Coronavirus-like particles comprising functionally deleted genomes
The invention relates to the field of coronaviruses and diagnosis, therapeutic use, and vaccines derived therefrom. The invention provides replicative coronaviruses and virus-like particles (VLPs) from which large parts of their genome are (at least functionally) deleted without abolishing their replicative capacities. The deletion preferably results in at least a functional deletion in that the corresponding gene is not or is only partly expressed wherein the resulting gene product is dysfunctional or at least functionally distinct from a corresponding wild-type gene product. One result seen with VLPs provided with deletions as provided herein is that the deleted VLP, albeit capable of replication in vitro and in vivo, are generally well attenuated, in that they do not cause disease in the target host, making them very suitable for therapeutic use, such as a delivery vehicle for genes and other cargo (wherein specific targeting may be provided as well when desired), and for use as a vaccine, being attenuated while carrying important immunogenic determinants that help elicit an immune response.
This application is a continuation of PCT International Patent Application No. PCT/NL/02/00318, filed on May 17, 2002, designating the United States of America, and published, in English, as PCT International Publication No. WO 02/092827 A2 on Nov. 21, 2002, the contents of the entirety of which is incorporated by this reference.
TECHNICAL FIELDThe invention relates to the field of coronaviruses and diagnosis, therapeutic use and vaccines derived thereof.
BACKGROUNDCoronavirions have a rather simple structure. They consist of a nucleocapsid surrounded by a lipid membrane. The helical nucleocapsid is composed of the RNA genome packaged by one type of protein, the nucleocapsid protein N. The viral envelope generally contains 3 membrane proteins: the spike protein (S), the membrane protein (M) and the envelope protein (E). Some coronaviruses have a fourth protein in their membrane, the hemagglutinin-esterase protein (HE). Like all viruses, coronaviruses encode a wide variety of different gene products and proteins.
Most important among these are the proteins responsible for functions related to viral replication and virion structure. However, besides these elementary functions, viruses generally specify a diverse collection of proteins the function of which is often still unknown but which are known or assumed to be in some way beneficial to the virus. These proteins may either be essential—operationally defined as being required for virus replication in cell culture—or dispensable. Coronaviruses constitute a family of large, positive-sense RNA viruses that usually cause respiratory and intestinal infections in many different species. Based on antigenic, genetic and structural protein criteria they have been divided into three distinct groups: Group I, II and III. Actually, in view of the great differences between the groups their classification into three different genera is presently being discussed by the responsible ICTV Study Group. The features that all these viruses have in common are a characteristic set of essential genes encoding replication and structural functions. Interspersed between and flanking these genes sequences occur that differ profoundly among the groups and that are, more or less, specific for each group.
Of the elementary genes, the most predominant one occupies about two-thirds of the genome. Located at the 5′ end, this so-called “polymerase gene” encodes two large precursors, the many functional cleavage products of which are collectively held responsible for RNA replication and transcription. The other elementary genes specify the basic structural proteins N, M, E, and S. The nucleocapsid (N) protein packages the viral RNA forming the core of the virion. This RNP structure is surrounded by a lipid envelope in which the membrane (M) protein abundantly occurs consituting a dense matrix. Associated with the M protein are the small envelope (E) protein and the spike (S) protein, the latter forming the viral peplomers that are involved in virus-cell and cell-cell fusion. The genes for these structural proteins invariably occur in the viral genome in the order 5′-S-E-M-N-3′.
In infected cells, the coronavirus nucleocapsids are assembled in the cytoplasm. The nucleocapsids interact with the viral envelope proteins which, after their synthesis in the endoplasmic reticulum, accumulate in the intermediate compartment, a membrane system localized between the endoplasmic reticulum (ER) and the Golgi complex. This membrane system acts as the budding compartment: the interaction of the nucleocapsids with the viral envelope proteins leads to the pinching off of virions that are then released from the cell by exocytosis.
We have recently demonstrated that the assembly of coronaviral particles does not require the involvement of nucleocapsids. Particles devoid of a nucleocapsid are assembled in cells when the viral envelope protein genes are co-expressed. The minimal requirements for the formation of virus-like particles (VLP's) are the M and E protein: the S protein is dispensable but is incorporated if present through its interactions with the M protein. Biochemical and electron microscopical analysis revealed that the VLPs are homogeneous in size and have similar dimensions as authentic corona virions. Clearly, the M and E protein have the capacity to associate in the plane of cellular membranes and induce curvature leading to the budding of specific “vesicles” which are subsequently secreted from the cells. An article describing these results has appeared in EMBO Journal (vol. 15, pp. 2020-2028, 1996).
In yet another article, coronavirus like particles were shown which were not devoid of a nucleocapsid, assembly here did not take place independent of a nucleocapsid (Bos et al., Virology 218, 52-60, 1996). Furthermore, packaging of RNA was not very efficient. Furthermore, neither of these two publications provides sufficient targeting and delivery features which would make the VLP's suitable as therapeutic carrier, for example being equipped with specific targeting information and/or with a genetic or non-genetic message.
However, coronaviruses do have several distinct theoretical advantages for their use as vectors over other viral expression systems (see, also, PCT International Publication WO98/49195): (i) coronaviruses are single-stranded RNA viruses that replicate within the cytoplasm without a DNA intermediary, making unlikely the integration of the virus genome into the host cell chromosome; (ii) these viruses have the largest RNA genome known having in principle room for the insertion of large foreign genes; (iii) since coronaviruses in general infect the mucosal surfaces, both respiratory and enteric, they may be used to induce a strong secretory immune response; (iv) the tropism of coronaviruses may be modified by the manipulation of the spike (S) protein allowing the engineering of the tropism and virulence of the vector; and, (v) non-pathogenic coronavirus strains infecting most species of interest are available to develop expression systems.
Two types of expression vectors have been developed based on coronavirus genomes. One requires two components (helper dependent) and the other a single genome that is modified either by targeted recombination or by engineering a cDNA encoding an infectious RNA. Helper dependent expression systems, also called minigenomes have been developed using members of the three groups of coronaviruses. Coronavirus derived minigenomes have a theoretical cloning capacity close to 25 kb, since minigenome RNAs of about 3 kb are efficiently amplified and packaged by the helper virus and the virus genome has about 30 kb. This is, in principle, the largest cloning capacity for a vector based on RNA virus genomes.
Reverse genetics has been possible by targeted recombination between a helper virus and either non-replicative or replicative coronavirus derived RNAs (9). Targeted recombination has been mediated by one or two cross-overs. Changes were introduced within the S gene that modified MHV pathogenicity. The gene encoding green fluorescent protein (GFP) was inserted into MHV between genes S and E by targeted recombination, resulting in the creation of a vector with the largest known RNA viral genome (1d). Mutations have also been created by targeted mutagenesis within the E and the M genes showing the crucial role of these genes in assembly.
DISCLOSURE OF THE INVENTIONThe construction of a full-length genomic cDNA clone could considerably improve the genetic manipulation of coronaviruses. The construction of an infectious TGEV cDNA clone has recently been made possible (1c). To obtain an infectious cDNA, three strategies have been combined (i) the construction of the full-length cDNA was started from a DI that was stably and efficiently replicated by the helper virus. Using this DI, the full-length genome was completed and the performance of the enlarged genome was checked after each step. This approach allowed for the identification of a cDNA fragment that was toxic to the bacterial host. This finding was used to advantage by reintroducing the toxic fragment into the viral cDNA in the last cloning step; (ii) in order to express the long coronavirus genome, and to add the 5′ cap, a two-step amplification system that couples transcription in the nucleus from the CMV promoter, with a second amplification in the cytoplasm, driven by the viral replicase, was uses; and, (iii) to increase viral cDNA stability within bacteria, the cDNA was cloned as a bacterial artificial chromosome (BAC), that produces only one or two plasmid copies per cell. The full-length cDNA was divided into two plasmids because their fusion into one reduced the stability of the cDNA. One plasmid contained all virus sequences except for a fragment Cla I to Cla I of abouw 5 kb that was included within a second BAC. A fully functional infectious cDNA clone, leading to a virulent virus able to infect both the enteric and respiratory tracts, was engineered by inserting the Cla I fragment into the rest of the TGEV cDNA sequence.
As said, both helper-dependent expression systems, based on two components and single genomes constructed by targeted recombination or by using an infectious cDNA have been developed. The sequences that regulate transcription have been characterized. Expression of high amounts of heterologous antigens (1 to 8 μg/106 cells) have been achieved, and the expression levels have been maintained for around 10 passages. These expression levels should be sufficient to elicite protective immune responses.
Single genome coronavirus vectors have been constructed efficiently expressing a foreign gene such as GFP. Thus, a new avenue has been opened for coronaviruses which have unique properties, such as a long genome size and enteric tropism, that makes them of high interest as expression vectors, be it that for vaccine development and gene therapy also other conditions need be met, notably host virulence of vector constructs, and viability of vector constructs in cell culture need to be within distinct limitations. One the one hand, the vector may not remain too virulent, but should have at least some attenuated properties rendering it useful for in vivo replication as gene delivery vehicle or vaccine for the host or subject undergoing therapy. On the other hand, the vector should replicate well in cell-culture, at least when commercial use is desired.
The invention provides replicative coronaviruses and replicative coronavirus-like particles (VLPs) from which large parts of their genome are (at least functionally) deleted and/or rearranged without abolishing their replicative capacities. The deletion is preferably resulting in at least a functional deletion in that the corresponding gene is not or only partly expressed wherein the resulting gene product is absent, dysfunctional or at least functionally distinct from a corresponding wild-type gene product. One striking result seen with VLPs provided with deletions and/or rearrangements as provided herein is that the deleted or rearranged VLP, albeit capable of replication in vitro and in vivo, is in general well attenuated, in that it does not cause disease (or do so much less) in the target host, making it very suitable for therapeutic use, for example as a delivery vehicle for genes and other cargo (wherein specific targeting may be provided as well when desired), or for use as a vaccine, being attenuated while carrying important immunogenic determinants that help elicit an immune response. Such determinants may be derived from a (homologous or heterologous) coronavirus, but may also be derived from other pathogens. In other words, the invention provides the use of a replicative VLP with a partly deleted and/or rearranged genome as a vector. Into the vector, a foreign nucleic acid sequence may be introduced. This foreign gene sequence encodes (part of) a protein. It is this protein, or part thereof, encoded by the inserted sequence, which may serve as an immunogen or a “targetting means” (ligand capable of binding to a receptor).
In a preferred embodiment, the VLP as provided herein are further modified in one of various ways, genomically or in their protein composition, thereby exposing at their surface various biological or target molecules and/or carrying within the particles molecules with biological activity which need to be protected or shielded and/or containing genomes into which foreign genes or sequences have been incorporated. One of the major needs in present-day medicine is systems for the targeted delivery of therapeutic agents in the body. By consequence, the development of carriers that can direct cargo to specified groups of cells and introduce this cargo into these cells such that it can exert its biological activity, is a major challenge in biomedical research. Tremendous efforts have already been spent in the development and testing of systems based on liposomes, microspheres, antibodies, etc. for delivery of drugs, genes, peptides and proteins. Though many of these approaches are promising, the actual successes so far are limited. The genome of a VLP as provided herein has been deleted and/or rearranged to such an extent without abolishing replication that it serves very well as delivery vehicle for the cargo. Providing the VLP with cargo or using the VLP as a vector is e.g. done by inserting a foreign gene sequence that encodes a desired molecule such as a ligand or binding molecule, whether this is an immunogen or receptor binding molecule or any other protein or peptide. In a preferred embodiment the cargo comprises a nucleic acid into which foreign genes or sequences of interest have been incorporated. Foreign genes can be expressed by a VLP both by the additional insertion of such a gene in its genome or by using the genetic space created by deletion of nonessential genes. To further increase the safety of gene delivery therapy with corona-like viruses, such as with VLP as provided herein, the invention also provides a method for inhibiting or blocking infection with coronaviruses in general and with a coronavirus-like particle in particular, comprising treatment of an organism or cells at risk for an infection or infected with such a coronavirus or VLP with a so-called heptad repeat peptide as provided herein. All coronaviruses have herein been found to contain one or two characteristic heptad repeat regions in their S protein, which are instrumental in coronavirus entry into cells. Peptides derived from the membrane-proximal heptad repeat region (HR2; e.g. for MHV strain A59 the peptide composed of amino acids 1216-1254 of the S protein, see also
In a further embodiment, the invention provides a replicative VLP according to the invention which is also modified at the ectodomain and/or the endodomain of a viral protein. For example, by modifying the ectodomain of the spike protein, the VLP is provided with modified biological molecules as targeting means that serve to direct the VLP to interact with other biological molecules that mirror or can interact, with the target means, such as receptor proteins on cells, be it hormone receptors, specific immunoglobulines on B-cells, MHC and MHC associated molecules present on T-cells and other cells, transfer proteins or other receptor molecules known to the person skilled in the field of cell surface receptors. The targeting means can also be provided to interact with known binding sites of selected enzymes on proteins or other molecules that serve as substrate for the selected enzyme.
In another embodiment, replicative VLPs are provided exposing an immunogenic determinant, such as a bacterial toxin or a heterologous viral or bacterial protein comprising a relevant antigenic determinant specific for a pathogen. This is an example where the VLPs serve as immunogen or vaccine, here, for example, directed against the bacterial toxin. B-lymphocytes carrying the corresponding immunoglobuline at their surface are in this case the target cells for the VLPs, once recognized by the B-lymfocyte, this cell(s) will multiply and produce the appropriate antibody.
Preparation of VLPs or coronaviruses with modified spikes can be achieved genetically by modification of the viral genome such that it expresses the modified S protein in infected cells. Here, we also provide the preparation of coronaviruses containing altered spikes in a different way by expressing modified S genes in cells which are in addition infected with coronavirus. The co-incorporation of the mutant spike provides the virus with new targeting means. In one embodiment, the invention provides a corona-like viral particle (VLP) comprising a genome from which at least a fragment of one of several genes or gene clusters not belonging to the genes specifying the polymerase functions (ORF1a/1b) or the structural proteins N, M, E, and S, are deleted without resulting in a total loss of replicative capacities, thereby providing these VLP with advantageous properties for therapeutic use, for example, as a vector for gene delivery purposes or for use as a vaccine delivering a suitable antigene or epitope for eliciting an immune response in the host of interest. In other words, the non-essential genes of coronaviruses are not crucial for in vitro growth but determine viral virulence. The attenuation acquired by their deletion thus provides excellent viral vaccines and therapeutic vectors. Gene delivery or vaccination with a coronavirus can now be achieved with the assuring knowledge that the virus-like particle delivering the gene or antigen of interest is sufficiently attenuated to not cause specific coronaviral disease. Of course, besides a VLP being only deleted in any one or several of the above mentioned non-essential genes, the invention also provides a VLP provided with a, preferably functional in that a substantial peptide fragment of at least 4 of the original amino acids, preferably of at least 40, is not expressed, deletion in any or the genes encoding the structural proteins, in particular, such a deletion in a structural protein leads to a VLP with modified spike protein (S) in which part of the nucleic acid encoding the spike is deleted and optionally replaced by a foreign gene sequence, thus for example providing the spike with another than the natural ectodomain (or endodomain) of the spike protein of the original coronavirus.
The viruses of group I, with the feline infectious peritonitis virus (FIPV) probably as the most complex member, have their typical genes located between genes S and E and downstream of the N-gene, i.e. between N-gene and 3′ UTR (untranslated region). Group II viruses, to which the mouse hepatitis viruses (MHV) belong, have their particular genes between the polymerase and S genes and between the genes for the S and E proteins. One of the encoded proteins characteristic for this group is the hemagglutinin-esterase (HE) protein, which is incorporated into virions and of which the hemagglutinating and esterase activities have been demonstrated. HE activities are not essential and their significance remains to be elucidated. Finally, also the group III viruses, with the prototype coronavirus infectious bronchitis virus (IBV) as the representative, have sequences between the S and E genes but also between the M and N genes. While for some of the group-specific genes no expression products could be detected in infected cells while for others (e.g., the HE gene in MHV) naturally occurring viral mutants carrying deletions in these genes have been observed, the possibility exists that for some of these genes not the protein products but other gene products, such as the nucleotide sequences (DNA or RNA) per se, are important or essential.
Considering that, based on their genome organisation three groups of coronaviruses can be distinguished the invention provides for group I (FCoV) a recombinant VLP from which preferably a fragment (preferably resulting in at least a functional deletion in that the corresponding gene is not or only partly expressed wherein the resulting gene product is absent, dysfunctional or at least functionally distinct from a corresponding wild-type gene product) from gene 3a, 3b, 3c, 7a or 7b, or the gene as a whole has been deleted. Such a deleted VLP, albeit capable of replication in vitro and in vivo, is well attenuated, in that it does essentially not cause disease in the target host, making it very suitable for therapeutic use, for example as a delivery vehicle for genes and other cargo (wherein specific targeting may be provided as well when desired), and for use as a vaccine, being attenuated while carrying important immunogenic determinants that help elicit an immune response. Such a deleted group I virus, especially a feline coronavirus, such as FIPV, from which a fragment corresponding to gene 3c, and/or 7b is deleted, is in particular immediately useful as a vaccine in that it expresses relevant antigenic and immunogenic determinants through its structural (among others, spike) proteins and is functionally deleted in such a way that attenuation is achieved, leading to a safe and efficacious vaccine. These live attenuated viruses still induce the fullest spectrum of humoral and cellular immune responses required to protect against infection and/or disease. Additionally, such a deleted VLP for the prevention of feline disease can be provided with heterologous proteins (or functional fragments thereon, such as (glyco)proteins of feline leukemia virus, feline calicivirus, feline herpes virus, allowing the production of a bivalent or even multivalent vaccine. When desired, other immunologically or therapeutically important polypeptides, such as cytokines, are incorporated instead or as well.
Furthermore, the invention provides for group II (MHV) a recombinant VLP from which preferably a fragment (preferably resulting in at least a functional deletion) from gene 2a, HE, 4a, 4b, or 5a, or the gene as a whole has been deleted. Such a deleted group II virus, especially a MHV, is provided with advantageous properties for therapeutic use, for example, especially as a vector for delivery purposes. Such a deleted VLP, albeit capable of replication in vitro and in vivo, is well attenuated, in that it does essentially not cause disease in the target host, making it very suitable for therapeutic use, as a delivery vehicle for genes and other cargo (wherein specific targeting may be provided as well when desired), and for use as a vaccine, being attenuated while carrying important immunogenic determinants that help elicit an immune response.
For group III (IBV), a recombinant VLP is provided from which preferably a fragment (preferably resulting in at least a functional deletion) from gene 3a, 3b, 5a or 5b, or the gene as a whole has been deleted. In another embodiment, for groups I, II, or III, the invention provides a virus-like particle capable of replication, the particle derived from a coronavirus wherein the genes for the structural proteins do not occur in the order 5′-S-E-M-N-3′. Such a replicative VLP with rearranged gene order has two important features. One is safety, resulting from the fact that homologous recombination of the VLP genome with that of an accidental field virus is unlikely to generate viable new progeny. The other is attenuation due to the shuffling of the genes. Such a replicative VLP with rearranged gene order provides well attenuated VLP's for vaccine or gene delivery use, and is herein provided bearing the deletions from the non-essential genes as well. It is shown herein that changes in the so-called invariable order of the genes specifying the polymerase functions (ORF1a/1b) and the structural proteins S, E, M, and N in the coronaviral genome is surprisingly well tolerated, for example VLP's with gene order S, M, E, N, or E, S, M, N are easily obtained. Also here, foreign genes can be inserted at different positions in the viral genome, either as an additional gene or replacing deleted non-essential genes; these genes are expressed and are stably maintained during passage of the virus.
In another embodiment, the invention provides a recombinant VLP where nucleic acid encoding the S-protein has been modified or at least partly deleted. The S protein of these viruses is responsible for binding to the cell receptor and for subsequent fusion of viral and cellular membrane during entry. These two functions occur in separate regions of the molecule: receptor binding in the amino-terminal and fusion in the carboxy-terminal part. Therefore, by replacing (parts of) the receptor binding domain by biological molecules with different targeting specificities coronaviruses can be directed to interact with a wide variety of target molecules that are for instance expressed on the surface of cells. Doing so without affecting the fusion function of the S-protein, the VLP according to the invention can fuse with or penetrate into cells not normally injectable by the original virus.
The invention provides virus-like particles (VLPs) derived from coronaviruses in which one or more copies of the viral membrane proteins have been modified so as to contain foreign protein moieties of viral (either coronaviral or non-coronaviral) or non-viral origin, which moieties either are replacing part(s) of the VLP membrane proteins or are incorporated within these membrane proteins thereby constituting an integral part of them. By this, the VLP is provided with novel biological properties such as new targeting means, or immunological information, or proteins with specific biological activity contained within the virus-like particle, which biological properties are associated with the VLP next to or in place of the natural spike protein of the original coronavirus.
In one embodiment, recombinant VLPs with deleted and/or rearranged genome are provided in which (a part of) the ectodomain (i.e., the part exposed at the outside of the viral particle) of the spike protein has been replaced by the corresponding domain (or part thereof) of the spike protein of another coronavirus. Hereby, the VLP has acquired another cell substrate specificity wherein the VPL is capable of entering cells otherwise not accessible or susceptible to the original coronavirus. In a further embodiment of the invention, VLPs are provided which are composed of the mouse hepatitis coronavirus (MHV) M and E proteins and which contain chimaeric spike molecules consisting of the transmembrane+carboxy-terminal domain of MHV S but the ectodomain of the spike protein of feline infectious peritonitis coronavirus (FIPV). These VLPs can now enter feline cells and deliver MHV-like particles. Particles with these chimaeric spikes are produced by making constructs of the coronavirus MHV S gene in which the region encoding the amino-terminal domain is replaced by the corresponding domain of FIPV. These constructs are inserted into plasmids behind a bacteriophage T7 polymerase promoter. The constructs are then co-transfected with plasmids carrying the MHV M and E genes, both also behind the T7 promotor, in OST-7 cells which have been infected with a recombinant vaccina virus expressing the T7 polymerase. The resulting VLPs contain the chimaeric MHV/FIPV S protein. In another embodiment of the invention, the VLP is provided by the methods used as above with ectodomains of the spike protein of infectious bronchitis coronavirus (IBV), or the ectodomain (or part thereof) of an envelope protein of any enveloped virus not belonging to the coronaviruses. For example, MHV-based VLPs are provided by the invention that carry at their surface the ectodomain of the pseudorabies virus (PRV) glycoprotein gD instead of the MHV spike ectodomain or the luminal (i.e., amino-terminal) domain (or part thereof) of any nonviral type I membrane protein. In this way, VLPs are provided that have a cell specificity for chicken cells, or pig cells, or cells reactive with the type I membrane protein.
In yet another embodiment, replicative VLPs are produced with modifications that are contained within the particles. This is achieved by the incorporation of modified constructs of any of the corona viral proteins S, M, E, and HE. In coronavirus particles, these proteins have their carboxy-terminal domain enclosed within the interior of the viral envelope. Thus, foreign protein sequences incorporated within, appended to or replacing the carboxy-terminal domain are enclosed as well. In this way, VLPs can be provided that contain protein moieties, or fragments thereof, from another virus, or non-viral proteins such as hormones, such as erythropoietin. This allows the production of VLPs containing a biological active protein or fragments thereof, which is/are shielded by the viral envelope and can be released and/or retrieved later, when the viral membrane is degraded or fused with another membrane. This allows the in vitro production in cells, or the in vivo production in secretory glands such as milk glands of biologically active substance which are otherwise harmful or toxic to the producing cells, or which for other reasons need to be produced in a shielded form.
In another embodiment, MHV-based VLPs are provided carrying on their surface or inside an enzymatically active molecule like furin, or a cytokine, or a hormone receptor, or another viral or nonviral polypeptide with biological activity. In these examples, VLPs are provided with (additional) targeting means that serve to direct the VLP to cells otherwise not accessible to the original coronavirus. The invention provides recombinantly obtained replicative VLPs which are further modified at the ectodomain and/or the ectodomain of any of the viral proteins. By modifying the ectodomain of the spike protein, the VLPs are provided with modified biological molecules as targeting means that serve to direct the VLP to interact with other biological molecules that mirror or can interact with the target means, such as receptor proteins on cells, be it hormone receptors, specific immunoglobulines on B-cells, MHC and MHC associated molecules present on T-cells and other cells, transfer proteins or other receptor molecules known to the person skilled in the field of cell surface receptors. The targeting means can also be provided to interact with known binding sites of selected enzymes on proteins or other molecules that serve as substrate for the selected enzyme.
Preparation of VLPs or coronaviruses with modified spikes can be achieved genetically by modification of the viral genome such that it expresses the modified S protein in infected cells. Here we also provide the preparation of coronaviruses containing altered spikes in a different way by expressing modified S genes in cells which are in addition infected with coronavirus. The co-incorporation of the mutant spike provides the virus with new targeting means. As an example we demonstrate the production of MHV particles containing the chimaeric MHV/FIPV S protein. The chimaeric S gene construct is expressed in L cells which are subsequently infected with wild-type MHV strain A59 (MHV-A59) or a mutant thereof. The progeny virus released by the cells contains the modified S protein. To demonstrate the altered targeting the virus was used to infect feline cells that are naturally not susceptible to MHV. The cells are now infected as shown by immunofluorescence and produce normal MHV. As another example we demonstrate the production of MHV containing chimaeric S proteins in which part of the S ectodomain has been replaced by the corresponding part (i.e. the luminal or amino-terminal domain) of the human CD4 molecule, as an example of a nonviral protein. These modified coronaviruses have acquired the property to infect HIV-infected cells and cells expressing HIV envelope glycoprotein through the specific recognition of the CD4 and HIV gp120 complex. As a result, the HIV-infected cells will undergo a lytic infection, effectively reducing the number of HIV-infected cells in the body and thereby reducing the severity of the disease or even terminating the infection.
As another example, we demonstrate the production of a VLP according to the invention containing spike molecules of which the amino-terminal part has been replaced by a single chain-antibody fragment recognizing a specific cell surface protein that is expressed on cells that can normally not be infected with the coronavirus laying at the basis of the VLP. The modified virus is able to infect these otherwise refractory cells. This example illustrates the principle that in this way, i.e., by inserting very specific targeting information into the viral spike, coronaviruses can be directed to selected cells or tissues. The single chain-antibody fragment can for instance be selected in phage-display systems, or in other clonal selection systems of single-chain antibody fragments known in the field.
Another aspect of the invention relates to the use of the replicative VLPs or coronaviruses as gene delivery vehicles. This can be achieved in different ways. One way is by incorporating foreign genes or sequences into the viral genome such that upon entry of the virus into cells these genes are expressed or that the inserted sequences become otherwise biologically active (as is the case with ribozymes or antisense RNAs generated by the virus within the cells). The other way uses VLPs to package foreign RNA into particles by making use of the coronaviral packaging signal(s). Incorporating foreign sequences into the coronaviral genome can be accomplished by genetic manipulation using an infectious (c-)DNA clone, a full-size DNA copy of the viral genome. It can also be achieved by RNA recombination in which case RNA representing part of the viral genome and containing the foreign sequences is introduced in infected cells allowing the foreign sequences to be incorporated through homologous recombination. Because coronaviruses will usually kill the cells they infect, it is important for most purposes to attenuate them so that they will not kill the cells with which they interact. Attenuation is here accomplished by genomically altering the virus through deletion or rearrangement.
As an example of the invention, attenuation is provided by the preparation of an MHV mutant from which an essential gene has been deleted by recombination. A mouse cell line is provided in which the MHV E gene has been chromosomally integrated allowing the E protein to be produced by the expression of the gene. MHV lacking an E gene has been produced in normal mouse cells by recombination using a synthetic RNA containing a perfect copy of the MHV genomic 3′-end except for the lack of an intact E gene. The E-defective virus is able to grow only in the cells complementing the defect. The virus produced is attenuated such that it can infect other mouse cells, but non-productively: the lack of an E protein prevents the assembly of progeny. As an example of the principle of incorporating foreign genetic sequences into attenuated or not-attenuated VLPs or coronaviruses and of their expression is the following provided by the invention. An MHV derived VLP is provided into which a reporter gene such as LacZ or green fluorescent protein has been recombined and one in which the chimaeric MHV/FIPV S gene has been incorporated. The expression of the genes is shown by blue or green-fluorescent staining of VLP infected cells and by the acquired ability to infect feline cells, respectively. The other way to obtain coronavirus-based delivery vehicles uses VLPs comprising foreign RNA sequences. Incorporation of foreign RNA sequences into these particles requires their packaging into nucleocapsids. Viral RNA-packaging by nucleocapsid (N) protein molecules occurs by the recognition of specific sequences, packaging signal(s) by the N protein. In MHV the packaging signal includes a 69 nucleotides long region in gene 1B. Foreign (noncoronaviral) RNAs containing the coronavirus packaging signal(s), or defective coronaviral genomes in which these signal(s) have been retained but into which foreign sequences have been incorporated, are assembled into VLPs when introduced into cells expressing the N, M and E (±S) genes. The VLP can introduce into a target cell a defined RNA that may have one of several functions. An example provided by the invention is a RNA acting as mRNA and specifying a particular protein such as a toxin or an inducer of apoptosis or an antibody fragment. Another example is an antisense RNA or an RNA with ribozyme activity. For most purposes it is essential to acquire multiple copies of the RNA in each cell to obtain the desired effect. This may not be feasible with VLPs which will only carry one or a few pseudo-NC. The invention thus provides the RNAs with amplification signals such that they will be multiplied in the target cell. To achieve this goal, Semliki Forest virus (SFV) replication sequences are used as the basis of the RNA construct. SFV-derived mRNA further comprising the coronavirus encapsidation sequences and specifying a reporter protein are assembled into VLPs. The SFV-driven amplification allows synthesis of the reporter protein in cells; in animals the appearance of antibodies to the reporter protein testifies to the productive delivery of the VLPs' content. The invention also provides a VLP which is an antigen or epitope delivery vehicle meant for the induction of specific immune responses, cellular and/or humoral, systemic and/or local, including the induction and production of specific antibodies against proteins, to achieve protection against infection by pathogens, of viral and nonviral origin. As an example the invention provides the induction of antibodies against the reporter protein derived from SFV-derived mRNA further comprising the coronavirus encapsidation sequences and specifying a reporter protein, as described above. As another example the induction of antibodies is demonstrated in mice to the FIPV spike and to PRV gD by immunization with the VLPs, also described hereinabove. Thus, immune responses can be elicited both against proteins which are encoded by the altered genome of the VLP and/or against proteins which have been incorporated as targeting means in the VLP, thereby partly or wholly replacing the original spike protein. The examples illustrate the applicability of the approach for the induction of immune responses against proteins as diverse as for instance viral, bacterial, parasitic, cellular and hormonal origins.
The invention also provides VLPs which have fully maintained the original spike protein, but which are altered genomically to attenuate the VLP and/or to encode nucleotide sequences that need to be delivered at the cells to which the original coronavirus was targeted. For example, in this way, intestinal epithelial cells, or respiratory epithelial cells, that are normally infected by TGEV, or PRCV, respectively, can now interact with VLPs derived from TGEV or PRCV, or other cell-specific coronaviruses if needed, to express proteins normally not expressed by the viruses. In this way, respiratory epithelial cells of cystic fibrosis patients can for instance be induced to express lung surfactant molecules that are encoded by the altered genome of the VLP. To further demonstrate the invention, various examples are provided in the detailed description which is not limiting the invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
B. The nucleotide sequences are shown of the new junctions created in the plasmid constructs (and the resulting viruses), the positions and numbers of which are indicated in part A (left side).
B. RT-PCR analysis of recombinant viruses carrying the RL gene. cDNA was prepared using primer 1412 by RT on viral RNA; for each virus two independently derived viruses (designated by the extensions A1A and B1A; A2 and B1; A7A and B2D) were analyzed in parallel. Subsequently, PCR was carried out on the resulting cDNA as well as on the plasmids used to generate the viruses (see
A. Growth curves of different viruses having the renilla luciferase gene as compared to MHV-WT.
B. The growth of two independently obtained clones of MHV-EFLM virus carrying the firefly luciferase gene is shown as compared to MHV-WT.
B. Plasmid pBRDI2 is a derivative of pBRDI1 in which the FIPV S gene has been replaced by a chimaeric S gene (designated mS) which encodes the MHV-A59 S ectodomain while having retained the sequences for the FIPV S protein transmembrane and endodomain. Plasmid pBRDI2 was obtained by substituting in pBRDI1 the SacI-AflII segment by the corresponding fragment of pTMFS1. The latter plasmid is a derivative of plasmid pTMFS (12) which contains the chimaeric gene sequence and into which a SacI-StuI fragment was cloned to extend the MHV S sequence at its 5′ end with sequences corresponding to the FIPV pol1B 3′ end.
A. The nucleotide sequence of pBRDI1 and pBRDI2 around the very 5′ end of the FIPV genome sequence: the XhoI site and the phage T7 sequence are followed by a G-triplet and subsequently by the FIPV sequence.
B. The sequence at the pol1A/pol1B junction.
C. The nucleotide sequence at the very 3′ end of the cDNA construct. The 3′ untranslated region (3′UTR) is followed by a stretch of adenine nucleotides and a NotI sequence.
D. Nucleotide sequence at the FIPV pol1B-MHV S transition in pTMFS1 and pBRDI2.
A. Schematic representation of the coronavirus MHV-A59 spike structure. The spike (S) glycoprotein contains an N-terminal signal sequence (SS) and a transmembrane domain (TM) close to its C-terminus. S is proteolytically cleaved (arrow) in an S1 and S2 subunit, which are non-covalently linked. S2 contains two conserved heptad repeat regions, HR1 and HR2, as indicated.
B. Sequence alignment of HR1 and HR2 domains of MHV-A59, HCV-OC43 (human coronavirus strain OC43), HCV-229E (human coronavirus strain 229E), FIPV and IBV (infectious bronchitis virus strain Beaudette). The alignment shows a remarkable insertion of exactly 2 heptad repeats (14 aa) in both HR1 and HR2 of HCV-229E and FIPV, which is present in S proteins of all group I viruses. The predicted hydrophobic heptad repeat a and d residues are indicated above the sequence. The frame shift of predicted heptad repeats in HR1 is caused by a stutter. Asteriks denote conserved residues. The amino acid sequences of the peptides HR1, HR1a, HR1b, HR1c and HR2 used in this study are presented in italics below the alignments. N-terminal residues derived from proteolytic cleavage site of the GST-fusion protein are between brackets.
FIGS. 29-31: RNA synthesis by recombinant MHVs. The genomes of the recombinant viruses are depicted at the top, their observed RNA expression patterns are shown below. Infected 17Cl1 cells were metabolically labeled with [33P]orthophosphate in the presence of actinomycin D essentially as described (4, 10). Samples of total cytoplasmic RNA, purified using Ultraspec reagent (Biotecx), were denatured with formaldehyde and formamide, separated by electrophoresis through 1% agarose containing formaldehyde, and visualized by fluorography. Note that the subgenomic RNA species in this figure are designated by their composition, rather than as RNA2 through RNA7, since the numerical designations would be ambiguous for the mutants.
Experimental data related to the patent:
- I. Mouse Hepatitis Virus (MHV), strain A59 (MHV-A59)
- 1. Generation of live attenuated viruses
- a. Construction of recombinant MHVs lacking genes
- b. Confirmation of the recombinant genotypes
- c. RNA synthesis by MHV deletion mutants
- d. Tissue culture growth phenotype
- e. Virulence of recombinant viruses in mice
- 2. Generation of recombinant viruses with rearranged gene order
- a. Construction of recombinant viruses with rearranged gene order
- b. RNA synthesis by MHV mutants with rearranged gene order
- C. Tissue culture growth phenotype
- d. Replication of recombinant viruses in mice
- 3. Generation of recombinant viruses expressing foreign genes
- a. Construction of recombinant viruses carrying reporter genes
- b. RNA synthesis by recombinant viruses
- c. Replication of viruses expressing renilla or firefly luciferase
- d. Expression of renilla and firefly luciferase in cell culture
- e. Maintenance of foreign genes during viral passage
- f. Expression of firefly luciferase in mice
- g. Generation of MHV expressing two foreign genes from one genome
- h. Generation of MHV expressing a chimaeric Spike-GFP gene
- 4. Inhibition of infection and of cell fusion by spike protein derived peptide
- 1. Generation of live attenuated viruses
- II. Feline Infectious Peritonitis Virus (FIPV), strain 79-1146
- 1. Generation of mFIPV, a feline coronavirus growing on murine cells
- a. Construction of a synthetic RNA transcription vector
- b. Generation of mFIPV by RNA recombination
- c. mFIPV protein analysis
- d. mFIPV growth characteristics
- 2. Generation of live attenuated FIPV vaccine by gene deletions
- a. Construction of synthetic RNA transcription vectors
- b. Generations of recombinant FIPVs lacking genes
- c. Genetic analysis of recombinant FIPVs lacking genes
- d. Growth characteristics of FIPVs lacking genes
- e. Virulence of recombinant viruses in cats
- f. Immune-response induced by recombinant viruses
- g. FIPV deletion viruses serve as attenuated, live vaccines
- 3. Insertion and expression of foreign genes
- a. Construction of recombinant viruses carrying reporter genes
- b. One-step growth of viruses
- c. Expression of renilla luciferase
- 4. Generation of multivalent FIPV-based vaccines
- a. Construction of a multivalent Feline leukemia virus (FeLV) vaccine based on FIPV vector
- b. Construction of a multivalent Feline immunodeficiency virus (FIV) vaccine based on a FIPV vector
- c. Construction of a multivalent Feline calicivirus (FCV) vaccine based on a FIPV vector
- d. Construction of a multivalent Feline panleucopenia virus (FPV) vaccine based on a FIPV vector
- e. Construction of a multivalent Feline herpes virus (FHV) vaccine based on a FIPV vector
- f. Construction of a multivalent FIPV serotype I and II vaccine based on a FIPV serotype II vector
- 5. Generation of recombinant viruses with rearranged gene order
- 6. Generation of FIPV based vaccines against canine pathogens
- a. Generation of FIPV based vaccine against canine distemper
- b. Generation of FIPV based vaccine against canine parvo disease
- c. Generation of FIPV based vaccine against infectious canine hepatitis
- d. Generation of FIPV based vaccine against hemorrhagic disease of pups
- 1. Generation of mFIPV, a feline coronavirus growing on murine cells
- III. Transmissible gastro-enteritis virus (TGEV)
- 1. Generation of a live attenuated vaccine against TGEV
- 2. Generation of multivalent TGEV-based vaccines
- a. Construction of a multivalent Porcine Parvovirus (PPV) vaccine based on a TGEV vector
- b. Construction of a multivalent swine influenza virus vaccine based on a TGEV vector
- C. Construction of a multivalent African swine fever virus vaccine based on a TGEV vector
- d. Construction of a multivalent Porcine circovirus type 2 vaccine based on a TGEV vector
- e. Construction of a multivalent Porcine respiratory and reproductive syndrome virus vaccine based on a TGEV vector
- 3. Generation of recombinant viruses with rearranged gene order
- IV. Avian Infectious Bronchitis Virus (IBV)
- 1. Generation of a live vaccine based on attenuated IBV
- 2. Generation of multivalent IBV-based vaccines
- aI. Construction of a multivalent vaccine based on an IBV vector that protects against more than one IBV serotype.
- aII. Construction of a multivalent vaccine based on an IBV vector that protects against Newcastle Disease.
- b. Construction of a multivalent vaccine based on an IBV vector that protects against Avian Influenza.
- c. Construction of a multivalent vaccine based on an IBV vector that protects against Chicken Anemia Virus (CAV) disease.
- d. Construction of a multivalent vaccine based on an IBV vector that protects against Avian reovirus disease.
- e. Construction of a multivalent vaccine based on an IBV vector that protects against Infectious Bursal Disease.
- f. Construction of a multivalent vaccine based on an IBV vector that protects against Marek's disease.
- g. Construction of a multivalent vaccine based on an IBV vector that protects against Infectious laryngotracheitis.
- 3. Generation of recombinant viruses with rearranged gene order
- V. Human Coronavirus (HCoV) strain 229E (HCoV-229E)
- 1. Generation of a live vaccine based on attenuated HCoV-229E
- 2. Generation of a live attenuated vaccine against HCoV strain OC43
- 3. Generation of multivalent HCoV-based vaccines
- a. Construction of a multivalent vaccine based on an HCoV vector that protects against Respiratory Syncytial Virus (RSV)
- b. Construction of a multivalent vaccine based on an HCoV vector that protects against rotavirus
- c. Construction of a multivalent vaccine based on an HCoV vector that protects against Norwalk-like viruses
- d. Construction of a multivalent vaccine based on an HCoV vector that protects against influenza virus
- 4. Generation of recombinant viruses with rearranged gene order
I. Mouse Hepatitis Virus (MHV), Strain A59 (MHV-A59)
I.1. Generation of Live Attenuated Viruses
General aim: establish whether deletion from the coronaviral (i.e. MHV-A59) genome of genes or gene clusters not belonging to the genes specifying the polymerase functions (ORF1a/1b) or the structural proteins N, M, E, and S, is tolerated and yields viable viruses even if these gene sequences are removed altogether; establish whether such deletions have an attenuating effect on the virus when inoculated into mice.
I.1.a. Construction of Recombinant MHVs Lacking Genes
Specific aim: generate MHV-A59 deletion mutants lacking genes 2a+HE (MHV-□Δ2aHE) genes 4a+4b+5a (MHV-□Δ45a), and genes 2a+HE+4a+4b+5a (MHV-min).
Approach: targeted RNA recombination (3, 9, 10) using fMHV, the MHV-A59 derivative infecting feline (FCWF) cells not murine (LR7) cells (7), and synthetic donor RNAs carrying the intended deletions (
Procedure: Transcription vectors for the production of synthetic donor RNAs were constructed from the plasmid pMH54 (7), which encodes a run-off transcript consisting of the 5′ end of the MHV-A59 genome (467 nt) fused to codon 28 of the HE gene and running to the 3′ end of the genome (
To generate the deletion mutant viruses, donor RNAs were transcribed from the (PacI-linearized) pMH54-derived plasmids and transfected by electroporation into feline FCWF cells that had been infected with fMHV. These infected and transfected cells were then plated onto a monolayer of mouse LR7 (7) cells. After 24 h of incubation at 37° C., progeny viruses were harvested by taking off the cell culture supernatant and candidate recombinants were selected by two rounds of plaque purification on LR7 cells.
Result: With each of the synthetic donor RNAs used, clear plaques were obtained.
Conclusion: Recombinant viruses had been obtained that had regained the ability to grow on murine cells.
I.1.b. Confirmation of the Recombinant Genotypes
Aim: Confirming by RT-PCR the genetic make-up of the recombinant viruses obtained.
Procedure and Results: Cloned recombinant viruses, one from each recombination experiment, were produced on LR7 cells, viral RNA was isolated and RT-PCR was done using standard methods on genomic RNA as shown in
Conclusion: The constructed viral mutants had the intended genetic deletions.
I.1.c. RNA Synthesis by MHV Deletion Mutants
Aim: Confirming the patterns of RNAs synthesized in cells infected by the mutant viruses.
Procedure: Infected 17Cl1 cells were metabolically labeled with [33P]orthophosphate in the presence of actinomycin D essentially as described (4, 10). Samples of total cytoplasmic RNA, purified using Ultraspec reagent (Biotecx), were denatured with formaldehyde and formamide, separated by electrophoresis through 1% agarose containing formaldehyde, and visualized by fluorography (
Result: For the recombinant MHV-WT the RNA pattern and the relative molar amounts of the six subgenomic (sg) RNA species and the genomic (g) RNA were very similar to those reported previously for MHV (10)(4)(5)(8), with one notable exception. The 4-5a/E-M-N sgRNA, which is usually denoted RNA4, was far more abundant than previously observed for this species in wild-type MHV (10)(4)(5)(8). This was presumably due to the three nucleotide changes at positions 13, 15, and 18 upstream of the consensus transcription regulatory signal, (5′AAUCUAAAC3′) that precedes gene 4 (
Conclusion: The results confirm the genotypes of the recombinant viruses and demonstrate their expected phenotypes at the RNA level.
I.1.d. Tissue Culture Growth Phenotype
Aim: Comparing the in vitro growth phenotypes.
Procedure and Results: Confluent LR7 cell monolayers grown in 35-mm dishes were infected with each recombinant virus (8 PFU/cell) and viral infectivity in culture media at different times post-infection (p.i.) was determined by titration on LR7 cells. TCID50 (50% tissue culture infective doses) values were calculated and plotted (
Conclusion: All deletion viruses multiply well in vitro although the deletion of genes 4 and 5a had a slightly negative effect.
I.1.e. Virulence of Recombinant Viruses in Mice
Aim: Establishing whether the genetic deletions affect viral virulence.
Procedure and Results: The recombinant viruses were characterized in their natural host, the mouse. As a first step, we determined the virulence of the recombinant viruses. An LD50 (50% lethal dose) assay was carried out by inoculating MHV-negative, C57B1/6 mice mice intracranially with four 10-fold serial dilutions (5×105-5×102) of recombinant viruses. Viruses were diluted using PBS containing 0.75% bovine serum albumin. A volume of 25 μl was used for injection into the left cerebral hemisphere. Five animals per dilution per virus were analyzed. LD50 values were calculated by the Reed-Muench method based on death by 21 days post-infection. Clearly, deletion mutant viruses were attenuated when compared to the recombinant MHV-WT. While the MHV-WT virus had an LD50 of 1.8×104 no LD50 could be derived for the deletion mutants. Although the animals inoculated with the higher doses showed some signs of illness, none of the animals infected with any of the deletion mutant viruses died up to input of 50,000 PFU/mouse. This implies that the LD50 for these viruses exceeds a value of 50,000 and may well be above 100,000.
Conclusion: Viruses with deletions of the sequences specifying the genes 2a+HE or genes 4a+4b+5a or of the combination of all these genes exhibit a significantly attenuated phenotype in mice. In other words, the non-essential genes of coronaviruses are not crucial for in vitro growth but determine viral virulence. The attenuation acquired by their deletion thus provides excellent viral vaccines and therapeutic vectors.
I.2. Generation of Recombinant Viruses with Rearranged Gene Order
General aim: establish whether the invariable order of the genes specifying the polymerase functions (ORF1a/1b) and the structural proteins S, E, M, and N in the coronaviral genome is essential for the viability of these viruses or whether rearrangement of this order is tolerated.
I.2.a. Construction of Recombinant Viruses with Rearranged Gene Order
Specific aim: generate MHV-A59 mutants in which the relative positions of structural protein genes in the MHV-A59 genome are changed by moving the M and/or E gene.
Approach: targeted RNA recombination using fMHV and synthetic donor RNAs carrying the intended rearrangements (
Procedure: Transcription vectors for the production of donor RNA for targeted recombination were constructed from plasmids pMH54 and pXH□2aHE (described above). In order to generate transcription vector pXHSM45N (
For the construction of pXHMSmN, first the ORFs 4, 5 and M were removed from pMH54 by restriction of this vector with enzymes Sse83871 and BssHII, followed by treatment with T4 DNA polymerase and religation of the remaining vector, which yielded pXH□45M5′. Next, the fragment resulting from treatment of pXH0302 with enzymes NheI and BssHII was cloned into pMH54 treated with the same enzymes, resulting in pXHMeN. Subsequently, the fragment obtained after restriction of pXHMeN with EcoRV was cloned into pXH1802 (described above), which was digested with HindIII and treated with Klenow fragment of DNA polymerase I, yielding pXH0305B. The fragment obtained by restriction of pMH54 with enzymes MluI and EcoRV was cloned into pB59 (2) treated with the same enzymes, which resulted in vector pXH2801. Next, the fragment resulting from the treatment of pXH2801 with restriction enzymes KpnI and PstI was treated with T4 DNA polymerase and cloned into pXH1802 treated with restriction enzyme HindIII and with Klenow fragment of DNA polymerase I, yielding pXH0806. Subsequently, the fragment obtained by digestion of pXH0305B with SpeI and AflII was cloned into pXH0806 treated with the same enzymes, resulting in pXH1506. Finally, pXHSmN was obtained by cloning the fragment resulting from restriction of pXH1506 with RsrII and AvrII into pXH□45M5′ treated with the same enzymes.
For the construction of transcription vector pXH1bMS, vector pXHMeN was restricted with EcoRV. The resulting fragment was removed and the vector was religated yielding pXH□M. Next, the fragment obtained by digestion of pXH0305B with RsrII and AvrII was cloned into pXH□M treated with the same enzymes, yielding pXH1bMS.
All constructs were confirmed by restriction and/or sequence analysis. They are depicted schematically in
Conclusion: The strict gene order of the coronaviruses is not an essential prerequisite for viability.
I.2.b. RNA Synthesis by MHV Mutants with Rearranged Gene Order
Aim: Confirming the patterns of RNAs synthesized in cells infected by the mutant viruses.
Procedure: Infected 17Cl1 cells were metabolically labeled with [33P}orthophosphate in the presence of actinomycin D essentially as described (4, 10). Samples of total cytoplasmic RNA, purified using Ultraspec reagent (Biotecx), were denatured with formaldehyde and formamide, separated by electrophoresis through 1% agarose containing formaldehyde, and visualized by fluorography (
Result: Coronaviruses express their genome via the generation of a 3′ co-terminal nested set of sg RNAs. Recombinant viruses with a rearranged genome organization are therefore predicted to synthesize patterns of viral RNAs that are distinctly different from that of the parent virus. For the reconstructed wild-type virus (MHV-WT) and for MHV-□2aHE, the RNA patterns and the amounts of the genomic (g) and sg RNA species appeared to be similar to those observed previously (
Conclusion: The results confirm the genotypes of the recombinant viruses and demonstrate their expected phenotypes at the RNA level.
I.2.c. Tissue Culture Growth Phenotype
Aim: Comparing the in vitro growth phenotypes of the mutant viruses.
Procedure and Results: Confluent LR7 cell monolayers grown in 35-mm dishes were infected with each recombinant virus (8 PFU/cell) and viral infectivity in culture media at different times post-infection was determined by titration on LR7 cells. TCID50 values were calculated and plotted (
Conclusion: Except for mutant virus MHV-SM45N, the gene rearrangement had no dramatic effect on their in vitro growth characteristics.
I.2.d. Replication of Recombinant Viruses in Mice
Aim: Establishing whether viruses with rearranged gene order are able to replicate in mice.
Procedure and Results: Eight weeks old, MHV-negative, female BALB/c mice were used in the experiment. Viruses were diluted in PBS and a total volume 100 μl (106 TCID50) was used for injection in the peritoneal cavity. Four animals per virus were inoculated. Mice were sacrificed and the livers were removed at day 4 post-infection. The livers were placed in 1.5 ml DMEM, weighed and then frozen at −80° C. until titred for virus. Virus titers were determined by plaque-assay on LR7 cell monolayers following homogenization of the organs. The replication of MHV-WT, MHV-□2aHE and MHV-MSmN was studied in their natural host, the mouse. While the 50% lethal dose of MHV-WT in mice was previously determined at 2.7×104 PFU, MHV-□2aHE was not virulent enough for a 50% lethal dose value determination (section I.1.e.). Therefore, we now decided to analyze the in vivo replication of the recombinant viruses. Mice inoculated intraperitoneally with 1×106 TCID50 were euthanized at day 4 post-infection and the viral replication in the liver was determined. The results are shown in
Conclusion: Viruses with a rearranged gene order, which lack the typical coronavirus genome organization, and viruses that lack ORFs 2a and HE are able to replicate in their natural host, the mouse.
I.3. Generation of Recombinant Viruses Expressing Foreign Genes
Aim: Establish whether foreign genes can be inserted at different positions in the viral genome, either as an additional gene or replacing deleted non-essential genes or in combination with a rearranged gene order; establish whether these genes are expressed and whether they are stably maintained during in vitro passage of the virus.
I.3.a. Construction of Recombinant Viruses Carrying Reporter Genes
Aim: Generate MHV-A59 viruses with foreign gene insertions.
Procedure: Several viruses were constructed containing a foreign reporter gene in their genome at different positions (see
As a first step, the MHV IGS was cloned in front of the RL gene. To this end, primer 1286 (5′-GGATACTAATCTAAACTTTAG-3′) (SEQ ID NO:_) and 1287 (5′-CTAGCTAAAGTTTAGATTAGATATCCTGCA-3′) (SEQ ID NO:_were annealed to each other and cloned into pRL-null (Promega) treated with NheI and PstI, resulting in pXH1909. For the construction of the transcription vector containing the RL gene between genes 2a and S (pXH22aRLS) the following steps were taken. Vector p96 (1) was restricted with HindIII and MluI and the resulting fragment was cloned into pXH1802 (described above) treated with HindIII and BssHII, yielding pXH2103. Next, the RL expression cassette was removed from pXH1909 by restriction with EcoRV and XbaI, treated with Klenow fragment of DNA polymerase I and cloned into pXH2103 digested with HindIII and treated with Klenow fragment, resulting in pXH2509A. Finally pXH22aRLS was constructed by cloning the fragment resulting from digestion of pXH2509A with RsrII and AvrII into pMH54 treated with the same enzymes.
For the construction of pXH2ERLM the same expression cassette, that was used to make pXH2509A, was cloned into pMH54 digested with EcoRV.
This same expression cassette was also cloned into pXHMeN (described above) treated with EcoRV, yielding pXH2ERLN. Subsequently, pXH2MRLN was constructed by cloning the fragment resulting from restriction of pXHMeN with EcoRV into pXH2ERLN treated with the same enzyme. pXHMSmNRL was constructed by cloning the renilla expression cassette into pXHMSmN (described above) treated with EcoRV.
For the construction of pXHEFLM, the FL gene was first cloned behind the same IGS as was used for the RL expression cassette. To this end, the luciferase gene was removed from pSP-Luc+(Promega) by restriction with AvrII and XbaI and cloned into pXH1909 treated with NheI and XbaI, yielding pXH2711. Subsequently, the FL expression cassette was cut out of pXH2711 by restriction with EcoRV and XbaI, treated with Klenow fragment of DNA polymerase I, and cloned into pMH54 restricted by EcoRV, resulting in pXHEFLM.
Plasmid pXHminFL was constructed by cloning the FL expression cassette into pXHmin (described above) digested with EcoRV. This plasmid lacks ORFs 2a/HE/4a/4b/5a and contains the FL gene between genes E and M.
After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the fMHV genome as described above. The resulting viruses were genetically confirmed by RT-PCR analysis.
Results of recombinant viruses containing the RL gene are shown in
Primer 1091 and primer 1173 correspond with the 3′end of gene 4b and gene 1b, respectively, while primer 1413 corresponds with the 5′ end of the RL gene. As positive controls, the appropriate transcription vectors were taken along. In all cases, PCR fragments were obtained of the same size as the positive controls, while the water control was negative. The observed (and predicted) fragment sizes were approx. 1,000 bp for MHV-2aRLS, approx. 670 bp for MBV-ERLM, and approx. 1,300 bp for MHV-MRLN (7B). For MHV-MSmNRL a PCR was performed with primer 1091 and primer 1413 and with primer 1173 and primer 1413. As positive controls, the appropriate transcription vector was taken along. In all cases, PCR fragments were obtained of the same size as the positive controls. The observed (and predicted) fragment sizes were approx. 670 bp for the PCR reaction with primers 1091 and 1413, and approx. 1,200 bp for the PCR reaction with primers 1173 and 1413 (7B) (note that in
Results of recombinant viruses containing the FL gene are shown in
Conclusion: Insertion of genetic modules into the coronaviral genome is tolerated at all positions tested; all the intended viruses were viable.
I.3.b. RNA Synthesis by Recombinant Viruses
Aim: Confirming the patterns of RNAs synthesized in cells infected by the mutant viruses.
Procedure: Infected 17Cl1 cells were metabolically labeled with [33P]orthophosphate in the presence of actinomycin D essentially as described (4, 10). Samples of total cytoplasmic RNA, purified using Ultraspec reagent (Biotecx), were denatured with formaldehyde and formamide, separated by electrophoresis through 1% agarose containing formaldehyde, and visualized by fluorography (
Result: For all the MHV mutants with the luciferase genes, all variant sg RNAs had mobilities that corresponded to their predicted sizes (
For the viruses containing the firefly luciferase gene two additional RNA species were observed, which correspond in size with transcription of sgRNAs from sequences in the firefly luciferase gene that are similar to the MHV IGS. Overall, the patterns of viral RNAs synthesized by the cells infected with the recombinant viruses nicely reflect the insertion of the luciferase expression cassettes into the coronavirus genome.
Conclusion: The results confirm the genotypes of the recombinant viruses and demonstrate their expected phenotypes at the RNA level.
I.3.c. Replication of Viruses Expressing Renilla or Firefly Luciferase
Aim: Compare the growth characteristics of the viruses with wild-type virus and with each other.
Procedure and Results: After two rounds of plaque purification virus stocks were prepared, titrated and used for high m.o.i (m.o.i. of 8) infection of LR7 cells after which the viral infectivities in the culture media were monitored. The results are represented by the growth curves shown in
Conclusion: The inserted expression cassettes hardly affected the in vitro growth characteristics of the recombinant viruses.
I.3.d. Expression of Renilla and Firefly Luciferase in Cell Culture
Aim: Establish whether the inserted expression cassettes were functional.
Procedure and Results: Confluent monolayer cultures of LR7 cells were infected at a m.o.i. of 8 and the production of luciferase activity in the cells was monitored over time. RL expression in cells was measured by using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Similarly, FL expression was measured by using the Luciferase Assay System (Promega) according to the manufacturer's instructions. RL and FL activity was measured in relative light units (RLU) using a luminometer (Lumac Biocounter M2500 or Turner Designs Model TD-20/20).
The results are shown graphically in
Conclusion: Foreign genes can be expressed by coronaviruses both by the additional insertion of such a gene, by using the genetic space created by deletion of nonessential genes, or in combination with a rearranged genome organization.
I.3.e. Maintenance of Foreign Genes During Viral Passage
Aim: Evaluate the stability of the inserted luciferase genes during viral passage.
Procedure and Results: The recombinant viruses MHV-ERLM and MHV-EFLM were passaged 8 times over LR7 cells at low m.o.i. (<0.05). After each passage the viral infectivity (TCID50) in the culture medium at 9 h post-infection was determined. Subsequently, the firefly and renilla luciferase activity was determined in a parallel expression experiment (m.o.i.=5) at 8 hr post-infection (
Conclusion: A foreign gene can be stably maintained in the coronaviral genome for at least 8 passages in vitro.
I.3.f. Expression of Firefly Luciferase in Mice
Aim: Establish whether the inserted luciferase gene is expressed in the natural host, the mouse.
Procedure and Results: Eight weeks old, MHV-negative, female BALB/c mice were used in the experiment. Mice were inoculated intranasally with 106 TCID50 MHV-EFLM. Four animals per virus were used. Mice were sacrificed and the livers and brains were removed at day 4 post-infection. Organs were quick-frozen in liquid nitrogen and homogenized in Cell Culture Lysis Reagent provided with the Luciferase Assay System (Promega). FL activity was measured according to the manufacturer's instructions using a luminometer (Lumac Biocounter M2500). Clearly, as shown in
Conclusion: A foreign gene can be expressed by coronaviruses in their natural host.
I.3.g. Generation of MHV Expressing Two Foreign Genes From One Genome
Aim: Establish whether two foreign genes can be expressed from a single genome.
Procedure and Results: pXH2aRLSEFLM was generated by cloning the FL expression cassette into pXH2aRLS digested with EcoRV. After confirmation of the construct by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the fMHV genome as described above. The resulting virus, MHV-RLFL, was genetically confirmed by RT-PCR analysis. Both renilla luciferase activity and firefly luciferase activity could be detected in individual plaques. After generation of a high titer stock, confluent monolayer cultures of LR7 cells were infected at a m.o.i. of 8 and the production of luciferase activity in the cells was monitored over time. RL expression in cells was measured by using the Renilla Assay System (Promega) according to the manufacturer's instructions. Similarly, FL expression was measured by using the Luciferase Assay System (Promega) according to the manufacturer's instructions at 8 hr post-infection. RL and FL activity was measured in relative light units (RLU) using a luminometer (Lumac Biocounter M2500). The results show that MHV-RLFL replicated to the same extent as MHV-2aRLS and MHVEFLM (
Conclusion: Two foreign genes can be expressed from a single coronavirus genome.
I.3.h. Generation of MHV Expressing a Chimaeric Spike-GFP Gene
Aim: Establish whether recombinant MHV can be generated that expresses and incorporates chimaeric spike-GFP proteins.
Procedure and Results: The GFP gene was cloned in frame with the S gene in pMH54. After confirmation of the construct by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the fMHV genome as described above. The resulting virus, MHV-SGFP, was genetically confirmed by RT-PCR analysis. Plaques were microscopically analyzed and showed expressing of GFP as evidenced by the green fluorescence. Immunoprecipitation analysis indicated that hybrid proteins were generated that could be precipitated with specific antibodies to MHV-S and GFP.
Conclusion: MHV can be generated that expresses a chimaeric S-GFP gene in stead of a wild-type S gene. This mutant virus is viable and could be propagated in cell culture indicating that these hybrid proteins are incorporated into the viral particle.
I.4. Inhibition of Infection and of Cell Fusion by Spike Protein Derived Peptide
General aim: inhibit coronaviral infection and the spread of an ongoing infection by interfering with membrane fusion using peptides.
Specific aim: produce a peptide constituting a sequence derived from the membrane-proximal heptad repeat region (HR2) of the MHV-A59 S protein and demonstrate its inhibitory effect on MHV-A59 entry into LR7 cells and on cell-cell fusion in an infected culture of these cells.
Procedures: a. plasmid constructions:
A PCR fragment from a template plasmid pTUMS (13) containing the MHV-A59 spike gene was obtained, corresponding to amino acid residues 1216-1254 (HR2) of the S protein (
b. bacterial protein expression and purification:
Freshly transformed BL21 cells (NOVAGEN) were grown in 2YT medium to log phase (OD600 of 1.0) and subsequently expression was induced by adding IPTG (GibcoBRL) to a final concentration of 0.4 mM. Two hours after the start of induction the cells were pelleted, resuspended in {fraction (1/25)} of culture volume 10 mM Tris pH (8.0), 10 mM EDTA, 1 mM PMSF and sonicated on ice (5 times for 2 min with 1-min intervals). Cell lysates were centrifuged at 20,000×g for 60 min at 4° C. Then, 2 ml glutathione-sepharose 4B (50% v/v in PBS) was added per 50 ml of supernatant and the suspension was incubated overnight (O/N) at 4° C. under rotation. Beads were washed three times with 50 ml PBS and resuspended in a final volume of 1 ml PBS. Peptides were cleaved from the GST moiety on the beads using 20 U of thrombin by incubation for 4 hours at room temperature (RT). Peptides in the supernatant were HPLC purified on a Phenyl column with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. Peptide containing fractions were vacuum-dried O/N and dissolved in water. Peptide concentration was determined by measuring the absorbance at A280 nm or by BCA protein analysis (Micro BCA™ Assay Kit, PIERCE).
c. virus-cell entry and cell-cell fusion inhibition assays:
The potency of the HR2 peptide in inhibiting viral infection was determined using the recombinant MHV-EFLM expressing the firefly luciferase. Confluent monolayers of LR7 cells in 96 wells plates were inoculated at 37° C. in DMEM at a multiplicity of infection of 5 for 1 h in the presence of varying concentrations of peptide ranging from 0.4-50μM. After 1 h, cells were washed with DMEM and medium was replaced by peptide-free DMEM. At 5 h post infection cells were lysed for 15 minutes at RT in 50 μl Lysis buffer, according to the manufacturer's protocol (Luciferase Assay System, Promega). Upon mixing of 10 μl cell lysate with 40 μl substrate, luciferase activity was measured immediately using a Wallace Betaluminometer. The 50% effective inhibitory concentration (EC50 value) was calculated by fitting the inhibition data to an equilibrium binding equation: % luciferase activity=100/(1+(C/EC50). The ability of the peptide HR2 to inhibit spike mediated cell-cell fusion was determined using a plaque assay. Monolayers of LR7 cells in 6 wells plates were inoculated with 50 PFU of MHV-A59 in DMEM at 37° C. After one hour the cells were washed with DMEM and an agar overlay was added containing the HR2 peptide at 50, 10, 2, 0.4 and 0.08 μM concentrations. Plaques were counted at 48 h p.i., after staining and fixing with 0.9% formaldehyde/0.75% crystal violet.
Results: The potency of the HR2 peptide to inhibit virus entry was tested using a virus expressing a luciferase reporter gene as this allows extremely sensitive detection of infection. Inoculations of cells with the virus were carried out in the presence of different concentrations of HR2 peptide. After 1 h of inoculation, cells were washed and incubated further in culture medium in the absence of peptide. At 4 h p.i., before syncytium formation normally takes place, cells were lysed and tested for luciferase activity (
The specificity of the inhibition was demonstrated in all these assays by testing in parallel the effect of other peptides (
Conclusion: The HR2 peptide is a potent inhibitor both of virus entry into cells and of MHV-A59 spike mediated cell-cell fusion.
II. Feline Infectious Peritonitis Virus (FIPV), Strain 79-1146
II.1. Generation of mFIPV, a Feline Coronavirus Growing on Murine Cells
General aim: To set up a targeted RNA recombination system for feline coronavirus FIPV 79-1146 similar to the one described above for the genetic manipulation of the murine coronavirus MHV-A59.
Specific aim: Generate mFIPV, a FIPV derivative in which the spike protein ectodomain has been replaced genetically by that of the MHV-A59 S protein, thereby shifting the tropism of the chimaeric virus to murine instead of feline cells.
I.1.a. Construction of a Synthetic RNA Transcription Vector
Aim: Prepare a plasmid construct from which synthetic donor RNA can be transcribed for targeted RNA recombination and which consists of sequences derived from the 5′ end of the FIPV genome fused to sequences derived from the 3′ part of the viral genome, i.e. all sequences downstream of (and including) the 3′-terminal end of the ORFIB. Also: prepare a derivative of this plasmid in which the sequence encoding the spike ectodomain has been replaced by that encoding the corresponding domain of the MHV-A59 spike protein.
Procedure and Results: Using standard DNA cloning techniques the vector pBRDI1 was constructed which contains a cDNA copy of the 5′-most 702 bases ligated to the 3′-most 9.262 bases of the FIPV 79-1146 genome (
Plasmid pBRDI1 was subsequently used to prepare pBRDI2 in which the FIPV S gene was replaced by a chimaeric spike gene (mS) composed of a part encoding the ectodomain of the MHV spike protein and a part encoding the transmembrane and endodomain of the FIPV spike protein. To introduce this hybrid gene into pBRDI1, first the 3′ end of the feline pol1B gene was fused to the 5′ end of the murine spike gene (see
II.1.b. Generation of mFIPV by RNA Recombination
Aim: Generate mFIPV, an FIPV derivative targeted to murine cells.
Procedure and Results: Capped, run-off donor RNA transcripts were synthesized from NotI-linearized pBRDI2 using a T7 RNA polymerase kit (Ambion) according to the instructions of the manufacturer. The transcripts were introduced into feline FCWF cells (80 cm2 culture flask) that had been infected before with FIPV 79-1146 (m.o.i. of 1), by electroporation (Gene pulser electroporation apparatus, Biorad, 2 consecutive pulses; 0.3 kV/960 microF). The electroporated cells were cocultured in a 25 cm2 flask with murine LR7 cells (50% confluency) to allow recombination of the synthetic and genomic RNA (
Conclusion: A virus with the intended murine cell tropism was obtained.
II.1.c. mFIPV Protein Analysis
Aim: Confirm the identity of mFIPV at the level of viral protein synthesis.
Procedure and Results: Murine LR7 cells were infected with mFIPV and the proteins were labeled with 35S-labeled amino acids for 2 h starting at 5 h p.i. As controls, we infected LR7 and FCWF cells with MHV and FIPV, respectively, and labeled them similarly from 5-7 h p.i. After the labeling, cell lysates were prepared and immunoprecipitations were carried out in the presence of detergent as described (12). The following antibodies were used (for references, see 12): G73 (αFIPV), an ascitis fluid obtained from an FIPV-infected cat (provided by H. Vennema); K134 (αMHV), a rabbit serum raised against purified MHV-A59; WA3.10 (αSm), a Mab against an epitope present in the MHV-A59 S ectodomain; 23F4.5 (αSf), a Mab against an epitope in the FIPV S ectodomain. The immunoprecipitated proteins were taken up in electrophoresis sample buffer and heated for 2 min at 95° C. except for one protein sample (lane 4 in
Conclusion: Cells infected by mFIPV express the predicted viral proteins, particularly the hybrid S protein with the MHV-derived ectodomain.
II.1.d. mFIPV Growth Characteristics
Aim: Compare titers obtained for mFIPV with those for FIPV and MHV-A59.
Procedure and Results: Infection and titration experiments revealed that the recombinant virus mFIPV was no longer able to infect feline FCWF cells but did grow efficiently in murine LR7 cells showing similar growth characteristics as MHV-A59. This is demonstrated, for instance, by a comparison of their one-step growth curves in these cells shown in
Conclusion: The replacement of the spike protein ectodomain has resulted in a recombinant mFIPV that replicates well in its “new” host cells and has apparently not lost much of its biological fitness.
II.2. Generation of Live Attenuated FIPV Vaccine by Gene Deletions
General aim: Delete gene sequences from the FIPV genome that are non-essential for viral replication in vitro to obtain deletion viruses that are attenuated in feline animals and are therefore viral (vector) vaccine candidates.
II.2.a. Construction of Synthetic RNA Transcription Vectors
Aim: Construct the plasmids for the synthesis of donor RNA transcripts lacking the genes 3ABC and/or 7AB for targeted recombination with mFIPV RNA (
Procedure and Results: Deletions of genes 3ABC and 7AB were introduced into the plasmid pBRDI1 using SOE-PCR. For the primers used, see Table 2 and
To delete the 3ABC cluster, combinations of primers 1 and 4 and of 2 and 3 were used to generate fragments of 375 bp (A) and 1012 bp (B), respectively (
To delete the 7AB genes, combinations of primers 5 and 8 and of primers 6 and 7 were used to generate fragments of 1215 bp (D) and of 324 bp (E), respectively (
To construct pBRDI1Δ3ABC+Δ7AB, the 1524 bp MluI/NotI fragment of pBRDI1Δ7AB was introduced into MluI/NotI-digested pBRDI1Δ3ABC (
Conclusion: The pBRDI1-derived donor RNA constructs lacking the gene clusters 3ABC and/or 7AB were obtained.
II.2.b. Generations of Recombinant FIPVs Lacking Genes
Aim: Generate the recombinant FIPVs that lack the sequences for the 3ABC genes, for the 7AB genes, and for both these gene clusters.
Procedure and Results: Capped, run-off donor transcripts were synthesized from NotI-linearized pBRDI1, pBRDI1Δ3ABC, pBRDI1Δ7AB and pBRDI1Δ3ABC+Δ7AB, respectively, using a T7 RNA polymerase kit (Ambion) as specified by the manufacturer. The donor transcripts were introduced into murine LR7 cells (80 cm2 flask), that had been infected before with mFIPV (m.o.i. of 0.4), by electroporation (Gene pulser electroporation apparatus, Biorad, 2 consecutive pulses; 0.85 kV/50 microF). The electroporated cells were cocultured in a 25 cm2 culture flask with feline FCWF cells (50% confluency). After 24 h incubation at 37° C. massive syncytia could be detected in both the murine LR7 cells and the feline FCWF cells. Candidate deletion viruses released into the mixed cell culture supernatant were purified by two rounds of plaque purification on FCWF cells.
Conclusion: Viruses that had acquired the ability to grow in feline FCWF cells had been obtained from the recombination experiment with mFIPV.
II.2.c. Genetic Analysis of Recombinant FIPVs Lacking Genes
Aim: Confirm the genetic make-up of the putative deletion viruses.
Procedure and Results: To evaluate whether the intended deletions indeed occurred in the various FIPV deletion mutants, RT-PCR on the genomic viral RNA's was performed focusing on the 3ABC and the 7AB region. The primers used and DNA sizes expected are indicated in Table 2 and
Conclusion: The deletion viruses had precisely the intended genomic deletions.
II.2.d. Growth Characteristics of FIPVs Lacking Genes
Aim: Check cell tropism of the deletion viruses and evaluate their in vitro growth.
Results: All 4 recombinant viruses were inoculated onto mouse LR7 cells but failed to produce any cytopathic effects. They did, however, grow efficiently in feline FCWF cells, as expected. The viruses r-wtFIPV, FIPVΔ3ABC and FIPVΔ7AB reached titers that were similar to those obtained with the parent FIPV strain 79-1146 on these cells. The titers were all in the order of 5.107 PFU/ml (
Conclusion: The sequences comprising the gene clusters 3ABC and 7AB are not essential for the viability of FIPV. Apparently, neither the nucleotide sequences nor the proteins encoded by the genes are essential for the replication of the virus.
II.2.e. Virulence of Recombinant Viruses in Cats
Aim: Establishing whether the genetic deletions affect virulence.
Procedure and Results: The recombinant deletion viruses were characterized in their natural host, the cat. To this purpose, 24 SPF cats (5 months old) were placed into 5 groups and inoculated oronasally (100 pfu) with FIPV strain 79-1146 (n=4), r-wtFIPV (n=5), FIPVΔ3ABC (n=5), FIPVΔ7AB (n=5) and FIPVΔ3ABC+Δ7AB (n=), respectively, and followed for (at least) 3 months. Clinical disease signs were scored as shown in Table 5. Inoculation of the cats with the deletion variants did not induce clinical signs of disease. Rather, all cats remained totally healthy throughout the experiment (Table 6 and
Conclusions: 1) FIPV 79-1146 and its wild type recombinant equivalent r-wtFIPV are equally virulent; and 2) Viruses with deletions of the sequences specifying the genes 3abc or 7ab or of the combination of some or all these genes exhibit a significantly attenuated phenotype in cats.
II.2.f. Immune Response Induced by Recombinant Viruses
Aim: Determining the FIPV-neutralizing activity in cat sera.
Procedure and Results: FIPV-specific antibody responses of the cats inoculated with the deletion viruses were characterized. To this purpose, blood samples were obtained at days 0, 21 and 90 post infection, and heat-inactivated sera were prepared and incubated with FIPV 79-1146 (10.000 PFU) after which the FIPV-neutralizing activity was determined using FCWF cells in a 96-well microplate assay. Titers were expressed as the reciprocal of the lowest dilution that no longer inhibited viral cytopathic effects. As expected, at day 0 none of the cat sera showed a significant FIPV-neutralizing activity. At day 21, all cats had sero-converted and showed high titers of neutralizing antibodies. The titers observed in cats inoculated with FIPV 79-1146, r-wtFIPV, FIPVΔ3ABC and FIPVΔ7AB were comparable whereas the titers observed in FIPVΔ3ABC+Δ7AB infected cats were approximately 50 fold lower (
Conclusion: Despite the absence of clinical disease signs, high titers of neutralizing antibodies are observed in cats that had been inoculated with FIPV deletion variants. This strongly suggests that the deletion viruses are viable and replicate in the cat leading to a strong immune response in the form of an antibody response.
II.2.g. FIPV Deletion Viruses Serve as Attenuated, Live Vaccines
Aim: To study whether previous inoculation with the attenuated deletion variants protects the cats against a FIPV 79-1146 challenge.
Procedure and Results: The cats previously inoculated with the attenuated deletion variants were challenged oranasally with FIPV 79-1146 (100 pfu) at day 90. As a control group, 4 untreated cats of similar age were challenged identically. The control group showed a rapid onset of clinical disease characterized by depression, anorexia, jaundice, weight loss and leukopenia (day 7). A similar rapid onset of symptoms was observed with 3 out 5 cats previously inoculated with FIPVΔ3ABC+Δ7AB, whereas all cats previously infected with FIPVΔ3ABC or FIPVΔ7AB remained generally healthy and without typical FIP symptoms throughout the experiment (Table 7), although 2 out 5 cats previously inoculated with FIPVΔ3ABC showed temporary weight loss.
Due to advanced symptoms of FIP, one cat out of the control group had to be euthanized at day 32, whereas the other cats in the control group recovered from their initial FIP symptoms. A lethality score of 25% is lower than observed in the previous experiments. This is supposedly due to the advanced age of the cats which is known to lead to reduced susceptibility for FIPV. The three FIPVΔ3ABC+Δ7AB vaccinated cats with initial symptoms remained ill and were euthanized between days 11 and 56 (
Conclusion: Prior vaccination with FIPVΔ3ABC or FIPVΔ7AB protects cats against disease caused by a FIPV challenge.
II.3. Insertion and Expression of Foreign Genes
Aim: Establish whether foreign genes can be inserted at different positions in the viral genome, either as an additional gene or replacing deleted non-essential genes; and establish whether these genes are expressed and whether they are stably maintained during in vitro passage of the virus.
II.3.a. Construction of Recombinant Viruses Carrying Reporter Genes
Aim: Generate FIPV 79-1146 viruses with foreign gene insertions.
Procedure: A virus was constructed containing a foreign reporter gene in its genome at the position of the 3abc genes. The reporter gene, renilla luciferase (RL), was placed under the transcriptional control of the IGS preceeding gene 3a. To delete the 3abc cluster and introduce the RL gene into the plasmid pBRDI1, combinations of primers 1244 (5′-GCCATTCTCATTGATAAC-3′) (SEQ ID NO:_) and 1514 (5′-CTGAGTCTAGAGTAGCTAGCTAATGACTAATAAGTTTAG-3′) (SEQ ID NO:_) and of 1245 (5′-GCTTCTGTTGAGTAATCACC-3′) (SEQ ID NO:_) and 1513 (5′-GCTAGCTACTCTAGACTCAGGCGGTTCTAAAC-3′) (SEQ ID NO:_) were used to generate fragments of 336 bp (A) and 1068 bp (B) via PCR, respectively. In primer 1513 and 1514, the underlined and bold sequences represent a NheI and XbaI restriction site, respectively. Fragments A and B were fused using the overlap between both fragments through primers 1514 and 1513 and amplified using primers 1244 and 1245, using SOE-PCR, resulting in a 1384 bp fragment (C). Fragment C was cloned into the pGEM-T Easy vector (Promega), resulting in pGEM-C. The RL gene derived from pRL-null (Promega) was introduced as a NheI/XbaI fragment into NheI and XbaI digested pGEM-C, resulting in pGEM-C+luc. Fragment C+luc was introduced as a AflII/SnaBI fragment into AflII and SnaBI-digested pBRDI1, resulting in pBRDI1Δ3ABC+luc.
After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the mFIPV genome as described above. The resulting viruses were genetically confirmed by RT-PCR analysis.
Conclusion: The foreign reporter gene renilla luciferase can be placed into the genome of the type I coronavirus FIPV. The intended recombinant viruses are viable.
II.3.b. One-Step Growth of Viruses
Aim: Compare the growth characteristics of the viruses with wild-type virus.
Procedure and Results: After two rounds of plaque purification virus stocks of 2 independently obtained recombinants under II.3.a were prepared, titrated and used for high m.o.i (m.o.i. of 8) infection of FCWF cells after which the viral infectivities in the culture media were monitored. The results are represented by the growth curves shown in
Conclusion: The recombinant viruses with inserted expression cassette grew normally in vitro but their yields were affected.
II.3.c. Expression of Renilla Luciferase
Aim: Establish whether the inserted expression cassette was functional.
Procedure and Results: Confluent monolayer cultures of FCWF cells were infected at a m.o.i. of 8 and the production of luciferase activity in the cells was monitored over time. RL expression in cells was measured by using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. RL was measured in relative light units (RLU) using a luminometer (Lumac Biocounter M2500 or Turner Designs Model TD-20/20).
The results are shown graphically in
Conclusion: Foreign genes can be expressed by coronaviruses by using the genetic space created by deletion of nonessential genes.
II.4 Generation of Multivalent FIPV-Based Vaccines
Aim: Development of multivalent vaccines based on a live attenuated FIPV strain as a vector.
Approach: Genes (or gene fragments) from other feline and canine pathogens encoding antigens known to induce protective immunity against these pathogens were selected. Expression cassettes of these genes were introduced into the FIPV genome in combination with attenuating deletions of non-essential genes. The expression cassettes contain the FIPV TRS in front of (parts of) the gene to be expressed.
II.4.a. Construction of a Multivalent Feline Leukemia Virus (FeLV) Vaccine Based on FIPV Vector
Aim: Development of FeLV vaccine based on a live attenuated FIPV strain as a vector.
Procedure: (Parts of) the protection related FeLV genes gag and env were placed into expression cassettes and subsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the mFIPV genome as described above. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of the FeLV gag and env genes was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related FeLV genes gag and env can be introduced into and expressed by a live attenuated FIPV strain. These recombinants can therefore function as a multivalent vaccine against a Feline leukemia virus and FIPV infection.
II.4.b. Construction of a Multivalent Feline Immunodeficiency Virus (FIV) Vaccine Based on a FJPV Vector
Aim: Development of FIV vaccine based on a live attenuated FIPV strain as a vector.
Procedure: (Parts of) the protection related FIV genes gag and env were placed into expression cassettes and subsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the mFIPV genome as described above. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of the FIV gag and env genes was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related FIV genes gag and env can be introduced into and expressed by a live attenuated FIPV strain. These recombinant viruses can therefore function as a multivalent vaccine against a Feline immunodeficiency virus and FIPV infection.
II.4.c. Construction of a Multivalent Feline Calicivirus (FCV) Vaccine Based on a FIPV Vector
Aim: Development of FCV vaccine based on a live attenuated FIPV strain as a vector.
Procedure: (Parts of) the protection related FCV capsid gene was placed into an expression cassette and subsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the mFIPV genome as described above. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of the FCV capsid gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related FCV capsid gene can be introduced into and expressed by a live attenuated FIPV strain. These recombinant viruses can therefore function as a multivalent vaccine against a Feline calicivirus and FIPV infection.
II.4.d. Construction of a Multivalent Feline Panleucopenia Virus (FPV) Vaccine Based on a FIPV Vector
Aim: Development of FPV vaccine based on a live attenuated FIPV strain as a vector.
Procedure: (Parts of) the protection related R3 gene, encoding the VP1 and VIP2 capsid proteins was placed into an expression cassette and subsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the mFIPV genome as described above. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of (parts of) the R3 gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related R3 gene can be introduced into and expressed by a live attenuated FIPV strain. These recombinant viruses can therefore function as a multivalent vaccine against a Feline panleucopenia virus and FIPV infection.
II.4.e. Construction of a Multivalent Feline Herpes Virus (FHV) Vaccine Based on a FIPV Vector
Aim: Development of FHV vaccine based on a live attenuated FIPV strain as a vector.
Procedure: (Parts of) the protection related gB, gC, gD, gE, gH, gI, gK, gL, gM, gM, ICP0, ICP1 and ICP4 feline herpes virus genes were placed into an expression cassette and subsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the mFIPV genome as described above. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of the inserted FHP genes was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related gB, gC, gD, gE, gH, gI, gK, gL, gM, ICP0, ICP1 and ICP4 feline herpes virus genes can be introduced and expressed in a live attenuated FIPV strain. These recombinant viruses can therefore function as a multivalent vaccine against Feline herpes virus and FIPV.
II.4.f. Construction of a Multivalent FIPV Serotype I and II Vaccine Based on a FIPV Serotype II vector.
Aim: Development of a vaccine protecting both against serotype I and against II feline coronaviruses, based on a live attenuated FIPV serotype II strain as a vector.
Procedure: (Parts of) the protection related spike gene of a serotype I feline coronavirus of which the region encoding the signal sequence was deleted was placed into an expression cassette and subsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. For example: in one case a spike gene construct was used from which the region encoding the signal sequence had been deleted; in another case a truncated spike gene was used, from which the region encoding the transmembrane domain+endodomain had been deleted. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the mFIPV genome as described above. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of the inserted the serotype I spike gene construct was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related spike gene of feline coronavirus serotype I can be introduced and expressed in a live attenuated FIPV serotype II strain and can therefore function as a multivalent vaccine against both serotype I and II feline coronaviruses.
II.5. Generation of Recombinant Viruses with Rearranged Gene Order
Aim: Development of a FIPV vaccine in which deletion of non-essential genes is combined with a rearranged gene order by moving the N gene upstream of the S gene in the FIPV genome.
Procedure and Results: Capped, run-off donor transcripts were synthesized from NotI-linearized pBRDI1Δ3ABC, pBRDI1Δ7AB and pBRDI1Δ3ABC+Δ7AB vectors in which the N gene, together with its TRS, was inserted at a position upstream of the S. The donor transcripts were introduced into murine LR7 cells (80 cm2 flask), that had been infected before with mFIPV (m.o.i. of 0.4), by electroporation (Gene pulser electroporation apparatus, Biorad, 2 consecutive pulses; 0.85 kV/50 microF). The electroporated cells were co-cultured in a 25 cm2 culture flask with feline FCWF cells (50% confluency). After 24 h incubation at 37° C. massive syncytia could be detected in the cell cultures. Deletion mutant viruses with rearranged genomes released into the mixed cell culture supernatant were purified by two rounds of plaque purification on FCWF cells.
Conclusion: Deletion mutant FIPVs with rearranged gene order could be generated. These viruses can function as live attenuated FIPV vaccines. By virtue of their rearranged gene order these viruses represent safer vaccines because of their reduced ability to generate viable progeny by recombination with viruses circulating in the field.
II.6 Generation of FIPV Based Vaccines Against Canine Pathogens
General aim: Development of vaccines based on a live attenuated FIPV serotype II strain as a vector against canine pathogens.
Approach: Since type II feline coronaviruses express canine coronavirus-like spike proteins (2a), such feline coronavirus vectors can be used as vaccines in canines.
Therefore, the live attenuated FIPV vaccine that we developed can also be used for vaccination of canines against canine coronavirus (CCV). Furthermore, multivalent vaccines can be generated by introducing expression cassettes of protection related genes of other canine pathogens into the FIPV genome in combination with attenuating deletions of non-essential genes and with genome rearrangements. The expression cassettes contain the FIPV TRS in front of (parts of) the gene to be expressed.
II.6.a. Generation of FIPV Based Vaccine Against Canine Distemper
Aim: Development of canine distemper vaccine based on a live attenuated FIPV strain as a vector.
Procedure: (Parts of) the protection related H and F genes were placed into an expression cassette and subsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the mFIPV genome as described above. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of (parts of) the H and F genes was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related H and F genes of canine distemper virus can be introduced into and expressed by a live attenuated FIPV strain. These recombinant viruses can therefore function as a vaccine against canine distemper virus and CCV infection.
II.6.b. Generation of FIPV Based Vaccine Against Canine Parvo Disease
Aim: Development of canine parvovirus vaccine based on a live attenuated FIPV strain as a vector.
Procedure: (Parts of) the protection related R3 gene, encoding the VP1 and VIP2 capsid proteins were placed into an expression cassette and subsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the mFIPV genome as described above. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of (parts of) the R3 gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related R3 gene of canine parvovirus can be introduced into and expressed by a live attenuated FIPV strain. These recombinant viruses can therefore function as a vaccine against canine parvovirus disease and CCV infection.
II.6.c. Generation of FIPV Based Vaccine Against Infectious Canine Hepatitis
Aim: Development of canine adenovirus 1 vaccine based on a live attenuated FIPV strain as a vector.
Procedure: (Parts of) the protection related structural protein genes of canine adenovirus 1 were placed into an expression cassette and subsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the mFIPV genome as described above. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of (parts of) the protection related structural protein genes of canine adenovirus 1 was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related structural protein genes of canine adenovirus 1 can be introduced into and expressed by a live attenuated FIPV strain. These recombinant viruses can therefore function as a vaccine against infectious canine hepatitis and CCV infection.
II.6.d. Generation of FIPV Based Vaccine Against Hemorrhagic Disease of Pups
Aim: Development of canine herpes virus 1 vaccine based on a live attenuated FIPV strain as a vector.
Procedure: (Parts of) the protection related gB, gC, gD, gE, gH, gI, gK, gL, gM, gM, ICP0, ICP1 and ICP4 canine herpes virus genes were placed into an expression cassette and subsequently introduced into pBRDI1Δ3ABC and pBRDI1Δ7AB, respectively. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by RNA-RNA recombination between transcription vector run-off transcripts and the mFIPV genome as described above. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of the inserted genes was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related gB, gC, gD, gE, gH, gT, gK, gL, gM, ICP0, ICP1 and ICP4 canine herpes virus genes can be introduced and expressed in a live attenuated FIPV strain. These recombinant viruses can therefore function as a multivalent vaccine against canine herpes virus 1 and CCV.
III. Transmissible Gastro-Enteritis Virus (TGEV)
III.1. Generation of a Live Attenuated Vaccine Against TGEV
Aim: Development of a live attenuated vaccine against TGEV.
Approach: To this aim recombinant TGEV deletion mutant viruses were generated that lack the non-essential genes 3ab and/or 7.
Procedure: Recombinant viruses were generated as described by Almazan et al. (2000). From pBAC-TGEVFL, an infectious TGEV cDNA clone placed behind a CMV promoter in pBeloBAC11, the 3ab and 7 genes were deleted via standard cloning techniques, leaving the surrounding open reading frames and transcription regulatory sequences intact. Epithelial swine testis (ST) cells were transfected with this pBAC-TGEVFL derivative which lacks the 3ab and 7 genes (PBAC-TGEVFL). Recombinant TGEV viruses were plaque purified and characterized by RT-PCR for the absence of the 3ab and/or 7 genes. Large amounts of the recombinant TGEV deletion viruses were generated by infecting ST cells.
Conclusion: Recombinant TGEV was generated that lacked the genes 3ab and 7. These viruses can function as a live attenuated vaccine against TGEV.
III.2 Generation of Multivalent TGEV-Based Vaccines
Aim: Development of multivalent vaccines based on a live attenuated TGEV as a vector.
Approach: Expression cassettes of protection related genes of porcine pathogens were introduced into the TGEV genome in combination with attenuating deletions of non-essential genes and genome rearrangements. The expression cassettes contain the TGEV TRS in front of (parts of) the gene to be expressed.
III.2.a. Construction of a Multivalent Porcine Parvovirus (PPV) Vaccine Based on a TGEV Vector
Aim: Development of a vaccine based on a live attenuated TGEV strain as a vector. Procedure: (Parts of) the protection related R3 gene, encoding the VP1 and VP2 capsid proteins, was placed into an expression cassette and subsequently introduced into a pBAC-TGEVFL derivative which lacks genes 3ab and 7. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by transfecting ST cells with this pBAC-TGEVFL deletion derivative which contains (parts of) the protection related R3 gene. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of the inserted R3 gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related R3 gene can be introduced into and expressed by a live attenuated TGEV strain. These recombinant viruses can therefore function as a multivalent vaccine against a Porcine Parvovirus and TGEV infection.
III.2.b. Construction of a Multivalent Swine Influenza Virus Vaccine Based on a TGEV Vector
Aim: Development of a swine influenza virus vaccine based on a live attenuated TGEV strain as a vector.
Procedure: (Parts of) the protection related hemagglutinin (HA) and neuraminidase (NA) genes were placed into an expression cassette and subsequently introduced into a pBAC-TGEVFL derivative which lacks the genes 3ab and 7. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by transfecting ST cells with this pBAC-TGEVFL deletion derivative which contains (parts of) the protection related hemagglutinin (HA) and neuraminidase (NA) genes. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of the inserted hemagglutinin (HA) and neuraminidase (NA) genes was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related hemagglutinin (HA) and neuraminidase (NA) genes can be introduced into and expressed by a live attenuated TGEV strain. These recombinant viruses can therefore function as a multivalent vaccine against a swine influenza virus and TGEV infection.
III.2.c. Construction of a Multivalent African Swine Fever Virus Vaccine Based on a TGEV Vector
Aim: Development of an African swine fever virus vaccine based on a live attenuated TGEV strain as a vector.
Procedure: (Parts of) the protection related structural protein encoding genes were placed into an expression cassette and subsequently introduced into a pBAC-TGEVFL derivative which lacks the genes 3ab and 7. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by transfecting ST cells with this pBAC-TGEVFL deletion derivative which contains (parts of) the protection related structural-protein encoding genes. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of the structural-protein encoding genes was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related structural protein encoding genes can be introduced into and expressed by a live attenuated TGEV strain. These recombinant viruses can therefore function as a multivalent vaccine against an African swine fever virus and TGEV infection.
III.2.d. Construction of a Multivalent Porcine Circovirus Type 2 Vaccine Based on a TGEV Vector
Aim: Development of a Porcine circovirus type 2 vaccine based on a live attenuated TGEV strain as a vector.
Procedure: (Parts of) the protection related C1, C2, V1 and V2 genes of the Porcine circovirus type 2 were placed into an expression cassette and subsequently introduced into a pBAC-TGEVFL derivative which lacks the genes 3ab and 7. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by transfecting ST cells with this pBAC-TGEVFL deletion derivative which contains (parts of) the protection related C1, C2, V1 and V2 genes of the Porcine circovirus type 2. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of the C1, C2, V1 and V2 genes of the Porcine circovirus type 2 was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related C1, C2, V1 and V2 genes of the Porcine circovirus type 2 can be introduced into and expressed by a live attenuated TGEV strain. These recombinant viruses can therefore function as a multivalent vaccine against a Porcine circovirus type 2, and TGEV infection.
III.2.e. Construction of a Multivalent Porcine Respiratory and Reproductive Syndrome Virus Vaccine Based on a TGEV Vector
Aim: Development of a Porcine respiratory and reproductive syndrome virus vaccine based on a live attenuated TGEV strain as a vector.
Procedure: (Parts of) the protection related ORF2 to ORF7 of the Porcine respiratory and reproductive syndrome virus were placed into an expression cassette and subsequently introduced into a pBAC-TGEVFL derivative which lacks the genes 3ab and 7. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by transfecting ST cells with this pBAC-TGEVFL deletion derivative which contains (parts of) the protection related ORF2 to ORF7 of the Porcine respiratory and reproductive syndrome virus. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of ORF2 to ORF7 of the Porcine respiratory and reproductive syndrome virus was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related ORF2 to ORF7 of the Porcine respiratory and reproductive syndrome virus can be introduced into and expressed by a live attenuated TGEV strain. These recombinant viruses can therefore function as a multivalent vaccine against a Porcine respiratory and reproductive syndrome virus and TGEV infection.
III.2.e. Construction of a Multivalent Foot-And-Mouth Disease Virus Vaccine Based on a TGEV Vector
Aim: Development of a food-and-mouth disease virus vaccine based on a live attenuated TGEV strain as a vector.
Procedure: (Parts of) the protection related sequences encoding VP1 to VP4 of the foot-and-mouth disease virus were placed into an expression cassette and subsequently introduced into a pBAC-TGEVFL derivative which lacks the genes 3ab and 7. After confirmation of all constructs by restriction and sequence analysis, recombinant viruses were generated by transfecting ST cells with this pBAC-TGEVFL deletion derivative which contains (parts of) the protection related sequences encoding VP1 to VP4 of the food-and-mouth disease virus. The resulting viruses were genetically confirmed by RT-PCR analysis. Expression of the sequences encoding VP1 to VP4 of the foot-and-mouth disease virus was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related sequences encoding VP1 to VP4 of the foot-and-mouth disease virus can be introduced into and expressed by a live attenuated TGEV strain. These recombinant viruses can therefore function as a multivalent vaccine against a foot-and-mouth disease virus and TGEV infection.
III.3. Generation of Recombinant Viruses with Rearranged Gene Order
Aim: Development of a TGEV vaccine in which deletion of non-essential genes is combined with a rearranged gene order by moving the N gene upstream of the S gene in the TGEV genome.
Procedure and Results: Recombinant viruses were generated as described by Almazan et al. (2000). From pBAC-TGEVFL, an infectious TGEV cDNA clone placed behind a CMV promoter in pBeloBAC11, the 3ab and 7 genes were deleted via standard cloning techniques, leaving the surrounding open reading frames and transcription regulatory sequences intact. In addition the N gene was cloned to a position in the genome upstream of the S gene. Epithelial swine testis (ST) cells were transfected with this pBAC-TGEVFL derivative which lacks the 3ab and 7 genes and has a rearranged genome. Recombinant TGEV viruses were plaque purified and characterized by RT-PCR for the absence of the 3ab and/or 7 genes. Large amounts of the TGEV deletion recombinants were generated by infecting ST cells.
Conclusion: Recombinant TGEV was generated that lacked the genes 3ab and 7, and which has a rearranged gene order. These viruses function as a live attenuated vaccine against TGEV, as well as against other porcine pathogens when genes encoding relevant antigens are appropriately incorporated.
IV. Avian Infectious Bronchitis Virus (IBV)
IV.1. Generation of a Live Attenuated Vaccine Against IBV
Aim: Development of a live attenuated vaccine against IBV.
Approach: Recombinant IBV deletion mutant viruses were generated that lack the non-essential genes 3a/b and/or 5a/b. In order to acquire vaccine viruses that protect against multiple IBV serotypes, spike gene constructs derived from such different viruses were incorporated.
Procedure: Recombinant viruses were generated as described by Casais et al. (2001). Infectious IBV cDNA clones were assembled in the vaccinia virus genome by using sequential in vitro ligation of cDNA fragments derived from pFRAG1, pFRAG2, and pFRAG3 derived plasmids, followed by direct cloning into the genome of vaccinia virus vNotI/tk as described (1a). Genes 3a/b and 5a/b were removed from pFRAG3, by PCR mutagenesis leaving the surrounding open reading frames and transcription regulatory sequences intact, resulting in pFRAG3Δ. The S gene in pFRAG3Δ could be replaced by S genes derived from different IBV serotypes by using RT-PCR. Recombinant vaccinia viruses were generated by recombination between the fowl pox virus HP1.441 and the vNotI/tk-IBV in vitro ligation mixture. Recombinant viruses were plaque purified and characterized by PCR and Southern blot analysis. Finally, infectious IBV lacking genes 3a/b and/or 5a/b was generated as follows: Chick kidney (CK) cells were infected with recombinant fowl pox virus expressing T7 RNA polymerase. Subsequently cells were transfected with DNA isolated from the recombinant vaccinia virus containing the IBV cDNA clone lacking genes 3a/b and 5a/b. At 3 days post-infection the culture medium was collected and used to infect CK cells to generate large amounts of recombinant IBV. The recombinant viruses were genetically confirmed by RT-PCR analysis.
Conclusion: Recombinant IBV was generated that lacked the genes 3a/b and/or 5a/b. These viruses can function as a live attenuated vaccine against IBV. By replacing the S gene, recombinant IBVs could be obtained that function as live attenuated vaccines against different IBV serotypes.
IV.2. Generation of Multivalent IBV-Based Vaccines
Aim: Development of multivalent vaccine based on a live attenuated IBV strain as a vector.
Approach: Expression cassettes of protection related genes of chicken pathogens were introduced into the IBV genome in combination with attenuating deletion of non-essential genes. The expression cassettes contain the IBV TRS in front of (parts of) the gene to be expressed.
IV.2.aI. Construction of a Multivalent Vaccine Based on an IBV Vector That Protects Against More Than One IBV Serotype.
Aim: Development of a IBV vaccine based on a live attenuated IBV strain that lacks non-essential genes and protects against more than one serotype.
Procedure: (Parts of) the protection related IBV S gene from a different IBV serotype were placed into expression cassettes, and subsequently introduced into pFRAG3Δ. The largest construct contained a complete extra copy of the S gene except for the sequence encoding the signal peptide; another construct had a carboxy-terminally truncated S gene lacking the code for the transmembrane domain and endodomain. After confirmation of all the constructs by restriction and sequence analysis, recombinant viruses were generated as described above. The recombinant viruses were genetically confirmed by RT-PCR analysis. The proper expression of the heterologous S gene constructs was verified by immunoprecipitation analysis with type-specific antisera using radiolabeled recombinant virus infected cell lysates.
Conclusion: (Parts of) the protection related IBV S gene from a different IBV serotype can be introduced in and expressed from a live attenuated IBV strain. These recombinant viruses can therefore function as multivalent vaccines to protect against infection by more than one IBV serotype.
IV.2.aII. Construction of a Multivalent Vaccine Based on an IBV Vector that Protects Against Newcastle Disease.
Aim: Development of a Newcastle Disease Virus (avian paramyxovirus type I; APMV-1) vaccine based on a live attenuated IBV strain that lacks non-essential genes.
Procedure: (Parts of) the protection related APMV-1 genes HN and F were placed in expression cassettes, and subsequently introduced into pFRAG3Δ. After confirmation of all the constructs by restriction and sequence analysis, recombinant viruses were generated as described above. The recombinant viruses were genetically confirmed by RT-PCR analysis. Expression of the foreign gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related APMV-1 genes HN and F can be introduced in and expressed from a live attenuated IBV strain. The recombinant viruses can therefore function as a multivalent vaccine to protect against APMV-1 and IBV infections.
IV.2.b. Construction of a Multivalent Vaccine Based on an IBV Vector that Protects Against Avian Influenza.
Aim: Development of an Avian Influenza virus vaccine based on a live attenuated IBV strain that lacks non-essential genes.
Procedure: (Parts of) the protection related Avian Influenza genes H and N were placed into expression cassettes, and subsequently introduced into pFRAG3Δ. After confirmation of all the constructs by restriction and sequence analysis, recombinant viruses were generated as described above. The recombinant viruses were genetically confirmed by RT-PCR analysis. Expression of the foreign gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related Avian Influenza genes H and N can be introduced in and expressed from a live attenuated IBV strain. These recombinant viruses can therefore function as a multivalent vaccine to protect against Avian Influenza and IBV infections.
IV.2.c. Construction of a Multivalent Vaccine Based on an IBV Vector that Protects Against Chicken Anemia Virus (CAV) Disease.
Aim: Development of a CAV vaccine based on a live attenuated IBV strain that lacks non-essential genes.
Procedure: (Parts of) the protection related CAV genes V1, V2, and V3 were placed into expression cassettes, and subsequently introduced into pFRAG3Δ. After confirmation of all the constructs by restriction and sequence analysis, recombinant viruses were generated as described above. The recombinant viruses were genetically confirmed by RT-PCR analysis. Expression of the foreign gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related CAV genes V1, V2, and V3 can be introduced in and expressed from a live attenuated IBV strain. These recombinant viruses can therefore function as a multivalent vaccine to protect against CAV and IBV infections.
IV.2.d. Construction of a Multivalent Vaccine Based on an IBV Vector that Protects Against Avian Reovirus Disease.
Aim: Development of an avian reovirus vaccine based on a live attenuated IBV strain that lacks non-essential genes.
Procedure: (Parts of) the protection related avian reovirus genes Φ1, Φ2, and 83 were placed into expression cassettes, and subsequently introduced into pFRAG3Δ. After confirmation of all the constructs by restriction and sequence analysis, recombinant viruses were generated as described above. The recombinant viruses were genetically confirmed by RT-PCR analysis. Expression of the foreign gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related avian reovirus genes Φ1, Φ2, and 83 can be introduced in and expressed from a live attenuated IBV strain. These recombinant viruses can therefore function as a multivalent vaccine to protect against avian reovirus and IBV infections.
IV.2.e. Construction of a Multivalent Vaccine Based on an IBV Vector that Protects Against Infectious Bursal Disease.
Aim: Development of an Infectious Bursal Disease Virus (IBDV) vaccine based on a live attenuated IBV strain that lacks non-essential genes.
Procedure: (Parts of) the protection related IBDV gene VP2 were placed into expression cassettes, and subsequently introduced into pFRAG3Δ. After confirmation of all the constructs by restriction and sequence analysis, recombinant viruses were generated as described above. The recombinant viruses were genetically confirmed by RT-PCR analysis. Expression of the foreign gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related IBDV gene VP2 can be introduced in and expressed from a live attenuated IBV strain. These recombinant viruses can therefore function as a multivalent vaccine to protect against avian IBDV and IBV infections.
IV.2.f. Construction of a Multivalent Vaccine Based on an IBV Vector that Protects Against Marek's Disease.
Aim: Development of a Gallid herpes virus 2 (Marek's disease virus) vaccine based on a live attenuated IBV strain that lacks non-essential genes.
Procedure: (Parts of) the protection related Gallid herpes virus 2 gene gB were placed into expression cassettes, and subsequently introduced into pFRAG3Δ. After confirmation of all the constructs by restriction and sequence analysis, recombinant viruses were generated as described above. The recombinant viruses were genetically confirmed by RT-PCR analysis. Expression of the foreign gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related Gallid herpes virus 2 gene gB can be introduced in and expressed from a live attenuated IBV strain. These recombinant viruses can therefore function as a multivalent vaccine to protect against Gallid herpes virus 2 and IBV infections.
IV.2.g. Construction of a Multivalent Vaccine Based on an IBV Vector that Protects Against Infectious Laryngotracheitis.
Aim: Development of a Gallid herpes virus 1 (Infectious laryngotracheitis virus) vaccine based on a live attenuated IBV strain that lacks non-essential genes.
Procedure: (Parts of) the protection related Gallid herpes virus 1 genes gB, gC, gD, gE, gH, gI, gK, gL, and gM were placed into expression cassettes, and subsequently introduced into pFRAG3Δ. After confirmation of all the constructs by restriction and sequence analysis, recombinant viruses were generated as described above. The recombinant viruses were genetically confirmed by RT-PCR analysis. Expression of the foreign gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related Gallid herpes virus 1 genes can be introduced in and expressed from a live attenuated IBV strain. These recombinant viruses can therefore function as a multivalent vaccine to protect against Gallid herpes virus 1 and IBV infections.
IV.3. Generation of Recombinant Viruses with Rearranged Gene Order
Aim: Development of an IBV vaccine in which deletion of non-essential genes is combined with a rearranged gene order by moving the N gene upstream of the S gene in the IBV genome.
Procedure: Recombinant viruses were generated as described by Casais et al. (2001). Infectious IBV cDNA clones were assembled in the vaccinia virus genome by using sequential in vitro ligation of cDNA fragments derived from pFRAG1, pFRAG2, and pFRAG3 derived plasmids, followed by direct cloning into the genome of vaccinia virus vNotI/tk as described (1a). Genes 3a/b and 5a/b were removed from pFRAG3, by PCR mutagenesis leaving the surrounding open reading frames and transcription regulatory sequences intact. In addition, the N gene together with its TRS was moved to a position upstream of the S gene resulting in pFRAG3Δrearranged. Recombinant vaccinia viruses were generated by recombination between the fowlpox virus HP1.441 and the vNotI/tk-IBV in vitro ligation mixture. Recombinant viruses were plaque purified and characterized by PCR and Southern blot analysis. Finally, infectious IBV lacking genes 3a/b and 5a/b and with a rearranged gene order was generated as follows: Chick kidney (CK) cells were infected with recombinant fowl pox virus expressing T7 RNA polymerase. Subsequently cells were transfected with DNA isolated from the recombinant vaccinia virus containing the IBV cDNA clone lacking genes 3a/b and 5a/b in combination with the rearranged gene order. At 3 days post-infection the culture medium was collected and used to infect CK cells to generate large amounts of recombinant IBV. The recombinant viruses were genetically confirmed by RT-PCR analysis.
Conclusion: Recombinant IBV was generated that lacked the genes 3a/b and 5a/b in combination with a rearranged gene order. These viruses function as a live attenuated vaccine against IBV. When combined with S gene constructs from other IBV serotypes and/or gene constructs from other avian pathogens additional, multivalent vaccines can be generated.
V. Human Coronavirus (HCOV) Strain 229E (HCoV-229E)
V.1. Generation of a Live Vaccine Based on Attenuated HCoV-229E
Aim: Development of a live attenuated vaccine against HCoV-229E.
Approach: Recombinant HCoV deletion mutant viruses were generated that lack the non-essential genes 4a/b.
Procedure: Recombinant viruses were generated essentially as described by Thiel et al. (2001). Infectious HCoV cDNA clones were assembled in the vaccinia virus genome. Vaccinia virus vDHCoV-vec-1 contains a 22.5 kbp cDNA fragment encoding the 5′end of the HCoV genome. This fragment was removed from the vaccinia genome by digestion with Bsp 120I, digested with MluI and ligated to a cDNA fragment of pMEΔ that was obtained by digestion with MluI/EagI. pMEΔ contains a cDNA fragment that encodes the 3′end of the HCoV genome, but lacks gene 4a/b, without disturbing the other genes or transcription regulatory sequences. pMEΔ was derived from pME by using conventional PCR mutagenesis methods. The ligation products were ligated to vNotI/tk vaccinia virus DNA. Recombinant vaccinia viruses were generated as described above. Recombinant viruses were plaque purified and characterized by PCR and Southern blot analysis. Finally, infectious HCoV lacking genes 4a/b was generated as follows: Chick kidney (CK) cells were infected with recombinant fowl pox virus expressing T7 RNA polymerase. Subsequently cells were transfected with DNA isolated from the recombinant vaccinia virus containing the HCoV cDNA clone lacking genes 4a/b. At 3 days post-infection the culture medium was collected and used to infect human long fibroblast cells (MRC-5) to generate large amounts of recombinant HCoV. The recombinant viruses were genetically confirmed by RT-PCR analysis.
Conclusion: Recombinant HCoV was generated that lacked the genes 4a/b. These viruses can function as a live attenuated vaccine against HCoV-229E.
V.2. Generation of a Live Attenuated Vaccine Against HCoV Strain OC43
Aim: Development of a live attenuated vaccine against HCoV-OC43.
Approach: Recombinant HCoV deletion mutant viruses were generated that lack the non-essential genes 4a/b, and contain a hybrid S protein gene, which encodes the ectodomain of HCoV-OC43.
Procedure: Recombinant viruses were generated essentially as described by Thiel et al. (2001). Infectious HCoV cDNA clones were assembled in the vaccinia virus genome. Vaccinia virus vHCoV-vec-1 contains a 22.5 kbp cDNA fragment encoding the 5′end of the HCoV genome. This fragment was removed from the vaccinia genome by digestion with Bsp 120I, digested with MluI and ligated to a cDNA fragment of pMEΔ-SOC43 that was obtained by digestion with MluI/EagI. pMEΔ-SOC43 contains a cDNA fragment that encodes the 3′end of the HCoV genome, lacks gene 4a/b, and contains a hybrid S protein gene, without disturbing the other genes or transcription regulatory sequences. The hybrid S protein gene contains the region of the S gene of HCoV-OC43 that encodes the S protein ectodomain, while the remainder of the gene is derived from the HCoV-229E gene. pMEΔ-SOC43 was derived from pMEΔ by using conventional (RT-)PCR mutagenesis methods. The ligation products were ligated to vNotI/tk vaccinia virus DNA. Recombinant vaccinia viruses were generated as described above. Recombinant viruses were plaque purified and characterized by PCR and Southern blot analysis. Finally, infectious HCoV lacking genes 4a/b was generated as follows: Chick kidney (CK) cells were infected with recombinant fowl pox virus expressing T7 RNA polymerase. Subsequently cells were transfected with DNA isolated from the recombinant vaccinia virus containing the HCoV cDNA clone lacking genes 4a/b. At 3 days post-infection the culture medium was collected and used to infect human long fibroblast cells (MRC-5) to generate large amounts of recombinant HCoV. The recombinant viruses were genetically confirmed by RT-PCR analysis.
Conclusion: Recombinant HCoV was generated that lacked the genes 4a/b. These viruses contain the ectodomain of the S protein of HCoV-OC43 and therefore function as a live attenuated vaccine against HCoV-OC43.
V.3. Generation of Multivalent HCoV-Based Vaccines
Aim: Development of multivalent vaccine based on a live attenuated HCoV strain as a vector.
Approach: Expression cassettes of protection related genes of human pathogens were introduced into the HCoV genome in combination with attenuating deletion of non-essential genes. The expression cassettes contain the HCoV TRS (TCTCAACT) in front of (parts of) the gene to be expressed.
V.3.a. Construction of a Multivalent Vaccine Based on an HCoV Vector that Protects Against Respiratory Syncytial Virus (RSV)
Aim: Development of a RSV vaccine based on a live attenuated HCoV strain that lacks non-essential genes.
Procedure: (Parts of) the protection related RSV genes HN and F were placed into expression cassettes, and subsequently introduced into pMEΔ. After confirmation of all the constructs by restriction and sequence analysis, recombinant viruses were generated as described above. The recombinant viruses were genetically confirmed by RT-PCR analysis. Expression of the foreign gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related RSV genes HN and F can be introduced in and expressed from a live attenuated HCoV strain. These recombinant viruses can therefore function as a multivalent vaccine to protect against RSV and HCoV infections.
V.3.b. Construction of a Multivalent Vaccine Based on an HCoV Vector that Protects Against Rotavirus
Aim: Development of a rotavirus vaccine based on a live attenuated HCoV strain that lacks non-essential genes.
Procedure: (Parts of) the protection related rotavirus genes VP4, VP6 and VP7 were placed into expression cassettes, and subsequently introduced into pMEΔ. After confirmation of all the constructs by restriction and sequence analysis, recombinant viruses were generated as described above. The recombinant viruses were genetically confirmed by RT-PCR analysis. Expression of the foreign gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related rotavirus genes VP4, VP6 and VP7 can be introduced in and expressed from a live attenuated HCoV strain. These recombinant viruses can therefore function as a multivalent vaccine to protect against rotavirus and HCoV infections.
V.3.c. Construction of a Multivalent Vaccine Based on an HCoV Vector that Protects Against Norwalk-Like Viruses
Aim: Development of a Norwalk-like virus vaccine based on a live attenuated HCoV strain that lacks non-essential genes.
Procedure: (Parts of) the protection related Norwalk-like virus capsid gene was placed into expression cassettes, and subsequently introduced into pMEΔ. After confirmation of all the constructs by restriction and sequence analysis, recombinant viruses were generated as described above. The recombinant viruses were genetically confirmed by RT-PCR analysis. Expression of the foreign gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related Norwalk-like virus capsid gene can be introduced in and expressed from a live attenuated HCoV strain. This recombinant virus can therefore function as a multivalent vaccine to protect against Norwalk-like virus and HCoV infections.
V.3.d. Construction of a Multivalent Vaccine Based on an HCoV Vector that Protects Against Influenza Virus
Aim: Development of an influenza virus vaccine based on a live attenuated HCoV strain that lacks non-essential genes.
Procedure: (Parts of) the protection related influenza virus genes H and N were placed into expression cassettes, and subsequently introduced into pMEΔ. After confirmation of all the constructs by restriction and sequence analysis, recombinant viruses were generated as described above. The recombinant viruses were genetically confirmed by RT-PCR analysis. Expression of the foreign gene was confirmed by immunoprecipitation analysis.
Conclusion: (Parts of) the protection related influenza virus genes H and N can be introduced in and expressed from a live attenuated HCoV strain. These recombinant viruses can therefore function as a multivalent vaccine to protect against influenza virus and HCoV infections.
V.4. Generation of Recombinant Viruses with Rearranged Gene Order
Aim: Development of a HCoV vaccine in which deletion of non-essential genes is combined with a rearranged gene order by moving the N gene upstream of the S gene in the HCoV genome.
Procedure: Recombinant viruses were generated essentially as described by Thiel et al. (2001). Infectious HCoV cDNA clones were assembled in the vaccinia virus genome. Vaccinia virus vHCoV-vec-1 contains a 22.5 kbp cDNA fragment encoding the 5′end of the HCoV genome. This fragment was removed from the vaccinia virus genome by digestion with Bsp 120I, digested with MluI and ligated to a cDNA fragment of pMEΔ rearranged that was obtained by digestion with MluI/EagI. pMEΔ rearranged contains a cDNA fragment that encodes the 3′end of the HCoV genome, but lacks gene 4a/b, without disturbing the other genes or transcription regulatory sequences, and in which the N gene, together with its TRS, was positioned upstream of the S gene. pMEΔ rearranged was derived from pME by using conventional PCR mutagenesis methods. The ligation products were ligated to vNotI/tk vaccinia virus DNA. Recombinant vaccinia viruses were generated as described above. Recombinant viruses were plaque purified and characterized by PCR and Southern blot analysis. Finally, infectious HCoV lacking genes 4a/b was generated as follows: Chick kidney (CK) cells were infected with recombinant fowlpox virus expressing T7 RNA polymerase. Subsequently cells were transfected with DNA isolated from the recombinant vaccinia virus containing the HCoV cDNA clone lacking genes 4a/b in combination with the rearranged gene order. At 3 days post-infection the culture medium was collected and used to infect human long fibroblast cells (MRC-5) to generate large amounts of recombinant HCoV. The recombinant viruses were genetically confirmed by RT-PCR analysis.
Conclusion: Recombinant HCoV was generated that lacked the genes 4a/b in combination with a rearranged gene order. These viruses can function as a live attenuated vaccine against HCoV-229E. When combined with S gene constructs from HcoV-OC43 and/or gene constructs from other human pathogens additional, multivalent vaccines can be generated.
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Claims
1. An isolated or recombinant virus-like particle capable of replication, said isolated or recombinant virus-like particle derived from a coronavirus from which at least a functional fragment from a nucleic acid sequence encoding a viral gene product other than a polymerase or a structural protein N, M, E, or S, is deleted.
2. The isolated or recombinant virus-like particle of claim 1, wherein the coronavirus from which the isolated or recombinant virus-like particle has been derived is selected from the group consisting of:
- (a) of group I coronavirus origin and wherein the nucleic acid encodes gene product 3a, 3b, 3c, 7a or 7b;
- (b) of group II coronavirus origin and wherein the nucleic acid encodes gene product 2a, HE, 4a, 4b, or 5a; and
- (c) of group III coronavirus origin and wherein the nucleic acid encodes gene product 3a, 3b, 5a or 5b.
3. An isolated or recombinant virus-like particle capable of replication, said isolated or recombinant virus-like particle derived from a coronavirus wherein the coronavirus' genes for structural proteins do not occur in the order 5′-S-E-M-N-3′.
4. The isolated or recombinant virus-like particle of claim 3 from which at least a functional fragment from a nucleic acid encoding a viral gene product other than a polymerase or a structural protein N, M, E, or S, is deleted.
5. The isolated or recombinant virus-like particle of claim 1, wherein said isolated or recombinant virus-like particle has been provided with at least one biologically active protein or fragment thereof associated with the surface of said isolated or recombinant virus-like particle other than the natural ectodomain of any one protein of the coronavirus from which the isolated or recombinant virus-like particle has been derived.
6. The isolated or recombinant virus-like particle of claim 1, wherein said isolated or recombinant virus-like particle has been provided with at least one biologically active protein or fragment thereof associated with the inside of said isolated or recombinant virus-like particle other than the natural endodomain of any one protein of the coronavirus from which the isolated or recombinant virus-like particle has been derived.
7. The isolated or recombinant virus-like particle of claim 2, wherein said isolated or recombinant virus-like particle has been provided with at least one biologically active protein or fragment thereof associated with the surface of said isolated or recombinant virus-like particle other than the natural ectodomain of any one protein of the coronavirus from which the isolated or recombinant virus-like particle has been derived.
8. The isolated or recombinant virus-like particle of claim 2, wherein said isolated or recombinant virus-like particle has been provided with at least one biologically active protein or fragment thereof associated with the inside of said isolated or recombinant virus-like particle other than the natural endodomain of any one protein of the coronavirus from which the isolated or recombinant virus-like particle has been derived.
9. The isolated or recombinant virus-like particle of claim 2, wherein said isolated or recombinant virus-like particle has been provided with a at least one functional targeting means associated with the surface of said isolated or recombinant virus-like particle other than the natural spike protein of the coronavirus from which the isolated or recombinant virus-like particle has been derived.
10. The isolated or recombinant virus-like particle of claim 3, wherein said isolated or recombinant virus-like particle has been provided with at least one biologically active protein or fragment thereof associated with the surface of said isolated or recombinant virus-like particle other than the natural ectodomain of any one protein of the coronavirus from which the isolated or recombinant virus-like particle has been derived.
11. The isolated or recombinant virus-like particle of claim 4, wherein said isolated or recombinant virus-like particle has been provided with at least one biologically active protein or fragment thereof associated with the surface of said isolated or recombinant virus-like particle other than the natural ectodomain of any one protein of the coronavirus from which the isolated or recombinant virus-like particle has been derived.
12. The isolated or recombinant virus-like particle of claim 3, wherein said isolated or recombinant virus-like particle has been provided with at least one biologically active protein or fragment thereof associated with the inside of said isolated or recombinant virus-like particle other than the natural endodomain of any one protein of the coronavirus from which the isolated or recombinant virus-like particle has been derived.
13. The isolated or recombinant virus-like particle of claim 3, wherein said isolated or recombinant virus-like particle has been provided with a at least one functional targeting means associated with the surface of said isolated or recombinant virus-like particle other than the natural spike protein of the coronavirus from which the isolated or recombinant virus-like particle has been derived.
14. The isolated or recombinant virus-like particle of any one of claim 1, wherein said isolated or recombinant virus-like particle has been provided with a coronavirus genome comprising a foreign gene or part thereof.
15. The isolated or recombinant virus-like particle of claim 1, which is attenuated.
16. The isolated or recombinant virus-like particle of claim 1, which is a gene delivery vehicle.
17. The isolated or recombinant virus-like particle of claim 1, which is an antigen or epitope delivery vehicle.
18. A immunogenic composition comprising:
- the isolated or recombinant virus-like particle of claim 1, and
- a pharmaceutically acceptable carrier.
19. A composition for diagnostic use comprising:
- the isolated or recombinant virus-like particle of claim 1 presented in a manner capable of diagnostic determination.
20. A method for inhibiting or blocking an infection with a coronavirus or coronavirus-like particle, said method comprising:
- treating an organism with a heptad repeat peptide or functional fragment thereof as shown in FIG. 20.
Type: Application
Filed: Nov 14, 2003
Publication Date: Jun 16, 2005
Patent Grant number: 7556957
Inventors: Petrus Rottier (Groenkan), Cornelis De Haan (Houten), Bert Haijema (Utrecht), Berend-Jan Bosch (Utrecht)
Application Number: 10/714,534
