Fusion proteins and use thereof for preparing vaccines

Abstract

An immunogenic fusion protein includes at least the following two peptides a) on the C-terminal side, a first peptide constituted of: the amino acid sequence of the protein S or the protein M of a human hepatitis B virus (HBV) isolate, which protein S or protein M is optionally deleted at the N-terminal end thereof, and b) on the N-terminal side, a second peptide constituted of: the sequence of amino acids of at least one transmembrane domain and the ectodomain of at least one protein of a Zika virus isolate selected from the envelope protein E or a fusion peptide including the envelope protein E and the protein prM.

Claims

1. An immunogenic fusion protein comprising at least two peptides: a) on the C-terminal side, a first peptide which consists of: an amino acid sequence of the protein S or the protein M of a human hepatitis B virus (HBV) isolate, wherein the sequence of the protein S is chosen from the group consisting of SEQ ID NO: 1 and 2, and the sequence of the protein M is chosen from the group consisting of SEQ ID NO: 3 and 4, or an amino acid sequence with a percent identity of at least 95% with said amino acid sequence of the protein S or the protein M, provided that said amino acid sequence maintains the ability to form subviral, non-infectious particles and/or immunogenic properties against the HBV virus, or, and b) on the N-terminal side, a second peptide which consists of: a sequence of amino acids comprising at least one transmembrane domain and the ectodomain of at least one protein of a Zika virus isolate, or an amino acid sequence with a percent identity of at least 95% with said amino acid sequence of at least one transmembrane domain and the ectodomain of at least one protein of a Zika virus isolate, provided that said amino acid sequence maintains the ability to form subviral, non-infectious particles and/or immunogenic properties against the Zika virus, said protein of a Zika virus isolate being chosen from among the envelope protein E represented by SEQ ID NO. 15 or a fusion peptide comprising the envelope protein E and the protein prM represented by SEQ ID NO. 50.

2. An immunogenic fusion protein according to claim 1, wherein the first peptide located on the C-terminal side thereof consists of: the amino acid sequence of the protein S of a human HBV isolate, wherein the sequence of the protein S is represented by of SEQ ID NO: 1, or an amino acid sequence presenting a percent identity of at least 95% with said amino acid sequence of the protein S represented by of SEQ ID NO: 1, provided that said amino acid sequence maintains the ability to form subviral, non-infectious particles and/or the immunogenic properties against the human HBV.

3. An immunogenic fusion protein according to claim 1, wherein the first peptide located on the C-terminal side thereof consists of: the amino acid sequence of the protein S of a human HBV isolate, wherein the sequence of the protein S is represented by of SEQ ID NO: 2, or an amino acid sequence presenting a percent identity of at least 95% with said amino acid sequence of the protein S represented by of SEQ ID NO: 2, provided that said amino acid sequence maintains the ability to form subviral, non-infectious particles and/or the immunogenic properties against the human HBV.

4. An immunogenic fusion protein according to claim 1, wherein the first peptide located on the C-terminal side thereof consists of: the amino acid sequence of the protein M of a human HBV isolate, wherein the sequence of the protein M is represented by of SEQ ID NO: 3, or an amino acid sequence presenting a percent identity of at least 95% with said amino acid sequence of the protein M deleted from a sequence of 1 to 54 amino acids located at the N-terminal end thereof, provided that said amino acid sequence maintains the ability to form subviral, non-infectious particles and/or the immunogenic properties against the human HBV.

5. An immunogenic fusion protein according to claim 1, wherein the first peptide located on the C-terminal side thereof consists of: the amino acid sequence of the protein M of a human HBV isolate, wherein the sequence of the protein M is represented by of SEQ ID NO: 4, or an amino acid sequence presenting a percent identity of at least 95% with said amino acid sequence of the protein M not deleted from a sequence of 1 to 54 amino acids located at the N-terminal end thereof, provided that said amino acid sequence maintains the ability to form subviral, non-infectious particles and/or the immunogenic properties against the human HBV.

6. An immunogenic fusion protein according to claim 1, wherein the second peptide located on the N-terminal side thereof consists of: the amino acid sequence of at least one transmembrane domain and the ectodomain of the envelope protein E of a Zika virus isolate, or an amino acid sequence presenting an identity percent of at least 95%, with said amino acid sequence of at least one transmembrane domain and the ectodomain of the envelope protein E of a Zika virus isolate, provided that said amino acid sequence maintains the ability to form subviral, non-infectious particles and/or the immunogenic properties against the Zika virus.

7. An immunogenic fusion protein according to claim 1, wherein the second peptide located on the N-terminal side thereof consists of: the amino acid sequence of a fusion peptide comprising at least one transmembrane domain and the ectodomain of the envelope protein E of a Zika virus isolate and the protein prM of a Zika virus isolate, wherein said amino acid sequence is chosen from among the envelope protein E represented by SEQ ID NO. 15 or a fusion peptide comprising the envelope protein E and the protein prM represented by SEQ ID NO. 50, or an amino acid sequence presenting an identity percent of at 95%, with said amino acid sequence of said fusion peptide, provided that said amino acid sequence maintains the ability to form subviral, non-infectious particles and/or the immunogenic properties against the Zika virus.

8. An immunogenic fusion protein according to claim 1, wherein said fusion protein comprises: the amino acid sequence represented by: SEQ ID NO. 7 or SEQ ID NO. 17, or SEQ ID NO. 8 or SEQ ID NO. 18, or SEQ ID NO. 9 or SEQ ID NO. 19, or SEQ ID NO. 10 or SEQ ID NO. 20, or SEQ ID NO. 11 or SEQ ID NO. 21, or SEQ ID NO. 12 or SEQ ID NO. 22, or SEQ ID NO. 13 or SEQ ID NO. 23, or SEQ ID NO. 14 or SEQ ID NO. 24, or an amino acid sequence presenting an identity percent of at least 95% with said SEQ ID NO. 7, SEQ ID NO. 17, SEQ ID NO. 8, SEQ ID NO. 18, SEQ ID NO. 9, SEQ ID NO. 19, SEQ ID NO. 10, SEQ ID NO. 20, SEQ ID NO. 11, SEQ ID NO. 21, SEQ ID NO. 12, SEQ ID NO. 22, SEQ ID NO. 13, SEQ ID NO. 23, SEQ ID NO. 14, SEQ ID NO. 24, provided that said amino acid sequence maintains the ability to form subviral, non-infectious particles and/or the immunogenic properties against the human HBV and/or the Zika virus, or the amino acid sequence of a synthetic variant derived from said SEQ ID NO. 7, SEQ ID NO. 17, SEQ ID NO. 8, SEQ ID NO. 18, SEQ ID NO. 9, SEQ ID NO. 19, SEQ ID NO. 10, SEQ ID NO. 20, SEQ ID NO. 11, SEQ ID NO. 21, SEQ ID NO. 12, SEQ ID NO. 22, SEQ ID NO. 13, SEQ ID NO. 23, SEQ ID NO. 14, SEQ ID NO. 24, provided that said amino acid sequence maintains the ability to form subviral, non-infectious particles and/or the immunogenic properties against the human HBV and/or the Zika virus.

9. Nucleic acid molecules coding a fusion protein according to claim 1.

10. A subviral, non-infectious and immunogenic particle comprising the following proteins: a protein comprising the wild-type domain S of the surface antigen of a hepatitis B virus isolate, and the fusion protein according to claim 1.

11. An immunogenic fusion protein according to claim 1, for its use as a medication, notably as a vaccine.

12. An immunogenic fusion protein according to claim 1, for its use in preventing and/or treating hepatitis B and/or Zika virus infections.

13. An immunogenic fusion protein according to claim 1, wherein the second peptide located on the N-terminal side thereof consists of: the sequence of amino acids of at least one transmembrane domain chosen from the group consisting of the amino acid sequences between positions 456-484 of SEQ ID NO: 15, 485-504 of SEQ ID NO: 15, 124-143 of SEQ ID NO: 50, and 150-164 of SEQ ID NO: 50, and the amino acid sequence of the ectodomain chosen from the group consisting of the amino acid sequences between positions 1-455 of SEQ ID NO: 15 and 1-123 of SEQ ID NO: 50 of at least one protein of a Zika virus isolate, or an amino acid sequence with a percent identity of at least 95% with said amino acid sequence of at least one transmembrane domain and the ectodomain of at least one protein of a Zika virus isolate, provided that said amino acid sequence maintains the ability to form subviral, non-infectious particles and/or immunogenic properties against the Zika virus, said protein of a Zika virus isolate being chosen from among the envelope protein E represented by SEQ ID NO. 15 and a fusion peptide comprising the envelope protein E and the protein prM represented by SEQ ID NO. 50.

Description

FIGURE LEGEND

(1) FIG. 1 illustrates the wild-type protein S of the HBV, which notably comprises four transmembrane domains, represented by black vertical rectangles; the N and C-terminal ends thereof are oriented towards the light of the endoplasmic reticulum (ER). In the case of the HBVadw isolate, this protein of approximately 45 kD comprises 226 amino acid residues.

(2) FIG. 2A illustrates the wild-type protein M of the HBV, which notably comprises four transmembrane domains, represented by black vertical rectangles; the N and C-terminal ends thereof are oriented towards the light of the endoplasmic reticulum (ER). In the case of the HBVadw isolate, this protein of approximately 33 kD comprises 281 amino acid residues. This FIG. 2A presents a first transmembrane topology of the protein M.

(3) FIG. 2B illustrates the protein M of the HBV, which comprises three transmembrane domains, represented by black vertical triangles; the C-terminal end thereof is oriented towards the light of the endoplasmic reticulum (ER). Unlike FIG. 2A, the N-terminal end is oriented towards the cytosol, and as a result thereof, the first transmembrane domain located on the N-terminal side is represented by a black horizontal rectangle. This FIG. 2B presents a second transmembrane typology of the protein M.

(4) FIG. 3 illustrates the wild-type envelope protein E of the Zika virus, which notably comprises two transmembrane domains, represented by dark gray vertical rectangles; the N-terminal end thereof is in the light of the endoplasmic reticulum (ER), but is oriented towards the cytosol, and the C-terminal end thereof is oriented towards the light of the endoplasmic reticulum (ER). This protein of approximately 54 kD comprises 504 amino acid residues.

(5) FIG. 4 illustrates the wild-type protein prM of the Zika virus, which notably comprises two transmembrane domain, represented by gray vertical rectangles; the N-terminal end thereof is in the light of the endoplasmic reticulum (ER) but is oriented towards the cytosol, and the C-terminal end thereof is oriented towards the light of the endoplasmic reticulum (ER). This protein of approximately 18 kD comprises 164 amino acid residues. The proteolytic cleavage site, allowing the pr portion and the M portion of the protein prM to be cleaved, is also indicated.

(6) FIG. 5 illustrates the configuration of certain proteins of the Zika virus, and notably the configuration of the capsid protein (C), of the protein prM (pr and M) and of the envelope protein E (E) in relation to the membrane.

(7) The capsid protein (C) comprises, on the C-terminal side, a transmembrane domain, represented by a light gray vertical rectangle; the N-terminal end thereof is oriented towards the cytosol.

(8) The proteins E and prM are as described in the legends of FIGS. 3 and 4.

(9) FIG. 6 illustrates the cartography of the synthesis gene allowing the fusion peptides prM+E to be obtained, i.e. a fusion peptide comprising the envelope protein E and the protein prM of Zika. The position of the transmembrane domains of the protein C, of the protein prM and of the envelope protein E is represented by short dark gray arrows.

(10) FIG. 7 illustrates the cartography of the synthesis gene allowing the fusion protein prM+deleted E+deleted S to be obtained, i.e. the fusion protein comprising the protein prM, the protein E deleted from a transmembrane domain and the protein S deleted from a transmembrane domain, and notably the fusion protein represented by SEQ ID NO. 8 or SEQ ID NO. 35 or SEQ ID NO. 18 or SEQ ID NO. 43. The position of the transmembrane domains of the protein prM and of the envelope protein E is represented by short arrows (indicated by the number 3 and in dark gray). The position of the initiation sequence is represented by a dark gray rectangle indicated by the number 1 and the position of the transfer initiation peptide is represented by a white arrow indicated by the number 2.

(11) FIG. 8 illustrates the transmembrane topology of the fusion protein prM+deleted E+deleted 5, i.e. the fusion protein comprising the protein prM, the protein E deleted from a transmembrane domain and the protein S deleted from a transmembrane domain, and notably the fusion protein represented by SEQ ID NO. 8 or SEQ ID NO. 35 or SEQ ID NO. 18 or SEQ ID NO. 43.

(12) FIG. 9 illustrates the cartography of the synthesis gene allowing the fusion protein prM+E+5 to be obtained, i.e. the fusion protein comprising the protein prM, the protein E and the protein S, and notably the fusion protein represented by SEQ ID NO. 10 or SEQ ID NO. 37 or SEQ ID NO. 20 or SEQ ID NO. 45. The position of the transmembrane domains of the protein prM and the envelope protein E is represented by short arrows (indicated by the number 3 and in dark gray). The position of the initiation sequence is represented by a gray rectangle indicated by the number 1 and the position of the transfer initiation peptide is represented by a white arrow indicated by the number 2.

(13) FIG. 10 illustrates the transmembrane topology of the fusion protein prM+E+5, i.e. the fusion protein comprising the protein prM, the protein E and the protein S, and notably the fusion protein represented by SEQ ID NO. 10 or SEQ ID NO. 37 or SEQ ID NO. 20 or SEQ ID NO. 45.

(14) FIG. 11 illustrates the cartography of the synthesis gene allowing the fusion protein prM+E+M to be obtained, i.e. the fusion protein comprising the protein prM, the protein E and the protein M, and notably the fusion protein represented by SEQ ID NO. 14 or SEQ ID NO. 41 or SEQ ID NO. 24 or SEQ ID NO. 49. The position of the transmembrane domains of the protein prM and of the envelope protein E is represented by short arrows (indicated by the number 3 and in dark gray). The position of the initiation sequence is represented by a gray rectangle indicated by the number 1 and the position of the transfer initiation peptide is represented by a white arrow indicated by the number 2.

(15) FIG. 12 illustrates the transmembrane topology of the fusion protein prM+E+M, i.e. the fusion protein comprising the protein prM, the protein E and the protein M, and notably the fusion protein represented by SEQ ID NO. 14 or SEQ ID NO. 41 or SEQ ID NO. 24 or SEQ ID NO. 49.

(16) FIG. 13 illustrates the cartography of the synthesis gene allowing the fusion protein prM+deleted E+M to be obtained, i.e. the fusion protein comprising the protein prM, the protein E deleted from a transmembrane domain and the protein M, and notably the fusion protein represented by SEQ ID NO. 12 or SEQ ID NO. 39 or SEQ ID NO. 22 or SEQ ID NO. 47. The position of the transmembrane domains of the protein prM and of the envelope protein E is represented by short arrows (indicated by the number 3 and in dark gray). The position of the initiation sequence is represented by a gray rectangle indicated by the number 1 and the position of the transfer initiation peptide is represented by a white arrow indicated by the number 2.

(17) FIG. 14 illustrates the transmembrane topology of the fusion protein prM+deleted E+M, i.e. the fusion protein comprising the protein prM, the protein E deleted from a transmembrane domain and the protein M, and notably the fusion protein represented by SEQ ID NO. 12 or SEQ ID NO. 39 or SEQ ID NO. 22 or SEQ ID NO. 47.

(18) FIG. 15 illustrates the cartography of the synthesis gene allowing the fusion protein deleted E+deleted 5 to be obtained, i.e. the fusion protein comprising the protein E deleted from a transmembrane domain and the protein S deleted from a transmembrane domain, and notably the fusion protein represented by SEQ ID NO. 7 or SEQ ID NO. 37 or SEQ ID NO. 17 or SEQ ID NO. 42. The position of the transmembrane domain of the envelope protein E is represented by a short arrow (indicated by the number 3 and in dark gray). The position of the initiation sequence is represented by a gray rectangle indicated by the number 1 and the position of the transfer initiation peptide is represented by a white arrow indicated by the number 2.

(19) FIG. 16 illustrates the transmembrane topology of the fusion protein deleted E+deleted 5, i.e. the fusion protein comprising the protein E deleted from a transmembrane domain and the protein S deleted from a transmembrane domain, and notably the fusion protein represented by SEQ ID NO. 7 or SEQ ID NO. 34 or SEQ ID NO. 17 or SEQ ID NO. 42.

(20) FIG. 17 illustrates the cartography of the synthesis gene allowing the fusion protein E+5 to be obtained, i.e. the fusion protein comprising the protein E and the protein S, and notably the fusion protein represented by SEQ ID NO. 9 or SEQ ID NO. 36 or SEQ ID NO. 19 or SEQ ID NO. 44. The position of the two transmembrane domains of the envelope protein E is represented by short arrows (indicated by the number 3 and in dark gray). The position of the initiation sequence is represented by a gray rectangle indicated by the number 1 and the position of the transfer initiation peptide is represented by a white arrow indicated by the number 2.

(21) FIG. 18 illustrates the transmembrane topology of the fusion protein E+S, i.e. the fusion protein comprising the protein E and the protein S, and notably the fusion protein represented by SEQ ID NO. 9 or SEQ ID NO. 36 or SEQ ID NO. 19 or SEQ ID NO. 44.

(22) FIG. 19 illustrates the cartography of the synthesis gene allowing the fusion protein E+M to be obtained, i.e. the fusion protein comprising the protein E and the protein M, and notably the fusion protein represented by SEQ ID NO. 13 or SEQ ID NO. 40 or SEQ ID NO. 23 or SEQ ID NO. 48. The position of the two transmembrane domains of the envelope protein E is represented by short arrows (indicated by the number 3 and in dark gray). The position of the initiation sequence is represented by a gray rectangle indicated by the number 1 and the position of the transfer initiation peptide is represented by a white arrow indicated by the number 2.

(23) FIG. 20 illustrates the transmembrane topology of the fusion protein E+M, i.e. the fusion protein comprising the protein E and the protein M, and notably the fusion protein represented by SEQ ID NO. 13 or SEQ ID NO. 40 or SEQ ID NO. 23 or SEQ ID NO. 48.

(24) FIG. 21 illustrates the cartography of the synthesis gene allowing the fusion protein deleted E+M to be obtained, i.e. the fusion protein comprising the protein E deleted from a transmembrane domain and the protein M, and notably the fusion protein represented by SEQ ID NO. 11 or SEQ ID NO. 38 or SEQ ID NO. 21 or SEQ ID NO. 46. The position of the two transmembrane domains of the envelope protein E is represented by short arrows (indicated by the number 3 and in dark gray). The position of the initiation sequence is represented by a gray rectangle indicated by the number 1 and the position of the transfer initiation peptide is represented by a white arrow indicated by the number 2.

(25) FIG. 22 illustrates the transmembrane topology of the fusion protein deleted E+M, i.e. the fusion protein comprising the protein E deleted from a transmembrane domain and the protein M, and notably the fusion protein represented by SEQ ID NO. 11 or SEQ ID NO. 38 or SEQ ID NO. 21 or SEQ ID NO. 46.

(26) FIG. 23 illustrates the immunofluorescence of cells transfected by the RNA transcribed in vitro from the plasmids pSFV1-HBV-S and/or pSFV1-prM+deleted E+deleted S and revealed by an anti-HBV-S antibody (ADRI-2F3) or an anti-Zika antibody (D1-4G2-4-15). In particular, the chimeric protein HBV-Zika-deleted E+deleted S is detected with comparable efficacy either by the antibody recognizing the envelope protein of the Zika virus or by the antibody recognizing the protein HBV-S (line 2). These two fluorescence signals are perfectly co-localized, which shows that the chimeric protein HBV-Zika-deleted E+deleted S is not degraded or cleaved during its expression.

(27) FIG. 24 illustrates the immunofluorescence of cells transfected by the RNA transcribed in vitro from the plasmids pSFV1-HBV-S and/or pSFV1-prM+E+S and revealed by an anti-HBV-S antibody (ADRI-2F3) or an anti-Zika antibody (D1-4G2-4-15). In particular, the chimeric protein HBV-Zika-E+S is detected with comparable efficacy either by the antibody recognizing the envelope protein of the Zika virus or by the antibody recognizing the protein HBV-S. These two fluorescence signals are perfectly co-localized, which shows that the chimeric protein HBV-Zika-E+S is not degraded or cleaved during its expression.

(28) FIG. 25 illustrates a western blot revealed by the anti-Zika antibody D1-4G2-4-15 (A) or the anti-HBV-S antibody 70-HG15 (B), on the lysates of cells transfected by the RNA transcribed in vitro from the plasmids pSFV1-prM+E+S, pSFV1-HBV-S or pSFV3. The lysate CHO-S is used as a positive control. The two glycolized forms of the protein HBV-S (p24 and p27) are specifically detected on the immunoblot incubated with the anti-HBV-S antibody. The chimeric protein HBV-Zika-E+S is specifically detected by the two antibodies, with the expected theoretical size of 80 kDa (p80). This confirms a satisfactory production as well as the integration of the chimeric protein HBV-Zika-E+S, which does not undergo cleavage during its expression.

(29) FIG. 26 illustrates a western blot revealed by the anti-HBV-S antibody (70-HG15) on the lysates of cells transfected by the RNA transcribed from the plasmids pSFV1-prM+deleted E+deleted S and/or pSFV1-HBV-S. The specific detection of the chimeric protein HBV-Zika-deleted E+deleted S with an expected theoretical size of 75 kDa (p75) shows that it is not degraded or cleaved during its expression. The protein HBV-S is also detected in its two glycosylation forms (p24 and p27).

(30) FIG. 27 corresponds to an ELISA analysis (HBV-S detection) of the fractions collected after differential ultracentrifugation of the lysates of the cells expressing the RNA transcribed in vitro from the plasmids pSFV1-prM+E+S and pSFV1-HBV-S. The chimeric particle-enriched fractions 8 and 9 were collected, dialyzed then observed through negative staining (FIGS. 28 and 29).

(31) FIG. 28 corresponds to a transmission electron microscopy observation with negative staining performed on the chimeric particles purified from the co-expression of the RNA transcribed from the plasmids pSFV-1-prM+E+S and pSFV1-HBV-S in BHK-21 cells. These chimeric particles are present in the form of bands and beads characteristic in size and structure of the assembly of the envelope proteins of the HBV (Patient R, Hourioux C, Sizaret P Y, Trassard S, Sureau C, Roingeard P. J Virol. 2007 April; 81(8):3842-51).

(32) FIG. 29 corresponds to a transmission electron microscopy observation with negative staining performed on the chimeric particles purified from the co-expression of the RNA transcribed from the plasmids pSFV-1-prM+E+S and pSFV1-HBV-S in BHK-21 cells. Immunolabeling (immunogold) by the anti-Zika antibody D1-4G2-15 demonstrates the effective incorporation of the chimeric protein HBV-Zika-E+S in the vaccine particles (presence of gold beads at the surface of particles, arrows).

(33) FIG. 30 illustrates a western blot revealed by the anti-HBV-S antibody 70-HG15 on the lysates of CHO-S cells transduced by pHR.sup.puro-HBV-Zika-prM+E+S, pHR.sup.puro-HBV-Zika-prM+deleted E+deleted S (clones 1 and 2) and pHR.sup.puro-HBV-Zika-prM+E+M (clones 1 and 2). The clone CHO-S is used as a positive control.

(34) FIG. 31 illustrates a western blot revealed by the anti-HBV-S antibody 70-HG15 on the supernatants of CHO-S cells transduced by pHR.sup.puro-HBV-Zika-prM+E+S, pHR.sup.puro-HBV-Zika-prM+deleted E+deleted S (clones 1 and 2) and pHR.sup.puro-HBV-Zika-prM+E+M (clones 1 and 2). The clone CHO-S is used as a positive control.

EXAMPLES

Example 1: Stable Production of the Wild-Type Envelope Protein S of Wild-Type HBV-S and of the Chimeric Protein HBV-Zika in Clones of the Cellular Line CHO

(35) Ovary cells of Chinese hamsters (CHO) are stably transduced using a strategy based on the lentiviral expression vector pHR (described by Dull et al., A third-generation lentivirus vector with a conditional packaging system, 1998, Journal of Virology, vol. 72, pp 8463-8471). The strategy based on the lentiviral expression vector pHR was described in the article Patient et al., Chimeric Hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies, 2009, New Biotechnology, vol. 25, no. 4, pp 226-234.

(36) The DNA sequence of the chimeric HBV-Zika envelope proteins is transferred to the restriction site BamH1 and/or XH01 of the plasmid pHR.sup.gfp (constructed from the plasmid pHR and coding the GFP as a screening marker) to generate the plasmids pHR.sup.gfp-HBV-Zika. The lentiviruses are produced in the HEK-293T cells (human embryo kidney cells), kept in Dulbecco's Modified Eagle Medium (DMEM).

(37) Twenty-four hours before the transfection, 310.sup.6 cells are used to inoculate a culture dish measuring 75 cm.sup.2 (Falcon). The cells are transfected with an equimolar mixture (1 pmol of each) of plasmid pHR.sup.gfP-HBV-Zika, of plasmid pHCMVG (referenced in the ATCC under the reference pHCMV-G (ATCC 754971) coding the VSV-G (envelope glycoprotein of the vesicular stomatitis virus) and the packaging construction p8.74 through the calcium phosphate method.

(38) The following day, the transfection solution is eliminated and replaced with the fresh complete medium.

(39) After 24 and 48 hours of culture, the supernatant is collected, filtered through a low protein binding filter with pores measuring 0.45 m (Sartorius) and concentrated through centrifugation on a 20% saccharose cushion at 4 C. for 90 minutes at 100,000g.

(40) The residue is resuspended in 500 L of PBS and stored at 80 C. until use.

(41) The transduction unit (TU) titer is determined by quantifying the protein p24 (Innotest HIV Antigen mAb Kit, Innogenetics).

(42) The clone CHO-S (stably producing the protein HBV-S, previously described in the article Patient et al., Chimeric Hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies, 2009, New Biotechnology, vol. 25, no. 4, pp 226-234), cultivated in the DMEM-F12 (commercialized by Fisher Scientific) is transduced with the recombining lentivectors HRR.sup.gfp-HBV-Zika.

(43) One day before transduction, 10.sup.5 CHO-S cells/well are used to inoculate a six-well cellular culture plate (Falcon).

(44) The cells are incubated with the vectors HR.sup.gfp-HBV-Zika (infection multiplicity: 2.5) and 4 g/mL of polybrene (Sigma) in the fresh complete medium.

(45) Three days after transduction, the cells are used to inoculate a 96-well cellular culture plate (Falcon) with a density of 1 cell/well.

(46) The plates are incubated for 3 weeks.

(47) The positive cellular clones of the GFP, named CHO-S+Zika-S or CHO-S+Zika-M are isolated and amplified.

(48) The intracellular production of the proteins HBV-S and chimeric HBV-Zika is analyzed with a western blot of the cellular lysates, as previously described in the article Patient et al., Chimeric Hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies, 2009, New Biotechnology, vol. 25, no. 4, pp 226-234.

(49) The membranes are incubated for one night at 4 C. with a polyclonal rabbit anti-HBsAg antibody (R247) or the monoclonal antibody directed to the Zika envelope protein (4G2).

Example 2: Analysis of the Supernatant of Cells Stably Coproducing the Envelope Proteins HBV-S and Chimeric HBV-Zika

(50) The subviral envelope particles secreted are purified from the supernatant of the cells through gradient centrifugation of cesium chloride (CsCl), as previously described in the article Patient et al., Chimeric Hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies, 2009, New Biotechnology, vol. 25, no. 4, pp 226-234.

(51) To summarize, 200 mL of supernatant are clarified and the total proteins are precipitated through the addition of a 45% solution of (NH.sub.4).sub.2SO.sub.4 (pH 7.5).

(52) The precipitate is collected through centrifugation at 4 C. for 15 minutes at 10,000g and the residue is dissolved in a minimum volume of Tris-NaCl-EDTA (TNE) pad (10 mM Tris/HCl pH 7.5/100 mM NaCl/1 mM EDTA).

(53) The solution is dialyzed against the TNE pad and cesium chloride is added until a density of 1.22 g/cm.sup.3 is achieved.

(54) Two successive cycles of isopycnic centrifugation are performed at 15 C. for 24 hours at 40,000 rpm in a 45Ti rotor (Beckman).

(55) The fractions are collected from above and tested for the HBsAg antigen using an ELISA test. The peak fractions are collected and dialyzed at 4 C. against the TNE pad.

(56) The final preparations are analyzed through negative staining electron microscopy and western blot as previously described.

Example 3: Transitory Production of the Wild-Type HBV Envelope Protein S (HBV-S) and the Chimeric HBV-Zika Protein (HBV-Zika-E+S, HBV-Zika-E+M or HBV-Zika-Deleted E+Deleted S) in Clones of the BKH-21 Cellular Line

(57) I.1) Construction of the Plasmids pSFV1-prM+E+S and pSFV1-prM+Deleted E+Deleted S

(58) The vector pSFV1 (Invitrogen) having a bicistronic structure of 11033 pb is used for the following constructions. This vector has a promoter sequence of SP6-ARN polymerase, inserted in 5 of the first cistron, to initiate the synthesis of a complete RNA with a positive polarity (RNA named 42S(+)) through in-vitro transcription. After transfection into mammal cells, these recombining RNA capped in vitro self-replicate in the presence of the replicase nsP1-4 of the SFV (Semliki Forest Virus) and serve to produce proteins of interest through the intermediate secondary mRNA named 26S(+).

(59) I.1.1) Cloning Sequences of Chimeric Proteins HBV-Zika-prM+E+S and HBV-Zika-prM+Deleted E+Deleted S in the Vector pSFV1

(60) The fragments of hybrid nucleic acid molecules are cloned at the BamHI site of the plasmid pSFV1, previously linearized with this enzyme. The different plasmids comprising the fusion proteins of the invention (notably the plasmids pSFV1-prM+E+S and pSFV1-prM+deleted E+deleted S) are magnified through bacterial transformation, then purified with DNA Maxiprep using phenol/chloroform. The orientation of the insertion is verified through enzyme restriction and all of the constructions are verified through sequencing.

(61) I.2) Obtaining of the Transitorily Transfected Cells by the RNA of Different Constructions Derived from the SFV

(62) The newborn hamster kidney cell (BHK-21) culture procedures as well as the in-vitro transcription protocols of the matrix plasmids SFV and the transfection plasmids of the self-replicating recombining RNA are identical to those previously described [Patient, R., Hourioux, C., Sizaret, P. Y., Trassard, S., Sureau, C., and Roingeard, P., (2007) Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking J Virol, 81(8):3842-51]. The construction pSFVHBVS, expressing the wild-type HBV protein S and previously described in the aforementioned article, was used as a control.

(63) I.3) Analysis of the Intracellular Production of the Wild-Type and Chimeric Envelope Proteins

(64) The procedures for the biochemical analysis of the proteins of interest (notably HBVS, HBV-Zika-E+S and HBV-Zika-deleted E+deleted S, HBV-Zika-E+M, etc.) through confocal microscopy immunofluorescence and western blot, the procedures for the ultrastructural analysis of the transfected cells with transmission electron microscopy as well as the procedures for the quantification (ELISA)/purification (sucrose gradient then dialysis) of the subviral particles of chimeric envelope HBV-Zika (HBV-Zika-E+S, HBV-Zika-deleted E+deleted S, HBV-Zika-E+M) are those previously described [Patient, R., Hourioux, C., Sizaret, P. Y., Trassard, S., Sureau, C., and Roingeard, P., (2007) Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking J Virol, 81(8):3842-51].

(65) The proteins HBV-S, HBV-Zika-E+S, HBV-Zika-deleted E+deleted S were detected with the anti-HBV-S antibody 70-HG15 (interchim), the anti-Zika antibody D1-4G2-15 (millipore) for western blog analyses. The proteins HBV-S, HBV-Zika-E+S, HBV-Zika-deleted E+deleted S were detected with the anti-HBV-S antibody ADRI-2F3 (Cerino A. et al., 2015, 10(4): e0125704) and the anti-Zika antibody D1-4G2-15 for immunofluorescence analyses.

(66) I.4) Analysis of the Culture Supernatant

(67) After transfection, the culture supernatant of approximately 10.sup.7 transfected cells is cleared via centrifugation for 10 minutes at 1500 g then ultracentrifuged at 4 C. for 16 hours at 35,000 rpm using an SW41 rotor (L70 Ultracentrifuge, Beckman). The residue is resuspended with 50 L of the lysis pad, then analyzed with a western blot.

(68) I.5) Production of the Fusion Proteins HBV-Zika-E+S and HBV-Zika-Deleted E+Deleted S of the Invention

(69) Sixteen hours after the transfection by the SFV RNA comprising the hybrid nucleic acid molecules of the invention and transcribed from the plasmids pSFV1-prM+E+S and pSFV1-prM+deleted E+deleted S, the BHK-21 cells were lysed then analyzed with a western blot using the antibodies D1-4G2-4-15 and 70-HG15. After transitory production in the BHK-21 cell, the size of the fusion proteins HBV-Zika-E+S and HBV-Zika-deleted E+deleted S is detected at around 80 kD for the protein HBV-Zika-E+S and at around 75 kD for the protein HBV-Zika-deleted E+deleted S, i.e. precisely at the sizes corresponding to those theoretically determined. These results show, moreover, that the cleavage between prM and the chimeric proteins is efficacious. Furthermore, the perfect co-localization of the immunofluorescence signals obtained through the detection of said fusion proteins of the invention with the anti-HBV-S antibody ADRI-2F3 and anti-Zika antibody D1-4G2-15 show that they are correctly produced in the cells and do not or only slightly undergo internal cleavage to their sequence after their translation (FIGS. 23, 24, 25A and B and 26).

(70) To restore the secretion abilities of the different fusion proteins of the invention (HBV-Zika-E+S and HBV-Zika-deleted E+deleted S), co-transfections are performed by providing the wild-type form of the protein HBV-S in trans to each of the fusion proteins of the invention (HBV-Zika-E+S and HBV-Zika-deleted E+deleted S). Sixteen hours after the transfection, the co-transfected cells are crushed and the intracellular subviral particles were purified through a sucrose gradient then affinity chromatography [Patient, R., Hourioux, C., Sizaret, P. Y., Trassard, S., Sureau, C., and Roingeard, P., (2007) Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking J Virol, 81(8):3842-51]. After detection through specific ELISA of the protein HBV-S and collection of the fractions enriched in subviral particles (FIG. 27), these particles are studied with a western blot using the anti-HBS antibody (70-HG-15) and electron microscopy with, if necessary, immunodetection using the antibody D1-4G2-1. The antibodies are as previously described in the example 1.3 (FIGS. 26, 28 and 29).

(71) The transmission electron microscopy images show that in all these experiments co-producing the wild-type protein HBV-S with one of the fusion proteins from the invention (HBV-Zika-E+S and HBV-Zika-deleted E+deleted S), it is possible to produce a significant quantity of spherical and filamentous subviral particles (FIG. 28). The western blot analyses show that these more or less filamentous subviral particles are rich in fusion proteins from the invention (HBV-Zika-E+S, HBV-Zika-deleted E+deleted S), which is confirmed, moreover, by the anti-Zika immunostaining (immunogold) specifically observed on the negative stainings presented in FIG. 29.

(72) The implementation of the present invention in an SFV system shows that the fusion proteins of the invention (HBV-Zika-E+S and HBV-Zika-deleted E+deleted S) containing nearly all or all of the protein E of the Zika virus gather into chimeric subviral particles of the same type as the subviral particles used in the production of vaccines against hepatitis B, thus facilitating the purification of said chimeric subviral particles from the invention and potentially the development of an industrial application of a vaccine against the Zika virus in perfect harmony with that of the vaccine against the HBV.

Example 4: Obtaining of Subviral Envelope Particles from the Zika Virus in a Lentiviral System

(73) Chinese hamster ovary cells (CHO) are stably transduced using a strategy based on the lentiviral expression vector pHR (described by Dull et al., A third-generation lentivirus vector with a conditional packaging system, 1998, Journal of Virology, vol. 72, pp 8463-8471). The strategy based on the lentiviral expression vector pHR was described in the article Patient et al., Chimeric Hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies, 2009, New Biotechnology, vol. 25, no. 4, pp 226-234.

(74) The DNA sequence of the chimeric envelope proteins HBV-Zika (prM+E+S, prM+E+M or prM+deleted E+deleted S) is introduced at the restriction site BamH1 of the linearized plasmid pHR,.sup.puro (constructed from the plasmid pHR, and coding the puromycin as a selection gene) to generate the chimeric plasmids pHR.sup.puro-HBV-Zika (pHR.sup.puro-HBV-prM+E+S, pHR.sup.puro-HBV-prM+deleted E+deleted S and pHR.sup.puro-HBV-prM+E+M). The lentiviruses are produced in the HEK-293T cells (human embryo kidney cells) and kept in the Dulbecco's Modified Eagle Medium (DMEM).

(75) Twenty-four hours before transfection, 310.sup.6 cells are used to inoculate a culture dish measuring 75 cm.sup.2 (Falcon). The cells are transfected with an equimolar mixture (1 pmol of each) of chimeric plasmid pHRPuro_HBV-Zika (pHR.sup.puro-HBV-prM+E+S, pHR.sup.puro-HBV-prM+deleted E+deleted S and pHR.sup.puro-HBV-prM+E+M), of plasmid pHCMVG (referenced in the ATCC under the reference pHCMV-G (ATCC 754971) coding the VSV-G (envelope glycoprotein of the vesicular stomatitis virus) and the encapsidation construction pCMVR8.74 (Addgene, reference #22036) using the calcium phosphate method.

(76) The following day, the transfection solution is eliminated and replaced by a fresh complete medium.

(77) After 24 and 48 hours of culture, the supernatant is collected, filtered through a low protein binding filter with pores measuring 0.45 m (Sartorius) and concentrated through centrifugation on a 20% saccharose cushion at 4 C. for 90 minutes at 26,000g.

(78) The residue is resuspended in 500 L of PBS and stored at 80 C. until use.

(79) The transduction unit (TU) titer is determined by quantifying the protein p24 (Innotest HIV Antigen mAb Kit, Innogenetics).

(80) The clone CHO-S (stably producing the protein HBV-S, previously described in the article Patient et al., Chimeric Hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies, 2009, New Biotechnology, vol. 25, no. 4, pp 226-234), cultivated in the medium DMEM-F12 (commercialized by Fisher Scientific), is transduced with the recombining lentivectors chimeric HR.sup.puro-HBV-Zika (obtained from the plasmids pHR.sup.puro-HBV-prM+E+S, pHR.sup.puro-HBV-prM+deleted E+deleted S and pHR.sup.puro-HBV-prM+E+M).

(81) One day before transduction, 10.sup.5 CHO-S cells/well are used to inoculate a six-well cellular culture plate (Falcon).

(82) The cells are incubated with the vectors chimeric pHR.sup.puro-HBV-Zika defined above (infection multiplicity: 2.5) and 4 g/mL of polybrene (Sigma) in the fresh complete medium.

(83) Three days after transduction, the cells are used to inoculate a 96-well cellular culture plate (Falcon) with a density of 1 cell/well.

(84) The plates are incubated for 3 weeks with puromycin selection (2.5 g/ml at the end in the culture supernatant).

(85) The cellular clones from the puromycin selection, named CHO-S+Zika-prM+E+S, CHO-S+Zika-prM+deleted E+deleted S or CHO-S+Zika-prM+E+M are isolated and amplified. The expression of HBV-S and chimeric HBV-Zika (HBV-Zika+E+S, HBV-Zika-deleted E+deleted S and HBV-Zika-E+M) is analyzed using immunofluorescence with a human monoclonal anti-HBs antibody and an anti-Zika antibody, as described in example 1.3.

(86) The intracellular production of the proteins HBV-S and chimeric HBV-Zika (HBV-Zika-E+S, HBV-Zika-deleted E+deleted S and HBV-Zika-E+M) is analyzed with a western blot of the cellular lysates, as previously described in the article Patient et al., Chimeric Hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies, 2009, New Biotechnology, vol. 25, no. 4, pp 226-234.

(87) The membranes are incubated for one night at 4 C. with an anti-HBV-S antibody (70-HG15) or the monoclonal antibody directed at the Zika envelope protein (D1-4G2-4-15). The sizes of the fusion proteins HBV-Zika-E+S and HBV-Zika-deleted E+deleted S are approximately 80 kD for the protein HBV-Zika-E+S and approximately 75 kD for the protein HBV-Zika-deleted E+deleted S and 85 kD for the protein HBV-Zika-E+M. These results show, moreover, that the cleavage between prM and the chimeric proteins is efficacious. The protein HBV-S presents two sizes based on its level of glycosylation, one shape of 24 kD (non-glycosylated) and one of 27 kD (glycosylated). These results, correlated with the intense immunofluorescence obtained through the detection of said fusion proteins from the invention with the anti-HBV-S antibody (ADRI-2F3) show that they are correctly produced (FIG. 30).

Example 5: Analysis of the Supernatant of Cells Stably Coproducing the Envelope Proteins HBV-S and Chimeric HBV-Zika (HBV-Zika-E+S, HBV-Zika-E+M or HBV-Zika-Deleted E+Deleted S)

(88) The supernatant is collected, filtered through a low protein binding filter with pores measuring 0.45 m (Sartorius) and concentrated through centrifugation on a 20% saccharose cushion at 4 C. for 90 minutes at 26,000g. The residue is resuspended in a minimum volume of PBS then undergoes a western blot analysis. The incubation of the antibody 70-HG15 shows that the chimeric proteins HBV-Zika are detected and consequently have co-assembled with the protein HBV-S into subviral particles (FIG. 31).

Example 6: Analysis of the Intracellular and Extracellular Production of Chimeric Envelope Proteins

(89) The procedures for the biochemical analysis of the proteins of interest (notably HBV-Zika-E+S, HBV-Zika-deleted E+deleted S and HBV-Zika-E+M) through confocal microscopy immunofluorescence and western blot, the procedures for the ultrastructural analysis of the transfected cells with transmission electron microscopy as well as the procedures for the quantification (ELISA)/purification (sucrose gradient then affinity chromatography) of the subviral particles of chimeric envelope HBV-Zika (HBV-Zika-E+S, HBV-Zika-deleted E+deleted S and HBV-Zika-E+M) are evaluated for their reactivity towards anti-Zika envelope antibodies. For example, the antibodies commercialized by Biofront Technologies (http://www.biofronttech.com/), like the antibodies from family 1176 (1176-46, 1176-56, 1176-66, 1176-76, 1176-86), or the antibodies 7E5 or 7G6 are used for qualitative analyses (immunofluorescence, immunomicroscopy, electronic/immunogold), qualitative and semi-qualitative analyses using western blot tests (analysis of the size of the proteins of interest and their production level) and quantitative analysis using a sandwich ELISA test allowing the quantity of proteins of interest secreted in the culture supernatant to be determined. The detection of the dimeric conformation of the protein sequence of the Zika envelope is evaluated through immunofluorescence detection using specific reference antibodies 747(4)B7, 752-2C8, 753(3)C10 and 747(4)A11 commercialized by the company Absolute antibody (http://absoluteantibody.com). The detection of the protein prM in the cells producing the chimeric proteins or in the subviral chimeric envelope particles of chimeric HBV-Zika is notably evaluated with the antibody GTX133305 commercialized by GeneTex (http://www.genetex.com).

Example 7: Immunization Assay in Small Animals and Analysis of the Immune Response

(90) A. Immunization Protocol

(91) Immunizations are performed in GLP conditions (good laboratory practices). The New Zealand rabbits are nave of all experimental protocols, in particular any previous immunization test. They are young and female, with homogeneous ages and weights. The animals are monitored with a daily clinical examination, monitoring of their mortality and weekly weighing.

(92) Batches of six animals are immunized for each vaccine preparation comprising the chimeric HBV-Zika proteins (HBV-Zika-E+M), HBV-Zika-E+S and HBV-Zika-deleted E+deleted S). A batch of 6 animals is used as a control, 3 animals receiving only the adjuvant and three animals receiving the control vaccine constituted of a commercial anti-HBV vaccine (preparation with only HBV-S protein). The injection is performed subcutaneously with a maximum volume not exceeding 1 ml.

(93) The vaccination schema comprises an initial injection on day 0 (D0) followed by two boosters on days D14 and D28. Three blood samples of at least 5 ml are taken from the animals before each injection; the sample on D0 is used as a negative control before immunization (pre-immune serum).

(94) To complete the study of the immune response development kinetics, samples are taken on D42, D56 and D70. The two animals in each batch that respond best to the anti-Zika and/or anti-HBV-S antibodies receive a fourth vaccine injection on D70, then are exsanguinated on D85.

(95) B. Analyses of the Anti-Zika and Anti-HBV Immune Response Induced in the Small Animal

(96) The analysis of the immune response takes place in vitro using standardized productions of authentic Zika virus. The cross-neutralization with other flaviviruses is measured in the same way with preparations of authentic dengue and West Nile viruses.

(97) 1. Production of Standardized Batches of Zika, Dengue and West Nile Viruses on Vero Cells

(98) These three viruses are produced in a BSL3 environment (Biosafety level 3). The Vero line (ATCC CCL81) is infected with each of the three viruses and the culture supernatants containing the virus are collected at different times ranging from 3 days (Zika virus), 4 days (West Nile virus) to 7 days for the dengue virus. For example, in the case of the Zika virus, the strain H/PF/2013 (genbank accession number: KJ776791) is used to infect sub confluent cells due to an MOI (multiplicity of infection) of 0.01 in the DMEM deprived of fetal bovine serum (FBS). After 24 h, the culture mediums are replaced by a complete nutritive medium (notably comprising 10% FBS). After 3 days of culture, the supernatants of these cells are collected, then clarified with centrifugation at 300g, and finally possibly ultracentrifuged on a saccharose gradient. The viral titers are determined with the cellular method (determination of the viral dose infecting 50% of the cellular tissues; TCID5.sub.0), according to the method described by Spearman-Karber (Krber, G. Archiv f. experiment. Pathol. u. Pharmakol. 1931; 162: 480), possibly completed with a specific quantitative method Q-RT-PCR.

(99) 2. Kinetics Analysis of the Reactivity of the Antibodies Induced in the Small Animal.

(100) The serums of the animals before and after immunization are dosed with the ELISA method to determine their reactivity to the Zika virus. For this, the ELISA anti-Zika virus test kit (IgA/IgG/IgM), commercialized by EUROIMMUN (euroimmun.com) is used to immunocapture anti-Zika antibodies in rabbits, which are then revealed using an anti-rabbit antibody coupled with peroxidase. The colorimetric revelation and the reading of the optical density (OD) allow the evolution of this anti-Zika response to be compared over time (D14 and D28, D42, D56, D70). The pre-immune serums as well as the serums of animals vaccinated only with the adjuvant or the anti-HBV commercial specialty are used as controls.

(101) All of the rabbit serums are also dosed for their anti-HBV reactivity. This is achieved using the commercial anti-HBs Architect System kit (Abbott laboratories).

(102) 3. Analysis of the Neutralizing Ability of the Antibodies Induced in the Small Animal Against the Zika Virus Infection

(103) Vero cells nave of all infection are inoculated 24 h before infection onto a 96-well plate at 210.sup.3 cells/well. Before infection, serum dilutions (e.g. , 1/25, 1/125, 1/625 and 1/3125) are incubated with constant virus quantities (110.sup.3) produced according to paragraph 1 for one hour at 37 C. These serum+virus mixtures are then deposited on the nave Vero cells and then cultured for 72 h. For each of the infection conditions with virus+serum dilution mixtures, the effective infection of the cell layer is measured through visual determination of the cytopathogenic effect (presence of cell lysis plaques). The results of three independent experiments are then considered to determine the neutralization percentage. This neutralization percentage, determined for each serum dilution, is defined according to the following method: 100% neutralization is determined when no lysis plaque is detected in any of the wells for a single dilution; 50% neutralization corresponds to the identification of lysis plaques in 50% of the wells. When all of the wells present lysis plaques, the neutralization percentage is 0%. For these experiments, different controls are implemented, like virus pre-incubation in the presence of pre-immune serums, virus pre-incubation with the serums of rabbits immunized only with the adjuvant and infection of the cell layer with the virus in the absence of pre-incubation with rabbit serums.