WO2016086980A1 - Vaccine composition - Google Patents

Abstract

A composition for use as a vaccine to protect against Chikungunya virus (CHIKV) infection is disclosed. The composition comprises a nucleic acid comprising a nucleotide sequence encoding an attenuated poxvirus vector and one or more CHIKV structural proteins. In preferred embodiments, the nucleotide sequence encodes all of the C, E3, E2, 6K, and E1 structural proteins. Dosage schedules are also described.

Description

Vaccine Composition

FIELD OF THE INVENTION

The present invention relates to a vaccine composition for protection against Chikungunya virus infection. Aspects of the invention relate to methods for protecting subjects against Chikungunya virus infection, and to compositions for use in the preparation of a vaccine composition.

BACKGROUND TO THE INVENTION

Chikungunya is a disease caused by the Chikungunya virus (CHIKV) with a mortality rate of about 1 :1000. The disease is characterized by skin rash, high fever, headache, vomiting, myalgia, and, mainly, polyarthralgia. Most of the symptoms resolve after 10 days, but the polyarthralgia can persist for months or years, and severe symptoms, such as encephalitis, hemorrhagic disease, and mortality, have also been described. The disease is transmitted to humans by Aedes mosquito vectors. CHIKV infection has been reported from Africa, islands in the Indian Ocean, India, Southeast Asia, and southern Europe, and recently in the Americas where over 700,000 individuals are estimated to have encountered CHIKV. In 2014 several mortality cases related to CHIKV infection have been reported in Venezuela. CHIKV contains a positive, single-stranded RNA genome of around 1 1 .8 kb which encodes four nonstructural and five structural proteins. The nonstructural proteins (nsP1 , nsP2, nsP3, and nsP4) are required for virus replication. The structural proteins are cleaved by capsid (C) autoproteinase and signalases from a polyprotein precursor to generate the C and envelope (pE2, 6K, and E1 ) proteins. The pE2 precursor is cleaved to E3 and E2 by the furin protease in the trans-Golgi during transport of the envelope complex to the cell surface where virus assembly occurs. pE2 is also known as p62. Virions are 70-nm enveloped particles containing 240 heterodimers of E1/E2 glycoproteins on their surfaces. There is currently no specific treatment for Chikungunya, nor are there currently any approved vaccines, although a number of candidate vaccines are in development. It would be desirable to develop a vaccine to protect against CHIKV.

Ross River Virus (RRV) is closely related to CHIKV, and causes an illness displaying similar symptoms. It has been shown that antibodies against RRV and CHIKV cross react. Therefore it is possible that a CHIKV vaccine could also provide protection against RRV.

SUMMARY OF THE INVENTION

Described herein is a vaccine candidate against CHIKV, based on an attenuated poxvirus vector expressing the CHIKV structural genes. The vaccine was described for the first time in J. Virol. 2014, 88(6):3527, Garcia-Arriaza et al, "A Novel Poxvirus Based Vaccine, MVA-CHIKV, Is Highly Immunogenic and Protects Mice against Chikungunya Infection", published ahead of print 8 January 2014. The contents of this publication are incorporated herein by reference.

According to a first aspect of the invention, there is provided a composition for use in the production of a vaccine, the composition comprising a nucleic acid comprising a nucleotide sequence encoding an attenuated poxvirus vector and one or more CHIKV structural proteins.

Preferably the nucleotide sequence encodes two, three, four, or five CHIKV structural proteins; most preferably five structural proteins. The structural proteins may be selected from the group consisting of the C, E3, E2, 6K, and E1 proteins. Preferably the nucleotide sequence encodes at least the E3 and E2 proteins; most preferably the nucleotide sequence encodes all of the C, E3, E2, 6K, and E1 proteins. The CHIKV sequences can be derived from virus isolates which may differ in nucleotide sequence from one another and from neutralization resistant mutants. The nucleotide sequence may further encode one or more replicase proteins (that is, one or more of nsP1 , nsP2, nsP3, and nsP4). The sequence may encode two, three, or four of said replicase proteins. It is possible that host immune system responses against the replicase may further enhance protectivity of the vaccine, for example by reducing long term infection of macrophages by CHIKV.

The CHIKV nucleotide sequences can be derived from one or more virus isolates. The isolates may be selected such that they differ from one another to the extent that a neutralisation response by sera generated from one isolate does not neutralise a second isolate. Including sequences from a number of isolates improves the chances of an immune response generated by the vaccine being effective against multiple strains of CHIKV. Sequences from different isolates may be combined in a single poxvirus vector, or multiple vectors representing different CHIKV isolates may be combined in a single vaccine composition. The nucleic acid may be DNA, RNA, or a combination thereof, and can include any combination of naturally occurring, chemically modified or enzymatically modified nucleotides. The nucleic acid of the invention may take the form of an expression vector that is capable of expression in an organism or in a cell of the organism, in culture or in vivo. An organism or cell in which the coding sequence of the vector can be expressed can be eukaryotic or prokaryotic, and can be, without limitation, a bacterium, a yeast, an insect, a protozoan, or animals, such as a human, a bird, or a non-human mammal. The nucleotide sequence of the nucleic acid may be codon optimised for expression in a particular organism or cell; for example, humans. It should be understood that nucleic acids of the invention can be single-stranded or double-stranded, and further that a single-stranded nucleic acid of the invention includes a polynucleotide fragment having a nucleotide sequence that is complementary to a nucleotide sequence that encodes said poxvirus vector and structural proteins according to the invention. As used herein, the term "complementary" refers to the ability of two single stranded polynucleotide fragments to base pair with each other.

Further, a single-stranded nucleic acid of the invention also includes a polynucleotide fragment having a nucleotide sequence that is substantially complementary to a nucleotide sequence that encodes said poxvirus vector and structural proteins according to the invention, or to the complement of the nucleotide sequence that encodes said poxvirus vector and structural proteins. Substantially complementary polynucleotide fragments can include at least one base pair mismatch, such that at least one nucleotide present on a first polynucleotide fragment will not base pair to at least one nucleotide present on a second polynucleotide fragment, however the two polynucleotide fragments will still have the capacity to hybridize. The present invention therefore encompasses polynucleotide fragments that are substantially complementary. Two polynucleotide fragments are substantially complementary if they hybridize under hybridization conditions exemplified by 2xSSC (SSC: 150 mM NaCI, 15 mM trisodium citrate, pH 7.6) at 55^eC. Substantially complementary polynucleotide fragments for purposes of the present invention preferably share at least about 85% nucleotide identity, preferably at least about 90%, 95% or 99% nucleotide identity. Locations and levels of nucleotide sequence identity between two nucleotide sequences can be readily determined using, for example, CLUSTALW multiple sequence alignment software.

The poxvirus vector may be a vaccinia virus; preferably a modified vaccinia virus Ankara (MVA). In preferred embodiments, the MVA vector is further modified by deletion of one or more vaccinia immunomodulatory genes; preferably one, two, or three of the genes C6L, K7R, and A46R. Most preferably, all three genes are deleted. Deletion of these genes has been shown to enhance the innate immune response triggered by the viral vector, and potentially also specific immune response. Alternative attenuated poxvirus vectors include NYVAC, an isolate of the Copenhagen vaccinia vaccine strain.

A further aspect of the invention provides an attenuated poxvirus vector expressing one or more CHIKV structural genes. Also provided is a vaccine composition comprising such an attenuated poxvirus vector. The vaccine composition may also be used in combination with other vectors expressing CHIKV genes and protein components, and/or as a combination vaccine against other infections. A combination vaccine may comprise one or more adjuvants. Preferred adjuvants include AS03, or Matrix - M.

A vaccine of the invention is conveniently administered to subjects using ingestion, topical administration, or direct injection, preferably subcutaneous or intramuscular injection. The subject is preferably mammalian, more preferably human, although alternative subjects may be treated, for example, in the course of vaccine or disease research. The dose of vaccine to be administered to a subject depends on the species and size of subject, the nature of the condition being treated, and can be readily determined by one of skill in the art.

The invention further provides an attenuated poxvirus vector expressing one or more CHIKV structural genes for use as a vaccine. Also provided is the use of an attenuated poxvirus vector expressing one or more CHIKV structural genes in the manufacture of a vaccine. The vaccine is intended for use in protecting a subject against CHIKV infection. The vaccine may also or instead be for use in protecting a subject against RRV infection.

A still further aspect of the invention provides a method of immunising a subject against CHIKV infection, the method comprising administering a vaccine comprising an attenuated poxvirus vector expressing one or more CHIKV structural genes to a subject. A single administration may be sufficient to provide suitable immunization, but in preferred embodiments, more than one dose of vaccine is administered. For example, a first dose may be followed by a booster dose after one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months or longer intervals.

In alternative embodiments, the first dose of the attenuated poxvirus vector vaccine may be followed by a second dose of an alternative vaccine; a so-called heterologous prime-boost protocol. Or the attenuated poxvirus vector vaccine may be administered second, after an initial dose of an alternative vaccine. The alternative vaccine may comprise a covalently linked p62-E1 heterodimer (E1 being the CHIKV E1 protein, and p62 being the CHIKV precursor protein which is cleaved during virus replication into the E2 and E3 proteins). Alternatively, the alternative vaccine may comprise a DNA- replicon (DREP) based vaccine; this may comprise a nucleotide sequence comprising CHIKV genomic sequences operably linked to a promoter sequence. The genomic sequences may comprise genes coding for the envelope proteins (E3, E2, 6K, and E1 ). Preferably the genomic sequences do not include genes coding for the capsid (C) protein; in the absence of this protein, the DREP-CHIKV is unable to form viral particles, and is only able to express envelope proteins on the host cell surface. In preferred embodiments of the invention the first dose is of DREP-CHIKV vaccine, and the second is of the attenuated poxvirus vector. The second dose may be combined with a dose of p62 - E1 vaccine. The invention may further provide a kit comprising first and second doses of CHIKV vaccine. The first dose is preferably a vaccine comprising an attenuated poxvirus vector expressing one or more CHIKV structural genes. The second dose may be the same as the first dose, or may be a vaccine comprising a covalently linked p62-E1 heterodimer, or a DREP-CHIKV vaccine. "First" and "second" here do not necessary refer to the order in which the vaccines are to be administered. The doses may be provided in a suitable pharmaceutical container; for example, sealed vials, or alternatively as preloaded syringes.

BRIEF DESCRIPTION OF THE DRAWINGS FIG 1 Generation and in vitro characterization of recombinant MVA-CHIKV. (A) Scheme of the MVA-CHIKV genome map. The different regions are indicated by capital letters. The right and left terminal regions are shown. Below the map, the deleted or fragmented genes are depicted as black boxes. The CHIKV C, E3, E2, 6K, and E1 structural genes (from CHIKV isolate LR2006-OPY1 ), driven by the synthetic early/late (sE/L) virus promoter and inserted within the VACV TK viral locus (J2R), are indicated. The deleted VACV C6L, K7R, and A46R genes are also indicated. TK-L, TK left; TK-R, TK right. (B) PCR analysis of the VACV TK locus. Viral DNA was extracted from chick DF-1 cells mock infected or infected at 5 PFU/cell with MVA-CHIKV, MVA-GFP, or MVA-WT. Primers spanning the TK locus-flanking regions were used for PCR analysis of the CHIKV genes inserted within the TK locus. DNA products with their corresponding sizes (base pairs) are indicated by an arrow on the right. A molecular size marker (1 -kb ladder) with the corresponding sizes (base pairs) is indicated on the left. (C) PCR analysis of the C6L, K7R, and A46R loci. Viral DNA was extracted from DF-1 cells mock infected or infected at 5 PFU/cell with MVA-CHIKV, MVA-GFP, or MVA-WT. Primers spanning C6L-, K7R-, and A46R-flanking regions were used for PCR analysis of the C6L, K7R, and A46R loci, respectively. DNA products with the corresponding size (base pairs) of the parental virus (wt) or the virus with the C6L, K7R, and A46R deletions are indicated by an arrow on the right. A molecular size marker (1 -kb ladder) with the corresponding sizes (base pairs) is indicated on the left. (C) Expression of CHIKV E1 and E2 proteins. DF-1 cells were mock infected or infected at 5 PFU/cell with MVA-CHIKV, MVA-GFP, or MVA-WT. At 24 h postinfection, cells were lysed in Laemmli buffer, fractionated by 10% SDS-PAGE, and analyzed by Western blotting using rabbit polyclonal antibody against E1 or mouse polyclonal antibody against E2. Arrows on the right indicate the positions of the CHIKV E1 and E2 proteins, with the corresponding sizes in kDa. The sizes of standards (in kDa) (Precision Plus protein standards; Bio-Rad Laboratories) are indicated on the left.

FIG 2 Viral growth kinetics and stability of MVA-CHIKV. (A) Viral growth kinetics. Monolayers of DF-1 cells were infected at 0.01 PFU/cell with MVA-WT or MVA-CHIKV. At different times postinfection (0, 24, 48, and 72 h), cells were collected and virus titers in cell lysates were quantified by a plaque immunostaining assay with anti-WR antibodies. The means of results from two independent experiments are shown. (B) Stability of MVA-CHIKV.MVA-CHIKV(P2 stock) was continuously grown to passage 9 in DF-1 cells. Then, DF-1 cells were mock infected or infected with MVA-CHIKV from the different passages or with MVA-WT. At 24h postinfection, cells were lysed in Laemmli buffer, fractionated by 10% SDS-PAGE, and analyzed by Western blotting using rabbit polyclonal antibody against E1 or mouse polyclonal antibody against E2. Rabbit anti-B-actin antibody was used as a protein loading control. Rabbit anti-VACV early E3 protein antibody was used as a VACV loading control. Arrows on the right indicate the positions of the CHIKV E1 and E2 proteins, B-actin, and the VACV E3 protein, with their corresponding sizes in kDa. The sizes of standards (in kDa) (Precision Plus protein standards; Bio-Rad Laboratories) are indicated on the left. (C) Genetic stability of MVA-CHIKV, as determined by plaque immunostaining. Virus titers of MVA-CHIKV (P2 stock, P3 stock, and passage 9) were quantified by a plaque immunostaining assay with anti-VACV and anti-CHIKV antibodies. The means and standard deviations of results from two independent determinations are shown. It should be noted that the P3 stock represents virus purified with two sucrose cushions, while P2 and passage 9 stocks are virus titers from crude cell lysates. FIG 3 Immunofluorescence analysis of CHIKV E2 protein produced in HeLa cells infected with MVA-CHIKV. Subconfluent HeLa cells mock infected or infected with MVA-CHIKV or MVA-WT were fixed at 6 and 16 hpi (MVA-WT- and mock-infected cells are represented at 16 hpi), labeled with the corresponding primary antibodies or probes, monitored for appropriate fluorescence from protein-conjugated secondary antibodies, and visualized by confocal microscopy. The antibodies or probes used were anti-E2 (D3-62; to detect CHIKV E2 protein, shown in green), phalloidin (to stain actin, shown in red), anti-calnexin (to detect the ER, shown in red), WGA(to detect Golgi apparatus membranes and the plasma membrane, shown in red), and DAPI (to mark DNA, shown in blue). Bar, 10 urn.

FIG 4 Architecture of HeLa cells following infection with MVA-CHIKV or MVA-WT. HeLa cells infected with MVA-WT or MVA-CHIKV were chemically fixed at 6 hpi and then processed for conventional embedding in an epoxy resin and observed by electron microscopy. (A) General overview of a cell infected with MVA-WT (left) and MVA-CHIKV (right); (B) two high-magnification panels of a cell infected with MVA- CHIKV. m, mitochondria; IV, immature virus; GM, Golgi apparatus membranes. Bars:

FIG 5 MVA-CHIKV triggers an innate immune response in human macrophages and dendritic cells, upregulating type I IFN, proinflammatory cytokines, and chemokine expression. Human THP-1 macrophages (A) and moDCs (B) were mock infected or infected with MVA-WT, MVA-GFP, or MVA-CHIKV at 5 PFU/cell (A) and 1 PFU/cell (B). At different times postinfection (3 h and 6 h in panel A, 6 h in panel B), RNA was extracted and the mRNA levels of IFN-, TNF-, MIP-1 , RANTES, IP-10, IFIT1 , IFIT2, RIG-I, MDA-5, and HPRT were analyzed by reverse transcription-PCR. Results are expressed as the ratio of the gene of interest to the hypoxanthine phosphoribosyltransferase (HPRT) mRNA level. A.U., arbitrary units. P values indicate significantly higher responses in comparisons of MVA-WT to MVA-GFP or MVA-CHIKV at the same hour (^*, P < 0.05; ^**, P < 0.005; ^***, P < 0.001 ). Data are means +/- standard deviations of results with duplicate samples from one experiment and are representative of two independent experiments.

FIG 6 Immunization with MVA-CHIKV induces strong, broad, and polyfunctional adaptive CHIKV-specific T cell immune responses. Splenocytes were collected from mice (5 per group) immunized with MVA-WT/MVA-WT or MVA-CHIKV/MVA-CHIKV 10 days after the last immunization. Next, adaptive, CHIKV-specific CD4 and CD8 T cell immune responses triggered by both immunization groups were measured by the ICS assay following stimulation of splenocytes with different CHIKV peptides (C, E1 , and E2). Values from unstimulated controls were subtracted in all cases. P values indicate significantly higher responses in comparisons of MVA-WT/MVA-WTto MVA- CHIKV/MVA-CHIKV (^***, P<0.001 ). Data are from one experiment and are representative of data from two independent experiments. (A) Overall magnitudes of CHIKV-specific CD4 and CD8 T cells. The values represent the sums of the percentages of T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 against the C, E1 , and E2 peptides. (B) Pattern of adaptive, CHIKV-specific CD8 T cell immune responses to C, E1 , and E2. Frequencies were calculated by reporting the number of CD8 T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 and are specific for each peptide to the total number of CD8 T cells in the MVA- CHIKV/MVA-CHIKV immunization group. (C) Magnitudes of C, E1 , and E2 CHIKV- specific CD8 T cells. Frequencies represent the sums of the percentages of CD8 T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 against the C, E1 , or E2 peptide. (D) Magnitudes of CHIKV-specific CD8 T cells producing CD107a, IFN-γ, TNF-a, or IL-2. Frequencies represent percentages of CD8 T cells producing CD107a, IFN-γ, TNF-a, or IL-2 against the C, E1 , and E2 peptides. (E) Polyfunctional profile of adaptive, CHIKV-specific CD8 T cell immune responses. All the possible combinations of the responses are shown on the x axis (15 different T cell populations), while the percentages of CD8 T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 against the C, E1 , and E2 peptides are shown on the y axis. Responses are grouped and color coded on the basis of the number of functions (4, 3, 2, or 1 ). The pie charts summarize the data. Each slice corresponds to the proportion of CHIKV-specific CD8 T cells exhibiting one, two, three, or four functions within the total population of CHIKV- specific CD8 T cells.

FIG 7 Adaptive, VACV-specific T cell immune responses. Splenocytes were collected from mice (5 per group) immunized with MVA-WT/MVA-WT or MVA-CHIKV/MVA- CHIKV 10 days after the last immunization. Next, adaptive, VACV-specific CD4 and CD8 T cell immune responses triggered by both immunization groups were measured by ICS assay following stimulation of splenocytes with MVA-infected EL4 cells. Values from unstimulated controls were subtracted in all cases. Data are from one experiment that is representative of two independent experiments. (A) Overall magnitude of VACV- specific CD4 and CD8 T cells. The values represent the sums of the percentages of T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 against MVA-infected EL4 cells. (B) Percentages of VACV-specific CD8 T cells producing CD107a, IFN-γ , TNF-a, or IL-2. Frequencies represent percentages of CD8 T cells producing CD107a, IFN-γ , TNF-a, or IL-2 against MVA-infected EL4 cells. (C) Functional profiles of adaptive, VACV-specific CD8 T cell immune responses. All the possible combinations of the responses are shown on the x axis (15 different T cell populations), while the percentages of CD8 T cells producing CD107a and/or IFN-γ and/or TNFa and/or IL-2 against MVA-infected EL4 cells are shown on the y axis. Responses are grouped and color coded on the basis of the number of functions (4, 3, 2, or 1 ). The pie charts summarize the data. Each slice corresponds to the proportion of VACV-specific CD8 T cells exhibiting one, two, three, or four functions within the total population of VACV- specific CD8 T cells. FIG 8 Immunization with MVA-CHIKV induces strong, broad, and polyfunctional CHIKV-specific memory T cell immune responses. Splenocytes were collected from mice (5 per group) immunized with MVA-WT/MVA-WT or MVA-CHIKV/MVA-CHIKV 52 days after the last immunization. CHIKV-specific CD4 and CD8 memory T cell immune responses triggered by both immunization groups were measured by ICS assay as described in the legend to Fig. 6. Values from unstimulated controls were subtracted in all cases. P values indicate significantly higher responses in comparisons of MVA- WT/MVA-WT to MVA-CHIKV/MVA-CHIKV (^***, P<0.001 ). Data are from one experiment that is representative of two independent experiments. (A) Overall magnitudes of CHIKV-specific CD4 and CD8 T cells. The values represent the sums of the percentages of T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 against C plus E1 plus E2. (B) Pattern of C, E1 , and E2 CHIKV-specific CD8 memory T cell immune responses. Frequencies were calculated by reporting the number of CD8 T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 and are specific for each peptide to the total number of CD8 T cells in the MVA-CHIKV/MVA-CHIKV immunization group. (C) Magnitudes of C, E1 , and E2 CHIKV-specific CD8 T cells. Frequencies represent the sums of the percentages of CD8 T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 against the C, E1 , or E2 peptide. (D) Magnitudes of CHIKV-specific CD8 T cells producing CD107a, IFN-γ, TNF-a or IL-2. Frequencies represent percentages of CD8 T cells producing CD107a, IFN-γ, TNF-a, or IL-2 against C plus E1 plus E2. (E) Polyfunctional profile of CHIKV-specific CD8 memory T cell immune responses. All the possible combinations of the responses are shown on the x axis (15 different T cell populations), while the percentages of CD8 T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 against C plus E1 plus E2 are shown on the y axis. Responses are grouped and color coded on the basis of the number of functions (4, 3, 2, or 1 ). The pie charts summarize the data. Each slice corresponds to the proportion of CHIKV-specific CD8 T cells exhibiting one, two, three, or four functions within the total population of CHIKV-specific CD8 T cells. (F) Phenotypic profile of CHIKV-specific CD8 memory T cells. CD127 and CD62L expression was used to identify central memory (TCM, CD127 CD62), effector memory (TEM, CD127 CD62L), and effector (TE, CD127CD62L) subpopulations. Magnitudes of TCM, TEM, and TE CHIKV-specific CD8 T cells are represented. The values represent percentages of TCM, TEM, and TE populations producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 against the C plus E1 plus E2 peptides. The pie charts summarize the data. Each slice corresponds to the proportion of the TCM, TEM, or TE population within the total CHIKV-specific CD8 T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2. P values indicate significantly higher responses in comparisons of MVA-WT/MVA-WT to MVA-CHIKV/MVA-CHIKV (^***, P< 0.001 ). FIG 9 VACV-specific memory T cell immune responses. Splenocytes were collected from mice (5 per group) immunized with MVA-WT/MVA-WT or MVA-CHIKV/MVA- CHIKV 52 days after the last immunization. Next, VACV-specific CD4 and CD8 memory T cell immune responses triggered by both immunization groups were measured by ICS assay following stimulation of splenocytes with MVA-infected EL4 cells. Values from unstimulated controls were subtracted in all cases. P values indicate significantly higher responses in comparisons of MVA-WT/MVA-WT to MVA- CHIKV/MVA-CHIKV (^***, P<0.001 ). Data are from one experiment that is representative of two independent experiments. (A) Overall magnitude of VACV- specific CD4 and CD8 memory T cells. The values represent the sums of the percentages of T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 against MVA-infected EL4 cells. (B) Percentage of VACV-specific CD8 T memory cells producing CD107a, IFN-γ, TNF-a, or IL-2. Frequencies represent percentages of CD8 memory T cells producing CD107a, IFN-γ, TNF-a, or IL-2 against MVA-infected EL4 cells. (C) Functional profiles of VACV-specific CD8 memory T cell immune responses. Responses are grouped and color coded on the basis of the number of functions (4, 3, 2, or 1 ). All possible combinations of the responses are shown on the x axis (15 different T cell populations), while the percentages of CD8 T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 against MVA-infected EL4 cells are shown on the y axis. The pie charts summarize the data. Each slice corresponds to the proportion of VACV-specific CD8 memory T cells exhibiting one, two, three, or four functions within the total population of VACV-specific CD8 T cells. (D) Phenotypic profile of VACV-specific CD4 and CD8 memory T cells. Fluorescence-activated cell sorter (FACS) plots of MVA-infected EL4 cells are represented. CD127 and CD62L expression was used to identify central memory (TCM, CD127 CD62), effector memory (TEM, CD127 CD62L), and effector (TE, CD127CD62L) subpopulations. The memory T cell subpopulations are depicted as density plots. Blue dots represent T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2. The graph represents the magnitudes of TCM, TEM, and TE VACV-specific CD8 T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2. Frequencies represent percentages of TCM, TEM, and TE populations producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2 against MVA-infected EL4 cells. The pie charts summarize the data. Each slice corresponds to the proportion of the TCM, TEM, or TE population within the total population of VACV-specific CD8 T cells producing CD107a and/or IFN-γ and/or TNF-a and/or IL-2.

FIG 10 Immunization with MVA-CHIKV induces neutralizing antibodies against CHIKV and protects mice against CHIKV infection. Five C57BL/6 mice per group were immunized as described in Materials and Methods. Data are from one experiment that is representative of two independent experiments. (A) Antibody titers against CHIKV E1 -p62 detected by ELISA at 3 weeks after the prime or 6 weeks after the boost in sera of animals immunized with one dose of MVA-WT or one or two doses of MVA-CHIKV. The detection limit is 100. (B) CHIKV-neutralizing antibody titers (NT50) detected at 6 weeks after the last immunization in sera of animals immunized with one dose of MVA- WT or one or two doses of MVA-CHIKV. The detection limit is 100. (C) Viremia detected in animals immunized with one dose of MVA-WT or one or two doses of MVA- CHIKV and challenged with a total of 10⁶ PFU of CHIKV subcutaneously in both feet. Blood samples were collected at days 1 to 3 postchallenge from tail bleedings, and viremia was analyzed by plaque assay (PFU/ml). The detection limit is 250 PFU/ml. (D) Foot swelling in animals immunized with one dose of MVA-WT or one or two doses of MVA-CHIKV and challenged with a total of 10⁶ PFU of CHIKV subcutaneously in both feet. Foot swelling was analyzed on the day of challenge and days 4 to 8 following challenge.

FIG 1 1 CHIKV specific antibody responses induced by different CHIKV vaccine candidates using various prime-boost immunization strategies. C57BL/6 mice (n=5) were immunized once or twice with three weeks immunization intervals with various combinations of the CHIKV vaccine candidates depicted in Table 1 . (A) Total IgG titers and (B) lgG1 (red) and lgG2c (blue) titers in serum collected six weeks post the last immunization. (C) 50% neutralization titers (NT50) on serum collected prior to challenge. Bars show mean values (n=5). A Kruskal-Wallis test followed by Dunn's posttest was used to compare responses from CHIKV (WT). ^*, ^** and ^*** indicate statistical differences of p<0.05, p<0.01 and p<0.001 , respectively.

FIG 12 Viremia and foot swelling after CHIKV challenge. Mice were challenged seven weeks post the last immunization with 10⁶ PFU of CHIKV in the feet. (A) Viremia in serum collected two days after challenge. (B) Peak foot swelling of each mouse (mean of height times breadth of both feet relative to day 0) day 4-9 after challenge. Bars show mean values (n=5). A Kruskal-Wallis test followed by Dunn's post-test was used to compare responses from nal^'ve mice (PBS) and a Spearman rank test was used to examine correlation between the magnitude of antibody responses prior to challenge and the level of viremia and foot swelling post challenge. ^*, ^** and ^*** indicate statistical differences of p<0.05, p<0.01 and p<0.001 , respectively. (C and D) Correlation between the magnitude of anti-CHI KV antibody responses prior to challenge and the level of viremia and foot swelling after challenge. A Spearman rank test was used to examine the correlations.

FIG 13 Induction of CD8+ T cells with different CHIKV vaccine candidates. Groups of 5 C57BL/6 mice were infected once with wild-type CHIKV, or primed with DREP-Env and boosted 3 weeks later with DREP-Env, p62-E1 protein antigen and/or MVA-CHIKV, as indicated. Mice were sacrificed, and spleens were collected 8 days after the last immunization. CHIKV E1 peptide (HSMTNAVTI)-specific CD8+ T cells were identified by pentamer staining and characterized for the expression of CD62L/CD127 and CD27/CD43. DETAILED DESCRIPTION OF THE INVENTION

EXAMPLE 1 - MVA-CHIKV is highly immunogenic and protects mice against Chikungunya infection MATERIALS AND METHODS

Viruses. The poxvirus strain used in this study as the parental virus for the generation of the recombinant MVA-CHIKV is a modified vaccinia virus Ankara (MVA) strain (obtained from the Ankara strain after 586 serial passages in CEFs; this strain is derived from clone F6 at passage 585, kindly provided by G. Sutter, Germany). We modified this virus by inserting the gene encoding the green fluorescent protein (GFP) into the thymidine kinase (TK) locus and by deleting the immunomodulatory vaccinia virus (VACV) genes C6L, K7R, and A46R. The GFP cassette in the TK locus was replaced by the CHIKV cassette to generate MVA-CHIKV. We also used in this study the parental attenuated MVA (referred to as wild-type MVA [MVA-WT]). All viruses were grown in primary CEFs, purified by centrifugation through two 36% (wt/vol) sucrose cushions in 10 mM Tris-HCI, pH 9, and titrated in DF-1 cells by a plaque immunostaining assay, as previously described. The titer determinations of the different viruses were performed at least three times. All viruses were free of contamination with mycoplasmas, fungi, or bacteria.

Construction of the infectious CHIKV clone LR2006-OPY1 has previously been described. Briefly, a plasmid for production of CHIKV was generated by cloning the CHIKV cDNA under the control of the SP6 RNA polymerase promoter. CHIKV was subsequently produced in BHK-21 cells as previously described.

Construction of the plasmid transfer vector pCyA20-ICRESl . The plasmid transfer vector pCyA-ICRES1 was generated and used for the construction of recombinant MVA-CHIKV, in which CHIKV structural genes (those for C, E3, E2, 6K, and E1 ) instead of the GFP gene were inserted into the TK locus of the parental MVA-GFP. In detail, a 3.7-kbp DNA fragment encoding the CHIKV structural proteins (C, E3, E2, 6K, and E1 ) was amplified with oligonucleotides ICRES1 upper (5'-ATATA GTTTAAACATGGAGTTCATCCCAACCCAAAC-3': Pmel site underlined) and ICRES1 lower (5'-TATATAGATCTTTAGTGCCTGCTGAACGACACG-3'; Bglll site underlined) from plasmid pSP6-ICRES1 , which contains the structural genes from the CHIKV genome (CHIKV isolate LR2006-OPY1 ). The amplification reaction was performed with Platinum Pfx DNA polymerase (Invitrogen) according to the manufacturer's recommendations. Then, this fragment was digested with Pmel and Bglll and cloned into the VACV insertion vector pCyA-20 (7,582 bp), previously digested with the same restriction enzymes and dephosphorylated by incubation with shrimp alkaline phosphatase, to generate the plasmid transfer vector pCyA-ICRES1 (1 1 ,324 bp). The plasmid pCyA-20, used for the generation of plasmid pCyA-ICRES1 , was previously described and contains a synthetic band containing the viral early/late promoter and a multiple-cloning site in the plasmid pLZAWI (provided by Sanofi-Pasteur). The correct generation of pCyA-ICRES1 was further confirmed by DNA sequence analysis. This plasmid was used in transient infection and transfection assays for the insertion of CHIKV structural genes (those for C, E3, E2, 6K, and E1 ) into the TK locus of the MVA genome under the transcriptional control of the viral synthetic early/late (sE/L) promoter. It contains a B-galactosidase (B-Gal) reporter gene sequence between two repetitions of the left TK-flanking arm, which allows the reporter gene to be deleted from the final recombinant virus by homologous recombination after successive passages.

Construction of recombinant MVA-CHIKV. DF-1 cells (3 x 10⁶ cells) were infected with parental MVA-GFP at a multiplicity of infection (MOI) of 0.05 PFU/cell and transfected 1 h later with 10 ug of DNA of plasmid pCyA-ICRES1 , using Lipofectamine reagent according to the manufacturer's recommendations (Invitrogen). At 48 h postinfection (hpi), the cells were harvested, lysed by freeze-thaw cycling, sonicated, and used for recombinant-virus screening. Recombinant MVAs containing the 3.7-kb CHIKV DNA fragment (encoding the structural proteins of CHIKV, C, E3, E2, 6K, and E1 ), with the GFP gene deleted, and transiently coexpressing the B-Gal marker gene (MVA-CHIKV, X-Gal+) were selected by consecutive rounds of plaque purification in DF-1 cells stained with X-Gal (5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside, 400 ug/ml, for three passages in total). In the following plaque purification steps, recombinant MVAs containing the 3.7-kb CHIKV DNA fragment and with the B-Gal gene deleted by homologous recombination from between the left TK arm and the short left TK arm repeat flanking the marker (MVA-CHIKV, X-Gal-) were isolated by three additional consecutive rounds of plaque purification screening for nonstaining viral foci in DF-1 cells in the presence of X-Gal (400 ug/ml). In each round of purification, the isolated plaques were expanded in DF-1 cells for 3 days, and the crude viruses obtained were used for the next plaque purification round. The resulting recombinant virus, MVA-CHIKV (passage 2 [P2] stock), was grown in CEFs, purified through two 36% (wt/vol) sucrose cushions, and titrated by plaque immunostaining assay.

Expression of CHIKV proteins from recombinant MVA-CHIKV by Western blotting. To check the correct expression of CHIKV antigens by the recombinant MVA- CHIKV, monolayers of DF-1 cells were mock infected or infected at 5 PFU/cell with MVA-WT, MVA-GFP, or MVACHIKV. At 24 hpi, cells were lysed in Laemmli buffer, and cells extracts were fractionated in 10% SDS-PAGE and analyzed by Western blotting with mouse polyclonal antibody against E2 (antibody 3E4, kindly provided by Philippe Despres from the Pasteur Institute, Paris, France; diluted 1 :500), mouse monoclonal antibody against E1 -E2 (antibody D3-62; kindly provided by Marc Lecuit and Therese Couderc from the Pasteur Institute, Paris, France; diluted 1 :500), or rabbit polyclonal antibody against E1 (kindly provided by Lisa F. P. Ng from the Singapore Immunology Network, Agency for Science, Technology and Research, Biopolis, Singapore; diluted 1 :1 ,000) to evaluate the expression of the different CHIKV structural proteins. The anti- rabbit HRP-conjugated antibody (Sigma; diluted 1 :5,000), or anti-mouse-HRP- conjugated antibody (Sigma; diluted 1 :2,000), were used as secondary antibodies. The immunocomplexes were detected using an enhanced HRP-luminol chemiluminescence system (ECL Plus; GE Healthcare).

Analysis of MVA-CHIKV

Analysis of genetic stability of recombinant MVA-CHIKV, immunofluorescence studies, and electron microscopy analysis were carried out as previously described in Gomez CE, Perdiguero B, Cepeda MV, Mingorance L, Garcia-Arriaza J, Vandermeeren A, Sorzano CO, Esteban M. 2013. High, broad, polyfunctioned and durable T cell immune responses induced in mice by a novel hepatitis C virus (HCV) vaccine candidate (MVA- HCV) based on modified vaccinia virus Ankara expressing the nearly full-length HCV genome. J. Virol. 87:7282-7300. http://dx.doi.org/10.1 128/JVI.03246-12. (reference 58).

Peptides. CHIKV peptides representative of the capsid (C), E1 , and E2 CHIKV proteins were used in the immunological analysis. Peptides sequences are as follows: ACLVGDJVM (capsid), HSMTNAAVTI (E1 ), and IILYYYELY (E2).

C57BL/6 mouse immunogenicity study. Female C57BL/6 mice (6 to 8 weeks old) were purchased from Harlan Laboratories and stored in a pathogen-free barrier area of the Centra Nacional de Biotecnologia (Madrid, Spain) in accordance with recommendations of the Federation of European Laboratory Animal Science Associations. A homologous MVA prime-MVA boost immunization protocol was performed to assay the immunogenicity of the recombinant MVA-CHIKV. Groups of animals (n 10) were immunized with 1 x10⁷ PFU of MVA-WT or MVA-CHIKV by the intraperitoneal (i.p.) route in 200 ul of PBS. Two weeks later, animals received 2 x 10⁷ PFU of MVA-WT or MVA-CHIKV by the i.p. route in 200ul of PBS. At 10 and 52 days after the last immunization, five mice in each group were sacrificed using carbon dioxide (C02), and their spleens were processed to measure the adaptive and memory immune responses, respectively, to the CHIKV and VACV antigens by an intracellular cytokine staining (ICS) assay. Two independent experiments were performed. The ability of MVA-CHIKV to induce antibody responses was studied in mice at the AFL at Karolinska Institutet, Stockholm, Sweden. Groups of 5- to 6-week-old female C57BL/6 mice (n 5) were immunized with 1 x10⁷ PFU of MVA-WT or MVA-CHIKV by the i.p. route in 200 ul PBS. MVA-WT was administered once, whereas MVA-CHIKV was administered once or twice with a 3-week immunization interval. Serum was collected by tail bleedings 3 weeks after the first immunization and 6 weeks after the second immunization, and antibody responses were analyzed by enzyme-linked immunosorbent assay (ELISA) and a neutralization assay.

CHIKV challenge study. A challenge study to evaluate the efficacy of the recombinant MVA-CHIKV was performed in a biosafety level 3 laboratory at the Astrid Fagraeus Laboratory at Karolinska Institutet, Stockholm, Sweden. Female C57BL/6 mice previously immunized with MVA-WT or MVA-CHIKV to study antibody responses were used (see "C57BL/6 mouse immunogenicity study" above [5 mice/group]). Seven weeks after the last immunization, mice were challenged with a total of 10⁶ PFU of CHIKV (CHIKV isolate LR2006-OPY1 ) via the subcutaneous route in the dorsal side of each hind foot. Blood samples were collected at days 1 to 3 post challenge from tail bleedings, and viremia was analyzed by plaque assay (PFU/ml). Foot swelling (height by breadth) was measured using a digital caliper before challenge and days 4 to 8 after challenge. Two independent experiments were performed.

ICS assay. The magnitudes, polyfunctionality, and phenotypes of the VACV- and CHIKV-specific adaptive and memory T cell responses were analyzed by ICS, as previously described, with some modifications. After an overnight rest, 4 x 10⁶ splenocytes (depleted of red blood cells) were seeded on M96 plates and stimulated for 6 h incomplete RPMI 1640 medium supplemented with 10% FCS containing 1 ul/ml GolgiPlug (BD Biosciences) (to inhibit cytokine secretion), anti-CD107a-Alexa Fluor 488 (BD Biosciences), Monensin (1 x; eBioscience), and 1 ug/ml of the different CHIKV peptides (C, E1 , or E2) or MVA-infected EL4 cells (4 x 10⁵ EL4 cells in 1 x 10⁶ splenocytes; the ratio of MVA infected EL4 cells to splenocytes was equal to 1 :2.5). Then, cells were washed, stained for the surface markers, fixed, permeabilized (Cytofix/ Cytoperm kit; BD Biosciences), and stained intracellular^ with the appropriate fluorochromes. Dead cells were excluded using the violet LIVE/ DEAD stain kit (Invitrogen). For functional analyses, the following fluorochrome-conjugated antibodies were used: CD3-phycoerythrin (PE)-CF594, CD4-allophycocyanin (APC)-Cy7, CD8- V500, IFN-Y-PE-Cy7, tumor necrosis factor alpha (TNF-a)-PE, and interleukin 2 (IL- 2)-APC. In addition, for phenotypic analyses, the following antibodies were used: CD62L-Alexa 700 and CD127-PerCP-Cy5.5. All antibodies were from BD Biosciences. Cells were acquired using a Gallios flow cytometer (Beckman Coulter). Analyses of the data were performed using FlowJo software version 8.5.3 (Tree Star, Ashland, OR). The number of lymphocyte- gated events was 10⁶. After gating, Boolean combinations of single functional gates were then created using FlowJo software to determine the frequency of each response based on all possible combinations of cytokine.

RESULTS

Generation and in vitro characterization of an MVA recombinant expressing CHIKV structural genes (MVA-CHIKV). Fig. 1 A shows a diagram of the MVA-CHIKV genome. The correct insertion and purity of recombinant MVA-CHIKV was confirmed by PCR and DNA sequence analysis. PCR using primers annealing in the VACV TK gene-flanking regions confirmed the presence of the CHIKV structural genes in MVA- CHIKV, with no wild-type contamination in the preparation (Fig. 1 B); in the parental virus MVA-GFP, the GFP was present, and in the MVA-WT, the VACV TK locus was amplified (Fig. 1 B). Moreover, PCRs using primers annealing in the C6L-, K7R-, and A46R-flanking regions confirmed the deletion of the VACV genes C6L, K7R, and A46R, respectively, from MVA-CHIKV (Fig. 1 C), also with no wild-type contamination in the preparation. The proper insertion and correct sequence of the CHIKV structural genes in the TK locus and the correct deletions of the VACV genes C6L, K7R, and A46R were also confirmed by DNA sequencing (data not shown). Additionally, to confirm that MVA-CHIKV constitutively expresses and correctly processes the CHIKV structural polyprotein C-E3-E2-6K-E1 , we carried out a Western blot analysis of DF-1 cells infected with MVA-CHIKV, MVA-GFP, or MVA-WT using specific antibodies that recognize the CHIKV E1 and E2 proteins. The results demonstrated that the viral polyprotein was correctly processed and that MVA-CHIKV expressed the CHIKV E1 and E2 proteins (Fig. 1 D). Expression of the C, E3, and 6K proteins is not shown, since we did not have specific antibodies. However, the proper processing of the polyprotein indicates that the other proteins should be correctly expressed by MVA-CHIKV.

MVA-CHIKV replicates in cell culture. To further determine whether expression of CHIKV structural proteins affects MVA replication in cell culture, we next analyzed the growth of MVACHIKV and MVA-WT in DF-1 cells. The results showed that the kinetics of viral growth were similar between the two viruses (Fig. 2A), indicating that the constitutive expression of CHIKV structural proteins does not impair vector replication under permissive conditions.

MVA-CHIKV is stable in cell culture. To ensure that MVACHIKV can be maintained in cultured cells without the loss of the CHIKV transgene, a stability test was performed. Here, the recombinant MVA-CHIKV was further grown in DF-1 cells infected at a low multiplicity during 9 consecutive passages, and expression of the CHIKV E1 and E2 proteins during the different passages was determined by Western blotting (Fig. 2B). The results showed that MVA-CHIKV efficiently expresses the CHIKV E1 and E2 proteins after successive passages, indicating that the recombinant MVACHIKV is genetically stable. Moreover, we also analyzed the genetic stability of MVA-CHIKV at passage 9 by a plaque immunostaining assay (Fig. 2C). The results showed that MVA-CHIKV titers (in the P2 stock, in the P3 stock, and at passage 9) were similar when plaques expressing VACV antigens or plaques expressing CHIKV antigens were analyzed, confirming the high genetic stability of MVA-CHIKV.

MVA-CHIKV expresses the CHIKV E2 protein in the cytoplasm and in the cell membrane. The expression and intracellular localization of the structural CHIKV E2 protein was also analyzed by immunofluorescence in human HeLa cells infected with MVA-WT or MVA-CHIKV using antibodies specific to E2 and specific probes or antibodies for different proteins and compartments, such as actin (phalloidin), ER (anticalnexin), and the Golgi apparatus or Golgi apparatus-derived membranes, as well as for the plasma membrane (WGA). As shown in Fig. 3, the CHIKV E2 protein is expressed mainly at the plasma membrane and throughout the cytoplasm, where it is localized in discrete accumulations at the viral factories. Moreover, no colocalization between E2 and the ER was observed. Nevertheless, partial colocalization between E2 and actin was found in discrete areas and spots, and it was also detected between E2 and the plasma membrane and between E2 and the Golgi apparatus or Golgi apparatus-derived membranes.

MVA-CHIKV induces specific morphological cell alterations, with the formation and accumulation of Golgi apparatus derived membranes. We next examined the impact of CHIKV structural proteins constitutively expressed by MVA-CHIKV on HeLa cell architecture using transmission electron microscopy analysis. Thus, ultrathin sections of HeLa cells infected with MVA-WT or MVA-CHIKV were visualized by electron microscopy at low and high magnifications (Fig. 4). Both in MVA-WT and in MVA-CHIKV-infected cells, the assembly of immature virus (IV) forms of MVA was detected, and remarkably, in MVA-CHIKV- infected cells, an atypical growth and accumulation of Golgi apparatus-like membranes was observed.

MVA-CHIKV triggers an innate immune response in human macrophages and dendritic cells, inducing type I IFN, proinflammatory cytokines, and chemokine expression. Type I IFN innate immune responses play a critical role in controlling CHIKV viral replication. Thus, to evaluate whether the presence of the CHIKV structural genes in the MVA genome is able to impair the response of innate immune cells to MVA infection, we analyzed by real-time PCR the expression of type I IFN (IFN-β), proinflammatory cytokines (TNF-a), chemokines (MIP-1 a, IP-10, and RANTES), IFN-inducible genes (IFIT1 and IFIT2), and some key cytosolic sensors that lead to antiviral IFN production (RIG-I and MDA-5) by human THP-1 macrophages that had been mock infected or infected for 3 and 6 h with 5 PFU/cell of MVA-WT, MVA-GFP, and MVA-CHIKV (Fig. 5A). The results showed that, compared to MVA- WT, MVA-CHIKV significantly upregulated the mRNA levels of most of these genes (Fig. 5A). To verify these results, we infected human moDCs with 1 PFU/ cell of MVA- WT, MVA-GFP, and MVA-CHIKV and measured IFN-β, TNF-a, MIP-1 a, IP-10, RANTES, IFIT1 , IFIT2, RIG-I, and MDA-5 mRNA levels at 6 h postinfection (Fig. 5B). In the same way as in human THP-1 cells, in moDCs, MVA-CHIKV strongly increased the mRNA levels of most of these genes, compared to levels in MVA-WT-infected cells (Fig. 5B). In conclusion, MVA-CHIKV promotes a robust innate immune response in human macrophages and moDCs by inducing the expression of type I IFN (IFN-β), proinflammatory cytokines (TNF-a), chemokines (MIP-1 a, IP-10, and RANTES), IFN- in-ducible genes (IFIT1 and IFIT2), and some key cytosolic sensors that lead to antiviral IFN production (RIG-I and MDA-5).

MVA-CHIKV induces strong, broad, and polyfunctional adaptive CHIKV-specific T cell immune responses. The role of T cell responses in controlling CHIKV infection is not well known. Thus, to study in vivo the effect of the presence of the CHIKV structural genes in the MVA genome on cellular immunogenicity against CHIKV antigens, we next analyzed the CHIKV specific immune responses induced by MVA- CHIKV in C57BL/6 mice by using a homologous MVA-CHIKV prime (1 x10⁷ PFU) and MVA-CHIKV boost (2x10⁷ PFU) immunization protocol (see Materials and Methods). Animals primed with nonrecombinant MVA-WT and boosted with MVA-WT were used as a control group. Adaptive, CHIKV-specific T cell immune responses elicited by both immunization groups (MVA-WT/MVA-WT and MVA-CHIKV/MVA-CHIKV) were measured 10 days after the last immunization by a polychromatic intracellular cytokine staining (ICS) assay, after the stimulation of splenocytes with specific peptides representative of the C, E1 , and E2 CHIKV proteins. Immunization with MVA- CHIKV/MVA-CHIKV elicited robust adaptive CHIKV-specific CD8⁺ T cell immune responses (determined as the sum of the individual responses obtained for the C, E1 , and E2 CHIKV peptides, producing IFN-γ , TNF-a, and/or IL-2 cytokines, as well as the expression of CD107a on the surfaces of activated T cells as an indirect marker of cytotoxicity) (Fig. 6A). The MVA-CHIKV/MVA-CHIKV immunization group triggered an overall CHIKV-specific immune response mediated only by CD8⁺ T cells, with no CHIKV-specific CD4⁺ T cells detected, indicating the selectivity for CD8⁺ T cells of the peptides used in the ICS assay (Fig. 6A). The pattern of adaptive, CHIKV-specific T cell immune responses showed that CD8+ T cell responses induced by MVACHIKV/ MVA-CHIKV were broad, with most of the responses directed mainly against the E1 and E2 peptides (80%) and to a lesser extent against C (Fig. 6B). Furthermore, compared to MVAWT/MVA-WT, MVA-CHIKV/MVA-CHIKV significantly enhanced the magnitude of C-, E1 - and E2-specific CD8+ T cell responses (P< 0.001 ) (Fig. 6C). Moreover, CHIKV-specific CD8+ T cells producing CD107a, IFN-γ, or TNF-a are the populations most induced by the MVACHIKV/MVA-CHIKV immunization group (Fig. 6D); levels were also of a significantly higher magnitude than those induced by MVAWT/MVA-WT (P< 0.001 ) (Fig. 6D). The quality of the adaptive, CHIKV-specific T cell immune response was characterized in part by the pattern of cytokine production and its cytotoxic potential. Thus, on the basis of the production of CD107a, IFN-y, TNF-a, and IL-2 from CHIKV-specific CD8⁺ T cells, 15 different CHIKV-specific CD8⁺ T cell populations could be identified (Fig. 6E). MVA-CHIKV/MVACHIKV induced a high polyfunctional profile, with 97% of CD8⁺ T cells exhibiting two, three, or four functions (Fig.6E). Furthermore, CD8⁺ T cells producing CD107a plus IFN-y plus TNF-a plus IL- 2, CD107a plus IFN-y plus TNF-a, or IFN-y plus TNF-a were the most abundant populations elicited by MVA-CHIKV/MVA-CHIKV, which, compared to MVA-WT/MVA- WT, also induced significantly higher increases in the percentages of most of the populations (P< 0.001 ) (Fig. 6E). Collectively, these results demonstrate that MVACHIKV induced strong, broad, and polyfunctional adaptive CHIKV-specific CD8⁺ T cell immune responses. Similar findings were observed in two independent experiments. MVA-WT and MVA-CHIKV induce similar magnitudes and polyfunctionalities of adaptive, VACV-specific T cell immune responses. To further characterize the immune responses elicited in mice by both immunization groups (MVA-WT/MVA-WT and MVA-CHIV/MVA-CHIKV), it was of interest to analyze the responses to the MVA vector. Thus, we next measured adaptive, VACV-specific T cell immune responses induced by MVA-WT and MVA-CHIKV 10 days after the boost by an ICS assay (similar to the protocol followed in the analysis of adaptive CHIKV-specific T cell immune responses) after the stimulation of splenocytes with MVA-infected EL4 cells. Both immunization groups triggered an overall adaptive, VACV-specific immune response mediated only by CD8⁺ T cells (determined as the sum of the individual responses producing IFN-γ, TNF-a, and/or IL-2 cytokines, as well as the expression of CD107a on the surfaces of activated T cells as an indirect marker of cytotoxicity); this experiment was carried out with MVA-infected EL4 cells, and the magnitudes of adaptive, VACV-specific CD8⁺ T cell immune responses were similar in the two immunization groups (Fig. 7A). Moreover, VACV-specific CD8⁺ T cells producing CD107a, IFN-γ, or TNF a-were the most induced populations in both immunization groups, and they were induced at similar magnitudes (Fig. 7B). The quality of adaptive, VACV-specific CD8⁺ T cell immune responses was characterized by the production of CD107a, IFN-γ, TNF-a, and/or IL-2, and 15 distinct VACV-specific CD8⁺ T cell populations could be identified (Fig. 7C). VACV-specific CD8⁺ T cell responses were similarly polyfunctional in the two immunization groups, with 88 to 91 % of the CD8⁺ T cells exhibiting two, three, or four functions. CD8+ T cells producing CD107a plus IFN-Y plus TNF-a plus IL-2, CD107a plus IFN-γ plus TNF-a, IFN-γ plus TNF-a, CD107a plus IFN-γ, or only CD107a were the most induced populations elicited by both immunization groups (at similar percentages) (Fig. 7C). In summary, these results showed that MVA-WT and MVACHIKV elicited similar magnitudes and levels of quality of adaptive, VACV-specific CD8⁺ T cell immune responses. Similar findings were observed in two independent experiments.

MVA-CHIKV induces strong, broad, polyfunctional, and durable CHIKV-specific memory T cell immune responses. The durability of a vaccine-induced T cell response is an important feature, since long-term protection is a requirement for prophylactic vaccination. Thus, we analyzed CHIKV-specific memory T cell immune responses elicited by both immunization groups 52 days after the last immunization by the ICS assay in a manner similar to that of the protocol followed in the adaptive phase. Immunization with MVA-CHIKV/MVA-CHIKV elicited robust CHIKV-specific CD8⁺ memory T cell immune responses (determined as the sum of the individual responses to the CHIKV C, E1 , and E2 peptides in splenocytes producing IFN-γ , TNF-a, and/or IL-2 cytokines, and the expression of CD107a on the surfaces of activated T cells was used as an indirect marker of cytotoxicity) (Fig. 8A). As with the results obtained in the adaptive phase, the MVA-CHIKV/MVA-CHIKV immunization group triggered an overall CHIKV-specific memory immune response mediated only by CD8⁺ T cells, with no CHIKV-specific CD4⁺ T cells detected, indicating the selectivity for CD8⁺ T cells of the peptides used in the ICS assay (Fig. 8A). The pattern of CHIKV-specific memory T cell immune responses showed that CD8 T cell responses induced by MVACHIKV/ MVA-CHIKV were broad, with most of the responses being directed mainly against the E1 and E2 peptides (77%) and, to a lesser extent, against C (Fig. 8B), as with the results obtained in the adaptive phase. Furthermore, compared to MVA-WT/MVAWT, MVA-CHIKV/MVA-CHIKV significantly enhanced the magnitudes of C-, E1 -, and E2- specific CD8⁺ T cell responses (PO.001 ) (Fig. 8C). Furthermore, CHIKV-specific CD8⁺ T cells producing CD107a, IFN-γ, or TNF-a are the populations most induced by the MVACHIKV/MVA-CHIKV immunization group (Fig. 8D), and their levels are also of a significantly higher magnitude than those induced by MVA-WT/MVA-WT (PO.001 ) (Fig. 8D), results similar to those obtained in the adaptive phase. The quality of the CHIKV-specific memory T cell immune responses was characterized by analyzing the simultaneous production of CD107a, IFN-γ , TNF-a, and/or IL-2 from CHIKV specific CD8⁺ T cells (Fig. 8E), where 15 distinct CHIKV-specific CD8⁺ T cell populations could be identified. MVA-CHIKV/MVA-CHIKV induced a polyfunctional profile, with 22% of CD8⁺ T cells exhibiting two, three, or four functions (Fig. 8E). Furthermore, again, CD8⁺ T cells producing CD107a plus IFN-γ plus TNF-a plus IL-2, CD107a plus IFN-y plus TNF-a, or CD107a were the most abundant populations elicited by MVACHIKV/ MVA-CHIKV, which also induced increases in the percentages of most of the populations that were significantly higher than those induced by MVA-WT/MVA-WT (P<0.001 ) (Fig. 8E), results similar to those obtained in the adaptive phase.

Moreover, we also determined the phenotype of the C-, E1 -, and E2-specific memory T cells by measuring the expression of CD127 and CD62L surface markers, which allowed us to define the different memory subpopulations: central memory (TCM; CD127/CD62L), effector memory (TEM; CD1277CD62L^"), and effector (TE;CD127^" /CD62L^") T cells (Fig. 8F). The results showed that MVA-CHIKV/MVA-CHIKV induced CHIKV-specific CD8⁺ memory T cells (determined as the sum of the individual responses, i.e., the levels of CD107a, IFN-γ , TNF-a, and/or IL-2 produced against the C plus E1 plus E2 peptides), mainly of the TEM phenotype (78%) (Fig.8F, right). Furthermore, immunization with MVA-CHIKV/MVA-CHIKV induced significantly higher increases in the percentages of CHIKV-specific CD8 TCM,TEM,and TE cells than immunization with MVA-WT (PO.001 ) (Fig. 8F, left). In summary, these results demonstrate that MVA-CHIKV induced strong, broad, polyfunctional, and durable CHIKV-specific CD8⁺ memory T cell immune responses. Similar findings were observed in two independent experiments.

MVA-WT and MVA-CHIKV induce similar magnitudes and polyfunctionalities of VACV-specific memory T cell immune responses. VACV-specific memory T cell immune responses elicited by both immunization groups (MVA-WT/MVA-WT and MVA- CHIV/MVA-CHIKV) were measured 52 days after the boost by ICS assay, which is similar to the protocol followed in the adaptive phase. Similarly to the results obtained in the adaptive phase, both immunization groups triggered overall VACV-specific memory immune responses mediated only by CD8⁺ T cells, determined as the sums of the individual levels of production of CD107a, IFN-γ, TNF-a, and/or IL-2 obtained in MVA-infected EL4 cells (Fig. 9A), with the magnitudes of VACV-specific CD8⁺ memory T cell immune responses being similar in the two immunization groups (Fig. 9A). Moreover, VACV-specific CD8⁺ T cells producing CD107a, IFN-γ, or TNF-a are the most induced populations in both immunization groups, and these populations were also produced at similar magnitudes (Fig. 9B). The quality of VACV-specific CD8⁺ memory T cell immune responses was characterized as the simultaneous production of CD107a, IFN- γ , TNF-α, and/or IL-2 and was assessed by using a protocol like that followed in the adaptive phase, where 15 distinct VACV-specific CD8⁺ T cell populations could be identified (Fig.9C). VACV-specific CD8⁺ T cell responses were similarly polyfunctional in the two immunization groups, with 95% of CD8⁺ T cells exhibiting two, three, or four functions. CD8⁺ T cells producing CD107a plus IFN-γ plus TNF-a plus IL-2, CD107a plus IFN- γ plus TNF-a, IFN-γ plus TNF-a, CD107a plus IFN- Y, or only CD107a were the most induced populations elicited by both immunization groups, and they were also induced at similar percentages (Fig. 9C). Additionally, the analysis of the memory phenotype of VACV specific CD8⁺ memory T cells by measurement of the expression of CD127 and CD62L surface markers showed that both immunization groups elicited mainly VACV-specific CD8⁺ memory T cells of the TEM phenotype and did so at similar magnitudes (Fig. 9D). In summary, these results showed that MVA-WT and MVACHIKV elicited similar magnitudes and levels of quality of VACV specific CD8⁺ memory T cell immune responses. Similar findings were observed in two independent experiments.

MVA-CHIKV induces high titers of neutralizing antibodies against CHIKV.

Antibodies against CHIKV are crucial to control CHIKV infection. Thus, to study the ability of MVA-CHIKV to elicit humoral immune responses against CHIKV, we analyzed the levels of CHIKV envelope-specific antibodies present in the sera of C57BL/6 mice immunized with one or two doses of MVA-CHIKV (see Materials and Methods). Animals immunized with nonrecombinant MVA-WT were used as a control group. The results show that one immunization with MVA-CHIKV elicited high titers of IgG antibodies against CHIKV that were further enhanced by the second immunization (Fig. 10A). Nevertheless, a single immunization with MVA-CHIKV was enough to induce high levels of IgG antibody responses to CHIKV. Next, we analyzed the titers of neutralizing antibodies against CHIKV present in the sera of immunized mice at 6 weeks postboost. The results showed that in contrast to infection with MVA-WT, immunization with one or two doses of MVA-CHIKV resulted in the production of high titers of CHIKV-neutralizing antibodies (Fig. 10B). In comparison to neutralizing antibody titers after a single immunization, titers after two doses were slightly increased. MVA-CHIKV protects mice against CHIKV infection. To study whether MVA-CHIKV protected mice against a CHIKV challenge, we immunized mice with one or two doses of MVACHIKV or one dose of MVA-WT (as a control). Seven weeks after the last immunization, mice were challenged with a high dose of CHIKV in their feet (see Materials and Methods). Protection was evaluated by determining viremia and analyzing foot swelling in CHIKV-challenged mice during the days following challenge. The results showed that no CHIKV infection was developed in MVACHIKV- vaccinated mice, as no viremia was detected postchallenge (Fig. 10C). However, MVA-WT-immunized mice developed CHIKV infection, with high levels of CHIKV (10⁴ to 10⁵ PFU/ml) detected in blood that peaked day 2 postchallenge (Fig. 10B). Moreover, MVA-CHIKV-vaccinated mice did not develop foot swelling after challenge, while MVA-WT-immunized mice developed severe foot swelling that peaked 6 days postchallenge (Fig. 10D). In conclusion, MVA-CHIKV is a highly effective vaccine that protected mice against challenge with a high dose of CHIKV, with no viremia detected in their blood and no signs of inflammation. Remarkably, just a single dose of MVA- CHIKV protected all mice from challenge with CHIKV.

EXAMPLE 2 - PRIME-BOOST IMMUNIZATION STRATEGIES AGAINST CHIKUNGUNYA VIRUS

In this study, in addition to the previously described attenuated A5nsp3 (Hallengard et al, 2013, J Virol epub ahead of print 26 December 2013, doi: 10.1 128/JVI.03453-13) and recombinant MVA-CHIKV vaccine candidates, novel p62- E1 protein and DNA replicon (DREP) based CHIKV vaccines were compared. We evaluated the immunogenicity and efficacy in mice immunized with several homologous and heterologous prime-boost immunization protocols using distinct CHIKV vaccine candidates representing different antigens and vaccine modalities. The DREP platform differs from conventional DNA plasmids in that it encodes the alphavirus (CHIKV) replicase, which drives the production of the subgenomic RNA and thus the expression of the encoded CHIKV antigen. Moreover, DREPs also possess intrinsic adjuvant properties as the replicase and RNA intermediates stimulate the production of type-1 interferons (IFNs) and apoptosis. Promising results have been reported for DNA replicons generated from other alphaviruses including Semliki Forest virus, Sindbis virus and Venezuelan equine encephalitis virus, when used for priming immunizations prior to boosting with other vaccine modalities.

The heterologous prime-boost approach takes advantage of the unique immune profile being induced by the different vaccine platforms. For example, both attenuated and genetic vaccines are being produced endogenously and can thus give rise to T cell- mediated immune responses. In contrast, protein antigens generally lack the ability to elicit cytotoxic T cell responses and are thus limited to the induction of humoral responses. Combining different vaccine strategies in heterologous prime-boost immunizations should induce a more balanced immune response in terms of cellular and humoral immune responses, and enhance the magnitude and quality of immune responses compared to homologous vaccination using a single vaccine modality alone.

The novel DREP and protein CHIKV vaccine candidates described in this study were both immunogenic and efficient when used in priming and boosting immunizations. Furthermore, we have identified several homologous and heterologous prime-boost immunization strategies that were able to elicit protective immune responses in the CHIKV mouse model. This important information will form the basis for immunogen selection in further preclinical and clinical testing of these CHIKV vaccine candidates. MATERIALS AND METHODS

CHIKV vaccine candidates. All vaccine constructs were based on the CHIKV clone LR2006-OPY1 . A DNA replicon vaccine encoding CHIKV envelope (termed DREP- Env) was constructed on the basis of cytomegalovirus (CMV) promoter launched infectious cDNA clone of CHIKV. Briefly, the region corresponding to the nucleotide residues 7565-8350 of CHIKV genome was replaced by sequence 5' CCTAGGCCACCATG 3' by PCR-based mutagenesis followed by multi-step subcloning procedure. The resulting deletion removed the CHIKV capsid coding sequence. In addition, the first amino acid residue of E3 protein (serine) was replaced by methionine.

Construction of the A5nsP3, MVA-CHIKV and p62-E1 CHIKV vaccine candidates have been described previously. Briefly, A5nsP3 is an attenuated CHIKV that was formed by attenuating an infectious CHIKV by a large deletion in the nsP3 gene, MVA-CHIKV was constructed by inserting the cDNA encoding for the structural polyprotein of CHIKV (C, E3, E2, 6K, E1 ) into MVA, and soluble recombinant p62-E1 CHIKV protein was formed by joining the ectodomains of CHIKV p62 and E1 proteins with a glycine serine linker. Wild type (WT) CHIKV was used for challenge and as a reference for immunizations.

Immunizations. Female C57BL/6 mice, 5-6 weeks old, were immunized once or twice with the different CHIKV vaccine candidates DREP-Env, A5nsP3, MVA-CHIKV or CHIKV p62-E1 , using various homologous or heterologous prime-boost immunization protocols with a three weeks immunization interval between prime and boost. A total of 10 μg of DREP-Env DNA diluted in 2x20 μΙ of PBS was injected intradermal^ followed by in vivo electroporation using the DermaVax™ electroporation device as previously described. 105 plaque-forming units (PFU) of A5nsP3 or WT CHIKV diluted in 2x50 μΙ of PBS was injected subcutaneously and 10⁷ PFU of recombinant MVA-CHIKV was diluted in 200 μΙ of PBS and injected intraperitoneally. 1 μg CHIKV p62-E1 protein was diluted in 2x50 μΙ of PBS and administered intramuscularly. CHIKV p62-E1 protein was injected alone or mixed with 25 μΙ of the adjuvant AS03 (1 :10 human dose) (GlaxoSmithKline Biologicals S.A., Rixensart, Belgium) or with 5 μg of the adjuvant Matrix-M (Novavax, MD, USA). Some groups of mice were immunized with two vaccine candidates at the same occasion but at different injection sites. All experiments were performed in at least two separate iterations. B and T cell responses. CHIKV-specific humoral and cellular immune responses induced prior to challenge by the different prime-boost immunization protocols were assessed by enzyme-linked immunosorbent assay (ELISA), neutralization assay and I FN-γ ELISpot (Mabtech AB, Nacka Strand, Sweden) as previously described. In the I FN-γ ELISpot assay, splenocytes from immunized mice were collected 8 days after the last immunization and 10⁵ splenocytes were plated per well. Then, cells were stimulated with 2.5 μς/ιτιΙ of a CD8 T cell-restricted CHIKV E1 peptide (HSMTNAVTI) or 10 μς/ιτιΙ of CHIKV p62-E1 protein. Responses over 25 Spot Forming Units (SFU)/10⁶ splenocytes and a minimum of four times above background were regarded as positive.

Epitope determination and structural localization. Specific linear B cell epitopes on CHIKV E2 glycoprotein were identified via peptide-based ELISA assays. Briefly, heat- inactivated pooled sera from vaccinated mice were screened using overlapping synthetic 18-mer biotinylated-peptides synthesized based on consensus E2 glycoprotein sequence (Mimotopes). Streptavidin-coated 96-wells plates (Nunc) were first blocked with 1 % w/v sodium caseinate (Sigma-Aldrich) in 0.1 % PBST (0.1 % Tween-20 in PBS) for 1 h at room temperature. Peptides were first dissolved in dimethyl sulphoxide (DMSO) to a concentration of 15 \g/m\ before being diluted 1 :1000 in 0.1 % PBST and subsequently coated onto the plates (100 μΙ/well). Heat-inactivated pooled sera were diluted 1 :500 in 0.1 % sodium caseinate in 0.1 % PBST and 100 μΙ were added into each well and incubated for 1 h at 37 °C. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG Abs (Santa Cruz) diluted 1 :10000 in 0.1 % sodium caseinate in 0.1 % PBST were used to detect the bound Abs. Reactions were developed with 3,3',5,5'-Tetramethylbenzidine substrate (Sigma-Aldrich) and terminated by Stop reagent (Sigma-Aldrich). Absorbance at 450 nm was measured using TECAN Infinite® M200 microplate reader and analyzed using Magellan™ software. Peptides were considered positive when the absorbance value is greater than mean + 6 SD that of the non-vaccinated mice. Positive peptides were plotted as signal relative to a positive peptide E2EP3 obtained with WT-CHIKV infected mice sera. Structural data of the E2 glycoprotein were retrieved from the Protein Data Bank (PDB) (ID: 3N42) and visualized using the UCSF CHIMERA software.

Challenge. Seven weeks after the last immunization mice were challenged with a total of 10⁶ PFU of WT CHIKV in 2x20 μΙ PBS subcutaneously in the dorsal side of the feet on both hind legs. Viremia and foot swelling post challenge was determined by plaque assay and by measuring the height and breadth of feet, respectively.

Prime-boost immunizations with different CHIKV vaccine candidates induce superior antibody responses. Homologous and heterologous prime-boost immunizations using different CHIKV vaccines representing different CHIKV vaccine candidates (Table 1 ) were studied in C57BL/6 mice. A single immunization with 10⁵ PFU of WT CHIKV was included for comparison. When combining different vaccine candidates they were either co-administered (at separate sites) in a single immunization (XY) or used in a prime-boost regimen (X,Y). The results showed that all vaccine candidates except p62-E1 protein generated high CHIKV-specific IgG titers (10⁵-10⁶) after a single immunization, and titers were further enhanced upon a booster immunization (titers of 10⁶-10⁷) (Fig.1 1 A). Simultaneous immunization with the p62-E1 protein antigen and either DREP-Env, A5nsP3 and MVA-CHIKV seemed to slightly augment IgG titers. However, using p62-E1 protein as a boost post prime immunization with the other CHIKV vaccine candidates induced even higher titers. Finally, most prime-boost immunized mice induced stronger antibody responses than mice inoculated once with WT CHIKV.

Vaccine Description Dose and route

D DREP-Env DNA replicwi (DIEP) encoding 10 u£ i.d.+EP

nsPl-nsP4 and E1-E3 (Ertv)

V A5nsP3 CHIKV attenuated by a large 10^s PFU s.c.

deletion in nsP3

M MVA-CHIKV Recombinant MVA encoding 10⁷ PFU «,p.

caps id and E1-E3

Table 1 Similarly to DREP-Env, the majority of CHIKV-specific antibody responses induced by the different prime-boost immunizations were of lgG2c rather than lgG1 isotype (Fig. 1 1 B) with the exception of mice immunized with p62-E1 protein, which had a balanced lgG1/lgG2c response, or even a slightly pronounced lgG1 response post homologous prime-boost immunizations. Furthermore, when DREP-Env was co-immunized with p62-E1 protein a balanced Th1/Th2 response was obtained. In contrast, when DREP- Env was first used as prime and then later boosted with p62-E1 protein a Th1 -type response was induced. Similar results were obtained when using AnsP3 plus p62-E1 vs AnsP3 prime followed by AnsP3 and p62-E1 boost.

Most vaccine candidates induced good neutralizing NT50 antibody titers in the range of 10³-10⁵. The DREP-Env induced NT50 titers in the range of 10²-10³ with some animals having no NT50 antibodies. These animals correspond to those that had binding (ELISA) lgG1 antibodies below or close to 10⁴ (Fig. 1 1 A,B). The lack of NT50 antibodies in some of the DREP-Env samples does not necessary mean that the animals were devoid of them but could be explained by the fact that the sensitivity of the assay is 1 :100 (first dilution). Another exception was p62-E1 protein alone given once that did not induce any detectable NT50 antibodies. Prime boost immunization generally augmented NT50 titers with titers being the highest when priming was performed with DREP-Env followed by boosting with MVA (around 10⁵). In contrast, boosting A5nsP3, DREP-Env or MVA-CHIK with p62-E1 protein did not significantly increase NT50 titers. CHIKV vaccine candidates protect against CHIKV infection. The efficacy of the different CHIKV vaccine candidates to protect from challenge with a high dose (10⁶ PFU) WT CHIKV was studied in a challenge model using the established immunization schedules. The results showed that all the immunized animals were protected from CHIKV and, with the exception of nal^'ve mice, only one out of five mice immunized with DREP-Env once and 80% mice immunized once with p62-E1 protein that induced detectable viremia post challenge, all other animals were completely protected (Fig. 12A). Similar to viremia, all the immunized animals developed low foot swelling, except mice immunized once with CHIKV p62-E1 protein that had pronounced foot swelling (Fig. 12B). Interestingly, there were clear correlations between both anti-CHIKV IgG / NT50 titers and vi rem ia / foot swelling (P < 0.001 ) (Fig. 12C and D), and an IgG titer of >10⁴ seemed to be protective.

EXAMPLE 3 - Kinetic and phenotypic analysis of CD8⁺ T cell responses after priming with alphavirus replicons and homologous or heterologous booster immunizations

In the present study, we demonstrate the induction of different subsets of CD8⁺ T cells by prime-boost immunization of various replication-proficient attenuated alphavirus (CHIKV) and booster immunizations with poxvirus vectors and / or protein antigen vaccine candidates. This may have implications for the design of efficient vaccination regimens in the clinic.

Materials and Methods

MVA-CHIKV, DREP, and p62-E1 vaccines used were as described in example 2. Immunisations and analysis were also as described.

We wanted to study whether immunization with a WT alphavirus that was capable of productive infection would result in the induction of memory T cells. We used a replication-competent CHIKV. Infection of adult immunocompetent mice by CHIKV has pathogenic consequences, but is not associated with mortality.

For this purpose, we infected C57BL/6 mice with CHIKV and characterized the phenotype of the CD8+ T cell response 8 days after the inoculation using an MHC class I pentamer loaded with a CHIKV E1 Env-derived peptide to identify antigen- specific CD8+ T cells. The phenotype of the CHIKV-specific CD8+ T cell response after wild-type CHIKV infection (Fig. 13) showed that most of the CD8+ T cells displayed a Te or Tern phenotype, and 12% were of the Tern phenotype (Fig. 13, top pie chart). Furthermore, half of the CD8+ T cells were CD27+CD43+, whereas CD27+CD43- T cells constituted one quarter of the CD8+ T cell response (Fig. 13, bottom pie chart).

Alphavirus replicon priming followed by heterologous booster immunizations induce different CD8+ T cell subpopulations.

Given the fact that there was very little difference between VREP and WT CHIKV infection, we wanted to analyze whether we would obtain different subpopulations of CD8+ T cells when boosting with different vaccine candidates following alphavirus immunization. Different vectors encoding a common immunogen can be combined in heterologous prime-boost regimens in order to increase immunogenicity and avoid buildup of antivector immunity. For this purpose, we studied the response induced by different booster vaccinations given after a DREP-Env prime. The DREP-Env vaccine candidate contains all the genomic sequence of CHIKV, but the gene encoding for the capsid protein. Hence, transfection with DREP-Env does not result in production of new virions. C57BL/6 mice were primed with DREP-Env and then boosted either homologously with DREP-Env, or heterologously with either CHIKV p62-E1 protein, MVA-CHIKV or both protein and MVA-CHIKV at the same time. The magnitude of the CHIKV-specific CD8+ T cell responses are greatly increased with heterologous prime-boost immunization. Compared to a homologous DREP-Env boost, the acute response is increased 9-fold with a CHIKV p62-E1 protein boost and 16-fold with an MVA-CHIKV boost. Boosting with both CHIKV p62-E1 protein and MVA-CHIKV results in an 18-fold increase. The phenotypes of CHIKV-specific CD8+ T cell responses were characterized 8 days after boost as described above. The results demonstrated that administering DREP-Env twice induced a CHIKV- specific CD8+ T cell response with a smaller proportion of the Te phenotype and instead a higher proportion of the Tern phenotype (Fig. 13, top pie chart). As the infection rate with viral particles is higher than the transfection rate with DNA (our observations in vitro), these results are in line with current knowledge that weaker signal strength induces a higher degree of Tern. Furthermore, the proportion of CD27+CD43+ CHIKV-specific CD8+ T cells was smaller whereas a higher proportion of CD27+CD43- cells was induced (Fig. 13, bottom pie chart).

Administering a heterologous boost revealed that the phenotype of the CHIKV-specific CD8+ T cell response was close to identical in groups boosted with MVA-CHIKV, either alone or together with CHIKV p62-E1 protein, with the CD8+ T cells mostly of the Tern phenotype, and only a small proportion of the Tern phenotype (Fig. 13, top pie charts). On the other hand, mice given a CHIKV p62-E1 protein boost, without MVA-CHIKV, developed a larger proportion of CD8+ T cells of the Tern phenotype and a smaller proportion of the Tern phenotype (Fig. 13, top pie charts), again in line with the hypothesis that a stronger signal favors Tern development . The proportion of Tern after a CHIKV p62-E1 protein boost was similar to that after a DREP-Env boost, about one third. Moreover, mice given a heterologous boost had similar proportions of CD8+ Te cells in the range of 22-29%, whereas mice given a homologous DREP-Env boost triggered only 7% of CD8+ Te cells (Fig. 13, top pie charts).

Furthermore, analysis of the CD27/CD43 staining revealed the induction of four distinct CHIKV-specific CD8+ T cell subpopulations that were of similar size after MVA-CHIKV or MVA-CHIKV + CHIKV p62-E1 protein boost (Fig. 13, bottom pie charts). However, boosting with CHIKV p62-E1 protein only also induced four CD8+ T cell subpopulations, although with a slightly larger proportion of the CD27+CD43- subset, similar to what was observed after a homologous DREP-Env boost (Fig. 13, bottom pie charts).

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Claims

CLAIMS:

1. A composition comprising a nucleic acid comprising a nucleotide sequence encoding an attenuated poxvirus vector and one or more CHIKV structural proteins.

2. The composition of claim 1 , wherein the nucleotide sequence encodes at least the E3 and E2 CHIKV structural proteins.

3. The composition of claim 1 or 2 wherein the nucleotide sequence encodes all of the C, E3, E2, 6K, and E1 CHIKV structural proteins.

4. The composition of any preceding claim wherein the nucleotide sequence further encodes one or more CHIKV replicase proteins.

5. The composition of any preceding claim wherein the poxvirus vector is a modified vaccinia virus Ankara (MVA) vector.

6. The composition of claim 5 wherein the MVA vector is further modified by deletion of one or more vaccinia immunomodulatory genes.

7. The composition of claim 6 wherein the MVA vector is modified by deletion of all of the immunomodulatory genes C6L, K7R, and A46R.

8. An attenuated poxvirus vector expressing one or more CHIKV structural genes.

9. The vector of claim 8, wherein the vector expresses at least the E3 and E2 CHIKV structural genes.

10 The vector of claim 8 or 9 wherein the vector expresses all of the C, E3, E2, 6K, and E1 CHIKV structural genes.

1 1 . The vector of any of claims 8 to 10 wherein the vector expresses one or more CHIKV replicase genes.

12. The vector of any of claims 8 to 1 1 wherein the poxvirus vector is a modified vaccinia virus Ankara (MVA) vector.

13. The vector of claim 12 wherein the MVA vector is further modified by deletion of one or more vaccinia immunomodulatory genes.

14. The vector of claim 13 wherein the MVA vector is modified by deletion of all of the immunomodulatory genes C6L, K7R, and A46R.

15. A vaccine composition comprising an attenuated poxvirus vector according to any of claims 8 to 14.

16. The vaccine composition of claim 15, further comprising a heterologous vaccine.

17. The vaccine composition of claim 16 wherein the heterologous vaccine is selected from a vaccine comprising a covalently linked p62-E1 heterodimer; or a DNA-replicon (DREP) vaccine comprising a nucleic acid comprising a nucleotide sequence comprising CHIKV genomic sequences operably linked to a promoter sequence.

18. A vector according to any of claims 8 to 14 for use as a vaccine.

19. The use of an attenuated poxvirus vector according to any of claims 8 to 14 in the manufacture of a vaccine.

20. A method of immunising a subject against CHIKV infection, the method comprising administering a vaccine comprising an attenuated poxvirus vector according to any of claims 8 to 14, or administering a vaccine composition according to any of claims 15 to

17, to a subject.

21 . The method of claim 20 further comprising administering a subsequent dose of a CHIKV vaccine to the subject.

22. The method of claim 21 wherein the subsequent dose is of a vaccine comprising an attenuated poxvirus vector according to any of claims 8 to 14, or a vaccine composition according to any of claims 15 to 17.

23. The method of claim 20 wherein the subsequent dose is of a heterologous vaccine.

24. The method of claim 23 wherein the heterologous vaccine is selected from a vaccine comprising a covalently linked p62-E1 heterodimer; or a DNA-replicon (DREP) vaccine comprising a nucleic acid comprising a nucleotide sequence comprising CHIKV genomic sequences operably linked to a promoter sequence.

25. The method of claim 24 wherein the vaccine is a DREP vaccine, and the CHIKV genomic sequences comprise genes coding for the envelope proteins (E3, E2, 6K, and

E1 ), but do not include genes coding for the capsid (C) protein.

26. A kit comprising first and second doses of CHIKV vaccine, wherein the first dose is of an attenuated poxvirus vector according to any of claims 8 to 14, or a vaccine composition according to any of claims 15 to 17.

27. The kit of claim 26 wherein the second dose is of an attenuated poxvirus vector according to any of claims 8 to 14, or a vaccine composition according to any of claims 15 to 17.

28. The kit of claim 26 wherein the second dose is of a vaccine comprising a covalently linked p62-E1 heterodimer; or a DNA-replicon (DREP) vaccine comprising a nucleic acid comprising a nucleotide sequence comprising CHIKV genomic sequences operably linked to a promoter sequence.

29. The kit of any of claims 26 to 28 wherein the doses are provided in sealed vials, or in preloaded syringes.