Complete genome sequence of a simian immunodeficiency virus from a red-capped mangabey
The nucleotide sequence and deduced amino acid sequences of the complete genome of a simian immunodeficiency virus isolate from a red-capped mangabey are disclosed. The invention relates to the nucleic acids and peptides encoded by and/or derived from these sequences and their use in diagnostic methods and as immunogens.
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[0001] This is a divisional application of non-provisional application U.S. Ser. No. 09/206,551, filed Dec. 7, 1998.
FEDERAL FUNDING LEGEND BACKGROUND OF THE INVENTION[0003] 1. Field of the Invention
[0004] The present invention is in the field of virology. The invention relates to the nucleic acid sequence of the complete genome of the new simian immunodeficiency virus isolate from a red-capped mangabey, sivrcm, and nucleic acids derived therefrom. The invention also relates to peptides encoded by and/or derived from sivrcm nucleic acid sequences, and host cells containing these nucleic acid sequences and peptides. The invention also relates to diagnostic methods, kits and immunogens which employ the nucleic acids, peptides and/or host cells of the invention.
[0005] 2. Description of the Related Art
[0006] Phylogenetic analyses of simian immunodeficiency virus (SIV) isolates reveal that they belong to five distinct lineages of the lentivirus family of retroviruses (47). These five SIV lentiviral lineages form a distinct sub-group because primate viruses are more closely related to each other than to lentiviruses from non-primate hosts (47). Importantly, only simian species indigenous to the African continent are naturally infected (4, 13, 28, 35). Thus far, natural SIV infections in Africa have been documented in the sooty mangabey (SM) (Cercocebus torquatus atys) (sivsm strains), in Liberia (30), in Sierra Leone (4, 5), and the Ivory Coast (43); in all four sub-species of African green monkeys (agm) (Cercopithecus aethiops) (1, 21, 22, 25, 33, 34, 39) (sivagm strains), in eastern, central and western Africa; in the Sykes monkey (syk) (Cercopithecus mitis) (sivsyk strains) in Kenya (9); in the mandrill (mnd) (Mandrillus sphinx) (sivmmd strains) (38, 50) in Gabon; and in chimpanzees (cpz) (Pan troglodytes) (sivcpz strains) (19, 20, 41, 42) also from Gabon. Because these sivs and their simian hosts are highly divergent from each other and widely distributed across Africa, it is believed that the SIV family evolved and established itself in African simians long before acquired immunodeficiency syndrome (AIDS) appeared in humans (4, 15, 18, 19, 21, 30, 37, 47).
[0007] One common characteristic among all sivs is that none are associated with immunodeficiency or any other disease in their natural hosts (9, 13, 22, 28, 30, 35, 38). This finding is in marked contrast to AIDS, which occurs in humans and macaques infected with primate lentiviruses (2, 7, 8, 27, 35). This lack of disease in the natural SIV hosts may be an example of long-term evolution toward avirulence (16), which supports the hypothesis that SIV has infected African simians for a relatively long time.
[0008] Human AIDS is caused by two distinct primate lentiviruses, human immunodeficiency virus (HIV), types 1 and 2 (2, 7). Interestingly, the phylogeny of HIV is markedly different from SIV, because genetic analyses have shown that the human viruses do not represent separate sixth and seventh lineages of primate lentiviruses, but instead, are members of two of the five existing SIV lineages (37, 46). HIV-1 is in the HIV-1/sivcpz group (19, 51) and HIV-2 belongs to the HIV-2/sivsm family (18, 23). These phylogenetic data on the HIV-1 and HIV-2 lineages have long suggested separate simian origins for HIV-1 and HIV-2 (37, 46).
[0009] Molecular studies of naturally occurring sivsm and HIV-2 strains from rural West Africa have provided convincing evidence for a simian origin of HIV-2. A close genetic relationship has been established between the HIV-2 D and E sub-types and sivsm strains found in household pet sooty mangabeys in West Africa (4, 14, 15). Moreover, all six known subtypes of HIV-2, including a new subtype F (3), are found only within the natural range of SIV-infected sooty mangabeys in West Africa. No other area of Africa or of the world has all six known HIV-2 subtypes. Together, these data provide strong support for independent transmissions of sivsm from naturally infected sooty mangabeys to humans.
[0010] In contrast, there is much less information to support a simian origin for HIV-1. Although sivcpz is the closest relative to HIV-1, there are only a few isolates, thus raising questions as to the likely primate reservoir. Only three sivcpz strains have thus far been identified (20, 41, 42, 51). The first one was isolated from a single, household pet chimpanzee in Gabon and was not part of a primate research colony (42). An additional sivcpz strain was found in a captive chimpanzee which was wild caught in Zaire and thus likely infected in Africa (41, 51). Finally, PCR data suggested the existence of a third sivcpz strain, again from a wild caught chimpanzee from Gabon (20). Thus, although based on limited data, the hypothesis that HIV-1 is derived from members of a larger sivcpz lineage remains plausible. However, additional sivs within the HIV-1/sivcpz lineage must be found to better understand the origin of the HIV-1 family.
[0011] The present invention is based on the genetic characterization of a new SIV isolate from a red-capped mangabey (RCM), Cercocebus torquatus torquatus. This RCM was a household pet in Lambarene, Gabon, and was not part of a primate colony, a zoo, or a research facility. Analysis of the full-length sequence of the sivrcm indicates that this virus is related to SIV from sooty mangabeys, albeit very distantly. Its genome organization contains a vpx gene which is unique to members of the sivsm/HIV-2 lineage. There is also phylogenetic evidence that sivrcm is a recombinant.
[0012] The sivrcm sequence(s) described herein will permit the development of new serological screening assays for testing of sivrcm infection of humans. Although such infections have not yet been documented, it should be noted that viruses from a second mangabey species (sivs from sooty mangabeys) have crossed the species barrier and have yielded a new human AIDS virus (HIV-2). It is thus conceivable that sivrcm is similarly infecting humans in Gabon and Cameroon. To test this possibility, strain specific reagents (antigens, polypeptides, etc.) Are required to test for sivrcm specific antibodies in people as a sign of viral infection. Such strain specific antigens can now be designed on the basis of the sivrcm sequence(s) described herein.
[0013] If evidence is found that humans in Africa are infected with sivrcm (regardless whether this infection is pathogenic or not), then new screening assays for the world's blood supply will have to be developed that specifically detect anti-sivrcm antibodies or sivrcm nucleic acids. Sivrcm sequences are necessary to design such strain-specific tests. Additionally, the new sequences will permit the development of assays for screening of primates, such as those in the wild, in zoos, and in research facilities, for sivrcm.
SUMMARY OF THE INVENTION[0014] The present invention pertains to the isolation and characterization of the genomic sequence of sivrcm, a new simian immunodeficiency virus identified from a Gabonese red-capped mangabey (RCM), and nucleic acids derived therefrom. In particular, the present invention relates to nucleic acids comprising the complete genomic sequence of sivrcm, as well as nucleic acids comprising the complementary (or antisense) sequence of the genomic sequence of sivrcm, and nucleic acids derived therefrom.
[0015] The invention also relates to vectors comprising the nucleic acid genomic sequence of sivrcm, as well as nucleic acids comprising the complementary (or antisense) sequence of the genomic sequence of sivrcm, and nucleic acids derived therefrom.
[0016] The invention also relates to cultured host cells comprising the nucleic acid genomic sequence of sivrcm, as well as nucleic acids comprising the complementary (or antisense) sequence of the genomic sequence of sivrcm, and nucleic acids derived therefrom.
[0017] The invention also relates to host cells containing vectors comprising the genomic sequence of sivrcm, as well as nucleic acids comprising the complementary (or antisense) sequence of the genomic sequence of sivrcm, and nucleic acids derived therefrom.
[0018] The invention also relates to synthetic or recombinant polypeptides encoded by or derived from the nucleic acid sequence of the genome of sivrcm, and fragments thereof.
[0019] The invention also relates to methods for producing the polypeptides of the invention in culture using the sivrcm virus or nucleic acids derived therefrom, including recombinant methods for producing the polypeptides of the invention.
[0020] The invention further relates to methods of using the polypeptides of the invention as immunogens to stimulate an immune response in a mammal, such as the production of antibodies, or the generation of cytotoxic or helper T-lymphocytes.
[0021] The invention also relates to methods of using the polypeptides of the invention to detect antibodies which immunologically react with the sivrcm virion and/or its encoded polypeptides, in a mammal or in a biological sample.
[0022] The invention also relates to kits for the detection of antibodies specific for sivrcm in a biological sample where said kit contains at least one polypeptide encoded by or derived from the sivrcm nucleic acid sequences of the invention.
[0023] The invention also relates to antibodies which immunologically react with the sivrcm virion and/or its encoded polypeptides.
[0024] The invention also relates to methods of detecting sivrcm virion and/or its encoded polypeptides, or fragments thereof, using the antibodies of the invention.
[0025] The invention also relates to kits for detecting sivrcm virion, and/or its encoded polypeptides, wherein the kit comprises at least one antibody of the invention.
[0026] The invention also relates to a method for detecting the presence of sivrcm virus in a mammal or a biological sample, said method comprising analyzing the DNA or RNA of a mammal or a sample for the presence of the rnas, cdnas or genomic dnas which will hybridize to a nucleic acid derived from sivrcm. Usually, when a completely complementary probe is used, high stringency conditions are desirable in order to prevent false positives. However, conditions of high stringency should only be used if the probes are complementary to target regions which lack heterogeneity. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, length of time, and concentration of formamide, if any. The nucleic acid sequences used in probes should be unique to sivrcm, i.e., The nucleic acid sequences should be absent from individual mammals not infected with sivrcm.
[0027] The invention also provides diagnostic kits for the detection of sivrcm in a mammal using the nucleic acids of the invention. In one embodiment, the kit comprises nucleic acids having sequences useful as hybridization probes in determining the presence or absence of sivrcm RNA, cdna or genomic DNA. In another embodiment, the kit comprises nucleic acids having sequences useful as primers for reverse-transcription polymerase chain reaction (RT-PCR) analysis of RNA for the presence of sivrcm in a biological sample.
[0028] The invention further relates to isolated and substantially purified nucleic acids, polypeptides and/or antibodies of the invention.
[0029] The invention further relates to compositions comprising one or more of the nucleic acids, polypeptides and/or antibodies of the invention.
BRIEF DESCRIPTION OF THE FIGURES[0030] FIG. 1. PCR primer pairs and sequencing strategy.
[0031] FIG. 2. Alignment of amino acid sequences of env proteins of sivrcm and other primate lentiviruses. The homologies among the sequences are indicated by dashes. Sequences of amino acids which are uniquely present in the various polypeptides (as compared to the corresponding amino acids of the sivrcm) are indicated by letters, i.e., The sequences themselves.
[0032] FIG. 3. Genomic organization of sivrcm. Schematic diagram of the three possible reading frames of the sivrcm genome. The location of stop codons are indicated by vertical lines, and the locations of the sivrcm genes are indicated.
[0033] FIG. 4. Duplication of the TAR stemloop structure in the sivrcm LTR.
[0034] FIG. 5. Phylogenetic analysis of sivrcm. (A) gag; (B) pol (5′ end); (C) env; and (D) nef genes. Phylogenies indicate discordant branching orders which strongly suggest a recombinant sivrcm genome.
[0035] FIG. 6. Alignment of the putative polypeptide products of the extended rev orfs in sivsm and sivrcm. Asterisks denote stop codons. Conservative amino acid changes are indicated as a colon (:); a vertical line depicts the position of the splice donor.
[0036] FIG. 7. Amino acid sequence alignment of the “extended rev ORF” in members of the HIV-2/sivsm/sivmac group. The vertical line indicates the position of the splice donor, usually used to express the spliced versions of tat and rev, respectively. Stop codons are indicated as asterisks. All sivsm/sivmac strains as well as HIV-2/FO784 have an uninterrupted extended rev ORF.
[0037] FIG. 8. Nucelotide sequence of the sivrcm genome (SEQ ID NO:1)
[0038] FIG. 9. Deduced amino acid sequence of the sivrcm Gag protein.
[0039] FIG. 10. Deduced amino acid sequence of the sivrcm Pol protein.
[0040] FIG. 11. Deduced amino acid sequence of the sivrcm Vif protein.
[0041] FIG. 12. Deduced amino acid sequence of the sivrcm Vpx protein.
[0042] FIG. 13. Deduced amino acid sequence of the sivrcm Vpr protein.
[0043] FIG. 14. Deduced amino acid sequence of the sivrcm Tat protein.
[0044] FIG. 15. Deduced amino acid sequence of the sivrcm Rev protein.
[0045] FIG. 16. Deduced amino acid sequence of the sivrcm Env protein.
[0046] FIG. 17. Deduced amino acid sequence of the sivrcm Nef protein.
[0047] FIG. 18. Partial nucleotide sequence of sivrcm gag gene and deduced amino acid sequence.
[0048] FIG. 19. Partial nucleotide sequence of sivrcm pol gene and deduced amino acid sequence.
DETAILED DESCRIPTION OF THE INVENTION[0049] The present invention relates to the determination of the nucleic acid sequence of the complete genome of sivrcm, an isolate of simian immunodeficiency virus identified from a Gabonese red-capped mangabey (RCM) and nucleic acids derived therefrom. The nucleotide sequence of the sivrcm is shown in the sequence listing as SEQ ID NO: 1.
[0050] The phrase “derived from” is used throughout the specification and claims with respect to nucleic acids to describe nucleic acid sequences which correspond to a region of the designated nucleotide sequence. Preferably, the sequence of the region from which the nucleic acid is derived is, or is complementary to, a sequence which is unique to the sivrcm genome. Whether or not a sequence is unique to the genome of sivrcm can be determined by techniques known to those of skill in the art. For example, the sequence can be compared to sequences in databanks, e.g., Genbank, to determine whether it is present in the uninfected host or other organisms. The sequence can also be compared to the known sequences of other viral agents, including other retroviruses. The correspondence or non-correspondence of the derived sequence to other sequences can also be determined by hybridization under the appropriate stringency conditions. Hybridization techniques for determining the complementarity of nucleic acid sequences are well known in the art. In addition, mismatches of duplex polynucleotides formed by hybridization can be determined by known techniques, including for example, digestion with a nuclease such as S1 that specifically digests single-stranded areas in duplex polynucleotides.
[0051] Regions of the viral genome from which nucleic acid sequences may be derived include, but are not limited to, regions encoding specific epitopes as well as non-transcribed and non-translated sequences. Preferably, the epitope is unique to a polypeptide encoded by the sivrcm genome. The uniqueness of the epitope may be determined by its degree of immunological cross-reactivity with other SIV viruses. Methods for determining immunological cross-reactivity are known in the art, e.g., Radioimmunoassay and ELISA and other assays mentioned herein. The uniqueness of an epitope can also be determined by computer searches of known databases, e.g., For the polynucleotide sequences which encode the epitope, and by amino acid sequence comparisons with other known proteins.
[0052] The derived nucleic acid is not necessarily physically derived from the nucleotide sequence shown, but may be generated in any manner, including for example, chemical synthesis or DNA replication or reverse transcription or transcription, which are based on the information provided by the sequence of bases in the region(s) from which the nucleic acid is derived. The derived nucleic acid is comprised of at least 6-12 bases, more preferably at least 15-19 bases, more preferably at least 30 bases. The derived nucleic acid may also be larger, e.g., At least 100 bases in length, depending on the desired use of the nucleic acid. In addition, regions or combinations of regions corresponding to that of the designated sequence may be modified in ways known in the art to be consistent with an intended use. The derived nucleic acid may be a polynucleotide or polynucleotide analog.
[0053] The term “recombinant nucleotide” or “recombinant nucleic acid” as used herein intends a nucleic acid of genomic, cdna, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the nucleic acid with which it is associated in nature; and/or (2) is linked to a nucleic acid other than that to which it is linked in nature.
[0054] The term “polynucleotide” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified, for example, by methylation and/or by capping, and unmodified forms of the polynucleotide.
[0055] The present invention relates to nucleic acids having the entire genomic sequence of the sivrcm isolate as shown in SEQ ID NO:1, as well as fragments (or partial sequences) thereof. The invention also relates to nucleic acids having complementary (or antisense) sequences of the sequence shown in SEQ ID NO:1, as well as fragments (or partial sequences) thereof. Partial sequences may be obtained by various methods, including restriction digestion of the complete sequence of sivrcm, PCR amplification, and direct synthesis. Partial sequences may be all or part of the LTR and/or other untranslated regions of the sivrcm genome, and/or all or part of the genes encoding the Gag, Pol, Vif, Vpr, Env, Tat, Rev, Nef and Vpx proteins and/or complementary (or antisense) sequences thereof. Nucleic acids of the invention also include cdna, mrna, and other nucleic acids derived from the sivrcm genomic sequence. Sequences of the ltrs and of the genes encoding Gag, Pol, Vif, Vpr, Env, Tat, Rev, Nef and Vpx are identified in FIGS. 9 to 17. Partial sequences of sivrcm gag and pol genes and encoded amino acid sequence are shown in FIGS. 18 and 19. These latter sequences are available in genbank, having Accession Numbers AF028608 and AF028607, respectively. Minor sequence variations between SEQ ID NO:1 and the pol gene of FIG. 19 are due to the fact that this partial pol gene sequence (in addition to the partial gag gene sequence) was obtained from a different clone of sivrcm than that represented by SEQ ID NO: 1.
[0056] The nucleic acids of the invention may be present in vectors or host cells in tissue culture or other media. The nucleic acids of the invention may also be isolated and substantially purified, by methods known in the art.
[0057] Nucleic acids of about 17 bases to about 35 bases in length are particularly preferred for use as primers in PCR amplification (see, e.g., The primers gag A and gag B (25mer and 32mer respectively)). Nucleic acids of about 14 to about 25 bases in length are particularly preferred for use in nucleotide arrays. (See, e.g., Ref. 80, which uses 20 to 25 mers).
[0058] The present invention also relates to vectors and host cells comprising the nucleic acids of the invention.
[0059] The present invention also relates to compositions comprising one or more of the nucleic acids, vectors, and/or host cells of the invention.
[0060] The present invention further relates to methods of using the nucleic acids, vectors, and/or host cells of the invention, and/or compositions thereof. For example, the invention relates to the use of nucleic acids of the invention as diagnostic agents to detect the presence or absence of sivrcm in a sample.
[0061] The present invention also relates to a method for detecting the presence of sivrcm in a mammal using the nucleic acids of this invention.
[0062] In one embodiment, the detection method involves analyzing DNA of a mammal suspected of harboring sivrcm. DNA can be isolated by methods well known in the art.
[0063] The methods for analyzing the DNA for the presence of sivrcm include Southern blotting (63), dot and slot hybridization (60), and nucleotide arrays (see, e.g., U.S. Pat. No. 5,445,934 and U.S. Pat. No. 5,733,729).
[0064] The nucleic acid probes used in the detection methods set forth above are derived from the nucleic acid sequence shown in SEQ ID NO: 1. The size of such probes is at least 10-12 bases long, more usually at least about 19 bases long, more usually from about 200 to about 500 bases, and often exceeding about 1000 bases.
[0065] The nucleic acid probes of this invention may be DNA or RNA. Nucleic acids can be synthesized using any of the known methods of nucleotide synthesis (see, e.g., Refs. 54, 55, 58), or they can be isolated fragments of naturally occurring or cloned DNA. In addition, those skilled in the art would be aware that nucleotides can be synthesized by automated instruments sold by a variety of manufacturers or can be commercially custom ordered and prepared. The probes of this invention may also be nucleotide analogs, such as nucleotides linked by phosphodiester, phosphorothiodiester, methylphosphonodiester, or methylphosphonothiodiester moieties (67) and peptide nucleic acids (pnas), in which the sugar-phosphate backbone of the polynucleotide is replaced with a polyamide or “pseudopeptide” backbone (68).
[0066] The nucleic acid probes can be labeled using methods known to one skilled in the art. Such labeling techniques can include radioactive labels, biotin, avidin, enzymes and fluorescent molecules (62).
[0067] The nucleic acid probes used in the detection methods set forth above are derived from sequences substantially homologous to the sequence shown in SEQ ID NO:1, or its complementary sequence. By “substantially homologous”, as used throughout the specification and claims to describe the nucleic acid sequences of the present invention, is meant a high level of homology between the nucleic acid sequence and the sequence of SEQ ID NO:1, or its complementary sequence. Preferably, the level of homology is in excess of 80%, more preferably in excess of 90%, with a preferred nucleic acid sequence being in excess of 95% homologous with a portion of SEQ ID NO:1, or its complement. The size of such probes is usually at least 20 nucleotides, more usually from about 200 to 500 nucleotides, and often exceeding 1000 nucleotides.
[0068] Although complete complementarity is not necessary, it is preferred that the probes are made completely complementary to the corresponding portion of the sivrcm genome, mrna or cdna target.
[0069] The probes can be packaged into diagnostic kits. Diagnostic kits may include ingredients for labelling and other reagents and materials needed for the particular hybridization protocol in addition to the probes.
[0070] In another embodiment of the invention, the detection method comprises analyzing the RNA of a mammal for the presence of sivrcm. RNA can be isolated by methods well known in the art.
[0071] The methods for analyzing the RNA for the presence of sivrcm include Northern blotting (66), dot and slot hybridization, filter hybridization (57), rnase protection (62), and reverse-transcription polymerase chain reaction (RT-PCR) (65). A preferred method is RT-PCR. In this method, the RNA can be reverse transcribed to first strand cdna using a nucleic acid primer or primers derived from the nucleotide sequence shown in SEQ ID NO:1. Once the cdnas are synthesized, PCR amplification is carried out using pairs of primers designed to hybridize with sequences in the genome of sivrcm which are an appropriate distance apart (at least about 50 bases) to permit amplification of the cdna and subsequent detection of the amplification product. Each primer of a pair is a single-stranded nucleic acid of about 20 to about 60 bases in length where one primer (the “upstream” primer) is complementary to the original RNA and the second primer (the “downstream” primer) is complementary to the first strand of cdna generated by reverse transcriptions of the RNA. The target sequence is generally about 100 to about 300 bases in length but can be as large as 500-1500 bases or more, e.g., 9,000 bases. Optimization of the amplification reaction to obtain sufficiently specific hybridization to the sivrcm nucleotide sequence is well within the skill in the art and is preferably achieved by adjusting the annealing temperature.
[0072] The amplification products of PCR can be detected either directly or indirectly. In one embodiment, direct detection of the amplification products is carried out via labeling of primer pairs. Labels suitable for labeling the primers of the present invention are known to one skilled in the art and include radioactive labels, biotin, avidin, enzymes and fluorescent molecules. The desired labels can be incorporated into the primers prior to performing the amplification reaction. Alternatively, the desired labels can be incorporated into the primer extension products during the amplification reaction in the form of one or more labeled dntps. In one embodiment of the present invention, the labeled amplified PCR products can be detected by agarose gel electrophoresis followed by ethidium bromide staining and visualization under ultraviolet light or via direct sequencing of the PCR-products. The labeled amplified PCR products can also be detected by binding to immobilized oligonucleotide arrays.
[0073] In yet another embodiment, unlabelled amplification products can be detected via hybridization with labeled nucleic acid probes in methods known to one skilled in the art, such as dot or slot blot hybridization or filter hybridization.
[0074] The invention also relates to methods of using these nucleic acids to produce polypeptides in vitro or in vivo. In one embodiment of the invention, a recombinant method of making a polypeptide of the invention comprises:
[0075] a) preparing of a nucleic acid capable of directing a host cell to produce a polypeptide encoded by the sivrcm genome;
[0076] b) cloning the nucleic acid into a vector capable of being transferred into and replicated in a host cell, such vector containing operational elements for expressing the nucleic acid, if necessary;
[0077] c) transferring the vector containing the nucleic acid and operational elements into a host cell capable of expressing the polypeptide;
[0078] d) growing the host under conditions appropriate for expression of the polypeptide; and
[0079] d) harvesting the polypeptide.
[0080] The present invention also relates to non-recombinant methods of making the polypeptides and nucleic acids of the invention. In addition to synthetic methods, the non-recombinant methods involve culturing sivrcm in cell lines, preferably in uninfected human peripheral blood mononuclear cells, under conditions appropriate for expression of the polypeptides and nucleic acids. This invention thus also relates to the polypeptides and nucleic acids produced by the virus in cell culture. The polypeptides and nucleic acids may be isolated and purified by methods known in the art.
[0081] The vectors contemplated for use in the present invention include any vectors into which a nucleic acid sequence as described above can be inserted, along with any preferred or required operational elements, and which vector can then be subsequently transferred into a host cell and, preferably, replicated in such cell. Preferred vectors are those whose restriction sites have been well documented and which contain the operational elements preferred or required for transcription of the nucleic acid sequence. Vectors may also be used to prepare large amounts of nucleic acids of the invention, which may be used, e.g., To prepare probes or other nucleic acid constructs.
[0082] When expression of a polypeptide is desired, the “operational elements” as discussed herein include at least one promoter sequence capable of initiating transcription of the nucleic acid sequence, at least one leader sequence, at least one terminator codon and/or termination signal, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the vector nucleic acid. In particular, it is contemplated that such vectors will preferably contain at least one origin of replication recognized by the host cell along with at least one selectable marker.
[0083] Preferred expression vectors of this invention are those which function in bacterial and/or eukaryotic cells. Examples of vectors which function in eukaryotic cells include, but are not limited to Venezuelan equine encephalitis virus vectors, simian virus vectors, vaccinia virus vectors, adenovirus vectors, herpes virus vectors, or vectors based on retroviruses, such as murine leukemia virus, or HIV or other lentivirus (76).
[0084] The selected expression vector may be transfected into a suitable bacterial or eukaryotic cell system for purposes of expressing the recombinant polypeptide. Eukaryotic cell systems include but are not limited to cell lines such as hela, COS-1, 293T, MRC-5, or CV-1 cells. Primary human cells, such as lymph node cells, macrophages, etc., Are also useful in practicing the invention.
[0085] The expressed polypeptides may be detected directly by methods known in the art including, but not limited to, Coomassie blue staining and Western blotting or indirectly, such as in detection of the expression product of a reporter gene, such as luciferase.
[0086] In another embodiment of the invention, the method comprises administering a composition comprising a vector comprising a nucleic acid of the invention to a mammal to produce a polypeptide in vivo.
[0087] The present invention also relates to polypeptides encoded by and/or derived from the nucleotide sequences of this invention. These polypeptides may be natural, synthetic or produced by recombinant methods. Polypeptides can be obtained as a crude lysate or can be purified by standard protein purification procedures known in the art which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity and immunoaffinity chromatography. The polypeptides may be purified by passage through a column containing a resin which has bound thereto antibodies specific for an open reading frame (ORF) polypeptide. The present invention also relates to compositions comprising one or more of the polypeptides of the invention.
[0088] A polypeptide or amino acid sequence derived from a designated nucleic acid sequence refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded by the sequence, or a portion thereof wherein the portion consists of at least 6-8 amino acids, and more preferably at least 10 amino acids, and more preferably at least 11-15 amino acids, and most preferably at least 30 amino acids or which is immunologically cross-reactive with a polypeptide encoded by the sequence. The polypeptide may also be larger, e.g., At least 100 amino acids in length, depending on the desired use of the polypeptide. Polypeptides from the V3-loop region and the “crown” of gp41 of Env are particularly preferred.
[0089] A recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence; it may be generated in any manner, including for example, chemical synthesis, or expression of a recombinant expression system, or isolation from sivrcm.
[0090] It should be noted that the nucleotide sequences described herein represent one embodiment of the present invention. Due to the degeneracy of the genetic code, it is to be understood that numerous choices of nucleotides may be made that will lead to a sequence capable of directing production of the polypeptides set forth above. As such, nucleic acid sequences which are functionally equivalent to the sequences described herein are intended to be encompassed within the present invention. For example, preferred codons which are appropriate to the host cell may be used (see, e.g., WO 98/34640), or the sequence may be modified to reduce the effect of any inhibitory/instability sequences and to provide for Rev-independent gene expression (77).
[0091] The polypeptides of this invention consist of at least 6-12 amino acids, more preferably at least 13-18 amino acids, even more preferably at least 19-24 amino acids and most preferably at least 25-30 amino acids encoded by, or otherwise derived from, the sivrcm genomic sequence.
[0092] The present invention further relates to the use of polypeptides of the invention as diagnostic agents. In one embodiment, the polypeptides of the invention can be used in immunoassays for detecting the presence of antibodies against sivrcm in a mammal and for diagnosing the presence of sivrcm infection in a mammal. For the purposes of the present invention, “mammal” as used throughout the specification and claims, includes, but is not limited to humans, chimpanzees, mangabeys, other primates and the like.
[0093] In a preferred embodiment, test serum is reacted with a solid phase reagent having a surface-bound polypeptide of this invention as an antigen. The solid surface reagent can be prepared by known techniques for attaching polypeptides to solid support material. These attachment methods include non-specific adsorption of the polypeptide to the support or covalent attachment of the polypeptide to a reactive group on the support. After reaction of the antigen with an antibody against any one of the viruses of this invention in the serum, unbound serum components are removed by washing and the antigen-antibody complex is reacted with a secondary antibody such as labeled anti-human antibody. The label may be an enzyme which is detected by incubating the solid support in the presence of a suitable fluorimetric or calorimetric reagent. Other detectable labels may also be used, such as radiolabels or colloidal gold, and the like.
[0094] Immunoassays of the present invention may be a radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay and the like. Standard techniques for ELISA are well known in the art. Such assays may be a direct, indirect, competitive, or noncompetitive immunoassay as described in the art (see, e.g., Ref. 61). Biological samples appropriate for such detection assays include, but are not limited to serum, liver, saliva, lymphocytes or other mononuclear cells.
[0095] Polypeptides of the invention may be prepared in the form of a kit, alone, or in combinations with other reagents such as secondary antibodies, for use in immunoassays. In yet another embodiment, the polypeptides of the invention can be used as immunogens to raise antibodies and/or stimulate cellular immunity in a mammal.
[0096] The immunogen may be a partially or substantially purified peptide. Alternatively, the immunogen may be a cell, cell lysate from cells transfected with a recombinant expression vector, or a culture supernatant containing the expressed polypeptide. The immunogen may comprise one or more structural proteins, and/or one or more non-structural proteins of sivrcm, or a mixture thereof.
[0097] The effective amount of polypeptide per unit dose sufficient to induce an immune response depends, among other things, on the species of mammal inoculated, the body weight of the mammal and the chosen inoculation regimen, as well as the presence or absence of an adjuvant, as is well known in the art. Inocula typically contain polypeptide concentrations of about 1 microgram to about 50 milligrams per inoculation (dose), preferably about 10 micrograms to about 10 milligrams per dose, most preferably about 100 micrograms to about 5 milligrams per dose.
[0098] The term “unit dose” as it pertains to the inocula refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of active material (polypeptide) calculated to produce the desired immunogenic effect in association with the required diluent. Inocula are typically prepared as a solution in a physiologically acceptable carrier such as saline, phosphate-buffered saline and the like to form an aqueous pharmaceutical composition. The route of inoculation of the polypeptides of the invention is typically parenteral and is preferably intramuscular, subcutaneous and the like. The dose is administered at least once. In order to increase the antibody level, at least one booster dose may be administered after the initial injection, preferably at about 4 to 6 weeks after the first dose. Subsequent doses may be administered as indicated.
[0099] To monitor the antibody response of individuals administered the compositions of the invention, antibody titers may be determined. In most instances it will be sufficient to assess the antibody titer in serum or plasma obtained from such an individual. Decisions as to whether to administer booster inoculations or to change the amount of the composition administered to the individual may be at least partially based on the titer. The titer may be based on an immunobinding assay which measures the concentration of antibodies in the serum which bind to a specific antigen. The ability to neutralize in vitro and in vivo biological effects of sivrcm may also be assessed to determine the effectiveness of the immunization.
[0100] For all therapeutic, prophylactic and diagnostic uses, the polypeptide of the invention, alone or linked to a carrier, as well as antibodies and other necessary reagents and appropriate devices and accessories may be provided in kit form so as to be readily available and easily used. Where immunoassays are involved, such kits may contain a solid support, such as a membrane (e.g., Nitrocellulose), a bead, sphere, test tube, microtiter well, rod, and so forth, to which a receptor such as an antibody specific for the target molecule will bind. Such kits can also include a second receptor, such as a labelled antibody. Such kits can be used for sandwich assays. Kits for competitive assays are also envisioned.
[0101] The immunogens of this invention can also be generated by the direct administration of nucleic acids of this invention to a subject. DNA-based vaccination has been shown to stimulate humoral and cellular responses to HIV-1 antigens in mice (69-72) and macaques (72, 73). More recent studies in infected chimpanzees have shown a possible application of this strategy in HIV-1-infected humans: DNA vaccination of HIV-1-infected chimpanzees with a construct that drives expression of HIV-1 env and rev appeared well-tolerated, and immunized animals demonstrated a boost in antibody response followed by a >1 log decrease in their virus loads (74). A DNA-based vaccine containing HIV-1 env and rev genes was injected into HIV-infected human patients in three doses (30, 100 or 300 micrograms) at 10-week intervals. Increased antibodies against gp120 were observed in the 100 and 300 &mgr;g groups. Increases were also noted in cytotoxic T lymphocyte (CTL) activity against gp160-bearing targets and in lymphocyte proliferative activity (78, 79).). DNA-based vaccines containing HIV gag genes, with modification of the viral nucleotide sequence to incorporate host-preferred codons (see, e.g., WO 98/34640), and/or to reduce the effect of inhibitory/instability sequences (see, e.g., Ref. 77), have likewise been described.
[0102] Therefore, it is anticipated that the direct injection of RNA or DNA vectors of this invention encoding viral antigen can be used for endogenous expression of the antigen to generate the viral antigen for presentation to the immune system without the need for self-replicating agents or adjuvants, resulting in the generation of antigen-specific ctls and protection from a subsequent challenge with a homologous or heterologous strain of virus.
[0103] Ctls in both mice and humans are capable of recognizing epitopes derived from conserved internal viral proteins and are thought to be important in the immune response against viruses. By recognition of epitopes from conserved viral proteins, ctls may provide cross-strain protection. Ctls specific for conserved viral antigens can respond to different strains of virus, in contrast to antibodies, which are generally strain-specific.
[0104] Thus, direct injection of RNA or DNA encoding the viral antigen has the advantage of being without some of the limitations of direct peptide delivery or viral vectors (see, e.g., Ref. 81 and the discussions and references therein). Furthermore, the generation of high-titer antibodies to expressed proteins after injection of DNA indicates that this may be a facile and effective means of making antibody-based vaccines targeted towards conserved or non-conserved antigens, either separately or in combination with CTL vaccines targeted towards conserved antigens. These may also be used with traditional peptide vaccines, for the generation of combination vaccines. Furthermore, because protein expression is maintained after DNA injection, the persistence of B and T cell memory may be enhanced, thereby engendering long-lived humoral and cell-mediated immunity.
[0105] Nucleic acids encoding a sivrcm polypeptide of this invention can be introduced into animals or humans in a physiologically or pharmaceutically acceptable carrier using one of several techniques such as injection of DNA directly into human tissues; electroporation or transfection of the DNA into primary human cells in culture (ex vivo), selection of cells for desired properties and reintroduction of such cells into the body, (said selection can be for the successful homologous recombination of the incoming DNA to an appropriate preselected genomic region); generation of infectious particles containing the sivrcm gag and/or other sivrcm genes, infection of cells ex vivo and reintroduction of such cells into the body; or direct infection by said particles in vivo. Substantial levels of polypeptide will be produced leading to an efficient stimulation of the immune system.
[0106] Also envisioned are therapies based upon vectors, such as viral vectors containing nucleic acid sequences coding for the polypeptides described herein. These molecules, developed so that they do not provoke a pathological effect, will stimulate the immune system to respond to the polypeptides.
[0107] The effective amount of nucleic acid immunogen per unit dose to induce an immune response depends, among other things, on the species of mammal inoculated, the body weight of the mammal and the chosen inoculation regimen, as is well known in the art. Inocula typically contain nucleic acid concentrations of about 1 microgram to about 50 milligrams per inoculation (dose), preferably about 10 micrograms to about 10 milligrams per dose, most preferably about 100 micrograms to about 5 milligrams per dose.
[0108] Immunization can be conducted by conventional methods. For example, the immunogen can be used in a suitable diluent such as saline or water, or complete or incomplete adjuvants. Further, the immunogen may or may not be bound to a carrier. While it is possible for the immunogen to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation.
[0109] The formulations of the present invention, both for veterinary and for human use, comprise an immunogen as described above, together with one or more physiologically or pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well-known in the pharmaceutical art.
[0110] The immunogen can be administered by any route appropriate for antibody production such as intravenous, intraperitoneal, intramuscular, subcutaneous, and the like. The immunogen may be administered once or at periodic intervals until a significant titer of anti-sivrcm antibody is produced. The antibody may be detected in the serum using an immunoassay. The host serum or plasma may be collected following an appropriate time interval to prove a composition comprising antibodies reactive with the sivrcm virus particle or encoded polypeptide. The gamma globulin fraction or the igg antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art.
[0111] In addition to its use to raise antibodies, the administration of the immunogens of the present invention may be for use as a vaccine for either a prophylactic or therapeutic purpose. When provided prophylactically, a vaccine(s) of the invention is provided in advance of any exposure to sivrcm or in advance of any symptoms due to sivrcm infection. The prophylactic administration of a vaccine(s) of the invention serves to prevent or attenuate any subsequent infection of sivrcm in a mammal. When provided therapeutically, a vaccine(s) of the invention is provided at (or shortly after) the onset of infection or at the onset of any symptom of infection or any disease or deleterious effects caused by sivrcm. The therapeutic administration of the vaccine(s) serves to attenuate the infection or disease. The vaccine(s) of the present invention may, thus, be provided either prior to the anticipated exposure to sivrcm or after the initiation of infection.
[0112] In another embodiment, the polypeptides of the invention can be used to prepare antibodies against sivrcm epitopes that are useful in diagnosis. The term “antibodies” is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fab′, F(ab′)2 and F(v) as well as chimeric antibody molecules.
[0113] An antibody of the present invention is typically produced by immunizing a mammal with an immunogen or vaccine of the invention. In one embodiment, the immunogen or vaccine contains one or more polypeptides of the invention, or a structurally and/or antigenically related molecule, to induce, in the mammal, antibody molecules having immunospecificity for the immunizing peptide or peptides. The peptide(s) or related molecule(s) may be monomeric, polymeric, conjugated to a carrier, and/or administered in the presence of an adjuvant. In another embodiment, the immunogen or vaccine contains one or more nucleic acids encoding one or more polypeptides of the invention, or one or more nucleic acids encoding structurally and/or antigenically related molecules, to induce, in the mammal, the production of the immunizing peptide or peptides. The antibody molecules may then be collected from the mammal if they are to be used in immunoassays or for providing passive immunity.
[0114] The antibody molecules of the present invention may be polyclonal or monoclonal. Monoclonal antibodies may be produced by methods known in the art. Portions of immunoglobulin molecules may also be produced by methods known in the art. The antibody of the present invention may be contained in various carriers or media, including blood, plasma, serum (e.g., Fractionated or unfractionated serum), hybridoma supernatants and the like. Alternatively, the antibody of the present invention is isolated to the extent desired by well known techniques such as, for example, by using DEAE SEPHADEX, or affinity chromatography. The antibodies may be purified so as to obtain specific classes or subclasses of antibody such as igm, igg, iga, igg1, igg2, igg3, igg4 and the like. Antibody of the igg class are preferred for purposes of passive protection.
[0115] The presence of the antibodies of the present invention, either polyclonal or monoclonal, can be determined by, but are not limited to, the various immunoassays described above. The antibodies of the present invention have a number of diagnostic and therapeutic uses. The antibodies can be used as an in vitro diagnostic agent to test for the presence of sivrcm in biological samples in standard immunoassay protocols. Preferably, the assays which use the antibodies to detect the presence of sivrcm in a sample involve contacting the sample with at least one of the antibodies under conditions which will allow the formation of an immunological complex between the antibody and the sivrcm antigen that may be present in the sample.
[0116] The formation of an immunological complex if any, indicating the presence of sivrcm in the sample, is then detected and measured by suitable means. Such assays include, but are not limited to, radioimmunoassays (RIA), ELISA, indirect immunofluorescence assay, Western blot and the like. The antibodies may be labeled or unlabeled depending on the type of assay used. Labels which may be coupled to the antibodies include those known in the art and include, but are not limited to, enzymes, radionucleotides, fluorogenic and chromogenic substrates, cofactors, biotin/avidin, colloidal gold and magnetic particles. Modification of the antibodies allows for coupling by any known means to carrier proteins or peptides or to known supports, for example, polystyrene or polyvinyl microtiter plates, glass tubes or glass beads and chromatographic supports, such as paper, cellulose and cellulose derivatives, and silica.
[0117] Such assays may be, for example, of direct format (where the labelled first antibody reacts with the antigen), an indirect format (where a labelled second antibody reacts with the first antibody), a competitive format (such as the addition of a labelled antigen), or a sandwich format (where both labelled and unlabelled antibody are utilized), as well as other formats described in the art. In one such assay, the biological sample is contacted with antibodies of the present invention and a labeled second antibody is used to detect the presence of sivrcm, to which the antibodies are bound.
[0118] The antibodies of the present invention are also useful as a means of enhancing the immune response. The antibodies may be administered with a physiologically or pharmaceutically acceptable carrier or vehicle therefor. A physiologically acceptable carrier is one that does not cause an adverse physical reaction upon administration and one in which the antibodies are sufficiently soluble and retain their activity to deliver a therapeutically effective amount of the compound. The therapeutically effective amount and method of administration of the antibodies may vary based on the individual patient, the indication being treated and other criteria evident to one of ordinary skill in the art. A therapeutically effective amount of the antibodies is one sufficient to reduce the level of infection by one or more of the viruses of this invention or attenuate any dysfunction caused by viral infection without causing significant side effects such as non-specific T cell lysis or organ damage.
[0119] The route(s) of administration useful in a particular application are apparent to one or ordinary skill in the art. Routes of administration of the antibodies include, but are not limited to, parenteral, and direct injection into an affected site. Parenteral routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal and subcutaneous.
[0120] The present invention includes compositions of the antibodies described above, suitable for parenteral administration including, but not limited to, pharmaceutically acceptable sterile isotonic solutions. Such solutions include, but are not limited to, saline and phosphate buffered saline for intravenous, intramuscular, intraperitoneal, or subcutaneous injection, or direct injection into a joint or other area.
[0121] Antibodies for use to elicit passive immunity in humans are preferably obtained from other humans previously inoculated with pharmaceutical compositions comprising one or more of the polypeptides of the invention. Alternatively, antibodies derived from other species may also be used. Such antibodies used in therapeutics suffer from several drawbacks such as a limited half-life and propensity to elicit an immune response. Several methods are available to overcome these drawbacks. Antibodies made by these methods are encompassed by the present invention and are included herein. One such method is the “humanizing” of non-human antibodies by cloning the gene segment encoding the antigen binding region of the antibody to the human gene segments encoding the remainder of the antibody. Only the binding region of the antibody is thus recognized as foreign and is much less likely to cause an immune response.
[0122] In providing the antibodies of the present invention to a recipient mammal, preferably a human, the dosage of administered antibodies will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history and the like. In general, it is desirable to provide the recipient with a dosage of antibodies which is in the range of from about 5 mg/kg to about 20 mg/kg body weight of the mammal, although a lower or higher dose may be administered. In general, the antibodies will be administered intravenously (IV) or intramuscularly (IM).
[0123] The invention also relates to the use of antisense nucleic acids to inhibit translation of peptides encoded by sivrcm. The antisense nucleic acids are complementary to sivrcm mrnas encoding peptides of this invention. The antisense nucleic acids may be in the form of synthetic nucleic acids or they may be encoded by a nucleotide construct, or they may be semi-synthetic. The antisense nucleic acids may be delivered to the cells using methods known to those skilled in the art.
[0124] Kits designed for diagnosis of sivrcm in a biological sample can be constructed by packaging the appropriate materials, including the nucleic acids and/or polypeptides of this invention and/or antibodies which specifically react with sivrcm antigens, along with other reagents and materials required for the particular assay. The following examples illustrate certain embodiments of the present invention, but should not be construed as limiting its scope in any way. Certain modifications and variations will be apparent to those skilled in the art from the teachings of the forgoing disclosure and the following examples, and these are intended to be encompassed by the spirit and scope of the invention.
EXAMPLE 1[0125] PCR Amplification, Molecular Cloning and Sequence Analysis of the Sivrcm Genome
[0126] We have PCR amplified, cloned, and sequenced a complete sivrcm proviral genome from a short-term PBMC culture originally established from the blood of a Gabonese red capped mangabey (82). Because of the extensive genetic diversity of this new SIV strain, we had to devise a novel PCR strategy to derive its genome. First, we amplified two small fragments in gag and pol, using primers corresponding to sequences highly conserved among all known primate lentiviral lineages. This allowed us to subsequently design strain-specific primers to amplify the region between gag and pol (gag/pol) as well as the regions outside (flanking) gag and pol by placing the primers in reverse and amplifying the rest of the genome from unintegrated circular DNA molecules. Strategy and primer pairs are shown in FIG. 1. Overlapping PCR fragments were sequenced in their entirety.
[0127] Sequence analysis of the entire provirus revealed a genomic organization previously found only in members of the HIV-2/sivsm/sivmac group of viruses. That is, in addition to gag, pol, vif, vpr, env, tat, rev and nef genes, sivrcm also encoded a vpx homologue thus far only been found in members of the HIV-2/sivsm/sivmac lineage (FIG. 3). Moreover, secondary structure analysis of its LTR sequences revealed a duplicated TAR stemloop structure, again a signature of sivsm/sivmac/HIV-2 viruses (FIG. 4). Based on these findings, we expected sivrcm to fall within a greater mangabey lineage, forming a distinct subcluster similar to what has been observed for the four species-specific subclusters of sivagm.
[0128] However, phylogenetic analyses failed to identify such a relationship of sivrcm with the HIV-2/sivsm/sivmac group (FIG. 5). Instead of grouping closely with other mangabey viruses, sivrcm clustered independently in most regions of its genome, forming a sixth lineage roughly equidistant from the other viruses (FIG. 5). In env and nef, sivsm, sivrcm and sivagm viruses appeared to be relatively more closely related to each other than they were to sivcpz, sivsyk and sivmnd; however, even in these regions there was no particularly close relationship between the two mangabey lineages (FIGS. 5C and 5D). Also, in trees derived from the 3′ and 5′ pol regions, sivrcm clustered with HIV-1 and sivcpz viruses, with significant bootstrap values (FIG. 5B). Finally, there was also a close relationship between sivrcm and sivagmsab, a virus we have previously reported to be mosaic with a divergent mangabey lineage in the 5′ half of its genome (21). In the 5′ half of gag, these two viruses clustered with significant bootstrap values (FIG. 5A), indicating that sivrcm likely represents this previously hypothesized divergent mangabey lineage. These results were confirmed by the use of maximum likelihood approaches to determine tree topologies.
[0129] The results suggest that sivrcm represents a highly divergent mangabey virus which forms an independent lineage (for most of its genome) roughly equidistant from all other primate lentiviruses. The extent of diversity between sivrcm and the other mangabey lineage (sivsm) is surprising, given the known relationships of sivagm strains from different African green monkey species. One explanation for this is that mangabeys acquired their SIV infection a very long time ago (millions of years), even before African green monkeys became infected with sivagm. Another is that these viruses have evolved with in their respective species with vastly different rates of evolution. Also of interest are the close phylogenetic relationships of sivrcm to sivcpz in the 3′ and 5′ pol regions, as well as to sivagmsab in the gag region, which are highly significant. These finding strongly suggest recombination events in the distant past. However, based on current data it is impossible to determine which of these lineages are mosaic. For example, it is quite conceivable the sivrcm is non-recombinant and that both sivcpz and sivagmsab viruses acquired sivrcm sequences independently through cross-species transmission events in the past. This would also mean that HIV-1 is mosaic with sivrcm related sequences. These findings are important because they indicate that primate lentiviral evolution is far more complex than previously appreciated, and that sequences from additional primate lentiviruses (in particular sivcpz, sivrcm, and sivagm strains from Gabon and Cameroon) are critically needed to resolve the unexpected phylogenetic relationships of sivrcm.
EXAMPLE 2[0130] Identification of a New Reading Frame in Sivrcm and Sivsm Viruses
[0131] Analysis of the sivrcm sequence revealed that there were about 100 bp of “non-coding sequences” between the splice donor site of the first tat and rev exons, and the initiation codon of the env open reading frame (ORF). Upon closer analysis, it was realized that the first exon of rev, instead of being terminated by a stop codon immediately downstream of the splice site, continued uninterrupted for about 100 more base pairs. Comparison of the genomic organization of members of all other major primate lentiviral lineages indicated that there was only one other group, i.e., The sivsm/sivmac group, that had a similarly “extended first rev exon”. All other viruses had stop codons shortly after the splice site (except for members of the HIV-1/sivcpz group, which encode the vpu gene in this region).
[0132] FIG. 6 shows an alignment of the deduced amino acid sequence of the sivsmpbj1.9 and sivrcmgb1 extended rev orfs. The putative pbj protein is 71 amino acids in length, while the putative sivrcm protein is shorter, i.e., Only 54 amino acids. However, this shorter length could be the result of inactivating mutations (frameshift and in frame stop codons). Assuming a “corrected” sequence, the sivrcm extended rev ORF would encode a 105 amino acid protein (FIG. 6) with considerable sequence homology to the corresponding sivsm product throughout its entire length. Correction of inactivating mutations would restore the predicted coding capacity of the sivrcm rev ORF to a protein of 105 amino acids, and nucleic acids containing repaired coding sequences, as well as the polypeptides encoded by the repaired coding sequences, are also considered to be a part of the invention.
[0133] The conservation of the extended rev orfs among different members of the HIV-2/sivsm/sivmac group of virus was also examined. FIG. 7 shows an amino acid sequence alignment, including all sequences from the Los Alamos HIV sequence database. The results were surprising: all sivsm/sivmac strains encoded a highly conserved and uninterrupted open reading frame; however, all HIV-2 strains, except one (FO784) which we was considered to represent an ill-adapted sooty managbey virus in humans, contained inactivating mutations (stop codons) in this “extended rev ORF”.
[0134] The finding of an “extended rev first exon ORF” in both sivrcm and sivsm lineages is significant for several reasons: (i) it suggests the existence of a “mangabey virus specific” protein, which likely plays an important role in mangabey virus replication (as do all other accessory proteins); (ii) it suggests the existence of a “mangabey virus specific” protein whose expression may be abrogated as a consequence of sivsm adaptation to the human host; (iii) it suggests the existence of a “managbey virus specific” protein, which could serve as a functional vpu equivalent (the position of this new ORF in the sivrcm/sivsm genome, as well as its overall length, are reminiscent of the vpu gene in HIV-1/sivcpz, although amino acid sequence homologies between the putative sivsm/sivrcm extended first rev exon product and the HIV-1/sivcpz Vpu protein were not found; however, this is not necessarily surprising since the Vpu proteins among different members of the HIV-1/sivcpz lineage are so divergent that they cannot be aligned (52). These findings are important for the current application because they highlight another potential structural similarity between sivrcm and sivsm lineages. However, additional sivrcm isolates need to be characterized to confirm the existence of this reading frame (and its putative protein product) among other viruses.
[0135] The following references are cited herein:
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[0218] Modification of the above described invention that are obvious to those of skill in the fields of genetic engineering, immunology, protein chemistry, medicine, and related fields are intended to be within the scope of the following claims. All of the references cited herein above are hereby incorporated by reference.
Claims
1. A nucleic acid comprising the nucleotide sequence of the genome of the simian immunodeficiency virus isolate SIVrcm shown in SEQ ID NO:1 or a complementary sequence thereof.
2. The nucleic acid of claim 1, wherein said nucleic acid comprises a nucleotide sequence of at least 12 contiguous bases of said nucleic acid or a complementary sequence thereof.
3. The nucleic acid of claim 1, wherein said nucleic acid comprises a nucleotide sequence of a LTR of said nucleic acid or a complementary sequence thereof.
4. The nucleic acid of claim 1, wherein said nucleic acid encodes a polypeptide selected from the group consisting of Gag, Pol, Vif, Vpr, Env, Tat, Rev, Nef and Vpx of SIVrcm.
5. The nucleic acid of claim 1, wherein said nucleic acid has a nucleotide sequence which is derived from SEQ ID NO:1.
6. A vector comprising the nucleic acid of claim 1.
7. A cell comprising the nucleic acid of claim 1.
8. A cell comprising the vector of claim 6.
9. A composition comprising the nucleic acid of claim 1 and a physiologically acceptable carrier.
10. A method for producing a polypeptide encoded by the nucleic acid of claim 1, comprising the step of growing a cell comprising the nucleic acid of claim 1 under conditions such that the encoded polypeptide is produced.
11. The method of claim 10, wherein said polypeptide comprises a contiguous sequence of at least 13 amino acids.
12. A composition comprising the polypeptide produced by the method of claim 10 and a physiologically acceptable carrier.
13. A method for producing a polypeptide encoded by the nucleic acid of claim 1, comprising the step of growing a cell comprising a vector, said vector comprising the nucleic acid of claim 1, under conditions such that the encoded polypeptide is produced.
14. The method of claim 13, wherein said polypeptide comprises a contiguous sequence of at least 13 amino acids.
15. A composition comprising the polypeptide produced by the method of claim 13 and a physiologically acceptable carrier.
16. A method of inducing serum antibodies that bind at least one polypeptide encoded by the nucleic acid of claim 1, said method comprising: administering to a mammal, in a physiologically acceptable carrier, an amount of said encoded polypeptide sufficient to elicit production of said antibodies.
17. An anti-SIVrcm antibody made by the method of claim 16.
18. A composition comprising an antibody according to claim 17 and a physiologically acceptable carrier.
19. A method for detecting the presence of SIVrcm in a sample comprising contacting said sample with the antibody of claim 17 under conditions that allow the formation of an antibody-antigen complex and detecting said complex.
20. A kit for detecting the presence of SIVrcm in a sample comprising an antibody of claim 17.
21. A method of inducing serum antibodies that bind at least one polypeptide encoded by the nucleic acid of claim 1, said method comprising: administering to a mammal, in a physiologically acceptable carrier, the nucleic acid of claim 1 encoding said polypeptide and which produces an immunologically sufficient amount of the encoded polypeptide to elicit said antibodies.
22. An antibody to SIVrcm made by the method of claim 21.
23. A composition comprising an antibody according to claim 22, and a physiologically acceptable carrier.
24. A method for detecting the presence of SIVrcm in a sample comprising contacting said sample with the antibody of claim 22 under conditions that allow the formation of an antibody-antigen complex and detecting said complex.
25. A kit for detecting the presence of SIVrcm in a sample comprising an antibody of claim 22.
26. A method for detecting the presence of antibodies to SIVrcm in a sample comprising contacting said sample with a polypeptide encoded by the nucleic acid of claim 1 under conditions that allow the formation of an antibody-antigen complex and detecting the complex.
27. A method for detecting the presence of SIVrcm in a sample comprising contacting said sample with the nucleic acid of claim 1 and detecting said nucleic acid bound to the genomic DNA, mRNA or cDNA of the SIVrcm virus.
28. A kit for detecting the presence of SIVrcm in a sample comprising the nucleic acid of claim 1.
29. A nucleic acid probe comprising a sequence of at least 19 contiguous nucleotides derived from the nucleic acid of claim 1, or the complementary sequence thereof.
30. A composition comprising a nucleic acid according to claim 29.
31. A method of detecting the presence of SIVrcm in a biological sample comprising:
- a.) contacting the nucleic acid of the biological sample with the nucleic acid probe of claim 29; and
- b.) detecting the presence or absence of complexes formed between said nucleic acid of the biological sample and said nucleic acid probe.
32. A method of detecting the presence of SIVrcm in a biological sample comprising:
- a). contacting said biological sample with at least two nucleic acid probes of claim 29;
- b). amplifying the RNA of the biological sample via reverse transcription-polymerase chain reaction to produce amplification products; and
- c.) detecting the presence or absence of amplification products.
33. A method for analyzing a first nucleotide sequence comprising comparing the nucleotide sequence of claim 1 with said first sequence.
Type: Application
Filed: Feb 18, 2003
Publication Date: Aug 28, 2003
Applicant: UAB Research Foundation
Inventors: Beatrice H. Hahn (Birmingham, AL), Feng Gao (Hoover, AL), George M. Shaw (Birmingham, AL), Preston A. Marx (Covington, LA), Stephen M. Smith (Essex Fells, NJ), Marie Claude Georges-Courbot (Paris XV), Chang Yong Lu (Forest Hills, NY)
Application Number: 10369294
International Classification: C12Q001/70; C07H021/04;
