGG TGGTGTCGCT
201  GTTTGAGATT GTGTTGGGCG GTTTGCGGAC GTATCTGTTT GCACATACGA
251  CTTCACGTAT TGATGTGGAA TTGGGCGCGC GTTTGTTCCG GCATCTGCTT
301  TCCCTGCCTT TATCCTATTT CGAGCACAGA CGAGTGGGTG ATACGGTGGC
351  TCGGGTGCGG GAATTGGAGC AGATTCGCAA TTTCTTGACC GGTCAGGCGC
401  TGACTTCGGT GTTGGATTTG GCGTTTTCGT TTATCTTTCT GGCGGTGATG
451  TGGTATTACA GCTCCACTCT GACTTGGGTG GTATTGGCTT CGTTG.....
     //
1451 .......... .......... .......... .......... ..........
1501 .......... .......... .......... .......... ..ATTTGCGC
1551 CAACCGGACG GTGCTGATTA TCGCCCACCG TCTGTCCACT GTTAAAACGG
1601 CACACCGGAT CATTGCCATG GATAAAGGCA GGATTGTGGA AGCGGGAACA
1651 CAGCAGGAAT TGCTGGCGAA CG..AACGGA TATTACCGCT ATCTGTATGA
1701 TTTACAGAAC GGGTAG

This corresponds to the amino acid sequence <SEQ ID 26; ORF39>:

1   ..KFDFTWFIPA VIKYRRLFFE VLVVSVVLQL FALITPLFFQ VVMDKVLVHR
51  GFSTLDVVSV ALLVVSLFEI VLGGLRTYLF AHTTSRIDVE LGARLFRHLL
101 SLPLSYFEHP RVGDTVARVR ELEQIRNFLT GQALTSVLDL AFSFIFLAVM
151 WYYSSTLTWV VLASL..... .......... .......... ..........
    //
501 .......... ....ICANRT VLIIAHRLST VKTAHRIIAH DKGRIVEAGT
551 QQELLANXNG YYRYLYDLQN G*

Further work revealed the complete nucleotide sequence <SEQ ID 27>:

1    ATGTCTATCG TATCCGCACC GCTCCCCGCC CTTTCCGCCC TCATCATCCT
51   CGCCCATTAC CACGGCATTG CCGCCAATCC TGCCGATATA CAGCATGAAT
101  TTTGTACTTC CGCACAGAGC GATTTAAATG AAACGCAATG GCTGTTAGCC
151  GCCAAATCTT TGGGATTGAA GGCAAAGGTA GTCCGCCAGC CTATTAAACG
201  TTTGGCTATG GCGACTTTAC CCGCATTGGT ATGGTGTGAT GACGGCAACC
251  ATTTCATTTT GGCCAAAACA GACGGTGAGG GTGAGCATGC CCAATTTTFG
301  ATACAGGATT TGGTTACGAA TAAGTCTGCG GTATTGTCTT TTGCCGAATT
351  TTCTAACAGA TATTCGGGCA AACTGATATT GGTTGCTTCC CGCGCTTCGG
401  TATTGGGCAG TTTGGCAAAG TTTGACTTTA CCTGGTTTAT TCCGGCGGTA
451  ATCAAATACC GCCGGTTGTT TTTTGAAGTA TTGGTGGTGT CGGTGGTGTT
501  GCAGCTGTTT GCGCTGATTA CGCCTCTGTT TTTCCAAGTG GTGATGGACA
551  AGGTGCTGGT ACATCGGGGA TTCTCTACTT TGGATGTGGT GTCGGTGGCT
601  TTGTTGGTGG TGTCGCTGTT TGAGATTGTG TTGGGCGGTT TGCGGACGTA
651  TCTGTTTGCA CATACGACTT CACGTATTGA TGTGGAATTG GGCGCGCGTT
701  TGTTCCGGCA TCTGCTTTCC CTGCCTTTAT CCTATTTCGA GCACAGACGA
751  GTGGGTGATA CGGTGGCTCG GGTGCGGGAA TTGGAGCAGA TTCGCAATTT
801  CTTGACCGGT CAGGCGCTGA CTTCGGTGTT GGATTTGGCG TTTTCGTTTA
951  TCTTTCTGGC GGTGATGTGG TATTACAGCT CCACTCTGAC TTGGGTGGTA
901  TTGGCTTCGT TGCCTGCCTA TGCGTTTTGG TCGGCATTTA TCAGTCCGAT
951  ACTGCGGACG CGTCTGAACG ATAAGTTCGC GCGCAATGCA GACAACCAGT
1001 CGTTTTTAGT AGAAAGCATC ACTGCGGTGG GTACGGTAAA GGCGATGGCG
1051 GTGGAGCCGC AGATGACGCA GCGTTGGGAC AATCAGTTGG CGGCTTATGT
1101 GGCTTCGGGA TTTCGGGTAA CGAAGTTGGC GGTGGTCGGC CAGCAGGGGG
1151 TGCAGCTGAT TCAGAAGCTG GTGACGGTGG CGACGTTGTG GATTGGCGCA
1201 CGGCTGGTAA TTGAGAGCAA GCTGACGGTG GGGCAGCTGA TTGCGTTTAA
1251 TATGCTCTCG GGACACGTGG CGGCGCCTGT TATCCGTTTG GCGCAGTTGT
1301 GGCAGGATTT CCAGCAGGTG GGGATTTCGG TGGCGCGTTT GGGGGATATT
1351 CTGAATGCGC CGACCGAGAA TGCGTCTTCG CATTTGGCTT TGCCCGATAT
1401 CCGGGGGGAG ATTACGTTCG AACATGTCGA TTTCCGCTAT AAGGCGGACG
1451 GCAGGCTGAT TTTGCAGGAT TTGAACCTGC GGATTCCGGC GGGGGAAGTG
1501 CTGGGGATTG TGGGACGTTC GGGGTCGGGC AAATCCACAC TCACCAAATT
1551 GGTGCAGCGT CTGTATGTAC CGGAGCAGGG ACGGGTGTTG GTGGACGGCA
1601 ACGATTTGGC TTTGGCCGCT CCTGCCTGGC TGCGGCGGCA GGTCGGCGTG
1651 GTCTTGCAGG AGAATGTGCT GCTCAACCGC AGCATACGCG ACAATATCGC
1701 GCTGACGGAT ACGGGTATGC CGCTGGAACG CATTATCGAA GCAGCCAAAC
1751 TGGCGGGCGC ACACGAGTTT ATTATGGAGC TGCCGGAAGG CTACGGCACC
1801 GTGGTGGGCG AACAAGGGGC CGGCTTGTCG GGCGGACAGC GGCAGCGTAT
1951 TGCGATTGCC CGCGCGTTAA TCACCAATCC GCGCATTCTG ATTTTTGATG
1901 AAGCCACCAG CGCGCTGGAT TATGAAAGTG AACGAGCGAT TATGCAGAAC
1951 ATGCAGGCCA TTTGCGCCAA CCGGACGGTG CTGATTATCG CCCACCGTCT
2001 GTCCACTGTT AAAACGGCAC ACCGGATCAT TGCCATGGAT AAAGGCAGGA
2051 TTGTGGAAGC GGGAACACAG CAGGAATTGC TGGCGAAGCC GAACGGATAT
2101 TACCGCTATC TGTATGATTT ACAGAACGGG TAG

This corresponds to the amino acid sequence <SEQ ID 28; ORF39-1>:

1   MSIVSAPLPA LSALIILAMY HGIAANPADI QHEFCTSAQS DLNETQWLLA
51  AKSLGLKAKV VRQPIKRLAM ATLPALVWCD DGNHFILAKT DGEGEHAQFL
101 IQDLVTNKSA VLSFAEFSNR YSGKLILVAS RASVLGSLAK FDFTWFIPAV
151 IKYRRLFFEV LVVSVVLQLF ALITPLFFQV VMDKVLVHRG FSTLDVVSVA
201 LLVVSLFEIV LGGLRTYLFA HTTSRIDVEL GARLFRHLLS LPLSYFEHPA
251 VGDTVARVRE LEQIRNFLTG QALTSVLDLA FSFIFLAVMW YYSSTLTWVV
301 LASLPAYAFW SAFISPILRT RLNDKFAPNA DNQSFLVESI TAVGTVKAMA
351 VEPQHTQRWD NQLAAYVASG FRVTKLAVVG QQGVQLIQKL VTVATLWIGA
401 RLVIESRLTV GQLIAFNMLS GQVAAPVIRL AQLWQDFQQV GISVARLGDI
451 VVAPTENASS HLALPDIRGE ITFEHVDFRY KADGRLILQD LNLRIRAGEV
501 LGIVGRSGSG KSTLTKLVQR LYVPEQGRVL VDGNDLALAA PAWLRRQVGV
551 VLOENVLLNR SIRDNIALTD TGNPLERIIE AAKLAGAHEF IMELPEGYGT
601 VVGEQGAGLS GGQRQRIAIA RALITNPRIL IFDEATSALD YESERAINQN
651 MQAICANRTV LIIAHRLSTV KTAHRIIAND KGRIVEAGTQ QELLAKPNGY
701 YRYLYDLQNG *

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF39 shows 100% identity over a 165 aa overlap with an ORF (ORF39a) from strain A of N. meningitidis:

          10        20        30
orf39.pep KFDFTWFIPAVIKYRRLFFEVLVVSVVLQL
          ||||||||||||||||||||||||||||||
orf39a    AVLSFAEFSNRYSGKLILVASRASVLGSLAKFDFTWFIPAVIKYRRLFFEVLVVSVVLQL
          110       120       130       140       150       160
          40        50        60        70        80        90
orf39.pep FALITPLFFQVVMDKVLVHRGFSTLDVVSVALLVVSLFEIVLGGLRTYLFAHTTSRIDVE
          ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a    FALITPLFFQVVMDKVLVHRGFSTLDVVSVALLVVSLFEIVLGGLRTYLFAHTTSRIDVE
          170       180       190       200       210       220
          100       110       120       130       140       150
orf39.pep LGARLFRHLLSLPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVM
          ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a    LGARLFRHLLSLPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVM
          230       240       250       260       270       280
          160       170       180       190       200       210
orf39.pep WYYSSTLTWVVLASLXXXXXXXXXXXXXXXXXXXXXXXXXXXXICANRTVLIIAHRLSTV
          |||||||||||||||
orf39a    WYYSSTLTWVVLASLPAYAFWSAFISPILRTRLNDKFARNADNQSFLVESITAVGTVKAM
          290       300       310       320       330       340

ORF39-1 and ORF39a show 99.4% identity in 710 aa overlap:

orf39-1.pep MSIVSAPLPALSALIILAHYHGIAANPADIQHEFCTSAQSDLNETQWLLAAKSLGLKAKV
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a      MSIVSAPLPALSALIILAHYHGIAANPADIQHEFCTSAQSDLNETQWLLAAKSLGLKAKV
orf39-1.pep VRQPIKRLAMATLPALVWCDDGNHFILAKTDGEGEHAQFLIQDLVTNKSAVLSFAEFSNR
            |||||||||||||||||||||||||||||||| |||||:|||||:|||||||||||||||
orf39a      VRQPIKRLAMATLPALVWCDDGNHFILAKTDGGGEHAQYLIQDLTTNKSAVLSFAEFSNR
orf39-1.pep YSGKLILVASRASVLGSLAKFDFTWFIPAVIKYRRLFFEVLVVSVVLQLFALITPLFFQV
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a      YSGKLILVASRASVLGSLAKFDFTWFIPAVIKYRRLFFEVLVVSVVLQLFALITPLFFQV
orf39-1.pep VMDKVLVHRGFSTLDVVSVALLVVSLFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLS
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a      VMDKVLVHRGFSTLDVVSVALLVVSLFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLS
orf39-1.pep LPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWVV
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a      LPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWVV
orf39-1.pep LASLPAYAFWSAFISPILRTRLNDKFARNADNQSFLVESITAVGTVKAMAVEPQMTQRWD
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a      LASLPAYAFWSAFISPILRTRLNDKFARNADNQSFLVESITAVGTVKAMAVEPQMTQRWD
orf39-1.pep NQLAAQVASGFRVTKLAVVGQQGVQLIQKLVTVATLWIGARLVIESKLTVGQLIAFNMLS
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a      NQLAAQVASGFRVTKLAVVGQQGVQLIQKLVTVATLWIGARLVIESKLTVGQLIAFNMLS
orf39-1.pep GQVAAPVIRLAQLWQDFQQVGISVARLGDILNAPTENASSHLALPDIRGEITFEHVDFRY
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a      GQVAAPVIRLAQLWQDFQQVGISVARLGDILNAPTENASSHLALPDIRGEITFEHVDFRY
orf39-1.pep KADGRLILQDLNLRIRAGEVLGIVGRSGSGKSTLTKLVQRLYVPEQGRVLVDGNDLALAA
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a      KADGRLILQDLNLRIRAGEVLGIVGRSGSGKSTLTKLVQRLYVPEQGRVLVDGNDLALAA
orf39-1.pep PAWLRRQVGVVLQENVLLNRSIRDNIALTDTGMPLERIIEAAKLAGAHEFIHELPEGYGT
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a      PAWLRRQVGVVLQENVLLNRSIRDNIALTDTGMPLERIIEAAKLAGAHEFIHELPEGYGT
orf39-1.pep VVGEQGAGLSGGQRQRIAIARALITNPRILIFDEATSALDYESERAIMQNMQAICANRTV
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a      VVGEQGAGLSGGQRQRIAIARALITNPRILIFDEATSALDYESERAIMQNMQAICANRTV
orf39-1.pep LIIAHRLSTVKTAHRIIAMDKGRIVEAGTQQELLAKPNGYYRYLYDLQNGX
            |||||||||||||||||||||||||||||||||||||||||||||||||||
orf39a      LIIAHRLSTVKTAHRIIAMDKGRIVEAGTQQELLAKPNGYYRYLYDLQNGX

The complete length ORF39a nucleotide sequence <SEQ ID 29> is:

1    ATGTCTATCG TATCCGCACC GCTCCCCGCC CTTTCCGCCC TCATCATCCT
51   CGCCCATYAC CACGGCATTG CCGCCAATCC TGCCGATATA CAGCATGAAT
101  TTTGTACTTC CGCACAGAGC GATTTAAATG AAACGCAATG GCTGTTAGCC
151  GCCAAATCTT TGGGATTGAA GGCAAAGGTA GTCCGCCAGC CTATTAAACG
201  TTTGGCTATG GCGACTTTAC CCGCATTGGT ATGGTGTGAT GACGGCAACC
251  ATTTTATTTT GGCTAAAACA GACGGTGGGG GTGAGCATGC CCAATATCTA
301  ATACAGGATT TAACTACGAA TAAGTCTGCG GTATTGTCTT TTGCCGAATT
351  TTCTAACAGA TATTCGGGCA AACTGATATT GGTTGCTTCC CGCGCTTCGG
401  TATTGGGCAG TTTGGCAAAG TTTGACTTTA CCTGGTTTAT TCCGGCGGTA
451  ATCAAATACC GCCGGTTGTT TTTTGAAGTA TTGGTGGTGT CGGTGGTGTT
501  GCAGCTGTTT GCGCTGATTA CGCCTCTGTT TTTCCAAGTG GTGATGGACA
551  AGGTGCTGGT ACATCGGGGA TTCTCTATTT TGGATGTGGT GTCGGTGGCT
601  TTGTTGGTGG TGTCGCTGTT TGAGATTGTG TTGGGCGGTT TGCGGACGTA
651  TCTGTTTGCA CATACGACTT CACGTATTGA TGTGGAATTG GGCGCGCGTT
701  TGTTCCGGCA TCTGCTTTCC CTGCCTTTAT CCTATTTCGA GCACAGACGA
751  GTGGGTGATA CGGTGGCTCG GGTGCGGGAA TTGGAGCAGA TTCGCAATTT
801  CTTGACCGGT CAGGCGCTGA CTTCGGTGTT GGATTTGGCG TTTTCGTTTA
951  TCTTTCTGGC GGTGATGTGG TATTACAGCT CCACTCTGAC TTGGGTGGTA
901  TTGGCTTCGT TGCCTGCCTA TGCGTTTTGG TCGGCATTTA TCAGTCCGAT
951  ACTGCGGACG CGTCTGAACG ATAAGTTCGC GCGCAATGCA GACAACCAGT
1001 CGTTTTTAGT AGAAAGCATC ACTGCGGTGG GTACGGTAAA GGCGATGGCG
1051 GTGGAGCCGC AGATGACGCA GCGTTGGGAC AATCAGTTGG CGGCTTATGT
1101 GGCTTCGGGA TTTCGGGTAA CGAAGTTGGC GGTGGTCGGC CAGCAGGGGG
1151 TGCAGCTGAT TCAGAAGCTG GTGACGGTGG CGACGTTGTG GATTGGCGCA
1201 CGGCTGGTAA TTGAGAGCAA GCTGACGGTG GGGCAGCTGA TTGCGTTTAA
1251 TATGCTCTCG GGACAGGTGG CGGCGCCTGT TATCCGTTTG GCGCAGTTGT
1301 GGCAGGATTT CCAGCAGGTG GGGATTTCGG TGGCGCGTTT CGGGGATATT
1351 CTGAATGCGC CGACCGAGAA TGCGTCTTCG CATTTGGCTT TGCCCGATAT
1401 CCGGGGGGAG ATTACGTTCG AACATGTCGA TTTCCGCTAT AAGGCGGACG
1451 GCAGGCTGAT TTTGCAGGAT TTGAACCTGC GGATTCGGGC GGGGGAAGTG
1501 CTGGGGATTG TGGGACGTTC GGGGTCGGGC AAATCCACAC TCACCAAATT
1551 GGTGCAGCGT CTGTATGTAC CGGCGCAGGG ACGGGTGTTG GTGGACGGCA
1601 ACGATTTGGC TTTGGCCGCT CCTGCTTGGC TGCGGCGGCA GGTCGGCGTG
1651 GTCTTGCAGG AGAATGTGCT GCTCAACCGC AGCATACGCG ACAATATCGC
1701 GCTGACGGAT ACGGGTATGC CGCTGGAACG CATTATCGAA GCAGCCAAAC
1751 TGGCGGGCGC ACACGAGTTT ATTATGGAGC TGCCGGAAGG CTACGGCACC
1801 GTGGTGGGCG AACAAGGGGC CGGCTTGTCG GGCGGACAGC GGCAGCGTAT
1851 TGCGATTGCC CGCGCGTTAA TCACCAATCC GCGCATTCTG ATTTTTGATG
1901 AAGCCACCAG CGCGCTGGAT TATGAAAGTG AACGAGCGAT TATGCAGAAC
1951 ATGCAGGCCA TTTGCGCCAA CCGGACGGTG CTGATTATCG CCCACCGTCT
2001 GTCCACTGTT AAAACGGCAC ACCGGATCAT TGCCATGGAT AAAGGCAGGA
2051 TTGTGGAAGC GGGAACACAG CAGGAATTGC TGGCGAAGCC GAACGGATAT
2101 TACCGCTATC TGTATGATTT ACAGAACGGG TAG

This encodes a protein having amino acid sequence <SEQ ID 30>:

1   MSIVSAPLPA LSALIILAHY HGIAANPADI QHEFCTSAQS DLNETQWLLA
51  AKSLGLKAKV VRQPIKRLAN ATLPALVWCD DGNHFILAKT DGGGEHAQYL
101 IQDLTTNKSA VLSFAEFSNR YSGKLILVAS RASVIASLAX FDFTWFIPAV
151 IKYRRLFFEV LVVSVVLQLF ALITPLFFQV VNDKVLVHRG FSTLDVVSVA
201 LLVVSLFEIV LGGLRTYLFA HTTSRIDVEL GARLFRHLLS LPLSYFEHRR
251 VGDTVARVRE LEQIRNFLTG QALTSVLDLA FSFIFLAVMW YYSSTLTWVV
301 LASLPAYAFW SAFISPILRT RLNDKFARNA ONQSFLVESI TAVGTVKAMA
351 VEPQNTQRWD NQLAAYVASG FRVTKLAVVG QQGVQLIQKL VTVATLWIGA
401 RLVIESKLTV GQLIAFNHLS GQVAAPVIRL AQLWQOFQQV GISVARLGDI
451 LNAPTENASS HLALPDIRGE ITFEHVDFRY KADGRLILQD LNLRIRAGEV
501 LGIVGRSGSG KSTLTKLVQR LYVPAQGRVL VDGNDLALAA PAWLREQVGV
551 VLQENVLLNR SIRDNIALTD TGMPLERIIE AAKLAGAHEF IHPLPEGYGT
601 VVGEQGAGLS GGQRQRIAIA RALITNPRIL IFDEATSALD YESERAIMQN
651 NQAICANRTV LIIAHRLSTV KTAMRIIAMD KGRIVEAGTQ QELLAKPNGY
701 YRYLYOLQNG *

ORF39a is homologous to a cytolysin from A. pleuropneumoniae:

sp|P26760|RT1B_ACTPL RTX-I TOXIN DETERMINANT B (TOXIN RTX-I SECRETION ATP-
BINDING PROTEIN) (APX-IB) (HLY-IB) (CYTOLYSIN IB) (CLY-IB)
>gi|97137|pir||D43599 cytolysin IB - Actinobacillus pleuropneumoniae
(serotype 9) >gi|36944 (X61112) ClyI-B protein [Actinobacillus
pleuropneumoniae] Length = 707 Score = 931 bits (2379), Expect =0.0
Identities = 472/690 (68%), Positives = 540/690 (77%),
Gaps = 3/690 (0%)
Query:  20 YHGIAANPADIQHEFCTSAQSDLNETQWXXXXXXXXXXXXVVRQPIKRLAMATLPALVWC 79
YH IA NP +++H+F    +  L+ T W             V++ I RLA   LPALVW
Sbjct:  20 YHNIAVNPEELKHKFDLEGKG-LDLTAWLLAAKSLELKAKQVKKAIDRLAFIALPALVWR 78
Query:  80 DDGNHFILAKTDGGGEHAQYLIQDLTTNKSAVLSFAEFSNRYSGKLILVASRASVLGSLA 139
+DG HFIL K D   E  +YLI DL T+   +L  AEF + Y GKLILVASRAS++G LA
Sbjct:  79 EDGKHFILTKIDN--EAKKYLIFDLETHNPRILEQAEFESLYQGKLILVASRASIVGKLA 136
Query: 140 KFDFTWFIPAVIKYRRXXXXXXXXXXXXXXXXXITPLFFQVVMDKVLVHRGFXXXXXXXX 199
KFDFTWFIPAVIKYR+                 ITPLFFQVVMDKVLVHRGF
Sbjct: 137 KFDFTWFIPAVIKYRKIFIETLIVSIFLQIFALITPLFFQVVMDKVLVHRGFSTLNVITV 196
Query: 200 XXXXXXXFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLSLPLSYFEHRRVGDTVARVR 259
FEIVL GLRTY+FAH+TSRIDVELGARLFRHLL+LP+SYFE+RRVGDTVARVR
Sbjct: 197 ALAIVVLFEIVLNGLRTYIFAHSTSRIDVELGARLFRHLLALPISYFENRRVGDTVARVR 256
Query: 260 ELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWVVLASLPAYAFWSAFISPILR 319
EL+QIRNFLTGQALTSVLDL FSFIF AVMWYYS  LT V+L SLP Y  WS FISPILR
Sbjct: 257 ELDQIRNFLTGQALTSVLDIMFSFIFFAVMWYYSPKLTLVILGSLPFYNGWSIFISPILR 316
Query: 320 TRLNDKFARNADNQSFLVESITAVGTVKAMAVEPQNTQRWDNQLAAYVASGFRVTKLAVV 379
RL++KFAR ADNQSFLVES+TA+ T+KA+AV PQMT  WD QLA+YV++GFRVT LA +
Sbjct: 317 RRLDEKFARGADNQSFLVESVTAINTIKALAVTPQMTNTWDKQLASYVSAGFRVTTLATI 376
Query: 380 GQQGVQLIQKLVTVATLWIGARLVIESKLTVGOLIAFNNLSGQVAAPVIRLAQLWQDFQQ 439
GQQGVQ IQK+V V TLW+GA LVI   L++GQLIAFNNLSGQV APVIRLAQLWQDFQQ
Sbjct: 377 GQQGVQFIQKVVNVITLWLGAMLVISGDLSIGQLIAFNNLSGQVIAPVIRLAQLWQDFQQ 436
Query: 440 VGISVAPLGDILNAPTENASSHLALPDIRGEITFEHVDFRYKADGRLILQDLNLRIRAGE 499
VGISV RLGD+LN+PTE+    LALP+I+G+ITF ++ FRYX D  +IL D+NL I+ GE
Sbjct: 437 VGISVTRLGDVLNSPTESYQGKLALPEIKGDITFRNIRFRYXPDAPVILNDVNLSIQQGE 496
Query: 500 VLGIVGRSGSGKSTLTKLVQRLYVPAOGRVLVDGNDLALAAPAWLRRQVGVVLQENVLLN 559
V+GIVGRSGSGKSTLTKL+QR Y+P  G+VL+DG+DLALA P WLRRQVGVVLQ+NVLLN
Sbjct: 497 VIGIVGRSGSGKSTLTKLIQRFYIPENGQVLIDGHDLALADPNWLRRQVGVVLQDNVLLN 556
Query: 560 RSIRDNIALTDTGMPLERIIEAAKLAGAIIEFINELPEGYGTVVGEQGNLSGGQRQRIAI 619
RSIRDNIAL D GMP+E+I+ AAKLAGAHEFI EL EGY T+VGEQGAGLSGGQRQRIAI
Sbjct: 557 RSIRDNIALADPGMPMEKIVHAAKLAGAHEFISELREGYNTIVGEOGAGLSGGQRQRIAI 616
Query: 620 ARALITNPRILIFDEATSALDYESERAIMQNMQAICANRTVLIIAHRLSTVKTAHRIIAM 679
ARAL+ NP+ILIFDEATSALDYESE IM+NM  IC  RTV+IIAHRLSTVK A RII M
Sbjct: 617 ARALVNNPKILIFDEATSALDYESEHIIMRNMHQICKGRTVIIIAHRLSTVKNADRIIVM 676
Query: 680 DKGRIVEAGTQQELLAKPNGYYRYLYDLQN 709
+KG+IVE G  +ELLA PNG Y YL+ LQ+
Sbjct: 677 EKGQIVEQGKHKELLADPNGLYHYLHQLQS 706

Homology with the HlyB Leucotoxin Secretion ATP-Binding Protein of Haemophilus actinomycetemcomitans (Accession Number X53955)

ORF39 and HlyB protein show 71% and 69% amino acid identity in 167 and 55 overlap at the N- and C-terminal regions, respectively:

Orf39 1 KFDFTWFIPAVIKYRRXXXXXXXXXXXXXXXXXITPLFFQVVMDKVLVHRGFXXXXXXXX 60
      KFDFTWFIPAVIKYR+                 ITPLFFQVVMDKVLVHRGF
HlyB  137 KFDFTWFIPAVIKYRKIFIETLIVSIFLQIFALITPLFFQVVMDKVLVHRGFSTLNVITV 196
Orf39 61 XXXXXXXFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLSLPLSYFEHRRVGDTVARVR 120
      FEI+LGGLRTY+FAH+TSRIDVELGARLFMLL+LP+SYFE RRVGDTVARVR
HlyB  197 ALAIVVLFEIILGGLRTYVFAHSTSRIDVELGARLFRHLLALPISYFEARRVGDTVARVR 256
Orf39 121 ELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWVVLASLIC 167
      EL+QIRNFLTGQALTS+LDL FSFIF AVMWYYS  LT VVL SL C
HlyB  257 ELDQIRNFLTGQALTSILDLLFSFIFFAVMWYYSPKLTLVVLGSLPC 303
      //
Orf39 166 ICANRTVLIIAHRLSTVKTAHRIIAMDKGRIVEAGTQQELLANXNGYYRYLYDLQ 220
IC NRTVLIIAHRLSTVK A RII MDKG I+E G  QELL +  G Y YL+ LQ
HlyB  651 ICQNRTVLIIAHRLSTVKNADRIIVMDKGEIIEQGKHQELLKDEKGLYSYLHQLQ 705

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 7

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 31>

1   ATGAAATACT TGATCCGCAC CGCCTTACTC GCAGTCGCAG CCGCCGGCAT
51  CTACGCCTGC CAACCGCAAT CCGAAGCCGC AGTGCAAGTC AAGGCTGAAA
101 ACAGCCTGAC CGCTATGCGC TTAGCCGTCG CCGACAAACA GGCAGAGATT
151 GACGGGTTGA ACGCCCAAAk sGACGCCGAA ATCAGA...

This corresponds to the amino acid sequence SEQ ID 32; ORF52>:

1  MKYLIRTALL AVAAAGIYAC QPQSEAAVQV KAZNSLTANR LAVADKQAEI
51 DGLNAQXDAE IR..

Further work revealed the complete nucleotide sequence <SEQ ID 33>:

1   ATGAAATACT TGATCCGCAC CGCCTTACTC GCAGTCGCAG CCGCCGGCAT
51  CTACGCCTGC CAACCGCAAT CCGAAGCCGC AGTGCAAGTC AAGGCTGAAA
101 ACAGCCTGAC CGCTATGCGC TTAGCCGTCG CCGACAAACA GGCAGAGATT
151 GACGGGTTGA ACGCCCAAAT CGACGCCGAA ATCAGACAAC GCGAAGCCGA
201 AGAATTGAAA GACTACCGAT GGATACACGG CGACGCGGAA GTGCCGGAGC
251 TGGAAAAATG A

This corresponds to the amino acid sequence <SEQ ID 34; ORF52-1>:

1  MKYLIRTALL AVAAAGIYAC QPQSEAAVQV KAENSLTAMR LAVADKQAEI
51 DGLNAQIDAE IRQREAEELK DThWIHGDAE VPELEK

Computer analysis of this amino acid sequence predicts a prokaryotic membrane lipoprotein lipid attachment site (underlined).

ORF52-1 (7 kDa) was cloned in the pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 4A shows the results of affinity purification of the GST-fusion. FIG. 4B shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF52-1.

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 8

The following DNA sequence was identified in N. meningitidis <SEQ ID 35>

1   ATGGTTATCG GAATATTACT CGCATCAAGC AAGCATGCTC TTGTCATTAC
51  TCTATTGTTA AATCCCGTCT TCCATGCATC CAGTTGCGTA TCGCGTTSGG
101 CAATACGGAA TAAAATCTGC TGTTCTGCTT TGGCTAAATT TGCCAAATTG
151 TTTATTGTTT CTTTAGGAGC AGCTTGCTTA GCCGCCTTCG CTTTCGACAA
201 CGCCCCCACA GGCGCTTCCC AAGCGTTGCC TTCCGTTACC GCACCCGTGG
251 CGATTCCCGC GCCCGCTTCG GCAGCCTGA

This corresponds to the amino acid sequence <SEQ ID 36; ORF56>:

1  MVIGILLASS KHALVITLLL NPVFHASSCV SRXAIRNKIC CSALAKFAKL
51 FIVSLGAACL AAFAFDNAPT GASQALPTVT APVAIPAPAS AA*

Further work revealed the complete nucleotide sequence <SEQ ID 37>:

1   ATGGCTTGTA CAGGTTTGAT GGTTTTTCCG TTAATGGTYA TCGGAATATT
51  ACTTGCATCA AGCAAGCCTG CTCCTTTCCT TACTCTATTG TTAAATCCCG
101 TCTTCCATGC ATCCAGTTGC GTATCGCGTT GGGCAATACG GAATAAAATC
151 TGCTGTTCTG CTTTGGCTAA ATTTGCCAAA TTGTTTATTG TTTCTTTAGG
201 AGCAGCTTGC TTAGCCGCCT TCGCTTTCGA CAACGCCCCC ACAGGCGCTT
251 CCCAAGCGTT GCCTACCGTT ACCGCACCCG TGGCGATTCC CGCGCCCGCT
301 TCGGCAGCCT GA

This corresponds to the amino acid sequence <SEQ ID 38; ORF56-1>:

1   MACTGLMVFP LNVZGILLAS SKPAPFLTLL LNPVFHASSC VSRWAIRNKI
51  CCSALAKFAK LFIVSLGAAC LAAFAFDNAP TGASQALPTV TAPVAIPAPA
101 SAA*

Computer analysis of this amino acid sequence predicts a leader peptide (underlined) and suggests that ORF56 might be a membrane or periplasmic protein.

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 9

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 39>

1   ATGTTCAGTA TTTTAAATGT GTTTCTTCAT TGTATTCTGG CTTGTGTAGT
51  CTCTGGTGAG ACGCCTACTA TATTTGGTAT CCTTGCTCTT TTTTACTTAT
101 TGTATCTTTC TTATCTTGCT GTTTTTAAGA TTTTCTTTTC TTTTTTCTTA
151 GACAGAGTTT CACTCCGGTC TCCCAGGCTG GAGTGCAAAT GGCATGACCC
201 TTTGGCTCAC TGGCTCACGG CCACTTCTGC TATTCTGCCG CCTCAGCCTC
251 CAGGG...

This corresponds to the amino acid sequence <SEQ ID 40; ORF63>:

1  MFSILNVFLR CILACVVSGE TPTIFGILAL FYLLYLSYLA VFKIFFSFFL
51 DRVSLRSPRL ECKWNDPLAH WLTATSAILP PQPPG...

Computer analysis of this amino acid sequence predicts a transmembrane region.

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 10

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 41>

1   ..GTGCGGACGT GGTTGGTTTT TTGGTTGCAG CGTTTGAAAT ACCCGTTGTT
51  GCTTTGGATT GCGGATATGT TGCTGTACCG GTTGTTGGGC GGCGCGGAAA
101 TCGAATGCGG CCGTTGCCCT GTGCCGCCGA TGACGGATTG GCAGCATTTT
151 TTGCCGGCGA TGGGAACGGT GTCGGCTTGG GTGGCGGTGA TTTGGGCATA
201 CCTGATGATT GAAAGTGAAA AAAACGGAAG ATATTGA

This corresponds to the amino acid sequence <SEQ ID 42; ORF69>:

1  ..VRTWLVFWLQ RLKYPLLLWI ADNLLYRLLG GAE1ECGRCP VPPMTDWQHF
51 LPANGTVSAW VAVIWAYLMI ESEKNGRY*

Computer analysis of this amino acid sequence predicts a transmembrane region.

A corresponding ORF from strain A of N. meningitidis was also identified:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF69 shows 96.2% identity over a 78 aa overlap with an ORF (ORF69a) from strain A of N. meningitidis:

          10        20        30        40        50        60
orf69.pep VRTWLVFWLQRLKYPLLLWIADMLLYRLLGGAEIECGRCPVPPMTDWQHFLPAMGTVSAW
          |||||||||||||||||| |||||||||||||||||||||||||||||||||:||||:||
orf69a    VRTWLVFWLQRLKYPLLLCIADMLLYRLLGGAEIECGRCPVPPMTDWQHFLPTMGTVAAW
          10        20        30        40        50        60
          70        79
orf69.pep VAVIWAYLMIESEKNGRYX
          |||||||||||||||||||
orf69a    VAVIWAYLMIESEKNGRYX
          70        79

The ORF69a nucleotide sequence <SEQ ID 43> is:

1   GTGCGGACGT GGTTGGTTTT TTGGTTGCAG CGTTTGAAAT ACCCGTTGTT
51  GCTTTGTATT GCGGATATGC TGCTGTACCG GTTGTTGGGC GGCGCGGAAA
101 TCGAATGCGG CCGTTGCCCT GTACCGCCGA TGACGGATTG GCAGCATTTT
151 TTGCCGACGA TGGGAACGGT GGCGGCTTGG GTGGCGGTGA TTTGGGCATA
201 CCTGATGATT GAAAGTGAAA AAAACGGAAG ATATTGA

This encodes a protein having amino acid sequence <SEQ ID 44>:

1  VRTWLVFWLQ RLKYPLLLCI ADMLLYRLLG GAEIECGRCP VPPNTDWQHF
51 LPTMGTVAAW VAVIWAYLMI ESEKNGRY*

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 11

The following DNA sequence was identified in N. meningitidis <SEQ ID 45>

1   ATGTTTCAAA ATTTTGATTT GGGCGTGTTC CTGCTTGCCG TCCTCCCCGT
51  GCTGCCCTCC ATTACCGTCT CGCACGTGGC GCGCGGCTAT ACGGCGCGCT
101 ACTGGGGAGA CAACACTGCC GAACAATACG GCAGGCTGAC ACTGAACCCC
151 CTGCCCCATA TCGATTTGGT CGGCACAATC ATCgTACCGC TGCTTACTTT
201 GATGTTCACG CCCTTCCTGT TCGGCTGGGC GCGTCCGATT CCTATCGATT
251 CGCGCAACTT CCGCAACCCG cGCCTTGCCT GGCGTTGCGT TGCCGCGTCC
301 GGCCCGCTGT CGAATCTAGC GATGGCTGTw CTGTGGGGCG TGGTTTTGGT
351 GCTGACTCCG TATGTCGGCG GGGCGTATCA GATGCCGTTG GCTCAAATGG
401 CAAACTACGG TATTCTGATC AATGCGATTC TGTTCGCGCT CAACATCATC
451 CCCATCCTGC CTTGGGACGG CGGCATTTTC ATCGACACCT TCCTGTCGGC
501 GAAATATTCG CAAGCGTTCC GCAAAATCGA ACCTTATGGG ACGTGGATTA
551 TCCTACTGCT GATGCTGACC SGGGTTTTGG GTGCGTTTAT wGCACCGATT
601 sTGCGGmTGc GTGATTGCrT TTGTGCAGAT GTwCGTCTGA CTGGCTTTCA
651 GACGGCATAA

This corresponds to the amino acid sequence <SEQ ID 46; ORF77>:

1   MFQNFDLGVF LLAVLPVLPS ITVSNVARGY TARYWGDNTA EQYGRLTLNP
51  LPHIDLVGTI IVPLLTLMFT PFLFGWPRPI PIDSRNFRNP RLAWRCVAAS
101 GPLSNLAMAV LWGVVLVLTP YVGGAYQMPL AQMANYGILI NAILFPLNII
151 PILPWDGGIF IDTFLSAKYS QAFRKIEPYG TWIILLLMLT XVLGAFIAPI
201 XRXRDCXCAD VRLTGFQTA*

Further work revealed the complete nucleotide sequence <SEQ ID 47>:

1   ATGTTTCAAA ATTTTGATTT GGGCGTGTTT CTGCTTGCCG TCCTGCCCGT
51  GCTGCTCTCC ATTACCGTCA GGGAGGTGGC GCGCGGCTAT ACGGCGCGCT
101 ACTGGGGAGA CAACACTGCC GAACAATACG GCAGGCTGAC ACTGAACCCC
151 CTGCCCCATA TCGATTTGGT CGGCACAATC ATCGTACCGC TGCTTACTTT
201 GATGTTCACG CCCTTCCTGT TCGGCTGGGC GCGTCCGATT CCTATCGATT
251 CGCGCAACTT CCGCAACCCG CGCCTTGCCT GGCGTTGCGT TGCCGCGTCC
301 GGCCCGCTGT CGAATCTAGC GATGGCTGTT CTGTGGGGCG TGGTTTTGGT
351 GCTGACTCCG TATGTCGGCG GGGCGTATCA GATGCCGTTG GCTCAAATGG
401 CAAACTACGG TATTCTGATC AATGCGATTC TGTTCGCGCT CAACATCATC
451 CCCATCCTGC CTTGGGACGG CGGCATTTTC ATCGACACCT TCCTGTCGGC
501 GAAATATTCG CAAGCGTTCC GCAAAATCGA ACCTTATGGG ACGTGGATTA
551 TCCTACTGCT GATGCTGACC GGGGTTTTGG GTGCGTTTAT TGCACCGATT
601 GTGCGGCTGG TGATTGCGTT TGTGCAGATG TTCGTCTGA

This corresponds to the amino acid sequence <SEQ ID 48; ORF77-1>:

1   MFQNFDLGVF LLAVLPVLLS ITVREVARGY TARYWGDNTA EQYGRLTLNP
51  LPHIDLVGTI IVPLLTLMFT PFLFGWARPI PIDSRNFRNP RLAWRCVAAS
101 GPLSNLAMAV LWGVVLVLTP YVGGAYQMPL AQMANYGILI NAILFALNII
151 PILPWDGGIF IDTFLSAXYS QAYRRIEPYG TWIILLLNLT GVLGAFIAPI
201 VRLVIAFVQH FV*

Computer analysis of this amino acid sequence reveals a putative leader sequence and several transmembrane domains.

A corresponding ORF from strain A of N. meningitidis was also identified:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF77 shows 96.5% identity over a 173 aa overlap with an ORF (ORF77a) from strain A of N. meningitidis:

          10        20        30        40        50        60
ort77.pep MFQNFDLGVFLLAVLPVLPSITVSHVARGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI
          |||||||||||||||||||||||||||||||||
orf77a    RGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI
          10        20        30
          70        80        90       100       110        120
orf77.pep IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLTP
          ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf77a    IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLTP
          40        50        60        70        80        90
          130       140       150       160       170       180
orf77.pep YVGGAYQMPLAQMANYGILINAILFALNIIPILPWDGGIFIDTFLSAKYSQAFRKIEPYG
          |||||||||||||||| ||||||| ||||||||||||||||||||||| |||||||||||
orf77a    YVGGAYQMPLAQMANYXILINAILXALNIIPILPWDGGIFIDTFLSAKXSQAFRKIEPYG
          100       110       120       130       140       150
          190       200       210       220
orf77.pep TWIILLLMLTGVLGAFIAPIVRLVIAFVQMFVX
          |||| |||||||||| |||||:|||||||||||
orf77a    TWIIXLLMLTGVLGAXIAPIVQLVIAFVQMFVX
          160       170       180

ORF77-1 and ORF77a show 96.8% identity in 185 aa overlap:

            10        20        30        40        50        60
orf77-1.pep MFQNFDLGVFLLAVLPVLLSITVREVARGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI
            |||||||||||||||||||||||||||||||||
orf77a      RGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI
            10        20        30
            70        80        90       100       110       120
orf77-1.pep IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLTP
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf77a      IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLTP
            40        50        60        70        80        90
            130       140       150       160       170       180
orf77-1.pep YVGGAYQMPLAQMANYGILINAILFALNIIPILPWDGGIFIDTFLSAKYSQAFRKIEPYG
            |||||||||||||||| ||||||| ||||||||||||||||||||||| |||||||||||
orf77a      YVGGAYQMPLAQMANYXILINAILXALNIIPILPWDGGIFIDTFLSAKXSQAFRKIEPYG
            100       110       120       130       140       150
            190       200       210
orf77-1.pep TWIILLLMLTGVLGAFIAPIVRLVIAFVQMFVX
            |||| |||||||||| |||||:|||||||||||
orf77a      TWIIXLLMLTGVLGAXIAPIVQLVIAFVQMFVX
            160       170       180

A partial ORF77a nucleotide sequence <SEQ ID 49> was identified:

1   ..CGCGGCTATA CAGCGCGCTA CTGGGGTGAC AACACTGCCG AACAATACGG
51  CAGGCTGACA CTGAACCCCC TGCCCCATAT CGATTTGGTC GGCACAATCA
101 TCGTACCGCT GCTTACTTTG ATGTTTACGC CCTTCCTGTT CGGCTGGGCG
151 CGTCCGATTC CTATCGATTC GCGCAACTTC CGCAACCCGC GCCTTGCCTG
201 GCGTTGCGTT GCCGCGTCCG GCCCGCTGTC GAATCTGGCG ATGGCTGTTC
251 TGTGGGGCGT GGTTTTGGTG CTGACTCCGT ATGTCGGTGG GGCGTATCAG
301 ATGCCGTTGG CNCAAATGGC AAACTACNNN ATTCTGATCA ATGCGATTCT
351 GTNCGCGCTC AACATCATCC CCATCCTGCC TTGGGACGGC GGCATTTTCA
401 TCGACACCTT CCTGTCGGCN AAATANTCGC AAGCGTTCCG CAAAATCGAA
451 CCTTATGGGA CGTGGATTAT CCNGCTGCTT ATGCTGACCG GGGTTTTGGG
501 TGCGTNTATT GCACCGATTG TGCAGCTGGT GATTGCGTTT GTGCAGATGT
551 TCGTCTGA

This encodes a protein having amino acid sequence <SEQ ID 50>:

1   ..RGYTARYWGD NTAEQYGRLT LNPLPHIDLV GTIIVPLLTL MFTPFLFGWA
51  RPIPIDSRNF RNPRLAWRCV AASGFLSNLA MAVLWGVVLV LTPYVGGAYQ
101 MPLAQNANYX ILINAILXAL NIIPILPWDG GIFIDTFLSA KXSQAFRKIE
151 PYGTWIIXLL MLTGVLGAXI APIVQLVIAF VQNFV*

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 12

The following partial DNA sequence was identified in N. meningitidis SEQ ID 51>

1   ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG TTATGGCGGT
51  TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT GAAATCCTGT
101 ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG GGAAATGCTG
151 GGCTACACCG CCCTCAAAAT GCCCGCCCGC GCCTACGAAC TGATTCCCCT
201 CGCCGTCCTT ATCGGCGGAC TGGTCTCCCT CAGCCAGCTT GCCGCCGGCA
251 GCGAACTGAC CGTCATCAAA GCCAGCGGCA TGAGCACCAA AAAGCTGCTG
301 TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA CCGTCGCGCT
351 CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA AACATCAAAG
401 CCGCCGCCAT CAACGGCAAA ATCAACACCG GCAATACCGG CCTTTGGCTG
451 AAAGAAAAAA ACAGCGTGAT CAATGTGCGC GAAATGTTGC CCGACCAT..

This corresponds to the amino acid sequence SEQ ID 52; ORF112>:

1   HNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG KGSYGIWEML
51  GYTALIQPAR AYELIPLAVL IGGLVSLSQL AAGSELTVIK ASGNSTKKLL
101 LILSQFGFIF AIATVPLGEW VAPTLSQKAE NIKAAAINGK ISTGNTGLWL
151 KEKNSVINVR EHLPDH...

Further work revealed further partial nucleotide sequence <SEQ ID 53>:

1   ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG TTATGGCGGT
51  TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT GAAATCCTGT
101 ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG GGAAATGCTG
151 GGCTACACCG CCCTCAAAAT GCCCGCCCGC GCCTACGAAC TGATTCCCCT
201 CGCCGTCCTT ATCGGCGGAC TGGTCTCCCT CAGCCAGCTT GCCGCCGGCA
251 GCGAACTGAC CGTCATCAAA GCCAGCGGCA TGAGCACCAA AAAGCTGCTG
301 TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA CCGTCGCGCT
351 CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA AACATCAAAG
401 CCGCCGCCAT CAACGGCAAA ATCAGCACCG GCAATACCGG CCTTGCTCTG
451 AAAGAAAAAA ACAGCTTAAT CAATGTGCGC GAAATGTTGC CCGACCATAC
501 GCTTTTGGGC ATCAAAATTT GGGCGCGCAA CGATAAAAAC GAATTGGCAG
551 AGGCAGTGGA AGCCGATTCC GCCGTTTTGA ACAGCGACGG CAGTTGGCAG
601 TTGAAAAACA TCCGCCGCAG CACGCTTGGC GAAGACAAAG TCGAGGTCTC
651 TATTGCGGCT GAAGAAAACT GGCCGATTTC CGTCAAACGC AACCTGATGG
701 ACGTATTGCT CGTCAAACCC GACCAAATGT CCGTCGGCGA ACTGACCACC
751 TACATCCGCC ACCTCCAAAA CAACAGCCAA AACACCCGAA TCTACGCCAT
601 CGCATGGTGG CGCAAATTGG TTTACCCCGC CGCAGCCTGG GTGATGGCGC
851 TCGTCGCCTT TGCCTTTACC CCGCAAACCA CCCGCCACGG CAATATGGGC
901 TTAAAACTCT TCGGCGGCAT CTGTSTCGGA TTGCTGTTCC ACCTTGCCGG
951 ACGGCTCTTT GGGTTTACCA GCCAACTCGG...

This corresponds to the amino acid sequence <SEQ ID 54; ORF112-1>:

1   MNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG KGSYGIWEHL
51  GYTALKMPAR AYELIPLAVL IGGLVSLSQL AAGSELTVIK ASGMSTKKLL
101 LILSQFGFIF AIATVALGEW VAPTLSQKAE NIKAAAINGK ISTGNTGLWL
151 KEKNSXINVR EHLPDHTLLG IKIWARNDKN ELAEAVEADS AVLNSDGSWQ
201 LKNIRRSTLG EDKVEVSIAA EENWPISVKR NLTDVLLVKP DQMSVGELTT
251 YIRHLONNSQ NTRIYAIAWW RKLVYPAAAW VMALVAFAFT PQTTRHGNMG
301 LKLFGGICXG LLFHLAGRLF GFTSQL...

Computer analysis of this amino acid sequence predicts two transmembrane domains.

A corresponding ORF from strain A of N. meningitidis was also identified:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF112 shows 96.4% identity over a 166 aa overlap with an ORF (ORF112a) from strain A of N. meningitidis:

           10        20        30        40        50        60
orf112.pep MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMLGYTALKMPAR
           |||||||||||||||||||||||||||||||||||||||||||||||| ||||||| ||
orf112a    MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMXGYTALKMXAR
           10        20        30        40        50        60
           70        80        90       100       110       120
orf112.pep AYELIPLAVLIGGLVSLSQLAAGSELTVIKASGMSTKKLLLILSQFGFIFAIATVALGEW
           ||||:||||||||||| |||||||||:|||||||||||||||||||||||||||||||||
orf112a    AYELMPLAVLIGGLVSXSQLAAGSELTVIKASGMSXKKLLLILSQFGFIFAIATVALGEW
           70        80        90       100       110       120
           130       140       150       160
orf112.pep VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSVINVREMLPDH
           |||||||||||||||||||||||||||||||||||:||||||||||
orf112a    VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSIINVREMLPDHTLLGIKIWARNDKN
           130       140       150       160
orf112a    ELAEAVEADSAVLNSDGSWQLKNIRRSTLGEDKVEVSIAAEEXWPISVKRNLMDVLLVKP
           190       200       210       220       230       240

A partial ORF112a nucleotide sequence <SEQ ID 55> was identified:

1    ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG TTATGGCGGT
51   TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT GAAATCCTGT
101  ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG GGAAATGNTG
151  GGNTACACCG CCCTCAAAAT GNCCGCCCGC GCCTACGAAC TGATGCCCCT
201  CGCCGTCCTT ATCGGCGGAC TGGTCTCTNT CAGCCAGCTT GCCGCCGGCA
251  GCGAACTGAN CGTCATCAAA GCCAGCGGCA TGAGCACCAA AAAGCTGCTG
301  TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA CCGTCGCGCT
351  CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA AACATCAAAG
401  CCGCGGCCAT CAACGGCAAA ATCAGTACCG GCAATACCGG CCTTTGGCTG
451  AAAGAAAAAA ACAGCATTAT CAATGTGCGC GAAATGTTGC CCGACCATAC
501  CCTGCTGGGC ATTAAAATCT GGGCCCGCAA CGATAAAAAC GAACTGGCAG
551  AGGCAGTGGA AGCCGATTCC GCCGTTTTGA ACAGCGACGG CAGTTGGCAG
601  TTGAAAAACA TCCGCCGCAG CACGCTTGGC GAAGACAAAG TCGAGGTCTC
651  TATTGCGGCT GAAGAAAANT GGCCGATTTC CGTCAAACGC AACCTGATGG
701  ACGTATTGCT CGTCAAACCC GACCAAATGT CCGTCGGCGA ACTGACCACC
751  TACATCCGCC ACCTCCAAAN NNACAGCCAA AACACCCGAA TCTACGCCAT
801  CGCATGGTGG CGCAAATTGG TTTACCCCGC CGCAGCCTGG GTGATGGCGC
851  TCGTCGCCTT TGCCTTTACC CCGCAAACCA CCCGCCACGG CAATATGGGC
901  TTAAAANTCT TCGGCGGCAT CTGTCTCGGP TTGCTGTTCC ACCTTGCCGG
951  NCGGCTCTTC NGGTTTACCA GCCAACTCTA CGGCATCCCG CCCTTCCTCG
1001 NCGGCGCACT ACCTACCATA GCCTTCGCCT TGCTCGCCGT TTGGCTGATA
1051 CGCAAACAGG AAAAACGCTA A

This encodes a protein having amino acid sequence <SEQ ID 56>:

1   MNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG KGSYGIWEMK
51  GYTALKMXAR AYELMPLAVL IGGLVSXSQL AAGSELXVIX ASGNSTKKLL
101 LILSQFGFIF AIATVALGEV VAPTLSQKAE NIKAAAINGK ISTGNTGLWL
151 KEKNSIINVR EMLPDHTLLG IKIWAIWDKN ELAEAVFADS AVLNSDGSWQ
201 LKNIRRSTLG EDKVEVSIAA EEXWPISVKR NLMDVLLVKP DQMSVGELTT
251 YIRHLQXXSQ NTRIYAIAWW RKLVYPAAAW VMALVAFAFT PQTTRHGNMG
301 LKXFGGICLG LLFHLAGRLF XFTSQLYGIP PFLXGALPTI AFALLAVWLI
351 RKQEKR*

ORF112a and ORF112-1 show 96.3% identity in 326 aa overlap:

orf112a.pep MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMXGYTALKMXAR
            ||||||||||||||||||||||||||||||||||||||||||||||||| ||||||| ||
orf112-1    MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMLGYTALKMXAR
orf112a.pep AYEIIPLAVLIGGLVSXSQLAAGSELXVIKASGHSTKKLLLILSQFGFIFAIATVALGEW
            ||||:||||||||||| |||||||||:|||||||||||||||||||||||||||||||||
orf112-1    AYEIIPLAVLIGGLVSLSQLAAGSELTVIKASGHSTKKLLLILSQFGFIFAIATVALGEW
orf112a.pep VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSIINVREMLPDHTLLGIKIWARNDKN
            ||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||
orf112-1    VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSXINVREMLPDHTLLGIKIWARNDKN
orf112a.pep ELAEAVEADSAVLNSDGSWQLKNIRRSTLGEDKVEVSIAAEEXWPISVKRNLMDVLLVKP
            |||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||
orf112-1    ELAEAVEADSAVLNSDGSWQLKNIRRSTLGEDKVEVSIAAEENWPISVKRNLMDVLLVKP
orf112a.pep DQMSVGELTTYIRHLQXXSQNTRIYAIAWWRKLVYPAAAWVMALVAFAFTPQTTRHGNMG
            ||||||||||||||||  ||||||||||||||||||||||||||||||||||||||||||
orf112-1    DQMSVGELTTYIRHLQXXSQNTRIYAIAWWRKLVYPAAAWVMALVAFAFTPQTTRHGNMG
orf112a.pep LKXFGGICLGLLFHLAGRLFXFTSQLYGIPPFLXGALPTIAFALLAVWLIRKQEXRX
            || ||||| ||||||||||| |||||
orf112-1    LKLFGGICXGLLFHLAGRLFGFTSQL

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 13

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 57>

1   ..GCAGTAGCCG AAACTGCCAA CAGCCAGGGC AAAGGTAAAC AGGCAGGCAG
51  TTCGGTTTCT GTTTCACTGA AAACTTCAGG CGACCTTTGC GGCAAACTCA
101 AAACCACCCT TAAAACTTTG GTCTGCTCTT TGGTTTCCCT GAGTATGGTA
151 TTGCCTGCCC ATGCCCAAAT TACCACCGAC AAATCACCAC CTAAAAACCA
201 GCAGGTCGTT ATCCTTAAAA CCAACACTGG TGCCCCCTTG GTGAATATCC
251 AAACTCCGAA TGGACGCGGA TTGAGCCACA ACCGCTA.TA CGCATTTGAT
301 GTTGACAACA AAGGGGCAGT GTTAAACAAC GACCGTAACA ATAATCCGTT
351 TGTGGTCAAA GGCAGTGCGC AATTGATTTT GAACGAGGTA CGCGGTACGG
401 CTAGCAAACT CAACGGCATC GTTACCGTAG GCGGTCAAAA GGCCGACGTG
451 ATTATTGCCA ACCCCAACGG CATTACCGTT AATGGCGGCG GCTTTAAAAA
501 TGTCGGTCGG GGCATCTTAA CTACCGGTGC GCCCCAAATC GGCAAAGACG
551 GTGCACTGAC AGGATTTGAT GTGCGTCAAG GCACATTGGA CCGTAGTAGC
601 AGCAGGTTGG AATGATAAAG GCGGAGCmrm yTACACCGGG GTACTTGCTC
651 GTGCAGTTGC TTTGCAGGGG AAATTwmmGG GTAAA.AACT GGCGGTTTCT
701 ACCGGTCCTC AGAAAGTAGA TTACGCCAGC GGCGAAATCA GTGCAGGTAC
751 GGCAGCGGGT ACGAAACCGA cTATTGCCCT TGATACTGCC GCACTGGGCG
901 GTATGTACGC CGACAGCATC ACACTGATTG CCAATGAAAA AGGCGTAGGC
951 GTCTAA

This corresponds to the amino acid sequence <SEQ ID 58; ORF114>:

1   ..AVAETANSQG KGKQAGSSVS VSLKTSGDLC GKLKTTLKTL VCSLVSLSHV
51  LPAHAQITTD KSAPKNQQVV ILKTNTGAPL VNIQTPNGRG LSHNRXYAFD
101 VDNKGAVLNN DRNNNPFVVK GSAQLILNEV RGTASKLNGI VTVGGQKADV
151 IIANPNGITV NGGGFXNVGR GILTTGAPQI GKDGALTGFD VVKAHVTVXA
201 AGWNDKGGAX YTGVLAPAVA LQGKXXGLXL AVSTGPQKVD YASGEISAGT
251 AAGTKPTIAL DTAALGGNYA DSITLIANEX GVGV*

Further work revealed the complete nucleotide sequence SEQ ID 59>:

1    ATGAATAAAG GTTTACATCG CATTATCTTT AGTAAAAAGC ACAGCACCAT
51   GGTTGCAGTA GCCGAAACTG CCAACAGCCA GGGCAAAGGT AAACAGGCAG
101  GCAGTTCGGT TTCTGTTTCA CTGAAAACTT CAGGCGACCT TTGCGGCAAA
151  CTCAAAACCA CCCTTAAAAC TTTGGTCTGC TTTTTGGTTT CCCTGAGTAT
201  GGTATTGCCT GCCCATGCCC AAATTACCAC CGACAAATCA GCACCTAAAA
251  ACCAGCAGGT CGTTATCCTT AAAACCAACA CTGGTGCCCC CTTGGTGAAT
301  ATCCAAACTC CGAATGGACG CGGATTGAGC CACAACCGCT ATACGCAGTT
351  TGATGTTGAC AACAAAGGGG CAGTGTTAAA CAACGACCGT AACAATAATC
401  CGTTTGTGGT CAAAGGCAGT GCGCAATTGA TTTTGAACGA GGTACGCGGT
451  ACGGCTAGCA AACTCAACGG CATCGTTACC GTAGGCGGTC AAAAGGCCGA
501  CGTGATTATT GCCAACCCCA ACGGCATTAC CGTTAATGGC GGCGGCTTTA
551  AAAATGTCGG TCGGGGCATC TTAACTACCG GTGCGCCCCA AATCGGCAAA
601  GACGGTGCAC TGACAGGATT TGATGTGCGT CAAGGCACAT TGACCGTAGG
651  AGCAGCAGGT TGGAATGATA AAGGCGGAGC CGACTACACC GGGGTACTTG
701  CTCGTGCAGT TGCTTTGCAG GGGAAATTAC AGGGTAAAAA CCTGGCGGTT
751  TCThCCGGTC CTCAGAAAGT AGATTACGCC AGCGGCGAAA TCAGTGCAGG
801  TACGGCAGCG GGTACGAAAC CGACTATTGC CCTTGATACT GCCGCACTGG
951  GCGGTATGTA CGCCGACACC ATCACACTGA TTGCCAATGA AAAAGGCGTA
901  GGCGTCAAAA ATGCCGGCAC ACTCGAAGCG GCCAAGCAAT TGATTGTGAC
951  TTCGTCAGGC CGCATTGAAA ACAGCGGCCG CATCGCCACC ACTGCCGACG
1001 GCACCGAAGC TTCACCGACT TATCTCTCCA TCGAAACCAC CGAAAAAGGA
1051 GCGGCAGGCA CATTTATCTC CAATGGTGGT CGGATCGAGA GCAAAGGCTT
1101 ATTGGTTATT GAGACGGGAG AAGATATCAG CTTGCGTAAC GGAGCCGTGG
1151 TGCAGAATAA CGGCAGTCTC CCAGCTACCA CGGTATTAAA TGCTGGTCAT
1201 AATTTGGTGA TTGAGAGCAA AACTAATGTG AACAATGCCA AAGGCCCGGC
1251 TACTCTGTCG GCCGACGGCC GTACCGTCAT CAAGGAGGCC AGTATTCAGA
1301 CTGGCACTAC CGTATACAGT TCCAGCAAAG GCAACGCCGA ATTAGGCAAT
1351 AACACACGCA TTACCGGGGC AGATGTTACC GTATTATCCA ACGGCACCAT
1401 CAGCAGTTCC GCCGTAATAG ATGCCAAAGA CACCGCACAC ATCGAAGCAG
1451 GCAAACCGCT TTCTTTGGAA GCTTCAACAG TTACCTCCGA TATCCGCTTA
1501 AACGGAGGCA GTATCAAGGG CGGCAAGCAG CTTGCTTTAC TGGCAGACGA
1551 TAACATTACT GCCAAAACTA CCAATCTGAA TACTCCCGGC AATCTGTATG
1601 TTCATACAGG TAAAGATCTG AATTIGAATG TTGATAAAGA TTTGTCIGCC
1651 GCCAGCATCC ATTTGAAATC GGATAACGCT GCCCATATTA CCGGCACCAG
1701 TAAAACCCTC ACTGCCTCAA AAGACATGGG TGTGGAGGCA GGCTCGCTGA
1751 ATGTTACCAA TACCAATCTG CGTACCAACT CGGGTAATCT GCACATTCAG
1801 GCAGCCAAAG GCAATATTCA GCTTCGCAAT ACCAAGCTGA ACGCAGCCAA
1851 GGCTCTCGAA ACCACCGCAT TGCAGGGCAA TATCGTTTCA GACGGCCTTC
1901 ATGCTGTTTC TGCAGACGGT CATGTATCCT TATCGGCCAA CGGTAATGCC
1951 GACTTTACCG GTCACAATAC CCTGACAGCC AAGGCCGATG TCAATGCAGG
2001 ATCGGTTGGT AAAGGCCGTC TGAAAGCAGA CAATACCAAT ATCACTTCAT
2051 CTTCAGGAGA TATTACGTTG GTTGCCGGCA ACGGTATTCA GCTTGGTGAC
2101 GGAAAACAAC GCAATTCAAT CAACGGAAAA CACATCAGCA TCAAAAACAA
2151 CGGTGGTAAT GCCGACTTAA AAAACCTTAA CGTCCATGCC AAAAGCGGGG
2201 CATTGAACAT TCATTCCGAC CGGGCATTGA GCATAGAAAA TACCAAGCTG
2251 GAGTCTACCC ATAATACGCA TCTTAATGCA CAACACGAAG GGGTAACGCT
2301 CAACCAAGTA GATGCCTACG CACACCGTCA TCTAAGCATT ACCGGCAGCC
2351 AGATTTGGCA AAACGACAAA CTGCCTTCTG CCAACAAGCT GGTGGCTAAC
2401 GGTGTATTGG CACTCAATGC GCGCTATTCC CAAATTGCCG ACAACACCAC
2451 GCTGAGAGCG GGTGCAATCA ACCTTATTGC CGGTACCGCC CTAGTCAAGC
2501 GCGGCAACAT CAATTGGAGT ACCGTTGCGA CCAAAACTTT GGAAGATAAT
2551 GCCGAATTAA AACCATTGGC CGGACGGCTG AATATTGAAG CAGGTAGCGG
2601 CACATTAACC ATCGAACCTG CCAACCGCAT CAGTGCGCAT ACCGACCTGA
2651 GCATCAAAAC AGGCGGAAAA TTGCTGTTGT CTGCAAAAGG AGGAAATGCA
2701 GGTGCGCCTA GTGCTCAAGT TTCCTCATTG GAAGCAAAAG GCAATATCCG
2751 TCTGGTTACA GGAGAAACAG ATTTAAGAGG TTCTAAAATT ACAGCCGGTA
2801 AAAACTTGGT TGTCGCCACC ACCAAAGGCA AGTTGAATAT CGAAGCCGTA
2951 AACAACTCAT TCAGCAATTA TTTTCCTACA CAAAAAGCGG CTGAACTCAA
2901 CCAAAAATCC AAAGAATTGG AACAGCAGAT TGCGCAGTTG AAAAAAAGCT
2951 CGCCTAAAAG CAAGCTGATT CCAACCCTGC AAGAAGAACG CGACCGTCTC
3001 GCTTTCTATA TTCAAGCCAT CAACAAGGAA GTTAAAGGTA AAAAACCCAA
3051 AGGCAAAGAA TACCTGCAAG CCAAGCTTTC TGCACAAAAT ATTGACTTGA
3101 TTTCCGCACA AGGCATCGAA ATCAGCGGTT CCGATATTAC CGCTTCCAAA
3151 AAACTGAACC TTCACGCCGC AGGCGTATTG CCAAAGGCAG CAGATTCAGA
3201 GGCGGCTGCT ATTCTGATTG ACGGCATAAC CGACCAATAT GAAATTGGCA
3251 AGCCCACCTA CAAGAGTCAC TACGACAAAG CTGCTCTGAA CAAGCCTTCA
3301 CGTTTGACCG GACGTACAGG GGTAAGTATT CATGCAGCTG CGGCACTCGA
3351 TGATGCACGT ATTATTATCG GTGCATCCGA AATCAAAGCT CCCTCAGGCA
3401 GCATAGACAT CAAAGCCCAT AGTGATATTG TACTGGAGGC TGGACAAAAC
3451 GATGCCTATA CCTTCTTAAA AACCAAAGGT AAAAGCGGCA AAATCATCAG
3501 AAAAACCAAG TTTACCAGCA CCCGCGACCA CCTGATTATG CCAGCCCCCG
3551 TCGAGCTGAC CGCCAACGGC ATAACGCTTC AGGCAGGCGG CAACATCGAA
3601 GCTAATACCA CCCGCTTCAA TGCCCCTGCA GGTAAAGTTA CCCTGGTTGC
3651 GGGTGAAGAG CTGCAACTGC TGGCAGAASA AGGCATCCAC AAGCACGAGT
3701 TGGATGTCCA AAAAAGCCGC CGCTTTATCG GCATCAAGGT AGGCAAGAGC
3751 AATTACAGTA AAAACGAACT GAACGAAACC AAATTGCCTG TCCGCGTCGT
3801 CGCCCAAACT GCAGCCACCC GTTCAGGCTG GGATACAGTG CTCGAAGGTA
3051 CCGAATTCAA AACCACGCTG GCCGGTGCGG ACATTCAGGC AGGTGTAGGC
3901 GAAAAAGCCC GTGCCGATGC GAAAATTATC CTCAAAGGCA TTGTGAACCG
3951 TATCCAGTCG GAAGAAAAAT TAGAAACCAA CTCAACCGTA TGGCAGAAAC
4001 AGGCCGGACG CGGCAGCACT ATCGAAACGC TGAAACTGCC CAGCTTCGAA
4051 AGCCCTACTC CGCCCAAACT GACCGCCCCC GGTGGCTATA TCGTCGACAT
4101 TCCGAAAGGC AATTTGAAAA CCGAAATCGA AAAGCTGGCC AAACAGCCCG
4151 AGTATGCCTA TCTGAAACAG CTCCAAGTAG CGAAAAACGT CAACTGGAAC
4201 CAGGTGCAAC TGGCTTACGA TAAATGGGAC TATAAGCAGG AAGGCTTAAC
4251 CAGAGCCGGT GCAGCGATTG TTACCATAAT CGTAACCGCA CTGACTTATG
4301 GATACGGCGC AACCGCAGCG GGCGGTGTAG CCGCTTCAGG AAGTAGTACA
4351 GCCGCAGCTG CCGGAACAGC CGCCACAACG ACAGCAGCAG CTACTACCGT
4401 TTCTACAGCG ACTGCCATGC AAACCGCTGC TTTAGCCTCC TTGTATAGCC
4451 AAGCAGCTGT ATCCATCATC AATAATAAAG GTGATGTCGG CAAAGCGTTG
4501 AAAGATCTCG GCACCAGTGA TACGGTCAAG CAGATTGTCA CTTCTGCCCT
4551 GACGGCGGGT GCATTAAATC AGATGGGCGC AGATATIGCC CAATTGAACA
4601 GCAAGGTAAG AACCGAACTG TTCAGCAGTA CGGGCAATCA AACTATTGCC
4651 AACCTTGGAG GCAGATTGGC TACCAATCTC AGTAATGCAG GTATCTCAGC
4701 TGGTATCAAT ACCGCCGTCA ACGGCGGCAG CCTGAAAGAC AACTTAGGCA
4751 ATGCCGCATT AGGAGCATTG GTTAATAGCT TCCAAGGAGA AGCCGCCAGC
4801 AAAATCAAAA CAACCTTCAG CGACGATTAT GTTGCCAAAC AGTTCGCCCA
4851 CGCTTTGGCT GGGTGTGTTA GCGGATTGGT ACAAGGAAAA TGTAAAGACG
4901 GGGCAATTGG CGCAGCAGTT GGGGAAATCG TAGCCGACTC CATGCTTGGC
4951 GGCAGAAACC CTGCTACACT CAGCGATGCG GAAAAGCATA AGGTTATCAG
5001 TTACTCGAAG ATTATTGCCG GCAGCGTGGC GGCACTCAAC GGCGGCGATG
5051 TGAATACTGC GGCGAATGCG GCTGAGGTGG CGGTAGTGAA TAATGCTTTG
5101 AATTTTGACA GTACCCCTAC CAATGCGAAA AAGCATCAAC CGCAGAAGCC
5151 CGACAAAACC GCACTGGAAA AAATTATCCA AGGTATTATG CCTGCACATG
5201 CAGCAGGTGC GATGACTAAT CCGCAGGATA AGGATGCTGC CATTTGGATA
5251 AGCAATATCC GTAATGGCAT CACAGGCCCG ATTGTGATTA CCAGCTATGG
5301 GGTTTATGCT GCAGGTTGGA CAGCTCCGCT GATCGGTACA GCGGGTAAAT
5351 TAGCTATCAG CACCTGCATG GCTAATCCTT CTGGTTGTAC TGTCATGGTC
5401 ACTCAGGCTG CCGAAGCGGG CGCGGGAATC GCCACGGGTG CGGTAACGGT
5451 AGGCAACGCT TGGGAAGCGC CTGTGGGGGC GTTGTCGAAA GCGAAGGCGG
5501 CCAAGCAGGC TATACCAACC CAGACAGTTA AAGAACTTGA TGGCTTACTA
5551 CAAGAATCAA AAAATATAGG TGGTGTAAAT ACACGAATAA ATATAGCGAA
5601 TAGTACTACT CGATATACAC CAATGAGACA AACGGGACAA CCGCTATCTG
5651 CTGGCTTTGA GCATGTFCTT GAGGGGGACT TCCATAGGCC TATTGCGAAT
5701 AACCGTTCAG TTTTTACCAT CTCCCCAAAT GAATTGAAGG TTATACTTCA
5751 AAGTAATAAA GTAGTTTCTT CTCCCGTATC GATGACTCCT GATGGCCAAT
5801 ATATGCGGAC TGTCGATGTA GGAAAAGTTA TTGGTACTAC TTCTATTAAA
5651 GAAGGTGGAC AACCCACAAC TACAATTAAA GTATTTACAG ATAAGTCAGG
5901 AAATTTGATT ACTACATACC CAGTAAAAGG AAACTAA

This corresponds to the amino acid sequence <SEQ ID 60; ORF114-1>:

1    HNKGLHRIIF SXKHSTMVAV AETANSQGKG KQAGSSVSVS LKTSGDLCGK
51   LKTTLKTLVC SLVSLSHVLP AHAQITTDKS APKNQQVVIL KFNTGAPLVN
101  IQTPNGRGLS HNRYTQFDVD NKGAVLNNDR NNNPFVVKGS AQLILNEVRG
151  TASKLNGIVT VGGQKADVII ANPNGITVNG GGFKNVGRGI LTTGAPQIGK
201  DGALTGFDVR QGTLTVGAAG WNDKGGADYT GVLAAAVALQ GKLQGKLLAV
251  STGFQKVDYA SGEISAGTAA GTKPTIALDT AALGGHYADS ITLIANEKGV
301  GVKNAGTLKA AXQLIVTSSG RIENSGRIAT TADGTFASPT YLSIETTEKG
351  AAGTFISNGG RIESKGLLVI ETGEDISLRN GAVVQINGSR PATTVLNAGH
401  HLVIESKTNV NNAKGFATLS ADGRTVIKEA SIQTGTTVYS SSKGTAELGN
451  NTRZTGADVT VLSNGTISSS AVIDAKOTAN IEAGKPLSLT ASTVTSDIRL
501  NGGSIKGGKQ LALLADDNIT AXTTNLNTPG NLYVHTGKDL NIMVDKDLSA
551  ASIHLKSDNA AHITGTSKTL TASKDMGVEA GSLNVTNTNL RTNSGNLHIQ
601  AAKGNIQLRN TKLNAAKALE TTALQGNIVS OGLHAVSADG HVSLLANGNA
651  DFTGHNTLTA KADVNAGSVG KGRLKADNTN ITSSSGDITL VAGNGIQLGD
701  GKQRNSINGK HISIKNNGGN ADLIQLNVHA KSGALNIHSD RALSIENTKL
751  ESTRNTHLNA QHERVTLNQV DAYAHRHLSI TGSQIWQNOK LPSANKLVTN
801  GVLALNARYS QIADNTTLRA GAINLTAGTA LVKRGNINWS TVSTKTLEDN
851  AELKPLAGRL NIEAGSGTLT IEPANRISAH TDLSIKTGGK LLLSAKGGNA
901  GAFSAQVSSL EAKGNIRLVT GETDLRGSKI TAGKNLVVAT TKGKLNIEAV
951  NNSFSNYFPT QKAAELNQKS KELEQQIAQL KKSSPKSKLI PTLQEERDRL
1001 AFYIQAINKE VKGKKPKGKE YLQAKLSAQN IDLISAQGIE ISGSDITASK
1051 KLNLHAAGVL PKAADSEAAA ILIDGITDQY EIGKPTYKSH YOKAALNKPS
1101 RLTGRTGVSI HAAAALDDAR IIIGASEIKA FSGSIDIKAH SDIVLEAGQN
1151 DAYTFLKTKG KSGKIIRXTK FTSTRDHLIM PAPVELTANG ITLQAGGNIE
1201 ANTTRFNAPA GKVTLVAGEE LQLLAEEGIH KHELDVQKSR RFIGIKVGKS
1251 NYSKNELNET KLPVRVVAQT AATRSGWDTV LEGTEFKTTL AGADIQAGVG
1301 EKAPADAKII LKGIVNRIQS EEKLETNSTV WQKQAGRGST IETLKLPSFE
1351 SPTPPKLTAP GGYIVDIPKG NLKTEIEKLA KQPEPEYLKQ LQVAKNVNWN
1401 QVQLAYDKWD YKQEGLTRAG AAIVTIIVTA LTYGYGATAA GGVAASGSST
1451 AAAAGTAATT TAAATTVSTA TANQTAALAS LYSQAAVSII NNKGDVGKAL
1501 KDLGTSDTVK QIVISALTAG ALNQMGADIA QLNSKVRTEL FSSTGVQTIA
1551 NLGGRLATNL SNAGISAGIN TAVNGGSLKD NLGNAALGAL VNSFQGEAAS
1601 KIKTTFSDDY VAKQFAHALA GCVSGLVQGK CKDGAIGAAV GEIVADSNLG
1651 GRNPATLSDA EKHKVISYSK IIAGSVAALN GGDVNTAANA AEVAVVNNAL
1701 NFDSTPTNAK KNQPQKPDKT ALEKIIQGIM PAHAAGAMTN PQDKDAAIWI
1751 SNIRNGITGP IVITSYGVYA AGWTAPLIGT AGKLAISTCM ANPSGCTVNV
1801 TQAAEAGAGI ATGAVTVGNA WEAPVGALSK AKAAKQAIPT QTVKELDGLL
1851 QESKNIGAVN TRINIANSTT RYTPNRQTGQ PVSAGFENVL EGHFHRPIAN
1901 NRSVFTXSPN ELKVILQSNK VVSSPVSMTP DGQYNRTVDV GKVIGTTSIK
1951 EGGQPTTTIK VFTDKSGNLI TTYPVKGN*

Computer analysis of this amino acid sequence predicts a transmembrane region and also gives the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF114 shows 91.9% identity over a 284 aa overlap with an ORF (ORF114a) from strain A of N. meningitidis:

           10        20        30        40
orf114.pep AVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC
           ||||||||||||||||||||||||||||||||||||||||||
orf114a    MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC
           10        20        30        40        50        60
           50        60        70        80        90       100
orf114.pep SLVSLSMVLPAHAQITTDKSAPKNQQVVILKTNTGAPLVNIQTPNGRGLSHNRXYAFDVD
           |||||||      ||||||||||| ||||||||||||||||||||||||||||   ||||
orf114a    SLVSLSMXXXXXXQITTDKSAPKNXQVVILKTNTGAPLVNIQTPNGRGLSHNRYTQFDVD
           70        80        90        100        110        120
           110       120       130       140       150       160
orf114.pep NKGAVLNNDRNNNPFVVKGSAQLILNEVRGTALKLNGIVTVGGQKADVIIANPNGITVNG
           |||||||||||||||:||||||||||||||||||||||||||||||||||||||||||||
orf114a    NKGAVLNNDRNNNPFLVKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG
           130       140       150       160       170       180
           170       180       190       200       210       220
orf114.pep GGFKNVGRGILTTGAPQIGKDGALTGFDVVKAHWTVXAAGWNDKGGAXYTGVLARAVALQ
|||||||||||| |||||||||||||||| ::  || |||||||||| ||||||||||||
orf114a    GGFKNVGRGILTIGAPQIGKDGALTGFDVRQGTLTVAAAGWNDKGGADYTGVLARAVALQ
           190       200       210       220       230       240
           230       240       250       260       270       290
orf114.pep GKXXGKXLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIANEKGV
           ||  || |||||||||||||||||||||||||||||||||||||||||||||||| ||||
orf114a    GKLQGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIAXEKGV
           250       260       270       280       290       300
orf114.pep GVX
           ||
orf114a    GVKNAGTLEAAKQLIVTSSGRIENSGRIATTADGTEASPTYLXIETTEKGAXGTFISNGG
           310       320       330       340       350       360

The complete length ORF114a nucleotide sequence <SEQ ID 61> is:

1    ATGAATAAAG GTTTACATCG CATTATCTTT AGTAAAAAGC ACAGCACCAT
51   GGTTGCAGTA GCCGAAACTG CCAACAGCCA GGGCAAAGGT AAACAGGCAG
101  GCAGTTCGGT TTCTGTTTCA CTGAAAACTT CAGGCGACCT TTGCGGCAAA
151  CTCAAAACCA CCCTTAAAAC CTTGGTCTGC TCTTTGGTTT CCCTGAGTAT
201  GGNATTNCNN NNCNNTNCCC AAATTACCAC CGACAAATCA GCACCTAAAA
251  ACCANCAGGT CGTTATCCTT AAAACCAACA CTGGTGCCCC CTTGGTGAAT
301  ATCCAAACTC CGAATGGACG CGGATTGAGC CACAACCGCT ATACGCAGTT
351  TGATGTTGAC AACAAAGGGG CAGTGTTAAA CAACGACCGT AACAATAATC
401  CGTTTCTGGT CAAAGGCAGT GCGCAATTGA TTTTGAACGA GGTACGCGGT
451  ACGGCTAGCA AACTCAACGG CATCGTTACC GTAGGCGGTC AAAAGGCCGA
501  CGTGATTATT GCCAACCCCA ACGGCATTAC TGTTAATGGC GGCGGCTTTA
551  AAAATGTCGG TCGGGGCATC TTAACTATCG GTGCGCCCCA AATCGGCAAA
601  GACGGTGCAC TGACAGGATT TGATGTGCGT CAAGGCACAT TGACCGTAGG
651  AGCAGCAGGT TGGAATGATA AAGGCGGAGC CGACTACACC GGGGTACTTG
701  CTCGTGCAGT TGCTTTGCAG GGGAAATTAC AAGGTAAAAA CCTGGCGGTT
751  TCTACCGGTC CTCAGAAAGT AGATTACGCC AGCGGCGAAA TCAGTGCAGG
801  TACGGCAGCG GGTACGAAAC CGACTATTGC CCTTGATACT GCCGCACTGG
951  GCGGTATGTA CGCCGAGAAA ATCACACTGA TTGCCAATGA AAAAGGCGTA
901  GGcGTCAAAA ATGCCGGCAC ACTCGAAGCG GCCAAGCAAT TGATTGTGAC
951  TTCGTCAGGC CGCATTGAAA ACAGCGGCCG CATCGCCACC ACTGCCGACG
1001 GCACCGAAGC TTCACCGACT TATCTNNCNA TCGAAACCAC CGAAAAAGGA
1051 GCNNCAGGCA CATTTATCTC CAATGGTGGT CGGATCGAGA GCAAAGGCTT
1101 ATTGGTTATT GAGACGGGAG AAGATATCAT CTTGCGTAAC GGAGCCGTGG
1151 TGCAGAATAA CGGCAGTCGC CCAGCTACCA CGGTATTAAA TGCTGGTCAT
1201 AATTTGGTGA TTGAGAGTAA AACTAATGTG AACAATGCCA AAGGCTCGNC
1251 TAATCTGTCG GCCGGCGGTC GTACTACGAT CAATGATGCT ACTATTCAAG
1301 CGGGCAGTTC CGTGTACAGC TCCACCAAAG GCGATACTGA NTTGGGTGAA
1351 AATACCCGTA TTATTGCTGA AAACGTAACC GTATTATCTA ACGGTAGTAT
1401 TGGCAGTGCT GCTGTAATTG AGGCTAAAGA CACTGCACAC ATTGAATCGG
1451 GCAAACCGCT TTCTTTAGAA ACCTCGACCG TTGCCTCCAA CATCCGTTTG
1501 AACAACGGTA ACATTAAAGG CGGAAAGCAG CTTGCTTTAC TGGCAGACGA
1551 TAACATTACT GCCAAAACTA CCAATCTGAA TACTCCCGGC AATCTGTATG
1601 TTCATACAGG TAAAGATCTG AATTTGAATG TTGATAAAGA TTTGTCTGCC
1651 GCCAGCATCC ATTTGAAATC GGATAACGCT GCCCATATTA CCGGCACCAG
1701 TAAAACCCTC ACTGCCTCAA AAGACATGGG TGTGGAGGCA GGCTTGCTGA
1751 ATGTTACCAA TACCAATCTG CGTACCAACT CGGGTAATCT GCACATTCAG
1801 GCAGCCAAAG GCAATATTCA GCTTCGCAAT ACCAAGCTGA ACGCAGCCAA
1851 GGCTCTCGAA ACCACCGCAT TGCAGGGCAA TATCGTTTCA GACGGCCTTC
1901 ATGCTGTTTC TGCAGACGGT CATGTATCCT TATTGGCCAA CGGTAATGCC
1951 GACTTTACCG GTCACAATAC CCTGACAGCC AAGGCCGATG TCNATGCAGG
2001 ATCGGTTGGT AAAGGCCGTC TGAAAGCAGA CAATACCAAT ATCACTTCAT
2051 CTTCAGGAGA TATTACGTTG GTTGCCGNNN NCGGTATTCA GCTTGGTGAC
2101 GGAAAACAAC GCAATTCAAT CAACGGAAAA CACATCAGCA TCAAAAACAA
2151 CGGTGGTAAT GCCGACTTAA AAAACCTTAA CGTCCATGCC AAAAGCGGGG
2201 CATTGAACAT TCATTCCGAC CGGGCATTGA GCATAGAAAA TACNAAGCTG
2251 GAGTCTACCC ATAATACGCA TCTTAATGCA CAACACGAGC GGGTAACGCT
2301 CAACCAAGTA GATGCCTACG CACACCGTCA TCTAAGCATT ANCGGCAGCC
2351 AGATTTGGCA AAACGACAAA CTGCCTTCTG CCAACAAGCT GGTGGCTAAC
2401 GGTGTATTGG CAATCAATGC GCGCTATTCC CAAATTGCCG ACAACACCAC
2451 GCTGAGAGCG GGTGCAATCA ACCTTACTGC CGGTACCGCC CTAGTCAAGC
2501 GCGGCAACAT CAATTGGATT ACCGTTTCGA CCAAGACTTT GGAAGATAAT
2551 GCCGAATTAA AACCATTGGC CGGACGGCTG AATATTGAAG CAGGTAGCGG
2601 CACATTAACC ATCGAACCTG CCAACCGCAT CAGTGCGCAT ACCGACCTGA
2651 GCATCAAAAC AGGCGGAAAA TTGCTGTTGT CTGCAAAAGG AGGAAATGCA
2701 GGTGCGCNTA GTGCTCAAGT TTCCTCATTG GAAGCAAAAG GCAATATCCG
2751 TCTGGTTACA GGAGNAACAG ATTTAAGAGG TTCTAAAATT ACAGCCGGTA
2901 AAAACTTGGT TGTCGCCACC ACCAAAGGCA AGTTGAATAT CGAAGCCGTA
2951 AACAACTCAT TCAGCAATTA TTTTCNTACA CAAAAAGNGN NNGNNCTCAA
2901 CCAAAAATCC AAAGAATTGG AACAACAGAT TGCGCAGTIG AAAAAAAGCT
2951 CGCNTAAAAG CAAGCTGATT CCAACCCTGC AAGAAGAACG CGACCGTCTC
3001 GCTTTCTATA TTCAAGCCAT CAACAAGGAA GTTAAAGGTA AAAAACCCAA
3051 AGGCAAAGAA TACCTGCAAG CCAAGCTTTC TGCACAAAAT ATTGACTTGA
3101 TTTCCGCACA AGGCATCGAA ATCAGCGGTT CCGATATTAC CGCTTCCAAA
3151 AAACTGAACC TTCACGCCGC AGGCGTATTG CCAAAGGCAG CAGATTCAGA
3201 GGCGGCTGCT ATTCTGATTG ACGGCATAAC CGACCAATAT GAAATTGGCA
3251 AGCCCACCTA CAAGAGTCAC TACGACAAAG CTGCTCTGAA CAAGCCTTCA
3301 CGTTTGACCG GACGTACGGG GGTAAGTATT CATGCAGCTG CGGCACTCGA
3351 TGATGCACGT ATTATTATCG GTGCATCCGA AATCAAAGCT CCCTCAGGCA
3401 GCATAGACAT CAAAGCCCAT AGTGATATTG TACTGGAGGC TGGACAAAAC
3451 GATGCCTATA CCTTCTTAAA AACCAAAGGT AAAAGCGGCA NAATNATCAG
3501 AAAAACNAAG TTTACCAGCA CCNGCGANCA CCTGATTATG CCAGCCCCNG
3551 TCGAGCTGAC CGCCAACGGT ATCACGCTTC ACGCAGGCGG CAACATCGAA
3601 GCTAATACCA CCCGCTTCAA TGCCCCTGCA GGTAAAGTIA CCCTGGTTGC
3651 GGGTGAANAG NTGCAACTGC TGGCAGAAGA AGGCATCCAC AAGCACGAGT
3701 TGGATGTCCA AAAAAGCCGC CGCTTTATCG GCATCAAGGT AGGTNAGAGC
3751 AATTACAGTA AAAACGAACT GAACGAAACC AAATTGCCTG TCCGCGTCGT
3801 CGCCCAAAAT GCAGCCACCC GTTCAGGCTG GGAThCCGTG CTCGAAGGTA
3851 CCGAATTCAA ATCCACGCTG GCCGGTGCCG ACATTCAGGC AGGTGTANGC
3901 GAAAAAGCCC GTGTCGATGC GAAAATCATC CTCAAAGGCA TTGTGAACCG
3951 TATCCAGTCG GAAGAAAAAT TAGAAACCAA CTCAACCGTA TGGCAGAAAC
4001 AGGCCGGACG CGGCAGCACT ATCGAAACGC TAAAACTGCC CAGCTTCGAA
4051 AGCCCTACTC CGCCCAAATT GTCCGCACCC GGCGGNTATA TCGTCGACAT
4101 TCCGAAAGGC AATCTGAAAA CCGAAATCGA AAAGCTGTCC AAACAGCCCG
4151 AGTATGCCtA TCTGAAACAG CTCCAAGTAG CGAAAAACAT CAACTGGAAT
4201 CAGGTGCAGC TTGCTTACGA CAGATGGGAC TACAAACAGG AGGGCTTAAC
4251 CGAAGCAGGT GCGGCGATTA TCGCACTGGC CGTTACCGTG GTCACCTCAG
4301 GCGCAGGAAC CGGAGCCGTA TTGGGATTAA ACGGTGCGNC CGCCGCCGCA
4351 ACCGATGCAG CATTCGCCTC TTTGGCCAGC CAGGCTTCCG TATCGTTCAT
4401 CAACAACAAA GGCGATGTCG GCAAAACCCT GAAAGAGCTG GGCAGAAGCA
4451 GCACGGTGAA AAATCTGGTG GTTGCCGCCG CTACCGCAGG CGTAGCCGAC
4501 AAAATCGGCG CTTCGGCACT GANCAATGTC AGCGATAAGC AGTGGATCAA
4551 CAACCTGACC GTCAACCTAG CCAATGNCGG GCAGTGCCGC ACTGAttaa

This encodes a protein having amino acid sequence <SEQ ID 62>:

1    MNKGLHRIIF SKKHSTMVAV AETANSQGKG KQAGSSVSVS LKTSGDLCGK
51   LKTTLKTLVC SLVSLSMXXX XXXQITTDKS APIDXQVVIL KTNTGAPLVN
101  IQTPNGRGLS HNRYTQFDVD NKGAVLNNDR NNNPFLVKGS AQLILNEVRG
151  TASKLNGIVT VGGQKADVII ANPNGITVNG GGFKNVGRGI LTIGAPQIGK
201  DGALTGFDVR QGTLTVGAAG WNDKGGADYT GVLARAVALQ GKLQGKNLAV
251  STGPQKVDYA SGEISAGTAA GTKPTIALDT AALGGMYADS ITLTAXEKGV
301  GVKNAGTLEA AKQLIVTSSG RIENSGRIAT TADGTEASPT YLXIETTEKG
351  AXGTFISNGG RIESKGLLVI ETGEDIXLPA GAVVQNNGSR PATTVLNAGH
401  NLVIESKTNV NNAXGSXNLS AGGRTTINDA TIQAGSSVYS STKGDTXLGE
451  NTRIIAENVT VLSNGSIGSA AVIEAKDTAN IESGKPLSLE TSTVASNIRL
501  NNGNIKGGKQ LALLADDNIT AKTTNLNTPG NLYVHTGKDL NLNVDKDLSA
551  ASIHLKSDNA AHITGTSKTL TASKDNGVEA GLLNVTNTNL RTNSGNLHIQ
601  AAKGNZQLRH TKLNAAKALE TTALQGNIVS DGLHAVSADG HVSLLANGNA
651  DFTGHNTLTA KADVXAGSVG KGRLKADNTN ITSSSGDITL VAXXGIQLGD
701  GKQRNSINGK HISIKNNGGN ADLKNLNVHA KSGALNIHSO RALSIENTKL
751  ESTHNTHLNA QHERVTLNQV DAYAHRHLSI XGSQIWQNDK LPSANKLVAN
801  GVLAXNARYS QIADNTTLRA CAINLTAGTA LVKRGNINWS TVSTKTLEDN
851  AELKPLAGRL NIEAGSGTLT IEFANRISAH TDLSIKTGGK LLLSAXGGNA
901  GAXSAQVSSL EAKGNIRLVT GXTDLRGSKI TAGKNLVVAT TKGKLNIEAV
951  NNSFSNYFXT QKXXXLNQKS KELEOQIAQL KKSSXKSKLI PTLQEERDRL
1001 AFYIQAINKE VKGKKPKGKE YLQAXLSAQN IDLISAQGIE ISGSDITASK
1051 KLNLHAAGVL PKAADSEAAA ILIDGITOQY EIGKPTYKSH YDKAALNKPS
1101 RLTGRTGVSI HAAAALDDAR IIIGASEIKA PSGSIDIKAR SDIVLEAGQN
1151 DAYTFLXTKG KSGXXIRKTK FTSTXXHLIM PAPVELTANG ITLQAGGNIE
1201 ANTTRFHAPA GKVTLVAGEK XQLLAEEGIK KHELDVQKSR RFIGIKVGXS
1251 NYSINELNET KLPVRVVAQX AATRSGWDTV LEGTEFKTTL AGADIQAGVX
1301 EKARVQAXII LKGIVNRIQS EEKLETNSTV WQKQAGRGST IETLKLPSFE
1351 SPTPPKLSAP GGYIVDIPKG NLKTEIEKLS KQPEYAYLKQ LQVAKNINWN
1401 QVQLAYQRWD YKQEGLTEAG AAIIALAVTV VTSGAGTGAV LGLNGAXAAA
1451 TDAAFASLAS QASVSFINNK GDVGKTLKEL GRSSTVKNLV VAAATAGVAD
1501 KIGASALXNV SDKQWINNLT VNLANXGQCR TD*

ORF114-1 and ORF114a show 89.8% identity in 1564 aa overlap

orf114a.pep MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf114-1    MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC
orf114a.pep SLVSLSMXXXXXXQITTDKSAPKNXQVVILKTNTGAPLVNIQTPNGRGLSHNRYTQFDVD
            |||||||      ||||||||||| |||||||||||||||||||||||||||||||||||
orf114-1    SLVSLSMXXXXXXQITTDKSAPKNQQVVILKTNTGAPLVNIQTPNGRGLSHNRYTQFDVD
orf114a.pep NKGAVLNNDRNNNPFLVKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG
            |||||||||||||||:||||||||||||||||||||||||||||||||||||||||||||
orf114-1    NKGAVLNNDRNNNPFVVKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG
orf114a.pep GGFKNVGRGILTIGAPQIGKDGALTGFDVRQGTLTVGAAGWNDKGGADYTGVLARAVALQ
            |||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||
orf114-1    GGFKNVGRGILTTGAPQIGKDGALTGFDVRQGTLTVGAAGWNDKGGADYTGVLARAVALQ
orf114a.pep GKLQGKNLAVSTGFQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIAXEKGV
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||
orf114-1    GKLOGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIANEKGV
orf114a.pep GVKNAGTLEAAXQLIVTSSGRIENSGRIATTADGTLASPTYLXIETTEKGAXGTFISNGG
            |||||||||||||||||||||||||||||||||||||||||| |||||||| ||||||||
orf114-1    GVKNAGTLEAAXQLIVTSSGRIENSGRIATTADGTLASPTYLSIETTEKGAAGTFISNGG
orf114a.pep RIESKGLLVIETGEDIXLRNGAVVQNNGSRPATTVLNAGHNLVIESKTNVNNAKGSXNLS
            |||||||||||||||| ||||||||||||||||||||||||||||||||||||||  :||
orf114-1    RIESKGLLVIETGEDISLRNGAVVQNNGSRPATTVLNAGHNLVIESKTNVNNAKGPANLS
orf114a.pep AGGRTTINDATIQAGSSVYSSTKGDTXLGENTRIIAENVTVLSNGSIGSAAVIEAKDTAH
            | |||:|::|:||:|::||||:||:: ||:|||| : :|||||||||:|:|||:||||||
orf114-1    ADGRTVIKEASIQTGTTVYSSSKGNAELGNNTRITGADVTVLSNGTISSSAVIDAKDTAH
orf114a.pep IESGKPLSLETSTVASNIRLNNGNIKGGKQLALLADDNITAKTTNLNTPGNLYVHTGKDL
            ||:|||||||||||:|||:|:||||:|:||||||||||||||||||||||||||||||||
orf114-1    IEAGKPLSLEASTVTSDIRLNGGSIKGGKQLALLADDNITAKTTNLNTPGNLYVHTGKDL
orf114a.pep NLNVDKDLSAASIHLKSDNAAHITGTSKTLTASKDMGVEAGLLNVTNTNLRTNSGNLHIQ
            ||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||
orf114-1    NLNVDKDLSAASIHLKSDNAAHITGTSKTLTASKDMGVEAGSLNVTNTNLRTNSGNLHIQ
orf114a.pep AAKGNIQLRNTKLNAAKALETTALQGNIVSDGLHAVSADGHVSLLANGNADFTGHNTLTA
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf114-1    AAKGNIQLRNTKLNAAKALETTALQGNIVSDGLHAVSADGHVSLLANGNADFTGHNTLTA
orf114a.pep KADVXAGSVGKGRLKADNTNITSSSGDITLVAXXGIQLGDGKQRNSINGKHISIKNNGGN
            |||| |||||||||||||||||||||||||||  ||||||||||||||||||||||||||
orf114-1    KADVNAGSVGKGRLKADNTNITSSSGDITLVAGNGIQLGDGKQRNSINGKHISIKNNGGN
orf114a.pep ADLKNLNVHAKSGALNIHSDRALSIENTKLESTHNTHLNAQHERVTLNQVDAYAHRHLSI
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf114-1    ADLKNLNVHAKSGALNIHSDRALSIENTKLESTHNTHLNAQHERVTLNQVDAYAHRHLSI
orf114a.pep XGSQIWQNDKLPSANKLVANGVLAXNARYSQIADNTTLRAGAINLTAGTALVKRGNINWS
            :||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||
orf114-1    TGSQIWQNDKLPSANKLVANGVLALNARYSQIADNTTLRAGAINLTAGTALVKRGNINWS
orf114a.pep TVSTKTLEDNAELKPLAGRLNIEAGSGTLTIEPANRISAHTDLSIKTGGKLLLSAKGGNA
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf114-1    TVSTKTLEDNAELKPLAGRLNIEAGSGTLTIEPANRISAHTDLSIKTGGKLLLSAKGGNA
orf114a.pep GAXSAQVSSLEAKGNIRLVTGXTDLRGSKITAGKNLVVATTKGKLNIEAVNNSFSNYFXT
            || |||||||||||||||||| |||||||||||||||||||||||||||||||||||| |
orf114-1    GAPSAQVSSLEAKGNIRLVTGETDLRGSKITAGKNLVVATTKGKLNIEAVNNSFSNYFPT
orf114a.pep QKXXXLNQKSKELEQQIAQLKKSSXKSKLIPTLQEERDRLAFYIQAINKEVKGKKPKGKE
            ||   ||||||||||||||||||| |||||||||||||||||||||||||||||||||||
orf114-1    QKAAELNQKSKELEQQIAQLKKSSPKSKLIPTLQEERDRLAFYIQAINKEVKGKKPKGKE
orf114a.pep YLQAKLSAQNIDLISAQGIEISGSDITASKKLNLHAAGVLPKAADSEAAAILIDGITDQY
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf114-1    YLQAKLSAQNIDLISAQGIEISGSDITASKKLNLHAAGVLPKAADSEAAAILIDGITDQY
orf114a.pep EIGKPTYKSHYDKAALNKPSRLTGRTGVSIHAAAALDDARIIIGASEIKAPSGSIDIKAH
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf114-1    EIGKPTYKSHYDKAALNKPSRLTGRTGVSIHAAAALDDARIIIGASEIKAPSGSIDIKAH
orf114a.pep SDIVLEAGQNDAYTFLXTKGKSGXXIRKTKFTSTXXHLIMPAPVELTANGITLQAGGNIE
            |||||||||||||||| ||||||  |||||||||  ||||||||||||||||||||||||
orf114-1    SDIVLEAGQNDAYTFLKTKGKSGKIIRKTKFTSTRDHLIMPAPVELTANGITLQAGGNIE
orf114a.pep ANTTRFNAPAGKVTLVAGEXXQLLAEEGIHKHELDVQKSRRFIGIKVGXSNYSKNELNET
            |||||||||||||||||||  ||||||||||||||||||||||||||| |||||||||||
orf114-1    ANTTRFNAPAGKVTLVAGEELQLLAEEGIHKHELDVQKSRRFIGIKVGKSNYSKNELNET
orf114a.pep KLPVRVVAQXAATRSGWDTVLEGTEFKTTLAGADIQAGVXEKARVDAKIILKGIVNRIQS
            |||||||||:||||||||||||||||||||||||||||| ||||:|||||||||||||||
orf114-1    KLPVRVVAQTAATRSGWDTVLEGTEFKTTLAGADIQAGVGEKARADAKIILKGIVNRIQS
orf114a.pep EEKLETNSTVWQKQAGRGSTIETLKLPSFESPTPPKLSAPGGYIVDIPKGNLKTEIEKLS
            ||||||||||||||||||||||||||||||||||||||:||||||||||||||||||||:
orf114-1    EEKLETNSTVWQKQAGRGSTIETLKLPSFESPTPPKLSTPGGYIVDIPKGNLKTEIEKLA
orf114a.pep KQPEYAYLKQLQVAKNINWNQVQLAYDRWDYKQEGLTEAGAAIIALAVTVVTSGAGTGAV
            ||||||||||||||||:||||||||||:|||||||||:|||||::: ||::| | |: |:
orf114-1    KQPEYAYLKQLQVAKNVNWNQVQLAYDKWDYKQEGLTRAGAAIVTIIVTALTYGYGATAA
orf114a.pep LGLNGA--------------XAATD----------AAFASLASQASVSFINNKGDVGKTL 1477
            |: ::              :||||          ||:||| |||:||:|||||||||:|
orf114-1    GGVAASGSSTAAAAGTAATTTAAATTVSTATAMQTAALASLYSQAAVSIINNKGDVGKAL 1500
orf114a.pep KELGRSSTVKNLVVAAATAGVADKIGA----------SALXNVSDKQWINNL----TVNL 1523
            |:|| |:|||::|::| |||: :::||          : | : : :| | ||    ::||
orf114-1    KDLGTSDTVKQIVTSALTAGALNQMGADIAQLNSKVRTELFSSTGNQTIANLGGRLATNL 1560
orf114a.pep ANXGQCRTDX
            :| |
orf114-1    SNAGISAGINTAVN...

Homology with pspA Putative Secreted Protein of N. meningitidis (Accession Number AF030941)

ORF114 and pspA protein show 36% aa identity in 302 aa overlap:

Orf114:	1	AVAETANSQGKGKQAGSSVSVSL----KTSGDXXXXXXXXXXXXXXXXXXXXXXXPAHAQ 56
		AVAE  +  GK  Q   + SV +      S                         PA A
pspA:	19	AVAENVHRDGKSMQDSEAASVRVTGAASVSSARAAFGFRMAAFSVMLALGVAAFSPAPAS 78
Orf114:	57	-ITTDKSAPKNQQVVILKTNTGAPLVNIQTPNGRGLSHNRXYAFDVDNKGAVLNNDRNN- 114
		I  DKSAPKNQQ VIL+T  G P VNIQTP+ +G+S NR   FDVD KG +LNN R+N
pspA:	79	GIIADKSAPKNQQAVILQTANGLPQVNIQTPSSQGVSVNRFKQFDVDEKGVILNNSRSNT 138
Orf114:	115	----------NPFVVKGSAQLILNEV-RGTASKLNGIVTVGGQKADVIIANPNGITVNGG 163
		NP + +G A++I+N++     S LNG + VGG++A+V++ANP+GI VNGG
pspA:	139	QTQLGGWIQGNPHLARGEARVIVNQIDSSNPSLLNGYIEVGGKRAEVVVANPSGIRVNGG 198
Orf114:	164	GFKNVGRGILTTGAPQIGKDGALTGFDVVKAHWTVXAAGWNDKGGAXYTGVLARAVALQG 223
		G  N     LT+G P +  +G LTGFDV      +   G  D   A YT +L+RA  +
papA:	199	GLINAASVTLTSGVPVL-NNGNLTGFDVSSGKVVIGGKGL-DTSDADYTRILSRAAEINA 256
Orf114:	224	KXXGKXLAVSTGPQKVDYASGEISAGTAAGTK----PTIALDTAALGGMYADSITLIANE 279
		GK + V +G  K+D+        +A  +     PT+A+DTA LGGMYAD ITLI+ +
pspA:	257	GVWGKDVKVVSGKNKLDFDGSLAKTASAPSSSDSVTPTVAIDTATLGGMYAQKITLISTD 316
Orf114:	280	KG 291
		G
papA:	317	NG 318

ORF114a is also homologous to pspA:

gil2623258 (AF030941) putative secreted protein
(Neisseria meningitidis) Length = 2273
Score +32 261 bits (659), Expect +32 3e−69
Identities = 203/663 (30%), Positives 314/663 (46%), Gaps 76/663 (11%)
Query:    1 MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLK-----TSGDXXXXXXXXX 55
            MNK  +++IF+KK S M+AVAE  +  GK  Q   + SV +      +S
Sbjct:    1 MNKRCYKVIFNKKRSCMMAVAENVHRDGKSMQDSEAASVRVTGAASVSSARAAFGFRMAA 60
Query:   56 XXXXXXXXXXXXXXXXXXQITTKDSAPKNXQVVILKTNTGAPLVNIQTPNGRGLSHNRYT 115
            I  DKSAPKN Q VIL+T  G P VNIQTP+ +G+S NR+
Sbjct:   61 FSVMLALGVAAFSPAPASGIIADKSAPKNQQAVILQTANGLPQVNIQTPSSQGVSVNRFK 120
Query:  116 QFDVDNKGAVLNNDRNN-----------NPFLVKGSAQLILNEV-RGTASKLNGIVTVGG 163
            QFDVD KG +LNN R+N-----------NP L +G A++I+N++     S LNG + VGG
Sbjct:  121 QFDVDEKGVILNNSRSNTQTQLGGWIQGNPHLARGEARVIVNQIDSSNPSLLNGYIEVGG 180
Query:  164 QKADVIIANPNGITVNGGGFKNVGRGILTIGAPQIGKDGALTGFDVRQGTLTVGAAGWND 223
            ++A+V++ANP+GI VNGGG  N     LT G P +  +G LTGFDV  G + +G  G  D
Sbjct:  181 KRAEVVVANPSGIRVNGGGLINAASVTLTSGVPVL-NNGNLTGFDVSSGKVVIGGKGL-D 238
Query:  224 KGGADYTGVLARAVALQGKLQGKNLAVSTGPQKVDYASGEISAGTAAGTK----PTIALD 279
            ADYT +L+RA  +   + GK++ V +G  K+D+        +A  +     PT+A+D
Sbjct:  239 TSDADYTRILSRAAEINAGVWGKDVKVVSGKNKLDFDGSLAKTASAPSSSDSVTPTVAID 298
Query:  280 TAALGGMYADSITLIAXEKGVGVKNAGTLEAAK-QLIVTSSGRIENSGRIATTADGTEAS 338
            TA LGGMYAD ITLI+ + G  ++N G + AA   + +++ G++ NSG I
Sbjct:  299 TATLGGMYADKITLISTDNGAVIRNKGRIFAATGGVTLSADGKLSNSGSI-------DAA 351
Query:  339 PTYLXIETTEKGAXGTFISNGGRIESKGLLVIETGEDIXLRNGAVVQNNGSRPATTVLNA 398
            +  +T +        +  G I S    V++  + I  + G +    GS     + +
Sbjct:  352 EITISAQTVD--------NRQGFIRSGKGSVLKVSDGINNQAGLI----GSAGLLDIRDT 399
Query:  399 GHNLVIESKTNVNNAKGS----XNLSAGGRTTINDATIQAGSSVYSSTKGDTXLGENTRI 454
            G     +S  ++NN  G+     ++S   ++  ND  + A   V S +  D   G+
Sbjct:  400 G-----KSSLHINNTDGTIIAGKDVSLQAKSLDNDGILTAARDV-SVSLHDDFAGKRDIE 453
Query:  455 IAENVTVLSNGSIGSAAVIEAKDTAHIESGKPLSLETSTVASNIRLNNGNIKGGKQLALL 514
            +T  + G + +  +I+A DT  + + +  +  +  + S  R       G     L+
Sbjct:  454 AGRTLTFSTQGRLKNTRIIQAGDTVSLTAAQIDNTVSGKIQSGNRTGLNGKNGITNRGLI 513
Query:  515 ADDNIT-----AKTTNLNTPGNLYVHTGKDLNLNVDKDLSAASIHLKSDNAAHITGTSKT 569
            + IT     AK+ N  T G +Y   G  + +  D  L+          AA
Sbjct:  514 NSNGITLLQTEAKSDNAGT-GRIY---GSRVAVEADTLLNREETVNGETKAA-------V 562
Query:  570 LTASKDMGVEAGXXXXXXXXXXXXSGNLHIQAA---KGNIQLRNTKL-NAAKALETTALQ 625
            + A + + + A             SG+LHI +A      +Q  NT L N + A+E++
Sbjct:  563 IAARERLDIGAREIENREAALLSSSGDLHIGSALNGSRQVQGANTSLHNRSAAIESS--- 619
Query:  626 GNI 628
            GNI
Sbjct:  620 GNI 622
Score +32 37.5 bits (65), Expect = 0.53
Identities = 87/432 (20%), Positives +32 159/432 (36%), Gaps = 62/432 (14%)
Query:  239 LQGKLQGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIAXEK 298
            LQG LQGKN+  + G    +  +G I A  A   K        A   + + S T     +
Sbjct: 1023 LQGDLQGKNIFAAAGSDITN--TGSIGAENALLLK--------ASNNIESRSETRSNQNE 1072
Query:  299 GVGVKNAGTLEAAKQLIVTSSGRI--ENSGRIATTADGTEASPTYLXIETTEKGAXG-TF 355
            V+N G + A   L    +G +  +    I  TA            E T +   G T
Sbjct: 1073 QGSVRNIGRV-AGIYLTGRQNGSVLLDAGNNIVLTAS-----------ELTNQSEDGQTV 1120
Query:  356 ISNGGRIESKGLLVIETGEDIXLRNGAVVQNNGSRPATTVLNAGHNLVIESK-------T 408
            ++ GG I S    +      I   +  V++   +   +T+   G NL + +K
Sbjct: 1121 LNAGGDIRSDTTGISRNQNTIFDSDNYVIRKEQNEVGSTIRTRG-NLSLNAKGDIRIPAA 1179
Query:  409 NVNNAKGSXNLSAGGRTTINDATIQAGSS--------VYSSTKGDTXLGENTRIIAENVT 460
            V + +G   L+AG      D  ++AG +         Y+   G     + TR +
Sbjct: 1180 EVGSEQGRLKLAAG-----RDIKVEAGKAHTETEDALKYTGRSGGGIKQKMTRHLKNQNG 1234
Query:  461 VLSNGSIGSAAVIEAKDTAHIESGKPLSLETSTVASWIRLNNGNIKGGKQLALLADDNIT 520
            +G++    +I         +G  +  +  T+ S    NN  +K  +  +  A+ N
Sbjct: 1235 QAVSGTLDGKEIILVSGRDITVTGSNIIADNHTILS--AKNNIVLKAAETRSRSAEMNKK 1292
Query:  521 AKTTNLNTPG-NLYVHTGKDLNLNVDKDLSAASIHLKSDN-------AAHITGTSKTLTA 572
            K+  + + G      + KD   N  + +S     + S N         H T T  T+++
Sbjct: 1293 EKSGLMGSGGIGFTAGSKKDTQTNRSETVSHTESVVGSLNGNTLISAGKHYTQTGSTISS 1352
Query:  573 SK-DMGVEAGXXXXXXXXXXXXSGNLHIQAAKG-----NIQLRNTKLNAAKALETTALQG 626
            + D+G+ +G              +  +   KG     ++ + NT + A  A++     G
Sbjct: 1353 PQGDVGISSGKISIDAAQNRYSQESKQVYEQKGVTVAISVPVVNTVMGAVDAVKAVQTVG 1412
Query:  627 NIVSDGLHAVSA 638
            +  ++A++A
Sbjct: 1413 KSKNSRVNAMAA 1424

Amino acids 1-1423 of ORF114-1 were cloned in the pGex vector and expressed in E. coli, as described above. GST-fusion expression was visible using SDS-PAGE, and FIG. 5 shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF114-1.

Based on these results, including the homology with the putative secreted protein of N. meningitidis and on the presence of a transmembrane domain, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 14

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 63>

1    CGCTTCATTC ATGATGAAGC AGTCGGCAGC AACATCGGCG GCGGCAAAAT
51   GATTGTTGCA GCCGGGCAGG ATATCAATGT ACGCGGCAnA AGCCTTATTT
101  CTGATAAGGG CATTGTTTTA AAAGCAGGAC ACGACATCGA TATTTCTACT
151  GCCCATAATC GCTATACCGG CAATGAATAC CACGAGAGCA wAAAwTCAGG
201  CGTCATGGGT ACTGGCGGAT TGGGCTTTAC TATCGGTAAC CGGAAAACTA
251  CCGATGACAC TGATCGTACC AATATTGTsC ATACAGGCAG CATTATAGGC
301  AGCCTGAaTG GAGACACCGT TACAGTTGCA GGAAACCGCT ACCGACAAAC
351  CGGCAGTACC GTCTCCAGCC CCGACGGGCG CAATACCGTC ACAGCCAAAw
401  GCATAGATGT AGAGTTCGCA AACAACCGGT ATGCCACTGA CTACGcCCAT
451  ACCCAGGGAA CAAAAAGGCC TTACCGTCGC CCTCAATGTC CCGGTTGTCC
501  AAGCTGCACA AAACTTCATA CAAGCAGCCC AAAATGTGGG CAAAAGTAAA
551  AATAAACGCG TTAATGCCAT GGCTGCAGCC AATGCTGCAT GGCAGAGTTA
601  TCAAGCAACC CAACAAATGC AACAATTTGC TCCAAGCAGC AGTGCGGGAC
651  AAGGTCAAAA CTACAATCAA AGCCCCAGTA TCAGTGTGTC CATTAC.TAC
701  GGCGAACAGA AAAGTCGTAA CGAGCAAAAA AGACATTACA CCGAAgCGGC
751  AgCAAGTCAA ATTATCGGCA AAGGGCAAAC CACACTTGCG GCAACAGGAA
801  GTGGGGAGCA GTCCAATATC AATATTACAG GTTCCGATGT CATCGGCCAT
951  GCAGGTACTC C.CTCATTGC AAGCAACCAT ATCAGACTCC AATCTGCCAA
901  ACAGGACGGC AGCGAGCAAA GCAAAAACAA AAGCAGTGGT TGGAATGCAG
951  GCGTACGTnn CAAAATAGGC AAcGGCATCA GGTTTGGAAT TACCGCCGGA
1001 GGAAATATCG GTAAAGGTAA AGAGCAAGGG GGAAGTACTA CCCACCGCCA
1051 CACCCATGTC GGCAGCACAA CCGGCAAAAC TACCATCCGA AGCGGCGGGG
1101 GATACCACCC TCAAAGGTGT GCAGCTCATC GGCAAAGGCA TACAGGCAGA
1151 TACGCGCAAC CTGCATATAG AAAGTGTTCA AGATACTGAA ACCTATCAGA
1201 GCAAACAGCA AAACGGCAAT GTCCAAGTTt ACTGTCGGTT ACGGATTCAG
1251 TGCAAGCGGC AGTTACCGCC AAAGCAAAGT CAAAGCAGAC CATGCCTCCG
1301 TAACCGGGCA AAgCGGTATT TATGCCGGAG AAGACGGCTA TCAAATyAAA
1351 GTyAGAGACA ACACAGACCT yAAGGGCGGT ATCATCACGT CTAGCCAAAG
1401 CGCAGAAGAT AAGGGCAAAA ACCTTTTTCA GACGGCCACC CTTACTGCCA
1451 GCGACATTCA AAACCACAGC CGCTACGAAG GCAGAAGCTT CGGCATAGGC
1501 GGCAGTTTCG ACCTGAACGG CGGCTGGGAC GGCACGGTTA CCGACAAACA
1551 AGGCAGGCCT ACCGACAGGA TAAGCCCGGC AGCCGGCTAC GGCAGCGACG
1601 GAGACAGCAA AAACAGCACC ACCCGCAGCG GCGTCAACAC CCACAACATA
1651 CACATCACCG ACGAAGCGGG ACAACTTGCC CGAACAGGCA GGACTGCAAA
1701 AGAAACCGAA GCGCGTATCT ACACCGGCAT CGACACCGAA ACTGCGGATC
1751 AACACTCAGG CCATCTGAAA AACAGCTTCG AC...

This corresponds to the amino acid sequence <SEQ ID 64; ORF116>:

1   ..RFIHDEAVGS NIGGGKNIVA AGQDINVRGX SLISDKGIVL KAGADIDIST
51  AHNRYTGNEY HESXXSGVMG TGGLGFTIGN RKTTDDTDRT NIVHTGSIIG
101 SLNGDTVTVA GNRYRQTGST VSSPEGRNTV TAKXIDVEFA NNRYATDYAH
151 TQEQKGLTVA LNVPVVQAAQ NFIQAAQNVG KSKNKRVNAM AAANAAWQSY
201 QATQQMQQFA PSSSAGQGQN YNQSPSISVS IXYGEQKSRN EQKRNYTEAA
251 ASQIIGKGQT TLAATGSGEQ SNINITGSDV IGHAGTXLIA DNHIRLQSAX
301 QDGSEQSKNK SSGWNAGVRX KIGNGIRFGI TAGGNIGKGK EQGGSTTHRH
351 THVGSTTGKT TIRSGGDTTL KGVQLIGXGI QADTRNLHIE SVQDTETYQS
401 KQQNGNVQVT VGYGFSASGS YRQSKVKADH ASVTGQSGIY AGEDGYQIKV
451 RDNTDLKGGI ITSSQSAEDK GKNLFQTATL TASDIQNHSR YEGRSFGIGG
501 SFDLNGGWDG TVTDKQGRPT DRISPAAGYG SDGDSKNSTT RSGVNTHNIH
551 ITDEAGQLAR TGRTAKETEA RIYTGIDTET ADQHSGHLKN SFD...

Computer analysis of this amino acid sequence gave the following results:

Homology with pspA Putative Secreted Protein of N. meningitidis (Accession Number AF030941)

ORF116 and pspA protein show 38% aa identity in 502 aa overlap:

Orf116: 6    EAVGSNIGGGKMIVAAGQDINVRGXSLISDKGIVLKAGHDIDISTAHNRYTGNEYHESXX 65
        +AV   + G ++I+ +G+DI V G ++I+D   +L A ++I +  A  R    E ++
PspA:   1235 QAVSGTLDGKEIILVSGRDITVTGSNIIADNHTILSAKNNIVLKAAETRSRSAEMNKKEK 1294
Orf116: 66   XXXXXXXXXXXXXXNRKXXXXXXRTNIVHTGSIIGSLNGDTVTVAGNRYRQTGSTVSSPE 125
        ++K         + HT S++GSLNG+T+  AG  Y QTGST+SSP+
PspA:   1295 SGLMGSGGIGFTAGSKKDTQTNRSETVSHTESVVGSLNGNTLISAGKHYTQTGSTISSPQ 1354
Orf116: 126  GRNTVTAKXIDVEFANNRYATDYAHTQEQKGLTVALNVPXXXX---XXXXXXXXXXXGKS 182
        G   +++  I ++ A NRY+ +     EQKG+TVA++VP               GKS
PspA:   1355 GDVGISSGKISIDAAQNRYSQESKQVYEQKGVTVAISVPVVNTVMGAVDAVKAVQTVGKS 1414
Orf116: 183  KNKRVXXXXXXXXXWQSYQATQQMQQFA--PSSSAGQGQNYNQSPSISVSIXYGEQKSRN 240
        KN RV          +   +   +   A  P  +AGQG        ISVS+ YGEQK+ +
PspA:   1415 KNSRVNAMAAANALNKGVDSGVALYNAARNPKKAAGQG--------ISVSVTYGEQKNTS 1466
Orf116: 241  EQKRHYTEAAASQIIGKGQTTLAATGSGEQSNINITGSDVIGHAGTXLIADNHIRLQSAK 300
        E +   T+    +I G G+ +L A+G+G+ S I ITGSDV G  GT L A+N +++++A+
PspA:   1467 ESRIKGTQVQEGKITGGGKVSLTASGAGKDSRITITGSDVYGGKGTRLKAENAVQIEAAR 1526
Orf116: 301  QDGSEQSKNKSSGWNAGVRXKIGNGIRFGITAXXXXXXXXXXXXSTTHRHTHVGSTTGKT 360
        Q   E+S+NKS+G+NAGV   I  GI FG TA             T +R++H+GS   +T
PspA:   1527 QTHQERSENKSAGFNAGVAIAINKGISFGFTAGANYGKGYGNGDETAYRNSHIGSKDSQT 1586
Orf116: 361  TIRSGGDTTLKGVQLIGKGIQADTRNLHIESVQDTETYQSKQQNGNVQVTVGYGFSASGS 420
        I SGGDT +KG QL GKG+     +LHIES+QDT  ++ KQ+N + QVTVGYGFS  GS
PspA:   1587 AIESGGDTVIKGGQLKGKGVGVTAESLHIESLQDTAVFKGKQENVSAQVTVGYGFSVGGS 1646
Orf116: 421  YRQSKVKADHASVTGQSGIYAGEDGYQIKVRDNTDLKGGIITSSQSAEDKGKNLFQTATL 480
        Y +SK  +D+ASV  QSGI+AG DGY+I+V   T L G  + S     DK KNL +T+ +
PspA:   1647 YNRSKSSSDYASVNEQSGIFAGGDGYRIRVNGKTGLVGAAVVSD---ADKSKNLLKTSEI 1703
Orf116: 481  TASDIQNHSRYEGRSFGIGGSF 502
        DIQNH+     + G+ G F
PspA:   1704 WHKDIQNHASAAASALGLSGGF 1725

Based on homology with pspA, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 15

The following partial DNA sequence was identified in N. meningitidis SEQ ID 65>

1   ..ACGACCGGCA GCCTCGGCGG CATACTGGCC GGCGGCGGCA CTTCCCTTGC
51  CGCACCGTAT TTGGACAAAG CGGCGGAAAA CCTCGGTCCG GCGGGCAAAG
101 CGGCGGTCAA CGCACTGGGC GGTGCGGCCA TCGGCTATGC AACTGGTGGT
151 AGTGGTGGTG CTGTGGTGGG TGCGAATGTA GATTGGAACA ATAGGCAGCT
201 GCATCCGAAA GAAATGGCGT TGGCCGACAA ATATGCCGAA GCCCTCAAGC
251 GCGAAGTTGA AAAACGCGAA GGCAGAAAAA TCAGCAGCCA AGAAGCGGCA
301 ATGAGAATCC GCAGGCAGAT ATGCGTTGGG TGGACAAAGG TTCCCAAGAC
351 GGCTATACCG ACCAAAGCGT CATATCCCTT ATCGGAATGA

This corresponds to the amino acid sequence <SEQ ID 66; ORF118>:

1   ..TTGSLGGILA GGGTSLAAPY LDKAAENLGP AGKAAVNALG GAAIGYATGG
51  SGGAVVGANV DWNNRQLHPK EMALADKYAE ALKREVEKRE GRKISSQEAA
101 MRIRRQICVG WTKVPKTAIP TKASYPLSE*

Computer analysis of this amino acid sequence reveals two putative transmembrane domains.

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 16

The following partial DNA sequence was identified in N. meningitidis SEQ ID 67>

1   ..CAATGCCGTC TGAAAAGCTC ACAATYTTAC AGACGGCATT TGTTATGCAA
51  GTACATATAC AGATTCCCTA TATACTGCCC AGrkGCGTGC GTgGCTGAAG
101 ACACCCCCTA CGCTTGCTAT TTGrAACAGC TCCAAGTCAC CAAAGACGTC
151 AACTGGAACC AGGTACWACT GGCGTACGAC AAATGGGACT ATAAACAGGA
201 AGGCTTAACC GGAGCCGGAG CAGCGATTAT TGCGCTGGCT GTTACCGTGG
251 TTACTGCGGG CGCGGGAgCC GGAGCCGCAC TGGGcTTAAA CGGCGCGGCc
301 GCAGCGGCAA CCGATGCCGC ATTCGCCTCG CTGGCCAGCC AGGcTTCCGT
351 ATCGCTCATC AaCAACAAAG GCAATATCGG TAaCACCCTG AAAGAGCTGG
401 GCAGAAGCAG CACGGTGAAA AATCTGATGG TTGCCGTCGc tACCGCAgGC
451 GTagCcgaCA AAATCGGTGC TTCGGCACTG AACAATGTCA GCGATAAGCA
501 GTGGATCAAC AACCTGACCG TCAACCTGGC CAATGCGGGC AGTGCCGCAC
551 TGATTAATAC CGCTGTCAAC GGCGGCAGCc tgAAAGACAA TCTGGAAGCG
601 AATATCCTTG CGGCTTTGGT GAATACTGCG CATGGAGAAG CAGCCAGTAA
651 AATCAAACAG TTGGATCAGC ACTACATTAC CCACAAGATT GCCCaTGCCA
701 TAGCGGGCTG TGCGGcTGCG GCGGCGAATA AGGGCAAGTG TCAGGATGGT
751 GCGATAgGTG CGGCTGTGGG CGAGATAGTC GGGGAgGCTT TGACAAACGG
801 CAAAAATCCT GACACTTTGA CAGCTAAAgA ACGCGaACAG ATTTTGGCAT
851 ACAGCAAACT GGTTGCCGGT ACGGTAAGCG GTGTGGTCGG CGGCGATGTA
901 AATGCGGCGG CGAATGCGGC TGAGGTAGCG GTGAAAAATA ATCAGCTTAG
951 CGACAAAtGA

This corresponds to the amino acid sequence <SEQ ID 68; ORF41>:

1   ..QCRLKSSQFY RRHLLCKYIY RFPIYCPXAC VAEDTPYACY LXQLQVTKDV
51  HWNQVXLAYD KWDYKQEGLT GAGAAIIALA VTVVTAGAGA GAALGLNGAA
101 AAATDAAFAS LASQASVSLI NNKGNIGNTL KELGRSSTVK NU4VAVATAG
151 VADKIGASAL NNVSDKQWIN NLTVNLANAG SAALINTAVN GGSLKDNLEA
201 NILAALVNTA HGEAASKIKQ LDQHYITHKI AHAIAGCAAA AANKGKCQDG
251 AIGAAVGEIV GEALTNGKNP DTLTAKEREQ ILAYSKLVAG TVSGVVGGDV
301 NAAANAAEVA VKNNQLSDK*

Further work revealed the complete nucleotide sequence <SEQ ID 69>:

1    ATGCAAGTAA ATATTCAGAT TCCCTATATA CTGCCCAGAT GCGTGCGTGC
51   TGAAGACACC CCCTACGCTT GCTATTTGAA ACAGCTCCAA GTCACCAAAG
101  ACGTCAACTG GAACCAGGTA CAACTGGCGT ACGACAAATG GGACTATAAA
151  CAGGAAGGCT TAACCGGAGC CGGAGCAGCG ATTATTGCGC TGGCTGTTAC
201  CGTGGTTACT GCGGGCGCGG GAGCCGGAGC CGCACTGGGC TTAAACGGCG
251  CGGCCGCAGC GGCAACCGAT GCCGCATTCG CCTCGCTGGC CAGCCAGGCT
301  TCCGTATCGC TCATCAACAA CAAAGGCAAT ATCGGTAACA CCCTGAAAGA
351  GCTGGGCAGA AGCAGCACGG TGAAAAATCT GATGGTTGCC GTCGCTACCG
401  CAGGCGTAGC CGACAAAATC GGTGCTTCGG CACTGAACAA TGTCAGCGAT
451  AAGCAGTGGA TCAACAACCT GACCGTCAAC CTGGCCAATG CGGGCAGTGC
501  CGCACTGATT AATACCGCTG TCAACGGCGG CAGCCTGAAA GACAATCTGG
551  AAGCGAATAT CCTTGCGGCT TTGGTGAATA CTGCGCATGG AGAAGCAGCC
601  AGTAAAATCA AACAGTTGGA TCAGCACTAC ATTACCCACA AGATTGCCCA
651  TGCCATAGCG GGCTGTGCGG CTGCGGCGGC GAATAAGGGC AAGTGTCAGG
701  ATGGTGCGAT AGGTGCGGCT GTGGGCGAGA TAGTCGGGGA GGCTTTGACA
751  AACGGCAAAA ATCCTGACAC TTTGACAGCT AAAGAACGCG AACAGATTTT
801  GGCATACAGC AAACTGGTTG CCGGTACGGT AAGCGGTGTG GTCGGCGGCG
851  ATGTAAATGC GGCGGCGAAT GCGGCTGAGG TAGCGGTGAA AAATAATCAG
901  CTTAGCGACA AAGAGGGTAG AGAATTTGAT AACGAAATGA CTGCATGCGC
951  CAAACAGAAT AATCCTCAAC TGTGCAGAAA AAATACTGTA AAAAAGTATC
1001 AAAATGTTGC TGATAAAAGA CTTGCTGCTT CGATTGCAAT ATGTACGGAT
1051 ATATCCCGTA GTACTGAATG TAGAACAATC AGAAAACAAC ATTTGATCGA
1101 TAGTAGAAGC CTTCATTCAT CTTGGGAAGC AGGTCTAATT GGTAAAGATG
1151 ATGAATGGTA TAAATTATTC AGCAAATCTT ACACCCAAGC AGATTTGGCT
1201 TTACAGTCTT ATCATTTGAA TACTGCTGCT AAATCTTGGC TTCAATCGGG
1251 CAATACAAAG CCTTTATCCG AATGGATGTC CGACCAAGGT TATACACTTA
1301 TTTCAGGAGT TAATCCTAGA TTCATTCCAA TACCAAGAGG GTTTGTAAAA
1351 CAAAATACAC CTATTACTAA TGTCAAATAC CCGGAAGGCA TCAGTTTCGA
1401 TACAAACCTA AAAAGACATC TGGCAAATGC TGATGGTTTT AGTCAAAAAC
1451 AGGGCATTAA AGGAGCCCAT AACCGCACCA ATTTTATGGC AGAACTAAAT
1501 TCACGAGGAG GACGCGTAAA ATCTGAAACC CAAACIGATA TTGAAGGCAT
1551 TACCCGAATT AAATATGAGA TTCCTACACT AGACAGGACA GGTAAACCTG
1601 ATGGTGGATT TAAGGAAATT TCAAGTATAA AAACTGTTTA TAATCCTAAA
1651 AAATTTTCTG ATGATAAAAT ACTTCAAATG GCTCAAAATG CTGCTTCACA
1701 AGGATATTCA AAAGCCTCTA AAATTGCTCA AAATGAAAGA ACTAAATCAA
1751 TATCGGAAAG AAAAAATGTC ATTCAATTCT CAGAAACCTT TGACGGAATC
1801 AAATTTAGAT CATATTTTGA TGTAAATACA GGAAGAATTA CAAACATTCA
1851 CCCAGAATAA

This corresponds to the amino acid sequence <SEQ ID 70; ORF41-1>:

1   MQVNIQIPYI LPRCVRAEDT PYACYLKQLQ VTKDVNWNQV QLAYOKWDYK
51  QEGLTGAGAA IIALAVTVVT AGAGAGAALG LNGAAAAATD AAFASLASQA
101 SVSLINNKGN IGNTLKELGR SSTVKNLHVA VATAGVADKI GASALNNVSD
151 KQWINNLTVN LANAGSAALI NTAVNGGSLX DNLEANILAA LVNTAHGEAA
201 SKIKQLDQHY ITHKIAHAIA GCAAAAANKG KCQDGAIGAA VGEIVGEALT
251 NGKNPDTLTA KEREQILAYS KLVAGTVSGV VGGDVNAAAN AAEVAVKNNQ
301 LSDKLGREFD NEMTACAKQN NPQLCRKNTV KKYQNVADKR LAASIAICTD
351 ISRSTECRTI RKQHLIDSRS LHSSWEAGLX GKDDEWYKLF SKSYTQADLA
401 LQSYHLNTAA KSWLOSGNTK PLSEWNSDQG YTLISGVNPR FIPIPRGFVK
451 QHTFITNVKY PEGISFDTNL KRMLANADGF SQKQGIKGAH NRTNFNAELN
501 SRGGRVKSET QTDIEGITRI KYEIPTLDRT GKPDGGFKEI SSIKTVYNPK
551 KFSDDKILQH AQNAASQGYS KASKIAQNER TKSISERKNV IQFSETFDGI
601 KFRSYFDVNT GRITNIHPE*

Computer analysis of this amino acid sequence predicts a transmembrane domain, and homology with an ORF from N. meningitidis (strain A) was also found.

ORF41 shows 92.8% identity over a 279 aa overlap with an ORF (ORF41a) from strain A of N. meningitidis:

          10        20        30        40        50        60        69
orf41.pep YRRHLLCKYIYRFPIYCPXACVAEDTPYACYLXQLQVTKDVNWNQVXLAYDKWDYKQEGL
          11 1111:1::11111 1111:11111111
orf41a    YLKQLQVAXNINWNQVQLAYDRWDYKQEGL
          10        20        30
          70        80        90       100       110       120       129
orf41.pep TGAGAAIIALAVTVVTAGAGAGAALGLNGAAAAATDAAFASLASQASVSLINNKGNIGNT
          | ||||||||||||||:|||:||:|||||| ||||||||||||||||||:|||||::|:|
orf41a    TEAGAAIIALAVTVVTSGAGTGAVLGLNGAXAAATDAAFASLASQASVSFINNKGDVGKT
          40        50        60        70        80        90
          130       140       150       160       170       180       189
orf41.pep LKELGRSSTVKNLMVAVATAGVADKIGASALNNVSDKQWINNLTVNLANAGSAALINTAV
          |||||||||||||:||:|||||||||||||| ||||||||||||||||||||||||||||
orf41a    LKELGRSSTVKNLVVAAATAGVADKIGASALXNVSDKQWINNLTVNLANAGSAALINTAV
          100       110       120       130       140       150
          190       200       210       220       230       240       249
orf41.pep NGGSLKDNLEANILAALVNTAHGEAASKIKQLDQHYITHKIAHAIAGCAAAAANKGKCQD
          ||||||| |||||||||||||||||||||||||||||:||||||||||||||||||||||
orf41a    NGGSLKDXLEANILAALVNTAHGEAASKIKQLDQHYIVHKIAHAIAGCAAAAANKGKCQD
          160       170       180       190       200       210
          250       260       270       280       290       300       309
orf41.pep GAIGAAVGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGVVGGDVNAAANAAEV
          ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf41a    GAIGAAVGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGVVGGDVNAAANAAEV
          220       230       240       250       260       270
          310       320
orf41.pep AVKNNQLSDKX
          |||||||||
orf41a    AVKNNQLSDXEGREFDNENTACAKQNXPQLCRXWIVKKYQNVADKRLAASIAICTDISRS
          280       290       300       310       320       330

A partial ORF41a nucleotide sequence <SEQ ID 71> is:

1    TATCTGAAAC AGCTCCAAGT AGCGAAAAAC ATCAACTGGA ATCAGGTGCA
51   GCTTGCTTAC GACAGATGGG ACTACAAACA GGAGGGCTTA ACCGAAGCAG
101  GTGCGGCGAT TATCGCACTG GCCGTTACCG TGGTCACCTC AGGCGCAGGA
151  ACCGGAGCCG TATTGGGATT AAACGGTGCG NCCGCCGCCG CAACCGATGC
201  AGCATTCGCC TCTTTGGCCA GCCAGGCTTC CGTATCGTTC ATCAACAACA
251  AAGGCGATGT CGGCAAAACC CTGAAAGAGC TGGGCAGAAG CAGCACGGTG
301  AAAAATCTGG TGGTTGCCGC CGCTACCGCA GGCGTAGCCG ACAAAATCGG
351  CGCTTCGGCA CTGANCAATG TCAGCGATAA GCAGTGGATC AACAACCTGA
401  CCGTCAACCT AGCCAATGCG GGCAGTGCCG CACTGATTAA TACCGCTGTC
451  AACGGCGGCA GCCTGAAAGA CANTCTGGAA GCGAATATCC TTGCGGCTTT
501  GGTCAATACC GCGCATGGAG AAGCAGCCAG TAAAATCAAA CAGTTGGATC
551  AGCACTACAT AGTCCACAAG ATTGCCCATG CCATAGCGGG CTGTGCGGCA
601  GCGGCGGCGA ATAAGGGCAA GTGTCAGGAT GGTGCGATAG GTGCGGCTGT
651  GGGCGAGATA GTCGGGGAGG CTTTGACAAA CGGCAAAAAT CCTGACACTT
701  TGACAGCTAA AGAACGCGAA CAGATTTTGG CATACAGCAA ACTGGTTGCC
751  GGTACGGTAA GCGGTGTGGT CGGCGGCGAT GTAAATGCGG CGGCGAATGC
801  GGCTGAGGTA GCGGTGAAAA ATAATCAGCT TAGCGACNAA GAGGGTAGAG
851  AATTTGATAA CGAAATGACT GCATGCGCCA AACAGAATAN TCCTCAACTG
901  TGCAGAAAAA ATACTGTAAA AAAGTATCAA AATGTTGCTG ATAAAAGACT
951  TGCTGCTTCG ATTGCAATAT GTACGGATAT ATCCCGTAGT ACTGAATGTA
1001 GAACAATCAG AAAACAACAT TTGATCGATA GTAGAAGCCT TCATTCATCT
1051 TGGGAAGCAG GTCTAATTGG TAAAGATGAT GAATGGTATA AATTATTCAG
1101 CAAATCTTAC ACCCAAGCAG ATTTGGCTTT ACAGTCTTAT CATTTGAATA
1151 CTGCTGCTAA ATCTTGGCTT CAATCGGGCA ATACAAAGCC TTTATCCGAA
1201 TGGATGTCCG ACCAAGGTTA TACACTTATT TCAGGAGTTA ATCCTAGATT
1251 CATTCCAATA CCAAGAGGGT TTGTAAAACA AAATACACCT ATTACTAATG
1301 TCAAATACCC GGAAGGCATC AGTTTCGATA CAAACCTANA AAGACATCTG
1351 GCAAATGCTG ATGGTTTTAG TCAAGAACAG GGCATTAAAG GAGCCCATAA
1401 CCGCACCAAT NTTATGGCAG AACTAAATTC ACGAGGAGGA NGNGTAAAAT
1451 CTGAAACCCA NACTGATATT GAAGGCATTA CCCGAATTAA ATATGATATT
1501 CCTACACTAG ACAGGACAGG TAAACCTGAT GGTGGATTTA AGGAAATTTC
1551 AAGTATAAAA ACTGTTTATA ATCCTAAAAA NTTTTNNGAT GATAAAATAC
1601 TTCAAATGGC TCAANATGCT GNTTCACAAG GATATTCAAA AGCCTCTAAA
1651 ATTGCTCAAA ATGAAAGAAC TAAATCAATA TCGGAAAGAA AAAATGTCAT
1701 TCAATTCTCA GAAACCTTTG ACGGAATCAA ATTTAGANNN TATNTNGATG
1751 TAAATACAGG AAGAATTACA AACATTCACC CAGAATAA

This encodes a protein having the partial amino acid sequence <SEQ ID 72>:

1   YLKQLQVAKN INWNQVQLAY DRWDYKQEGL TEAGAAIIAL AVTVVTSGAG
51  TGAVLGLNGA XAAATDAAFA SLASQASVSF INNKGDVGKT LKELGRSSTV
101 KNLVVAAATA GVADKIGASA LXNVSDKQWI NNLTVNLANA GSAALINTAV
151 NGGSLKDXLE ANILAALVNT AHGEAASKIK QLDQHYIVRK IAHAIAGCAA
201 AAANKGKCQD GAIGAAVGEI VGEALTNGKN PDTLTAKERE QILAYSKLVA
251 GTVSGVVGGD VNAAANAAEV AVKNNQLSDX EGREFONENT ACAKQNXPQL
301 CRKNTVKKYQ NVADKRLAAS IAICTDISRS TECRTIRKQH LIDSRSLHSS
351 WEAGLIGKDD EWYKLFSKSY TQADLALQSY BLNTAAKSWL QSGNTKPLSE
401 VNSDQGYTLI SGVNPRFIFI PRGFVKQNTP ITNVKYPEGI SFDTNLXRHL
451 ANADGFSQEQ GIKGAHNRTN XMAELNSRGG XVKSETXTDI EGITRIKYEI
501 PTLDRTGKPD GGFKEISSIK TVYNPKXFXD DKILQMAQXA XSQGYSKASK
551 IAQNERTKSI SERKNVIQFS ETFDGIKFRX YXDVNTGRIT NIHPE*

ORF41a and ORF41-1 show 94.8% identity in 595 aa overlap:

           10        20        30
orf41a.pep YLKQLQVAKNINWNQVQLAYDRWDYKQEGLTEAGAA
           |||||||:|::||||||||||:||||||||| ||||
orf41-1    MQVNIQIPYILPRCVRAEDTPYACYLKQLQVTKDVNWNQVQLAYDKWDYKQEGLTGAGAA
           10        20        30        40        50        60
           40        50        60        70        80        90
orf41a.pep IIALAVTVVTSGAGTGAVLGLNGAXAAATDAAFASLASQASVSFINNKGDVGKTLKELGR
           ||||||||||:|||:||:|||||| ||||||||||||||||||:|||||::|:|||||||
orf41-1    IIALAVTVVTAGAGAGAALGLNGAAAAATDAAFASLASQASVSLINNKGNIGNTLKELGR
           70        80        90       100       110       120
           100       110       120       130       140       150
orf41a.pep SSTVKNLVVAAATAGVADKIGASALXNVSDKQWINNLTVNLANAGSAALINTAVNGGSLK
           |||||||:||:|||||||||||||| ||||||||||||||||||||||||||||||||||
orf41-1    SSTVKNLMVAVATAGVADKIGASALNNVSDKQWINNLTVNLANAGSAALINTAVNGGSLK
           130       140       150       160       170       180
           160       170       180       190       200       210
orf41a.pep DXLEANILAALVNTAHGEAASKIKQLDQHYIVHKIAHAIAGCAAAAANKGKCQDGAIGAA
           | |||||||||||||||||||||||||||||:||||||||||||||||||||||||||||
orf41-1    DNLEANILAALVNTAHGEAASKIKQLDQHYITHKIAHAIAGCAAAAANKGKCQDGAIGAA
           190       200       210       220       230       240
           220       230       240       250       260       270
orf41a.pep VGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGVVGGDVNAAANAAEVAVKNNQ
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf41-1    VGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGVVGGDVNAAANAAEVAVKNNQ
           250       260       270       280       290       300
           280       290       300       310       320       330
orf41a.pep LSDXEGREFDNEMTACAKQNXPQLCRKNTVKKYQNVADKRLAASIAICTDISRSTECRTI
           ||| |||||||||||||||| |||||||||||||||||||||||||||||||||||||||
orf41-1    LSDKEGREFDNEMTACAKQNNPQLCRKNTVKKYQNVADKRLAASIAICTDISRSTECRTI
           310       320       330       340       350       360
           340       350       360       370       380       390
orf41a.pep RKQHLIDSRSLHSSWEAGLIGKODEWYKLFSKSYTQADLALQSYHLNTAMCSWLQSGNTK
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf41-1    RKQHLIDSRSLHSSWEAGLIGKDDEWYKLFSKSYTQADLALQSYHLNTAAXSWLOSGNTK
           370       380       390       400       410       420
           400       410       420       430       440       450
orf41a.pep PLSEWMSDQGYTLISGVNPRFIPIPRGFVKQNTPITNVKYPEGISFDTNLXRHLANADGF
           |||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||
orf41-1    PLSEWMSDQGYTLISGVNPRFIPIPRGFVKQNTPITNVKYPEGISFDTNLKRHLANADGF
           430       440       450       460       470       480
           460       470       480       490       500       510
orf41a.pep SQEQGIKGAHNRTNXMAELNSRGGXVKSETXTDIEGITRIKYEIPTLDRTGKPDGGFKEI
           ||:||||||||||| ||||||||| ||||| |||||||||||||||||||||||||||||
orf41-1    SQKQGIKGAHNRTNFMAELNSRGGRVKSETQTDIEGITRIKYEIPTLDRTGKPDGGFKEI
           490       500       510       520       530       540
           520       530       540       550       560       570
orf41a.pep SSIKTVYNPKXFXDDKILQMAQXAXSQGYSKASKIAQNERTKSISERKNVIQFSETFDGI
           |||||||||| | ||||||||| | |||||||||||||||||||||||||||||||||||
orf41-1    SSIKTVYNPKKFSDDKILQMAQNAASQGYSKASKIAQNERTKSISERKNVIQFSETFDGI
           550       560       570       580       590       600
           580       590
orf41a.pep KFRXYXDVNTGRITNIHPEX
           ||| | ||||||||||||||
orf41-1    KFRSYFDVNTGRITNIHPEX
           610       620

Amino acids 25-619 of ORF41-1 were amplified as described above. FIG. 6 shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF41-1.

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 17

The following DNA sequence was identified in N. meningitidis <SEQ ID 73>

1   ATGGCAATCA TTACATTGTA TTATTCTGTC AATGGTATTT TAAATGTATG
51  TGCAAAAGCA AAAAATATTC AAGTAGTTGC CAATAATAAG AATATGGTTC
101 TTTTTGGGTT TTTGGSmrGC ATCATCGGCG GTTCAACCAA TGCCATGTCT
151 CCCATATTGT TAATATTTTT GCTTAGCGAA ACAGAAAATA AAAATcgTAT
201 CGTAAAATCA AGCAATCTAT GCTATCTTTT GGCGAAAATT GTTCAAATAT
251 ATATGCTAAG AGACCAGTAT TGGTTATTAA ATAAGAGTGA ATACGdTTTA
301 ATATTTTTAC TGTCCGTATT GTCTGTTATT GGATTGTATG TTGGAATTCG
351 GTTAAGGACT AAGATTAGCC CAaATTTTTT TAAAATGTTA ATTTTTATTG
401 tTTTATTGGT ATTGGCtCTG AAAATCGGGC AttCGGGTTT AAtCAAACTT
451 TAA

This corresponds to the amino acid sequence <SEQ ID 74; ORF51>:

1   HAIITLYYSV NGILNVCAKA KNIQVVANNK NMVLFGFLXX IICGSTNANS
51  PILLIFLLSE TENKNRIVKS SNLCYLLAKI VQIYMLRDQY WLLNKSEYXL
101 IFLLSVLSVI GLYVGIRLRT KISPNFFKML IFIVLLVLAL KIGHSGLIKL
151 *

Further work revealed the complete nucleotide sequence <SEQ ID 75>:

1   ATGCAAGAAA TAATGCAATC TATCGTTTTT GTTGCTGCCG CAATACTGCA
51  CGGAATTACA GGCATGGGAT TTCCGATGCT CGGTACAACC GCATTGGCTT
101 TTATCATGCC ATTGTCTAAG GTTGTTGCCT TGGTGGCATT ACCAAGCCTG
151 TTAATGAGCT TGTTGGTTCT ATGCAGCAAT AACAAAAAGG GTTTTTGGCA
201 AGAGATTGTT TATTATTTAA AAACCTATAA ATTGCTTGCT ATCGGCAGCG
251 TCGTTGGCAG CATTTTGGGG GTGAAGTTGC TTTTGATACT TCCAGTGTCT
301 TGGCTGCTTT TACTGATGGC AATCATTACA TTGTATTATT CTGTCAATGG
351 TATTTTAAAT GTATGTGCAA AAGCAAAAAA TATTCAAGTA GTTGCCAATA
401 ATAAGAATAT GGTTCTTTTT GGGTTTTTGG CAGGCATCAT CGGCGGTTCA
451 ACCAATGCCA TGTCTCCCAT ATTGTTAATA TTTTTGCTTA GCGAAACAGA
501 AAATAAAAAT CGTATCGTAA AATCAAGCAA TCTATGCTAT CTTTTGGCGA
551 AAATTGTTCA AATATATATG CTAAGAGACC AGTATTGGTT ATTAAATAAG
601 AGTGAATACG GTTTAATATT TTTACTGTCC GTATTGTCTG TTATTGGATT
651 GTATGTTGGA ATTCGGTTAA GGACTAAGAT TAGCCCAAAT TTTTTTAAAA
701 TGTTAATTTT TATTGTTTTA TTGGTATTGG CTCTGAAAAT CGGGCATTCG
751 GGTTTAATCA AACTTTAA

This corresponds to the amino acid sequence <SEQ ID 76; ORF51-1>:

1   MQEIMQSIVF VAAAILHGIT GMGFPMLGTT ALAFIMPLSK VVALVALPSL
51  LMSLLVLCSN NKKGFWQEIV YYLKTYKLLA IGSVVGSILG VKLLLILPVS
101 WLLLLMAIIT LYYSVNGILN VCAKAKNIQV VANNKNNVLF GFLAGIIGGS
151 TNAMSPILLI FLLSETENKN RIVKSSNLCY LLAKIVQIYN LRDQYWLLNK
201 SEYGLIFLLS VLSVIGLYVG IRLRTKISPN FFKMLIFIVL LVLALKIGHS
251 GLIKL*

Computer analysis of this amino acid sequence reveals three putative transmembrane domains. A corresponding ORF from strain A of N. meningitidis was also identified:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF51 shows 96.7% identity over a 150 aa overlap with an ORF (ORF51a) from strain A of N. meningitidis:

          10        20        30
orf51.pep MAIITLYYSVNGILNVCAKAKNIQVVANNK
          ||||||||||||||||||||||||||||||
orf51a    YKLLAIGSVVGSILGVKLLLILPVSWLLLLMAIITLYYSVNGILNVCAKAKNIQVVANNK
          80        90       100       110       120       130
          40        50        60        70        80        90
orf51.pep NMVLFGFLXXIIGGSTNAMSPILLIFLLSETENKNRIVKSSNLCYLLAKIVQIYMLRDQY
          |||||||| ||||||||||||||||||||||||||||:||||||||||||||||||||||
orf51a    NMVLFGFLAGIIGGSTNAMSPILLIFLLSETENKNRIAKSSNLCYLLAKIVQIYMLRDQY
          140       150       160       170       180       190
          100       110       120       130       140       150
orf51.pep WLLNKSEYXLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVLLVLALKIGHSGLIKL
          |||||||| ||||||||||||||||||||||||||||||||||||||||||||:||||||
orf51a    WLLNKSEYGLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVLLVLALKIGYSGLIKL
          200       210       220       230       240       250

ORF51-1 and ORF51a show 99.2% identity in 255 aa overlap:

orf51a.pep MQEIMQSIVFVAAAILHGITGMGFPMLGTTALAFIMPLSKVVALVALPSLLMSLLVLCSN
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf51-1    MQEIMQSIVFVAAAILHGITGMGFPMLGTTALAFIMPLSKVVALVALPSLLMSLLVLCSN
orf51a.pep NKKGFWQEIVYYLKTYKLLAIGSVVGSILGVKLLLILPVSWLLLLMAIITLYYSVNGILN
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf51-1    NKKGFWQEIVYYLKTYKLLAIGSVVGSILGVKLLLILPVSWLLLLMAIITLYYSVNGILN
orf51a.pep VCAKAKNIQVVANNKNMVLFGFLAGIIGGSTNAMSPILLIFLLSETENKNRIAKSSNLCY
           ||||||||||||||||||||||||||||||||||||||||||||||||||||:|||||||
orf51-1    VCAKAKNIQVVANNKNMVLFGFLAGIIGGSTNAMSPILLIFLLSETENKNRIVKSSNLCY
orf51a.pep LLAKIVQIYMLRDQYWLLNKSEYGLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVL
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf51-1    LLAKIVQIYMLRDQYWLLNKSEYGLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVL
orf51a.pep LVLALKIGYSGLIKLX
||||||||:|||||||
orf51-1    LVLALKIGHSGLIKLX

The complete length ORF51a nucleotide sequence SEQ D 77> is:

1   ATGCAAGAAA TAATGCAATC TATCGTTTTT GTTGCTGCCG CAATACTGCA
51  CGGAATTACA GGCATGGGAT TTCCGATGCT CGGTACAACC GCATTGGCTT
101 TTATCATGCC ATTGTCTAAG GTTGTTGCCT TGGTGGCATT ACCAAGCCTG
151 TTAATGAGCT TGTTGGTTCT ATGCAGCAAT AACAAAAAGG GTTTTTGGCA
201 AGAGATTGTT TATTATTTAA AAACCTATAA ATTGCTTGCT ATCGGCAGCG
251 TCGTTGGCAG CATTTTGGGG GTGAAGTTGC TTTTGATACT TCCAGTGTCT
301 TGGCTGCTTT TACTGATGGC AATCATTACA TTGTATTATT CTGTCAATGG
351 TATTTTAAAT GTATGTGCAA AAGCAAAAAA TATTCAAGTA GTTGCCAATA
401 ATAAGAATAT GGTTCTTTTT GGGTTTTTGG CAGGCATCAT CGGCGGTTCA
451 ACCAATGCCA TGTCTCCCAT ATTGTTAATA TTTTTGCTTA GCGAAACAGA
501 GAATAAAAAT CGTATCGCAA AATCAAGCAA TCTATGCTAT CTTTTGGCAA
551 AAATTGTTCA AATATATATG CTAAGAGACC AGTATTGGTT ATThAATAAG
601 AGTGAATACG GTTTAATATT TTTACTGTCC GTATTGTCTG TTATTGGATT
651 GTATGTTGGA ATTCGGTTAA GGACTAAGAT TAGCCCAAAT TTTTTTAAAA
701 TGTTAATTTT TATTGTTTTA TTGGTATTGG CTCTGAAAAT CGGGTATTCA
751 GGTTTAATCA AACTTTAA

This encodes a protein having amino acid sequence <SEQ ID 78>:

1   MQEIMQSIVF VAAAILHGIT GMGFPNLGTT ALAFIMPLSK VVALVALPSL
51  LMSLLVLCSN NKKGFWQEIV YYLKTYKLLA IGSVVGSILG VKLLLILPVS
101 WLLLLMAIIT LYYSVNGILN VCAKAKNIQV VANNKNMVLF GFLAGIIGGS
151 TNAMSFILLI FLLSETENKN RIAXSSNLCY LLAKIVQIYM LRDQYWLLNK
201 SEYGLIFLLS VLSVIGLYVG IRLRTKISPN FFKMLIFIVL LVLALKIGYS
251 GLIKL*

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 18

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 79>

1   ATGAGACATA TGAAAATACA AAATTATTTA CTAGTATTTA TAGTTTTACA
51  TATAGCCTTG ATAGTAATTA ATATAGTGTT TGGTTATTTT GTTTTTCTAT
101 TTGATTTTTT TGCGTTTTTG TTTTTTGCAA ACGTCTTTCT TGCTGTAAAT
151 TTATTATTTT TAGAAAAAAA CATAAAAAAC AAATTATTGT TTTTATTGCC
201 GATTTCTATT ATTATATGGA TGGTAATTCA TATTAGTATG ATAAATATAA
251 AATTTTATAA ATTTGAGCAT CAAATAAAGG AACAAAATAT ATCCTCGATT
301 ACTGGGGTGA TAAAACCACA TGATAGTTAT AATTATGTTT ATGACTCAAA
351 TGGATATGCT AAATTAAAAG ATAATCATAG ATATGGTAGG GTAATTAGAG
401 AAACACCTTA TATTGATGTA GTTGCATCTG ATGTTAAAAA TAAATCCATA
451 AGATTAAGCT TGGTTTGTGG TATTCATTCA TATGCTCCAT GTGCCAATTT
501 TATAAAATTT GTCAGG..

This corresponds to the amino acid sequence <SEQ ID 80; ORF82>:

1   MRHMKIQNYL LVFIVLHIAL IVINIVFGYF VFLFDFFAFL FFANVFLAVN
51  LLFLEKNIKN KLLFLLPISI IIWMVIHISM INIKFYKFEH QIKEQNISSI
101 TGVIKPNDSY NYVYDSNGYA KLKDWHRYGR VIRETPYIDV VASDVKNKSI
151 RLSLVCGIHS YAPCANFIKF VR..

Further work revealed the complete nucleotide sequence SEQ ID 81>:

1   ATGAGACATA TGAAAAATAA AAATTATTTA CTAGTATTTA TAGTTTTACA
51  TATAGCCTTG ATAGTAATTA ATATAGTGTT TGGTTATTTT GTTTTTCTAT
101 TTGATTTTTT TGCGTTTTTG TTTTTTGCAA ACGTCTTTCT TGCTGTAAAT
151 TTATTATTTT TAAAAAAAAA CATAAAAAAC AAATTATTGT TTTTATTGCC
201 GATTTCTATT ATTATATGGA TGGTAATTCA TATTAGTATG ATAAATATAA
251 AATTTTATAA ATTTGAGCAT CAAATAAAGG AACAAAATAT ATCCTCGATT
301 ACTGGGGTGA TAAAACCACA TGATAGTTAT AATTATGTTT ATGACTCAAA
351 TGGATATGCT AAATTAAAAG ATAATCATAG ATATGGTAAG GTAATTAGAG
401 AAACACCTTA TATTGATGTA GTTGCATCTG ATGTTAAAAA TAAATCCATA
451 AGATTAAGCT TGGTTTGTGG TATFCATTCA TATGCFCCAT GTGCCAATTT
501 TATAAAATTT GCAAAAAAAC CTGTTAAAAT TTATTTTTAT AATCAACCTC
551 AAGGAGATTT TATAGATAAT GTAATATTTG AAATTAATGA TGGAAACAAA
601 AGTTTGTACT TGTTAGATAA GTATAAAACA TTTTTTCTTA TTGAAAACAG
651 TGTTTGTATC GTATTAATTA TTTTATATTT AAAATTTAAT TTGCTTTTAT
701 ATAGGACTTA CTTCAATGAG TTGGAATAG

This corresponds to the amino acid sequence <SEQ ID 82; ORF82-1>:

1   MRHMKNKNYL LVFIVLHIAL IVINIVFGYF VFLFDFFAFL FFANVFLAVN
51  LLFLEKNIKH KLLFLLPISI IIWMVIHISH INIKFYKFEH QIKEQNISSI
101 TGVIKPHDSY NYVYDSNGYA KLKDNHRYGR VIRETPYIDV VASDVKNKSI
151 RLSLVCGIHS YAPCANFIKF AKKPVKIYFY NQPQGDFIDN VIFEINDGNK
201 SLYLLDKYKT FFLIENSVCI VLIILYLKFN LLLYRTYFNE LE*

Computer analysis of this amino acid sequence reveals a predicted leader peptide.

A corresponding ORF from strain A of N. meningitidis was also identified:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF82 shows 97.1% identity over a 172 aa overlap with an ORF (ORF82a) from strain A of N. meningitidis:

           10        20        30        40        50        60
orf82 pep  MRHMKIQNYLLVFIVLHIALIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN
           ||||| :|||||||||||:|||||||||||||||||||||||||||||||||||||||||
orf82a     MRHMKIKNYLLVFIVLHITLIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN
           10        20        30        40        50        60
           70        80        90       100       110       120
orf82 pep  KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf82a     KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKFHDSYNYVYDSNGYA
           70        80        90       100       110       120
130       140       150       160       170
orf82 pep  KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFVR
           ||||||||||||||||||||||||||||||||||||||||||||||||||::
orf82a     KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY
           130       140       150       160       170
orf82a.pep MRHMKNKNYLLVFIVLHITLIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN
           ||||||||||||||||||:|||||||||||||||||||||||||||||||||||||||||
orf82-1    MRHMKNKNYLLVFIVLHIALIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN
orf82a.pep KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf82-1    KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA
orf82a.pep KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf82-1    KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY
orf82a.pep NQPQGDFIDNVIFEINKGKKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf82-1    NQPQGDFIDNVIFEINKGKKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE
orf82a.pep LEX
           |||
orf82-1    LEX

ORF82a and ORF82-1 show 99.2% identity in 242 aa overlap:

orf82a.pep MRHMKNKNYLLVFIVLHITLIVINIVFGYGVFLFDFFAFLFFANVFLAVNLLFLEKNIKN
           ||||||||||||||||||:|||||||||||||||||||||||||||||||||||||||||
orf82-1    MRHMKNKNYLLVFIVLHIALIVINIVFGYGVFLFDFFAFLFFANVFLAVNLLFLEKNIKN
orf82a.pep KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf82-1    KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA
orf82a.pep KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
orf82-1    KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY
orf82a.pep NQPQGDFIDNVIFEINDGKKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE
           ||||||||||||||||||:|||||||||||||||||||||||||||||||||||||||||
orf82-1    NQPQGDFIDNVIFEINDGNKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE
orf82a.pep LEX
           |||
orf82-1    LEX

The complete length ORF82a nucleotide sequence <SEQ D 83> is:

1   ATGAGACATA TGAAAAATAA AAATTATTTA CTAGTATTTA TAGTTTTACA
51  TATAACCTTG ATAGTAATTA ATATAGTGTT TGGTTATTTT GTTTTTCTAT
101 TTGATTTTTT TGCGTTTTTG TTTTTTGCAA ACGTCTTTCT TGCTGTAAAT
151 TTATTATTTT TAGAAAAAAA CATAAAAAAC AAATTATTGT TTTTATTGCC
201 GATTTCTATT ATTATATGGA TGGTAATTCA TATTAGTATG ATAAATATAA
251 AATTTTATAA ATTTGAGCAT CAAATAAAGG AACAAAATAT ATCCTCGATT
301 ACTGGGGTGA TAAAACCACA TGATAGTTAT AATTATGTTT ATGACTCAAA
351 TGGATATGCT AAATTAAAAG ATAATCATAG ATATGGTAGG GTAATTACAG
401 AAACACCTTA TATTGATGTA GTIGCATCTG ATGTTAAAAA TAAATCCATA
451 AGATIAAGCT TGGTTTGTGG TATTCATTCA TATGCTCCAT GTGCCAATTT
501 TATAAAATTT GCAAAAAAAC CTGTTAAAAT TTATTTTTAT AATCAACCTC
551 AAGGAGATTT TATAGATAAT GTAATATTTG AAATTAATGA TGGAAAAAAA
601 AGTTTGTACT TGTTAGATAA GTATAAAACA TTTTTTCTTA TTGAAAACAG
651 TGTTTGTATC GTATTAATTA TTTTATATTT AAAATTTAAT TTGCTTTTAT
701 ATAGGACTTA CTTCAATGAG TTGGAATAG

This encodes a protein having amino acid sequence <SEQ ID 84>:

1   MRHMKNKNYL LVFIVLHITL IVINIVFGYF VFLFDFFAFL FFANVFLAVN
51  LLFLEKNIKN KLLFLLPISI IIWMVIHISM INIKFYKFEH QIKEQNISSI
101 TGVIKPHDSY NYVYDSNGYA KLKDNHRYGR VIRETPYIDV VASDVIQKSI
151 RLSLVCGIHS YAPCANFIKF AXKPVKIYFY NQPQGDFXDN VIFEINDGKK
201 SLYLLDKYKT FFLIENSVCI VLIILYLKFN LLLYRTYFNE LE*

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 19

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 85>

1   ..ACCCCCAACA GCGTGACCGT CTTGCCGTCT TTCGGCGGAT TCGGGCGTAC
51  CGGCGCGACC ATCAATGCAG CAGGCGGGGT CGGCATGACT GCCTTTTCGA
101 CAACCTTAAT TTCCGTAGCC GAGGGCGCGG TTGTAGAGCT GCAGGCCGTG
151 AGAGCCAAAG CCGTCAATGC AACCGCCGCT TGCATTTTTA CGGTCTTGAG
201 TAAGGACATT TTCGATTTCC TTTTTATTTT CCGTTTTCAG ACGGCTGACT
251 TCCGCCTGTA TTTTCGCCAA AGCCATGCCG ACAGCGTGCG CCTTGACTTC
301 ATATTTAAAA GCTTCCGCGC GTGCCAGTTC CAGTTCGCGC GCATAGTTTT
351 GAGCCGACAA CAGCAGGGCT TGCGCCTTGT CGCGCTCCAT CTTGTCGATG
401 ACCGCCTGCA GCTTCGCAAA TGCCGACTFG TAGCCTTGAT GGTGCGACAC
451 AGCCAAGCCC GTGCCGACAA GCGCGATAAT GGCAATCGGT TGCCAGTAAT
501 TCGCCAGCAG TTTCACGAGA TTCATTCTCG ACCTCCTGAC GCTTCACGCT
551 GA

This corresponds to the amino acid sequence <SEQ ID 86; ORF124>:

1   ..TPNSVTVLPS FGGFGRTGAT INAAGGVGMT AFSTTLISVA EGAVVELQAV
51  RAKAVNATAA CIFTVLSKDI FDFLFIFRFQ TADFRLYFRQ SHADSVRLDF
101 IFKSFRACQF QFARIVLSRQ QQGLRLVALH LVDORLQLRX CRLVALMVRH
151 SQARADKRDN GNRLPVIRQQ FHEIHSRPPD ASR*

Computer analysis of this amino acid sequence predicts a transmembrane domain.

Further work revealed the complete nucleotide sequence SEQ ID 87>:

1   ATGACTGCCT TTTCGACAAC CTTAATTTCC GTAGCCGAGG GCGCGGTTGT
51  AGAGCTGCAG GCCGTGAGAG CCAAAGCCGT CAATGCAACC GCCGCTTGCA
101 TTTTTACGGT CTTGAGTAAG GACATTTTCG ATTTCCTTTT TATTTTCCGT
151 TTTCAGACGG CTGACTTCCG CCTGTTTTTT CGCCAAAGCC ATGCCGACAG
201 CGTGCGCCTT GACTTCATAT TTTTTAGCTT CCGcGCGTGC CAGTTCCAGT
251 TCGCGCGCAT AGTTTTGAGC CGACAACAGC AGGGCTTGCG CCTTGTCGCG
301 CTCCATCTTG TCGATGACCG CCTGCTGCTT CGCAAATGCC GACTTGTAGC
351 CTTGATGGTG CGACACAGCC AAGCCCGTGC CGACAAGCGC GATAATGGCA
401 ATCGGTTGCC AGTTATTCGC CAGCAGTTTC ACGAGATTCA TTCTCGACCT
451 CCTGACGCTT CACGCTGA

This corresponds to the amino acid sequence SEQ ID 88; ORF124-1>:

1   MTAFSTTLIS VAEGAVVELQ AVRAKAVNAT AACIFTVLSK DIFDFLFIFR
51  FQTADFRLFF RQSHADSVRL DFIFFSFRAC QFQFARIVLS RQQQGLRLVA
101 LHLVDDRLLL RKCRLVAIMV RHSQARADKR DNGNRLPVIR QQFHEIHSRP
151 PDASR*

A corresponding ORF from strain A of N. meningitidis was also identified:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF124 shows 87.5% identity over a 152 aa overlap with an ORF (ORF124a) from strain A of N. meningitidis:

           10        20        30        40        50        60
orf124.pep TPNSVTVLPSFGGFGRTGATINAAGGVGMTAFSTTLISVAEGAVVELQAVRAKAVNATAA
           |||||||||||||||:|||||| |||||:|||
orf124a    MTAFSTTLISVAEGALVELQAVMAKAVNTTAA
           10        20        30
           70        80        90       100       110       120
orf124.pep CIFTVLSKDIFDFLFIFRFQTADFRLYFRQSHADSVRLDFIFKSFRACQFQFARIVLSRQ
           ||||||||||||||||||||||||||:|||||||:||||||| |||:  |||| :|||||
orf124a    CIFTVLSKDIFDFLFIFRFQTADFRLFFRQSHADGVRLDFIFFSFRTRLFQFAGVVLSRQ
           40        50        60        70        80        90
           130       140       150       160       170       180
orf124.pep QQGLRLVALHLVDDRLQLRKCRLVALMVRHSQARADKRDNGNRLPVIRQQFHEIHSRPPD
           ||||||||||:::||| ||| ||||||||| |:||||||:||||||||||||||||||||
orf124a    QQGLRLVALHFLNDRLLLRKSRLVALMVRHRQTRADKRDDGNRLPVIRQQFHEIHSRPPD
           100       110       120       130       140       150
orf124.pep ASRX
           :
orf124a    VX

ORF124a and ORF124-1 show 89.5% identity in 152 aa overlap:

orf124-1.pep MTAFSTTLISVAEGAVVELQAVRAKAVNATAACIFTVLSKDIFDFLFIFRFQTADFRLFF
             |||||||||||||||:|||||| |||||:|||||||||||||||||||||||||||||||
orf124a      MTAFSTTLISVAEGALVELQAVMAKAVNTTAACIFTVLSKDIFDFLFIFRFQTADFRLFF
orf124-1.pep RQSHADSVRLDFIFFSFRACQFQFARIVLSRQQQGLRLVALHLVDDRLLLRKCRLVALMV
             ||||||:|||||||||||:  |||| :|||||||||||||||:::||||||| |||||||
orf124a      RQSHADGVRLDFIFFSFRTRLFQFAGVVLSRQQQGLRLVALHFLNDRLLLRKSRLVALMV
orf124-1.pep RHSQARADKRDNGNRLPVIRQQFHEIHSRPPDASRX
             || |:||||||:||||||||||||||||||||:
orf124a      RHRQTRADKRDDGNRLPVIRQQFHEIHSRPPDVX

The complete length ORF124a nucleotide sequence <SEQ ID 89> is:

1   ATGACCGCCT TTTCGACAAC CTTAATTTCC GTAGCCGAGG GCGCGCTTGT
51  AGAGCTGCAA GCCGTGATGG CCAAAGCCGT CAATACAACC GCCGCCTGCA
101 TTTTTACGGT CTTGAGTAAG GACATTTTCG ATTTCCTTTT TATTTTCCGT
151 TTTCAGACGG CTGACTTCCG CCTGTTTTTT CGCCAAAGCC ATGCCGACGG
201 CGTGCGCCTT GACTTCATAT TTTTTAGCTT CCGCACGCGC CTGTTCCAGT
251 TCGCGGGCGT AGTTTTGAGC CGACAACAGC AGGGCTTGCG CCTTGTCGCG
301 CTTCATTTTC TCAATGACCG CCTGCTGCTT CGCAAAAGCC GACTTGTAGC
351 CTTGATGGTG CGACACCGCC AAACCCGTGC CGACAAGCGC GATGATGGCA
401 ATCGGTTGCC AGTTATTCGC CAGCAGTTTC ACGAGATTCA TTCTCGACCT
451 CCTGACGTTT GA

This encodes a protein having amino acid sequence <SEQ ID 90>:

1   MTAFSTTLIS VAEGALVELQ AVNAXAVNTT AACIFTVLSK DIFDFLFIFR
51  FQTADFRLFF RQSHADGVRL DFIFFSFRTR LFQFAGVVLS RQQQGLRLVA
101 LHFLNDELLL RKSRLVALHV RHRQTRADKR DDGNRLPVIR QQFHEIHSRP
151 PDV*

ORF124-1 was amplified as described above. FIG. 7 shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF124-1.

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 20

Table III lists several Neisseria strains which were used to assess the conservation of the sequence of ORF 40 among different strains.

TABLE III
List of Neisseria Strains Used for Gene Variability Study of ORF 40
Identification
number	Strains	Source/reference
Group B
zn02_1	BZ198	R. Moxon/Seiler et al., 1996
zn03_1	NG3/88	R. Moxon/Seiler et al., 1996
zn04_1	297-0	R. Moxon/Seiler et al., 1996
zn06_1	BZ147	R. Moxon/Seiler et al., 1996
zn07_1	BZ169	R. Moxon/Seiler et al., 1996
zn08_1	528	R. Moxon/Seiler et al., 1996
zn10_1	BZ133	R. Moxon/Seiler et al., 1996
zn11_1ass	NGE31	R. Moxon/Seiler et al., 1996
zn14_1	NGH38	R. Moxon/Seiler et al., 1996
zn16_1	NGH15	R. Moxon/Seiler et al., 1996
zn18_1	BZ232	R. Moxon/Seiler et al., 1996
zn19_1	BZ83	R. Moxon/Seiler et al., 1996
zn20_1	44/76	R. Moxon/Seiler et al., 1996
zn21_1	MC58	R. Moxon
Group A
zn22_1	205900	R. Moxon
zn23_1	F6124	R. Moxon
z2491_1	Z2491	R. Moxon/Maiden et al., 1998
Group C
zn24_1	90/18311	R. Moxon
zn25_1ass	93/4286	R. Moxon
Others
zn28_1ass	860800 (group Y)	R. Moxon/Maiden et al., 1998
zn29_1ass	E32 (group Z)	R. Moxon/Maiden et al., 1998
References:
Seiler A. et al., Mol. Microbiol., 1996, 19(4): 841-856.
Maiden et al., Proc. Natl. Acad. Sci. USA, 1998, 95: 3140-3145.

The amino acid sequences for each listed strain are as follows:

>Z2491 <SEQ ID 91>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNVSPSKADEMDETV
NINAGNNIEISRNGKNIDIATSNAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RINVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVWDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN02_1 <SEQ ID 92>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVATAVLATLLFATVQANATDDDDLYLE
PVQRTAVLSFRSDKEGTGEKEGTEDSHGGAVYFDEKRVLKAGAITLKAGDNLKIKQNTNE
NTNDSSFTYSLKKDLTDLTSVETEKLSFGAAGNKVNITSDTKGLNFAKETAGTAGDPTVH
LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV
DFVRTYDTVEFLSADTKTTTNVSKDNGKKTEVICIGAIVTSVIKEKDGKLVTGKGKDENG
SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA
TVSKODQGNITVRYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGRMDE
TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK
PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG
KSMMAIGGDTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN03_1 <SEQ ID 93>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVATAVLATLLFATVQASTTDDDDLYLE
PVQRTAPVLSFHADSEGTGEKEVTEDSNWGVYFDKKGVLTAGTITLKAGDNLKIKQNTDE
NTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVH
LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV
DFVRTYDTVEFLSADTRTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGKDENG
SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA
TVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDE
TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK
PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG
KSMMAIGGDTYRGEAGYAIGYSSISDGGNMIIKGTASGNSRGHFGASASVGYQW*
>ZN04_1 <SEQ ID 94>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVATAVLATLLFATVQASTTDDDDLYLE
PVQRTAPVLSFHADSEGTGEKEVTEDSNWGVYFDKKGVLTAGTITLKAGDNLKIKQNTDE
NTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVH
LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV
DFVRTYDTVEFLSADTRTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGKDENG
SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA
TVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDE
TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK
PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG
KSMMAIGGDTYRGEAGYAIGYSSISDGGNMIIKGTASGNSRGHFGASASVGYQW*
>ZN06_1 <SEQ ID 95>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNVSPSKADEMDETV
NINAGNNIEISRNGKNIDIATSNAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RINVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVWDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN07_1 <SEQ ID 96>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNVSPSKADEMDETV
NINAGNNIEISRNGKNIDIATSNAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RINVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVWDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN08_1 <SEQ ID 97>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVETAVLATLLFATVQANATDTDEDDEL
EPVVRSALVLQFMIDKEGNGEIESTGDIGWSIYYDDHNTLHGATVTLKAGDNLKIKQNTD
ENTNASSFTYSLKKDLTDLTSVGTEELSFGANGNKVNITSDTKGLNFAKKTAGTNGDTTV
HLNGIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSEN
VDFVRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGEN
GSSTEDGEGELVTAKEVIDAVNKAGWRMKTTANGQTGQADKFETVTSGTNVTFASGKGTT
ATVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNAVSPSKGKMD
ETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPLTLSVDDEALNVGSKDAN
KPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNHIDNVDGNARAGIAQAIATAGLVQAYLP
GKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN10_1 <SEQ ID 98>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGYTRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN11_1 ASS <SEQ ID 99>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVATAVLATLLFATVQASTTDDDDLYLE
PVQRTAPVLSFHADSEGTGEKEVTEDSNWGVYFDKKGVLTAGTITLKAGDNLKIKQNTDE
NTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVH
LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV
DFVRTYDTVEFLSADTRTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGKDENG
SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA
TVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDE
TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK
PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG
KSMMAIGGDTYRGEAGYAIGYSSISDGGNMIIKGTASGNSRGHFGASASVGYQW*
>ZN14_1 <SEQ ID 100>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVETAVLATLLFATVQANATDTDEDDEL
EPVVRSALVLQFMIDKEGNGEIESTGDIGWSIYYDDHNTLHGATVTLKAGDNLKIKQNTD
ENTNASSFTYSLKKDLTDLTSVGTEELSFGANGNKVNITSDTKGLNFAKKTAGTNGDTTV
HLNGIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSEN
VDFVRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGEN
GSSTEDGEGELVTAKEVIDAVNKAGWRMKTTANGQTGQADKFETVTSGTNVTFASGKGTT
ATVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNAVSPSKGKMD
ETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPLTLSVDDEALNVGSKDAN
KPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNHIDNVDGNARAGIAQAIATAGLVQAYLP
GKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN16_1 <SEQ ID 101>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVATAVLATLLFATVQANATDDDDLYLE
PVQRTAVVLSFRSDKEGTGEKEGTEDSNWAVYFDEKRVLKAGAITLKAGDNLKIKQNTNE
NTNENTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGD
PTVHLNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTA
SDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGK
DENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTKVTFASGN
GTTATVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKG
KMDETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDDEGALNVGSK
DANKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLAQA
YLPGKSMMAIGGGTYRGEAGYAIGYSSISDTGNWVIKGTASGNSRGHFGASASVGYQW*
>ZN18_1 <SEQ ID 102>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVATAVLATLLFATVQASTTDDDDLYLE
PVQRTAPVLSFHADSEGTGEKEVTEDSNWGVYFDKKGVLTAGTITLKAGDNLKIKQNTDE
NTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVH
LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV
DFVRTYDTVEFLSADTRTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGKDENG
SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA
TVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDE
TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK
PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG
KSMMAIGGDTYRGEAGYAIGYSSISDGGNMIIKGTASGNSRGHFGASASVGYQW*
>ZN19_1 <SEQ ID 103>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL
YLDPVQRTVAVLIVNSDKEGTGEKEKVEENSDWAVYFNEKGVLTAREITLKAGDNLKIKQ
NGTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKDKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGDALNVGSKKDNKPVR
ITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKSM
MAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN20_1 <SEQ ID 104>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL
YLDPVQRTVAVLIVNSDKEGTGEKEKVEENSDWAVYFNEKGVLTAREITLKAGDNLKIKQ
NGTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKDKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGDALNVGSKKDNKPVR
ITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKSM
MAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN21_1 <SEQ ID 105>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL
YLDPVQRTVAVLIVNSDKEGTGEKEKVEENSDWAVYFNEKGVLTAREITLKAGDNLKIKQ
NGTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKDKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGDALNVGSKKDNKPVR
ITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKSM
MAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN22_1 <SEQ ID 106>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGYTRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN23_1 <SEQ ID 107>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGYTRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN24_1 <SEQ ID 108>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVATAVLATLLSATVQANATDTDEDEEL
ESVVRSALVLQFMIDKEGNGEIESTGDIGWSIYYDDHNTLHGATVTLKAGDNLKIKQSGK
DFTYSLKKELKDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVHLNGIG
STLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDFVRT
YDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSSTDE
GEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTKVTFASGNGTTATVSKD
DQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETVNIN
AGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPVRIT
NVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLAQAYLPGKSMMA
IGGGTYRGEAGYAIGYSSISDTGNWVIKGTASGNSRGHFGTSASVGYQW*
>ZN25_ASS <SEQ ID 109>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVATAVLATLLSATVQANATDTDEDEEL
ESVVRSALVLQFMIDKEGNGEIESTGDIGWSIYYDDHNTLHGATVTLKAGDNLKIKQSGK
DFTYSLKKELKDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVHLNGIG
STLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDFVRT
YDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSSTDE
GEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTKVTFASGNGTTATVSKD
DQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETVNIN
AGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPVRIT
NVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLAQAYLPGKSMMA
IGGGTYRGEAGYAIGYSSISDTGNWVIKGTASGNSRGHFGTSASVGYQW*
>ZN28_ASS <SEQ ID 110>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGYTRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*
>ZN29_ASS <SEQ ID 111>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVETAVLATLLFATVQANATDTDEDDEL
EPVVRTAPVLSFHSDKEGTGEKEEVGASSNLTVYFDKNRVLKAGTITLKAGDNLKIKQNT
NENTNENTNASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTN
GDPTVHLNGIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTT
GQSENVDFVRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGK
GKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTKVTFAS
GNGTTATVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPS
KGKMDETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVVEAGALNVG
SKDANKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLV
QAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW
*

FIG. 8 shows the results of aligning the sequences of each of these strains. Dark shading indicates regions of homology, and gray shading indicates the conservation of amino acids with similar characteristics. As is readily discernible, there is significant conservation among the various strains of ORF 40, further confirming its utility as an antigen for both vaccines and diagnostics.

It will be appreciated that the invention has been described by means of example only, and that modifications may be made whilst remaining within the spirit and scope of the invention.

Appendix 1

Scarlato, Continuation of U.S. App. Ser.
No. 10/695,499, filed herewith   Ruelle, U.S. Pat. No. 6,780,419
18. (New) An isolated polypeptide comprising 1. An isolated polypeptide comprising a
a member selected from the group consisting of member selected from the group consisting of
(a) the amino acid sequence of SEQ ID (a) the amino acid sequence of SEQ ID
NO: 4; and                       NO: 2;
(b) an immunogenic fragment of at (b) an immunogenic fragment of at least
least 15 contiguous amino acids of SEQ ID 15 contiguous amino acids of SEQ ID NO: 2;
NO: 4, wherein the immunogenic fragment, wherein the immunogenic fragment, when
when administered to a subject in a suitable administered to a subject in a suitable
composition which can include an adjuvant, or composition which can include an adjuvant, or
a suitable carrier coupled to the polypeptide, a suitable carrier coupled to the polypeptide,
induces an antibody or T-cell meditated induces an antibody or T-cell meditated
immune response that recognizes the isolated immune response that recognizes the isolated
polypeptide SEQ ID NO: 4.        polypeptide SEQ ID NO: 2.
19. (New) The isolated polypeptide of claim 2. The isolated polypeptide of claim 1,
18, wherein the polypeptide is according to (a). wherein the polypeptide is according to (a).
20. (New) The isolated polypeptide of claim 3. The isolated polypeptide of claim 1,
18, wherein the polypeptide is according to (b). wherein the polypeptide is according to (b).
21. (New) The isolated polypeptide of claim 4. The isolated polypeptide of claim 1,
18, wherein the immunogenic fragment of (b) wherein the immunogenic fragment of (b)
comprises at least 20 contiguous amino acids of comprises at least 20 contiguous amino acids of
SEQ ID NO: 4; wherein the immunogenic SEQ ID NO: 2; wherein the immunogenic
fragment, when administered to a subject in a fragment, when administered to a subject in a
suitable composition which can include an suitable composition which can include an
adjuvant, or a suitable carrier coupled to the adjuvant, or a suitable carrier coupled to the
polypeptide, induces an antibody or T-cell polypeptide, induces an antibody or T-cell
meditated immune response that recognizes the meditated immune response that recognizes the
isolated polypeptide SEQ ID NO: 4. isolated polypeptide SEQ ID NO: 2.
22. (New) The isolated polypeptide of claim 5. The isolated polypeptide of claim 1,
18, wherein the isolated polypeptide consists of wherein the isolated polypeptide consists of
SEQ ID NO: 4.                    SEQ ID NO: 2.
23. (New) A fusion protein comprising the 6. A fusion protein comprising the isolated
isolated polypeptide of claim 18. polypeptide of claim 1.
24. (New) An immunogenic composition 7. An immunogenic composition comprising
comprising the polypeptide of claim 18, and a the polypeptide of claim 1, and a
pharmaceutically acceptable carrier. pharmaceutically acceptable carrier.
25. (New) The isolated polypeptide of claim 9. The isolated polypeptide of claim 1,
18, wherein the isolated polypeptide is a wherein the isolated polypeptide is a
recombinant polypeptide.         recombinant polypeptide.
26. (New) The isolated polypeptide of claim 10. The isolated polypeptide of claim 2,
19, wherein the isolated polypeptide is a wherein the isolated polypeptide is a
recombinant polypeptide.         recombinant polypeptide.
27. (New) The isolated polypeptide of claim 11. The isolated polypeptide of claim 3,
20, wherein the isolated polypeptide is a wherein the isolated polypeptide is a
recombinant polypeptide.         recombinant polypeptide.
28. (New) An immunogenic composition 12. An immunogenic composition comprising
comprising the isolated polypeptide of claim 19. the isolated polypeptide of claim 2.
29. (New) An immunogenic composition 13. An immunogenic composition comprising
comprising the isolated polypeptide of claim 20. the isolated polypeptide of claim 3.
30. (New) A fusion protein comprising the 14. A fusion protein comprising the isolated
isolated polypeptide of claim 19. polypeptide of claim 2.
31. (New) A fusion protein comprising the 15. A fusion protein comprising the isolated
isolated polypeptide of claim 20. polypeptide of claim 3.

Appendix 2

	Written Description Support
	in the Current Application	Written Description
	(Continuation of Application	Support in Application
Added Claim #	No. 10/695,499)	No. PCT/IB99/00103
Claims 18-31	Throughout the	Throughout the
	application and at least at	application and at least at
	the following citations:	the following citations:
	Page 3, lines 2-24;	Page 2, line 29 to page 3,
	Page 31, line 7 to page 34,	line 20;
	line 17;	Page 30, line 6 to page
	Page 52, lines 10-18;	33, line 11;
	Page 65, line 3 to page 70,	Page 50, lines 12-20;
	line 3.	Page 61, line 11 to page
		66, line 6.
Claims 23, 30,	Throughout the	Throughout the
and 31	application and at least at	application and at least at
	the following citations:	the following citations:
	Page 3, lines 24-27;	Page 3, lines 21-24;
	Page 9, line 26 to page 10,	Page 9, lines 11-18;
	line 4;	Page 20, line 6 to page
	Page 21, lines 1-22.	21, line 4.
Claims 25-27	Throughout the	Throughout the
	application and at least at	application and at least at
	the following citations:	the following citations:
	Page 3, lines 24-27;	Page 3, lines 17-20;
	Page 8, line 15 to page 28,	Page 8, line 1 to page 27,
	line 23.	line 25.

Appendix 3

Disclosure of Constructive Reductions to Practice within the Scope of the Interfering Subject Matter in Application No. GB 9800760.2, filed Jan. 14, 1998

Number of
Amino Acids	Location in ORF40 of	Location in SEQ 2
in Fragment	Application No. GB 9800760.2	of ‘419 Patent
25	Residues 85-109	Residues 127-151
16	Residues 111-126	Residues 153-168
98	Residues 131-228	Residues 173-270
16	Residues 230-245	Residues 272-287

Claims

1-17. (canceled)

18. An isolated polypeptide comprising a member selected from the group consisting of

(a) the amino acid sequence of SEQ ID NO: 4; and

(b) an immunogenic fragment of at least 15 contiguous amino acids of SEQ ID NO: 4, wherein the immunogenic fragment, when administered to a subject in a suitable composition which can include an adjuvant, or a suitable carrier coupled to the polypeptide, induces an antibody or T-cell meditated immune response that recognizes the isolated polypeptide SEQ ID NO: 4.

19. The isolated polypeptide of claim 18, wherein the polypeptide is according to (a).

20. The isolated polypeptide of claim 18, wherein the polypeptide is according to (b).

21. The isolated polypeptide of claim 18, wherein the immunogenic fragment of (b) comprises at least 20 contiguous amino acids of SEQ ID NO:4; wherein the immunogenic fragment, when administered to a subject in a suitable composition which can include an adjuvant, or a suitable carrier coupled to the polypeptide, induces an antibody or T-cell meditated immune response that recognizes the polypeptide SEQ ID NO: 4.

22. The isolated polypeptide of claim 18, wherein the isolated polypeptide consists of SEQ ID NO: 4.

23. A fusion protein comprising the isolated polypeptide of claim 18.

24. An immunogenic composition comprising the polypeptide of claim 18, and a pharmaceutically acceptable carrier.

25. The isolated polypeptide of claim 18, wherein the isolated polypeptide is a recombinant polypeptide.

26. The isolated polypeptide of claim 19, wherein the isolated polypeptide is a recombinant polypeptide.

27. The isolated polypeptide of claim 20, wherein the isolated polypeptide is a recombinant polypeptide.

28. An immunogenic composition comprising the isolated polypeptide of claim 19.

29. An immunogenic composition comprising the isolated polypeptide of claim 20.

30. A fusion protein comprising the isolated polypeptide of claim 19.

31. A fusion protein comprising the isolated polypeptide of claim 20.