The present invention relates to recombinant viral pseudo-particles of a virus of the Parvoviridae family or of a related virus, useful in particular for inducing cytotoxic CD8+ T lymphocyte (CTL) and CD4+ helper T lymphocyte responses in vivo at a high level.
Another object is the use of these pseudo-particles for the production of antitumoral vaccines or drugs.
It also covers compositions comprising said pseudo-particles in a physiologically acceptable excipient and/or diluent.
According to Fernandez et al. (Mxc3xa9decine/Sciences, 1995, 11, 975-983), the response to cellular mediation, essential in the case of a tumor, can be divided into two parts:
on the one hand the initiation of the response which causes the involvement of the CD4+ T lymphocytes and the cells presenting the antigen (CPA), and
on the other hand the cytotoxic effector response in which the CD8+ T lymphocytes are involved.
The activated CD4+ T lymphocytes proliferate and secrete cytokines while the activated CD8+ T lymphocytes proliferate and differentiate into cytotoxic T lymphocytes (CTL).
These two families of lymphocytes thus have very different functions and are activated by distinct mechanisms. The stimulation of the CD4+ Lymphocytes requires the association of a peptide arising from the degradation of the antigen to class II molecules of the Major Histocompatibility Complex (MHC), while those of the CD8+ cells require the association of a peptide to class I molecules of the MHC.
A number of prophylactic or therapeutic strategies have already been proposed in attempts to control various human and animal diseases caused by viruses, bacteria, parasites, or cancerous or tumoral diseases.
For example, the induction of CTL responses in vivo by xe2x80x9clivexe2x80x9d vectors is known. However, the safety of these vectors is not guaranteed, particularly towards immunocompromised individuals. In addition, the systems using the replication of these xe2x80x9clivexe2x80x9d vectors do not retain their activity in immune individuals.
Martinez et al., teach in Vaccine (vol. 10, pp. 684-690, 1992) the use of empty capsids of pig parvovirus (PPV) in pig vaccination. The Parvoviridae family contains viruses very widely distributed in mammals such as pigs, cattle, cats, rabbits, rats and man. However, the parvoviruses are relatively specific to their host mammals. Pig parvovirus (PPV), in particular, is responsible for many infections in industrial pig breeding. Pig parvovirus (PPV) is composed of a non-enveloped isometric particle of diameter 20 nm with icosahedral symmetry, containing a single-strand DNA molecule. It is composed of two capsid proteins, VP1 (83 kDa) and VP2 (64 kDa), and a third protein, VP3, resulting from the proteolysis of VP2.
The VP2 protein can be produced by insect cells using a recombinant baculovirus system, and the VP2 proteins produced are capable of auto-assembly to form viral pseudo-particles which have the same size as the native virion. The vaccination of pigs against PPV is performed by immunizing them with a mixture of said capsids in combination with the adjuvant Alhydrogel (50%) +Quil A 500 xcexcg (Superfos). A comparable teaching to that of this article is found in applications EP-551.449 and EP-554.414, which also disclose that antigenic determinants corresponding to other viral proteins may be incorporated into the capsids, without however specifying the nature of these antigenic determinants.
Application EP-647.655 relates to synthetic peptides corresponding to the antigenic sites of VP2. It thus does not describe the complete protein.
Attempts to modify the pseudo-particles with a view to incorporating heterologous antigenic determinants come up against difficulties such as the inhibition of the formation of the pseudo-particles. Even when the latter are formed, the heterologous antigenic determinant is not necessarily immunogenic; the antigenic determinant for example may be located in a site where it is incapable of taking up its natural conformation, or of being produced by the cells presenting the antigen.
In other respects, in the same way as vaccines with xe2x80x9clivexe2x80x9d vectors, the safety of these hybrid particles combined with adjuvants is not completely guaranteed, since a toxicity may arise from some properties of the adjuvants able to induce secondary effects which are harmful to the organism.
It is thus apparent from the state of the art that there is no known reliable and effective system for stimulating the cytotoxic T-lymphocyte response and or cytokine secretion, without risk to the health of the individual treated and without use of immunostimulant adjuvants.
The applicant has shown that it is possible to induce specifically a CD8+ lymphocyte response, in other words a cytotoxic response, and/or a cytokine secretion by the CD4+ T cells, using pseudo-particles containing antigenic determinants able to associate with at least one class I or class II molecule, respectively, of the MHC.
The object of the present invention is recombinant viral pseudo-particles produced by auto-assembly of at least one viral structural protein, having a size of between 20 and 60 nm and forming an approximately icosahedral structure, said protein being modified by the presence of at least one antigenic determinant comprising a sequence of 8 to 25 amino acids, able to associate with at least one class I or II molecule of the major histocompatibility complex (MHC), or of a combination of several of these antigenic determinants.
The CD8+ T lymphocytes according to the invention are able to recognize at least one antigenic determinant comprising a sequence of 8 to 9 amino acids able to associate with at least one class I MHC molecule.
The CD4+ T lymphocytes according to the invention are able to recognize at least one antigenic determinant comprising a sequence of between 8 and 25 amino acids able to associate with at least one class II MHC molecule.
Advantageously, the structural proteins comprise at least one parvovirus protein of the Parvoviridae family, or of a related virus, and preferentially at least one VP2 protein.
The particles according to the invention may also be particles composed of surface or core proteins of the hepatitis B virus, particles of poliovirus, of rabbit hemorrhagic virus, of Norwalk virus, of rotavirus, of retrovirus gag proteins (HIV, FIV, FeLV) or, in general, particles of any non-enveloped virus able to auto-assemble in vitro by the expression of one or more viral proteins.
The particles according to the invention may comprise proteins of various types, and form hybrid particles. They may for example be composed of VP1 and VP2 proteins.
The association formed between the amino acid sequence comprising the antigenic determinant and the class I and/or II molecules is recognized by the CD8+ or CD4+ T lymphocyte receptor and induces a differentiation and proliferation of these lymphocytes.
Advantageously, said sequence is bounded by regions known as flanking regions able to modulate its degradation, in other words to facilitate its production during the degradation of the antigen, or to facilitate its binding to class I or II MHC molecules.
The flanking regions may correspond to amino acids flanking the CD8+ or CD4+ T antigenic determinant in its natural environment. In general, they may be composed of several amino acids, and in particular alanine or lysine.
The pseudo-particles according to the invention have a first advantage, arising from the safety of this method of immunization, the vector used being non-replicating, consisting of a single type of viral protein and being able to be administered even to immunocompromised individuals.
A second advantage of the pseudo-particles according to the invention lies in the absence of cross-reactions with human parvovirus and thus in the absence of problems associated with a pre-immunization of the individuals, possibly leading to a weak immunogenicity of this type of recombinant vaccine.
In addition the pseudo-particles according to the invention do not require the replication of the vector and should thus retain their effectiveness in immune individuals.
Also, the pseudo-particles according to the invention have an excellent immunogenicityxe2x80x94only a single immunization is neededxe2x80x94and can thus be used in the absence of an immunostimulant adjuvant.
The parvovirus is advantageously PPV, CPV (Canine Parvovirus), or another related virus such as FPLV (Feline Panleukopenia virus) or MEV (Mink Enteritis virus).
The antigenic determinant may be composed of any sequence binding to a class I MHC molecule and able to induce a T lymphocyte cytotoxicity. It may also be composed of a sequence binding to a class II molecule in which case it is able to induce a helper response and/or cytokine production. It may in particular be a CD8+ T antigenic determinant of a virus, such as HIV-1 or HIV-2, or influenza virus, or a tumoral antigenic determinant or one expressed by cancer cells.
According to an advantageous embodiment, the pseudo-particles according to the invention are characterized in that the selected antigenic determinant is the CD8+ CTL antigenic determinant contained in the 118-132 region of the nucleoprotein of lymphocytic choriomeningitis virus (LCMV).
The antigenic determinant may however be any other antigenic determinant able to induce a CTL response, such as for example one of the following antigenic determinants:
The antigenic determinants of the Env gp120, Env gp41, Gag p17, Gag p24, Gag p15, Pol and Nef proteins of the HIV virus, and the antigenic determinants of the Gag and nef proteins of the SIV virus and the Gag protein of the HIV-2 virus, as defined by Venet and Walker (AIDS 1993, Vol. 7, suppl. 1, 119-120).
The antigenic determinants of the proteins expressed by tumors which develop in man, such as the proteins expressed by the MAGE-1, MAGE-3, BAGE, GAGE-1,2, HER-2/neu genes, and melanocytic differentiation antigens, such as tyrosinase, Pmel17gp100, Melan-AMART-1, and gp75TRP1, defined by Van Den Eynde and Brichard (Current Opinion in Immunology, 1995, 7, 674-681).
The antigenic determinant may also be an antigenic determinant able to induce a CD4+ helper T response, such as for example one of the following antigenic determinants:
The antigenic determinants expressed by viruses such as poliovirus, for example the C3:T antigenic determinant of the PV1 poliovirus contained in the 103-115 region, or the viral hepatitis virus, such as the PreS:T antigenic determinant contained in the PreS2 120-132 region of the hepatitis B virus.
The pseudo-particle may also comprise a combination of antigenic determinants, and in particular:
at least one CD4+ T antigenic determinant and at least one CD8+ T antigenic determinant
a combination of multicopies of CD4+ or CD8+ T antigenic determinants
a combination of four copies of the Mut CD8+ T antigenic determinants of Lewis carcinoma.
By a combination of antigenic determinants should be understood a combination comprising at least two antigenic determinants and preferably between two and ten antigenic determinants of identical proteins, such as for example a multicopy of the Mut 1 antigenic determinants of Lewis carcinoma, or of different proteins. Thus a combination of multicopies of CD8+ T antigenic determinants may comprise at least two antigenic determinants and preferably between two and ten antigenic determinants originating from different proteins, such as the combination of a CD8+ T antigenic determinant of the nucleoprotein of lymphocytic choriomeningitis virus and an Env gp 41 CD8+ T antigenic determinant of HIV.
Said recombinant viral pseudo-particles may advantageously be obtained by a process comprising a step of expression in a baculovirus of the chimeric protein consisting of the VP2 protein and said antigenic determinant.
The pseudo-particles according to the invention are obtained, even more preferably, by auto-assembly of the parvovirus VP2 protein modified by the presence, at its N-terminal end, of at least one antigenic determinant of the LCMV nucleoprotein or of at least one antigenic determinant of HBV, poliovirus, or a combination of multicopies of antigenic determinants.
It should be noted that any other molecule, and in particular any other protein, with properties similar to those of VP2, such as auto-assembly, and able to be modified by an antigenic determinant, may be used within the scope of the present invention instead of VP2.
Another object of the present invention is the use of the pseudo-particles in an effective quantity for immunization, to induce CTL and/or CD4+ helper T responses in vivo at a high level.
By the expression xe2x80x9ceffective quantity for immunizationxe2x80x9d should be understood a quantity of the pseudo-particles according to the invention chosen as a function of the administration route and of the weight of the individual. The quantity administered is advantageously between 10 and 500 xcexcg of pseudo-particles per individual.
A further object of the present invention is a composition comprising the pseudo-particles in an immunologically excipient and/or diluent from the immunological point of view. As diluent may be used an aqueous solution buffered to around pH 7 (physiological solution).
Advantageously, the composition according to the present invention is free from immunostimulant adjuvant. It may nevertheless contain, if necessary, such an adjuvant which may be aluminum hydroxide.
The pseudo-particles according to the present invention may also be used in an in vitro stimulation process of the cytotoxic T responses of lymphocytes originating from subjects suffering from viral or tumoral infections, comprising bringing the subjects"" lymphocytes into contact with the pseudo-particles. In this case, the cells are, after treatment, readministered to the patients, for example by injection.
Another object of the present invention is the use of the pseudo-particles according to the invention in the preparation of a vaccine, preferably a single-dose vaccine, and in particular antitumoral or antiviral. It may be administered by normal routes for vaccines, i.e. via intramuscular, intradermal, subcutaneous or possibly oral routes, or any other route leading to a CTL and/or CD4+ T response.