HK1211951B - Recombinant measles virus expressing chikungunya virus polypeptides and their applications - Google Patents

Description

Recombinant measles virus expressing chikungunya virus polypeptide and its application

Technical Field

The present invention relates to recombinant measles viruses expressing Chikungunya virus polypeptides, and more particularly to virus-like particles (VLPs) containing Chikungunya virus envelope and capsid proteins on their surfaces. These particles are recombinant infectious particles capable of replicating in a host after administration. The present invention provides tools for producing these recombinant infectious particles, particularly nucleic acids, vectors, cells, and rescue systems. The present invention also relates to the use of these recombinant infectious particles, particularly in the form of compositions, and more particularly in the form of vaccines, for treating or preventing Chikungunya virus infection.

Background Art

Chikungunya virus (CHIKV) is a positive-strand RNA virus of the genus Alphavirus in the family Togaviridae. It was first isolated in Tanzania in 1952.

Infection with this virus causes a human disease characterized by symptoms similar to dengue fever, with an acute febrile phase of 2-5 days followed by a prolonged arthralgia affecting the joints of the limbs. CHIKV is endemic to Africa, India, and Southeast Asia, and is transmitted by Aedes mosquitoes through urban or sylvatic transmission cycles. In 2006, CHIKV fever broke out in several islands of the Indian Ocean (Comoros, Mauritius, Seychelles, Madagascar, Reunion Island...), and subsequently spread to India, where an estimated 1.4 million cases were reported. Recently, imported infections have been reported in Europe, with approximately 200 endemic cases reported in Italy (Jose, J. et al., A structural and functional perspective of alphavirus replication and assembly. Future Microbiol, 2009. 4(7): p. 837-56). Clinically, this CHIKV epidemic is accompanied by more severe symptoms than previous outbreaks, with reports of severe polyarthralgia and myalgia, complications, and death.

The CHIKV genome is an 11.8 kb single-stranded RNA molecule with positive polarity. The virus is closely related to Semliki Forest virus (SFV), Sindbis virus (SINV), and other Old World alphaviruses, but is more distant from New World alphaviruses such as Venezuelan equine encephalitis virus (Griffin, D.E., Alphaviruses, in Fields Virology, 5th ed., D.M. Knipe, Editor 2007, Wolters Kluwer, Lippincott Williams & Wilkins. p. 1023-1067). The genomic RNA is capped and directly translated into a full-length nonstructural polyprotein (nsP) named P1234, which is encoded by the 5' two-thirds of the genome (Jose, J., J.E. Snyder, and R.J. Kuhn, A structural and functional perspective of alphavirus replication and assembly. Future Microbiol, 2009. 4(7): p.837-56; Kuhn, R.J., Togaviridae: the viruses and their replication, in Fields Virology, 5th ed., D.M. Knipe, Editor 2007, Wolters Kluwer, Lippincott Williams & Wilkins. p.1001-1022). This precursor is self-cleaved to produce P123 and nsP4, which has RNA-dependent RNA polymerase activity. These proteins, together with cellular cofactors, assemble into a replication complex that produces antisense genomic RNA molecules. P123 is subsequently cleaved into nsP1 and P23, generating a polymerase complex that produces both sense and antisense genomic RNA. P23 is further cleaved into nsP2 and nsP3, generating a polymerase complex that produces only positive-sense genomic RNA molecules. In addition to replicating the viral genome, this viral protein complex transcribes the 26S subgenomic RNA from the 3′ end of the viral genome. This messenger RNA is translated into a polyprotein precursor, which is cleaved by a combination of viral and cellular enzymes to produce the capsid protein (C), two major envelope proteins (E1 and E2), and two smaller accessory peptides, E3 and 6k. Once assembled, CHIKV virions are spherical particles 65–70 nm in diameter, consisting essentially of genomic RNA molecules associated with capsid proteins and enclosed in a host-derived lipid membrane decorated by E1-E2 heterodimers assembled into an icosahedral lattice (Voss, J.E., et al., Glycoprotein organization of Chikungunyavirus particles revealed by X-ray crystallography. Nature, 2010. 468(7324): p. 709-12).

The severity of the disease represented by the evolution of viruses and their spread to new geographical areas is a serious public health problem that needs to be addressed. To address this problem, vaccines with attenuated live viruses, with chimeric alphaviruses, with recombinant DNA or with virus-like particles have been developed.

A formalin-inactivated vaccine has been shown to be immunogenic in non-human primates and humans, but the large amount of antigen required for large-scale immunization under BSL-3 conditions has limited the development of this strategy (Tiwari, M., et al., Assessment of immunogenic potential of Vero adapted formalin inactivated vaccine derived from novel ECSA genotype of Chikungunya virus. (Vaccine, 2009. 27(18): p. 2513-22). The live attenuated TSI-GSD-218 CHIKV vaccine developed by the US Army is immunogenic, but side effects related to virulence reversion occurred in Phase II clinical trials, raising safety concerns. Therefore, although the results obtained with vaccines based on obtaining live attenuated viruses indicate that effective immunity can be achieved in this way, these vaccines are still questioned because of the risk of possible side effects (Edelman R et al., Am J Trop Med Hyg. 2000 Jun; 62(6): 681-685). Chimeric alphavirus vaccine strategies encoding E1, E2, and capsid proteins from CHIKV are immunogenic in mice (Wang, E., et al., Chimeric alphavirus vaccine candidates for Chikungunya. (Vaccine, 2008. 26(39): p. 5030-9), but the ability of alphaviruses to readily recombine has raised safety concerns that have impacted the development of these strategies (Weaver, S.C., et al., Recombinational history and molecular evolution of western equine encephalomyelitis complex alphaviruses. J Virol, 1997. 71(1): p. 613-23).

Another strategy that has been developed is to design a recombinant DNA construct for use as a vaccine. DNA-based CHIKV vaccines encoding E1, E2, and capsid proteins have been shown to be immunogenic in mice and nonhuman primates (Muthumani, K., et al., Immunogenicity of novel consensus-based DNA vaccines against Chikungunya virus. Vaccine, 2008. 26(40): p.5128-34; Mallilankaraman, K., et al., A DNA vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates. PLoS Negl Trop Dis, 2011. 5(1): p.e928), but the DNA strategy does not induce the strong neutralizing immune response required to eliminate CHIKV in humans. The disadvantage of DNA vaccines is that large amounts of DNA are required to induce an immune response, and multiple booster vaccinations must be performed. The need for multiple boosters and the injection of large amounts of DNA into the nuclei of many cells raises concerns that DNA vaccines can be integrated into host DNA and cause insertional mutations. Therefore, a recent study reported the use of DNA vaccines in combination with live attenuated viruses (WO 2011/082388). Although this technology allows to reduce the disadvantages of live attenuated viruses and DNA vaccines, there is still a need to provide vaccines with reduced side effects.

To avoid the shortcomings of live attenuated viruses and DNA vaccines, other types of vaccines have been developed, such as vaccines based on virus-like particles (VLPs), which are obtained by expressing Chikungunya virus structural proteins. These structural proteins can self-assemble in virus-like particles. On this basis, vaccines containing polynucleotides encoding all Chikungunya virus structural proteins have been developed (Akahata, W., et al., A virus-like particle vaccine for epidemic Chikungunyavirus protects nonhuman primates against infection. Nat Med, 2010. 16(3): p. 334-8). However, the preparation of in vitro produced VLPs is expensive, and full immunity requires three administrations, so these vaccines are expensive. The CHIKV VLP strategy disclosed in Akata et al. requires several immunizations with adjuvants to induce protection. For this reason, there is still a need to design improved vaccines that can produce CHIKV VLPs in infected cells, especially infected host cells, in vivo and thereby provide effective and long-lasting immunity, especially vaccines that induce long-lasting immunity after only a single or two administration steps.

SUMMARY OF THE INVENTION

To this end, the present inventors have achieved the production of a vaccine based on a recombinant infectious replicating measles virus, the measles virus being recombined from a polynucleotide encoding a Chikungunya virus antigen, and the vaccine being recovered after administration, particularly when the recombinant virus replicates in a host. Thus, the present invention relates to a live CHIKV vaccine active ingredient based on the widely used Schwarz measles vaccine. In a preferred embodiment, the recombinant live MV-CHIKV vaccine produces CHIK virus-like particles by replicating in infected cells.

Measles virus is the non-segmented single-stranded antisense enveloped RNA virus of the family Morbillivirus of Paramyxoviridae. This virus is separated from (Enders, J.F., and T.C.Peebles.1954.Propagation in tissue cultures of cytopathogenic agents from patients with measles.Proc.Soc.Exp.Biol.Med.86:277-286.) in 1954, and live attenuated vaccine derives from this virus, produces vaccine strain thus, and especially from Schwarz strain. Over the past 30 years, measles vaccine has been applied to hundreds of millions of children, and its effectiveness and safety have been confirmed. It is mass-produced and distributed at a low price in many countries. For all these reasons, the present inventor uses attenuated measles virus to produce the recombinant measles virus particle, particularly VLP, of stably expressing chikungunya virus structural antigen.

Therefore, the present invention relates to a nucleic acid construct comprising a polynucleotide encoding at least one Chikungunya virus (CHIKV) structural antigen, which is operably linked, in particular cloned, into a cDNA molecule encoding the full-length, infectious antigenomic (+) RNA chain of measles virus (MV).

The nucleic acid constructs of the present invention, particularly purified DNA molecules, are obtained or obtainable recombinantly from polynucleotides of various origins operably linked together.

The expression "operably linked" refers to the presence of a functional linkage between the different polynucleotides of the nucleic acid construct of the invention, whereby the different polynucleotides and the nucleic acid construct are efficiently transcribed and, where appropriate, translated, in particular in a cell or cell line, in particular in a cell or cell line or host cell used as part of a rescue system to produce the chimeric infectious MV particles of the invention.

In a specific embodiment of the present invention, a construct is prepared by cloning a polynucleotide encoding one or more CHIKV structural proteins into a cDNA molecule encoding the full-length antigenomic (+) RNA of measles virus. Alternatively, the nucleic acid construct of the present invention can be synthesized using nucleic acid fragments or polymerized from a template, including by PCR.

In a specific embodiment of the present invention, the polynucleotide encoding at least one CHIKV protein or each of these polynucleotides is cloned into an ATU (additional transcription unit) inserted into the measles virus cDNA. The ATU sequence is known to the skilled person and is used for cloning into the MV cDNA and includes the cis-acting sequences necessary for MV-dependent expression of the transgene, such as a gene promoter, located in front of the insert in the MV cDNA, said insert being represented by the polynucleotide encoding the CHIKV protein incorporated into the multiple cloning site cassette.

When used in the present invention, ATU is advantageously located in the N-terminal sequence of the cDNA molecule encoding the full-length (+) RNA chain of the MV antigenome, and in particular, between the P and M genes or between the H and L genes of the virus. It has been observed that MV viral RNA transcription follows a gradient from the 5' to the 3' end. This explains why, when inserted at the 5' end of the cDNA coding sequence, ATU allows for more efficient expression of the heterologous DNA sequence it contains (e.g., a polynucleotide encoding at least one CHIKV structural protein).

Therefore, the polynucleotide encoding at least one CHIKV structural protein can be inserted into any intergenic region of the measles virus cDNA molecule, in particular, ATU. Specific constructs of the present invention are exemplified in the Examples.

In a specific embodiment, when several different polynucleotides are present in a DNA construct, each of these polynucleotides encoding at least one CHIKV structural protein can be inserted into a different site of the MV cDNA, possibly a different ATU of the measles virus cDNA.

In a preferred embodiment of the present invention, the polynucleotide encoding at least one CHIKV structural protein is inserted into the intergenic region of the P and M genes of the measles virus cDNA molecule, in particular into ATU.

The expression "encoding" used in this application defines the ability of a nucleic acid molecule to be transcribed and (if appropriate) translated in a selected cell or cell line to express a product. Accordingly, the nucleic acid construct of the present invention may comprise regulatory elements that control transcription of the coding sequence, particularly transcription promoters and termination sequences, as well as possible enhancers and other cis-acting elements. These regulatory elements may be heterologous to the CHIK polynucleotide sequence.

The term "protein" is used interchangeably with the term "antigen" or "polypeptide" to define a molecule produced by the connection of amino acid residues. In particular, the proteins disclosed in the present application are derived from CHIKV and are structural proteins, which may be identical to the native protein or alternatively may be derived from the native protein by mutation, including substitution (particularly by conserved amino acid residues) or addition of amino acids or post-translational secondary modifications, or deletion of part of the native protein to produce fragments with a shortened size relative to the native protein used as a reference. The present invention comprises fragments having epitopes of native proteins that are suitable for eliciting an immune response in a host, particularly a human host, preferably such a response is capable of preventing CHIKV infection or CHIKV-related disease. The epitope is particularly a B-type epitope that participates in eliciting a humoral immune response by activating antibody production in a host to which the protein is administered or in a host expressing the protein after administration of the infectious replicating particles of the present invention. Alternatively, the epitope may be a T-type epitope that participates in eliciting a cell-mediated immune response (CMI response). The fragment may have a size that represents more than 50% of the size of the amino acid sequence of the native CHIKV protein, preferably at least 90% or 95%. Alternatively, a fragment may be a short polypeptide of at least 10 amino acid residues that has an epitope of the native protein. In this regard, a fragment also includes a polytope as defined herein.

In a specific embodiment of the present invention, the cDNA encoding the nucleotide sequence of the full-length infectious antigenome (+) RNA strand of MV in the nucleic acid construct conforms to the rule of six of the measles virus genome.

The organization of the measles virus genome and its replication and transcription processes have been well established in the prior art, in particular, the Schwarz inoculation strain of the virus disclosed by Horikami S.M. and Moyer S.A. (Curr. Top. Microbiol. Immunol. (1995) 191, 35-50 or Combredet C. et al (Journal of Virology, Nov 2003, p11546-11554), or the negative sense RNA virus widely identified in Neumann G. et al (Journal of General Virology (2002) 83, 2635-2662).

The "6-fold rule" means that the total number of nucleotides present in a nucleic acid representing the MV (+) chain RNA genome or a nucleic acid construct comprising the MV (+) chain RNA genome is a multiple of 6. The "6-fold rule" has been recognized in the art as a requirement regarding the total number of nucleotides in the measles virus genome that allows for efficient or optimized replication of the MV genomic RNA. In embodiments of the present invention that define nucleic acid constructs that satisfy the 6-fold rule, the rule applies to nucleic acid constructs that define cDNAs encoding the full-length MV (+) chain RNA genome. In this regard, the 6-fold rule applies solely to cDNAs encoding the nucleotide sequence of the full-length infectious antigenomic (+) RNA chain of the measles virus, but does not necessarily apply to polynucleotides cloned into the cDNA and encoding one or more CHIKV proteins.

According to a specific aspect of the present invention, the nucleic acid construct comprises the following gene transcription unit, from 5' to 3':

(a) a polynucleotide encoding the N protein of MV,

(b) a polynucleotide encoding the P protein of MV,

(c) a polynucleotide encoding at least one CHIKV structural protein,

(d) a polynucleotide encoding the M protein of MV,

(e) a polynucleotide encoding the F protein of MV,

(f) a polynucleotide encoding the H protein of MV, and

(g) a polynucleotide encoding the L protein of MV,

The polynucleotides and nucleic acid constructs are operably linked and under the control of viral replication and transcription regulatory sequences, such as the MV leader and trailer sequences.

The expressions "N protein," "P protein," "M protein," "F protein," "H protein," and "L protein" refer to the nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H), and large RNA polymerase protein (L) of measles virus, respectively. These components have been identified in the prior art and are disclosed in particular in Fields, Virology (Knipe & Howley, 2001).

In a preferred embodiment of the present invention, the cDNA molecule encoding the full-length infectious antigenomic (+) RNA strand of measles virus has characteristics of or is obtained from an attenuated strain of MV.

An "attenuated strain" of measles virus is defined as a strain that is avirulent or less virulent than the parent strain in the same host and retains immunogenicity and possible adjuvant properties when administered to the host, i.e., retains immunodominant T and B cell epitopes and possible adjuvant properties such as induction of T cell co-stimulatory proteins or the cytokine IL-12.

An attenuated strain of measles virus refers to a strain that has been serially passaged on selected cells and, if applicable, adapted to other cells to produce a seed strain suitable for preparing a vaccine strain, and has a stable genome that does not allow reversion to pathogenicity or integration into the host chromosome. Specifically, an "attenuated strain" is a strain that has been confirmed for use in a vaccine and is suitable for the present invention and meets the regulations defined by the FDA (U.S. Food and Drug Administration), i.e., safety, efficacy, quality, and reproducibility requirements after rigorous review of laboratory and clinical data ( www.fda.gov/cber/vaccine/vacappr.htm ) .

Specific strains that can be used to implement the present invention and in particular to obtain MV cDNA for nucleic acid constructs are Schwarz MV strain, Zagreb strain, AIK-C strain and Moraten strain. All of these strains have been described in the prior art and are available, in particular, as commercial vaccines.

According to a specific embodiment of the present invention, the cDNA molecule is placed under the control of a heterologous expression control sequence.

Insertion of cDNA expression controls is advantageous when expression of the cDNA is sought in a cell type that has a cDNA's native control sequences that are incapable of fully transcribing the cDNA.

According to a specific embodiment of the present invention, the heterologous expression control sequence comprises a T7 promoter and a T7 terminator sequence, each of which is located 5' and 3' to the coding sequence of the full-length antigenomic (+) RNA strand of MV and is adjacent to the sequence surrounding the coding sequence.

In a particular embodiment of the invention, the cDNA molecule as defined above is modified, ie comprises additional nucleotide sequences or motifs.

In a preferred embodiment, the cDNA molecule of the present invention further comprises a GGG motif at its 5' end (adjacent to the first nucleotide of the nucleotide sequence encoding the full-length antigenomic (+) RNA strand of the MV validated vaccine strain) followed by a hammerhead ribozyme sequence and a ribozyme sequence at its 3' end (adjacent to the last nucleotide of the nucleotide sequence encoding the full-length antigenomic (+) RNA strand). The hepatitis delta virus ribozyme (δ) is suitable for carrying out the present invention.

The GGG motif, located at the 5' end, adjacent to the first nucleotide of the coding sequence, improves the transcription efficiency of the cDNA coding sequence. For proper assembly of measles virus particles, the cDNA encoding the antigenomic (+) RNA should conform to the six-fold rule. When the GGG motif is added, a ribozyme is also added to the 5' end of the cDNA coding sequence, located 3' to the GGG motif, to enable cleavage of the transcript at the first coding nucleotide of the full-length antigenomic (+) RNA chain of MV.

In a specific embodiment of the present invention, in order to prepare the nucleic acid construct of the present invention, the preparation of a cDNA molecule encoding the full-length antigenomic (+) RNA of the measles virus disclosed in the prior art is achieved by known methods. When the cDNA is inserted into a vector such as a plasmid, a genomic vector is particularly provided.

Specific cDNA molecules suitable for preparing the nucleic acid constructs of the present invention are cDNA molecules obtained using the Schwarz strain of measles virus. Accordingly, the cDNA used in the present invention can be obtained as disclosed in WO2004/000876, or obtained from the plasmid pTM-MVSchw (deposit number 1-2889, deposit date June 12, 2012) deposited by the Pasteur Institute of the CNCM, the sequence of which is disclosed in WO2004/000876 and incorporated herein by reference. Plasmid pTM-MVSchw is obtained from the Bluescript plasmid and comprises a polynucleotide encoding the full-length measles virus (+) RNA chain of the Schwarz strain under the control of the T7 RNA polymerase promoter. It has 18967 nucleotides and the sequence is shown in SEQ ID NO: 1. cDNA molecules from other MV strains (for convenience, also named measles virus cDNA or MV cDNA) can be similarly obtained from nucleic acids purified from viral particles of the attenuated MV described herein, for example.

The nucleic acid constructs of the present invention are suitable for and used to prepare recombinant infectious replicating measles-chikungunya virus (MV-CHIKV), and accordingly, the nucleic acid constructs are used to insert into a transfer genome vector, whereby it contains a cDNA molecule of a measles virus, in particular a Schwarz strain, for producing the MV-CHIKV virus and producing CHIKV structural proteins, in particular CHIKV VLPs. The pTM-MVSchw plasmid is suitable for preparing a transfer vector by inserting CHIKV structural proteins, in particular CHIKV polynucleotides necessary for expression of CHIKV VLPs.

The present invention therefore relates to transfer vectors which, when rescued from helper cells, are used to prepare recombinant MV-CHIKV particles. The transfer vector of the present invention is preferably a plasmid, in particular a plasmid obtained from the Bluescript plasmid.

The present invention also relates to the use of transfer vectors to transform cells suitable for rescuing viral MV-CHIKV particles, in particular to transfect or transduce said cells using a plasmid or a viral vector containing the nucleic acid construct of the invention, respectively, and to select said cells for their ability to express the desired measles virus proteins for the proper replication, transcription and encapsidation of the viral recombinant genome, which corresponds to the nucleic acid construct of the invention in the recombinant infectious replicating MV-CHIKV particles.

The present invention also relates to cells or cell lines transformed with the transfer vectors of the present invention and other polynucleotides providing auxiliary functions and proteins.

Thus, a polynucleotide is located in the cell that encodes proteins that particularly include the N, P and L proteins of the measles virus (i.e., native MV proteins or functional variants thereof that are capable of forming ribonucleoprotein (RNP) complexes), preferably as stably expressed proteins, at least the N and P proteins that play a role in the transcription and replication of the recombinant viral MV-CHIKV particles. The N and P proteins can be expressed in the cell from a plasmid containing their coding sequences, or can be expressed from a DNA molecule inserted into the cell genome. The L protein can be expressed from a different plasmid. It can be expressed transiently. The helper cell can also express an RNA polymerase that is suitable for synthesizing recombinant RNA from the nucleic acid construct of the present invention, possibly as a stably expressed RNA polymerase. The RNA polymerase can be T7 bacteriophage polymerase or its nuclear form (nlsT7).

In one embodiment, the measles virus cDNA clone is from the same measles virus strain as the N protein and/or P protein and/or L protein. In another embodiment, the measles virus cDNA clone is from a different virus strain than the N protein and/or P protein and/or L protein.

The present invention therefore relates to a method for preparing recombinant infectious measles virus particles, comprising:

1) transfer, in particular transfection, of a nucleic acid construct of the invention or a transfer vector containing the nucleic acid construct in a helper cell line, said helper cell line also expressing proteins from MV cDNA under conditions enabling assembly of viral particles, said proteins being necessary for transcription, replication and encapsidation of MV antigenomic (+) RNA sequences; and

2) Recovering recombinant infectious MV-CHIKV virus expressing at least one CHIKV structural protein.

According to a specific embodiment, the method comprises:

1) using a transfer vector to transfect a helper cell line with the nucleic acid construct according to the present invention, wherein the helper cell is capable of expressing helper function to express RNA polymerase and express N, P and L proteins of MV virus;

2) co-culturing the transfected helper cells of step 1) with passage cells suitable for passage of the attenuated MV strain from which the cDNA is derived;

3) Recovering the recombinant infectious MV-CHIKV virus expressing at least one CHIKV structural protein.

According to another embodiment of the present invention, the method for producing recombinant infectious MV-CHIKV virus comprises:

1) recombining cells or cell cultures with the nucleic acid construct of the present invention and a vector comprising a nucleic acid encoding the large protein (L) of measles virus RNA polymerase, which stably produces RNA polymerase, measles virus nucleoprotein (N) and measles virus polymerase cofactor phosphoprotein (P); and

2) Recovering infectious MV-CHIKV virus from the recombinant cell or recombinant cell culture.

According to a specific embodiment of the method, a recombinant MV expressing CHIKV structural proteins, in particular CHIKV VLPs, is produced, wherein the particles express a combination of antigens, such as the CE3E26KE1 antigens of CHIK virus. As an example, a method for rescuing a recombinant MV expressing CHIKV structural proteins, in particular CHIKV VLPs, comprises the following steps:

1) co-transfecting helper cells stably expressing T7 RNA polymerase and measles N and P proteins, in particular HEK293 helper cells, with (i) a transfer vector, in particular a plasmid, comprising a cDNA encoding the full-length antigenomic (+) RNA of measles virus, recombined with at least one polynucleotide encoding a CHIKV structural protein, such as encoding the CHIKV-CE3E26KE antigen, and (ii) a vector, in particular a plasmid encoding the MV L polymerase cDNA;

2) culturing the co-transfected helper cells under conditions capable of producing MV-CHIKV recombinant virus;

3) co-culturing the helper cells of step 2) with cells capable of virus proliferation, such as Vero cells, to propagate the recombinant virus thus produced;

4) Recovering the replicated MV-CHIKV recombinant virus and CHIKV structural proteins, particularly CHIKV virus-like particles, particularly CHIKV-CE3E26KE1 VLPs.

The method is exemplified in Figure 1B along with the constructs and conditions used.

As used herein, "recombinant" refers to the introduction of at least one polynucleotide into a cell, for example in the form of a vector, with or without integration (in whole or in part) into the genome of the cell (eg, as described above).

According to a specific embodiment, recombination can be achieved using a first polynucleotide that is a nucleic acid construct of the present invention. Recombination can also or alternatively include the introduction of a polynucleotide that is a vector encoding the large protein (L) of measles virus RNA polymerase, the definition, properties and expression stability of which have been described herein.

According to the present invention, cells, cell lines, or cell cultures that stably produce RNA polymerase, measles virus nucleoprotein (N), and measles virus polymerase cofactor phosphoprotein (P) are cells, cell lines, or cell cultures defined herein, i.e., recombinant cells, modified by the introduction of one or more polynucleotides described above. In specific embodiments of the present invention, cells, cell lines, or cell cultures that stably produce RNA polymerase, N, and P proteins do not produce measles virus L protein or do not stably produce measles virus L protein, for example, transiently express or produce L protein.

The production of MV-CHIKV virus of the present invention may involve the transfer of transformed cells as described herein. "Transfer" as used herein refers to placing recombinant cells on different types of cells, especially on a monolayer of different types of cells. These latter cells can support the replication and production of infectious MV-CHIKV viruses, i.e., form infectious viruses intracellularly, and possibly release these infectious viruses extracellularly. This transfer causes the recombinant cells of the present invention to be co-cultured with the competent cells defined in the preceding sentence. When the recombinant cells are not effective virus production cultures, i.e., when infectious MV-CHIKV can not be effectively recovered from these recombinant cells, the above-mentioned transfer can be an additional, i.e., optional step. This step is introduced after the recombinant cells of the present invention and the nucleic acid construct of the present invention, and optionally a vector encoding the large protein (L) of measles virus RNA polymerase, are further recombined.

In a specific embodiment of the present invention, the transfer step is required because the recombinant cells that are typically selected for their ability to facilitate recombination are not efficient enough to support and produce recombinant infectious MV-CHIKV viruses. In such an embodiment, the cell or cell line or cell culture of step 1) of the above method is a recombinant cell or cell line or recombinant cell culture according to the present invention.

Cells suitable for preparing the recombinant cells of the present invention are prokaryotic or eukaryotic cells, particularly animal or plant cells, more particularly mammalian cells such as human cells or non-human mammalian cells or avian cells or yeast cells. In a specific embodiment, the cells are isolated from a primary culture or cell line before their genome is recombined. The cells of the present invention may be dividing or non-dividing cells.

According to a preferred embodiment, the helper cells are derived from the human embryonic kidney cell line 293, which has been deposited with the ATCC under accession number CRL- 1573. The specific cell line 293 is the cell line disclosed in WO 2008/078198 and is cited in the subsequent examples.

According to another aspect of the method, the cells suitable for passaging are CEF cells.CEF cells can be obtained from fertilized chicken eggs obtained from EARL Morizeau, 8 rue Moulin, 28190 Dangers, France, or any other fertilized chicken egg producer.

The methods disclosed herein are advantageously used to produce infectious, replicating MV-CHIKV viruses suitable for use as immunogenic compositions.

The present invention therefore relates to immunogenic compositions, the active ingredients of which comprise infectious replicating MV-CHIKV viruses rescued from the nucleic acid constructs of the invention and in particular obtained by the disclosed methods.

As defined herein, the nucleic acid construct of the invention and the MV-CHIKV virus of the invention encode or express at least one CHIKV structural protein.

"Chikungunya virus structural protein" refers to a "protein" as defined herein, whose sequence is identical to the corresponding one in the CHIKV strain, including polypeptides, which are naturally occurring mature CHIKV structural proteins or CHIKV structural protein precursors, or fragments thereof or mutants thereof as defined herein, in particular fragments or mutants having at least 50%, at least 80%, particularly advantageously at least 90% or preferably at least 95% amino acid sequence identity with naturally occurring Chikungunya virus capsid or envelope proteins. Amino acid sequence identity can be determined by one skilled in the art using manual alignment or by alignment using various available alignment programs (e.g., BLASTP - http://blast.ncbi.nlm.nih.gov/ ). Fragments or mutants of the CHIKV structural proteins of the present invention can be defined relative to the specific amino acid sequences exemplified herein.

According to the present invention, the polynucleotide encoding at least one CHIKV structural protein encodes one or more of the following proteins: a structural glycoprotein, a structural polypeptide, and a capsid protein.

In a specific embodiment, the glycoprotein comprises envelope glycoproteins E1, E2, and E3 envelope glycoproteins. The polypeptide comprises a 6K polypeptide and a capsid protein. In the following paragraphs, the terms protein or glycoprotein are used interchangeably to designate E1, E2, or E3 glycoproteins or a combination thereof.

According to a specific embodiment of the present invention, the polynucleotide encoding the CHIKV structural protein is selected from the following group:

- a polynucleotide encoding one of the E1, E2, E3, 6K or C proteins;

- a fusion polynucleotide encoding several proteins selected from the group consisting of E1, E2, E3, 6K and C proteins;

- a polynucleotide encoding the E3-E2-6K-E polyprotein;

- a polynucleotide encoding the C-E3-E2-6K-E1 polyprotein and, in particular, an open reading frame (ORF) from the CHIKV genome or the corresponding cDNA encoding said polyprotein;

- Any of these polynucleotides, which have been modified to encode a mutant form of one or more of these proteins, in particular a mutant form of the E2 protein.

In this regard, specific polynucleotides encode a soluble form of the E2 protein (sE2) or encode the extracellular domain of the E2 protein or a soluble form thereof (sE2Δstem). In specific embodiments, the polynucleotide encodes one of the following polypeptides: E3-sE2-6K-E1, E3-sE2Δstem-6K-E1, C-E3-sE2-6K-E1, and C-E3-sE2Δstem-6K-E1.

As used herein, the term "ectodomain" refers to the domain of glycoprotein E2 that extends out of the viral particle and is responsible for attachment and entry into cells during CHIKV virion infection.

According to a specific embodiment, the polynucleotide encodes a single epitope of a CHIKV structural protein or encodes multiple epitopes resulting from the expression of repeated epitopes (having the same or similar sequence) or multiple different epitopes of one or more CHIKV structural proteins.

For example, a polytope is formed by fusion of repeated polynucleotides encoding a single E2EP3 epitope located at the N-terminus of the E2 glycoprotein, adjacent to the furin E2/E3 cleavage site. The amino acid sequence of the E2EP3 epitope is disclosed in Kam Y.W et al. (EMBO Mol Med 4, 330-343), i.e., SEQ ID NO: 33.

According to a specific embodiment of the present invention, several polynucleotides (wherein each polynucleotide encodes at least one CHIKV structural protein) are combined or fused to form a polynucleotide encoding several CHIKV structural proteins. These polynucleotides may be different from each other, encoding proteins of different strains of CHIKV. Thus, the polynucleotides encode multiple epitopes described herein, such as multiple epitopes of a single E2EP3 epitope.

A polynucleotide encoding at least one CHIKV structural protein is cloned into a cDNA molecule (encoding the full-length infectious antigenomic (+) RNA strand of measles virus), possibly at different sites, to generate the nucleic acid construct of the present invention.

According to one aspect of the present invention, the polynucleotide encoding at least one Chikungunya virus (CHIKV) structural protein is derived from the genome of an isolated and purified wild-type CHIKV strain. The wild-type CHIKV strain can be, for example, the Ross strain (GenBank: AF490259.3), or the S27 strain (GenBank: AF339485.1), both of which were isolated from patients during the 1952 Tanzania outbreak, or the strain named Ae.furcifer (GenBank: AY726732.1), which was isolated during the 1983 Senegal outbreak.

According to another aspect of the present invention, the polynucleotide encoding at least one CHIKV structural protein is derived from the following purified and isolated wild CHIKV strains: 05.61, 05.115, 05.209, 06.21, 06.27 and 06.49, which are described in WO2007/105111. The almost complete genome sequences of these CHIKV isolates representing different geographical origins, time points and clinical forms of the Indian Ocean outbreak of chikungunya virus have been sequenced and disclosed in WO2007/105111. 11,601 nucleotides were determined, corresponding to positions 52 (5'NTR) to 11,667 (3'NTR, the end of the 3 repeat sequence segment) in the nucleotide sequence of the 1952 Tanzanian isolate S27 (full length 11,826 nt) used as a reference.

The genomic sequences of isolates 05.61, 05.115, 05.209, 06.21, 06.27, and 06.49, as described in WO2007/105111, and the polynucleotides according to the invention that may be derived therefrom in specific embodiments of the invention, are organized as follows. The coding sequence consists of two large open reading frames (ORFs) of 7,422 nt and 3,744 nt, encoding the nonstructural polyprotein (2,474 amino acids) and the structural polyprotein (1,248 amino acids), respectively. The nonstructural polyprotein is the precursor of proteins nsP1 (535 aa), nsP2 (798 aa), nsP3 (530 aa), and nsP4 (611 aa), while the structural polyprotein is the precursor of proteins C (261 aa), p62 (487 aa, E3 (64 aa), and E2 (423 aa), 6K (61 aa), and E1 (439 aa). The cleavage sites in the nonstructural and structural polyproteins that are characteristic of the alphavirus family are conserved. The glycosylation sites in E3, E2, and E1 are also conserved. The published genomic sequences are incorporated herein by reference.

According to one embodiment, the polynucleotide encoding the CHIKV structural protein is derived from the genome of the wild CHIKV strains designated as 06.115, 06.21, 06.27 and 06.49 as described above.

The term "derived" in the definition of a polynucleotide only indicates that the sequence of the polynucleotide may be identical to or different from the corresponding sequence in a CHIKV strain to encode a CHIKV structural protein that meets the definition of a "protein" of the present invention. Accordingly, the term does not limit the mode of production of the polynucleotide.

The polynucleotides and nucleic acid constructs of the present invention may be prepared according to any method known in the art and may in particular be cloned, obtained by polymerisation, in particular using PCR methods, or may be synthesized.

The nucleic acid construct of the present invention is further defined as comprising one of the following polynucleotides encoding at least one CHIKV structural protein.

According to a specific embodiment, the polynucleotide encoding one or more CHIKV structural proteins encodes a soluble form of glycoprotein E2. As an example, the polynucleotide comprises a coding domain having a sequence of SEQ ID NO: 2, 4, 6, or 8.

In a specific embodiment, the polynucleotide encoding one or more CHIKV structural proteins encodes the E2 extracellular domain. As an example, the polynucleotide comprises a coding domain having a sequence of SEQ ID NO: 10, 12, or 14.

In another embodiment of the present invention, the polynucleotide encoding the soluble form of glycoprotein E2 encodes a polypeptide having an amino acid sequence selected from SEQ ID NO: 3, 5, 7, 9.

In another embodiment of the present invention, the polynucleotide encoding the extracellular domain of glycoprotein E2 encodes a polypeptide having an amino acid sequence selected from SEQ ID NO: 11, 13, and 15.

According to a specific embodiment of the present invention, the polynucleotide encoding the CHIKV structural protein comprises one of the nucleotide sequences defined above, which encodes a soluble form of the E2 glycoprotein or the extracellular domain of the protein, and further comprises a polynucleotide encoding one of the E3, E1, 6K or C proteins, or a polynucleotide encoding any combination thereof.

Accordingly, the specific polynucleotides of the invention encoding the structural polyprotein E2-6K-E1 of the France/2010 strain isolated from Fréjus are selected from the polynucleotides comprising the coding domains having the sequences of SEQ ID NO: 16 (GenBank: CCA61130.1) and 20 (GenBank: CCA61131.1).

Preferred polynucleotides of the present invention encode the structural protein C-E3-E2-6K-E1 from a CHIKV strain. In a specific embodiment, the polynucleotide encodes the polyprotein C-E3-E2-6K-E1 of one of the strains designated S27 or 06.49 and has the sequence SEQ ID NO: 20 and 27, respectively.

In another embodiment of the present invention, the polynucleotide used encodes the E2EP3 epitope or a polytope formed by or comprising a repeat of the epitope. The polynucleotide particularly has the nucleotide sequence disclosed in SEQ ID No: 32.

According to a preferred embodiment, the present invention also relates to the modification and optimization of polynucleotides to allow efficient expression of Chikungunya virus proteins on the surface of MV-CHIKV chimeric infectious particles in the host.

According to this embodiment, the optimization of the polynucleotide sequence can be performed to avoid the cis-active domains of the nucleic acid molecule: internal TATA boxes, chi sites and ribosome entry sites; AT-rich or GC-rich sequence segments; ARE, INS, CRS sequence elements; repetitive sequences and RNA secondary structures; hidden splicing donor and acceptor sites, branch points.

The optimized polynucleotides can also be codon optimized for expression in specific cell types, in particular they can be modified for Maccaca codon usage or human codon usage. This optimization allows for increased efficiency of production of chimeric infectious particles in cells without affecting the expressed protein.

In particular, optimization of a polynucleotide encoding a CHIKV protein can be performed by modifying the wobble position in a codon without affecting the identity of the amino acid residue translated from the codon relative to the original amino acid residue.

Optimization was also performed to avoid editing-like sequences from measles virus. Editing of measles virus transcripts is a process that occurs specifically in transcripts encoded by the measles virus P gene. This editing, by inserting an extra G residue at a specific site in the P transcript, produces a new protein that is truncated compared to the P protein. Adding only a single G residue results in the expression of a V protein that contains a unique carboxyl terminus (Cattaneo R et al., Cell. 1989 Mar 10; 56(5): 759-64).

In the polynucleotide according to this embodiment of the present invention, the following editing-like sequences from measles virus can be mutated: AAAGGG, AAAAGG, GGGAAA, GGGGAA, and their complementary sequences: TTCCCC, TTTCCC, CCTTTT, CCCCTT. For example, AAAGGG can be mutated to AAAGGC, AAAAGG can be mutated to AGAAGG or TAAAGG or GAAAGG, and GGGAAA can be mutated to GCGAA.

One embodiment of the modified and optimized polynucleotide according to the present invention is defined by SEQ ID NO: 29. This polynucleotide encodes a soluble form of the envelope protein E2 without the stem region.

One embodiment of a modified and optimized polynucleotide encoding all structural proteins C-E3-E2-6K-E1 is defined by SEQ ID NO:31.

The optimized polynucleotides according to this embodiment of the invention defined by SEQ ID NOs: 29 and 31 contain mutations in the sequence of the BsiWI and BssHII sites but not at the ends of the sequence in order to preserve the sites for cloning purposes.

Thus, according to this specific embodiment, the present invention provides a nucleic acid construct comprising a polynucleotide that increases the efficiency of chimeric MV-CHIKV infectious particle production.

Other optimized polynucleotides that contain sequences encoding CHIKV structural proteins and are also suitable for use in the nucleic acid constructs of the present invention are optimized fusion polynucleotides that encode one of the following combinations of structural proteins: E3-E2-6K-E1, E3-sE2-6K-E1, E3-sE2Δstem-6K-E1, C-E3-E2-6K-E1, C-E3-sE2-6K-E1 and C-E3-sE2Δstem-6K-E1.

The present invention also relates to a nucleic acid construct, wherein the polynucleotide encoding at least one CHIKV structural protein encodes one of the following polypeptides.

Examples of amino acid sequences of CHIK virus structural proteins and related soluble forms of glycoprotein E2 from strains designated 05.115, 06.21, 06.27 and 06.49 according to preferred embodiments of the present invention are defined by SEQ ID NOs: 3, 5, 7 and 9.

Examples of CHIK virus structural proteins according to preferred embodiments of the present invention and amino acid sequences related to the extracellular domain of glycoprotein E2 from strains designated 05.115, 06.21, 06.27 and 06.49 are defined by SEQ ID NOs: 11, 13 and 15.

In a specific embodiment, the present invention relates to a fusion protein composed of the structural proteins E2-6K-E1 of the France/2010 strain isolated from Fréjus. These proteins are derived from the expression of polynucleotides having the sequences SEQ ID NOs: 16 (GenBank: CCA61130.1) and 18 (GenBank: CCA61131.1), respectively. Their sequences are defined by SEQ ID NOs: 17 and 19, respectively.

In a particularly preferred embodiment, the present invention relates to a fusion protein composed of all CHIKV structural proteins, which are derived from the expression of polynucleotides encoding all structural proteins C-E3-E2-6K-E1 and are defined by SEQ ID NOs: 21, 22, 23, 24, 25, 26, and 28.

The obtained structural protein C-E3-E2-6K-E1 can self-assemble into CHIKV virus-like particles (VLPs) in CHIKV-MV particles.

As used herein, the term "virus-like particle (VLP)" refers to a structure that has at least one property similar to a virus but has not been shown to be infectious. According to the present invention, virus-like particles do not carry genetic information encoding virus-like particle proteins. Generally, virus-like particles lack a viral genome and are therefore non-infectious and non-replicating. According to the present invention, virus-like particles can be produced in large quantities and expressed with recombinant CHIKV-MV particles.

In a specific embodiment, the present invention relates to a soluble form of glycoprotein E2 from a CHIKV strain designated 06.49, without a stem region, which is expressed from a modified and optimized polynucleotide defined by SEQ ID NO: 29, whose sequence is defined by SEQ ID NO: 30.

According to another aspect, the present invention relates to recombinant CHIKV-Measles virus particles expressing Chikungunya virus structural proteins as defined herein, with particular reference to their nucleic acid and polypeptide sequences. The recombinant CHIKV-MV virus advantageously expresses the CHIKV structural proteins as VLPs.

The present invention also relates to virus-like particles of CHIKV structural proteins, in particular C-E3-E2-6K-E1 VLPs, in combination with CHIKV-MV infectious replicating virus particles in a composition.

According to a preferred embodiment of the present invention, the recombinant measles virus vector is designed in such a way that the production process involves cells, whereby viral particles from a measles virus strain suitable for vaccination are produced in helper cells transfected or transformed with said vector, resulting in the production of recombinant measles-chikungunya infectious and replicating viruses, and the production of CHIKV-VLPs for use in immunogenic compositions, preferably protective or even vaccine compositions.

Advantageously, the genome of the recombinant measles-chilam virus of the present invention is replication-competent. "Replication-competent" means that the nucleic acid is transcribed and expressed when transduced into a helper cell line expressing the N, P, and L proteins of MV, thereby producing new virus particles.

The replication of the recombinant virus of the present invention obtained by using MV cDNA for preparing the MV-CHIKV recombinant genome can also be achieved in a host, particularly a human host to which the recombinant MV-CHIKV is administered.

The present invention also relates to compositions or combinations of active ingredients comprising a recombinant measles-chikungunya virus in combination with VLPs of CHIKV structural proteins, such as VLPs of the CE3E26KE1 protein. These compositions or combinations induce an immune response, particularly a protective immune response, against chikungunya virus, and in particular induce the production of antibodies against chikungunya virus structural proteins and/or induce a cellular immune response against CHIKV infection. These compositions may accordingly comprise a suitable carrier for administration to a host, particularly a human host, such as a pharmaceutically acceptable carrier, and may further comprise, but not necessarily, an adjuvant to enhance the immune response in the host. The inventors have in fact demonstrated that administration of the active ingredients of the present invention can elicit an immune response without the need for adjuvanting.

The present invention particularly relates to compositions for administration to children.

The present invention also relates to immunogenic compositions, particularly vaccine compositions, and in particular to vaccine compositions for administration to children. The compositions or vaccines are useful in preventing CHIKV infection in prophylactic therapy. The vaccine compositions advantageously have an active ingredient comprising recombinant measles-chilovirus infectious replicating virions rescued from a vector, as defined herein, in combination with VLPs of CHIKV structural proteins, such as VLPs of the CE3E26KE1 protein.

In the context of the present invention, the term "combined" or "associated" refers to the simultaneous presence of MV-CHIKV recombinant virus particles and CHIKV structural proteins in a unique composition, in particular as VLPs, usually as physically separate entities.

The present invention also relates to recombinant MV-CHIKV infectious replicating virus particles in combination with CHIKV viral structural proteins, in particular CHIKV virus-like particles expressing CHIKV structural proteins, in particular CHIKV-CE3E26KE1VLPs, or a composition of the present invention, for treating or preventing Chikungunya virus infection in a subject, in particular a human.

The present invention also relates to MV-CHIKV infectious, replicating viruses and combined CHIKV viral structural proteins, in particular combined CHIKV structural protein VLPs, such as CE3E26KE1 protein VLPs, for use in an administration regimen, and for use according to a dosing regimen, which elicits an immune response against CHIKV viral infection or induced disease, advantageously a protective immune response, particularly in a human host.

Administration regimens and dosing schedules require unique administration of selected doses of MV-CHIKV infectious, replicating virus and associated CHIKV viral structural proteins, particularly associated CHIKV structural protein VLPs, such as CE3E26KE1 protein VLPs.

Alternatively, multiple doses may be administered in an immunization-boosting regimen. Immunization and boosting may be achieved using the same active ingredient, which consists of MV-CHIKV infectious, replicating virus and a combination of CHIKV viral structural proteins, particularly a combination of CHIKV structural protein VLPs, such as CE3E26KE1 protein VLPs.

Alternatively, immunization and boosting administration may be achieved using different active ingredients comprising MV-CHIKV infectious, replicating virus and associated CHIKV structural proteins, particularly associated CHIKV structural protein VLPs, such as CE3E26KE1 protein VLPs, in at least one administration step, and other active immunogens of CHIKV, such as CHIKV proteins or VLPs expressing the C-E3-E2-6K-E1 polyprotein, in other administration steps.

The present invention also relates to a combination of different active ingredients, comprising one of the following active ingredients: an MV-CHIKV infectious and replicating virus and a CHIKV structural protein, in particular a CHIKV structural protein VLP, such as a CE3E26KE1 protein VLP. This combination of active ingredients is advantageously used for immunization of a host, in particular a human host.

The present inventors have demonstrated that administration of MV-CHIKV infectious, replicating viruses and, in combination, CHIKV structural protein VLPs elicits an immune response, and in particular, elicits antibodies that cross-react with different CHIKV strains, at least strains of the ECSA genotype. Accordingly, administration of the active ingredient of the present invention (when prepared using coding sequences of a specific CHIKV strain) has been demonstrated to elicit an immune response against a group of CHIKV strains, in particular the group of strains of the ECSA genotype, in particular the group of strains comprising CHIKV India, CHIKV Congo, CHIKV Thailand, and CHIKV Reunion.

Taking into account the available knowledge about vaccines suitable for other pathogens (such as HBV or HPV) (which involve the administration of virus-like particles (VLPs)) and also suitable for the dosage of known human MV vaccines, the inventors determined that the recovery of CHIKV-VLPs with recombinant MV-CHIKV viruses enables the administration of effectively low doses of active ingredients. In fact, considering that each MV-CHIKV replicating particle of the recombinant MV-CHIKV virus is capable of producing approximately 10 ⁴ CHIKV-VLPs, and considering that the currently known human MV vaccine dose is 10 ³ to 10 ⁴ pfu, a suitable dose of the recombinant MV-CHIKV virus to be administered is 0.1 to 10 ng, in particular 0.2 to 6 ng, and possibly as low as 0.2 to 2 ng. For comparison, the VLP doses administered in HBV or HPV vaccines are in the range of 10 μg, which means that a dose of the recombinant MV-CHIKV vaccine can contain about 2000 or as many as 5000 to 10000 times less VLPs.

According to a particular embodiment of the invention, the immunogenic composition of the vaccine defined herein may also be used to prevent measles virus infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 : Schematic diagram of the MV-CHIKV construct showing Chikungunya virus structural proteins and ORFs of the MV genome backbone - including the CHIK virus antigens expressed by measles virus.

Figure 1B: Rescue of recombinant MV expressing CHIKV VLPs

FIG2 : Immunofluorescence detection of E2 antigen in Vero cells infected with recombinant MV-CHIKV at an MOI of 0.1 for 24 h.

E2 was detected using anti-E2 monoclonal antibody 3E4 (1/100 dilution), and the secondary antibody was used at a dilution of 1/5000.

Figure 3: MV-CHIKV vector expresses E2 and capsid proteins.

Western blot analysis of cell lysates (cells) and supernatants (SN) from Vero cells infected with MV-sE2Δstem and MV-CE3E26KE1 for 24 h. E2 was detected using 3E4 mAb, and protein C was detected using an anti-capsid mAb (from P. Desprès, 1/100 dilution). Secondary antibodies were used at a 1/5000 dilution.

Figure 4: Electron microscopy analysis of CHIKV VLPs secreted from the supernatant of Vero cells infected with MV-CE3E26KE1 recombinant virus at an MOI of 0.1. Scale bars are 200 nm (left) and 100 nm (right). The red arrows represent the specific arrangement of spikes on the particle surface and the icosahedral symmetry of the capsid protein within the particle.

Figure 5: Sequence of truncated sE2 (156 aa, 19 kDa) expressed by MV-sE2 recombinant virus.

Figure 6: Growth kinetics of recombinant MV-sE2Δstem and MV-CE3E26KE1 compared to standard MV on Vero cells (MOI 0.01). Cell-associated virus titers are expressed as TCID50.

Figure 7: Immunization and challenge schedule of Example 2.

FIG8 : Survival curves of mice lethally challenged with 100 PFU CHIKV-06-49 after two immunizations with MV-CE3E26KE1 recombinant virus.

Figure 9: Immunization and challenge schedule of Example 3.

Figure 10: Survival curves of mice lethally challenged with 100 PFU CHIKV-06-49 after a single immunization with MV-CE3E26KE1 recombinant virus.

Figure 11: Immunization and challenge schedule of Example 4.

FIG12 : Survival curves of mice immunized with different doses of MV-CE3E26KE1 recombinant virus and lethally challenged with 100 PFU CHIKV-06-49.

Figure 13: Passively transferred immune serum and challenge schedule of Example 5.

FIG14 : Survival curves of mice lethally challenged with 100 PFU CHIKV-06-49 after passive transfer of MV-CE3E26KE1 immune serum.

FIG15 : Cell-mediated immune responses elicited in splenocytes of CD46-IFNAR mice immunized with a single injection of 10 6 TCID 50 of MV-CHIKV.

Figure 16: Immunization and challenge schedule of Example 6.

Figure 17: Survival curves of pre-immunized mice lethally challenged with 100 PFU of CHIKV-06-49 after immunization with MV-CE3E26KE1.

Figure 18: PRNT assay against CHIK was performed on day 90 before the first immunization (before boost) and day 111 (day 21 after boost).

Example

Construction and characterization of a recombinant measles virus vector expressing chikungunya virus protein

The present inventors designed three chikungunya virus antigens based on peptide sequences of natural proteins of chikungunya virus strain 06-49. The natural proteins that can produce these peptide sequences are five structural proteins, which are composed of the capsid (C) envelope and the accessory proteins E1, E2, E3 and 6K.

The first construct involves the expression of a soluble form of the envelope protein E2 (sE2), the second construct involves the expression of sE2 without the stem region (sE2Δstem), and the third construct involves the expression of all viral structural proteins (C-E3-E2-6K-E1) (Figure 1). The experimental protocol described herein is based on this latter construct.

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

pTM-MVSchw-CE3E26KE1 was constructed. The pTM-MVSchw plasmid 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). It contains infectious MV cDNA corresponding to the antigenome of the Schwarz MV vaccine strain. The cDNA encoding the structural CE3E26KE1 CHIKV antigen was prepared by chemical synthesis (GenScript, USA). It contains the sequence of the viral structural proteins C-E3-E2-6K-E1 from CHIKV strain 06-49 (WO2007/105111). The complete sequence complies with the "6-fold rule", which stipulates that the number of nucleotides entering the MV genome must be a multiple of 6 and contain a BsiWI restriction enzyme site at the 5' end and a BssHII restriction enzyme site at the 3' end. The sequence has been optimized for expression of measles virus in mammalian cells. The cDNA is inserted into BsiWI/BssHII-digested pTM-MVSchw-ATU2, which contains an additional transcription unit (ATU) between the phosphoprotein (P) and 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 is named pTM-MVSchw-CE3E26KE1.

Rescue of recombinant MV-CE3E26KE1. Recombinant Schwarz MV-CHIKV was rescued from plasmid pTM-MVSchw-CE3E26KE1 using the rescue system described previously (Radecke, F., et al., Rescue of measlesviruses from cloned DNA. J Virol, 1995. 14(23): p. 5773-84; WO 2008/078198). Viral titers were determined by endpoint limiting dilution assay on Vero cells, and TCID50 was calculated using the RT-PCR method.

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 Cy3-conjugated IgG antibody (Jackson Immunoresearch Laboratories) was used as a secondary antibody.

Western blot assay. Protein lysates from Vero cells infected with the recombinant virus were separated by SDS-PAGE gel electrophoresis and transferred to a cellulose membrane (Amersham Pharmacia Biotech). The blot was probed with mouse anti-E2 monoclonal antibody 3E4 and an anti-capsid antibody. Goat anti-mouse immunoglobulin G (IgG)-horseradish peroxidase (HRP) conjugate (Amersham) was used as a secondary antibody. Peroxidase activity was visualized using an enhanced chemiluminescence detection kit (Pierce).

VLP production was analyzed by electron microscopy. Vero cells (3 x T-150 flasks) were infected with MV–CHIKV recombinant viruses at an MOI of 1. Supernatants collected 36 h postinfection were clarified by centrifugation at 3000 rpm for 30 min, overlaid on a 20% sucrose cushion in PBS, and centrifuged at 41,000 rpm for 2 h in an SW41 rotor. The pellet was resuspended in PBS containing 1% BSA and analyzed by electron microscopy. Carbon-coated copper grids were negatively stained with 2% uranyl acetate and glow-discharged before use. Samples were observed using a Jeol JEM1200 (Tokyo, Japan) transmission electron microscope at 80 kV. Images were recorded using an Eloise Keenview camera and Analysis Pro software version 3.1 (Eloise SARL, Roissy, France).

Mouse experiments. CD46-IFNAR susceptible to MV infection was prepared as previously described (Combredet, C., et al., Amolecularly 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 in the Pasteur Institute animal facility under specific pathogen-free conditions. For immunization, 6-week-old CD46-IFNAR mice were inoculated intraperitoneally (ip) with 10 ⁵ TCID50 recombinant MV-CE3E26KE1 or MV. For protection assays, immunized mice were inoculated intraperitoneally with 100 pfu of CHIKV 06-49 strain, and mortality was followed for 2 weeks. All experiments were approved and performed in accordance with the guidelines of the Pasteur Institute's Office of Experimental Animal Care. For passive transfer studies, CD46-IFNAR mice were inoculated intraperitoneally with 20 μl of pooled serum from six mice immunized with ¹⁰ TCID50 of MV-CE3E26KE1. Control mice received 20 μl of pooled serum from mice immunized with ¹⁰ TCID50 of empty MV Schw or 20 μl of anti-CHIKV HMAF. Sera were diluted in a total volume of 100 μl of PBS 24 hours before passive transfer, 16 hours before challenge with 100 pfu of CHIKV 06-49 strain, and then 12 hours after challenge to simulate the presence of antibodies in infected animals. Mice were analyzed for mortality for 2 weeks to determine protection.

Analysis of humoral immune response. To evaluate specific antibody responses, mice were bled by the retroorbital route at different times after immunization. Serum was 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 a secondary antibody. Anti-CHIKV antibodies were detected using a specific ELISA. Briefly, 96-well plates were coated with recombinant CHIKV-E2 protein produced in Escherichia coli. HRP-conjugated anti-mouse immunoglobulin was used as a secondary antibody. The endpoint titer of the mixed serum was calculated as the reciprocal of the final dilution that produced twice the absorbance of the serum from the MV-vaccinated mouse as a negative control. Anti-CHIKV neutralizing antibodies were measured using the plaque reduction neutralization test (PRNT). Vero cells were seeded in 12-well plates for 24 h. Serum samples were serially diluted in DMEM Glutamax/2% FCS. 100 μl of the dilution was incubated with an equal volume of CHIKV containing 100 pfu of strain 06-49 at 37°C with gentle agitation for 2 hours. Residual infectivity was then determined on Vero cell monolayers overlaid with DMEM GlutaMAX ^™ /2% FCS containing a final volume of 0.8% (weight/volume) carboxymethylcellulose. After 3 days of incubation, cells were fixed and stained with crystal violet, and plaque counts were determined. Endpoint neutralization titers were calculated as the highest serum dilution tested that reduced the number of plaques by at least 50% (PRNT50).

Analysis of cell-mediated immune responses. Six-week-old CD46+/-IFNα/βR-/- mice were intraperitoneally inoculated with ¹⁰⁶ TCID50 of MV-CHIKV recombinant virus. Control mice were immunized with ¹⁰⁶ TCID50 of empty MV vector. Mice were euthanized 7 days after infection, and spleens were harvested. Splenocytes from immunized mice were incubated in RPMI, 10% FCS, and 10 IU of recombinant human interleukin-2 (rh-IL-2; Boehringer Mannheim). Their ability to secrete IFN-γ upon stimulation was assessed by enzyme-linked immunoblot (ELISPOT) assay. Cells were stimulated for 18 h with concanavalin A (5 μg/ml; Sigma) as a positive control and RPMI–IL-2 (10 U/ml) as a negative control, CHIKV (MOI 1), or MV (MOI 1). Multiscreen-HA 96-well plates were coated with 5 μg of anti-mouse IFN-γ/ml (R4-6A2; Pharmingen) in PBS overnight at 4°C, washed, and then incubated with 100 μl of RPMI and 10% FCS at 37°C for 1 hour. The medium was replaced with 100 μl of cell suspension (5×10 ⁵ splenocytes per well, triplicate) and 100 μl of stimulator. After 2 hours at 37°C, heated FCS (10%) was added and the plates were incubated at 37°C for 18 hours. After washing, biotinylated anti-mouse IFN-γ antibody (XMG1.2; Pharmingen) was added and the plates were incubated at room temperature for 2 hours. Streptavidin-alkaline phosphatase conjugate (Roche) was used as a second step. Spots were formed using BCIP/NBT (Promega) and counted (ELISpot Reader; Bio-Sys).

Recombinant MV vectors express CHIKV virus-like particles. CHIKV VLPs have been shown to induce 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 from this ability, the present inventors designed a recombinant MV vector capable of inducing CHIKV VLP secretion. For this purpose, the cDNA encoding the C-E3-E2-6K-E1 structural proteins required for CHIKV VLP production was chemically synthesized (Genscript) and optimized for measles virus expression in mammalian cells, and then the additional transcription unit (ATU) of the Schwarz MV vaccine infectious cDNA was introduced (Figure 1). MV-CHIKV was obtained by transfecting this plasmid into HEK-293 helper cells and propagating it on Vero cells. The virus stock was grown on Vero cells and the titer was determined.

Immunofluorescence, western blotting, and electron microscopy confirmed that high levels of CHIKV VLPs were secreted into the culture medium of infected cells.

This strategy provides a live recombinant MV vaccine virus that secretes CHIKV VLPs during each round of replication. Assembly of native alphavirus particles was not expected to occur, and these VLPs did not interfere with the simultaneous replication of paramyxoviruses. This was demonstrated for the first time here. Since the MV vaccine is industrially prepared from crude viral extracts, batches of recombinant MV-CHIKV contain live MV virus and non-replicating CHIKV VLPs. This strategy allows the use of the favorable immunogenic properties of the multimeric antigens displayed on the VLPs without the need for harsh and expensive purification and concentration processes. Moreover, since the VLPs benefit from the favorable immunogenic characteristics of live vaccines, such as a balanced Th1 response and long-term memory, no adjuvant is required.

Expression of CHIKV E2 and capsid antigens was confirmed in infected Vero cells by immunofluorescence using an antibody specific for the CHIKV E2 protein (mAb 3E4) ( Figure 2 ). To search for the presence of secreted VLPs, culture medium from infected cells was clarified by low-speed centrifugation, then plated on a 20% sucrose cushion and concentrated by centrifugation at 41,000 rpm in an SW41 rotor for 2 h. The precipitate was dissolved in PBS containing 1% BSA. Proteins extracted from the cell lysate and concentrated culture medium were separated by SDS-PAGE gel electrophoresis and transferred to a cellulose membrane. The blot was labeled with the 3E4 mouse mAb for E2 and an anti-capsid mAb, produced by a hybridoma deposited on September 6, 2007, at the CNCM (Collection Nationale de Cultures de Microorganismes, Paris, France) under the name of the Institut Pasteur and designated I-3824. The E2 protein of the correct size (46KDa) was found in both cell lysates and concentrated infected cell supernatants, indicating that the MV-CHIKV virus induces the secretion of high-density particles comprising the E2 protein. Capsid protein was also found in the high-density particles, confirming the formation of CHIKV-VLP. The presence of both C and E2 proteins in the concentrated supernatant of infected cells indicates the formation of CHIKV VLP. In order to observe its physical presence, the inventors analyzed the concentrated precipitation from the supernatant of MV-CHIKV infected cells by electron microscopy. Images showed the presence of a high content of particles with a size and phenotype similar to those described following infection with wild-type CHIKV (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 Sindbisvirus. J Virol, 2002. 76(22): p. 11645-58) (Figure 4). The observed particles exhibited an outer diameter of 65 nm and a core diameter of 40 nm. Surface organization revealed the presence of spikes on the VLP surface, similar to the arrangement of 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 finding confirmed that infection of Vero cells with the recombinant MV-CHIKV virus resulted in the secretion of high levels of self-assembled CHIK VLPs.

The E2 protein was also expressed and secreted at the correct size (46 kDa) by the measles virus-sE2Δstem recombinant virus and the measles virus-CE3E26KE1 recombinant virus.

Unfortunately, analysis of MV-sE2-infected cells again confirmed the expression of a truncated form of the E2 protein. The inventors sequenced the E2 mRNA produced by the MV-sE2 virus after RT-PCR amplification of infected cells. The analysis confirmed the presence of a mutation that generates a STOP codon, thereby leading to truncation (Figure 5).

The inventors then compared the replication rates of measles virus-sE2, measles virus-sE2Δstem, and measles virus-CE3E26KE1 recombinant viruses on Vero cells relative to those produced from a standard measles virus stock using a low MOI (0.01) ( Figure 6 ). MV-sE2 growth was similar to that of the control MV. Growth of measles virus-sE2Δstem and measles virus-CE3E26KE1 recombinant viruses was slightly delayed, but their final titers were in the same range as those of empty measles virus.

Immunogenicity of MV-sE2 and MV-CE3E26KE1 and protection in CD46-IFNAR mice

CD46-IFNAR, which is susceptible to MV infection, is used to evaluate the immunogenicity and protective efficacy of recombinant MV-CHIKV virus. These mice express the human CD46 gene, which has human-like tissue specificity and lacks type I interferon receptors. Mice were raised in the Pasteur Institute animal facility under specific pathogen-free conditions, and all experiments were approved and carried out in accordance with the guidelines of the Pasteur Institute's Laboratory Animal Care Office. 6-week-old CD46-IFNAR mice were inoculated intraperitoneally (ip) with 10 ³ to 10 ⁵ TCID50 doses of MV-CHIKV recombinant virus and boosted with the same dose of recombinant virus after 1 month. Control mice were immunized with the same dose of empty MVSchw vector. For antibody determination, blood samples were collected by orbital route 1 month after the initial inoculation and then 2 or 4 weeks after the boost.

Previous studies have demonstrated that IFNAR mice are susceptible to lethal Chikungunya virus infection, display pathological manifestations of infection, and provide a model for evaluating immune mechanisms of protection (Couderc et al; 2008).

CD46-IFNAR mice, susceptible to measles virus infection, were used to evaluate the immunogenicity and protective efficacy of recombinant measles-chikungunya virus. These mice express the human CD46 gene, have human-like tissue specificity, and lack type I interferon receptors. Mice were maintained under specific pathogen-free conditions at the Institut Pasteur's animal facility. All experiments were approved and performed in accordance with the guidelines of the Pasteur Office of Graduate Animal Care.

Experiment 1: Analysis of the immunogenicity and protective efficacy of measles virus-CE3E26KE1 recombinant virus in CD46-IFNAR mice

Six-week-old CD46-IFNAR mice susceptible to measles virus infection were inoculated intraperitoneally with ^2.10 _TCID₅⁰ of measles virus–Chikungunya virus recombinant virus and boosted with the same dose of recombinant virus one month later. Control mice were immunized with the same dose of empty measles virus Schwarz vector (MV Schw). For antibody determination, blood samples were collected retro-orbitally one month after the primary vaccination and then two weeks after the boost. Mice were then challenged with 100 pfu of Chikungunya virus strain 06-49 via intraperitoneal injection to assess protection (immunization and challenge schedule are shown in Figure 7).

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

A single injection of the measles virus-CE3E26KE1 recombinant virus induced high antibody titers, which were strongly boosted by a second injection (Table 1-Figure 8). After two immunizations, high neutralizing titers (PRNT50 = 450-4050, PRNT90 = 50-450) were induced. All animals immunized with ¹⁰⁴ or ¹⁰⁵ TCID50 were protected from a lethal CHIKV challenge with 100 PFU of CHIKV-06-49, while immunization with a lower dose ( ¹⁰³ TCID50) protected 83% of the animals.

Table 1. Antibody responses of CD46-IFNAR mice to immunization with MV-sE2 and MV-CE3E26KE1 (determined in pooled mouse sera)

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

Experiment 2 : Analysis of the immunogenicity and protective efficacy of a single dose of measles virus-CE3E26KE1 recombinant virus in CD46-IFNAR mice

Six-week-old CD46-IFNAR mice were inoculated intraperitoneally with ¹⁰ _TCID50 of measles virus-CE3E26KE1 recombinant virus. Control mice were immunized with the same dose of empty measles virus Schw vector. Blood samples were collected retro-orbitally two weeks after immunization for antibody determination, and mice were then challenged with 100 pfu of Chikungunya virus 06-49 intraperitoneally (immunization and challenge schedule shown in Figure 9).

Measles virus-CE3E26KE1 recombinant virus induced high antibody titers after a single injection in IFNAR mice (Table 2 - Figure 10), as well as neutralizing titers sufficient to confer protection against a lethal challenge with 100 pfu Chikungunya virus 06-49.

Table 2. Antibody responses elicited after a single immunization with MV-CE3E26KE1 virus in CD46-IFNAR mice

Experiment 3: Determination of the protective dose of measles virus-CE3E26KE1 recombinant virus in CD46-IFNAR mice

Six-week-old CD46-IFNAR mice were inoculated intraperitoneally (ip) with 10 ³ to 10 ⁵ TCID 50 doses of MV–CHIKV recombinant virus and boosted with the same dose one month later. Control mice were immunized with the same dose of empty MV Schw vector. One month after the last immunization, mice were challenged by intraperitoneal injection of 100 pfu CHIKV 06-49 (immunization and challenge schedule are shown in Figure 11). For antibody determination, blood samples were collected by retroorbital route one month after the primary vaccination and then one month after the boost, before challenge. Specific ELISAs were performed to detect anti-MV and anti-CHIKV binding antibodies. Anti-CHIKV neutralizing antibody titers were determined by plaque reduction neutralization test (PRNT) on Vero cells.

The results are shown in Table 3 and Figure 12.

Table 3. Antibody responses after vaccination with different doses of MV-CE3E26KE1

As the dose of recombinant MV increased, both anti-MV and anti-CHIKV antibody titers increased. A single immunization with MV-CE3E26KE1 virus induced high antibody titers, which were boosted by a second injection. After two immunizations, high neutralizing titers (PRNT50 = 450-4050, PRNT90 = 50-450) were induced. All animals immunized with ¹⁰⁴ or ¹⁰⁵ _TCID50 were protected from a lethal CHIKV challenge with 100 PFU of CHIKV-06-49, while immunization with a lower dose ( ¹⁰³ TCID50) protected 83% of animals (Figure 12).

Experiment 4 Evaluation of the protection conferred by passive transfer of serum from mice immunized with recombinant MV-CE3E26KE1 virus

Six-week-old CD46-IFNAR mice were inoculated intraperitoneally (ip) with 20 μl of pooled serum from mice immunized with ^{10 5} TCID50 of recombinant MV-CE3E26KE1. Control mice received 20 μl of pooled serum from mice immunized with 10 5 TCID50 of empty measles virus Schwarz or 20 μl of anti-CHIKV HMAF. Sera were diluted in a total volume of 100 μl of PBS. Sera were transferred 24 hours and 16 hours before challenge with 100 pfu of Chikungunya virus 06-49, and then 12 hours after challenge to simulate the presence of antibodies in infected animals. Mouse mortality was analyzed for 2 weeks to determine protection (immunization and challenge schedule are shown in Figure 13).

Passive transfer of immune serum from mice immunized with MV-CE3E26KE1 virus protected 83% of recipient mice from a lethal Chikungunya virus challenge, while all mice receiving anti-Chikungunya virus HMAF were protected. In contrast, all mice receiving immune serum from mice immunized with empty measles virus died. These results indicate that the humoral immune response induced by the measles virus-CE3E26KE1 recombinant virus confers protection against Chikungunya virus in CD46-IFNAR mice (Figure 14).

Experiment 5 Induction of specific cell-mediated immune response

In order to determine whether MV-CHIKV immunity triggers a cell-mediated immune response, we measured the ability of splenocytes from immune mice to secrete IFN-γ when specifically stimulated in vitro by ELISPOT. Splenocytes were collected 7 days after a single immunization and MV-specific and CHIKV-specific responses were evaluated. 1MOI of CHIKV and MV were used for splenocyte stimulation. A significant number of CHIKV-specific cells were detected (up to 300/10 ⁶ splenocytes, an average of 150/10 ⁶ ) (Figure 15), which represents one-third of the MV-specific response under similar stimulation conditions (up to 600/10 ⁶ splenocytes, an average of 500/10 ⁶ ). All eight mice immunized with MV-CHKV, except one, had a significant CMI response to CHIKV. In contrast, control mice immunized with empty MVSchw had similar MV-specific responses, but did not have CHIKV-specific responses. These results indicate that a single inoculation of MV-CHIKV induced high levels of CHIKV- and MV-specific cellular immune responses in the spleens of immunized mice.

Experiment 6 : Effects of measles virus pre-immunization on the immunogenicity and protective efficacy of recombinant MV-CE3E26KE1 virus in CD46-IFNAR mice

Six-week-old CD46-IFNAR mice were inoculated intraperitoneally (ip) with 5.10 ³ TCID ₅₀ of empty measles virus Schw (Figure 16 Group 1) to simulate pre-immunization. Three months later, these mice were injected twice with 10 ⁵ TCID ₅₀ of measles virus-CE3E26KE1 recombinant virus, one month apart. Control mice were immunized with 10 ⁵ TCID ₅₀ of measles virus-CE3E26KE1 recombinant virus (Group 2) or 10 ⁵ TCID ₅₀ of empty measles virus Schw (Group 3). For antibody determination, blood samples were collected via the retroorbital route as shown in Figure 16, and then mice were challenged with 100 pfu of Chikungunya virus 06-49 strain via intraperitoneal injection for protection assays.

This experiment demonstrated that CD46-IFNAR mice pre-immunized with 5.10 ³ TCID50 of empty measles virus were able to mount a protective Chikungunya virus immune response following immunization with the measles virus-CE3E26KE1 recombinant virus. ELISA and PRNT titers (Table 4–Figure 17) remained high and within the same range as titers induced in naive mice (ELISA titers remained unchanged, PRNT titers were reduced by 1-fold dilution). In both pre-immunized and naive groups of animals immunized with the measles virus-CE3E26KE1 recombinant virus, 100% of vaccinated animals were protected from lethal Chikungunya virus challenge.

Table 4. Antibody responses of CD46-IFNAR mice to MV-CE3E26KE1 in the presence of pre-immunization with MV vectors

Experiment 7 : Antibody cross-reactivity induced by vaccination

To determine whether MV-CHIKV immunization elicits cross-reactive antibody responses to different primary CHIKV isolates, sera obtained from animals in Experiment 4 were tested for their ability to neutralize different primary CHIKV isolates. Four strains belonging to the ECSA genotype were selected.

CHIKV Indian strain, clinical isolate n°3710 (NRC for Arbovirus, France), isolated in 2011. Passaged once on Vero cells (November 2011), virus titer 6.3 log PFU/ml

CHIKV Congo strain, clinical isolate n°525 (NRC for Arbovirus, France), isolated in 2011. Passaged once on Vero cells (June 2011), virus titer 6.5 log PFU/ml

CHIKV Thai strain, clinical isolate n°1499 (NRC for Arbovirus, France), isolated in 2009. Passaged on C6/36 cells (December 2009), virus titer 6.3 log PFU/ml

CHIKV Réunion strain, clinical isolate 2006.49 (NRC for Arbovirus, France), isolated in 2006. Passaged 3 times on Vero cells (April 4, 2011), virus titer 7.3 log PFU/ml

Anti-CHIKV neutralizing antibodies were detected using the plaque reduction neutralization test (PRNT). Vero cells were seeded in 12-well plates for 24 h. Serum samples were serially diluted in DMEM Glutamax/2% FCS. 100 μl of the dilutions were incubated with an equal volume of CHIKV containing 100 pfu of strain 06-49 at 37°C with gentle agitation for 2 h. Residual infectivity was then determined on Vero cell monolayers covered with DMEM GlutaMAX ^™ /2% FCS containing a final 0.8% (weight/volume) carboxymethylcellulose. After 3 days of incubation, cells were fixed and stained with crystal violet, and plaque counts were determined. Endpoint neutralization titers were calculated as the highest serum dilution tested that reduced the number of plaques by at least 50% (PRNT ₅₀ ).

Table 5. Cross-reactivity of neutralizing antibodies from MV-CHIKV-immunized mice

The results showed that immunization of mice with MV-CHIKV induced cross-reactive antibodies capable of neutralizing several primary isolates of CHIKV from different countries. Interestingly, challenging the animals with 06-49 Réunion virus resulted in an even more extended response, enhancing neutralization to very high levels against isolates from India and Thailand. Only the ECSA genotype was tested because it is available at the Institut Pasteur.

Experiment 8 : Immunogenicity of MV-CHIKV in cynomolgus monkeys

The immunogenicity of a recombinant measles virus vaccine against Chikungunya was tested in nonhuman primates. Two groups of four cynomolgus macaques (Macaca fascicularis), preselected to be seronegative for flaviviruses and measles virus, were subcutaneously inoculated with ^10⁴ or ^10⁵ _TCID₅₀ of MV-CHIKV on day 0 and then boosted with the same dose on day 90. Serum and plasma were collected and stored at -20°C for subsequent analysis. Neutralizing antibodies against Chikungunya virus were detected using the PRNT assay.

Table 6

The results shown in Table 6 show that all monkeys developed high titers of antibodies against CHIKV. The highest dose was more effective than the lower doses. As shown in Figure 18, boosting was effective in most animals (day 90, when boosting was performed, compared to day 111, 21 days after boosting). These results confirm the immunogenicity of the MV-CHIKV vaccine candidate in non-human primates.

Overall conclusion

The inventors generated a recombinant MV-CHIK virus that stably expresses the complete structural protein CE3E26KE1 of CHIKV strain 06.49. Vero cells infected with this recombinant virus expressed high levels of CHIKV proteins and secreted high densities of VLPs. The growth kinetics of the recombinant virus were slightly delayed, but titers similar to those of the empty MV vector were produced. Evaluation in CD46-IFNAR mice, which are susceptible to MV infection, showed that this vaccine candidate induced high levels of CHIKV neutralizing antibodies (PRNT50 = 450-4050; PRNT90 = 150-450) depending on the dose and number of administrations. All immunized mice were reproducibly protected, even after a single administration, demonstrating the robust immunocompetence of this vaccine candidate. Passive transfer of immune sera in naive animals conferred protection against lethal challenge, even in these highly susceptible mice. Finally, the inventors demonstrated that pre-immunization with the MV vector in CD46-IFNAR mice did not preclude the induction of protective immunity following immunization with the MV-CE3E26KE1 vaccine candidate. Based on these results, the resulting recombinant vectors deserve evaluation in a reliable nonhuman primate infection model.

Claims (26)

1. A nucleic acid construct comprising a polynucleotide encoding a C-E3-E2-6K-E1 structural protein of flexure virus (CHIKV), the polynucleotide being operatively linked to a cDNA molecule encoding a nucleotide sequence of a full-length, infectious antigenomic (+) RNA strand of measles virus (MV). 2. The nucleic acid construct of claim 1, characterized in that the nucleic acid construct conforms to the 6-fold rule of the measles genome. 3. The nucleic acid construct of claim 1, comprising a gene transcription unit, from 5' to 3' including: (a) Polynucleotides encoding the N protein of MV (b) Polynucleotides encoding the P protein of MV (c) Polynucleotides encoding the C-E3-E2-6K-E1 structural protein of CHIKV (d) Polynucleotides encoding the M protein of MV (e) Polynucleotides encoding the F protein of MV (f) Polynucleotides encoding the H protein of MV, and (g) Polynucleotides encoding the L protein of MV The polynucleotide is operatively linked to the nucleic acid construct and is controlled by viral replication and transcription regulatory sequences. 4. The nucleic acid construct of claim 3, characterized in that the viral replication and transcription regulatory sequences are MV leader and tail sequences. 5. The nucleic acid construct of claim 1, characterized in that the measles virus is an attenuated viral strain. 6. The nucleic acid construct of claim 5, characterized in that the attenuated viral strain is selected from the Schwarz strain, the Zagreb strain, the AIK-C strain, and the Moraten strain. 7. The nucleic acid construct of claim 1, characterized in that the polynucleotide encoding the C-E3-E2-6K-E1 structural protein of CHIKV has been optimized for Macacca codon usage or human codon usage. 8. The nucleic acid construct of claim 1, characterized in that the polynucleotide encoding the C-E3-E2-6K-E1 structural protein of CHIKV comprises at least one measles-edit-like sequence selected from mutated AAAGGG, AAAAGG, GGGAAA, and GGGGAA. 9. The nucleic acid construct of claim 1, characterized in that the flexure virus is derived from a strain named 06-49. 10. The nucleic acid construct of claim 1, characterized in that the extracellular domain of the envelope protein E2 has the sequences of SEQ ID NO: 11, 13, 15. 11. The nucleic acid construct of claim 1, characterized in that the C-E3-E2-6K-E1 structural protein is defined by SEQ ID NO: 21, 22, 23, 24, 25, 26 and 28. 12. The nucleic acid construct of claim 1, characterized in that the nucleic acid construct comprises the sequences defined by SEQ ID NO: 20, 27 and 31. 13. A transfer vector plasmid comprising the nucleic acid construct of any one of claims 1-12. 14. Cells, characterized in that the cells are transformed with the nucleic acid construct of any one of claims 1-12 or the transfer vector plasmid of claim 13. 15. The cell of claim 14, wherein it is a eukaryotic or avian or CEF cell, a mammalian cell or a yeast cell. 16. A recombinant infectious replicating MV-CHIK virus particle comprising the nucleic acid construct of any one of claims 1-12 as its genome. 17. The recombinant infectious replicating MV-CHIK virus particle of claim 16, characterized in that the genome of the virus particle contains a polynucleotide sequence comprising the sequence defined by SEQ ID NO:27 or 31. 18. An active ingredient composition or combination thereof comprising the recombinant infectious replicating MV-CHIK virus particles of claim 16 or 17 in combination with CHIKV-C-E3-E2-6K-E1VLP and a pharmaceutically acceptable carrier. 19. Use of the active ingredient composition or combination of claim 18 in the preparation of a vaccine, wherein the vaccine is used to induce a protective immune response against the CHIK virus in a host by triggering an antibody and/or cellular immune response against the CHIKV protein. 20. The use of claim 19, wherein the host is a human host in need. 21. Use of the recombinant infectious replicating MV-CHIK virus particles of claim 16 or 17, or the active ingredient composition or combination of claim 18, in combination with CHIKV-C-E3-E2-6K-E1VLP in the preparation of a vaccine for the prevention of infection with flexnervous virus in a subject. 22. The use of claim 21, wherein the subject is a human. 23. A method for rescuing recombinant measles virus (MV) expressing the C-E3-E2-6K-E1 structural protein of curvature virus (CHIKV) or the CHIKV-C-E3-E2-6K-E1 virus-like particle (VLP), comprising the following steps: 1) Co-transfect helper cells stably expressing T7 RNA polymerase and measles N and P proteins with (i) the transfer vector plasmid of claim 13 and (ii) the vector encoding MV L polymerase; 2) Culture the co-transfected helper cells under conditions that enable the production of MV-CHIKV recombinant virus; 3) The recombinant virus produced therefrom is proliferated by co-culturing the helper cells described in step 2) with cells capable of viral proliferation; 4) Recover replicated MV-CHIKV recombinant virus and CHIKV C-E3-E2-6K-E1 structural protein or CHIKV-C-E3-E2-6K-E1VLP. 24. The method of claim 23, wherein the transfer vector plasmid comprises the sequence defined by SEQ ID NO:27 or 31. 25. The method of claim 23, wherein the helper cell in step 1) is a HEK293 helper cell. 26. The method of claim 23, wherein the cell capable of viral proliferation in step 3) is a Vero cell.