Recombinant measles virus expressing chikungunya virus polypeptides and their applications

Abstract

The invention relates to recombinant Measles virus expressing Chikungunya virus polypeptides, and concerns in particular virus like particles (VLP) that contain envelope and capsid proteins of a Chikungunya virus at their surface. These particles are recombinant infectious particles able to replicate in a host after an administration. The invention provides means, in particular nucleic acids, vectors, cells and rescue systems to produce these recombinant infectious particles. The invention also relates to the use of these recombinant infectious particles, in particular under the form of a composition, more particularly in a vaccine formulation, for the treatment or prevention of an infection by Chikungunya virus.

Claims

1. A nucleic acid construct which comprises: (1) a polynucleotide encoding the C-E3-E2-6K-E1 structural proteins of a Chikungunya virus (CHIKV); and (2) a cDNA molecule which encodes a full-length, infectious antigenomic (+) RNA strand of a measles virus (MV); wherein the polynucleotide encoding the C-E3-E2-6K-E1 structural proteins and the cDNA molecule are operatively linked.

2. The nucleic acid construct according to claim 1, wherein said cDNA molecule consists of a number of nucleotides that is a multiple of six.

3. The nucleic acid construct according to claim 1, comprising the following polynucleotides from 5 to 3: (a) a polynucleotide encoding a full length of N protein of the MV, (b) a polynucleotide encoding a full length of P protein of the MV, (c) the polynucleotide encoding the C-E3-E2-6K-E1 structural proteins of CHIKV, (d) a polynucleotide encoding a full length of M protein of the MV, (e) a polynucleotide encoding a full length of F protein of the MV, (f) a polynucleotide encoding a full length of H protein of the MV, and (g) a polynucleotide encoding a full length of 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.

4. The nucleic acid construct according to claim 1, wherein said Measles virus is an attenuated virus strain selected from the group consisting of the Schwarz strain, the Zagreb strain, the AIK-C strain and the Moraten strain.

5. The nucleic acid construct according to claim 1, wherein said polynucleotide encoding the C-E3-E2-6K-E1 structural proteins of CHIKV has been optimized for a Macacca codon usage or has been optimized for a human codon usage.

6. The nucleic acid construct according to claim 1, wherein said polynucleotide encoding the C-E3-E2-6K-E1 structural proteins comprises at least one measles editing-like sequence selected from AAAGGG, AAAAGG, GGGAAA, and GGGGAA that has been mutated.

7. The nucleic acid construct according to claim 1, wherein said Chikungunya virus is from the strain designated 06-49 strain.

8. The nucleic acid construct according to claim 1, wherein the encoded E2 structural protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15.

9. The nucleic acid construct according to claim 1, wherein said nucleic acid construct encodes C-E3-E2-6K-E1 structural proteins having a sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 28.

10. 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: 20, SEQ ID NO: 27, and SEQ ID NO: 31.

11. A transfer vector plasmid, comprising the nucleic acid construct of claim 1.

12. The vector according to claim 11, wherein said vector is pMV-CHIKV.

13. Transformed eukaryotic cells comprising the nucleic acid construct according to claim 1.

14. Recombinant infectious replicating MV-CHIKV particles produced by a method comprising expressing the nucleic acid construct according to claim 1 in a host cell comprising an RNA polymerase recognized by said host cell, a nucleoprotein (N) of a MV, and a phosphoprotein (P) of a MV.

15. The recombinant infectious replicating MV-CHIKV particles of claim 14, wherein said virus particles are encoded by a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 31.

16. A composition comprising the recombinant infectious replicating MV-CHIKV particles according to claim 14, CHIKV-C-E3-E2-6K-E1 Virus Like Particles (VLPs) and a pharmaceutically acceptable vehicle.

17. A method of inducing a protective immune response against CHIKV in a host, comprising administering the composition according to claim 16 to the host.

18. A method of treating or preventing an infection by CHIKV in a host, comprising administering the composition according to claim 16 to the host.

19. A process to rescue recombinant measles virus (MV) expressing the C-E3-E2-6K-E1 structural proteins of a Chikungunya virus (CHIKV) and CHIKV-C-E3-E2-6K-E1 Virus Like Particles (VLPs), comprising: 1) cotransfecting helper cells that stably express T7 RNA polymerase, and measles N and P proteins with (i) a transfer vector plasmid according to claim 14, and (ii) a vector, encoding the MV L polymerase; 2) cultivating said cotransfected helper cells in conditions enabling the production of MV-CHIKV recombinant virus; 3) propagating the thus produced recombinant virus by co-cultivating said helper cells of step 2) with cells enabling said propagation; and 4) recovering replicating MV-CHIKV recombinant virus expressing the C-E3-E2-6K-E1 structural proteins of CHIKV and CHIKV-C-E3-E2-6K-E1 VLPs.

20. The process according to claim 19, wherein the transfer vector plasmid comprises a sequence selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 31.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 Schematic representation of ORFs for structural proteins of Chikungunya virus and for the backbone of MV genome-MV-CHIKV constructs including antigens of said CHIK virus for expression by Measles virus are proposed.

(2) FIG. 1B. Rescue of recombinant MV expressing CHIKV VLP

(3) FIG. 2 Immunofluorescence detection of E2 antigen in Vero cells infected by recombinant MV-CHIKV for 24 h at MOI 0.1 E2 was detected using the anti-E2 Mab 3E4 used at 1/100 dilution and secondary antibody were used at 1/5000 dilution.

(4) FIG. 3: Expression of E2 and capsid proteins by MV-CHIKV vectors. Cell lysates (cells) and supernatants (SN) of Vero cells infected for 24 h by MV-sE2stem, and MV-CE3E26KE1 analyzed by western blot. E2 was probed with the 3E4 Mab, and C protein was detected using an anti-capsid Mab (from P. Desprs) used at 1/100 dilution and secondary antibodies were used at 1/5000 dilution.

(5) FIG. 4. Electron microscopy analysis of CHIKV VLPs secreted in the supernatant of Vero cells infected by MV-CE3E26KE1 recombinant virus at MOI 0.1. Scale bar 200 nm (left) and 100 nm (right). Red arrows indicate the specific arrangement of spikes on particles surface and the icosahedral symmetry of the capsid protein inside the particles.

(6) FIG. 5. Sequence of the truncated sE2 expressed by MV-sE2 recombinant virus (156 aa, 19 kDa).

(7) FIG. 6: Growth kinetics of recombinant MV-sE2stem, and MV-CE3E26KE1 viruses compared with standard MV on Vero cells (MOI 0.01). Cell-associated virus titers are indicated in TCID50.

(8) FIG. 7: Immunization and challenge schedule of example 2.

(9) FIG. 8: Survival curve of mice lethally challenged with 100 PFU of CHIKV-06-49 after two immunizations with MV-CE3E26KE1 recombinant virus.

(10) FIG. 9: Immunization and challenge schedule of example 3.

(11) FIG. 10: Survival curve of mice lethally challenged with 100 PFU of CHIKV-06-49 after a single immunization with MV-CE3E26KE1 recombinant virus.

(12) FIG. 11: Immunization and challenge schedule of example 4.

(13) FIG. 12: Survival curve of mice lethally challenged with 100 PFU of CHIKV-06-49 after immunization with different doses of MV-CE3E26KE1 recombinant virus.

(14) FIG. 13: Passive transfer of immune sera and challenge schedule of example 5.

(15) FIG. 14: Survival curve of mice lethally challenged with 100 PFU of CHIKV-06-49 after passive transfer of MV-CE3E26KE1 immune sera.

(16) FIG. 15: Cell-mediated immune responses elicited in splenocytes of CD46-IFNAR mice immunized by a single injection of 106 TCID50 of MV-CHIKV.

(17) FIG. 16: Immunization and challenge schedule of example 6.

(18) FIG. 17: Survival curve of MV pre-immune mice lethally challenged with 100 PFU of CHIKV-06-49 after immunization with MV-CE3E26KE1.

(19) FIG. 18: PRNT assays performed against CHIK prior to first immunization on day 90 (prior to boost) and day 111 (21 days after boost).

EXAMPLES

(20) Construction and Characterization of Recombinant Measles Virus Vectors Expressing Chikungunya Virus Proteins.

(21) The inventors designed three Chikungunya Virus antigens based on peptide sequences from native proteins of the strain 06-49 of Chikungunya virus. The native proteins enabling preparation of these peptide sequences were the five structural proteins which consist of capsid (C) envelope and accessory proteins E1, E2, E3 and 6K.

(22) The first construct was directed to the expression of the soluble form of the envelope protein E2 (sE2), the second construct to the expression of the sE2 without the stem region (sE2stem), and the third construct was directed to the expression of all viral structural proteins (C-E3-E2-6K-E1) (FIG. 1). The experiment protocols have been described herein with respect to this latter construct.

(23) Cell culture. Vero (African green monkey kidney) cells were maintained in DMEM GlutaMAX (Gibco-BRL) supplemented with 5% heat-inactivated fetal calf serum (FCS, Invitrogen, Frederick, Md.). HEK-293-T7-MV helper cells (WO2008/078198) used for recombinant measles virus rescue were grown in DMEM supplemented with 10% FCS.

(24) Construction of pTM-MVSchw-CE3E26KE1. The plasmid pTM-MVSchw, which contains an infectious MV cDNA corresponding to the anti-genome of the Schwarz MV vaccine strain, has been described elsewhere (Combredet, C., et al., A molecularly cloned Schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice. J Virol, 2003. 77(21): p. 11546-54). The cDNA encoding for the structural CE3E26KE1 CHIKV antigens was generated by chemical synthesis (GenScript, USA). It contains the sequence for viral structural proteins C-E3-E2-6K-E1 from CHIKV strain 06-49 (WO2007/105111). The complete sequence respects the rule of six, which stipulates that the number of nucleotides into the MV genome must be a multiple of 6, and contains BsiWI restriction site at the 5 end, and BssHII at the 3 end. The sequence was optimized for measles virus expression in mammalian cells. This cDNA was inserted into BsiWI/BssHII-digested pTM-MVSchw-ATU2, which contains an additional transcription unit (ATU) between the phosphoprotein (P) and the matrix (M) genes of the Schwarz MV genome (Combredet, C., et al., A molecularly cloned Schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice. J Virol, 2003. 77(21): p. 11546-54). The resulting plasmid was designated as pTM-MVSchw-CE3E26KE1.

(25) Rescue of recombinant MV-CE3E26KE1. Rescue of recombinant Schwarz MV-CHIKV from the plasmid pTM-MVSchw-CE3E26KE1 was performed as previously described using a rescue system previously described (Radecke, F., et al., Rescue of measles viruses from cloned DNA. Embo J, 1995. 14(23): p. 5773-84; WO2008/078198). Viral titers were determined by endpoint limit dilution assay on Vero cells and TCID50 was calculated by using the Krber method.

(26) Immunofluorescence. Immunofluorescence staining was performed on infected cells, as described elsewhere (Lucas, M., et al., Infection of mouse neurons by West Nile virus is modulated by the interferon-inducible 2-5 oligoadenylate synthetase 1b protein. Immun. Cell Biol., 2003. 81: p. 230-236). Cells were probed with mouse anti-E2 (3E4) and anti-capsid antibodies. Cy3-conjugated goat anti mouse IgG antibody Cy3 conjugated (Jackson Immunoresearch laboratories), was used as secondary antibody.

(27) Western blot assays. Protein lysates from Vero cells infected with recombinant virus were fractionated by SDS-PAGE gel electrophoresis and transferred to cellulose membranes (Amersham Pharmacia Biotech). The blots were probed with mouse Mab 3E4 anti-E2 and anti-capsid. A goat anti-mouse immunoglobulin G (IgG)-horseradish peroxidase (HRP) conjugate (Amersham) was used as a secondary antibody. Peroxidase activity was visualized with an enhanced chemiluminescence detection kit (Pierce).

(28) Analysis of VLP production by electron microscopy. Vero cells (3T-150 flasks) were infected with MV-CHIKV recombinant virus at MOI 1. Supernatants collected after 36 h of infection were clarified by centrifugation at 3000 rpm for 30 min, layered on 20% sucrose cushion in PBS and centrifuged at 41,000 rpm for 2 h in a SW41 rotor. Pellets were resuspended in PBS with 1% BSA and analysed by electron microscopy. Negative staining was made by 2% uranyl acetate on copper grids coated with carbon and glow discharged just before use. The samples were observed at 80 kV with a Jeol JEM1200 (Tokyo, Japan) transmission electron microscope. Images were recorded using an Eloise Keenview camera and the Analysis Pro-software version 3.1 (Eloise SARL, Roissy, France).

(29) Mice experiments. CD46-IFNAR mice susceptible to MV infection were produced as previously described (Combredet, C., et al., A molecularly cloned Schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice. J Virol, 2003. 77(21): p. 11546-54). Mice were housed under specific pathogen-free conditions at the Pasteur Institute animal facility. For immunization, six-week-old CD46-IFNAR mice were inoculated intraperitoneally (i.p.) with 10.sup.5 TCID50 of recombinant MV-CE3E26KE1 or MV. For protection assays, immunized mice were i.p inoculated with 100 pfu of CHIKV 06-49 strain and mortality was followed for 2 weeks. All experiments were approved and conducted in accordance with the guidelines of the Office of Laboratory Animal Care at Pasteur Institute. For passive transfer study, CD46-IFNAR mice were inoculated intraperitoneally with 20 l of pooled sera from 6 mice immunized with 10.sup.5 TCID50 of MV-CE3E26KE1. Control mice received either 20 l of pooled sera from mice immunized with 10.sup.5 TCID50 of empty MVSchw or 20 l of anti-CHIKV HMAF. The sera were diluted in a total volume of 100 l in PBS before passive transfer at 24 h and 16 h before challenge with 100 pfu of CHIKV 06-49 strain, and then 12 h post-challenge to mimic antibody persistence in infected animals. Mice mortality was analyzed for 2 weeks to determine protection.

(30) Analysis of humoral immune responses. To evaluate the specific antibody responses, mice were bled via the periorbital route at different time after immunization. Sera were heat inactivated at 56 C. for 30 min and anti-MV antibodies were detected by ELISA (ENZYGNOST-Siemens). HRP-conjugated anti-mouse immunoglobulin (Jackson Immuno Research) was used as secondary antibody. Anti-CHIKV antibodies were detected with a specific ELISA. Briefly, 96-wells plates were coated with a recombinant CHIKV-E2 protein produced in E. Coli. HRP-conjugated anti-mouse immunoglobulin was used as secondary antibody. The endpoint titers of pooled sera were calculated as the reciprocal of the last dilution giving twice the absorbance of sera from MV inoculated mice that served as negative controls. Anti-CHIKV neutralizing antibodies were measured by using a plaque reduction neutralization test (PRNT). Vero cells were seeded into 12-well plates for 24 h.

(31) Serum samples were serially diluted in DMEM Glutamax/2% FCS. Dilutions of 100 l were incubated for 2 h at 37 C., under gentle agitation, with an equal volume of CHIKV containing 100 pfu of 06-49 strain. Remaining infectivity was then assayed on Vero cell monolayers overlaid with DMEM GlutaMAX/2% FCS containing 0.8% final (wt/vol) carboxy methylcellulose. After 3 days of incubation, cells were fixed and stained with crystal violet for plaque count determination. The endpoint neutralization titer was calculated as the highest serum dilution tested that reduced the number of plaques by at least 50% (PRNT50).

(32) Analysis of cell mediated immune response. Six-week-old CD46+/IFN/R/ mice were inoculated intraperitoneally with 10.sup.6 TCID50 of MV-CHIKV recombinant virus. Control mice were immunized with 10.sup.6 TCID50 of empty MV vector. Mice were euthanized at 7 days post-infection and spleens were collected. Splenocytes from immunized mice were incubated in RPMI, 10% FCS, and 10 IU of recombinant human interleukin-2 (rh-IL-2; Boehringer Mannheim). Their capacity to secrete IFN- upon stimulation was tested by enzyme-linked immunospot (ELISPOT) assay. Cells were stimulated 18 h by concanavalin A (5 g/ml; Sigma) as a positive control, RPMI-IL-2 (10 U/ml) as a negative control, CHIKV (MOI 1), or MV (MOI 1). Multiscreen-HA 96-well plates were coated overnight at 4 C. with 5 g of anti-mouse IFN-/ml (R4-6A2; Pharmingen) in PBS, washed, and then incubated with 100 l of RPMI and 10% FCS for 1 h at 37 C. The medium was replaced by 100 l of cell suspension (510.sup.5 splenocytes per well in triplicate) and 100 l of stimulating agent. After 2 h at 37 C., heated-FCS (10%) was added, and the plates were incubated for 18 h at 37 C. After washing, biotinylated anti-mouse IFN- antibody (XMG1.2; Pharmingen) was added and the plates were incubated for 2 hours at room temperature. Streptravidin-alkaline phosphatase conjugate (Roche) was used as secondary step. Spots were developed with BCIP/NBT (Promega) and counted (ELISpot Reader; Bio-Sys).

(33) Expression of CHIKV virus-like particles by recombinant MV vector. CHIKV VLPs have been shown to elicit protective immunity against CHIKV infection (Akahata, W., et al., A virus-like particle vaccine for epidemic Chikungunya virus protects nonhuman primates against infection. Nat Med, 2010. 16(3): p. 334-8). To benefit of this capacity, the inventors designed a recombinant MV vector able to induce the secretion of CHIKV VLPs. To this aim, a cDNA encoding the C-E3-E2-6K-E1 structural proteins required for CHIKV VLPs production was chemically synthetized (Genscript) and optimized for measles virus expression in mammalian cells, then introduced into an additional transcription unit (ATU) of the Schwarz MV vaccine infectious cDNA (FIG. 1). The recombinant MV-CHIKV virus was obtained by transfecting this plasmid into HEK-293 helper cells and propagation on Vero cells. Virus stocks were grown on Vero cells and titer was determined.

(34) High amounts of CHIKV VLPs were secreted in the culture medium of infected cells, as demonstrated by immunofluorescence, western blot and electron microscopy.

(35) This strategy provides a live recombinant MV vaccine virus secreting CHIKV VLPs at each round of replication. It was not expected that the assembly of native alphavirus particles would take place and that these VLPs would not hamper the simultaneous replication of a paramyxovirus. This is demonstrated here for the first time. Because MV vaccine is industrially produced as a crude viral extract, the batches of recombinant MV-CHIKV contain both live MV virus and non-replicating CHIKV VLPs. This strategy allows benefiting of the advantageous immunogenic property of multimeric antigens displayed on VLPs with no need of fastidious and expensive purification and concentration process. Moreover, no adjuvant is needed as the VLPs benefit of the advantageous immunogenic characteristics of live vaccines, such as balanced Th1 response and long-term memory.

(36) The expression of CHIKV E2 and capsid antigens was demonstrated in infected Vero cells by immunofluorescence using a specific antibody (Mab 3E4) directed against the E2 protein of CHIKV (FIG. 2). To look for the presence of secreted VLPs, the culture medium of infected cells was clarified by low-speed centrifugation, then layered onto a 20% sucrose cushion and concentrated by centrifugation at 41,000 rpm for 2 h in a SW41 rotor. Pellets were dissolved in PBS with 1% BSA. Proteins extracted from cell lysates and from concentrated culture media were fractionated by SDS-PAGE gel electrophoresis and transferred to cellulose membranes. The blots were probed with the 3E4 mouse Mab produced by hybridoma deposited at the CNCM (Collection Nationale de Cultures de Microorganismes, Paris, France) on Sep. 6, 2007 under number 1-3824, in the name of Institut Pasteur for the detection of E2 and an anti-capsid Mab (FIG. 3).

(37) The E2 protein was found both in cell lysates and in concentrated supernatant of infected cells at the correct size (46 KDa), indicating the capacity of MV-CHIKV virus to induce the secretion of high-density particles containing the E2 protein. The capsid protein was also found in high-density particles, confirming the formation of CHIKV-VLPs. The presence of both C and E2 proteins in concentrated supernatant of infected cells suggests the formation of CHIKV VLPs. To observe their physical presence, the inventors analyzed by electron microscopy the pellets concentrated from supernatant of MV-CHIKV infected cells. The images revealed the presence of high amount of particles of size and morphology similar to those described after wild type CHIKV infection (Pletnev, S. V., et al., Locations of carbohydrate sites on alphavirus glycoproteins show that E1 forms an icosahedral scaffold. Cell, 2001. 105(1): p. 127-36; Zhang, W., et al., Placement of the structural proteins in Sindbis virus. J Virol, 2002. 76(22): p. 11645-58) (FIG. 4). The observed particles present an external diameter of 65 nm and a core diameter of 40 nm. The surface organization indicates the presence of spikes on the surface of the VLPs, similarly arranged than for other alphaviruses (Pletnev, S. V., et al., Locations of carbohydrate sites on alphavirus glycoproteins show that E1 forms an icosahedral scaffold. Cell, 2001. 105(1): p. 127-36; Zhang, W., et al., Placement of the structural proteins in Sindbis virus. J Virol, 2002. 76(22): p. 11645-58). This observation confirms that infection of Vero cells by recombinant MV-CHIKV virus enables the secretion of high amounts of CHIK VLPs that self assemble.

(38) The E2 protein was also expressed and secreted at the correct size (46 KDa) by cells infected with Measles virus-sE2stem recombinant viruses, and Measles virus-CE3E26KE1 recombinant viruses.

(39) Unfortunately, the analysis of MV-sE2 infected cells showed repeatedly the expression of a truncated form of the E2 protein. The inventors sequenced the E2 mRNA produced by MV-sE2 virus after RT-PCR amplification of infected cells. The analysis demonstrated the presence of a mutation generating a STOP codon, responsible for the truncation (FIG. 5).

(40) The inventors then compared the replication rate of Measles Virus-sE2, Measles Virus-sE2stem, and Measles Virus-CE3E26KE1 recombinant viruses on Vero cells to standard Measles virus stock production, using a low MOI (0.01) (FIG. 6). The growth of MV-sE2 was similar to that of control MV. The growth of Measles virus-sE2stem and Measles virus-CE3E26KE1 recombinant viruses was slightly delayed, but their final titers were in the same range as that of empty Measles virus.

(41) Immunogenicity of MV-sE2 and MV-CE3E26KE1 and Protection in CD46-IFNAR Mice.

(42) CD46-IFNAR mice susceptible to MV infection were used to assess the immunogenicity of the recombinant MV-CHIKV viruses and their protective efficacy. These mice express the human CD46 gene with human-like tissue specificity and lack the type-I interferon receptors. Mice were housed under specific pathogen-free conditions at the Pasteur Institute animal facility and all experiments were approved and conducted in accordance with the guidelines of the Office of Laboratory Animal Care at Pasteur Institute. Six-week-old CD46-IFNAR mice were inoculated intraperitoneally (i.p.) with doses ranging from 10.sup.3 to 10.sup.5 TCID50 of MV-CHIKV recombinant viruses and boosted 1 month later with the same dose of recombinant viruses. Control mice were immunized with the same dose of empty MVSchw vector. For antibody determination, blood samples were collected via the periorbital route 1 month after the first inoculation, then at 2 or 4 weeks after boosting.

(43) Previous studies have shown that IFNAR mice are susceptible to lethal Chikungunya virus infection, showing pathological manifestations of infection and providing a model to evaluate immune mechanisms of protection (Couderc et al; 2008).

(44) CD46-IFNAR mice susceptible to Measles virus infection were used to assess the immunogenicity of the recombinant Measles-Chikungunya viruses and their protective efficacy. These mice express the human CD46 gene with human-like tissue specificity and lack the type-I interferon receptors. Mice were housed under specific pathogen-free conditions at the Pasteur Institute animal facility and all experiments were approved and conducted in accordance with the guidelines of the Office of Laboratory Animal Care at Pasteur Institute.

Experiment 1

Analysis of the Immunogenicity and the Protective Efficacy of Measles Virus-CE3E26KE1 Recombinant Viruses in CD46-IFNAR Mice

(45) Six-week-old CD46-IFNAR mice susceptible to Measles virus infection were intraperitoneally inoculated with 2.10.sup.4 TCID.sub.50 of Measles virus-Chikungunya virus recombinant viruses and boosted 1 month later with the same dose of recombinant viruses. Control mice were immunized with the same dose of empty Measles virus Schwarz vector (MV Schw). For antibody determination, blood samples were collected via the periorbital route 1 month after the first inoculation, then at 2 weeks after boosting. Mice were then challenged by i.p. injection of 100 pfu of Chikungunya virus 06-49 strain for evaluating protection (Immunization and challenge schedule is given in FIG. 7).

(46) To evaluate the specific antibody responses, mice were bled at different time-points after inoculation. Sera were heat inactivated at 56 C. for 30 min and anti-Chikungunya virus antibodies were detected by ELISA. 96-well plates were coated with recombinant Chikungunya virus-E2 protein produced in E. coli. HRP-conjugated anti-mouse immunoglobulin was used as secondary antibody and mouse antibodies anti-Chikungunya virus was used as a positive control. Anti-Chikungunya virus neutralizing antibodies were detected by a plaque reduction neutralization test (PRNT) (Warter L et al. JIM 2011 (D4 enclosed) and Russell P K et al. JIM 1967) on Vero cells using 50 PFU of Chikungunya virus-06-49 (produced on Vero cells). The endpoint titer was calculated as the highest serum dilution tested that reduced the number of PFU by at least 50% (PRNT50) or 90% (PRNT90).

(47) A single injection of Measles virus-CE3E26KE1 recombinant viruses induced high antibody titers, which were strongly boosted by a second injection (table1FIG. 8). After two immunizations, high neutralizing titers were induced (PRNT50=450-4050 and PRNT90=50-450). All animals immunized with 104 or 105 TCID50 were protected from CHIKV lethal challenge with 100 PFU of CHIKV-06-49, whereas immunization with the lower dose (103 TCID50) protected 83% of the animals.

(48) TABLE-US-00001 TABLE 1 Antibody response of CD46-IFNAR mice to immunization with MV-sE2 and MV-CE3E26KE1 (determined in pooled mice sera) Elisa 1 Elisa 2 dose dose PRNT50 PRNT90 MV <100 <100 <50 <50 MV-CHIK.SE2 450 4000 <50 <50 MV-CHIK.CE3E26KE1 4000 >12000 1350 150 Anti-CHIKV HMAF ND ND 4050 450

(49) After two immunizations, high neutralizing titers were induced (PRNT50=1350 and PRNT90=150) that protected mice from a lethal challenge with 100 PFU of Chikungunya virus-06-49.

Experiment 2

Analysis of the Immunogenicity and the Protective Efficacy of a Single Dose of Measles Virus-CE3E26KE1 Recombinant Virus in CD46-IFNAR Mice

(50) Six-week-old CD46-IFNAR mice were i.p. inoculated with 10.sup.5 TCID.sub.50 of Measles virus-CE3E26KE1 recombinant viruses. Control mice were immunized with the same dose of empty Measles virus Schw vector. Blood samples were collected via the periorbital route 2 weeks after immunization for antibody determination, and then mice were challenged by i.p. injection of 100 pfu of Chikungunya virus 06-49 (Immunization and challenge schedule is given in FIG. 9).

(51) The Measles virus-CE3E26KE1 recombinant viruses induced high antibody titers after a single injection (table 2FIG. 10), and neutralizing titers that were sufficient to confer protection against a lethal challenge with 100 PFU of Chikungunya virus-06-49 in IFNAR mice.

(52) TABLE-US-00002 TABLE 2 Antibody response elicited in CD46-IFNAR mice after a single immunization with MV-CE3E26KE1 virus MV CHIKV Elisa Elisa CHIKV CHIKV titer titer PRNT50 PRNT90 MV 10 000 <100 <50 <50 MV-CHIKV 10 000 4 050 150 50 Anti-CHIKV HMAF ND ND 12150 450

Experiment 3

Determination of the Protective Dose of Measles Virus-CE3E26KE1 Recombinant Virus in CD46-IFNAR Mice

(53) Six-week-old CD46-IFNAR mice were inoculated intraperitoneally (i.p.) with doses ranging from 10.sup.3 to 10.sup.5 TCID50 of MV-CHIKV recombinant virus and boosted one month later with the same dose. Control mice were immunized with the same dose of empty MVSchw vector. One month after the last immunization, mice were challenged by i.p. injection of 100 pfu of CHIKV 06-49 (Immunization and challenge schedule is given in FIG. 11). For antibody determination, blood samples were collected via the periorbital route 1 month after the first inoculation, then 1 month after boosting, just before challenge. Specific Elisa's were performed to detect anti-MV and anti-CHIKV binding antibodies. Anti-CHIKV neutralizing antibodies titers were determined by a plaque reduction neutralization test (PRNT) on Vero cells.

(54) The results are given in table 3 and FIG. 12.

(55) TABLE-US-00003 TABLE 3 Antibody response after immunization with different doses of MV-CE3E26KE1 1st immunisation 2.sup.nd immunisation MV CHIKV CHIKV CHIKV MV CHIKV CHIKV CHIKV Elisa titer Elisa titer PRNT50 PRNT90 Elisa titer Elisa titer PRNT50 PRNT90 MV 10.sup.5 10 000 <100 <50 <50 300 000 <100 <50 <50 MV-CHIKV 10.sup.3 1 000 1 350 50 <50 3 000 2 700 450 50 MV-CHIKV 10.sup.4 3 000 4 050 150 50 30 000 12 150 1 350 150 MV-CHIKV-10.sup.5 3 000 12 150 450 150 300 000 48 600 4 050 450

(56) Both anti-MV and anti-CHIKV antibody titers increased when the dose of recombinant MV increased. A single immunization with MV-CE3E26KE1 virus induced high antibody titers, which were boosted by the second injection. After two immunizations, high neutralizing titers were induced (PRNT50=450-4050 and PRNT90=50-450). All animals immunized with 10.sup.4 or 10.sup.5 TCID.sub.50 were protected from CHIKV lethal challenge with 100 PFU of CHIKV-06-49, whereas immunization with the lower dose (10.sup.3 TCID50) protected 83% of the animals (FIG. 12).

Experiment 4

Evaluation of the Protection Conferred by Passive Transfer of Sera from Mice Immunized with Recombinant MV-CE3E26KE1 Virus

(57) Six-week-old CD46-IFNAR mice were i.p. inoculated with 20 l of pooled sera from mice immunized with 10.sup.5 TCID50 of recombinant MV-CE3E26KE1. Control mice received either 20 l of pooled sera from mice immunized with 10.sup.5 TCID50 of empty Measles virus Schwarz or 20 l of anti-Chikungunya virus HMAF. The sera were diluted in a total volume of 100 l PBS. The sera were transferred at 24 h and 16 h before challenge with 100 pfu of Chikungunya virus 06-49, and then 12 h post-challenge to mimic antibody persistence in infected animals. Mice mortality was analyzed for 2 weeks to determine protection (Immunization and challenge schedule is given in FIG. 13).

(58) Passive transfer of immune sera of mice immunized with MV-CE3E26KE1 virus protected 83% of recipient mice from lethal Chikungunya virus challenge, while mice that received anti-Chikungunya virus HMAF were fully protected. In contrast, mice that received immune sera from mice immunized with empty Measles viruses all died. These results indicate that humoral immune responses induced by Measles virus-CE3E26KE1 recombinant viruses confer protection against Chikungunya virus infection in CD46-IFNAR mice (FIG. 14).

Experiment 5

Induction of Specific Cell-mediated Immune Responses

(59) To determine whether immunization with MV-CHIKV elicited cell-mediated immune responses, we measured by ELISPOT assay the capacity of splenocytes from immunized mice to secrete IFN- upon specific ex-vivo stimulation. Splenocytes were collected 7 days after a single immunization and both MV-specific and CHIKV-specific responses were evaluated. CHIKV and MV were used at an MOI of 1 for splenocytes stimulation. A significant number of CHIKV-specific cells (up to 300/106 splenocytes, mean 150/106) were detected (FIG. 15), which represents one third of MV-specific response in similar stimulation condition (up to 600/106 splenocytes, mean 500/106). All mice immunized with MV-CHKV, except one out of eight, had a significant CMI response to CHIKV. In contrast, control mice immunized with empty MVSchw had a similar MV-specific response but no CHIKV-specific response. These results show that a single inoculation of MV-CHIKV induced high levels of CHIKV- and MV-specific cellular immune response in the spleen of immunized mice.

Experiment 6

Analysis of Measles Virus Pre-immunity Impact on the Immunogenicity and Protective Efficacy of Recombinant MV-CE3E26KE1 Virus in CD46-IFNAR Mice

(60) Six-week-old CD46-IFNAR mice were i.p. inoculated with 5.10.sup.3 TCID.sub.50 of empty Measles virus Schwarz (Group1 FIG. 16) to mimic pre-immunity. Three months later, these mice were injected twice with 10.sup.5 TCID.sub.50 of Measles virus-CE3E26KE1 recombinant viruses at one month of interval. Control mice were immunized with 10.sup.5 TCID.sub.50 of Measles virus-CE3E26KE1 recombinant viruses (Group 2) or 10.sup.5 TCID.sub.50 of empty Measles virus Schw (Group 3). For antibody determination, blood samples were collected via the periorbital route as indicated in FIG. 16, and mice were then challenged by i.p. injection of 100 pfu of Chikungunya virus 06-49 strain for protection assays.

(61) This experiment demonstrates that CD46-IFNAR mice previously immunized with 5.10.sup.3 TCID50 of empty Measles viruses are able to mount a protective Chikungunya virus immune response after immunization with Measles virus-CE3E26KE1 recombinant viruses. The ELISA and PRNT titers (Table 4FIG. 17) remain high and in the same range of that induced in naive mice (ELISA titer unchanged and PRNT titers reduced by one-fold dilution). 100% of the vaccinated animals were protected from Chikungunya virus lethal challenge in both pre-immune and naive groups of animals immunized with Measles virus-CE3E26KE1 recombinant viruses.

(62) TABLE-US-00004 TABLE 4 Antibody response of CD46-IFNAR mice to MV-CE3E26KE1 in the presence of pre-immunity to MV vector Immunizations 1.sup.st 2.sup.nd 3.sup.rd MV MV CHIKV CHIKV CHIKV MV CHIKV CHIKV CHIKV Elisa Elisa Elisa PRNT50 PRNT90 Elisa Elisa PRNT50 PRNT90 MV + MV-CHIKV 3 000 30 000 12 150 150 50 30 000 150 000 12 150 150 MV-CHIKV ND 10 000 12 150 450 150 100 000 150 000 12 150 450 MV ND 10 000 <50 <50 <50 100 000 <50 <50 <50

Experience-7

Cross-reactivity of Abs Elicited by Vaccination

(63) To determine whether immunization with MV-CHIKV elicited cross-reactive antibody response to different CHIKV primary isolates, sera obtained from animals of experiment 4 were tested for their ability to neutralize different CHIKV primary isolates. Four strains belonging to the ECSA genotype were chosen. CHIKV strain India, clinical isolate no 3710 (NRC for Arbovirus, France), isolated in 2011. Passage 1 on Vero cells (December 2011), virus titer 6.3 log PFU/ml CHIKV strain Congo, clinical isolate no 525 (NRC for Arbovirus, France), isolated in 2011. Passage 1 on Vero cells (June 2011), virus titer 6.5 log PFU/ml CHIKV strain Thailand, clinical isolate no 1499 (NRC for Arbovirus, France), isolated in 2009. Passage on C6/36 cells (December, 2009), virus titer 6.3 log PFU/ml CHIKV strain La Reunion, clinical isolate 2006.49 (NRC for Arbovirus, France), isolated in 2006. Passage 3 on Vero cells (Apr. 4, 2011), virus titer 7.3 log PFU/ml

(64) Anti-CHIKV neutralizing antibodies were detected by use of a plaque reduction neutralization test (PRNT). Vero cells were seeded into 12 well plates for 24 h. Serum samples were serially diluted in DMEM Glutamax/2% FCS. Dilutions 100 l were incubated for 2 h at 37 C., under gentle agitation, with an equal volume of CHIKV containing 100 pfu of 06-49 strain. Remaining infectivity was then assayed on Vero cell monolayers overlaid with DMEM GlutaMAX/2% FCS containing 0.8% final (wt/vol) carboxy methylcellulose. After 3 days of incubation, cells were fixed and stained with crystal violet for plaque count determination. The endpoint neutralization titer was calculated as the highest serum dilution tested that reduced the number of plaques by at least 50% (PRNT.sub.50).

(65) TABLE-US-00005 TABLE 5 Cross-reactivity of neutralizing Ab from mice immunized with MV-CHIKV PRNT50 PRNT50 PRNT50 post 1st post 2nd post challenge MV-CHIKV MV MV-CHIKV MV MV-CHIKV MV 06-49 50 <50 1350 <50 450 ND Inde 150 <50 1350 <50 13250 ND Congo <50 <50 1350 <50 1350 ND Thai 150 <50 4050 <50 12150 ND

(66) The result shows that immunization of mice with MV-CHIKV induces cross-reactive antibodies that are able to neutralize several primary isolates of CHIKV from different countries. Interestingly, the challenge of animals with the 06-49 La Reunion virus results in even broadening the response and boosts the neutralization of Indian and Thai isolates to very strong levels. Only ECSA genotype was tested because of its availability at Institut Pasteur.

Experience-8

Immunogenicity of MV-CHIKV in Cynomolgus Macaques

(67) The immunogenicity of recombinant measles virus vaccine against Chikungunya was tested in non-human primates. Two groups of four cynomolgus macaques (macaca fascicularis) previously selected to be seronegative for flaviviruses and measles virus were vaccinated subcutaneously with 10.sup.4 or 10.sup.5 TCID.sub.50 MV-CHIKV on day 0 then boosted on day 90 with the same dose. Serum and plasma were collected and stored at 20 C. for later analysis. Neutralizing antibodies to Chikungunya virus were detected by using a PRNT assay.

(68) TABLE-US-00006 TABLE 6 Reciprocal CHIK-PRNT Ani- Day Day Day Day Group mal Code Vaccine Dose 0 21 90 111 D D1 12/9 MV-CHIK 10.sup.4 <10 574 <10 14 D2 12/4 MV-CHIK 10.sup.4 <10 142 73 97 D3 12/5 MV-CHIK 10.sup.4 <10 16 17 121 D4 12/10 MV-CHIK 10.sup.4 <10 70 83 118 E E1 12/16 MV-CHIK 10.sup.5 <10 247 101 382 E2 12/23 MV-CHIK 10.sup.5 <10 103 178 355 E3 12/26 MV-CHIK 10.sup.5 <10 6173 151 652 E4 12/22 MV-CHIK 10.sup.5 <10 97 386 228

(69) Results presented in Table 6 show that all monkeys developed high titers of antibodies that neutralized CHIKV. The highest dose was more efficient than the lower dose. In most animals, boosting was efficient, as shown on FIG. 18 (day 90, the day of boost versus day 111, 21 days after boost). These results demonstrate the immunogenicity of MV-CHIKV vaccine candidate in non-human primates.

GENERAL CONCLUSION

(70) The inventors have generated a recombinant MV-CHIK virus expressing stably the complete structural proteins CE3E26KE1 of CHIKV strain 06.49. Vero cells infected by this recombinant virus expressed high levels of CHIKV proteins and secreted high density VLPs. The recombinant virus was slightly delayed in growth kinetics but yielded similar titers that empty MV vector. Evaluated in CD46-IFNAR mice susceptible to MV infection, this vaccine candidate induced high levels of neutralizing antibodies to CHIKV depending on the dose and number of administrations (PRNT50 =450-4050; PRNT90=150-450). All immunized mice were repeatedly protected, even after a single administration, demonstrating the strong immune capacity of this vaccine candidate. The passive transfer of immune sera in nave animals conferred protection from lethal challenge, even in these highly susceptible mice. Lastly, the inventors demonstrated that the presence of pre-existing immunity to MV vector in CD46-IFNAR mice did not prevent the induction of protective immunity after immunization with MV-CE3E26KE1 vaccine candidate. In view of the results, the recombinant vector thus obtained deserves to be evaluated in a reliable non-human primate model of infection.