Pharmaceutical compositions comprising a polypeptide comprising at least one CXXC motif and heterologous antigens and uses thereof

Abstract

The invention relates to pharmaceutical compositions using a polypeptide comprising at least one CXXC motif, such as Giardia parasite's variable surface proteins (VSP) or a fragment thereof to raise by oral or mucosal vaccination an immune response against a heterologous selected antigen, such as tumor antigen, microbial antigen or other antigen.

Claims

1. An immunogenic composition against an antigen comprising: an antigen bound to a polypeptide, wherein the polypeptide: is heterologous to the antigen, comprises 10 to 14 CXXC motifs, wherein C represents a cysteine residue and X any amino acid residue, is a variable surface protein (VSP), a VSP-like protein of a microorganism or a fragment thereof, and is able to attach to epithelial cells of the gut, and wherein CXXC corresponds to SEQ ID NO 1, wherein the microorganism is selected from the group consisting of Giardia, Tetrahymena, Paramecium and Entamoeba species, wherein the polypeptide is bound to a vector particle containing said antigen, and wherein the vector particle is a viral particle, a viral-like particle (VLP) or a nanoparticle.

2. The immunogenic composition according to claim 1 for use in oral administration.

3. The immunogenic composition according to claim 1, wherein the polypeptide is resistant to enzymatic and/or chemical degradation of the upper gastrointestinal tract.

4. The immunogenic composition according to claim 1, wherein the polypeptide is the extracellular domain of VSP or a fragment thereof.

5. The immunogenic composition according to claim 1, wherein the polypeptide is fused to said antigen.

6. The immunogenic composition according to claim 1, wherein The polypeptide is bound to a vector particle containing said antigen, and wherein the vector particle is a VLP displaying at its surface the polypeptide.

7. The immunogenic composition according to claim 6, wherein the antigen is contained inside or on the surface of the VLP.

8. The immunogenic composition according to claim 1, wherein the vector particle is a nanoparticle displaying at its surface the polypeptide and the heterologous antigen.

9. A pharmaceutical composition comprising at least the vaccine according to claim 1.

10. A method of immunization of a subject against an influenza A H5N1, wherein said method comprises administering to said subject said antigen bound to a polypeptide, wherein the polypeptide: is heterologous to said antigen, comprises 10 to 14 CXXC motifs, wherein C represents a cysteine residue and X any amino acid residue, is a variable surface protein (VSP), a VSP-like protein of a microorganism or a fragment thereof, and is able to attach to epithelial cells of the gut, and wherein CXXC corresponds to SEQ ID NO 1, wherein the microorganism is selected from the group consisting of Giardia, Tetrahymena, Paramecium and Entamoeba species, wherein the polypeptide is bound to a vector particle containing said antigen, and wherein the vector particle is a viral particle, a viral-like particle (VLP) or a nanoparticle.

11. The method of claim 10, wherein said antigen bound to a polypeptide is administered by oral route.

Description

FIGURES

(1) FIG. 1: CFSE labeled splenocytes from SFE-TCR mice (10.sup.6/well) were cultured with CD11c purified DCs (5T:1DC ratio) for 72 h in medium alone, VLP-VSP-HA or recombinant SFE peptide (positive control) at the indicated concentrations. Histograms show T cell proliferation by CFSE dilution of SFE-specific T cells (gated using an anti-CD4+ antibody and the 6.5 anti-clonotypic antibody that specifically recognizes the transgenic T cells). Numbers indicate the percentage of divided cells.

(2) FIG. 2: ELISPOT Assay (Oral immunization with HA-VSP fusion protein). HA-specific IFN-γ production was determined by a standard ELISPOT assay (Mabtech, Sophia Antipolis, France). Splenocytes (5×105 cells/well) were stimulated overnight at 37° C. in 5% CO2 with 1 μg/mL of HA protein. PBS or concanavalin A (5 μL/mL; ConA; Sigma-Aldrich) were used as negative and positive controls, respectively. After revelation, spots were counted using the AID ELISPOT reader (ELR03, AID AutoimmunDiagnostika, Strassberg, Germany) and unspecific spots detected in the negative controls were substracted. Symbols represent individual mice and horizontal lines represent the geometric mean of each group. *p<0.05, **p<0.01.

(3) FIG. 3: Humoral immune response in mice orally vaccinated with the VSP-HA fusion protein. Enzyme-linked immunosorbent assay (ELISA). 96-well microtiter plates were coated with recombinant HA H5N1. Serial dilutions of sera were added and incubated for 2 h at RT, revealed for 1 h at RT with biotin-labeled goat Goat anti mouse Ig (H+L) (Biot. Human adsorbed. Southern Biotech Cat #1010-08), and for 1 h at RT with an ultrasensitive streptavidin-peroxidase polymer (Sigma-Aldrich). Peroxidase activity was measured using TMB substrate (Sigma-Aldrich) and optical densities were read at 450 nm (OD450) after blocking the reaction by adding HCl. The amount of anti HA were calculated based in a standard monoclonal antibody anti H5N1 HA (Mouse anti Influenza A, Avian H5N1 hemagglutinin (HA) Cat #17649-55B. USBiological).

(4) FIG. 4: ELISPOT Assay (Oral immunization with VLPs pseudotyped with HA and VSP). HA-specific IFN-γ (A) and IL-4-(B) production was determined by a standard ELISPOT assay (Mabtech, Sophia Antipolis, France). Splenocytes (5×105 cells/well) were stimulated overnight at 37° C. in 5% CO2 with 20 ng of HIV-Gag based particles pseudotyped with HA and NA†. Medium alone or concanavalin A (2 μL/mL; ConA; Sigma-Aldrich) were used as negative and positive controls, respectively. After revelation, spots were counted using the AID ELISPOT reader (ELR03, AID AutoimmunDiagnostika, Strassberg, Germany) and unspecific spots detected in the negative controls were subtracted. Symbols represent individual mice and horizontal lines represent the geometric mean of each group. *p<0.05, **p<0.01, *** p<0.001.

(5) †: HIV-Gag based lentiviral particles were generated by transfection of 293T cells with expression vectors encoding the viral components (pCMV9 (Gag)+HA (pXD14)+NA (pXD15). An HIV p24-specific ELISA assay (Kit RETRO-TEK#HIV-1p24 Antigen ELISA;ZeptoMetrixCorp., New-York, USA) was used to determine the p24 concentrations in the lentiviral vector samples, according to the manufacturer's instructions.

(6) FIG. 5: Humoral immune response in mice orally vaccinated with the VLP-VSP-HA/NA. Hemagglutination inhibition (HI) antibody responses. Sera samples were serially diluted and incubated 1 h at 37° C. with 4 HA units of H5N1-pseudotyped MLV-Gag based particles in 25 μL PBS. Then, 50 μL of a 0.5% chicken erythrocyte suspension was added to each well. HI antibody titers are expressed as the reciprocal of the highest dilution of samples inhibiting agglutination. Symbols represent individual mice and horizontal lines represent the geometric mean of each group.

EXAMPLES

(7) To obtain a proof of principle and, simultaneously, to develop a potential vaccine candidate, we used flu hemaggluttinin (HA) as a model vaccinal antigen.

(8) Therefore, to determine whether a protective and complete (T and B) immune response could be elicited by oral delivery of HA antigens carried by VSP, we constructed several vectors which would permit the concomitant expression of the VSP and the HA antigens. The HA protein from Influenza A H5N1/Hong Kong virus was used as antigen to induce B cell- and T cell-specific immune responses.

Example 1

Material & Methods

(9) Generation of Three Different VSP/HA Constructions to be Used as Oral Vaccines:

(10) To determine whether a protective and complete (T and B) immune response could be elicited by oral delivery of influenza hemagglutinin (HA) antigens carried by Giardia VSP, several constructions are made in order to permit the concomitant expression of the VSP and the HA antigens. The HA protein and its immunodominant SFE peptide are used as heterologous antigens to induce B cell- and T cell-specific immune responses, respectively.

(11) These VSP/HA constructions include fusion proteins and virus-like particle (VLP) on which VSP and HA antigens are bound.

(12) More precisely, three different VSP/HA constructions are thus generated, namely:

(13) 1—VSP fused to the SFE peptide to monitor T-cell specific immune responses.

(14) 2—VSP fused to the extracellular portion of HA (AHA) to monitor B-cell and T-cell specific immune responses

(15) 3—A virus-like particle (VLP) displaying VSP and HA proteins (full-length form) at its surface and/or the SFE peptide inside the particle as a gag fusion protein. VLP are formed by MLV Gag proteins fused with the SFE peptide. For displaying the VSP protein at the surface of VLPs, VSP are fused to the VSV-G transmembrane (TM) region.

(16) Production of the VSP Fusion Proteins and Biochemical Characterization:

(17) Control: ΔHA Alone and Polypeptide of the Invention: VSP Ex Fused to the Extracellular Portion of HA (ΔHA).

(18) More particularly, the cDNA sequences encoding the ΔHA were derived from Influenza A H5N1 (Hong Kong). Genscript Company was hired for the production of the protein. The recombinant protein DNA sequence was codon optimized and transformed in bacteria using the company's expression vectors. Recombinant HA was obtained in the bacteria expression system using E. coli BL21 (DE3) strain. For purification of the HA protein a Q-column under denaturating conditions was used and the resulted protein was refolded by dialysis against 50 mM Tris-HCl, 5% Glicerol, pH 8.0.

(19) For the production of the VSP fused to the extracellular part of the HA protein, we used HA in its soluble form—the TM region was deleted-(ΔHA). Protein sequences were analyzed using the online topology prediction platform Phobius (http://phobius.sbc.su.se/).

(20) The full-length VSP contains a cysteine-rich extracellular region containing numerous CXXC (SEQ ID NO.: 1) motifs. The signal peptide, the transmembrane region and the cytoplasmic 5 residues were eliminated (VSP Ex). VSP 1267 was used in these experiments.

(21) The sequence of the fusion protein was codon optimized for the cloning into the baculovirus system. The protein was expressed and purified by one step affinity purification using the His tag present in the carboxyl terminal portion of the protein

(22) Production of the Retroviral Particles (VLP) Pseudotyped with VSP and HA at the Surface:

(23) VLPs made from a Gag and Pseudotyped with HA and NA (Neuraminidase):

(24) For the HA vector, the HA H5N1 (Hong Kong) sequence was cloned including its own TM domain (pXD14 vector). Wild type HA is naturally and very efficiently pseudotyped onto viral or pseudoviral particles. For NA expression the pXD15 vector was used.

(25) VLPs Made from a Gag and Pseudotyped with VSP and HA and NA:

(26) For pseudotyping VSP sequences onto the VLPs, the extracellular domain of the VSP was fused to the transmembrane domain (TM) of the G protein of vesicular stomatitis virus (VSV-G), which is known to be efficiently exported at the plasma membrane in mammalian cells, co-localized with Gag proteins, and be pseudotyped onto newly formed viral or pseudoviral particles (pCP1267 vector).

(27) Note: for some experiments a Gag-Gp33-41 fusion protein was used instead of GAG, so as to measure the CD8 response. For displaying the Gp33-41 peptide inside the particle, Gp33-41 was fused to the carboxyl terminus of MLV Gag (pEB1 vector).

(28) DNA Production:

(29) Once all constructs were validated, plasmids were amplified and purified. The production of plasmid was done using endotoxin-free preparation kits (Nucleobond® PC 2000 EF; Macherey-Nagel, Hoerd, France).

(30) VLP Production:

(31) To generate recombinant retroviral particles, 293T cells were transfected with the generated expression vectors (pEB1, pCP1267, pXD15 and pXD14). Supernatants containing particles were concentrated and purified by ultracentrifugation, with or without an additional purification step by FPLC. Each batch of VLPs was submitted to quality control analysis to validate the presence of MLV-Gag, HA or VSP by different techniques: The functionality of the VSP-TM construct is demonstrated by its proper expression at the surface of cells transfected by the pCP1267 vector

(32) The efficiency of the VLPs production and incorporation of VSP into or onto the VLPs was assessed by Western blot on ultracentrifuged supernatant.

(33) Results:

(34) The correct pseudotyping of HA onto the VLPs was assessed by a hemaglutination assay. Chicken red blood cells (RBC) were incubated in presence of serial dilutions of different VLPs to evaluate agglutination in presence of a good conformational HA protein. We used VLP-HAH5N1/NA as positive control and VLP-Gag-GFP as negative control. Incubation of RBC with PBS serves to evaluate sedimentation time. By this test we showed that the VLP-VSP-HA/NA that we have developed during this study were able to promote chicken RBC agglutination.

Example 2

Material & Methods

(35) In Vitro Validation of the Three Constructions:

(36) 2.1—At the Biochemical Level:

(37) VSP-specific immunoprecipitation are made in order to validate the VSP-AHA and VLP constructs. Immunoprecipitates are analyzed by Western-blot. The detection of HA and the detection of HA/Gag proteins are made in order to validate the construct respectively.

(38) 2.2—At the Immunological Level:

(39) To determine if the HA antigens can be recognized by HA-specific T cells, in vitro proliferation tests are performed with SFE-specific CD4+ T cells (obtained from the SFE transgenic mice as described in Kirberg et al. 1994), in the presence of DC sensitized with each of the three constructions or loaded with purified recombinant proteins.

(40) Results:

(41) We confirmed that the HA antigen present in the VLPs was correctly processed and activated HA-specific T cells by an in vitro proliferation test using CFSE labeled SFE-specific CD4+ T cells transgenic for a TCR that specifically recognizes the SFE110-119 peptide form HA (obtained from the SFE transgenic mice (Kirberg J., 1994). As observed in FIG. 1, the transgenic cells actively divide in the presence of dendritic cells (DC) pulsed with the VLP-VSP-HA, indicating that the HA protein present in the VLP has been correctly processed and presented in an MHC cII restricted way.

Example 3

Characterization of the Immune Response Anti-HA and Anti-VSP in Mice Orally Immunized by the VSP/HA Constructs

(42) The local and systemic immune responses in mice are analyzed at different time points after oral vaccination.

(43) Model:

(44) Mice (H-2.sup.d) are immunized orally with VSP-AHA, or HA-VLPs (VSP-). As control, mice are immunized orally with ΔHA or HA-VLP (VSP-) and immunized sub-cutaneously with HA-VLPs in Al(OH).sub.3. [positive control].

(45) Analysis: Systemic T cell responses: Frequency of HA-specific T cells are analyzed by IFN-γ ELISPOT after HA-specific re-stimulation of the spleen cells. Systemic B cell responses: the HA-specific antibody response are studied in serum by ELISA or inhibition hemagglutination assays.

(46) Oral Immunization with the VSP-HA Fusion Protein:

(47) Immunization Protocol:

(48) Female Balb/c (H-2d) mice, 7 weeks-old received three successive oral administrations of 35 μg of the recombinant ΔHA protein or the recombinant ΔHA-VSP protein suspended in sterile PBS-Tween 20, 0.01% 3 days apart. As control, mice received vehicle only (negative control), or were once immunized s.c. with 35 ug of ΔHA in alum (positive control).

(49) The anti-HA T cell response was analyzed in a group of mice sacrificed at day 17 (10 days after the last oral dose). The anti-HA B cell response was studied in another group of mice at day 21 (14 days after the last immunization).

(50) Results:

(51) Analysis of the T Cell Response:

(52) As observed in FIG. 2, immunization with the VSP-HA fusion protein successfully induced an HA specific IFN-γ T cell response in 2 out of 2 immunized mice, as opposed to the oral immunization with the HA protein alone, which induced no significant response in 3 out of 3 vaccinated mice.

(53) These results establish that fusion of the HA antigen to the VSP protein endows the fusion protein with the unique capacity to generate an HA antigen specific systemic T cell response when administered by the oral route.

(54) Analysis of the B cell Response:

(55) The generation of anti-HA specific Ab was analyzed by ELISA. Oral administration of the HA protein was unable to induce anti-HA Ab, whereas the VSP-HA fusion protein induced high titers of anti-HA Abs in one of the orally immunized mice, indicating that in the presence of the VSP, a systemic B cell response can be generated against HA (FIG. 3).

(56) Oral Immunization with the VLPs Pseudotyped with VSP and HA at the Surface:

(57) Immunization Protocol:

(58) Female Balb/c (H-2d) mice, 7 weeks-old received three successive oral administrations of 35 μg of the VLP-HA/NA, VLP-VSP-HA/NA suspended in sterile PBS-Tween 20, 0.01% 3 days apart. As control, mice received vehicle only (negative control), or were once immunized s.c. with 35 ug of the VLP-VSP-HA/NA in alum (positive control).

(59) Mice were sacrificed at day 17 (10 days after the last oral dose) and the T and B cell response to HA was analyzed.

(60) Results:

(61) Analysis of the T cell Response:

(62) As observed in FIG. 4, immunization with the VLP-VSP-HA/NA fusion protein successfully induced an HA specific IFN-γ T cell response in 3 out of 3 immunized mice, as opposed to the oral immunization with the VLP-HA/NA, which induced no significant response in 3 out of 3 vaccinated mice. No significant HA-specific IL-4 production was detected, suggesting that the immune response generated by the fusion protein is of the Th1 type. These results establish that shielding the VSP-HA with the VSP protein endows the particle with the unique capacity to generate an HA antigen specific systemic T cell response when administered by the oral route.

(63) Analysis of the B cell Response:

(64) For the analysis of the systemic B cell responses, we quantified the HA-specific antibody response in serum using an inhibition hemagglutination assay as shown in FIG. 5. It can be seen that only VLP pseudotyped with HA and VSP can generate specific anti HA antibodies, indicating that the presence of VSP onto the VLP was necessary for the generation of a systemic anti-HA B cell response.

Conclusion

(65) We have produced the oral vaccines composed of VSP-HA chimerical proteins or HA-expressing VLPs covered with VSPs and HA and the corresponding controls. We have biochemically validated that the VSPs and the HA proteins keep their correct conformation in the corresponding constructions. We have scaled up the production to orally immunize animals. In these experiments we have observed that contrary to the oral administration of the HA protein alone, which does not induce an specific T or B cell response, the oral administration of HA shuttled by VSPs—be as a fusion protein or in a VLP formulation-generates a HA-specific humoral and cellular response.

(66) This work proves the validity of our strategy.

(67) The development of this universal platform for oral delivery of vaccines should have a broad application to different infectious diseases. A great interest exists in the oral administration route, in particular for prophylactic vaccines for mass vaccination.

(68) It results of the experiments that Giardia VSPs and more generally polypeptides comprising at least one CXXC motif according to the present invention seem to represent an excellent carrier to shuttle a candidate antigen trough the digestive tube to the intestine, where it may stay for a time allowing for the development of an immune response. And not least, the VSP may also act as a mucosal adjuvant, as suggested by its capacity to induce an immune response (antibodies response) by its own.

(69) Indeed, as proof-of-principle the extracellular domain of the intestinal parasite Giardia VSPs as carrier to shuttle candidate antigens for oral vaccines has been shown to have the capacity to induce an effective immune response to the flu HA by oral vaccination.

REFERENCES

(70) Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. Adam R D, Nigam A, Seshadri V, Martens C A, Farneth G A, Morrison H G, Nash T E, Porcella S F, Patel R; The Giardia lamblia vsp gene repertoire: characteristics, genomic organization, and evolution; BMC Genomics. 2010 Jul. 9; 11:424. Franzén O, Jerlström-Hultqvist J, Castro E, Sherwood E, Ankarklev J, Reiner D S, Palm D, Andersson J O, Andersson B, Svärd S G; Draft genome sequencing of giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species?; PLoS Pathog. 2009 August; 5(8):e1000560. Hlaysa M C, Watson J C, Beach M J; Giardiasis surveillance—United States, 1998-2002; MMWR Surveill Summ. 2005 Jan. 28; 54(1):9-16. Jerlström-Hultqvist J, Franzen O, Ankarklev J, Xu F, NohýnkováE, Andersson J O, Svärd S G, Andersson B; Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate; BMC Genomics. 2010 Oct. 7; 11:543. Kirberg J, Baron A, Jakob S, Rolink A, Karjalainen K, von Boehmer H; Thymic selection of CD8+ single positive cells with a class II major histocompatibility complex-restricted receptor; J Exp Med. 1994 Jul. 1; 180(1):25-34. Lavelle E C, O'Hagan D T; Delivery systems and adjuvants for oral vaccines; Expert Opin. Drug Deliv. 3(6), 747-762 (2006). Morrison H G, McArthur A G, Gillin F D, Aley S B, Adam R D, Olsen G J, Best A A, Cande W Z, Chen F, Cipriano M J, Davids B J, Dawson S C, Elmendorf H G, Hehl A B, Holder M E, Huse S M, Kim U U, Lasek-Nesselquist E, Manning G, Nigam A, Nixon J E, Palm D, Passamaneck N E, Prabhu A, Reich C I, Reiner D S, Samuelson J, Svard S G, Sogin M L; Genomic minimalism in the early diverging intestinal parasite Giardia lamblia; Science. 2007 Sep. 28; 317(5846):1921-6. Nash T. E; Antigenic variation in Giardia lamblia and the host's immune response. Philos Trans R Soc Lond B Biol Sci 352, 1369-1375 (1997). Nash T. E; Surface antigenic variation in Giardia lamblia; Mol. Microbiol. 2002 August; 45(3):585-90. Rivero F D, Saura A, Prucca C G, Carranza P G, Torri A, Lujan H D; Disruption of antigenic variation is crucial for effective parasite vaccine; Nat Med 2010 May; 16(5):551-7.