Recombinant measles virus expressing zika virus prM and E proteins

Abstract

The present invention relates to recombinant measles virus expressing Zika virus proteins and their applications, in particular in inducing preventive protection against Zika virus. The present invention is directed to recombinant measles virus (MV) expressing at least (i) the precursor of membrane (prM) protein of a Zika virus (ZIKV), and the envelope (E) protein of a ZIKV or a truncated version thereof, or (ii) the E protein of a ZIKV or a truncated version thereof, and concerns recombinant infectious particles of said MV-ZIKV able to replicate in a host after an administration, and also Virus Like Particles (VLPs) that contain these ZIKV proteins at their surface. The present invention provides means, in particular nucleic acids, vectors, cells and rescue systems to produce these recombinant infectious particles and VLPs. The present invention also relates to the use of these recombinant infectious particles and/or VLPs, in particular under the form of a composition, more particularly in a vaccine formulation, for the prevention of an infection by ZIKV or for the preventive protection against clinical outcomes of ZIKV infection.

Claims

1. A nucleic acid construct which comprises: (1) a polynucleotide encoding at least (i) the precursor of membrane (prM) protein of a Zika virus (ZIKV), and the envelope (E) protein of a ZIKV or a truncated version thereof of SEQ ID NO: 26, SEQ ID NO: 29 and SEQ ID NO: 32, or (ii) the E protein of a ZIKV or the truncated version thereof of SEQ ID NO: 26, SEQ ID NO: 29 and SEQ ID NO: 32; and (2) a cDNA molecule encoding a full-length, infectious antigenomic (+) RNA strand of a live-attenuated measles virus (MV) vaccine strain; wherein the polynucleotide encoding at least (i) the prM protein of the ZIKV, and the E protein of the ZIKV or the truncated version thereof, or (ii) the E protein of the ZIKV or the truncated version thereof and the cDNA molecule are operatively linked; the nucleic acid construct comprising the following polynucleotides from 5 to 3: (a) a polynucleotide encoding the N protein of the MV; (b) a polynucleotide encoding the P protein of the MV; (c) the polynucleotide encoding at least (i) the prM protein of the ZIKV, and the E protein of the ZIKV or the truncated version thereof, or (ii) the E protein of the ZIKV or the truncated version thereof; (d) a polynucleotide encoding the M protein of the MV; (e) a polynucleotide encoding the F protein of the MV; (f) a polynucleotide encoding the H protein of the MV; and (g) a polynucleotide encoding the L protein of the MV; wherein said polynucleotides are operably linked in the nucleic acid construct and under a control of viral replication and transcription regulatory sequences.

2. The nucleic acid construct according to claim 1, characterized in that the polynucleotide of (1) and the cDNA molecule of (2) together consist of a number of nucleotides that is a multiple of six.

3. The nucleic acid construct according to claim 1, wherein said live-attenuated MV vaccine strain is selected from the group consisting of the Schwarz strain, the Zagreb strain, the AIK-C strain and the Moraten strain.

4. The nucleic acid construct according to claim 1, wherein said polynucleotide encoding at least (i) the prM protein of the ZIKV, and the E protein of the ZIKV or the truncated version thereof, or (ii) the E protein of the ZIKV or the truncated version thereof, has been optimized for a Macaca codon usage or has been optimized for a human codon usage.

5. The nucleic acid construct according to claim 1, wherein measles editing-like sequences have been deleted from said polynucleotide encoding at least (i) the prM protein of the ZIKV, and the E protein of the ZIKV or the truncated version thereof, or (ii) the E protein of the ZIKV or the truncated version thereof.

6. The nucleic acid construct according to claim 1, wherein said ZIKV is from the African lineage, or from the Asian strain.

7. The nucleic acid construct according to claim 1, wherein said polynucleotide encoding at least (i) the prM protein of the ZIKV, and the E protein of the ZIKV or the truncated version thereof, further encodes (iii) the signal peptide from the capsid of the ZIKV and the signal peptide from the membrane protein of the ZIKV, or wherein said polynucleotide encoding at least (ii) the E protein of the ZIKV or the truncated version thereof, further encodes (iii) the signal peptide from the capsid of the ZIKV or the signal peptide from the membrane protein of the ZIKV.

8. The nucleic acid construct according to claim 1, wherein the polynucleotide encoding the E protein encodes either the full-length E protein or its soluble form lacking the two C-terminal transmembrane domains of the full-length E protein.

9. The nucleic acid construct according to claim 1, wherein the polynucleotide encoding the truncated version of the E protein is selected from the group consisting of (i) the polynucleotide encoding the E protein truncated at amino acid position 456 of the full-length E protein of the ZIKV of SEQ ID NO: 23, (ii) the polynucleotide encoding the E protein truncated at amino acid position 445 of the full-length E protein of the ZIKV of SEQ ID NO: 23 and (iii) the polynucleotide encoding the E protein truncated at amino acid position 404 of the full-length E protein of the ZIKV of SEQ ID NO: 23.

10. The nucleic acid construct according to claim 1, wherein the polynucleotide encodes the prM protein of the ZIKV whose sequence is SEQ ID NO: 20, and the polynucleotide encodes the E protein of the ZIKV or the truncated version thereof whose sequence is selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29 and SEQ ID NO: 32.

11. The nucleic acid construct according to claim 1, wherein the polynucleotide encoding the prM protein of the ZIKV has the sequence of SEQ ID NO: 19, and the polynucleotide encoding the E protein of the ZIKV or the truncated version thereof has a sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28 and SEQ ID NO: 31.

12. The nucleic acid construct according to claim 1, wherein said nucleic acid construct comprises a sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 70, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 168 and SEQ ID NO: 170.

13. The nucleic acid construct according to claim 1, which comprises the sequence from nucleotide at position 83 to nucleotide at position 18404 in the sequence of SEQ ID NO: 165, or the sequence from nucleotide at position 83 to nucleotide at position 18074 in the sequence of SEQ ID NO: 166, or the sequence from nucleotide at position 83 to nucleotide at position 17702 in the sequence of SEQ ID NO: 167.

14. A transfer vector plasmid, comprising the nucleic acid construct according to claim 1.

15. The transfer vector plasmid according to claim 14, whose sequence is SEQ ID NO: 165, SEQ ID NO: 166 or SEQ ID NO: 167.

16. Isolated transformed eukaryotic cells comprising inserted in their genome the nucleic acid construct according to claim 1.

17. Isolated recombinant infectious replicating measles virus-Zika virus (MV-ZIKV) particles, which comprise as their genome a nucleic acid construct according to claim 1.

18. The isolated recombinant infectious replicating MV-ZIKV particles according to claim 17, which are rescued from a helper cell line expressing an RNA polymerase recognized by said cell line, a nucleoprotein (N) of a MV, a phosphoprotein (P) of a MV, and an RNA polymerase large protein (L) of a MV.

19. The isolated recombinant infectious replicating MV-ZIKV particles according to claim 17, wherein said particles comprise in their genome a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 70, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 168 and SEQ ID NO: 170.

20. A pharmaceutical composition comprising the recombinant infectious replicating MV-ZIKV particles according to claim 17, in association with ZIKV-Virus Like Particles (VLPs) expressing the same ZIKV protein(s) as said MV-ZIKV particles, and a pharmaceutically acceptable vehicle.

21. A method of inducing a ZIKV-specific immune response in a host, comprising administering the pharmaceutical composition of claim 20 to the host.

22. A method of treating ZIKV infection or inhibiting ZIKV infection in a host, comprising administering the recombinant infectious replicating MV-ZIKV particles according to claim 17 in association with ZIKV-VLPs expressing the same ZIKV protein(s), or the pharmaceutical composition according to claim 20 to the host.

23. A process to rescue recombinant infectious measles virus-Zika virus (MV-ZIKV) particles expressing at least (i) the precursor of membrane (prM) protein of a ZIKV, and the envelope (E) protein of a ZIKV or a truncated version thereof, or (ii) the E protein of a ZIKV or a truncated version thereof, and ZIKV Virus Like Particles (VLPs) expressing the same ZIKV protein(s), comprising: 1) co-transfecting helper cells that stably express T7 RNA polymerase, and measles N and P proteins with (i) the transfer vector plasmid according to claim 14 and with (ii) a vector, encoding the MV L polymerase; 2) cultivating said co-transfected helper cells in conditions enabling the production of recombinant MV-ZIKV particles; 3) propagating the thus produced recombinant MV-ZIKV particles by co-cultivating said helper cells of step 2) with cells enabling said propagation; 4) recovering recombinant infectious replicating MV-ZIKV particles expressing at least (i) the prM protein of the ZIKV, and the E protein of the ZIKV or the truncated version thereof, or (ii) the E protein of the ZIKV or the truncated version thereof, and ZIKV VLPs expressing the same ZIKV protein(s).

24. The process according to claim 23, wherein the transfer vector plasmid has the sequence of SEQ ID NO: 165, SEQ ID NO: 166 or SEQ ID NO: 167, preferably has the sequence of SEQ ID NO: 165.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Schematic representation of Zika virus genome.

(2) FIG. 2. Phylogenetic trees of the main human pathogenic flaviviruses based on the amino acid sequences of the E protein (left) and of the polymerase NS5 protein (right). JEV, Japanese encephalitis virus; MVEV, Murray Valley encephalitis virus; POWV, Powassan virus; SLEV, Saint Louis encephalitis virus; TBEV, tick-borne encephalitis virus; YFV, yellow fever virus; WNV, West Nile virus.

(3) FIG. 3. Schematic representation of Zika virus antigens. Protein domains are drawn to scale. Zika, Zika virus; JEV, Japanese encephalitis virus; MV, Measles virus. A. 12 variants of the Zika antigen, where the native signal peptide from the capsid (sp) or from the membrane protein (sp) of Zika virus is used. B. 8 variants of the chimeric JEV-Zika antigen, where a signal peptide of the capside of JEV is used. C. 10 variants of the MV-Zika antigen, where the signal peptide of the fusion protein of MV (MVsp) is used. D. 10 variants of the MV Zika antigen, where a modified signal peptide of the fusion protein of MV (MVsp) is used.

(4) FIG. 4. Schematic representation of MV Vector. MV genes are indicated: N (nucleoprotein), PVC (phoshoprotein and V/C proteins), M (matrix), F (fusion), H (hemagluttinin), L (polymerase), T7 (T7 RNA polymerase promoter), hh (hammerhead ribozyme), T7t (T7 RNA polymerase terminator), a (hepatitis delta virus ribozyme), red arrows (additional transcription units).

(5) FIG. 5. Single immunization in mice. A) Zika antibody response measured in mice sera by ELISA at one month after a single immunization. MV-prMEd404 (native sequence, insert 4); MV-ssEd445 (native sequence, insert 5). B) Survival of immunized mice after challenge by Zika virus. C) Zika virus viremia in serums of immunized mice (determined by RT-qPCR) at different days after challenge. D) IFN-gamma Elispot detected in splenocytes of mice one week after immunization with MV-Zika or control MVSchw viruses. Elispots are detected against MV (Schwarz), Zika virus (Zika) and Concanavalin A as a control.

(6) FIG. 6. Prime-boost immunization in mice. A) Zika antibody response measured in mice sera by ELISA at day 30, 45 and 55 after two immunizations. B) Detection of Zika virus neutralizing antibodies in the sera of mice immunized with two injections of MV-prMEd404 (native sequence, insert 4), MV-ssEd445 (native sequence, insert 5). C) Survival of immunized mice after challenge with low dose of Zika virus. D) Zika virus viremia in serums of immunized mice (determined by RT-qPCR) at different days after challenge.

(7) FIG. 7. Recombinant MV expressing the full-length prME Zika antigen (construct A1) produces Zika VLPs. Vero cells were infected with three different clones of rMV-Zika_A1 (1, 2, 3) for 48 hours. Cell lysates and medium were collected. Supernatant medium was clarified by low-speed centrifugation (1500 rpm) then concentrated by ultracentrifugation on a 20% sucrose cushion for 3 hours (36000 rpm). All material was analyzed by western blot to detect the Zika E protein (50 kD) with 4G2 panflavi monoclonal antibody. (A) Cell lysates, (B) Concentrated medium, (C) Non-concentrated medium and positive and negative controls. Positive control is a lysate of Vero cells transfected for 48 hours with pcDNA5 plasmid expressing the Zika A1 antigen. The positive E protein recovered in panel B after ultracentrifugation demonstrates that high density VLPs were produced in the supernatant of infected Vero cells.

(8) FIG. 8. Zika virus antigen expression assay. HEK293T cells were transfected with each codon-optimized construct, and cell lysates and medium were collected after 48 h. Supernatant medium was clarified by low-speed centrifugation (1500 rpm), and then a fraction was concentrated by ultracentrifugation on a 20% sucrose cushion for 3 hours (36000 rpm). All material was analyzed by western blot to detect the Zika virus E protein (50 kD) with the 4G2 pan-flavivirus antibody. (L) Cell lysates, (S) non-concentrated medium, and (U) ultracentrifugated medium.

(9) FIG. 9. Expression of Zika virus antigen A1 from measles vector and growth curve of recombinant MV-Zika-A1. (A) Immunofluorescence analysis showed large syncytia in Vero cells infected for 24 hours with MV-Zika-A1 (the Zika virus E protein was detected with the 4G2 pan-flavivirus antibody). (B) Replication of recombinant MV-Zika-A1 virus on Vero cells at 32 C. after infection with a multiplicity of infection of 0.01 (titers were determined by limiting dilution and the Karber method).

(10) FIG. 10. Antibody response to ZIKV in immunized CD46-IFNAR.sup./ mice. The antibody titers against ZIKV EDIII were determined using indirect ELISA in mice sera collected after prime and boost with MV-ZIKV-A1, MV-prMEd404 (native sequence, insert 4), MV-ssEd445 (native sequence, insert 5), MV-ZIKV-A12 or control empty MV-Schwarz. Readings from wells coated with mock antigens were subtracted from wells with ZIKV-EDIII and the ZIKV specific IgG titers were calculated as the reciprocal of the highest dilution of an individual serum giving an absorbance of 0.5. A strong antibody response to ZIKV was induced in immunized mice with slightly higher values for A1 (highly reproducible) and A12 (more variability).

(11) FIG. 11. ZIKV neutralizing antibody titers in immunized CD46-IFNAR.sup./ mice. Neutralizing antibody titers against ZIKV were determined by using plaque reduction neutralizing tests (PRNT.sub.50) in mice sera collected after last boost with MV-ZIKV-A1, MV-prMEd404 (native sequence, insert 4), MV-ssEd445 (native sequence, insert 5), MV-ZIKV-A12 or control empty MV-Schwarz and before challenge. The strongest neutralizing titers were observed with the MV-ZIKV-A1 construct.

(12) FIG. 12. Protection of immunized CD46-IFNAR.sup./ mice from ZIKV non-lethal challenge. Mice immunized twice with MV-ZIKV-A1, MV-ZIKV-A1 2 or control empty MV-Schwarz were challenged with 10.sup.3 ffu of ZIKV (Asian-South American lineage, isolated in December 2015) one month after the last immunization. Viral loads were determined by RT-qPCR. LOD indicates the limit of detection of the RT-qPCR. Mice immunized with construct MV-ZIKV-A1 were all protected from viremia while mice immunized with MV-ZIKV-A12 or empty MV Schwarz control were infected.

(13) FIG. 13. Protection of immunized CD46-IFNAR.sup./ mice from ZIKV lethal challenge. Mice immunized twice with MV-ZIKV-A1, or control empty MV-Schwarz were challenged with 10.sup.3 ffu of ZIKV (Mouse adapted strain of the African lineage) one month after the last immunization. Animals were monitored for morbidity and mortality for 15 days. All animals immunized with MV-ZIKV-A1 survived without presenting signs of disease, while all control mice died by day 8.

EXAMPLES

(14) Generation of Vaccine Candidates

(15) Previous experiences with different flaviviruses (dengue, West Nile, Japanese encephalitis, tick-borne encephalitis) widely demonstrated that the flaviviral surface envelope (E) proteins are able to elicit protective neutralizing antibodies that allow reducing virus infectivity. The ZIKV genome consists of a single-stranded positive sense RNA molecule of 10800 kb of length with 2 flanking non-coding regions (5 and 3 NCR) and a single long open reading frame encoding a polyprotein that is cleaved into three structural proteins (capsid (C), precursor of membrane (prM), envelope (E)) and seven non-structural proteins (NS) (FIG. 1). The E protein (53 kDa) is the major virion surface protein involved in various aspects of the viral cycle, mediating binding to target cells and membrane fusion.

(16) The inventors therefore chose to express the Zika virus E protein. Several forms of E protein were selected in order to express either soluble secreted proteins or anchored proteins onto the surface of VLPs. The following Zika virus antigens were cloned and expressed from a mammalian expression plasmid in human cells: prM-E and different forms of E with or without the stem or anchor region. These proteins contain either the original signal peptide sequence of Zika virus E or a heterologous signal peptide sequence from JEV or MV fusion protein. These proteins contain the signalase cleavage site located between the prM and the E sequences (FIGS. 3A, 3B, 3C, 3D).

(17) Antigens Selection and Design

(18) The Zika antigens were selected based on previous works concurring into suggesting that envelope antigens of flaviviruses may be able to elicit neutralizing antibodies and T cell responses. Selecting a suitable antigen should however take into consideration the evolution of the virus over time and the variety of existing virus strains. To this end, the inventors reconstructed the phylogeny of representative members of the flavivirus family, including Zika virus, using only the amino acid region of the flavivirus polyprotein corresponding to the envelope (E) gene. Unlike phylogenetic analyses based on the full genome, or the polymerase (NS5) of flaviviruses, where the closest relative of Zika virus are neurotropic viruses such as Saint-Louis Encephalitis virus, the inventors noticed that Zika E appeared closer to DENV E (FIG. 2) (Barba-Spaeth, et al. Nature 2016, 536, 48-53). The inventors then proceeded to identify the different domains of Zika membrane (M), its precursor (prM) and E proteins through structural homology modelling based on available data on DENV (Ekins et al. Illustrating and homology modeling the proteins of the Zika virus, F1000Research 2016, 5:275). The inventors also identified the signal peptides at the end of the Capsid (C) gene, just upstream of prM, using again homology modelling with dengue virus as a reference, as well as publicly available algorithms to predict signal peptide sequences (sigpep. services. came. sbg. ac. at/sidnalblast. html; cbs. dtu. dk/services/SignalP/; predisi. de/). The inventors chose to include the signal peptide sequence to induce the export and secretion of the candidate antigen, either the full-length prM-E, or the E only, outside the cells. For the E antigen, the inventors also predicted the signal peptide at the end of M, just upstream of E, and designed versions of the antigen using this native signal (FIG. 3A). In addition, the inventors also designed chimeric antigens where the native signal peptide of Zika virus was replaced with the signal peptide present at the end of JEV C (FIG. 3B), or the signal peptide present at the N-terminal of the fusion protein (F) of MV (FIG. 3C), hypothesizing that these sequences would provide enhanced export of the candidate antigens. The inventors designed an additional version of the chimeric antigen including the signal peptide of F from MV, where two amino acids corresponding to the junction between the end of the signal peptide of F and the beginning of F itself were removed (FIG. 3D).

(19) Secondly, the inventors also designed shorter variations of the antigens by removing C terminal fragments of the E protein corresponding to the predicted stem and/or anchor domains, including the intermediate region between the stem and anchor (as predicted by comparison to DENV). The aim of these modifications that reduced antigens size was to generate antigens that were able to form VLPs. For a third variant, the inventors removed the anchor, the intermediate domain between the anchor and the stem, as well as a fragment of the second helix that composed the stem, this time in homology modelling with WNV (variant Ed445).

(20) Finally, the inventors designed chimeric prM-E and E antigens using the signal peptide from MV F protein, and replacing Zika E anchor by the transmembrane (TM) and intracytoplasmic tail of MV F protein (FIGS. 3C and 3D).

(21) For the selection sequence of the antigen itself, the inventors analyzed all publicly available sequences of Zika virus (both Asian and African lineages), as well as unpublished sequences generated by the inventors, from the epidemic in South America and Pacific. Based on the epidemiological data reporting an association of congenital syndromes and neurological afflictions in adults with only the Asian lineage, the inventors designed an antigen using the consensus amino acid sequence of Zika viruses as observed circulating from 2015 and onward, notably to include the S139N change that generated a novel potential N glycosylation site in prM that was absent from the African lineage, and the V763M in E.

(22) The sequences were codon-optimized for Homo sapiens expression and adapted to measles vector cloning and to the rule of six (total number of nucleotides divisible by 6). Regions very rich (>80%) or very poor (<30%) in GC were avoided to increase RNA stability, a high CAI value (0.97) was obtained to increase translation efficacy, the following CIS active sequences were avoided: internal TATA-boxes, chi-sites, ribosomal entry sites, AT- or GC-rich sequence stretches, ARE, INS, CRS elements, repeat sequences, RNA secondary structures, cryptic splice donor and acceptor sites, branch points. The following measles virus editing sequences were avoided where possible: AAAGGG, AAAAGG, GGGAAA, GGGGAA, TTAAA, AAAA, and also their complementary sequences on the same strand: TTCCCC, TTTCCC, CCTTTT, CCCTT, TTTAA, TTTT. The enzyme restriction sites BssHII, BsiWI were avoided internally and inserted at both ends for cloning purpose.

(23) Antigen Expression in Mammalian Cells

(24) The optimized antigen sequences were cloned into pcDNA5 mammalian expression plasmid and transfected into HEK293 cells. The size and level of expression of each antigen were characterized after western blotting using appropriate antibodies for detection.

(25) Antigen Expression in Measles Vector

(26) The optimized Zika antigen sequences were inserted into the MV vector in different additional transcription units, according to the desired level of expression. After sequencing of the measles vector plasmids expressing the different Zika antigens, the replicating recombinant vectors were generated by reverse genetics using a cell-based system previously developed (Combredet, C. et al., 2003, J Virol, 77(21): 11546-11554), and the rescued viruses were amplified and titrated on Vero cells. The recombinant viruses were grown on Vero cells to document the expression of Zika proteins detected both in supernatants and in cells by using Western Blot and indirect immunofluorescence staining with appropriate antibodies. The presence of Zika virus VLPs (in prM/E expressing vectors) was identified after ultracentrifugation of culture medium and western blot (FIG. 7). The correct processing of antigens in infected cells was checked by Western Blot. The vectors with the best expression capacity of Zika antigens were isolated by serial dilution and single plaque cloning before amplification on Vero cells.

(27) Growth Capacity of Recombinant Vaccine Virus

(28) The growth capacity of selected vaccine viruses was compared with standard MV Schwarz. Growth curve analysis was performed in Vero cell culture by using different multiplicity of infection then titration.

(29) Stability of Recombinant Vaccine Virus

(30) The best vaccine vectors selected were tested for their genetic stability by serial passaging over 10 cell culture passages on Vero cell culture followed by western blot for antigen expression and full sequencing analysis.

(31) Preclinical Evaluation of First MV-Zika Recombinant in Mice

(32) Single Immunization

(33) The two recombinant vectors MV-prMEd404 (native sequence, insert 4) and MV-ssEd445 (native sequence, insert 5) were evaluated in CD46/IFNAR mice susceptible to measles infection. Mice were immunized with one or two intraperitoneal injections with defined infectious units of vaccine virus and functional antibodies and cell-mediated immune responses were analysed using both standard and specifically developed assays. Binding antibodies to Zika virus were determined with ELISA and neutralizing antibodies with specific plaque reduction neutralization test (PRNT). The T cell responses were analysed by Elispot assay using Zika virus-specific peptides for ex vivo stimulation of splenic cells. The vaccine vectors were then tested for protective efficacy: immunized mice were challenged with a lethal dose of Zika virus. A dose-response challenge was previously established in CD46/IFNAR mice showing that doses between 10.sup.2 and 10.sup.6 focus forming unit (ffu) of Zika virus African strain HD78788 (adapted to mouse) efficiently kill these mice.

(34) In a first experiment 6 mice per group were immunized with a single intraperitoneal injection of 10.sup.6 TCID50 of MV-prMEd404 (native sequence, insert 4), MV-ssEd445 (native sequence, insert 5) or empty MVSchw as a control. Blood was taken before immunization and at day 30 after immunization, and Zika virus ELISA titers were determined (FIG. 5A).

(35) The immunized mice were then challenged at day 30 by intraperitoneal injection of 10.sup.6 ffu of Zika virus African strain HD78788 (mouse adapted). Morbidity and mortality were controlled during 12 days (FIG. 5B) and Zika virus viremia was determined in serum by qRT-PCR (FIG. 5C).

(36) To determine T-cell response to the vaccine, another group of CD46/IFNAR mice were immunized by MV-prMEd404 (insert 4) or empty MVSchw and spleens were collected at 8 days after immunization. Elispot assay was performed on freshly extracted splenocytes using MVSchw or Zika virus to re-stimulate T-cells or concanavalin A as a control (FIG. 5D).

(37) Prime-Boost Immunization

(38) In a second set of experiments, groups of CD46/IFNAR mice were immunized with two successive intraperitoneal injections of 10.sup.6 TCID50 of MV-prMEd404 (native sequence, insert 4), MV-ssEd445 (native sequence, insert 5) or empty MVSchw as a control. Blood was taken before immunization and at day 30, 45 and 55 after immunizations and Zika virus ELISA titers were determined (FIG. 6A). Neutralizing antibodies were determined in sera collected at day 50 using a specific neutralization test of Zika virus (FIG. 6B). The immunized mice were then challenged at day 60 by intraperitoneal injection of 10.sup.6 ffu of Zika virus African strain HD78788 (mouse adapted). Morbidity and mortality were controlled during 12 days (FIG. 6C) and Zika virus viremia was determined in serum by qRT-PCR at days 2, 4 and 6 post infection (FIG. 6D).

(39) Preclinical Evaluation in Non-Human Primates (NHP)

(40) Validation of the ZIKV Strain Used in the NHP Challenge Study

(41) Because little is known about the physiopathology of ZIKV in cynomolgus macaque (Macaca fascicularis), two animals were inoculated in a preliminary assay with three doses of Zika wild-type virus (10.sup.4, 10.sup.5 and 10.sup.6 pfu) to assess the viral stock and associated clinics in macaques. These two animals were submitted to the same follow-up than vaccinated and challenged animals but for a 6-month period. The following points were addressed: Virology (qRT-PCR; clinics (Rash, Fever); Blood cell count (Lymphocytes, Monocytes, Granulocytes, platelettes); Biochemistry (ASAT, ALAT, CRP); Non-specific (innate and inflammatory) and specific immune response: Cytokines/chemokines by luminex, NK, B and T cell profile (14 colors flow cytometry), Antibodies (neutralizing, binding) on serial sera samples, T cells functional response and memory cells (ELISpot, ICS). Shedding of the virus in biological fluid (saliva, tears, genital fluids) was assessed by qRT-PCR and/or isolation methods at various time-points.

(42) Vaccine Immunogenicity Study in NHP

(43) Macaques were immunized with one or two subcutaneous injections at 3 months interval of defined infectious units of vaccine virus. Humoral and cell-mediated immune responses were determined at different times post immunization. Macaques were then challenged with infectious doses of ZIKV. Infectious viremia and clinical signs were determined. For this task, twenty-one adult cynomolgus macaques were selected to be negative for anti-flaviviruses and anti-measles antibodies; Two groups of 7 animals were vaccinated with a single dose or a prime boost regiment with the best MV-ZIKV recombinant virus (MV-prMEd404 native) selected. Immunity (Humoral and cell associated) was explored and virology was followed up to 1 month post vaccination. Clinics and biological parameters are assessed in parallel to a third group of 7 animals vaccinated with the control empty MVSchw strain following the prime boost schedule. Antibody neutralization titer was determined.

(44) Vaccine Efficacy Study in NHP

(45) Immunized NHP were challenged with ZIKV two months after immunization. ZIKV viremia level (qRT-PCR) was analyzed in blood, saliva and tears. Inflammation and immune response was assessed in plasma (neutralizing Ab, cytokines).

(46) Expression Assays

(47) The expression assays performed for all constructs generated (FIG. 8) showed a strong expression for several of them. Signal was detected in the ultracentrifugated fraction, which was compatible with the generation of virus-like particles, in varying amounts for some candidate antigens, notably A1 and A12. These two antigens were thus further cloned into the measles vector and demonstrated high-level expression as shown by immunofluorescence (FIG. 9A). The recombinant MV-ZIKV-A1 vector replicated similarly to standard MV Schwarz virus, although with a lower final titer (FIG. 9B).

(48) Tested for their immunogenicity in CD46/IFNAR mice, MV-ZIKV-A1 and MV-ZIKV-A12 vectors elicited strong immune responses following a prime and boost regimen with 1-month interval, comparable to MV-prMEd404 and MV-ssEd445 vectors, as detected by ELISA (FIG. 10). However, different amounts of neutralizing antibodies were induced (FIG. 11). Only the candidate MV-ZIKV-A1 induced a strong neutralizing response (2 log stronger). This correlated with the complete protection conferred to mice by immunization with MV-ZIKV-A1 (FIG. 12) against viremia, as well as protection from a lethal challenge (FIG. 13).

(49) In conclusion, this study demonstrated that the A1 full-length Zika antigen expressed in MV vector was able to provide sterile protection from infectious and lethal challenge of immunized animals, correlating with strong neutralizing antibody induction.