Adjuvant viral particle

p00001Adjuvant Viral Particle

p00002Technical field

p00003The present invention relates to a viral particle that carries immunogens and that has immunopotentiation or adjuvant activity. The invention particularly relates to recombinant viral particles, in particular for use in improving an immune response in a human being or an animal by means of these particles.

p00004Background Technique

p00005Vaccination is the most effective method to fight infectious diseases. The emergence of new viral diseases (for example, the Hepatitis C virus, the Human Immunodeficiency virus) and the resistance of pathogenic bacteria (Salmonella typhii) to antibiotics are alarming. Therefore, vaccination becomes an effective alternative to help control these diseases.

p00006In the last 15 years, genetic engineering has allowed the precise identification of protein fragments that are responsible for the protective immune response. Therefore, new vaccination strategies emerged. Immunization of animals with appropriate immunogenic peptides allowed the production of neutralizing antibodies that can control diseases. The expression of these immunogenic peptides in heterologous systems provided the basis for subunit vaccines.

p00007Although it has been shown that certain chemically synthesized oligopeptides are capable of stimulating the production of antibodies against the protein from which they are derived, it has generally been observed that the peptides themselves are insufficiently immunogenic to serve as vaccines. This is why there is considerable interest in developing epitope presentation systems in which the peptide sequence is fused to a transporter molecule capable of forming a macromolecular structure.

p00008Specific immunity can be enhanced by the use of immunopotentiators, such as adjuvants, when an antigen is administered to a host. The immune response is mediated by a variety of cells of the immune system. There are two types of immune response: humoral immunity mediated by antibodies, and cellular immunity mediated primarily by cytotoxic T lymphocytes. The antigen presenting cells ("APC") process and present the antigen to both B cells and T cells. B cells secrete specific antibodies as a result of activation and T cells or become auxiliary cells of the humoral response or in cytotoxic cells and directly attack the antigen. It has been shown that adjuvants increase these immune responses.

p00009The initial presentation of an antigen induces both IgM and IgG antibodies, which constitute the primary response. However, this antibody production may decline over time. The secondary response, which mainly involves the production of IgG antibodies, can be triggered by the presentation of the secondary or subsequent antigen in time. A secondary or even primary response, however, is not guaranteed merely by the presentation of the antigen to the host.

p00010A difficulty that frequently arises in the administration of an antigen is the extent to which the immune system will respond. Certain antigens are not very immunogenic because after administration they cause a weak primary response or do not cause any response. In those cases, the immune system may not respond to secondary exposure, and for example, the host may suffer the disease or condition that was intended to be prevented by immunization with the antigen.

p00011In these situations, it is common to administer a physiological response modulator (“PRM”). A PRM is generally defined as an immunopotentiating compound. It can come from bacteria, such as Bordella pertussis or Corynebacterium parvum. PRMs can also include chemicals such as polynucleotides, physiologically active molecules such as thymic hormones and adjuvants.

p00012Adjuvants are compounds that enhance the response of immune systems when administered with an antigen, producing higher antibody titers and a prolonged host response. Commonly used adjuvants include Freund's Incomplete Adjuvant, which consists of a water-in-oil emulsion, Freund's Complete Adjuvant, comprising the former with the addition of Mycobacterium tuberculosis, and alum. The difficulty, however, of using these materials in humans, for example, is that they are toxic or can cause the host to develop lesions at the injection site.

p00013Kawamura and Berzofsky described another approach in J. Immunol., 136: 58 (1986). In this approach, anti-Ig antibodies were conjugated, which are reactive with the immunoglobulins present in certain B cells, with ferritin and myoglobin, and were administered to mice with Freund's Incomplete Adjuvant. The immunogenicity of the mixture improved, but there was no evidence of the immunogenicity of the sample without the addition of the adjuvant. In addition, although adjuvants such as Freund's Complete Adjuvant, Freund's Incomplete Adjuvant and Montanide can greatly improve the immune response to an antigen, they suffer some disadvantages. When used with an antigen in an injectable form, large lesions usually form at the injection site, a situation that makes them unsatisfactory for this use in humans, pets or meat animals. In addition, these adjuvants cannot act as immunopotentiating agents when administered orally or enterically.

p00014It is known in the art that carriers of immunogens or antigens of different nature can be genetically modified relatively easily. Those systems that can be produced in plants and easily adapt to this application are plant viruses. It is known that cowpea mosaic virus (CPMV), tobacco mosaic virus (TMV) and alfalfa mosaic virus (AIMV) have been modified for the presentation of epitopes of interest. It is known that another viral plant vector, potato X virus (PVX), a member of the potexvirus group, tolerates the transport of a complete protein coat. Also, United States Patents

p000156,232,099 and 6,042,832, Publications of International Patent Applications under number WO 97/39134, WO 02/04007, WO 01/66778, WO 02/00169, application EP 1167530, and Porta, C. et al. . (Reviews in Medical Virology, 1998, 8: 25-41), all of them, describe different variations of virus-like particles that carry foreign proteins fused with endogenous proteins. In addition, the use of papaya mosaic virus (PapMV) has been described as an epitope presentation system (Ikegami, R., M.Sc. Thesis, National Library of Canada, Ottawa, Ontario).

p00016Considering the state of the art described herein, there is still a great need for compounds and transport particles that allow a strong immunization of the human being and animals while also avoiding the use of adjuvants and second vaccinations as is currently practiced.

p00017Disclosure of the invention

p00018The scope of the present invention is defined by the appended claims. In the embodiments of the invention disclosed herein, the reference to a virus or a virus-like particle (VLP) is understood as a reference to PapMV or VLP PapMV.

p00019An object of the present invention is to provide an immunogen-transporter complex having an immunopotentiation property, consisting of a virus-like particle (VLP) that carries at least one immunogen fused with a protein or fragment thereof of said VLP, which can be used in the preparation of a composition to induce an immune response against the protein or fragment thereof.

p00020Another object of the present invention is to provide a composition comprising a virus-like particle (VLP) and a protein or extract derived from a virus, bacteria or parasite, for use as a vaccine.

p00021A method to immunopotentiate an immune response in a human being is disclosed herein.

p00022or an animal, comprising administering to said human or animal an immunogen carrier consisting of a virus-like particle (VLP) that carries at least one immunogen fused with a protein or fragment thereof of said VLP, or administering a VLP or a fragment thereof concomitantly with an antigen not directly bound to said VLP.

p00023The present invention also relates to polynucleotides encoding an immunogen-transporter complex consisting of a virus-like particle (VLP) that carries at least one fused immunogen to the Cterminal end of the envelope protein of said VLP, said complex having of immunogen-transporter the ability to assemble when expressed in a plant cell, an animal cell or a microorganism.

p00024In addition, the use of a papaya mosaic virus as an adjuvant is disclosed herein.

p00025For the purposes of the present invention, the following terms are defined below.

p00026The term "chimeric protein" is created when two or more genes that normally encode two different proteins recombine, either naturally or as a result of human intervention, to encode a protein that is a combination of all or part of each of those two proteins.

p00027The term "capsid fusion protein" means a fusion protein in which one of the fusion genes encodes a capsid protein from a plant virus.

p00028It is understood that the term "protective immunity", as used herein, means the ability of an animal, such as a mammal, bird or fish, to resist (delayed occurrence of symptoms or reduced severity of the symptoms), as a result of its exposure to a pathogen's antigen, to the disease or death that would occur otherwise after contact with the pathogen. Protective immunity is achieved by one or more of the following mechanisms: mucosal, humoral or cellular immunity. Mucosal immunity is primarily the result of secretory IgA antibodies (sIGA) on the mucous surfaces of the respiratory, gastrointestinal and genitourinary tracts. SIGA antibodies are generated after a series of events mediated by antigen-processing cells, B and T lymphocytes, which result in the production of sIGA by B lymphocytes in mucosal coated body tissues. Mucosal immunity can be stimulated by an oral vaccine. The primary result of protective immunity is the destruction of the pathogen or the inhibition of its ability to replicate.

p00029The term "humoral immunity," as used herein, means the result of IgG antibodies and IgM antibodies in serum.

p00030The term "cellular immunity", as used herein, can be achieved through cytotoxic T lymphocytes or through delayed type hypersensitivity in which macrophages and T lymphocytes are involved, as well as through other mechanisms in those that are involved T cells without the need for antibodies.

p00031A "recombinant virus" is one in which the genetic material of a virus has been combined with other genetic material.

p00032It is understood that the terms "polypeptide" or "peptide", as used herein, mean a molecule in which there are at least four amino acids linked by peptide bonds.

p00033The term "viral nucleic acid", as used herein, may be the genome (or most of it) of a virus, or a complementary nucleic acid molecule in the base sequence to said genome. A DNA molecule that is complementary to viral RNA is also considered viral nucleic acid. An RNA molecule that is complementary in the base sequence to the viral DNA is also considered viral nucleic acid.

p00034The term "virus-like particle" (VLP), as used herein, refers to self-assembling particles that have a physical appearance similar to viral particles and include pseudoviruses. Virus-like particles may lack or possess dysfunctional copies of certain natural virus genes, and this may make the virus-like particle unable to perform any function that is characteristic of the natural virus, such as replication and / or movement from one cell to another.

p00035The term "vaccine", as used herein, is intended to mean the fusion protein, any particle of which that protein is a part, or any preparation such as plant material of which that protein is a part.

p00036The term "immunopotentiator", as used herein, is intended to mean a substance that, when mixed with an antigen, enhances immunogenicity or antigenicity and provides a superior immune response. It will be recognized that it can enhance the expression of costimulators in macrophages and other antigen presenting cells.

p00037Brief description of the drawings

p00038Figure 1 illustrates an electron micrograph of purified PapMV;

p00039Figure 2 illustrates the tricine SDS-PAGE analysis of the PapMV CP (A) and the labeling by immuno-gold techniques showing that the fusion is exposed on the surface of the PapMV VLP (B);

p00040Figures 3A to 3F illustrate electron micrographs of PapMV and VLP PapMV assembled in vitro;

p00041Figure 4 illustrates the accumulation of leukocytes induced by PapMV in the air bag model;

p00042Figure 5 illustrates the immune response to PapMV. In mice (6 for each concentration) IP was injected once PapMV or ISS (isotonic saline);

p00043Figure 6 illustrates an immune response to PapMV. In mice (6 for each concentration) IP was injected once PapMV or ISS (isotonic saline);

p00044Figure 7 illustrates an evaluation of the potency of PapMV as an adjuvant to ovalbumin;

p00045Figures 8A and 8B illustrate the characterization of the immune response to PapMV and HCV peptides derived from HCV surface glycoproteins E1 and E2; and

p00046Figure 9 illustrates a Western blot showing the specific migration of the PapMV CP into the lymph node and spleen with an intraperitoneal injection.

p00047Method for carrying out the invention

p00048This document discloses a virus-like particle that carries an immunogen fused with viral endogenous proteins, which therefore forms a new type of immunogen-transporter complex that is also capable of performing immunopotentiation or having an adjuvant effect.

p00049This document discloses a class of transporters that when genetically bound to an immunogen or hapten can enhance the host's immune response against the immunogen or hapten, regardless of whether the complex is administered parenterally, enterically or orally. In addition, its use does not result in the formation of large lesions at the injection sites.

p00050Accessory cells such as macrophages, B lymphocytes and dendritic cells are essential for the induction of T-cell-dependent immune responses. Accessory cells present antigens to MHC-restricted T cells and produce membrane-associated and secreted costimulators that enhance proliferation. and differentiation of T lymphocytes. Therefore, the presence of competent accessory cells stimulates T-cell-dependent immune responses, and their absence leads to poor responses. It is possible that resting macrophages and virgin B lymphocytes, without stimulation, presented by such antigen presenting cells (APC) do not stimulate virgin CD4 + T cells and may even induce T-cell tolerance. In contrast, dendritic cells and activated macrophages and B cells express costimulators, as well as high levels of APC. One mechanism of action of the immunogen-transporter disclosed herein is to enhance the expression of costimulators in macrophages and other APCs. Therefore, the administration of immunogens or protein antigens with the immunogen carriers disclosed herein, which act simultaneously as an adjuvant, promotes cell-mediated immunity and the production of T-cell-dependent antibodies. Immunogens are extremely effective. to generate systemic immunity when administered in conjunction with an immunogen-transporter disclosed herein.

p00051In a first embodiment, the invention provides a complex comprising an immunogen coupled to a transporter that is a virus-like particle (VLP) at the C-terminal end of the envelope protein, such that the transporter molecule potentiates the Immune response of a host to the immunogen when the complex is administered to said host, where the immunogen may comprise an antigen or a hapten and the transport molecule comprises an integral particle of PapMV.

p00052One way to get a good response from B cells is to present the antigen in an organized way. It has been shown that repetitively organized epitopes form crosslinks with the B cell receptor effectively and induce a rapid IgM response, independent of T lymphocytes, followed after an IgG response. Therefore, a good strategy to increase the immunogenicity of epitopes and recognition and presentation to the immune system is the expression of immunodominant epitopes in an organized manner as on the surface of a plant virus such as PapMV. Particularly, PapMV fulfills several characteristics of a good adjuvant and transporter because it is a phylogenetically distant antigen, it is exogenous to the mammalian immune system, it is very complex from a molecular point of view and it is an organized structure that has a high molecular weight.

p00053The applicant has recognized that, surprisingly, a crystalline and repetitive structure is not only recognized by the innate immune system, but also has an adjuvant effect on the immune system of an immunized host.

p00054In one embodiment of the present invention, a method is provided in which the use of a high number of copies of the benign rod-shaped papaya mosaic virus (PapMV) produces immunogens connected to subunits of shell proteins. of the virus When assembled, the virus particles comprise long helical formations of more than 1,000 identical fusion proteins (which are typically fusion proteins of shell proteins - foreign proteins) per virion. Generally, the part of the immunogen will be exposed on the outer surface of the virus particles at the C-terminal end of the envelope protein.

p00055The capsid protein structure of plant and animal viruses meets these requirements and can be engineered to present pathogen-derived immunogenic peptides or other sources.

p00056with which the vaccine adjuvant is produced. Papaya mosaic virus (PapMV) envelope protein, according to the present invention, is an excellent candidate for the development of said immuno-transporter immunopotentiator. This virus is shaped like a crystalline rod and is very repetitive (1,200 copies of the same subunit per virion). Recent immunization experiments with PapMV indicate that this virus induces a very strong immune response in mice and is an excellent vector for the development of a vaccine. This immunogen transporter virus can be engineered with several immunogenic peptides, for example, from HCV surface envelope proteins, peptides derived from Salmonella typhii porine protein, and insulin α9-23 peptide . The set of fusion coat proteins carried by virus-like particles (VLP) is defined as an immunogen-transporter that has adjuvant or immunopotentiating properties.

p00057In accordance with the present invention, it is possible to provide a composition to immunopotentiate or strengthen an immune reaction against a given antigen. It is particularly known that small molecules often act only poorly as immunogens in their ability to induce antibodies in an in vivo system. When they bind to an immunogen transporter virus, which in itself is antigenic, they will result in an improved antibody response against smaller molecules. The small molecule bound to the immunogen transporter of this system may be called hapten or antigen, and may vary in size from small to quite large. In an example of this combination, of interest to the health care field, a small part of the Hepatitis B surface antigen, which comprises a sequence of certain amino acids, which is not antigenic by itself, can covalently bind to VLP, and the resulting conjugate induces antibodies in an in vivo system that can cross-react with the native surface antigen of the VLP and also strongly with the complete hepatitis virus. This immunogen-transport system can be the basis for an effective vaccine against a disease encoded by the hapten or antigen.

p00058Immunogen transporter systems are disclosed herein, as described below, which are conveniently produced by recombinant DNA techniques, which are useful for providing both univalent and multivalent immunogenic vaccines, and which employ the immunogen transporter concept described above. .

p00059An immunogen is coupled to a transporter VLP to form an immunogen-transporter complex and can then be used in a host to elicit an immune response. The immunogen can be specific or recognized by surface structures present in T cells, B cells, NK cells and macrophages, but not by cell surface structures associated with class I or class II APCs.

p00060The immunogen to which the transporter VLP is coupled can comprise peptides, haptens, carbohydrates, proteins, nucleic acids, and parts of viruses, bacteria, parasites and other whole organisms. Regardless of the immunogen selected, it must be coupled to the transport VLP in such a way that it does not interfere with the recognition of the immunogen by the host's immune system as an antigenic entity.

p00061The immunogen-transporter complex can be used as a vaccine to induce an immune response in the host. The complex may initially be administered in an appropriate dosage to induce an immune response. This can be continued by boosting with the complex or immunogen alone. A variation of this approach may include the formation of one or more immunogen-transporter complexes in which one or more forms of an immunogen are coupled to one or more transporter VLPs and a plurality of said complexes are administered.

p00062The objective of administering the immunogen-transporter complex is to provide protection to the host in the form of immunity against the antigen and avoid the use of adjuvants that have unwanted side effects.

p00063In one embodiment, the antigen can be an immunogen as small as a hapten or it can be relatively large, such as part of a virus. The size and type of antigen is not critical for the implementation of this invention. Any antigen for which an immune response is desired in a host can be used. However, the invention is especially useful for weakly immunogenic small haptens.

p00064Once the immunogen-transporter complex or complexes have been formed, the complex or complexes can be administered to the host. It is not necessary for the administration regimen to differ from any other generally accepted vaccination program. A single administration may be used in an amount sufficient to induce an effective immune response. Alternatively, other initial administration regimens of the complex may be used followed by the reinforcement with antigen alone or one or more complexes. Similarly, reinforcement with the complex or antigen can be performed at times well after initial administration if antibody titers are below acceptable levels.

p00065Another embodiment of the present invention is that since VLPs have a true regular and helical multivalent structure that can be more immunogenic than protein-free aggregation or protein-free subunits, they can also be easily assembled from a coding nucleic acid. In addition, the greater stability of the particle can provide long-term exposure of the immunogen part to the immune system.

p00066The part of the virus to which the immunogen binds is preferably arranged on the outer surface of the PapMV VLP to be used as an adjuvant in vaccination to enhance an immune response. Thus, when the particle comes from PapMV, the part of the transporter is disposed at the carboxy end. This may result in a better assembly compared to the assembly of particles that have the second part in another location of the CP, and may enhance the immune recognition of the second part on the surface of the particle.

p00067The development of peptide vaccines using a plant virus vector allows mass production of vaccines in safe conditions. With recombinant PapMV, it is expected to obtain up to 1 gram of recombinant virus per kilogram of infected fresh leaves.

p00068The administration of 200 pg of recombinant virus, or immunogen-transporter complex, corresponding to 14 μg of peptide, may be sufficient for immunization. One hectare of infected papaya could then be enough for the vaccination of 5 million patients. In addition, plant cultivation is cheap and effective. Agriculture is the cheapest way to produce a biomass because it does not need sophisticated equipment.

p00069The virus or pseudovirus can be assembled into the host cell to produce infectious virus particles comprising nucleic acid and fusion protein. This may allow infection of adjacent cells by the pseudovirus or infectious virus particle and the expression of the fusion protein in them.

p00070The host cell may initially be infected with virus or pseudovirus in the form of particles (ie, in assembled rods comprising nucleic acid and a protein) or, alternatively, in the form of nucleic acid (ie, RNA such as viral RNA; CDNA or run-off transcripts (transcripts obtained by a transcription protocol in which the plasmid is cut after the inserted DNA) prepared from cDNA) provided that the virus nucleic acid used for the initial infection can replicate and cause production of whole virus particles that have the chimeric protein.

p00071The first (viral) part of the fusion protein can be any protein, polypeptide or parts thereof, coming from the papaya mosaic virus including any genetically modified version thereof (such as deletions, insertions, amino acid replacements and Similar). According to the appended claims, the first part will come from a papaya mosaic virus envelope protein (or a genetically modified version thereof). A fusion protein molecule can be assembled with other fusion protein molecules or with the natural coat protein in an immunogen transporting virion.

p00072According to the compositions and fusion proteins of the invention, the particle is derived from the PapMV potexvirus, and the second part is arranged at the C-terminal end of the envelope protein. In PapMV, the Cterminal end of the envelope protein forms a domain outside the virion.

p00073According to the polynucleotides of the invention, a polynucleotide encoding the immunogen part is inserted into the end of the polynucleotide encoding the C-terminal end of the viral part, such that after translation, the fusion protein has the viral part at one end and the immunogen part at the opposite end. It is not necessary for the viral part to comprise a protein from the entire virus envelope, but it remains an alternative choice.

p00074Another embodiment of the present invention is constituted by a genetically modified virus or pseudovirus to express the fusion protein. A host cell infected with said virus or pseudovirus is also disclosed herein.

p00075Preferably, the host cell used to replicate the virus or pseudovirus is a bacterium, where the virus is a plant virus, although plant cells, insect cells, mammalian cells and bacteria with viruses that replicate in said cells can be used. The cell is preferably a bacterium such as E. coli, although other forms of bacteria and other cells, such as the cells mentioned above, may be useful. The cell may be a natural host cell for the virus from which the virus-like particle is derived, but this is not necessary.

p00076The whole virus-like particle is used for stable and long-term presentation of peptide epitopes for vaccination of animals.

p00077PapMV and particles similar to PapMV virus appear to be very stable and can be easily stored at room temperature. They withstand very high temperatures and adverse conditions since plant viruses have evolved to withstand very difficult conditions found in the environment. This is a very important advantage when the vaccine has to reach people who are living in poor countries, in regions where access is difficult, or for conservation for a prolonged period.

p00078Alternatively, the VLP described herein can be used alone as an immunopotentiator or adjuvant to enhance an immune response in humans or animals against target antigens. It is preferable that the adjuvant or immunopotentiating VLP be administered concomitantly with the antigen against which an immune response must be induced. However, adjuvant VLP can be administered sooner or later, depending on the needs, of the administration of the antigen to patients, humans or animals.

p00079The present invention will be more readily understood by reference to the following examples that are provided to illustrate the invention rather than to limit its scope.

p00080Example I

p00081Preparation of immunogen-VLP transporter

p00082The avidity of the selected affinity peptides described herein will be improved by multimerization of the peptides. Multimerization will be performed on the surface of the papaya mosaic virus (PapMV), which is a member of the potexvirus group. PapMV has a rod type structure that is formed by assembling the subunits of the CP. A virus particle contains 1,200 subunits. A fusion of the selected peptide with the PapMV CP will be performed. Fusion will be performed to expose the peptide on the surface of the PapMV particles after in vitro assembly from a PapMV CP expressed and purified from an E. coli expression system. The assembly of the viral CP then ensures the multimerization of the peptide and has a considerably improved avidity.

p00083The envelope protein (CP) gene was cloned and developed in an in vitro assembly system using the papaya mosaic virus (PapMV) envelope protein (CP) (Fig. 1). PapMV CP was produced in E. coli in a large amount (Fig. 2a) and produced particles similar to PapMV virus in vitro that are very similar to those of the natural virus (Fig. 2b). For the first time it is demonstrated that a recombinant PapMV CP can be assembled into virus-like particles in vitro. The fusion of several peptides to the C-terminus of the CP is allowed by in vitro assembly and results in virus-like particles that are larger than the natural virus due to fusion (Fig. 3).

p00084Example II

p00085Immuno-potentiation effect of immunogen-transporter VLP

p00086An adjuvant is often used to increase the immune response of a candidate vaccine. The potentiation of the inflammatory response favors the migration of more phagocytes to the injection site, which, in turn, results in a better presentation of the antigen by the antigen presenting cells (APC). To increase the immune response of the candidate vaccine, alum, emulsions, microparticles and cytokines such as GM-CSF have been used. It was confirmed that PapMV induced an inflammatory episode by itself, thus eliminating the need for additional adjuvants. The air bag model was used to examine whether PapMV induced a proinflammatory event in vivo. In this model, sterile air is injected under the backs of mice on days 0 and 3. On day 7, proinflammatory agents can be injected into the air bag and the inflammatory response measured. This model closely represents subcutaneous injection sites.

p00087Injection of PapMV in the murine air bag resulted in the accumulation of approximately 8.5 x 106 leukocytes, compared with 0.8 x 106 leukocytes in the mice in which vehicle (PBS) was injected (Fig. 4) . Neutrophils (85%) and monocytes (15%) accumulated in the air bag 6 hours after PapMV injection. Although amounts as low as 1 μg of PapMV were sufficient to induce leukocyte accumulation, maximum accumulation occurred when 100 μg of PapMV was injected. This accumulation was similar to that induced by the injection of 1 μg of LPS, a potent pro-inflammatory factor. These results clearly demonstrate that PapMV can effectively induce an inflammatory episode. This observation clearly shows that PapMV is perceived by the immune system that induces the signaling and recruitment of cells involved in the defense of our body. PapMV is likely to induce signaling through innate immunity.

p00088In addition, in the present experiment it was shown that PapMV induces a strong and long-lasting humoral response in mice (Fig. 5). In 10 mice three concentrations of PapMV were injected; 1, 10 and 100 μg. A primary antibody response was effectively induced in BALB / c mice immunized with PapMV regardless of the route of immunization (Fig. 5). High titers were detected on day 5 after immunization. A classic curve of a primary IgM response was observed. Around day 20, there was no

p00089IgM response, even after the reinforcement of mice with more viruses. The IgG response in immunized mice follows classical kinetics. High titers of anti-PapMV were detected on day 12 after immunization and increased proportionally after booster with this virus. The analysis of the IgG isotypes showed a preference in the production of IgG2b and IgG1 during the primary and secondary phase of the antibody response (Ab). IgG3 titers increased during the memory phase of the Ab response. These data show that PapMV can induce an effective Ab response in mice. Primary and secondary responses were effectively induced as well as a long-lasting Ab memory. Preferred production of IgG1 suggests a preferential release of IL-4. IL-4 favors class change to this type of IgG. Therefore, a balance towards a TH2 response could be anticipated in these mice. The absence of IgG2a indicates the absence of IFN-α release, since this cytokine has been directly implicated in the class change towards this IgG isotype. Taken together, these data showed PapMV's ability to induce an effective and long-lasting antibody response. This result suggests that PapMV particles are an excellent vector for the development of a humoral vaccine. Then, the fusion of an immunogen of interest with the VLP will also be recognized by the immune system and will trigger a strong immune response against the epitope of interest.

p00090In addition, it was found that PapMV VLPs specifically migrate to lymph nodes and spleen after intraperitoneal or subcutaneous injection in BALB / c mice (Fig. 6). This result indicates that PapMV VLPs are excellent transporters because they migrate effectively to the sites of immune response.

p00091Experimental data demonstrate that the PapMV antigen induces an effective antibody response in mice (Fig. 5). In fact, primary and secondary responses are effectively induced as well as a long-lasting antibody memory (Fig. 6). Several immunization pathways produced effectively large amounts of antibodies. Only oral immunization did not result in an immune response. It is likely that the NaHCO3 used to neutralize stomach acid will damage the virus particles and affect the immunogenicity of the particles. Amounts of IgG1, IgG2a, IgG2b and IgG3 were present even 350 days after a 100 μg injection of PapMV (Fig. 6). Since IgG2a and IgG3 are present and persist, it can be deduced that a TH1 response is induced with PapMV. This suggests that PapMV particles are excellent vectors for the development of a humoral immune response to a foreign antigen. The fusion of an epitope of interest to the PapMV particle should help trigger a humoral immune response against the epitope of interest.

p00092Experimental data obtained using the air bag model on the back of mice showed that PapMV potentiates the inflammatory response and favors the migration of phagocytes to the inoculation site (Fig. 6). This result confirms that PapMV induces an inflammatory episode by itself, thus eliminating the need for additional adjuvant strategies that aim to improve antigen presentation by antigen presenting cells. Similar results were obtained with virus-like particles (VLP) that carried the fusion of specific peptides generated in vitro from recombinant proteins (Fig. 6). The recruitment was very fast since the maximum number of cells was observed between 6 and 9 hours after treatment (data not shown). In addition, PapMV particles are effective in inducing an immune response to ovalbumin, a protein known to be non-immunogenic (Fig. 7). This was proven by injecting 2 mg of ovalbumin intraveitoneally into mice (Balb / C), a protein known to be a very weak immunogen, or in combination with 50 or 100 μg of PapMV. Injections were made in 6 mice per treatment and samples were collected 0, 4, 8, 12 and 20 days after the injection. Only one injection was made for each treatment. A twice as strong immune response to ovalbumin was detected in the presence of PapMV even though ovalbumin was a weak immunogen.

p00093These observations demonstrate that PapMV particles are quickly perceived as foreign by the mammalian immune system, which in turn induces signaling and recruitment of cells involved in the defense of the organism.

p00094Example III

p00095Hepatitis C virus as a vaccination target

p00096Hepatitis C virus (HCV) is a positive chain RNA virus that causes acute and chronic liver diseases. The acute phase of the infection is usually associated with mild symptoms, but it can lead to cirrhosis and hepatocellular carcinoma. Worldwide, more than 170 million people are infected, which is four times more than the case of HIV. In the coming years, the number of deaths from diseases associated with HCV may even exceed the death rate caused by AIDS. At this time, current therapies against HCV are not satisfactory. The only therapy available is interferon (IFN), but most HCVs are resistant due to inhibition of interferon-inducible protein kinase (PKR) by HCV E2 protein.

p00097It is known that 20% of patients infected with HCV naturally eliminate the virus. This observation suggests that the immune system can eliminate viruses if it reacts effectively. This also suggests that chronically infected patients could be helped by strengthening their immune system with a therapeutic vaccine against HCV that could help eliminate viral infection by inducing neutralizing antibodies against the virus.

p00098The two epitopes chosen are found on the surface of the HCV virion. It has been shown that the E1 epitope (amino acids 285-303) and the E2 epitope (amino acids 512-536) are strongly immunogenic in patients who have eliminated the viral infection (David et al., 2001). PapMV was modified by genetic engineering to take at its C-terminal end the fusion of HCV E1 and E2 peptide, which can be assembled to form particles similar to PapMV virus in vitro (Fig. 3).

p00099Three epitopes that are found on the surface of the HCV virion of E1 and E2 outside of HVR-1 in the conserved region of the viral envelope glycoproteins were chosen. It was shown that one epitope of E1 (amino acids 285303) and two epitopes of E2 (amino acids 512-536 and 528-546) were strongly immunogenic in patients who eliminated viral infection. In addition, it was shown that an epitope of E2 (amino acids 512-536) triggered the production of neutralizing antibodies found in the sera of patients eliminating the infection. These three regions are good candidates for the development of an HCV vaccine because they are conserved between HCV subtypes and strains and are located outside the hypervariable region of the envelope glycoproteins. The PapMV-E1 and PapMV-E2 constructs were expressed in E. coli. Recombinant proteins were purified and assembled in vitro. The assembly of the recombinant CP with the HCV E2 fusions generates rVLPs that are similar to the natural recombinant control CP except that they appear slightly larger due to the fusion.

p00100Mice were immunized with the recombinant VLPs that were produced in vitro. LPS were extracted using a polymyxin column and injected into mice intraperitoneally and subcutaneously. 1, 10 and 100 μg of VLP were used and three mice were injected for each treatment. The immune response to the peptide and PapMV was analyzed by ELISA. It was observed that the IgG was directed at the peptide as well as at the surface of the VLPs (Fig. 8). This result shows that recombinant PapMV can be used to trigger an excellent immune response on the surface of epitopes and be used as a vaccine without the aid of adjuvants.

p00101Example IV

p00102Immunization against typhoid fever

p00103Typhoid fever is an acute infection of the reticuloendothelial system, intestinal lymphoid tissue and gallbladder that is caused by the bacterium Salmonella typhii. Worldwide it is still a significant disease that affects more than 16 million people, of which 600,000 do not survive the infection. Most infections affect children and young adults, and can be prevented by vaccination. Different types of vaccines are currently available: 1) Parenteral whole cell vaccine preserved with phenol, thermally inactivated (Wyeth-Amherst) administered intramuscularly or subcutaneously. 2) Parenteral whole cell vaccine inactivated with acetone and dehydrated. 3) Parental vaccine of purified (undenatured) Vi polysaccharide (Aventis) that is administered by injection into the deltoid. 4) Ty21a gal E strain Vi-negative attenuated, used as a live oral vaccine .

p00104Inactivated parental bacteria (type 1-3) may produce unwanted immune responses due to the complexity of lipopolysaccharide (LPS) and the number of antigens presented that induce undesirable side effects. In addition, Vi polysaccharides are thymus-independent antigens (Robins and Robins, 1984) which, as demonstrated, had good efficacy in field trials but, as is also known, are ineffective in inducing immune memory. Several exposures to the antigen are needed to maintain protection, which makes this approach appropriate only for travelers visiting endemic areas. Currently available vaccines are not adapted for people who live permanently in contaminated areas. The vaccine based on attenuated bacteria (type 4) can cause nausea, vomiting and abdominal pain. It is also not recommended to administer this vaccine to patients suffering from immunosuppression, intestinal diseases, diarrhea, who take antibiotics or are pregnant women or children under 6 years. This vaccine has to be stored at 4 ° C because it is sensitive to heat and should not be frozen. The sensitivity of ty21A to adverse conditions is a problem when it is desired to reach populations that live in poor countries with tropical climates, which are the regions most affected by typhus.

p00105It was shown that an S. typhii membrane protein called porin was a good immunogen because it induces both an antibody immune response and a cellular immune response in mice and humans and could protect mice against S. typhii. Porins are the most abundant proteins in the membrane of Gram-negative bacteria and function as passive diffusion channels for low molecular weight molecules. These proteins have a high degree of structural and functional homology and, therefore, are supposed to have a common ancestor. It was shown that two small epitopes corresponding to loop 6 and 7 of the S. typhii porin that are exposed on the surface of the bacteria were involved in protective mechanisms induced by porin immunization. Those regions are specific to S. typhii and are excellent epitopes for the development of a recombinant subunit vaccine. The present applicants have cloned loop 6 of the porin S. typhii at the C-terminal end of the PapMV CP. The recombinant protein was purified and PapMV virus-like particles were produced in vitro with RNA as described above (Fig. 3F).

p00106It is understood that the invention is not restricted to the above preferred embodiments and that modifications are possible provided they are within the scope of the appended claims.