The present invention relates to an immunogen-carrier having immunopotentiating or adjuvant properties. More particularly, the immunogen-carrier is a virus-like particle (VLP) from the family of potexvirus, and most particularly the papaya mosaic virus. The VLP produced by recombinant techniques is in fusion with one of its own proteins a protein immunogen. The above VLP and a protein or a protein extract from a viral, bacterial or parasital pathogen may be used as a vaccine.

Description of the Invention

SUMMARY OF THE INVENTION

One aim of the present invention is to provide an immunogen-carrier complex having an immupotentiation property, consisting of a viral-like particle (VLP) carrying at least one immunogen in fusion with a protein or fragment thereof of said VLP, that may be used in the preparation of a composition for inducing an immune response against the protein or fragment thereof.

Another aim of the present invention is to provide a composition comprising a viral-like particle (VLP) and a protein or an extract derived from a virus, bacteria or parasite, that may be used as a vaccine.

In accordance with the present invention there is also provided a method for immunopotentiating an immune response in a human or an animal which comprises administering to said human or animal an immunogen-carrier consisting of a viral-like particle (VLP) carrying at least one immunogen in fusion with a protein or fragment thereof of said VLP, or administering a VLP or a fragment thereof concomitantly with an antigen not directly linked to said VLP.

The present invention also relates to polynucleotide encoding a immunogen-carrier complex consisting of a viral-like particle (VLP) carrying at least one immunogen in fusion with a protein or A polynucleotide encoding a immunogen-carrier complex consisting of a fragment thereof of said VLP, or a VLP alone, said immunogen-carrier complex having the capacity of being assembled when expressed in a plant cell, an animal cell or a microorganism.

The invention also provides for the use of a papaya mosaic virus as an adjuvant.

DESCRIPTION OF PREFERRED EMBODIMENTS

In accordance with the present invention, there is provided a virus-like particle carrying immunogen in fusion with endogenous viral proteins, therefore forming a new type of immunogen-carrier being also capable of immunopotentiation or having an adjuvant effect.

In one embodiment of the present invention, there is provided a class of carriers which when linked genetically to an immunogen or hapten can enhance the host's immune response to the immunogen or hapten regardless of whether the complex is administered parenterally, enterally or orally. In addition their use does not result in the formation of large lesions at injection sites.

Accessory 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 the proliferation and differentiation of T lymphocytes. Therefore, the presence of competent accessory cells stimulates T cell-dependent immune responses, and their absence leads to deficient responses. Resting macrophages and naive, unstimulated B lymphocytes sented by such antigen-presenting cells (APCs) may fail to stimulate naive CD4+ T cells, and may even induce T cell tolerance. In contrast, dendritic cells and activated macrophages and B cells do express costimulators, as well as high levels of APCs. A mechanism of action of the immunogen-carrier of the present invention, is to enhance the expression of costimulators on macrophages and other APCs. Because of this, the administration of immunogens or protein antigens with the immunogen carriers of the invention, acting simultaneously as an adjuvant, promotes cell-mediated immunity and T cell-dependent antibody production. Immunogens are most effective for generating systemic immunity when administered coupled together with an immunogen-carrier of the present invention.

In a first embodiment, the invention provides a complex comprising an immunogen coupled to a carrier virus-like particle (VLP), such that the carrier molecule causes the immune response of a host to the immunogen to be enhanced when the complex is administered to said host, wherein the immunogen may comprise either an antigen or a hapten and the carrier molecule comprises an integral particle of a virus. More particularly, the virus of the present invention is a plant virus.

One way to obtain a good response of B cells is to present the antigen in an organized manner. It is shown that repetitively arranged epitopes cross-link to B cell receptor efficiently and induce a prompt T-independent IgM response followed later by an IgG response. Therefore, a good strategy to increase the immunogenicity of the epitopes and the recognition and presentation to the immune system is the expression of the immunodominant epitopes in an organized fashion like on the surface of a plant virus like PapMV. Particularly, PapMV filfils several characteristics of a good adjuvant and carrier because it is a phylogenetically distant antigen, it is exogenous to the mammal immune system, it is molecularly very complex and it is an organized structure that has a high molecular weight.

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

In one embodiment of the present invention, there is provided a method in which the use of benign high copy number rod-shaped viruses, preferably plant viruses such as papaya mosaic virus (PapMV), produce immunogen connected to viral coat protein subunits. When assembled, the virus particles comprise long helical arrays of more than 1000 identical fusion proteins (which are typically coat protein--foreign protein fusion molecules) per virion. Generally, the immunogen portion will be displayed on the outer surface of the virus particles.

According to the present invention, the structure of the capsid proteins of plant and animal viruses fulfil these requirements and can be engineered to present immunogenic peptides derived from the pathogen or other sources with which a vaccine-adjuvant is produced. The coat protein of papaya mosaic virus (PapMV), for example, but without limiting it thereto, is an excellent candidate for the development of such a immunogen-carrier-immunopotentiator. This virus harbours a crystalline rod shape and is very repetitive (1200 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 carrier virus can be engineered with several immunogenic peptides of the HCV surface envelope proteins, such as for example, Salmonella typhii peptides derived from the porin protein, and with the peptide .alpha.9-23 of insulin. The assembly of fusion coat proteins carried by virus like particles (VLPs) are defined as an immunogen-carrier having adjuvant or immunopotentiating properties.

According to the present invention, it is possible to immunopotentiate, or boost an immune reaction against a given antigen. It is known particularly that small molecules often act only poorly as immunogens in their ability to elicit antibodies in an in vivo system. When attached to a immunogen-carrier virus of the present invention, that itself is antigenic, it will give rise to improved antibody response to the smaller molecule. The small molecule attached to the immunogen-carrier in this system, may be called a hapten or antigen, and can vary in size from small to quite large. In one example of this combination, of interest to the health care field, a small portion of the Hepatitis B surface antigen, comprising a sequence of determined amino acids, which is not itself antigenic, can be covalently bound to the VLP, keyhole limpet immunogen-carrier, and the resulting conjugate elicites antibodies in an in vivo system that may cross-react with the native surface antigen of the VLP and also strongly with the whole hepatitis virus. This system of immunogen-carrier can be the basis for an effective vaccine against a disease for which the hapten or antigen codes.

In the present invention, there are provided certain novel immunogen-carriers, as described below, which are conveniently produced by recombinant DNA techniques, which are useful in providing univalent as well as multivalent immunogenic vaccines, and which employ the immunogen-carrier concept described above.

An immunogen is coupled to a carrier VLP to form an immunogen-carrier complex and may then be used in a host in order to provoke an immune response. The immunogen may be specific or recognised for surface structures on T cells, B cells, NK cells and macrophages but not for Class I or Class II APC associated cell surface structures.

The immunogen to which the carrier VLP is coupled may comprise peptides, haptens, carbohydrates, proteins, nucleic acids, and part of viruses, bacteria, parasites and other whole microorganisms. Regardless of the immunogen selected, it must be coupled to the carrier VLP in such a way as not to interfere with the recognition of the immunogen by the host's immune system as an antigenic entity.

The immunogen-carrier complex may be used as a vaccine to raise an immune response in the host. The complex initially may be given in an appropriate dosage in order to elicit an immune response. This may be followed by boosting with the complex or immunogen alone. A variation of this approach may include the formation of one or more immunogen-carrier complexes wherein one or more forms of an immunogen are coupled to one or more carrier VLPs and a plurality of such complexes is administered.

The purpose of administering the immunogen-carrier complex is to provide protection to the host in the form of immunity to the antigen and to avoid the use of adjuvants which have undesired side affects.

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

Once the immunogen-carrier complex or complexes are formed, the complex or complexes may be administered to the host. The administration regime need not differ from any other generally accepted vaccination programs. A single administration in an amount sufficient to elicit an effective immune response may be used. Alternatively, other regimes of initial administration of the complex followed by boosting with antigen alone or one or more complexes may be used. Similarly, boosting with either the complex or antigen may occur at times that take place well after the initial administration if antibody titres fall below acceptable levels.

A further embodiment of the present invention is that as the VLPs have a regular multivalent and true helical structure which can be more immunogenic than aggregation of protein or free subunits of proteins, it can be easily assembled from an encoding nucleic acid. Also the greater stability of the particle can provide a long lasting exposure of the immunogen portion to the immune system.

The virus portion on which the immunogen is attached, is preferably disposed on the outer surface of the VLP. Thus where the particle is derived from PapMV, the carrier's portion can be disposed on the amino or carboxy terminus, or inserted in an internal loop disposed on the outer surface of the CP. This can result in improved assembly as compared with the assembly of particles having a second portion on another location of the CP, and can enhance immune recognition of the second portion on the particle surface.

In one embodiment of the present invention, the development of peptide vaccines using a plant viral vector allows to mass-produce vaccines under safe conditions. As much as 1 gram of recombinant virus per kilogram of fresh infected leaves is expected with the recombinant PapMV.

In another embodiment of the present invention, the administration of 200 .mu.g of recombinant virus, or immunogen-carrier complex, which corresponds to 14 .mu.g of peptide, can be sufficient for immunization. One hectare of infected papaya can then potentially be sufficient for the vaccination of 5 million patients. Furthermore, to grow plants is cheap and efficient. Agriculture is the cheapest way to produce a biomass because it does not necessitate sophisticated equipment.

The virus or pseudovirus can be assembled in the host cell to produce infective virus particles which comprise nucleic acid and fusion protein. This can enable the infection of adjacent cells by the infective virus or pseudovirus particle and expression of the fusion protein therein.

The host cell can be infected initially with virus or pseudovirus in particle form (i.e. in assembled rods comprising nucleic acid and a protein) or alternatively in nucleic acid form (ie RNA such as viral RNA; cDNA or run-off transcripts prepared from cDNA) provided that the virus nucleic acid used for initial infection can replicate and cause production of whole virus particles having the chimeric protein.

The first (viral) portion of the fusion protein may be any protein, polypeptide or parts thereof, derived from a viral source including any genetically modified versions thereof (such as deletions, insertions, amino acid replacements and the like). In certain embodiments, the first portion will be derived from a viral coat protein (or a genetically modified version thereof). Mention may be made of the coat protein of Papaya Mosaic virus as being suitable for this purpose. A fusion protein molecule can assemble with other fusion protein molecules or with wild-type coat protein into a immunogen-carrier virion.

In a preferred embodiment of the invention, the particle is derived from a potyvirus or even more preferably a potexvirus such as PapMV, and in such an embodiment, the second portion is preferably disposed at or adjacent the C-terminus of the coat protein. In PapMV, the C-terminus of the coat protein forms a domain on the outside of the virion.

Preferably, a polynucleotide coding for the immunogen portion is inserted at or adjacent a terminus of the polynucleotide coding for the viral portion, such that upon translation, the fusion protein has the viral portion at one end and the immunogen portion at the opposite end. It is not necessary for the viral portion to comprise a whole virus cost protein, but this remains an alternative choice.

A virus or pseudovirus genetically modified to express the fusion protein forms a further embodiment of the present invention, as does any host cell infected with such a virus or pseudovirus.

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

According to a particular embodiment of the present invention, the whole virus-like particle is used for stable and long lasting presentation of peptide epitopes for the vaccination of animals.

According to another embodiment of the present invention, PapMV and PapMV virus like particles appear to be very stable and can be stored easily at room temperature. They resist very high temperature and adverse conditions since plant viruses has evolved to resist very difficult conditions that we find in the environment. This is a very important advantage when the vaccine must reach people that are living in poor countries, in regions where access is difficult or for the storage of a diagnostic test for a long period.

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

Claim 1 of 35 Claims

1. A method of potentiating an immune response against an antigen comprising one or more B-cell antigenic epitopes and/or one or more T-cell antigenic epitopes in an animal, said method comprising the step of administering to said animal said antigen and an effective amount of an adjuvant, wherein said adjuvant is a papaya mosaic virus (PapMV), or a virus-like particle (VLP) comprising PapMV coat protein, said PapMV coat protein being capable of assembling to form said VLP, wherein said antigen is fused to the C-terminus of a coat protein of said VLP, such that said antigen is disposed on the outer surface of the PapMV or VLP, and wherein said immune response is a humoral and/or cellular response.

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