Methods for treating or preventing HBV infection or HBV related diseases

Abstract

The present invention relates to polymerase HBV mutant polypeptides comprising a mutated polymerase domain which is functionally disrupted for polymerase activity and fusion proteins comprising such polymerase mutant polypeptide. The present invention also relates to a nucleic acid molecule and an expression vector for expressing said polymerase mutant polypeptide as well as a composition which can be used for eliciting an immune response to HBV with the goal of providing a protective or therapeutic effect against HBV infection.

Claims

1. A method for treating or inhibiting or delaying an HBV infection, a chronic HBV infection or HBV-associated liver lesions, liver cancer, liver inflammation, chronic liver disease, comprising one or more administration(s), to a subject in need thereof, of a therapeutically effective amount of composition comprising a nucleic acid molecule coding for a mutant polypeptide, a vector comprising said nucleic acid molecule, a host cell comprising said nucleic acid molecule or said vector, or any combination thereof, wherein said mutant polypeptide comprises a mutated HBV polymerase domain with an internal deletion that functionally disrupts the polymerase activity, wherein said internal deletion is of at least 4 amino acid residues and at most 30 amino acid residues, said mutated polymerase domain comprising: the amino acid sequence shown in SEQ ID NO:1 but lacking at least the Tyr residue in position 203, the Met residue in position 204, the Asp residue in position 205, the Asp residue in position 206, the Val residue in position 207, the Val residue in position 208, and the Leu residue in position 209.

2. The method according to claim 1, wherein said nucleic acid molecule encodes a mutant polypeptide which, in addition to the deletion of residues 203 to 209 of SEQ ID NO:1: comprises a polymerase domain having the amino acid sequence shown in SEQ ID NO:2; comprises a RNaseH domain having the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4; comprises an amino acid sequence which exhibits at least 90% of identity with the amino acid sequence shown in SEQ ID NO:5; or comprises an amino acid sequence selected from which exhibits at least 90% of identity with the amino acid sequence shown in any of SEQ ID NOs 6-12.

3. The method according to claim 1, wherein said nucleic acid molecule encodes a mutant polypeptide which, in addition to the deletion of residues 203 to 209 of SEQ ID NO:1, comprises a nucleotide sequence exhibiting at least 90% of identity with the nucleotide sequence shown in any of SEQ ID NOs 13-17.

4. The method according to claim 1, wherein said vector is a plasmid or a viral vector for expression in higher eukaryotic cells or organisms.

5. The method according to claim 4, wherein said vector is a viral vector originating from a retrovirus, adenovirus, adenovirus-associated virus (AAV), poxvirus, herpes virus, measles virus, foamy virus, alphavirus, or vesicular stomatis virus.

6. The method according to claim 5, wherein said vector is a replication-defective adenoviral vector originating from a human or from a chimpanzee adenovirus.

7. The method according to claim 6, wherein the nucleic acid molecule is inserted in the adenoviral E1 region and placed under the control of a CMV promoter.

8. The method according to claim 5, wherein said vector is a poxviral vector originating from a canarypox, a fowlpox, or a vaccinia virus.

9. The method according to claim 1, wherein said vector is selected from the group consisting of: A defective Ad vector comprising inserted in place of the E1 region a nucleic acid molecule placed under the control of a promoter such as the CMV promoter, and encoding a mutant polypeptide comprising an amino acid sequence as shown in SEQ ID NO:5 or a fusion protein comprising an amino acid sequence as shown in SEQ ID NO:6 or SEQ ID NO:8; A replication-defective Ad vector comprising inserted in place of the E1 region a nucleic acid molecule placed under the control of a promoter such as the CMV promoter, and comprising the nucleotide sequence shown in SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; A replication-defective Ad vector, especially a defective AdCh3 comprising inserted in place of the E1 region a nucleic acid molecule placed under the control of a promoter such as the CMV promoter and comprising the nucleotide sequence shown in SEQ ID NO:16 or SEQ ID NO:17; A MVA vector comprising a nucleic acid molecule placed under the control of a vaccinia promoter such as the 7.5K or pH5R promoter, and encoding a mutant polypeptide comprising an amino acid sequence as shown in SEQ ID NO:5 or SEQ ID NO:10 or a fusion protein comprising an amino acid sequence as shown in SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:12; and A MVA vector comprising a nucleic acid molecule placed under the control of a vaccinia promoter such as the 7.5K or pH5R promoter, and comprising the nucleotide sequence shown in SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

10. The method according to claim 1, wherein said vector is in the form of infectious viral particles.

11. The method according to claim 1, wherein the mutant polypeptide encoded by said nucleic acid molecule is fused in frame to a signal peptide and to a trans-membrane peptide.

12. The method according to claim 1, wherein said composition further comprises a pharmaceutically acceptable vehicle.

13. The method according to claim 1, wherein said composition is formulated for intramuscular, subcutaneous, intradermal administration or scarification.

14. The method according to claim 1, wherein said composition comprises doses of about 510.sup.8, about 10.sup.9, about 510.sup.9, about 10.sup.10, about 510.sup.10 vp or about 10.sup.11 vp of an adenoviral vector.

15. The method according to claim 1, wherein said composition comprises doses of about 510.sup.8, about 10.sup.7, about 510.sup.7, about 10.sup.8, or about 510.sup.8 pfu of an MVA vector.

16. The method according to claim 1, for treating a chronic HBV infection.

17. The method according to claim 1, for eliciting or stimulating an immune response in the treated organism.

18. The method according to claim 17, wherein said elicited or stimulated immune response is specific and/or non-specific, humoral and/or cellular.

19. The method according to claim 18, wherein said immune response is a T cell response CD4+ or CD8+-mediated or both, directed to an HBV polypeptide/epitope.

20. The method according to claim 1, wherein said vector is an adenoviral vector and said method comprises one or two intramuscular or subcutaneous administrations.

21. The method according to claim 1, which is carried out in combination with the standard of care.

22. The method according to claim 1, which is carried out according to prime boost modality.

23. The method according to claim 22, wherein the priming is carried out with an MVA vector and the boosting with an Ad vector.

24. The method according to claim 23, wherein the MVA and/or the Ad vector encodes the fusion protein shown in SEQ ID NO: 8.

25. The method according to claim 24, comprising at least 3 subcutaneous administrations of the MVA vector separated by a period of time varying from 3 days to 3 months followed by an intramuscular or subcutaneous boost of the adenovirus vector.

26. The method according to claim 1, wherein said HBV-associated liver lesions, liver cancer, liver inflammation, chronic liver disease is selected from the group consisting of: cirrhosis, steatosis, fibrosis, hepatocellular carcinoma and liver carcinoma.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the mutant polymerase polypeptides, fusion proteins and antigenic combinations described in the invention.

(2) FIG. 2 illustrates Elispot IFNg assays carried out following immunization of HLA-A2 transgenic mice with plasmids pTG18188 (Core-Pol-Env1-Pol-Env2-Pol), pTG18194 (Core-Pol) or pTG13135 (Empty). Results are presented as the number of spots for 10.sup.6 cells corresponding to the frequency of IFNg producing cells specific of each HBV HLA-A2 peptides evaluated in the experiment for 10.sup.6 spleen cells of immunized mice. Each bar represent an individual mouse vaccinated by one or the other plasmid and indicated by its reference number (1.1 or 2.3 . . . etc) and the mean of all mice immunized with the same plasmid is also represented for each group (bar indicated as mean on the graph). For individual mouse and the means, frequency of IFNg producing cells specific of the different tested peptides are piled. Bars are filled with different symbols, each symbol representing the response against one specific HBV peptide as indicated by the legend on the graph.

(3) FIGS. 3A-F illustrates ICS assays carried out following immunization of HLA-A2 transgenic mice with plasmids pTG18188 (Core-P-E1-P-E2-P or Core-Pol-Env1-Pol-Env2-Pol), pTG18194 (Core-Pol) or pTG13135 (empty). Results are presented as the percentage of CD8 (FIGS. 3A, 3B, 3D and 3F), or CD4 (FIGS. 3C and 3E) T cells producing IFNg (alone or combined with TNFa) specific of each HBV HLA-A2 peptides (FIG. 3A) or peptide pools covering HBV core (FIGS. 3B and 3C), polymerase (FIGS. 3D and 3E) and env (FIG. 3F) antigens. Each bar represent an individual mouse vaccinated by one or the other plasmid and indicated by its reference number (1.1 or 2.3 . . . etc) and the mean of all mice immunized with the same plasmid is also represented for each group (bar indicated as mean on the graph). For individual mouse and the means, frequency of CD8 or CD4 T cells specific of the different tested peptides are piled. Bars are filled with different symbols, each symbol representing the response against one specific HBV peptide as indicated by the legend on the graph.

(4) FIG. 4 illustrates Elispot IFNg assays carried out following immunization of HLA-A2 transgenic mice with Adenovirus AdTG18201 (Core-Pol-Env Ad), AdTG18202 (Core-Pol Ad), AdTG18203 (Pol Ad) and AdTG15149 (Empty Ad). Results are presented as the number of spots for 10.sup.6 cells corresponding to the frequency of IFNg producing cells specific of each peptide pools covering the antigen of interest, evaluated in the experiment for 10.sup.6 spleen cells of immunized mice. Each bar represent an individual mouse vaccinated by one or the other plasmid and indicated by its reference number (1.1 or 2.3 . . . etc) and the mean of all mice immunized with the same plasmid is also represented for each group (bar indicated as mean on the graph). For individual mouse and the means, frequency of IFNg producing cells specific of the different tested peptides are piled. Bars are filled with different symbols, each symbol representing the response against one specific HBV peptide as indicated by the legend on the graph.

(5) FIG. 5 illustrates Elispots IFNg assays carried out following immunization of HLA-A2 transgenic mice with AdTG18201 (Core-Pol-Env Ad). Results are presented as the number of spots for 10.sup.6 cells corresponding to the frequency of IFNg producing cells specific of each HBV HLA-A2 epitope, or of an irrelevant peptide, evaluated in the experiment for 10.sup.6 spleen cells of immunized mice. Each bar represents an individual mouse vaccinated by the AdTG18201 (indicated with its reference number, 3.1 to 3.8) and the median of all these mice immunized with AdTG18201 is also represented (bar indicated as Median on the graph). For individual mouse and median, frequency of IFNg producing cells specific of the different HBV HLA-A2 epitopes are piled whereas the frequency of IFNg producing cells observed in presence of an irrelevant peptide is represented on a separate bar (indicated Irrel on the graph for each mouse). Bars are filled with different symbols, each symbol representing the response against one specific HBV peptide as indicated by the legend on the graph.

(6) FIG. 6 illustrates ICS assays carried out following immunization of HLA-A2 mice with AdTG18201 (Core-Pol-Env Ad) or AdTG15149 (Empty Ad). Results are presented as the percentage of CD8 T cells producing IFNg alone or in combination with TNFa, specific of two selected peptide pools, called PP8 and PC1, and covering respectively a part of the HBV polymerase (amino acids 725 to 835) and a part of the HBV Core protein (amino acids 1 to 100). Each bar represents an individual mouse vaccinated by one or the other adenovirus and indicated by its reference number (1.1 or 3.2 . . . etc) and the median of all mice immunized with the same adenovirus is also represented for each group (bar indicated as median on the graph). Bars are filled with different symbols, each symbol representing the cytokine(s) produced by the detected CD8 T cells as indicated by the legend on the graph.

(7) FIGS. 7A-B illustrates in vivo CTL assays carried out following immunization of HLA-A2 mice with AdTG18201 (Core-Pol-Env Ad) or AdTG15149 (Empty Ad). Results are presented as the percentage of in vivo specific lysis, ie lysis specific of the HBV HLA-A2 epitopes that were tested. Each square or triangle symbols represents an individual mouse and the mean of all mice immunized with the same adenovirus is also represented for each group (represented by a thick bar symbol on the graph).

(8) FIG. 8 illustrates ICS assays carried out following immunization of HBV transgenic mice with AdTG18201 (Core-Pol-Env Ad) or AdTG15149 (Empty Ad). Results are presented as the percentage of CD8 T cells producing both IFNg and TNFa specific of epitopes from the HBV polymerase (mix of 2 peptides, called VSA and N13F) or from the HBV envelope (1 peptide called F13L) and found both in spleens and in livers of vaccinated mice. Each bar represents an individual mouse vaccinated by one or the other adenovirus and indicated by its reference number (1.1 or 3.2 . . . etc) and the median of all mice immunized with the same adenovirus is also represented for each group (bar indicated as median on the graph). The dotted line represented on graphs corresponds to the cut-off of the experiment, ie the threshold above which observed percentage of CD8 T cells is considered as a positive immune response.

(9) FIG. 9 illustrates Elispots IFNg assays carried out following immunization of HLA-A2 transgenic mice with AdTG18201 (Core-Pol-Env Ad) with different doses, from 10.sup.5 iu to 10.sup.9 iu. Results are presented as the number of spots for 10.sup.6 cells corresponding to the frequency of IFNg producing cells specific of each HBV HLA-A2 epitope, or of an irrelevant peptide, or in presence of medium only, evaluated in the experiment for 10.sup.6 spleen cells of immunized mice. Each solid or dotted bar represents an individual mouse vaccinated by the AdTG18201 and the mean of all mice immunized with AdTG18201 at one specific dose is also represented (hatched bars). The different doses are represented by different colors or symbols as indicated by the legend on the graph. The dotted-line represents the cut-off value, defined as described in Material and Methods, above which observed T cell responses are considered as positive.

(10) FIG. 10 illustrated Elispots IFNg assays carried out following immunization of HLA-A2 transgenic mice with AdTG18202 (Core-Pol Ad) according to different schedules of injections Mice were immunized either once (squares) or received 3 injections at 1 week interval (triangles) or 6 injections at 1 week interval (circles). Results are presented as the number of spots for 10.sup.6 cells corresponding to the frequency of IFNg producing cells specific of each HBV HLA-A2 epitope, or of the adenovirus vector or an irrelevant peptide or in presence of medium only evaluated in the experiment for 10.sup.6 spleen cells of immunized mice. Each symbol (square, triangle or circle) represents an individual mouse vaccinated by the AdTG18202 and the mean of all mice immunized with AdTG18202 with one of the tested schedules is represented by a solid thick line. The dotted-line represents the cut-off value, defined as described in Material and Methods, above which observed T cell responses are considered as positive.

(11) FIGS. 11A-B illustrated Elispots IFNg assays carried out following immunization of HLA-A2 transgenic mice with AdTG18202 (Core-Pol Ad) according to different schedules of injections Mice were immunized either once 2 weeks before the monitoring of T cell responses (group 1 represented by squares) or once 20 weeks before the monitoring of T cell responses (group 2 represented by triangles), or twice at 2 months interval (group 3 represented by circles), or twice at 4 months interval (group 4 represented by crosses) or three times at 2 month interval (group 5 represented by rhombuses). For all groups except group 2, T cell responses were monitored 2 weeks after the last injection. Results are presented as the number of spots for 10.sup.6 cells corresponding to the frequency of IFNg producing cells specific of each HBV HLA-A2 epitope or an irrelevant peptide or in presence of medium only, evaluated in the experiment for 10.sup.6 spleen cells of immunized mice. Each symbol (square, triangle, circle, cross, rhombus) represents an individual mouse vaccinated by the AdTG18202 and the mean of all mice immunized with AdTG18202 with one of the tested schedules is represented by a solid thick line. The dotted-line represents the cut-off value, defined as described in Material and Methods, above which observed T cell responses are considered as positive.

EXAMPLES

(12) 1. Material and Methods

(13) The constructions described below (see FIG. 1) are carried out according to the general genetic engineered and molecular cloning techniques detailed in Maniatis et al. (1989, Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. or subsequent editions) or according to the manufacturer's recommendations when a commercial kit is used. PCR amplification techniques are known to the person skilled in the art (see for example PCR protocolsA guide to methods and applications, 1990, published by Innis, Gelfand, Sninsky and White, Academic Press). The recombinant plasmids carrying the ampicillin resistance gene are replicated in the E. coli C600 (Stratagene) on agar or liquid medium supplemented with 100 g/ml of antibiotic.

(14) MVA vector construction are generated by homologous recombination between a shuttle plasmid and the MVA genome as previously described in Erbs et al. (2008, Cancer gene Ther. 15: 18). The basic shuttle plasmid contains a multiple cloning site, a vaccinia virus (VV) promoter surrounded by the flanking sequences of deletion III as well as the E. Coli xanthine-guanine phosphoribosyl-transferase (GPT) selection gene under the control of p11K7.5 vaccinia promoter (Falkner and Moss, 1988). Briefly, CEF cells were infected with a genomic MVA without any inserted transgene (MVA-null) and then transfected by CaCl.sub.2 precipitation with the shuttle plasmid carrying the gene of interest cloned downstream the VV promoter. Homologous recombination occurred between MVA-null and the shuttle plasmid and recombinant viruses were isolated by multiple steps of mycophenolic acid selection. Recombinant MVA viruses were controlled by PCR, amplified in CEF and virus stocks were titrated on CEF by plaque assay.

(15) For adenoviral vector construction, an adenoviral shuttle plasmid is first constructed by inserting the nucleic acid molecule of interest into the basic shuttle plasmid pTG13135. Typically, the nucleic acid molecule is inserted into the NheI and NotI restriction sites of pTG13135 containing a CMV-driven expression cassette surrounded by adenoviral sequences (adenoviral nucleotides 1-454 and nucleotides 3513-5781 respectively) to allow further generation of the vector genome by homologous recombination (Chartier et al., 1996, J. Virol. 70:4805). The adenoviral vector is then obtained by homologous recombination between the recombinant shuttle vector digested by Bst1107I and PacI and pTG15375 (encoding the complete adenoviral genome) linearized by ClaI digestion. The resulting adenoviral vector is E3 (nucleotides 27867-30743) and E1 (nucleotides 455-3512) deleted, with the E1 region replaced by the expression cassette containing, from 5 to 3, the CMV immediate-early enhancer/promoter, a chimeric human -globin/IgG intron (as found in pCI vector available in Promega), the nucleic acid molecule of interest and the SV40 late polyadenylation signal. Adenoviral particles are then obtained by transfecting the PacI linearized viral genome into an E1 complementation cell line. Virus propagation, purification and titration is made as described previously (Erbs et al., 2000, Cancer Res. 60:3813)

(16) 1.1. Vectors Constructions and Production

(17) The vectors illustrated hereinafter have been engineered to express the mutant polymerase polypeptide eventually fused to the Core polypeptide and/or immunogenic domains of the envelope protein. All HBV sequences originate from HBV strain Y07587 which sequence is described in international databases (Genbank Y07587) and in different publications. It is a genotype D virus of serotype ayw.

(18) The following examples illustrate the fusion of a truncated Core polypeptide (aa 1-148) with a mutated polymerase polypeptide (designated Pol*) comprising two internal deletions (from positions 538 to 544 and from positions 710 to 742) and 4 amino acid substitutions (D689H, V769Y, T776Y and D777H respectively) as represented in SEQ ID NO: 6 as well as a longer fusion further comprising two immunogenic Env domains (Env1 and Env2 respectively extending from amino acids 14 to 51 and from amino acids 165 to 194 of the HBs protein) inserted in place of the deleted pol regions as represented in SEQ ID NO: 8.

(19) 1.1.1. Construction and Production of Plasmid and Adenovirus Vectors Expressing Truncated Core-Pol*-Env1-Env2 (or Core-Pol-Env1-Pol-Env2-Pol) Fusion

(20) A synthetic gene (3024 nucleotides described in SEQ ID NO: 15) encoding the truncated Core-Pol*-Env1-Env2 fusion protein (amino acid sequence is shown in SEQ ID NO: 8) was synthesized by GENEART (Regensburg, Germany). The synthetic fragment was inserted into the NheI and NotI restriction sites of pTG13135 shuttle plasmid, providing pTG18188. An adenoviral vector was then obtained by homologous recombination between pTG18188 digested by Bst1107I and PacI and pTG15375 linearized by ClaI digestion. The resulting adenoviral vector pTG18201 is E3 and E1 deleted, with the E1 region replaced by the expression cassette containing the synthetic sequence encoding the truncated Core-Pol*-Env1-Env2 driven by the CMV promoter. Adenoviral particles (AdTG18201) were obtained by transfecting the Pad linearized viral genome into an E1 complementation cell line.

(21) 1.1.2. Construction and Production of Plasmid and Adenovirus Vectors Expressing Truncated Core-Pol*

(22) A synthetic gene (2820 nucleotides described in SEQ ID NO: 14) encoding a truncated Core-Pol* fusion protein was synthesized by GENEART (Regensburg, Germany). The synthetic fragment was inserted into the NheI and NotI restriction sites of pTG13135 shuttle plasmid, providing pTG18194. An adenoviral vector was then obtained by homologous recombination between pTG18194 digested by Bst1107I and PacI and pTG15375 linearized by ClaI digestion. The resulting adenoviral vector pTG18202 is E3 and E1 deleted, with the E1 region replaced by the expression cassette containing the synthetic sequence encoding the truncated Core-Pol* driven by the CMV promoter. Adenoviral particles (AdTG18202) were obtained by transfecting the PacI linearized viral genome into an E1 complementation cell line.

(23) 1.1.3. Construction and Production of Plasmid and Adenovirus Vectors Expressing Pol*

(24) A synthetic gene (2379 nucleotides described in SEQ ID NO: 13) encoding the Pol mutant polypeptide was synthesized by GENEART (Regensburg, Germany). The synthetic fragment was inserted into the NheI and NotI restriction sites of pTG13135 shuttle plasmid, providing pTG18195. An adenoviral vector was then obtained by homologous recombination between pTG18195 digested by Bst1107I and PacI and pTG15375 linearized by ClaI digestion. The resulting adenoviral vector pTG18203 is E3 and E1 deleted, with the E1 region replaced by the expression cassette containing the synthetic sequence encoding Pol* driven by the CMV promoter. Adenoviral particles (AdTG18203) were obtained by transfecting the PacI linearized viral genome into an E1 complementation cell line.

(25) 1.2. Immunogenicity Evaluation in a Mouse Model

(26) Antigen immunogenicity was evaluated in vivo by Elispot IFN assays and Intracellular cytokine staining (ICS) following immunization of HLA transgenic mice.

(27) 1.2.1 Mouse Model

(28) The HLA-A2.1 transgenic mice used in the study were described by Pascolo et al. (1997, J. Exp. Med. 185:2043). These mice have the H-2D.sup.b and murine .sub.2-microglobulin genes knocked-out and express a transgenic monochain histocompatibility class I molecule (HHD molecule) in which the C-terminus of the human 2m is covalently linked to the N-terminus of a chimeric heavy chain (HLA-A*0201 1-2, H-2D.sup.b 3 transmembrane and intracytoplasmic domains). Seven to 10 weeks-old mice (male and female) were immunized. Average weight of the mice is around 25-30 g.

(29) The HBV transgenic mice used in the study were described by Halverscheid et al (2008, J. Med. Virol. 80: 583-590) and kindly provided by Reinhold Schirmbeck. These mice are on a C57Bl/6J genetic background and transgenic for the HBV genome (1.4 copy of the HBV genome with a mutation at position 1438 (T to C) which avoid the expression of the small form of the HBsAg protein and inhibit the formation of HBV infectious particles). Ten to 16 weeks-old mice (male and female) were immunized. Average weight of the mice is around 25-30 g.

(30) 1.2.2. Immunization Protocols

(31) 1.2.2.1 DNA Immunization Protocols

(32) DNA immunization protocols were run in order to evaluate the immunogenicity of the different fusion proteins encoded by the plasmids illustrated in Example 1.1. The DNA used for immunization was produced in endotoxin-free conditions. Mice were immunized twice at 15-day interval with 100 g/injection of each tested plasmid via intramuscular route in the tibialis anterior muscle. A cardiotoxin injection was done prior to the 1rst DNA injection in order to favor DNA immunogenicity. Cellular immune response was evaluated 15 days following the last DNA injection.

(33) 1.2.2.2 Adenovirus Immunization Protocols

(34) Adenovirus immunization protocols were run in order to compare the immunogenicity of the different fusion proteins encoded by the Ad vectors which were produced as described in Example 1.1. Mice were immunized once with the adenovirus encoding the different fusion proteins (10.sup.8 iu/mouse/injection) via sub-cutaneous route at the base of the tail. Cellular immune response was evaluated 15 days following the last adenovirus injection.

(35) Different doses of adenoviruses were also evaluated with the AdTG18201. Mice were immunized once with 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8 or 10.sup.9 iu of AdTG18201 by sub-cutaneous route at the base of the tail.

(36) Different schedules of immunization were also tested with the AdTG18202 and mice were injected one, two, three or 6 times at different time interval. Each injection was performed with 10.sup.8 iu/mouse via sub-cutaneous route at the base of the tail. One, 3 or 6 injections at 1 week interval were compared side by side. One injection 2 weeks or 20 weeks before the time of monitoring of induced T cell responses, 2 injections at 2 or 4 month interval and 3 injections at 2 month interval were also compared side by side.

(37) 1.2.3 Peptides

(38) Peptides used for cells stimulation in vitro are either short peptides of 9 to 10 amino acids which are described or predicted as HLA-A2 restricted epitopes or long peptides of 15 amino acids included in peptide libraries covering all the antigens of interest.

(39) Short peptides corresponding to described or predicted HLA-A2 restricted epitopes of Core protein, Pol protein or Env domains were synthesized by Eurogentec (Belgium) and were dissolved in 100% DMSO (sigma, D2650) at a concentration of 10 mM.

(40) Peptides libraries covering the whole Core, Pol and Envelope domains were synthesized by ProImmune (Oxford, United Kingdom). The Core, Pol and Env libraries were composed of 15 mer peptides overlapping by 11 amino acids. Each crude peptide was dissolved in 100% DMSO (sigma, D2650) at a concentration of 50 mg/ml. For each library, peptides were pooled to a concentration of 2 mg/ml per peptide: HBV Core protein was covered by 2 pools of 21 and 22 peptides (Pool 1 (PC1): 22 peptides covering Core residues 1 to 100; Pool 2 (PC2): 21 peptides covering Core residues 89 to 183); HBV Pol protein was covered by 8 pools of 24 peptides (Pool 1 (PP1): 24 peptides covering aa 45 to 151; Pool 2 (PP2): 24 peptides covering aa 141 to 251 (peptide from aa 205 to 219 was excluded because of insolubility in 100% DMSO or DMSO+Tris 100 mM pH9; peptide from aa 221 to 235 was dissolved in DMSO+Tris 100 mM pH9 because of insolubility in 100% DMSO); Pool 3 (PP3): 24 peptides covering aa 241 to 347; Pool 4 (PP4): 24 peptides covering aa 337 to 447 (peptide from aa 373 to 387 was excluded because of insolubility in 100% DMSO or DMSO+Tris 100 mM pH9); Pool 5 (PP5): 24 peptides covering aa 437 to 543; Pool 6 (PP6): 24 peptides covering aa 533 to 639; Pool 7 (PP7): 24 peptides covering aa 629 to 735; Pool 8 (PP8): 24 peptides covering aa 725 to 835); Env domains were covered by 2 pools of 9 and 10 peptides (Pool 1 (PE1): 10 peptides covering HBs residues 9 to 59; Pool 2 (PE2): 9 peptides covering HBs residues 157 to 194).

(41) For experiments performed in HBV transgenic mice, with a C57BL/6J genetic background, HBV peptides described in the literature or identified in previous experiments as being reactive in mice with a C57Bl/6J genetic background were used for cell stimulation in vitro. They are either short peptide (VSAAFYHLPL for polymerase; SEQ ID NO: 24) or long peptides (NLNVSIPWTHKVGNF called N13F for polymerase (SEQ ID NO: 25) and FLWEWASARFSWLSL called F13L for envelope protein (SEQ ID NO: 26)). They were synthesized by Eurogentec (Belgium) or by ProImmune (Oxford, United Kingdom). Each peptide was dissolved in 100% DMSO (sigma D2650) at a concentration of 10 mM. They were used at a concentration of 10 M during the ICS assays (even when tested as a mix of 2 peptides).

(42) 1.2.4. IFNg Elispot Assays

(43) Splenocytes from immunized mice were collected and red blood cells were lysed (Sigma, R7757). 2.10.sup.5 cells per well were cultured in triplicate for 40 h in Multiscreen plates (Millipore, MSHA S4510) coated with an anti-mouse IFN monoclonal antibody (BD Biosciences; 10 g/ml, 551216) in MEM culture medium (Gibco, 22571) supplemented with 10% FCS (JRH, 12003-100M), 80 U/mL penicillin/80 g/mL streptomycin (PAN, P06-07-100), 2 mM L-glutamine (Gibco, 25030), lx non-essential amino acids (Gibco, 11140), 10 mM Hepes (Gibco, 15630), 1 mM sodium pyruvate (Gibco, 31350) and 50 M -mercaptoethanol (Gibco, 31350) and in presence of 10 units/ml of recombinant murine IL2 (Peprotech, 212-12), alone as negative control, or with: 10 M of a selected HLA-A2 restricted peptide present in HBV antigens encoded by plasmids (FLP, ILC for Core, VLQ, FLG and GLS for Env and SLY for Pol) described in SEQ ID NO: 18-23) or an irrelevant one; a pool of peptides at a final concentration of 5 g/ml per peptide 5 g/ml of Concanavalin A (Sigma, C5275) for positive control.

(44) IFNg-producing T cells were quantified by Elispot (cytokine-specific enzyme linked immunospot) assay as previously described (Himoudi et al., 2002, J. Virol. 76:12735). The number of spots (corresponding to the IFNg-producing T cells) in negative control wells were subtracted from the number of spots detected in experimental wells containing HBV peptides. Results are shown as the mean value obtained for triplicate wells. An experimental threshold of positivity for observed responses (or cut-off) was determined by calculating a threshold value which corresponds to the mean value of spots observed with medium alone+2 standard deviations, reported to 10.sup.6 cells. A technical cut-off linked to the CTL Elispot reader was also defined as being 50 spots/10.sup.6 cells (which is the value above which the CV (coefficient of variation) of the reader was systematically less than 20%). The highest value between the technical cut-off and the experimental threshold calculated for each experiment was taken into account to define the cut-off value of each experiment. Statistical analyses of Elispot responses were conducted by using a Mann-Whitney test. P value equal or inferior to 0.05 will be considered as significant.

(45) 1.2.5. Intracellular Cytokine Staining (ICS) Assays

(46) ICS were performed on splenocytes from each animal of each group. Following red blood cells lysis with lysis buffer (Sigma, R7757), 210.sup.6 cells per well in flat-bottom 96-well plate were incubated in complete alpha MEM culture medium (Gibco BRL, 22571) in the presence of 10 units/ml of murine recombinant IL-2 (Peprotech, 212-12) alone as negative control or with 1 M of specific HBV peptide or with a pool of peptides at a final concentration of 5 g/ml per peptide or with 1 M of an irrelevant peptide. The GolgiPlug (BD Biosciences, 555029) was immediately added at a 1 l/ml final concentration for 5 h. Then, cells were harvested in V-bottom 96-well plates and washed with 1% FCS-PBS. Staining was performed using monoclonal antibodies against CD3 (hamster MAb anti-CD3e-PE, dilution 1/200), CD8 (rat MAb anti CD8a-APC, dilution 1/600) and CD4 (rat MAb anti-CD4-PerCP, dilution 1/600) (all from BD Biosciences, 553063, 553035 and 553052 respectively) in 50 l of 1% FCS-PBS for 15 min at room temperature. After washing, cells were fixed and permeabilized with Cytofix/Cytoperm and washed with Perm/Wash solution (BD Biosciences, 554714). Then, the anti-mouse IFNg-PE antibodies (BD Biosciences, 554412557724) and anti-mouse TNFa-Alexa488 antibodies (BD Biosciences, 557719) or the anti-mouse IFNg-PE antibodies (BD Biosciences, 554412557724) were added for 15 min at room temperature and after washing with Perm/Wash, cells were resuspended in 1% FCS-PBS and analysed by flow cytometry using a FacsCalibur (Becton Dickinson). CD3e+, CD8a+ cells or CD3e+, CD4+ cells were gated to determine percentages of IFNg+ CD8+ or IFNg+ CD4+ T or TNFa+ CD8+ or TNFa+ CD4+ T or IFNg+ TNFa+ CD8+ or IFNg+ TNFa+ CD4+ T cell population. The percentage obtained in medium only was considered as background.

(47) For experiments performed in HBV transgenic mice, ICS were also performed on liver cells of each animal of each group. After euthanasia of the mouse, the liver was perfused in situ by the hepatic portal vein with cold PBS until the organ becomes pale. The liver was harvested, placed in PBS+FCS 2% solution, cut into small pieces, pressed gently through a 70 m cell-strainer and then suspended in cold PBS+2% FCS solution. After centrifugation, cells were washed again with cold PBS+2% FCS solution. After a new centrifugation, the pellet containing cells was resuspended in 10 mL of Percoll solution, centrifuged for 12 minutes at 700 g at room temperature and washed again with PBS+2% FCS solution. Then red blood cells were lysed as described before for splenocytes and all subsequent steps were performed as described in the previous paragraph for splenocytes. Of note, for the liver, as the quantity of T lymphocytes collected is limited, number of cells per well is variable: all obtained cells were cultured in a way that an equivalent quantity of cells was dispatched in all wells.

(48) 1.2.6 In Vivo CTL Assays

(49) In vivo CTL assay was performed as described by Fournillier et al. (2007, Vaccine, 25: 7339-53) in HLA-A2 transgenic mice. Splenocytes suspensions were obtained from syngenic mice spleens and adjusted to 2010.sup.6 cells/mL after lysis of red blood cells. Half of the cells were incubated with the HBV peptide of interest (SLY, FLP or ILC) at 10 M final concentration for 1 h at 37 C. and half of the cells was left unpulsed. 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular probes, C1157) was then added at 10 M (CFSE-high) to unpulsed cells and 1 M (CFSE-low) to HBV-peptide pulsed cells for 10 min. After washing with PBS, all populations were mixed and 2010.sup.6 total cells were injected to anaesthetized mice via the retro-orbital sinus, mice being previously immunized (2 weeks earlier) by AdTG18201 or AdTG15149. Thus, CFSE-low population represented specific targets supposed to be lysed by cytotoxic T cells induced by the vaccination and CFSE-high population was an internal reference allowing assay normalization. Splenocytes from recipient mice were analyzed 24 h later by flow cytometer to detect the CFSE-labeled cells. For each animal, ratio between peptide-pulsed targets and unpulsed targets was calculated (R=Number CFSE-low cells/Number CFSE-high cells). The percentage of specific lysis for each animal was determined by the following formula: % lysis=(1R.sub.mouse/R.sub.reference)100 where R.sub.reference is the mean R obtained for 2 nave HLA-A2 mice which were injected with the same suspension of CFSE-labeled targets. A response was considered positive if the percentage of specific lysis was higher than 10%.

(50) 1.3 In Vitro Analysis of AdTG18201 by Electron Microscopy

(51) A549 cells (Human lung adenocarcinoma epithelial cell line) were infected in suspension and under reduced medium volume conditions with AdTG18201 at different MOI (25 to 100) and then cultured for 16H, 24H or 48H before being collected for analysis. Cells were collected at these different timepoints and then fixed using glutaraldehyde 2% diluted in sodium cocadylate buffer 0.2M. Cells were then dried, included in blocks of resin and then cut in ultra-thin sections. Obtained grids were then stained using uranyl acetate and lead citrate and observed by electron microscopy.

(52) 2. Results

(53) 2.1. Immunogenicity of HBV Fusion Proteins Expressed by DNA Plasmids pTG18188 and pTG18194

(54) The immunogenicity of the HBV fusion proteins expressed by DNA plasmids was assessed in HLA-A2 transgenic mice. Following two intramuscular injections of either pTG18188 (tCore-Pol*-Env1-Env2) or pTG18194 (tCore-Pol*) or pTG13135 as negative control (empty plasmid), specific T cell responses were evaluated by Elispot IFNg and ICS (IFNg/TNFa) using known HLA-A2 epitopes present in Polymerase, Core or the envelope domains and/or pools of overlapping peptides covering the HBV antigens of interest.

(55) 2.1.1. HBV Specific IFN Producing Cell Evaluation by Elispot Assays

(56) As illustrated in FIG. 2, immunization with the plasmid pTG18194 encoding the HBV fusion protein tCore-Pol* induced IFNg producing cells specific of the HLA-A2 restricted SLY epitope (SEQ ID NO: 23) located within the HBV polymerase (positions 816-824). Immunization with the plasmid pTG18194 also resulted in the induction of high frequency of IFNg producing cells specific for 2 Core HLA-A2 restricted epitopes FLP (SEQ ID NO: 18, located within the HBV Core protein at position 18-27) and ILC (SEQ ID NO: 19 located within the HBV Core protein at position 99-108). Positive responses were observed in 4 out of the 8 vaccinated mice.

(57) As illustrated in FIG. 2, the plasmid pTG18188 encoding the HBV fusion protein Core-Pol*-Env1-Env2 also induced IFNg producing cells specific of the pol HLA-A2 epitope SLY and of the 2 Core HLA-A2 restricted epitopes FLP and ILC. Positive responses were observed in 4 out of the 8 vaccinated mice. In addition, IFNg-producing cells specific of HLA-A2 GLS epitope (SEQ ID NO: 22 located within Env2 at positions 185-194 of HBsAg) were also detected although at a weak frequency and in 1 vaccinated mouse.

(58) 2.1.2. Evaluation of Induced HBV Specific IFNg Producing T CD8+ and CD4+ Cells by Intracellular Staining Assays

(59) 2.1.2.1. CD8 T Cell Response Specific of HLA-A2 Restricted Epitopes

(60) The percentage of CD8 T cells producing either IFNg alone or combined with TNFa targeting HLA-A2 restricted epitopes included into polymerase (SLY), Core (FLP and ILC) and envelope domains (VLQ, FLG and GLS) was evaluated by ICS assay. The results are shown in FIG. 3A as percentages of CD8+ T cell specific of these epitopes and producing IFNg (sum of single IFNg producing cells or double IFNg and TNFa producing cells). Four out of 8 animals immunized with pTG18194 (expressing tCore-Pol*) mounted IFNg producing CD8+ T cells specific of FLP, ILC and SLY HLA-A2 restricted epitopes located respectively in Core and Pol antigens. Similarly, 4 out of 8 animals immunized with pTG18188 expressing tCore-Pol*-Env1-Env2) also mounted IFNg producing T CD8+ T cells specific of FLP, ILC and SLY epitopes. In addition as already observed in ELISPOT assay, 1 out of 8 mice immunized with the plasmid pTG18188 displayed a response specific of the GLS HLA-A2 restricted epitope located within the Env2 domain mediated by IFNg producing CD8+ T cells. Immunization with pTG13135 did not induce any specific response as expected.

(61) 2.1.2.2 CD8 and CD4 T Cell Response Specific of Pools of Peptides Covering the Core Protein, Polymerase Protein and Env Domains.

(62) Responses Specific of Pools of Peptides Covering the Core Protein

(63) The percentage of CD8 and CD4 T cells able to produce either IFNg alone or combined with TNFa in response to pools of peptides covering the Core protein (PC) was evaluated by ICS assay. The results are expressed as percentages of CD8+ or CD4+ T cell specific of these pools of peptides and producing IFNg (sum of single IFNg producing cells or double IFNg and TNFa producing cells).

(64) As shown in FIG. 3B, a positive percentage of CD8+ T cells producing IFNg was detected against the 2 pools of peptides covering the Core protein (PC1 and PC2), with a CD8+ T cell response mainly focused on peptides of Pool Core 1. The percentage of reactive CD8+ T cells observed in mice vaccinated by either the pTG18194 or the pTG18188 was significantly different from the percentage that was observed for mice vaccinated with the negative control (pTG13135) (p<0.05, Mann Withney test) for both peptide pools (1 and 2).

(65) As shown in FIG. 3C, positive percentage of CD4+ T cells producing IFNg was also detected against one pool of peptides covering the Core protein, the pool Core 2 in the two groups of mice vaccinated with pTG18194 or pTG18188. The percentage of reactive CD4+ T cells observed in mice vaccinated by the pTG18188 was significantly different from the percentage that was observed for mice vaccinated with the negative control (pTG13135) (p<0.05, Mann Withney test) for pool Core 2.

(66) Responses Specific of Pools of Peptides Covering the Polymerase Protein

(67) The percentage of CD8 and CD4 T cells able to produce either IFNg alone or combined with TNFa in response to pools of peptides covering the polymerase protein was evaluated by ICS assay. The results are expressed as percentages of CD8+ or CD4+ T cell specific of these pools of peptides and producing IFNg (sum of single IFNg producing cells or double IFNg and TNFa producing cells).

(68) As shown in FIG. 3D, a positive percentage of CD8+ T cells producing IFNg was mainly detected against one pool of peptides, PP8. Specifically for PP8, the percentage of reactive CD8+ T cells observed in mice vaccinated by either the pTG18194 or the pTG18188 was significantly different from the percentage that was observed for mice vaccinated with the negative control (pTG13135) (p<0.05, Mann Withney test). Of note, one mouse in the group of mice vaccinated with pTG18188 also displayed positive percentage of IFNg producing CD8+ T cells against pool 4, pool 5 and pool 6.

(69) As shown in FIG. 3E, a weak but positive percentage of CD4+ T cells producing IFNg was detected against 4 pools of peptides covering the Pol protein, the pool Pol 1, pool Pol 4, pool Pol 5 and pool Pol 6 in the two groups of mice vaccinated with pTG18194 or pTG18188, with at least 3 out of the 8 tested mice in each group displaying responses.

(70) Responses Specific of Pools of Peptides Covering the Envelope Domains

(71) The percentage of CD8 and CD4 T cells able to produce either IFNg alone or combined with TNFa in response to pools of peptides covering the Envelope domains, Env1 and Env2, was evaluated by ICS assay. No specific CD4+ T cell response was detected during this experiment. The results for CD8+ T cell response are shown in FIG. 3F as percentages of CD8+ T cells specific of these pools of peptides and producing IFNg (sum of single IFNg producing cells or double IFNg and TNFa producing cells). Specifically, a weak but positive percentage of CD8+ T cells producing IFNg was detected for 1 mouse vaccinated with the pTG18188 against one pool of peptides, pool Env2.

(72) 2.2. Immunogenicity of HBV Fusion Proteins Expressed by Adenovirus AdTG18201, AdTG18202 and AdTG18203

(73) 2.2.1. Evaluation of HBV-Specific IFNg Producing T Cells by Elispots IFNg Using Pools of Overlapping Peptides

(74) The immunogenicity of the HBV Pol mutant and fusion proteins expressed by human adenovirus 5 was assessed in HLA-A2 transgenic mice immunized with either AdTG18201 or AdTG18202 or AdTG18203 or AdTG15149 (empty adenovirus used as negative control). Specific T cell responses induced following one subcutaneous injection of adenovirus were evaluated by Elispot IFNg using pools of overlapping peptides covering the HBV antigens of interest, Core (PC1-2), Polymerase (PP1-8) and Env (PE1-2) domains.

(75) As illustrated in FIG. 4, AdTG18203 encoding the HBV mutant polymerase polypeptide alone is able to induce IFNg producing cells specific of polymerase peptide pools 4, 5, 6 and 8. All immunized mice displayed specific T cell responses with a high frequency of IFNg producing cells mainly against the polymerase peptide pools 4 and 8.

(76) As illustrated in FIG. 4, AdTG18202 encoding the HBV fusion protein tCore-Pol* induced IFNg producing cells specific of peptide pools PP2, PP3, PP4, PP5 and PP8, the polymerase-specific response being mainly focused on against PP2, PP3 et PP8. Immunization with AdTG18202 also resulted in the induction of high frequency of IFNg producing cells specific for the 2 Core peptide pools PC1 and PC2 with a higher frequency of T cells targeting PC1. Positive responses targeting both the polymerase and core antigens were observed in 5 out of the 5 vaccinated mice.

(77) AdTG18201 encoding the HBV fusion protein Core-Pol*-Env1-Env2 was also found immunogenic as illustrated in FIG. 4. More specifically, IFNg producing cells specific of polymerase peptide pools PP2, PP3, and PP8 were induced in all vaccinated mice, as well as against PP4 and PP5 although with weaker spots and lower responding mice frequencies. Immunization with AdTG18201 also resulted in the induction of high frequency of IFNg producing cells specific for the 2 Core peptide pools PC1 and PC2, in all vaccinated mice (5/5). Immunisation with AdTG18201 also induced specific T cell responses against the Env domains, even if those responses are weak and sporadic with 1 out of 5 mice displaying responses targeting PE1 and 2 out of 5 mice displaying responses targeting PE2.

(78) 2.2.2. Evaluation of HBV Specific IFNg Producing T Cells by Elispots IFNg Using HLA-A2 Peptides Following Immunization with AdTG18201.

(79) The immunogenicity of one of the HBV fusion protein expressed by AdTG18201 was assessed in HLA-A2 transgenic mice. The animals were immunized by one subcutaneous injection of either AdTG18201 or AdTG15149 (empty adenovirus used as negative control). Specific T cell responses were evaluated by Elispot IFNg using HLA-A2 restricted epitopes contained in Polymerase (SLY), Core (FLP and ILC) and Envelope (VLQ and GLS).

(80) As illustrated in FIG. 5, AdTG18201 encoding the HBV fusion protein Core-Pol*-Env1-Env2 was found immunogenic. More specifically, IFNg producing cells specific of the polymerase HLA-A2 epitope, SLY, were induced in all AdTG18201-vaccinated mice. At the same time, AdTG18201 also induced IFNg-producing cells specific of the 2 HLA-A2 epitopes of the Core protein, FLP and ILC, with high frequencies. Immunization with AdTG18201 also induced specific T cell responses against the Env domains, although the frequencies and number of responding mice are lower, with 2 out of 8 tested mice displaying positive T cell response against the VLQ peptide and 5 out of 8 tested mice displaying positive T cell response against the GLSP peptide.

(81) 2.2.3. Evaluation of HBV Specific IFNg and/or TNFa Producing CD8+ T Cells by Intracellular Staining Assays Following Immunization of HLA-A2 Mice with the AdTG18201 and Using Selected Pools of Peptides.

(82) The percentage of CD8+ T cells able to produce either IFNg alone or combined with TNFa in response to selected pools of peptides, covering a part of the polymerase protein (PP8, amino acids 725 to 835) and a part of the HBV Core protein (PC1, amino acids 1 to 100), was evaluated by ICS assay. The result is expressed as percentage of CD8+ T cells specific of these pools of peptides and producing IFNg alone and IFNg combined with TNFa.

(83) As shown in FIG. 6, AdTG18201 is specifically capable of inducing high percentages of CD8+ T cells producing both IFNg alone as well as IFNg combined with TNFa recognizing peptides of PP8 and PC1 pools. All vaccinated mice displayed a high percentage of both single producing (IFNg alone) and double producing (IFNg and TNFa) specific CD8+ T cells.

(84) Of note, similar experiments performed in another mouse model, C57B16 mice, displayed similar results of immunogenicity of the AdTG18201 (not shown)

(85) 2.2.4. Evaluation of the Induction of In Vivo Functional CD8+ T Cells Using an In Vivo CTL Assays Following Immunization of HLA-A2 Mice with the AdTG18201.

(86) The capacity of AdTG18201 to induce in vivo functional CD8 T cells displaying cytolytic activity was evaluated by in vivo cytolytic (or CTL) assay in HLA-A2 mice following immunization with AdTG18201 or AdTG15149 (as negative control) and using 3 of the HLA-A2 epitopes already shown as being targeted by induced CD8+ T cells producing IFNg (SLY, FLP and ILC).

(87) As illustrated by FIG. 7, the AdTG18201 is able to induce high percentage of in vivo specific lysis against the polymerase epitope, SLY, with a specific lysis detected for all immunized mice and a percentage ranging from 42% to 75% (FIG. 7a). It was also shown that the AdTG18201 is able to induce high percentage of in vivo specific lysis against the 2 tested HLA-A2 epitopes of the Core protein, FLP (FIG. 7a) and ILC (FIG. 7b), with percentages ranging from 32% to 69% and 3% to 64% respectively. Response against env epitope was detectable but at low levels.

(88) These data clearly demonstrate the ability of the AdTG18201 to induce in vivo functional CD8+ T cells displaying cytolytic activity and targeting both the HBV polymerase and the HBV core proteins.

(89) 2.2.5. Evaluation of the Induction of Functional CD8+ T Cells in HBV Transgenic Mice Following Immunization with AdTG18201 and Using an ICS Assay.

(90) The capacity of AdTG18201 to induce functional T cells in a tolerant mouse model was evaluated in HBV transgenic mice. In fact, these mice are transgenic for the HBV genome and, thus, tolerant to HBV antigens mimicking, to some extent, the tolerance encountered in HBV chronic patients. The HBV transgenic mice were immunized by one subcutaneous injection of AdTG18201 (10.sup.8 iu) or AdTG15149 as negative control. Induced T cells were monitored both in spleens and livers of vaccinated mice by ICS (detection of CD8+ T cells producing both IFNg and TNFa). In this specific model, peptides identified to be reactive in C57Bl/6J mice were used to screen the induced T cell response: a pool of the VSA and the N13F peptides for the polymerase and the F13L peptide for the envelope.

(91) As illustrated in FIG. 8, functional CD8+ T cells producing both IFNg and TNFa were detected in spleens and livers of AdTG18201-vaccinated mice, with 4 out of 5 tested mice displaying functional IFNg/TNFa producing CD8+ T cells specific of polymerase and with 2 out of 5 tested mice displaying functional IFNg/TNFa producing CD8+ T cells specific of Envelope in both organs. As expected, no responses were detected in mice immunized with the empty AdTG15149 or when stimulation is using an irrelevant peptide.

(92) All together, these data demonstrate the ability of the viral vector AdTG18201 expressing a fusion protein containing a RNaseH-defective and YMDD-deleted pol mutant, env domains and core to induce functional CD8+ T cells, producing both IFNg and TNFa, in a HBV tolerant model.

(93) 2.3 Evaluation of Different Doses and Schedules of Immunization with the AdTG18201 or the AdTG18202.

(94) 2.3.1. Adenovirus Dose Evaluation.

(95) The immunogenicity of the HBV fusion protein expressed by AdTG18201 was assessed in HLA-A2 transgenic mice at different doses. The animals were immunized by one subcutaneous injection of either AdTG18201 at a dose of 10.sup.5 iu or 10.sup.6 iu or 10.sup.7 iu or 10.sup.8 iu or 10.sup.9 iu or AdTG15149 at 10.sup.9 iu (empty adenovirus used as negative control). Specific T cell responses were evaluated by Elispot IFNg using HLA-A2 restricted epitopes contained in Polymerase (SLY), Core (FLP and ILC) and Envelope (VLQ and GLS).

(96) As illustrated in FIG. 9, AdTG18201 encoding the HBV fusion protein Core-Pol*-Env1-Env2 was found immunogenic when injected at doses of 10.sup.7 iu, 10.sup.8 iu and 10.sup.9 iu. More specifically, no IFNg producing cells specific of the tested HLA-A2 epitopes of Core, Polymerase or Env domains were detected with the doses of 10.sup.5 and 10.sup.6 iu. Specific IFNg producing cells targeting the 2 tested core epitopes and the tested epitope of Pol were detected for doses of 10.sup.7, 10.sup.8 and 10.sup.9 iu. A dose effect is observed for the 3 epitopes (SLY, FLP and ILC). For the 2 epitopes of the Env domains (VLQ and GLS), frequencies of IFNg producing cells are low for doses of 10.sup.7 and 10.sup.8 whereas frequencies are clearly increased with a dose of 10.sup.9 iu.

(97) 2.3.2. Evaluation of Multiple Immunization Schedule at Short Term Interval.

(98) The immunogenicity of one of the HBV fusion protein expressed by AdTG18202 was assessed in HLA-A2 transgenic mice according to different schedules of immunization. AdTG18202 was either administered once or 3 times (1 injection/week during 3 weeks) or 6 times (1 injection/week during 6 weeks) and the induced immune T cell responses was assessed 2 weeks after the last injection by an Elispots IFNg assay and using HLA-A2 restricted epitopes, SLY (Pol) and FLP and ILC (Core). Some mice were immunized 6 times with an empty adenovirus as a negative control (not shown)

(99) As illustrated in FIG. 10, AdTG18202 encoding for the fusion protein Core-Pol* was found immunogenic whatever the tested schedules. More particularly, no specific T cell response was detected with medium alone and irrelevant peptide whereas high and similar frequencies of IFNg producing cells were detected in presence of the 3 tested HBV epitopes, SLY, FLP and ILC. Frequencies of detected IFNg producing T cells appeared comparable between groups of mice injected once, 3 times or 6 times at 1 week interval, without the appearance, at the IFNg production level, of a T cell exhaustion due to a too high number of immunizations in a short time-interval. The adenovirus specific T cell responses appeared higher when mice were injected 6 times than when they were injected once or 3 times. As expected, no HBV-specific T cell responses were observed following immunization with an empty adenovirus.

(100) 2.3.3. Evaluation of Multiple Immunization Schedule at Long Term Interval.

(101) The immunogenicity of one of the HBV fusion protein expressed by AdTG18202 was assessed in HLA-A2 transgenic mice according to different schedules of immunization. AdTG18202 was either administered once (2 (group 1) or 20 (group 2) weeks before the monitoring of T cell responses) or twice (2 injections at 2 (group 3) or 4 (group 4) month interval, monitoring of T cell responses 2 weeks after the last immunization) or three times (at 2 month interval (group 5), monitoring of T cell responses 2 weeks after the last injection). Induced T cells were monitored by an Elispots IFNg assay and using HLA-A2 restricted epitopes, SLY (Pol) and FLP and ILC (Core). Some mice were immunized either once or three times at 2 month interval with an empty adenovirus as a negative control (not shown).

(102) As illustrated by FIGS. 11A-B, AdTG18202 encoding for the fusion protein Core-Pol* was found immunogenic whatever the tested schedule whereas no specific T cell response was detected following immunizations with AdTG18202 in presence of medium alone or of an irrelevant peptide. More particularly, observed specific T cell responses in group 2 showed that even if lower than those observed in group 1, 2 weeks after 1 immunization, induced T cell responses after one injection of AdTG18202 still exist 20 weeks after the injection. Observed T cell responses in group 3 and 4 showed that a 2.sup.nd immunization 2 or 4 months after the first one was able to recall T cell responses specific of HBV epitopes at least at the level of the primary immune response observed in group 1, even slightly higher for the SLY epitope. A similar observation was done with mice immunized three times at 2 month interval (group 5) with a recall of induced T cell responses through the 2.sup.nd and 3.sup.rd injections to a level similar to the one observed in group 1. As expected, no HBV-specific T cell responses were observed following immunization with an empty adenovirus

(103) 2.4 Electron Microscopy Observation

(104) A549 cells were infected in vitro by AdTG18201 at MOI 25, 50 or 100 and cells were collected at either 16 h, 24 h or 48 post-infection. Collected cells were then treated to be observed by electron microscopy.

(105) Some virus-like particles (VLP) were observed in the nucleus and cytoplasm of AdTG18201 infected cells whereas none of these structures were observed in cells infected by an empty adenovirus. These VLP were mainly located within the nucleus. In some cells both protein aggregates and VLP were observed.