# Patent

## Title
Isolated and purified strains of Chikungunya virus and polynucleotides and polypeptides sequences, diagnostic and immunogenical uses thereof

## Abstract

The present invention concerns wild-strains of Chikungunya virus isolated from patients exhibiting severe forms of infection and stemming from a human arbovirosis epidemy. The present invention also concerns polypeptide sequences and fragment thereof derived from their genome, the polynucleotide encoding same and their use as diagnostic products, as vaccine and/or as immunogenic compositions.

## Background
This is a Division of application Ser. No. 12/225,111, filed Sep. 29, 2009, which is a §371 of PCT/IB2007/001716, which claims the benefit of Canadian Application No. 2,545,597, filed Apr. 4, 2006 and Canadian Application No, 2,538,898, filed Mar. 15, 2006, all of which are incorporated herein by reference.


FIELD OF THE INVENTION
The present invention concerns wild-strains of Chikungunya virus isolated from patients exhibiting severe forms of infection and stemming from a human arbovirosis epidemy. The present invention also concerns polypeptide sequences and fragment thereof derived from their genome, the polynucleotide encoding same and their use as diagnostic products, as vaccine and/or as immunogenic compositions.
BACKGROUND OF THE INVENTION
Chikungunya virus (CHIKV) is a mosquito-transmitted Alphavirus belonging to family Togaviridae [1,2]. It was isolated for the first time from a Tanzanian outbreak in 1952 [3]. It is responsible for an acute infection of abrupt onset, characterized by high fever, arthralgia, myalgia, headache and rash [4,5]. Poly-arthralgia, the pathognomonic sign of the disease, is very painful. Symptoms are generally self-limiting and last 1 to 10 days. However, arthralgia or arthritic symptoms may persist for months or years. In some patients, minor hemorrhagic signs such as epistaxis or gingivorrhagia have also been described.
CHIKV is geographically distributed in Africa, India and South East Asia. In Africa, the virus is maintained through a sylvatic transmission cycle between wild primates and mosquitoes such as Aedes luteocephalus, Ae. furcifer or Ae. taylori [4]. In Asia, CHIKV is mainly transmitted from human to human by Ae. aegypti and to a lesser extent by Ae. albopictus through an urban transmission cycle. Since the 1952 Tanzania outbreak, CHIKV has caused outbreaks in East Africa (Tanzania, Uganda), in Austral Africa (Zimbabwe, South Africa), in West Africa (Senegal, Nigeria) and in Central Africa (Central African Republic, Democratic Republic of the Congo) [4]. The most recent epidemic re-emergence was documented in 1999-2000 in Kinshasa, where an estimated 50,000 persons were infected [6]. Since the first documented Asian outbreak in 1958 in Bangkok, Thailand, outbreaks have been documented in Thailand, Cambodia, Vietnam, Laos, Myanmar, Malasia, Philippines and Indonesia [4,5]. The most recent epidemic re-emergence was documented in 2001-2003 in Java after 20 years [7]. Either in Africa or Asia, the re-emergence was unpredictable, with intervals of 7-8 years to 20 years between consecutive epidemics.
Since the end of 2004, Chikungunya virus (CHIKV) has emerged in the islands of the south-western Indian Ocean. Between January and March 2005, more than 5,000 cases were reported in Comoros. Later in 2005, the virus has circulated in the other islands, i.e Mayotte, Seychelles, Réunion and Mauritius. Starting in December 2005, the rainy season gave rise to a renewed epidemic circulation of the virus. Between January 1st and Mar. 1, 2006, 2,553, 3,471, and 4,650 cases have been reported in Mauritius, Mayotte and Seychelles (Mar. 12, 2006). The most affected island is Reunion with an estimated 212,000 cases until Mar. 12, 2006 (total population: 770,000). More recently, circulation of the virus has been documented in Madagascar.
In Reunion Island, the first documented cases were patients coming 1 ng back from Comoros in March 2005. More than 3,000 cases were reported from March to June. The transmission was limited during the winter season of the southern hemisphere and a major upsurge has been observed since mid-December, with an estimated 210,000 cases between January and March 2006 [8]. Since March 2005, 85 patients with a confirmed CHIKV infection have developed severe clinical signs (meningoencephalitis or fulminant hepatitis) which justified hospitalization in an intensive care unit. Several cases of meningo-encephalitis and major algic syndrome have been associated with vertical transmission of the virus 9.
To date, two CHIKV complete nucleotide sequences have been determined, for the strains Ross (accession no: AF490259) and S27 [9], both isolated from patients during the 1952 Tanzania outbreak. Another complete nucleotide sequence has been determined for a strain isolated in Ae. furcifer during the Senegal 1983 outbreak (accession no AY726732). Khan and coworkers [9] showed that the S27 genome was similar in its structure to that of other alphaviruses and that O'nyong-nyong virus (ONN) was the closest relative to CHIKV. In addition, phylogenetic analyses based on partial E1 sequences from African and Asian isolates revealed the existence of three distinct CHIKV phylogroups, one containing all isolates from West Africa, one containing isolates from Asia, and one corresponding to Eastern, Central and Southern African isolates [10]. Strains isolated in 1999-2000 in the Democratic Republic of the Congo belonged to the latter phylogroup [6].
SUMMARY OF THE INVENTION
An aspect of the invention is to provide new diagnostic and immunologic tools against CHIK virus associated diseases, such as arbovirosis.
Such an aspect is particularly achieved by providing an isolated and purified wild strain of Chikungunya virus (CHIK) capable of in vitro infecting human cells; and its use for the detection of a CHIKV associated to an arbovirus, or for the preparation of a composition that prevents and/or treats an arbovirus.
Another aspect of the invention concerns an isolated and purified strain of CHIKV comprising at least one mutation in structural protein E1 and/or structural protein E2; and its use for the detection of a CHIKV associated to an arbovirus, or for the preparation of a composition that prevents and/or treats an arbovirus.
Another aspect of the invention concerns an isolated and purified polynucleotide comprising all or part of the sequence of SEQ ID NOS: 1, 2, 3, 4, 5 or 6; and its use for the detection of a CHIKV associated to an arbovirus, or for the preparation of a composition that prevents and/or treats an arbovirus.
Another aspect of the invention concerns a fragment of the polynucleotide of the invention wherein it codes for the ectodomain of glycoprotein E2 or E1; and its use for the detection of a CHIKV associated to an arbovirus, or for the preparation of a composition that prevents and/or treats an arbovirus.
Other aspects of the invention concern a vector or plasmid comprising a polynucleotide or fragment contemplated by the present invention, and host cell comprising said vector or plasmid; and its use for the detection of a CHIKV associated to an arbovirus, or for the preparation of a composition that prevents and/or treats an arbovirus.
Yet another aspect of the invention concerns a purified polypeptide encoded by a polynucleotide or fragment of the invention; and its use for the detection of a CHIKV associated to an arbovirus, or for the preparation of a composition that prevents and/or treats an arbovirus.
A further aspect of the invention concerns a monoclonal or polyclonal antibody or fragment thereof that specifically binds to a polypeptide of the invention; and its use for the detection of a CHIKV associated to an arbovirus, or for the preparation of a composition that prevents and/or treats an arbovirus.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Localization of the E1 changes on the 3D structure modeled from the crystal structure of SFV E1 [43] [19].
A) Ribbon diagram of E1, with domain I colored red, domain II yellow and domain III, blue. Green tubes mark the disulfide bonds. The fusion peptide, at the tip of the molecule (in domain II) is colored orange and labeled. The N-terminus and the C-terminus observed in the crystal (which is 30 aa upstream of the transmembrane region) are also labeled. The 2 unique changes observed in the Indian Ocean isolates are indicated by stars and labeled: positions 226 (white) and 284 (magenta).
B) Partial representation (one octant, slightly extended) of the icosahedral E1 scaffold at the surface of the virion, viewed down a 5-fold symmetry axis. One E1 protomer is highlighted in colors, as in A); all the others are represented in grey. The location of some of the icosahedral symmetry axes are drawn as solid black symbols: pentagon for 5-fold axis, triangle for 3-fold axes, ellipse for 2-fold axes (which in the T=4 lattice of alphaviruses are coincident with quasi 6-fold axes). Open triangles indicate roughly the location of the E2 trimers that interact tightly with E1, covering domain II and the fusion peptide, and presenting the main antigenic sites. The open triangles mark also quasi 3-fold symmetry axes of the T=4 surface icosahedral lattice. A magenta ball marks the location of Glu 284, at an inter-E1 protomer contact site. This contact is propagated 240 times at the surface lattice (note all pink balls drawn on the grey protomers). Note that the fusion peptide, in orange, is pointing up and away from contacts with other E1 protomers. This is more easily seen at the periphery of the virion, where one of them is labeled (FP). In the virion, this region of E1 is not accessible, covered underneath the E2 molecule [19].
FIG. 2: Phylogenetic relationships among chikungunya isolates based on partial E1 nucleotide sequences. Isolates from the Indian Ocean outbreak (Reunion, Seychelles, Mayotte, Mauritius, Madagascar) represent a distinct Glade within a large East, Central and South African (ECSA) phylogroup. Bootstrap resampling values are indicated at major nodes. The branch leading to West-African phylogroup (of length approx. 15%) was shortened for convenience.
FIG. 3: Proposed evolutionary scenario of chikungunya virus isolates from 1 the Indian Ocean outbreak. The scenario is based on six genome sequences determined by direct sequencing of RT-PCR products obtained using RNA extracts as templates; the sequences thus correspond to consensus sequences (Cons. Seq.) of the possible mixture of coexisting genomes (quasispecies). Inset: number of cases of E1-226A and E1-226V at different time intervals in Reunion Island, based on partial E1 sequences. E1-226V was observed in consensus sequences 2, 3 and 4, and therefore most E1-226V isolates genotyped based on partial E1 sequences are likely related to these genotypes. However, the independent appearance of E1-226V in other genotypes cannot be excluded. Int.: intermediate sequence. The location, size and relative position of the Islands and the African border are indicative. Consensus sequence 1 was obtained from a Reunion patient who traveled back from Comoros in March 2005, and from a Reunion Island patient. Sequences 2 to 4 were sampled in Reunion Island; sequence 5 was sampled in the Seychelles.
FIG. 4 shows the nucleotide sequence of the genome of a CHIK virus strain according to a preferred embodiment of the invention and more specifically for the preferred strain named 05.115 (SEQ ID NO:1).
FIG. 5 shows the nucleotide sequence of the genome of a CHIK virus strain according to a preferred embodiment of the invention and more specifically for the preferred strain named 05.209 (SEQ ID NO:2).
FIG. 6 shows the nucleotide sequence of the genome of a CHIK virus strain according to a preferred embodiment of the invention and more specifically for the preferred strain named 06.21 (SEQ ID NO:3).
FIG. 7 shows the nucleotide sequence of the genome of a CHIK virus strain according to a preferred embodiment of the invention and more specifically for the preferred strain named 06.27 (SEQ ID NO:4).
FIG. 8 shows the nucleotide sequence of the genome of a CHIK virus strain according to a preferred embodiment of the invention and more specifically for the preferred strain named 06.49 (SEQ ID NO:5).
FIG. 9 shows the nucleotide sequence of the genome of a CHIK virus strain according to a preferred embodiment of the invention and more specifically for the preferred strain named 05.61 (SEQ ID NO:6).
FIG. 10 shows a nucleotide sequence of a fragment of a CHIK virus according to a preferred embodiment of the invention, and more specifically a fragment which codes for the ectodomain of the glycoprotein E2 of the preferred strain named 06.21 (SEQ ID NO:7).
FIG. 11 shows a nucleotide sequence of a fragment of a CHIK virus according to a preferred embodiment of the invention, and more specifically a fragment which codes for the ectodomain of the glycoprotein E2 of the preferred strain named 06.27 (SEQ ID NO:8).
FIG. 12 shows a nucleotide sequence of a fragment of a CHIK virus according to a preferred embodiment of the invention, and more specifically a fragment which codes for the ectodomain of the glycoprotein E2 of the preferred strain named 06.49 (SEQ ID NO:9).
FIG. 13 shows a nucleotide sequence of a fragment of a CHIK virus according to a preferred embodiment of the invention, and more specifically a fragment which codes for the ectodomain of the glycoprotein E2 of the preferred strain named 06.115 (SEQ ID NO:10).
FIG. 14 shows a nucleotide sequence of a fragment of a CHIK virus according to a preferred embodiment of the invention, and more specifically a fragment which codes for a soluble form of the glycoprotein E2 of the preferred strain named 06.21 (SEQ ID NO:11).
FIG. 15 shows a nucleotide sequence of a fragment of a CHIK virus according to a preferred embodiment of the invention, and more specifically a fragment which codes for a soluble form of the glycoprotein E2 of the preferred strain named 06.27 (SEQ ID NO:12).
FIG. 16 shows a nucleotide sequence of a fragment of a CHIK virus according to a preferred embodiment of the invention, and more specifically a fragment which codes for a soluble form of the glycoprotein E2 of the preferred strain named 06.49 (SEQ ID NO:13).
FIG. 17 shows a nucleotide sequence of a fragment of a CHIK virus according to a preferred embodiment of the invention, and more specifically a fragment which codes for a soluble form of the glycoprotein E2 of the preferred strain named 06.115 (SEQ ID NO:14).
FIG. 18 shows an amino acid sequence of a preferred CHIK virus polypeptide according to a preferred embodiment of the invention, and related more specifically to the ectodomain of the glycoprotein E2 of the preferred strain named 06.21 (SEQ ID NO:15).
FIG. 19 shows an amino acid sequence of a preferred CHIK virus polypeptide according to a preferred embodiment of the invention, and related more specifically to the ectodomain of the glycoprotein E2 of the preferred strain named 06.27 (SEQ ID NO:16).
FIG. 20 shows an amino acid sequence of a preferred CHIK virus polypeptide according to a preferred embodiment of the invention, and related more specifically to the ectodomain of the glycoprotein E2 of the preferred strain named 06.49 (SEQ ID NO:17).
FIG. 21 shows an amino acid sequence of a preferred CHIK virus polypeptide according to a preferred embodiment of the invention, and related more specifically to the ectodomain of the glycoprotein E2 of the preferred strain named 06.115 (SEQ ID NO:18).
FIG. 22 shows an amino acid sequence of a preferred CHIK virus polypeptide according to a preferred embodiment of the invention, and related more specifically to a soluble form of the glycoprotein E2 of the preferred strain named 06.21 (SEQ ID NO:19).
FIG. 23 shows an amino acid sequence of a preferred CHIK virus polypeptide according to a preferred embodiment of the invention, and related more specifically to a soluble form of the glycoprotein E2 of the preferred strain named 06.27 (SEQ ID NO:20).
FIG. 24 shows an amino acid sequence of a preferred CHIK virus polypeptide according to a preferred embodiment of the invention, and related more specifically to a soluble form of the glycoprotein E2 of the preferred strain named 06.49 (SEQ ID NO:21).
FIG. 25 shows an amino acid sequence of a preferred CHIK virus polypeptide according to a preferred embodiment of the invention, and related more specifically to a soluble form of the glycoprotein E2 of the preferred strain named 06.115 (SEQ ID NO:22).
FIG. 26: Repeat Sequence Elements found in the 3′NTR region
A. Alignment of Repeat Sequence Elements found in the 3′NTR region of chikungunya virus genome. All sequences form conserved and stable stem-loop structures in which the less conserved nucleotides around position 20 constitute the loop. Three RSE are found in all chikungunya genomes. The first one (RSE1) is inserted before the internal poly-A sequence of S27 genome [9], whereas the two others are found downstream this motif.
B. Predicted secondary structure for RSE1 of isolate 05-115.
FIG. 27: Focus phenotype of chikungunya viruses on AP61 cells by Focus Immunoassay
Mosquito AP61 cells in 24-well plates were infected with CHIK virus stocks grown on mosquito cells (virus titers 2−5×10%8 FFU. mL-1) at 0.0001 (top well) or 0.00001 (bottow well) multiplicity of infection. Infected cells were overlaid with CMC in Leibovitz L15 growth medium with 2% FBS for 2 days to allow focus development at 28° C. The cells were fixed with 3% PFA in PBS, permeabilized with Triton X-100 in PBS, and foci of CHIK virus replication were immunostained with mouse anti-CHIK HMAF (dilution 1:2,000) and peroxidase-conjugated goat anti-mouse Ig (dilution 1:100).
FIG. 28: Viral preparation containing pE2. pE2 proteins detected by anti-CHIK antibodies.
FIG. 29: Alignment of nucleotide sequences encoding soluble form of E2 glycoprotein (E2-1 to E2-361) from Indian Ocean CHIK virus strains -21, -27, -49 and -115.
FIG. 30: Primer sequences (SEQ ID NO:79 and 80) used for the amplification and cloning of the soluble form of the E2 (E2-1 to E2-364) (N-terminal and C-terminal nucleic acid fragment: SEQ ID NO:81 and 82; N-terminal and C-terminal protein fragment: SEQ ID NO:83 and 84) from CHIK virus into the shuttle vector pMT2/BiP/V5-HisA.
FIG. 31: SDS-PAGE showing CHIK-sE2 staining by Coomassie blue.
FIG. 32: Immunoblot analysis of highly purified CHIK sE2 protein.
FIG. 33: Construct of the TRIP vector expressing the secreted soluble form of the E2 glycoprotein (sE2) from Chikungunya virus La Reunion 05 strains.
FIG. 34 shows the nucleotide sequence coding the secreted soluble form of the E2 glycoprotein (sE2) into the TRIP vector (SEQ ID NO:85).
FIG. 35: Immunofluorescent (IF) assay using anti-CHIK antibodies on TRIP/CHIK.sE2-transduced 293 cells.
FIG. 36 shows a direct ELISA with 10−4 mL of enriched pE2 protein per well. Antigens were tested respectively with a mouse anti-DEN1 (dilution 1:1000), anti-WN (dilution 1:1000) and anti-CHIK (dilution 1:10 000).
FIG. 37 shows an amino acid sequence of the ORF 2 (structural proteins) of the CHIK S27 strain (GenBank AF339485; SEQ ID NO: 23).
FIG. 38 shows an amino acid sequence of the ORF 2 (structural proteins) of a preferred CHIK virus according to a preferred embodiment of the invention, namely the 05.61 strain (SEQ ID NO: 24).
FIG. 39 shows an amino acid sequence of the ORF 2 (structural proteins) of a preferred CHIK virus according to a preferred embodiment of the invention, namely the 05.209 strain (SEQ ID NO: 25).
FIG. 40 shows an amino acid sequence of the ORF 2 (structural proteins) of a preferred CHIK virus according to a preferred embodiment of the invention, namely the 05.115 strain (SEQ ID NO: 26).
FIG. 41 shows an amino acid sequence of the ORF 2 (structural proteins) of a preferred CHIK virus according to a preferred embodiment of the invention, namely the 06.49 strain (SEQ ID NO: 27).
FIG. 42 shows an amino acid sequence of the ORF 2 (structural proteins) of a preferred CHIK virus according to a preferred embodiment of the invention, namely the 06.27 strain (SEQ ID NO: 28).
FIG. 43 shows an amino acid sequence of the ORF 2 (structural proteins) of a preferred CHIK virus according to a preferred embodiment of the invention, namely the 06.21 strain (SEQ ID NO: 29).
FIG. 44 shows an amino acid sequence of the ORF 1 (non-structural proteins) of a preferred CHIK virus according to a preferred embodiment of the invention, namely the 05.61 strain (SEQ ID NO: 30).
FIG. 45 shows an amino acid sequence of the ORF 1 (non-structural proteins) of a preferred CHIK virus according to a preferred embodiment of the invention, namely the 05.209 strain (SEQ ID NO: 31).
FIG. 46 shows an amino acid sequence of the ORF 1 (non-structural proteins) of a preferred CHIK virus according to a preferred embodiment of the invention, namely the 05.115 strain (SEQ ID NO: 32).
FIG. 47 shows an amino acid sequence of the ORF 1 (non-structural proteins) of a preferred CHIK virus according to a preferred embodiment of the invention, namely the 06.49 strain (SEQ ID NO: 33).
FIG. 48 shows an amino acid sequence of the ORF 1 (non-structural proteins) of a preferred CHIK virus according to a preferred embodiment of the invention, namely the 06.27 strain (SEQ ID NO: 34).
FIG. 49 shows an amino acid sequence of the ORF 1 (non-structural proteins) of a preferred CHIK virus according to a preferred embodiment of the invention, namely the 06.21 strain (SEQ ID NO: 78).
FIG. 50: Evaluation of anti-CHIK E2 Mab reactivity by ELISA.
FIG. 51: Evaluation of anti-CHIK E2 MAb reactivity on CHIK virions by ELISA.
FIG. 52: Immunofluorescence (IF) analysis of anti-CHIK E2 Mab reactivity on CHIKV-infected Vero cells.
FIG. 53: Immunofluorescence (IF) analysis of anti-CHIK E2 Mab reactivity on TRIP/CHIK.sE2-transduced 293A cells.
FIG. 54: Anti-CHIK E2 Mab binding on cell surface of CHIK virus-infected Vero cells by FACS analysis.
FIG. 55: Western blot analysis of CHIKsE2 expression in TRIP/CHIK.sE2-transduced 293A cells.



DETAILED DESCRIPTION OF THE INVENTION
In the present study, the inventors determined the nearly complete nucleotide sequences of viruses isolated from six patients originating from Reunion and Seychelles Islands. The present invention allows to determine the genome structure as well as the unique molecular features of the Indian Ocean outbreak isolates, which distinguish them from other reported CHIKV and alphavirus sequences.
As one in the art may appreciate, the originality of the present invention is the identification of novel strains of the Chikungunya (CHIK) virus which are distinguished from CHIK virus of the prior art, and the use of these CHIK strains and the polypeptides and the polynucleotides encoding same derived from their genome in the diagnostic, prevention and/or treatment of arbovirosis.
According to a first aspect, the present invention concerns an isolated and purified wild strain of chikungunya virus (CHIKV) capable of in vitro infecting human cells. Preferably, the present invention concerns a wild strain of CHIK virus which exhibits the same characteristics than those selected from the group consisting of the isolates 05.115, 05.61, 05.209, 06.21, 06.27 and 06.49. According to a preferred embodiment, the strains that are within the scope of the present invention are characterized in that their genome comprises at least one mutation when compared to the sequence of the genome of the CHIK virus strain S-27 (GenBank AF339485). Also within the scope of the invention, is any strain grown or obtained by cell culture from a sample of a preferred CHIK strain of the invention. The genome of the preferred strains according to the present invention comprises a sequence as shown in FIG. 4, 5, 6, 7, 8 or 9 (SEQ ID NO: 1, 2, 3, 4, 5 or 6).
According to another aspect, the present invention provides an isolated and purified strain of chikungunya virus (CHIKV) comprising at least one mutation in structural protein E1 and/or in structural protein E2, and more particularly in their ectodomain region. According to a preferred embodiment, the strain of the invention is characterized by the fact that its genome comprises at least one mutation in the E2 protein at a position homologous to amino acid position 382, 399, 404, 485, 489, 506, 536, 624, 637, 669, 700 or 711 of SEQ ID NO: 23 (FIG. 37). More particularly, the mutation is preferably selected from the group consisting of G382K, 1399M, G404E, N485T, A489T, L506M, 1536T, S624N, T637M, A669T, S700T and V711A as shown in Table 6. According to another preferred embodiment, the strain of the invention is characterized by the fact that its genome comprises at least one mutation in the E1 protein at a position homologous to amino acid position 1035, 1078, 1093 or 1131 of SEQ ID NO: 23. More particularly, the mutation is preferably selected from the group consisting of A1035V, M1078V, D1093E and V1131A as shown in Table 6. Most preferably, the mutation in the E1 protein is A1035V.
As use herein, the expression “at a position homologous to an amino acid position” of a protein, refers to amino acid positions that are determined to correspond to one another based on sequence and/or structural alignments with a specified reference protein. For instance, in a position corresponding to an amino acid position of a CHIK virus structural protein set forth as SEQ ID NO: 1 can be determined empirically by aligning the sequences of amino acids set forth in SEQ ID NO: 1 with a particular CHIK virus structural protein. Homologous or corresponding positions can be determined by such alignment by one of skill in the art using manual alignments or by using the numerous alignment programs available (for example, BLASTP). Homologous or corresponding positions also can be based on structural alignment, for example by using computers simulated alignments of protein structure. Recitation that amino acids of a polypeptide correspond to amino acids in a disclosed sequence refers to amino acids identified upon alignment of the polypeptide with the disclosed sequence to maximize identity or homology (where conserved amino acids are aligned) using a standard algorithm, such as the GAP algorithm. As used herein, “at a position homologous to” refers to a position of interest (i.e., base number or residue number) in a nucleic acid molecule or protein relative to the position in another reference nucleic acid molecule or protein. The position of interest to the position in another reference protein can be in, for example, an amino acid sequence from the same protein of another CHIK strain. Homologous positions can be determined by comparing and aligning sequences to maximize the number of matching nucleotides or residues, for instance, such that identity between the sequences is greater than 95%, preferably greater than 96%, more preferably greater than 97%, even more preferably greater than 98% and most preferably greater than 99%. The position of interest is then given the number assigned in the reference nucleic acid molecule.
Another aspect of the invention concerns an isolated and purified polynucleotide comprising all or part of the sequence as shown in FIG. 4, 5, 6, 7, 8 or 9 (SEQ ID NO: 1, 2, 3, 4, 5 or 6).
Another aspect of the invention concerns a fragment of the polynucleotide of the invention characterized by the fact that it codes for the glycoprotein E1 or E2, and more preferably for their ectodomain region. Advantageously, the fragment of the invention when coding for the E2 ectodomain, comprises, or more preferably, consists of a nucleotide sequence as shown in FIG. 10, 11, 12 or 13 (SEQ ID NO: 7, 8, 9 or 10).
Yet another aspect of the invention concerns a fragment of the polynucleotide of the invention characterized by the fact that it codes for a soluble form of glycoprotein E2. According to a preferred embodiment, the soluble fragment of glycoprotein E2 comprises or more preferably consists of a nucleotide sequence as shown in FIG. 14, 15, 16 or 17 (SEQ ID NO. 11, 12, 13 or 14).
As one skilled in the art may appreciate, a fragment as contemplated by the present invention may be obtained by:

    
    
        use of restriction enzymes wherein their cleavage sites are present in the polynucleotide comprising said fragment;
        amplification with specific primers for said fragment;
        in vitro transcription; or
        chemical synthesis.
    
    


According to another aspect, the present invention is concerned with an isolated and purified polypeptide encoded by a polynucleotide or by a fragment of the invention. As used herein, the terms “polypeptide” and “protein” are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers.
By “isolated” is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro-molecules of the same type. The term “isolated” with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
Broadly defined, the terms “purified polypeptide” or “purified polynucleotide” refer to polypeptides or polynucleotides that are sufficiently free of other proteins or polynucleotides, or carbohydrates, and lipids with which they are naturally associated. The polypeptide or polynucleotide may be purified by any process by which the protein or polynucleotide is separated from other elements or compounds on the basis for instance, of charge, molecular size, or binding affinity.
The preferred peptides of the invention comprise at least one amino acid substitution compared with the amino acid sequence of strain S-27 (GenBank AF339485) and are derived from the sequence of a protein coded by a fragment of the invention. Preferably, a purified polypeptide of the invention comprises all or part of the amino acid sequence of a CHIK virus ORF 1 or 2 contemplated by the present invention such as one defined in any one of SEQ ID NOS 24 to 29 (ORF 2) or of SEQ ID NOS 30 to 34 and 78 (ORF 1). More preferably, a purified polypeptide of the invention comprises all or part of the amino acid sequence of a glycoprotein E2 contemplated by the present invention such as one defined in any one of SEQ ID NOS 15 to 18 (FIGS. 18 to 21). Even more preferably, a purified polypeptide of the invention comprises all or part of the amino acid sequence of a soluble form of glycoprotein E2 contemplated by the present invention such as one defined in any one of SEQ ID NOS 19 to 22 (FIGS. 22 to 25).
The present invention is also concerned with a vector comprising a polynucleotide of the invention or a fragment of a polynucleotide of the invention. As used herein, the term “vector” refers to a polynucleotide construct designed for transduction/transfection of one or more cell types. Vectors may be, for example, “cloning vectors” which are designed for isolation, propagation and replication of inserted nucleotides, “expression vectors” which are designed for expression of a nucleotide sequence in a host cell, or a “viral vector” which is designed to result in the production of a recombinant virus or virus-like particle, or “shuttle vectors”, which comprise the attributes of more than one type of vector. Preferred vector are those deposited at the CNCM (Collection Nationale de Cultures de Microorganismes), 28 rue du Docteur Roux, 75724 PARIS Cedex 15, France, on Mar. 15, 2006 under accession numbers I-3587, I-3588, I-3589 and I-3590.
Another preferred vector contemplated by the present invention is the plasmid called TRIP-CHIK.sE2 which has been deposited at the CNCM (Collection Nationale de Cultures de Microorganismes), 28 rue du Docteur Roux, 75724 PARIS Cedex 15, France, on Mar. 14, 2007, under accession number I-3733. Such a vector comprises a fragment which codes for a soluble form of the glycoprotein E2 of the invention. This preferred vector has been optimised for efficient production of the recombinant E2 protein into mammalian cells. As used herein, the term “optimised” means that the vector incorporates regulation sequences, such as a signal peptide sequence, in order to provide adequate expression of the desired encoded protein.
In a related aspect, the present invention provides a host cell comprising a vector as defined above. The term “host cell” refers to a cell that has a new combination of nucleic acid segments that are not covalently linked to each other in nature. A new combination of nucleic acid segments can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art. A host cell can be a single eukaryotic cell, or a single prokaryotic cell, or a mammalian cell. The host cell can harbor a vector that is extragenomic. An extragenomic nucleic acid vector does not insert into the cell's genome. A host cell can further harbor a vector or a portion thereof that is intragenomic. The term intragenomic defines a nucleic acid construct incorporated within the host cell's genome. A preferred host cell of the invention E. coli such as the one containing a vector of the invention and deposited at the CNCM (Collection Nationale de Cultures de Microorganismes), 28 rue du Docteur Roux, 75724 PARIS Cedex 15, France, on Mar. 15, 2006 under accession numbers I-3587, I-3588, I-3589 and I-3590 and on Mar. 14, 2007 under accession number I-3733.
The present invention is further concerned with a monoclonal antibody or polyclonal antibodies, or fragments thereof, that specifically bind to a polypeptide of the invention. As used herein, the term “specifically binds to” refers to antibodies that bind with a relatively high affinity to one or more epitopes of a protein of the invention, but which do not substantially recognize and bind to molecules other than the one(s) of interest. As used herein, the term “relatively high affinity” means a binding affinity between the antibody and the protein of interest of at least 10−6 M, and preferably of at least about 10−7 M and even more preferably 10−8 M to 10−10 M. Determination of such affinity is preferably conducted under standard competitive binding immunoassay conditions which is common knowledge to one skilled in the art.
As used herein, the term “antibody” refers to a glycoprotein produced by lymphoid cells in response to a stimulation with an immunogen. Antibodies possess the ability to react in vitro and in vivo specifically and selectively with an antigenic determinant or epitope eliciting their production or with an antigenic determinant closely related to the homologous antigen. The term “antibody” is meant to encompass constructions using the binding (variable) region of such an antibody, and other antibody modifications. Thus, an antibody useful in the method of the invention may comprise a whole antibody, an antibody fragment, a polyfunctional antibody aggregate, or in general a substance comprising one or more specific binding sites from an antibody. The antibody fragment may be a fragment such as an Fv, Fab or F(ab′)2 fragment or a derivative thereof, such as a single chain Fv fragment. The antibody or antibody fragment may be non-recombinant, recombinant or humanized. The antibody may be of an immunoglobulin isotype, e.g., IgG, IgM, and so forth. In addition, an aggregate, polymer, derivative and conjugate of an immunoglobulin or a fragment thereof can be used where appropriate.
Another aspect of the invention is the use of an element selected from the group consisting of a strain, a polynucleotide, a fragment, a vector, a host cell, a polypeptide and an antibody of the invention for either the detection of a CHIKV associated to an arbovirosis, or for the preparation of a composition that prevents and/or treats an arbovirosis.
Another aspect of the present invention relates to a composition for treating and/or preventing an arbovirosis. The composition of the present invention advantageously comprises at least one element selected from the group consisting of a strain, a polynucleotide, a fragment, a vector, a host cell, a polypeptide and an antibody of the invention. The composition of the invention may further comprise an acceptable carrier. In a related aspect, the invention provides a method for treating and/or preventing an arbovirosis. The method comprises the step of administering to a subject in need thereof a composition of the invention.
As used herein, the term “treating” refers to a process by which the development of an infection from a CHIKV is affected or completely eliminated. As used herein, the term “preventing” refers to a process by which the CHIKV infection is obstructed or delayed.
As used herein, the expression “an acceptable carrier” means a vehicle for containing the components (or elements) of the composition of the invention that can be administered to a animal host without adverse effects. Suitable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions. Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.
The amount of components of the composition of the invention is preferably a therapeutically effective amount. A therapeutically effective amount of components of the composition of the invention is the amount necessary to allow the same to perform their preventing and/or treating role against a CHIKV infection without causing overly negative effects in the host to which the composition is administered. The exact amount of components to be used and the composition to be administered will vary according to factors such as the mode of administration, as well as the other ingredients in the composition.
The composition of the invention may be given to a host (such as a human) through various routes of administration. For instance, the composition may be administered in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents. They may be given parenterally, for example intravenously, intramuscularly or sub-cutaneously by injection, by infusion or per os. Suitable dosages will vary, depending upon factors such as the amount of each of the components in the composition, the desired effect (short or long term), the route of administration, the age and the weight of the host to be treated. Any other methods well known in the art may be used for administering the composition of the invention.
Yet another aspect of the invention is the use of a composition as defined hereabove for the preparation of a medicament for treating and/or preventing an arbovirosis in a subject in need thereof.
Yet another aspect of the invention is to provide a kit for the detection of a CHIKV associated to an arbovirosis, comprising at least one element selected from the group consisting of a strain, a polynucleotide, a fragment, a vector, a host cell, a polypeptide and an antibody of the invention. Kits according to this embodiment of the invention may comprise packages, each containing one or more of the above mentioned elements (typically in concentrated form) which are required to perform the respective diagnostic tests.
EXAMPLES
The examples here below will highlight other characteristics and advantages of the present invention, and will serve to illustrate the scope of the use of the present invention and not to limit its scope. Modifications and variations may be made without departing from the spirit and the scope of the invention. Although it is possible to use other methods or products equivalent to those that are found here below to test or to realize the present invention, the preferred material and methods are described.
Example 1
Identification and Characterization of CHIK Viruses Causing the Indian Ocean Outbreak
The inventors (as sometimes referred therein as “we”) report the nearly complete genome sequence of six selected clinical isolates, along with partial sequences of glycoprotein E1 from a total of 60 patients from Reunion, Seychelles, Mauritius, Madagascar and Mayotte Islands. The present results indicate that the outbreak was initiated by a strain related to East-African isolates, from which viral variants have evolved following a traceable microevolution history. Unique molecular features of the outbreak isolates were identified. Notably, in the region coding for the non-structural proteins, ten amino acid changes were found, three of which being located in alphavirus conserved positions of nsP2 (which contains helicase, protease and RNA triphosphatase activities) and of the polymerase nsP4. The sole isolate obtained from the cerebrospinal fluid of a patient showed unique changes in nsP1 (T301I), nsP2 (Y642N) and nsP3 (E460 deletion). In the structural protein region, two noteworthy changes (A226V and D284E) were observed in the membrane fusion glycoprotein E1. Homology 3D modelling allowed mapping of these two changes to regions that are important for virion assembly and for membrane fusion. Change E1-A226V was absent in the initial strains but was observed in >85% of subsequent viral sequences from Reunion, denoting evolutionary success possibly due to adaptation to the mosquito vector.
Material and Methods
Patients.
The 60 patients for whom partial or complete CHIKV nucleotide sequences were determined originated from Reunion (N=43), Seychelles (N=3), Madagascar (N=7), Mayotte (N=4) and Mauritius (N=3). Characteristics of the patients and biological samples are listed in Table 1.
Virus Isolation and RNA Extraction.
Viruses were isolated either from serum or cerebrospinal fluid (CSF) (Table 1). Briefly, C6-36 Aedes albopictus cells were inoculated with 1 ml of serum or CSF diluted 1:10 in L15 medium (Gibco). The cells were grown at 28° C. in L15 supplemented with 5% foetal bovine serum and 10% tryptose-phosphate. Cells and supernatants were harvested after the first passage (5 days) and the second passage (7 days). The virus isolates were identified as CHIKV by indirect immunofluorescence, using CHIKV hyper immune ascitic fluid. In the case of isolates 05.115, 06.21, 06.27 and 06.49 whose genomes were sequenced, absence of yellow fever, dengue and West Nile viruses was confirmed by indirect immunofluorescence using specific sera. RNA was extracted using the QIAAmp Viral Minikit (Qiagen, France).
Nucleotide Sequencing.
Primers (Table 4) were designed based on the nucleotide sequence 20 of the S27 strain. RT-PCR was performed using the Titan One Tube RT-PCR kit (Roche, France). RT-PCR fragments were purified by ultrafiltration prior to sequencing (Millipore, France). Sequencing reactions were performed using the BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems, USA) and purified by ethanol precipitation. Sequence chromatograms were obtained on automated sequence analysers ABI3100 or ABI3700 (Applied Biosystems). All amplicons were sequenced on both strands.
Assembly of Genome Sequences and Sequence Analysis.
Contig assembly was performed independently by distinct operators and software, using either BioNumerics version 4.5 (Applied-Maths, Sint-Martens-Latem, Belgium) or PhredPhrap/Consed [11]. Both analyses yielded exactly the same consensus sequence for all strains. A single contig of 11,601 nt was obtained for five isolates, whereas for strain 05.61, a sequence portion was missing, between S27 positions 5,246 to 5,649 (positions 390 to 524 of nsP3). Sequence alignments and computation of substitution tables were performed using programs BioNumerics, DNASP version 4.10 [12] and DAMBE version 4.2.13 [13]. Alignments of nucleotide and amino acid sequences against selected alphavirus sequences were performed with the ClustalW1.7 software [14]. Sequence identities were computed with the Phylip package [15]. RNA secondary structure was predicted with the Vienna RNA secondary structure server [16]. Neighbor-joining trees were constructed using MEGA version 3.1 [17] with the Kimura-2 parameter corrections of multiple substitutions. Reliability of nodes was assessed by bootstrap resampling with 1,000 replicates. Amounts of synonymous substitutions per synonymous site (Ks) and of non synonymous substitutions per non synonymous site (Ka) were estimated using DNASP. RDP2 [18] was used to detect putative mosaic sequences.
3D Structure Modeling.
The crystallographic structure of the ectodomain of the glycoprotein E1 of Semliki Forest Virus (SFV) at neutral pH [19]; Protein Data Bank code 2ALA) was used as a template to model and analyze the two amino acid mutations of the Indian Ocean isolates. FIG. 2 was prepared using the program RIBBONS [20].
Detection of Viral Foci by Immunological Staining.
Aedes pseudoscutellaris AP61 cells were grown in a 24-well tissue culture plates in Leibovitz L-15 growth medium with 10% heat inactivated fetal calf serum (FCS) for 24 h. Mosquito cell monolayers were washed once with Leibovitz L-15 and 0.2 ml Leibovitz L-152% FCS were added. Cells were infected with CHIK virus in 0.2 ml of Leibovitz L-152% FCS and incubated at 28° C. for 1 h. Overlay medium consisting of 0.4 ml of Leibovitz L-152% FBS and carboxymethylcellulose (CMC) (1.6%) was then added and the tissue culture plates were incubated at 28° C. for 2 days. Foci of infected cells were visualized by focus immunoassay (FIA). The cells were washed with PBS, fixed with 3% paraformaldehyde (PFA) in PBS for 20 min, and permeabilized with 0.5% Triton X-100 in PBS for 4 min at room temperature. The fixed cells were incubated for 20 min at 37° C. with 1:2,000 dilution of hyperimmune mouse ascitic fluid (HMAF) directed against CHIKV. Goat anti-mouse IgG, horseradish peroxidase conjugated was used as the second antibody (1:100 dilution) at 37° C. for 20 min. Foci were visualized with DAB. Peroxidase Substrate (Sigma).
1. Genome structure and molecular signatures of the Indian Ocean outbreak chikungunya viruses
Genome Organization.
We determined the nearly complete genome sequences of six CHIKV isolates (05.115, 05.61, 05.209, 06.21, 06.27 and 06.49) representing distinct geographic origins, time points and clinical forms (Table 1) of the Indian Ocean outbreak of chikungunya virus. 11,601 nucleotides were determined, corresponding to positions 52 (5′NTR) to 11,667 (3′NTR, end of third Repeat Sequence Element) in the nucleotide sequence of the 1952 Tanzanian isolate S27 (total length 11,826 nt). There were three insertion/deletion events between S27 and Réunion isolates, two of which were observed in the 3′NTR. First, the internal poly-A stretch of 14 nucleotides observed in S27 (11,440-11,443) and corresponding to a probable internal poly-A site [9] was replaced by a stretch of only 5 A in Indian Ocean isolates, similar to what was observed in other chikungunya viruses, e.g. the Ross strain (accession no.: AF490259). Second, one A was missing in Indian Ocean isolates in a 5-A stretch at S27 position 11,625. Finally, one codon was missing in isolate 06.27, corresponding to nsP3 codon 460, at which all other Indian Ocean isolates analyzed and available alphavirus sequences are GAA, coding for Glu.
The genome sequences of the six isolates presented therein was similar to those previously reported for alphaviruses [9, 21, 22]. Coding sequences consisted of two large open reading frames (ORF) of 7,422 nt and 3,744 nt encoding the non-structural polyprotein (2,474 amino-acids) and the structural polyprotein (1,248 amino-acids), respectively. The non structural polyprotein is the precursor of proteins nsP1 (535 aa), nsP2 (798 aa), nsP3 (530 aa) and nsP4 (611 aa), and the structural polyprotein is the precursor of proteins C (261 aa), p62 (487 aa, precursor to E3-64 aa- and E2-423 aa), 6K (61 aa), and E1 (439 aa). Cleavage sites characteristic of the alphavirus family in the non-structural and structural polyproteins were conserved. Glycosylation sites in E3, E2 and E1 were also conserved. A 65 nt junction sequence was identified between the stop codon (TAG, 7499-7501) of the non-structural ORF and the start codon (7567-7569) of the structural ORF. The 5′ non-translated region (5′NTR) ended at position 76. The 3′NTR region started at position 11,314 and contained three repeat sequence elements (RSE) with predicted secondary structures (FIG. 26) that were consistent with previous work [9].
Differences Between Indian Ocean Outbreak Isolates and Strain S27.
Compared to strain S27, Reunion isolate 05.115 showed 28 aa changes (1.13%) in the non-structural proteins (Table 5, with the highest proportion in nsP3 (2.26%) and the lowest in nsP2 (0.6%). Ten out of 12 amino acid changes in nsP3 were concentrated between positions 326 and 524 (5.0% variation), similar to findings in ONN viruses [23]. One important difference with S27 was that the Indian Ocean isolates exhibited an opal stop codon (UGA) at nsP3 codon 524, instead of Arg (CGA) in S27. This opal codon was observed in related alphaviruses [9, 22, 23], and is believed to regulate the expression of nsP4, the putative RNA polymerase, by a read-through mechanism [21, 24].
Compared to S27, the structural proteins showed 21 (1.68%, for 05.115) to 22 (1.76%, for other isolates) amino-acid substitutions in Indian Ocean isolates (Table 6). Notably, envelope protein E2 showed the highest variation, with 14 (3.3%) aa changes, higher than envelope protein E1 (0.68%) and the capsid protein (0.38%). The ratio of rates of evolution of synonymous and non-synonymous sites (Ks/Ka) between S27 and 05.115 isolates was 11.0 for the whole polyprotein, whereas it was only 6.12 for protein E2, probably indicative of a positive selection in favor of amino-acid changes in this immunogenic protein. By comparison, Ks/Ka was 18.75 for the non-structural polyprotein.
Indian Ocean Outbreak Molecular Signatures in Non-Structural Proteins and Phenotypic Variation.
Ten positions (excluding polymorphic positions) had aa that were unique to the non-structural proteins of outbreak isolates, when compared to other CHIKV sequences (Table 2). First, nsP2-54 was Asn in Indian Ocean isolates and in SFV, but was Ser in all other sequences. Second, nsP2-374 was Tyr in Indian Ocean isolates, but was His or Asn in other alphavirus sequences (Table 2). Third, position 500 in nsP4 was Leu in the Indian Ocean sequences instead of Gln in the four other reported CHIKV sequences. Interestingly, this position, which is about 30 aa from the catalytic “GDD” motif, is a strictly conserved Glu in all other alphaviruses. The remaining seven changes took place in relatively variable regions.
Additional specific changes were observed in isolates 05.209 (S358P) and 06.27 (nsP1-T301I, nsP2-Y642N, and nsP3-460del). Notably, our phenotypic assays conducted in parallel showed differences for strain 06.27. Focus immunoassay showed that CHIKV stocks 05.115, 06.21, 06.27 and 06.49 formed mixtures of foci with different sizes on Ae. Albopictus C636 (data not shown) and Ae. pseudoscuterallis AP61 cells (FIG. 27). Interestingly, only isolate 06-27 formed medium foci, whereas others formed minutes and small foci. The particular phenotype of 06-27 could be linked to the observed aa differences in the non structural proteins, which are involved in the viral replication [21].
Indian Ocean Molecular Signatures in Structural Proteins and 3D Modelling.
When analyzing the aa sequences of the structural proteins, seven positions (four in E2, one in 6K and two in E1) were found to be unique to isolates from the Indian Ocean outbreak (Table 2). Two of these were located in the E2 ectodomain, with Thr 164 and Met 312 being identified in our isolates instead of Ala and Thr, respectively, in all other available CHIKV sequences (Table 2). The first of these two positions is variable in alphaviruses; it lies in a region defined previously as containing neutralizing epitopes [5, 25]. At position 312, Thr is present in other CHIKV, in ONNV and in SFV, but varies in other alphaviruses; it lies in a region identified as important for E1-E2 oligomerization [5, 25].
In E1, two crucial substitutions were observed, one at residue 284, specific to Indian Ocean isolates, and one at residue 284, present in 3 out of 6 Indian isolates (06.21, 06.27 and 06.49). Both mutations were mapped on the 3D structure (modeled from the crystal structure of SFV E1) in FIG. 1. Interestingly, residue 226 is Ala in all reported CHIKV sequences (Table 2), and was also Ala in the first of our Indian Ocean isolates sequenced here (05.61 and 05.115, obtained at the beginning of the outbreak). All subsequent isolates (obtained from patients collected in November and December 2005) displayed a Val residue at this position. Although position 226 is relatively variable among alphaviruses, it was observed that a single mutation at this position (Pro to Ser) allowed SFV to adapt to growth in cholesterol-depleted insect cells [26, 27].
The other unique aa observed in E1 from Indian Ocean isolates was Glu 284. This is a highly conserved position in E1, which displays an Asp in the majority of alphaviruses or an Asn in SIN (Table 2). This amino acid is located at the interface between E1 protomers at the surface of the virion, participating in contacts that make up the icosahedral E1 scaffold (FIG. 1).
2. Phylogenetic Analysis
Previous work based on E1 protein sequences showed strong phylogeographic structure of the chikungunya virus species [6, 10]. In order to determine the progenitor phylogroup from which the Indian Ocean outbreak isolates emerged, we compared a 1,044 nt region within the E1 coding sequence (positions 271 to 1314, i.e., codons 91 to 438) from 63 biological specimens from 60 patients from Reunion, Seychelles, Madagascar, Mayotte and Comoros (Table 1) with 29 other available chikungunya sequences (Table 7). Phylogenetic analysis (FIG. 2) clearly demonstrated that the current Indian Ocean isolates represent a homogeneous clade within a broad group (group ECSA) comprising isolates from East, Central and South Africa (ECSA, FIG. 2). The isolates from an outbreak in Democratic Republic of the Congo [6] also formed a homogeneous Glade within group ECSA. There was no ECSA group member showing a significantly closer relationship with the Indian Ocean isolates. Asian isolates were less related to Indian Ocean isolates and constituted the sister group of group ECSA, whereas West-African isolates were even more divergent. Inclusion of other alphaviruses, including the closest relative ONN, placed the root of the chikungunya isolates on the branch leading to the West-African phylogroup (data not shown).
Comparison of the sequences of Indian Ocean outbreak isolates to the S27 sequence revealed 316 (2.7%) nucleotide substitutions in isolate 05.115 (Table 8). The Asian Glade Nagpur strain showed 5.1% average nucleotide divergence from 05.115, whereas the West-African Glade Senegal strain 37997 displayed 15% difference (Table 8). Interestingly, the latter strain showed complete conservation of an 87 nucleotides portion (9,958-10,045, at the junction between structural proteins 6K and E1) with East-African and Indian Ocean outbreak isolates. Sequence identity in this portion may reflect a past event of genetic recombination between West-African and East/Central-African strains. Differently, we did not find statistical support (P>7E-2) for sequence mosaicism or recombination since the split between S27 and Reunion isolates, although some genomic regions differed in their density of nucleotide polymorphisms.
3. Genotypic and Phenotypic Variation Among Indian Ocean Outbreak Isolates and Microevolutionary Scenario
Specific aa changes in the non-structural proteins were observed in the isolates 05209 (S358P) and 06.27 (nsP1-T3011, nsP2-Y642N, and nsP3-460del). In the structural proteins, change E1-A226V was observed in isolates 06.21, 06.27 and 06.49, and change E2-Q146R in the Seychelles isolate 05.209. In addition to these non-synonymous changes, there were 8 silent substitutions, observed in 05.209, 06.27 and 06.49 (Table 3).
A history of probable sequence evolution that occurred during the outbreak (FIG. 3) was deduced from the 14 amino-acid variations observed among the six complete genomes (Table 3). Isolate 05.61 was initially selected for genome analysis because it was isolated in March 2005, at the onset of the outbreak, from a Reunion patient returning 1 turning from Comoros Island, where the outbreak had been going on since January 2005. Remarkably, the isolates 05.61 and 05.115 (which was the second earliest isolate analyzed), the African isolate S27 and previous unrelated chikungunya isolates from Africa and Asia were identical at all 14 polymorphic sites. Therefore, the consensus sequence of isolates 05.61 and 05.115 (consensus sequence 1) likely represents the ancestral genotype of the Reunion outbreak. Distribution of the 14 polymorphisms suggested that this founder gave rise to three consensus sequences that likely evolved in four steps. First, substitution at genome position 10,670 (causing the E1 A226V change) gave rise to consensus sequence 2, represented by the late-November 2005 isolate 06.21. Second, a G to A synonymous substitution at position 6,547 (nsP4) led to an intermediate sequence, which itself gave rise to two late sequences: consensus sequence 3 (isolate 06.27), following four additional substitutions and one codon deletion (Table 3), and consensus sequence 4 (06.49), which arose after three distinct synonymous substitutions (Table 3). A fifth consensus sequence was represented by the Seychelles isolate 05.209 alone, which exhibited four substitutions (two of them causing aa changes in nsP3-S358P and in E2-Q146R) compared to consensus sequence 1 (FIG. 3).
Since Reunion isolates had E1-226A at the beginning of the outbreak and E1-266V A at the beginning of the outbreak and E1-266V later in the epidemics, we compared residue 226 in 57 additional sequences (57 sequences from 54 sera and 3 CSF) from the Indian Ocean epidemic. Remarkably, the nature of E1-226 differed totally on Reunion Island before and after the winter season. Five sequences from patients sampled from March to June 2005 (including the sequence originating from a traveller back from Comoros) had E1-226A. Between September and end December 2005, 21 sequences showed E1-226V. Among 17 Reunion sequences from 2006, E1-226V was observed 12 times and E1-226A 5 times (Table 1). On Madagascar and Seychelles sequences, for which the samples were collected when the first clinical cases were suspected (i.e probably at the beginning of the outbreaks), only the E1-226 Ala was observed. On Mayotte 2006 sequences, only the E1-226 V was observed. On Mauritius 2006 sequences, both E1-226 Ala and Val were observed.
To date, only CHIKV laboratory strains, passaged many times on mosquito or mammalian cells, had been entirely sequenced [9]. We provide for the first time nearly complete nucleotide sequences of six clinical isolates passaged in-vitro only once or twice (see M&M section). The presence in infected patients of a mixed viral population, called quasispecies [31-33], with genotypes co-existing in an equilibrium governed by a balance between mutation and natural selection. The presence in S27 of an Arg codon instead of the opal stop codon in Indian Ocean isolates is probably explained by numerous in-vitro passages of S27, as evolution of opal to Arg was observed experimentally in ONN viruses [23]. Whereas it may be advantageous for viral quasispecies to maintain the opal codon in-vivo, an Arg codon probably confers a selective advantage in-vitro, as observed for the closely related Semliki Forest virus [34]. Chikungunya virus quasispecies situation in-vivo could also explain the nsP1-T3011 polymorphism observed for the LCR isolate 06.27. Indeed, it is likely that selection for a subset of genotypes harboring this change may be associated with invasion of the LCR [33]. These results underscore that the genome sequence of laboratory “reference” strains may not accurately reflect the natural situation, as the genotypic complexity of quasispecies in-vivo is subject to erosion by in-vitro selection. Since the Indian Ocean isolates sequenced here were subjected to in-vitro selection for only a few generations, they probably correspond more closely to the in-vivo genotypes than previously sequenced chikungunya strains.
The amino acid (aa) differences detected among the outbreak 1 isolates may relate to biological or pathogenic characteristics of the virus. Although our viral culture results are preliminary, they clearly show phenotypic differences between the unique isolate from CSF (06.27), isolated from a neonatal encephalopathy case, and three other isolates, associated with either the classical form of the disease or encephalopathy. The larger foci observed in culture with 06.27 could reflect a higher replication rate of the virus and be linked to the specific amino acid changes identified in nsP1, nsP2 and nsP3. Single amino-acid changes in nsP1, including a Thr/Ile change (residue 538 of Sindbis virus) [35,36] and a 18-nt deletion in nsP3 have previously been shown to affect neurovirulence in other alphaviruses [35-37]. However, in the absence of nsP1 structural data, it is difficult to predict the structural or functional impact of the I301T change observed in 06.27 isolate. It should also be noted that all the viral sequences determined from either the serum or the isolates from three neonatal encephalopathy cases and an adult meningo-encephalitis case had E1-226 Val. However, as this genotype is observed also in classical forms of the disease, a potential link of E1-226 Val with neuropathogenesis needs further studies. Host factors have to be considered in the occurrence of neurological forms of the disease. For example, the blood-brain crossing may be favoured by young age or hypertension.
Unique molecular signatures of the Indian Ocean outbreak genomes were identified when they were compared to all other reported alphavirus sequences. These features represent interesting targets for future functional studies, as well as for epidemiological follow-up. One particularly interesting feature was the E1-226 Val residue (see above). Another interesting molecular signature of Indian Ocean outbreak genomes was E1-284 Asp. Although pseudo-atomic model of the scaffold used is of modest resolution (the resolution of the crystal structure is limited—approaching 3 Å—and the model results of fitting this structure into a 9 Å resolution cryo-electron microscopy reconstruction), it appears that the side-chain of Asp 284 interacts with the main chain of an adjacent E1 polypeptide in the virion. Indeed, it is in a position compatible with acceptance of a hydrogen bond from main chain amide 379 from the neighboring E1 protomer. Because the packing is very tight (see FIG. 1B), it is possible that the longer Glutamic acid side chain (which has an extra CH2 group compared to Asp or Asn) may introduce a slight distortion at the contact sites, an effect that is propagated by the icosahedral T=4 symmetry of the virion. Thus, a cooperative effect due to this change at position Asp 284 may play a role in either allowing a less efficient assembly of new particles in infected cells, or a more efficient particle disassembly process during invasion of a new cell, or a combination of both. This information 1 tion can guide new site-directed mutagenesis studies, using reverse genetics, to test the effect of the Asp/Glu replacement on the virus cycle.
Example 2
Identification and Characterization of a Soluble Form of E2 (sE2) of the CHIK Virus
The TOPO/CHIK-21.pE2 (CNCM I-3587) plasmid containing the cDNA coding for the pE2 glycoprotein (E3+E2) from the CHIK 21 virus strain (Schuffenecker et al., Plos Med., 3:1058, 2006) was used as a template for the amplification by PCR of the ectodomain sequence of the E2 envelope glycoprotein (FIG. 29). The ectodomain of gp-E2 (E2-1 to E2-364; 85% of E2) is strictly conserved among the CHIK-21, -27, 49 and 115 cell lines isolated in the Indian Ocean during the epidemic outbreak of 2005-06 (see FIG. 29). The soluble form of the sE2 corresponds to the gp-E2 ectodomain which is deleted at the carboxylic terminal of its transmembrane anchor region. It is of interest that the soluble form carries the main epitopes eliciting virus-neutralizing antibodies. The PCR primers are described in FIG. 30 (SEQ ID NO:79 and 80): they allow the cloning of the sE2 sequence between the unique sites Bg/II and NotI of the pMT/BiP/V5-HisA vector (Invitrogen), on the one hand in a phase dependent on the BiP peptide signal at the N-terminal, and on the other hand in joining successive V5 (His)6 tags at its carboxylic terminal.
Drosophila S2 cells were transfected with the recombinant plasmid pMT/BiP/CHIK-sE2 in the presence of the plasmid coding for the blasticidin resistance gene. The S2/CHIK-sE2 stable cell line was obtained by successive passages in presence of blasticidin. The cell line was selected for its capacity to promote efficient secretion of the CHIK-sE2 virus following the activation of the metallothioneine promoter.
The S2/CHIK-sE2 cells in suspension were induced for the secretion of sE2 during 21 days in the presence of Cu2+. The cellular supernatant is filtered at 0.22 μM and concentrated for 16 hours on an affinity column of 5 ml HiTrap Chelating HP (Amersham Biosciences) with the help of a peristaltic pump. The CHIK sE2 protein is eluded from the affinity column in the presence of increasing concentrations of imidazole (50, 100 and 500 mM, pH 8). The CHIK sE2 protein is specifically eluded at a concentration of 500 mM imidazole (E3 elution) from the E37 fraction (FIG. 31). The sE2 protein is detected as being highly purified in PAGESDS following Coomassie Blue coloration. The sE2 protein eluded in the E39 fraction is specifically immunodetected by an ascite (HMAF) of a mouse hyperimmunized against the CHIK virus which was produced at the IMFH unit (FIG. 32). No cross-reactivity was observed with the anti-dengue (DEN) or anti-West Nile (WN) HMAHF and the monoclonal antibody 9D12 anti-DEN E. The soluble DEN sE proteins (DEN-3 and DEN-4) and WN sE purified from the supernatants of the S2 cellular clones induced according to the protocol described hereinabove are used as control viral antigens for the specificity of anti-CHIK murine antibodies.
Example 3
Construction of the TRIP Vector Expressing the Soluble Form of E2 (sE2) of the CHIK Virus According to the Present Invention
The gene coding the CHIK sE2 protein has been optimised by the Genecust firm so as to provide a synthetic DNA with an enriched G+C content in comparison to the cDNA obtained from the viral genomic RNA. The G+C rich codons (amino acids E2-1 to E2-364, soluble gp-E2 ectodomain, sE2) were fused to the signal peptide sequence of the human calreticuline (ssCRT) MLLSVPLLLLGLLGLAA (SEQ ID NO: 77) for translocation of the viral protein into the secretion pathway. The enzyme restriction sites BamHI in 5′ and XhoI in 3′ have been added at their respective ends of the sequences coding for the fusion ssCRT+sE2 protein.
The synthetic gene was cloned into the TRIP vector between the BamHI and XhoI sites under the transcription of the ieCMV promoter. The non-replicative and integrative TRIP/CHIK.sE2 plasmid thus produced was validated for the expression of the sE2 protein following transduction of 293 cells.
As shown in FIG. 33, the inventors have constructed a vector expressing the CHIK sE2. As mentioned above, the original CHIK sE2 sequence cloned into the TRIP vector has been modified for improving expression in mammalian cells (FIG. 34).
FIG. 35 shows mammalian cells, such as the 293 cells, transduced with the TRIP-CHIK.sE2 vector. The expressed sE2 protein has been revealed by IF with anti-CHIK antibodies.
Example 4
Production of Recombinant Protein sE2 and Specific Monoclonal Antibodies
The inventors have generated the stable inducible S2/CHIK.sE2 cell line which releases the soluble form of the envelope E2-glycoprotein (sE2) from Reunion CHIK virus strains. The inventors have also generated a stable cell line 293A/CHIK.sE2 which was transducted by the recombinant lentiviral vector TRIP/CHIK.sE2. A synthetic sE2 gene that was modified for optimal codon usage in mammalian cells had to be used in order to obtain efficient expression of CHIK virus sE2 in human fibroblastic 293A cells. The TRIP/CHIK.sE2 vector is currently assessed for its capability to induce protective immunity in a murine model of experimental infection. Viral suspension mainly enriched in CHIK pE2 (E2 precursor or E3E2) was obtained by solubilizing CHIK virions grown in mosquito cells with Triton X-100. Adult mice were hyperimmunized with CHIK pE2 in the presence of adjuvant in order to generate hybridoma directed against CHIK structural proteins. Anti-CHIK E2 monoclonal antibodies produced by mouse hybridoma were characterized by ELISA assay on highly purified CHIK virion and Western blot on secreted sE2 from stable cell line S2/CHIK.sE2. (FIGS. 50, 51 and Table 9). Fluorescent immunodetection assays of intracellular or surface viral antigens were also established on CHIK virus-infected VERO cells and stable 293A/CHIK.sE2 transduced cell line (FIGS. 52-55). Anti-CHIK.sE2 MAbs of the inventors find a potential use in developing early viral diagnosis of CHIK disease based on immunocapture of CHIK virions in viremic blood of patients, and as tools for immunological as well as virological studies.







TABLE 1







Characteristics of the patients















Patients

Region or


Sampling

Virus Isolate



No.
Island
Island
Town or Locality
Sample (b)
Date
Clinical signs (c)
No. (d)
E1-226 (e)


















1
Réunion
—
—
S
16-Mar-05
Classical
05.61 (G)
A (*)



(Comoros) (a)


2
Réunion
West
St Gilles Les Bains
S
11-Apr-05
Classical
05.55
A (*)


3
Réunion
South
Saint Pierre
S
2-May-05
Classical
05.107
A (*)


4
Réunion
West
Mare Sèche Cilaos
S
4-May-05
Classical
05.111
A (*)


5
Réunion
South
La Riviève St Louis
S
6-May-05
Classical
05.115 (G)
A (*)


6
Réunion
South
St Louis
CSF
7-Sep-05
Neonatal
05.223
V (**)








encephalopathy


7
Réunion
South
La Riviève St Louis
S
11-Oct-05
Classical
06.55
V (**)


8
Réunion
South
St Louis
S
21-Oct-05
Classical
06.59
V (**)


9
Réunion
South
La Riviève St Louis
S
21-Oct-05
Classical
06.53
V (**)


10
Réunion
South
La Riviève St Louis
P
26-Oct-05
Classical
n.i.
V (**)


11
Réunion
South
St Joseph
P
9-Nov-05
Classical
n.i.
V (**)


12
Réunion
South
La Riviève St Louis
P
10-Nov-05
Classical
n.i.
V (**)


13
Réunion
South
St Louis
P
20-Nov-05
Classical
n.i.
V (**)


14
Réunion
South
La Riviève St Louis
P
21-Nov-05
Classical
n.i.
V (**)


15
Réunion
South
La Rivieve St Louis
S
23-Nov-05
Classical
06.45
V (**)


16
Réunion
South
La Riviève St Louis
S
28-Nov-05
Neonatal ME
06.21 (G)
V (***)





(parents)


17
Réunion
South
St Joseph
S
23-Nov-05
Classical
06.47
V (**)


18
Réunion
South
La Riviève St Louis
P
24-Nov-05
Classical
n.i.
V (**)


19
Réunion
South
Le Tampon
P
26-Nov-05
Classical
n.i.
V (**)


20
Réunion
South
Ravine des Cabris
P
25-Nov-05
Classical
n.i.
V (**)


21
Réunion
South
St Joseph (parents)
S
29-Nov-05
Neonatal ME
06.25
V (**)


21
Réunion
South
St Joseph (parents)
CSF
29-Nov-05
Neonatal ME
06.27 (G)
V (***)


22
Réunion
South
St Louis
S
2-Dec-05
Classical
06.49 (G)
V (***)


23
Réunion
South
St Louis
P
8-Dec-05
Classical
n.i.
V (**)


24
Réunion
South
Ravine des Cabris
S
9-Dec-05
ME
06.17
V (**)


25
Réunion
South
St Louis
P
13-Dec-05
Classical
n.i.
V (**)


26
Réunion
South
St Pierre
P
2-Jan-06
Classical
n.i.
A (**)


27
Réunion
South
St Pierre
P
4-Jan-06
Algic symdrome
n.i.
A (**)


28
Réunion
East
St André
S
4-Jan-06
Classical
n.i.
A (**)


29
Réunion
South
St Louis
P
29-Dec-05
Algic syndrome
n.i.
V (**)


29
Réunion
South
St Louis
CSF
29-Dec-05
Algic syndrome
n.i.
V (**)


30
Réunion
South
La Riviève St Louis
P
29-Dec-05
Classical
n.i.
V (**)


31
Réunion
South
La Riviève St Louis
P
27-Dec-05
Classical
n.i.
V (**)


32
Réunion
South
St Pierre
P
27-Dec-05
Severe vesicular
n.i.
V (**)








rash lower limbs


32
Réunion
South
St Pierre
L
28-Dec-05
Severe vesicular
n.i.
V (**)








rash lower limbs


33
Réunion
South
Ravine des Cabris
P
4-Jan-06
n.d.
n.i.
A (**)


34
Réunion
South
St Joseph
P
3-Jan-06
Algic syndrome
n.i.
V (**)


35
Réunion
South
St Louis
P
2-Jan-06
Algic syndrome
n.i.
V (**)


36
Réunion
South
St Joseph
P
5-Jan-06
Algic syndrome
n.i.
To be










determined


37
Réunion
South
Ravine des Cabris
P
6-Jan-06
Classical
n.i.
V (**)


38
Réunion
South
St Louis
P
6-Jan-06
Algic syndrome
n.i.
V (**)


39
Réunion
West
Saint-Paul hospital
S
5-Jan-06
n.d.
n.i.
A (**)


40
Réunion
West
St Leu
S
19-Jan-06
Classical
n.i.
V (**)


41
Réunion
South
Les Avirons
S
30-Jan-06
Classical
06.97
V (**)


42
Réunion
East
St Benoit
S
3-Feb-06
Hepatitis
n.i.
V (**)


43
Réunion
n.d.
n.d.
S
22-Feb-06
Classical
n.i.
V (**)


44
Seychelles
Mahe
Anse aux Pins
S
9-Aug-05
Classical
05.209 (G)
A (*)




island


45
Seychelles
Mahe
Anse aux Pins
S
10-Aug-05
Classical
negative
A (**)




island


46
Seychelles
Mahe
Anse aux Pins
S
10-Aug-05
Classical
negative
A (**)




island


47
Madagascar
East
Toamasina
S
1-Feb-06
Classical
06.103
A (**)


48
Madagascar
East
Toamasina
S
8-Feb-06
Classical
06.99
A (**)


49
Madagascar
East
Toamasina
S
9-Feb-06
Classical
06.101
A (**)


50
Madagascar
East
Toamasina
S
15-Feb-06
Classical
n.i.
A (**)


51
Madagascar
North
Ampany
S
14-Feb-06
Classical
n.i.
A (**)


52
Madagascar
North
Djamandjary
S
14-Feb-06
Classical
n.i.
A (**)


53
Madagascar
North
Djamandjary
S
15-Feb-06
Classical
n.i.
A (**)


54
Mayotte
n.d.
n.d.
S
7-Feb-06
Classical
06.111
To be










determined


55
Mayotte
n.d.
n.d.
S
11-Feb-06
Classical
n.i.
V (**)


56
Mayotte
n.d.
n.d.
S
13-Feb-06
Classical
n.i.
V (**)


57
Mayotte
n.d.
n.d.
S
13-Feb-06
Classical
n.i.
V (**)


58
Mauritius
n.d.
n.d.
S
12-Feb-06
Classical
06.93
V (*)


59
Mauritius
n.d.
n.d.
S
27-Feb-06
Classical
n.i.
A (**)


60
Mauritius
n.d.
n.d.
S
1-Mar-06
Classical
n.i.
A (**)





(a) This patent traveled back from Comoros and is believed to have been infected there.


(b) S: serum; P: plasma; CSF: cerebrospinal fluid


(c) ME: meningo-encephalitis


(d) Isolates labeled with (G) correspond to those for which the nearly complete genome sequence was established


(e) A: Alanine; V: Valine; (*): Sequence determined from virus isolates; (**): Sequence determined from biological samples; (***): Sequence determined from both virus isolates and biological samples


n.i.: isolation of virus not intended;


n.d.: not determined













TABLE 2





Relevant amino acid changes identified between Indian Ocean isolates versus a selection of Alphavirus sequences.

















Non-structural proteins





















Protein
nsP1
nsP1
nsP2
nsP2
nsP2
nsP2
nsP3
nsP3
nsP3
nsP3
nsP3
nsP4
nsP4
nsP4





Polypeptide
301
488
589
909
1177
1328
1550
1670
1691
1793
1804
1938
2117
2363


position (a)


Protein
301
488
 54
374
 642
 793
 217
 337
 358
 460
 471
 75
 254
 500


position (a)


05.115
T
R
N
Y
Y
V
H
I
S
E
S
A
A
L


(Genotype 1)


06.21
T
R
N
Y
Y
V
H
I
S
Nd
nd
A
A
L


(Genotype 2)


06.27
I
R
N
Y
N
V
H
I
S
Del
S
A
A
L


(Genotype 3)


06.49
T
R
N
Y
Y
V
H
I
S
E
S
A
A
L


(Genotype 4)


05.209
T
R
N
Y
Y
V
H
I
P
E
S
A
A
L


(Genotype 5)


S27
T
Q
S
H
C
A
Y
T
S
L
P
T
T
Q


Ross
T
Q
S
H
C
A
Y
T
S
L
P
T
T
Q


37997
T
K
S
H
Y
A
Y
T
S
P
P
T
T
Q


(West-African


phylogroup)


Nagpur (Asian
nd
nd
nd
nd
nd
nd
nd
nd
nd
Nd
nd
nd
nd
nd


phylogroup)


ONNV
S
Q
S
N
H
A
Y
**
S
**
**
T
T
E


EEV
S
Q
S
N
E
R
N
**
**
**
**
I
V
E


SFV
V
S
N
H
Y
A
L
**
S
**
**
T
T
E


RRV
V
N
S
H
Y
G
S
**
**
**
**
V
T
E


SINV
V
M
S
H
E
R
K
**
**
**
**
I
T
E












Structural proteins


















Protein
E2
E2
E2
E2
E2
6K
E1
E1
E1







Polypeptide
471
489
637
700
711
756
1035
1078
1093



position (a)



Protein
146
164
312
375
386
 8
 226
 269
 284



position (a)



05.115
Q
T
M
T
A
I
A
V
E



(Genotype 1)



06.21
Q
T
M
T
A
I
V
V
E



(Genotype 2)



06.27
Q
T
M
T
A
I
V
V
E



(Genotype 3)



06.49
Q
T
M
T
A
I
V
V
E



(Genotype 4)



05.209
R
T
M
T
A
I
A
V
E



(Genotype 5)



S27
Q
A
T
S
V
I
A
M
D



Ross
Q
A
T
S
V
V
A
M
D



37997
Q
A
T
S
V
A
A
V
D



(West-African



phylogroup)



Nagpur (Asian
Q
A
T
S
G
V
A
M
D



phylogroup)



ONNV
H
A*
T
S
L*
T*
A
V
D



EEV
E
G*
S
A
T*
D*
A
E
D



SFV
H
V*
T
S
C*
A*
P
M
D



RRV
H
D*
D
S
C*
A*
P
M
D



SINV
V
A*
V
T
V*
S*
A
V
N







(a) S27 reference numbering



*Variable position,



**Hypervariable position



ONV: o'nyong-nyong virus;



SFV: Semliki Forest virus;



RRV: Ross River virus;



SINV: Sindbis virus;



EEV: Eastern-Equine Encephalitis virus.



nd: not determined.



Note that the opal stop codon observed in nsP3-524 of Indian Ocean outbreak isolates, but not in S27, is not represented in the Table.













TABLE 3





Polymorphisms observed among Indian Ocean isolates

















Genome position (b)
















978
1378
3605
4705
5147
5452
5453-5455
6547





Protein
nsP1
nsP1
nsP2
nsP3
nsP3
nsP3
nsP3
nsP4


Protein position (for aa change)
301
—
642
—
358
—
460
—


05.115 (Genotype 1)
C
G
T
T
T
G
GAA (Glu)
G


05.61 (Genotype 1)
C
G
T
T
T
n.d.
n.d.
G


06.21 (Genotype 2)
C
G
T
T
T
G
GAA (Glu)
G


06.27 (Genotype 3)
T (Thr→Ile)
G
A (Tyr→Asn)
A
T
A
Deleted
A


06.49 (Genotype 4)
C
A
T
T
T
G
GAA (Glu)
A


05.209 (Genotype 5)
C
G
T
T
C (Ser→Pro)
G
GAA (Glu)
G


S27 (a)
C
G
T
T
T
G
GAA (Glu)
G












Genome position (b)
















7045
8978
9600
10670
11295
11421







Protein
nsP4
E2
E2
E1
E1
3′NTR



Protein position (for aa change)
—
146
—
226
—
—



05.115 (Genotype 1)
C
A
T
C
G
C



05.61 (Genotype 1)
C
A
T
C
G
C



06.21 (Genotype 2)
C
A
T
T (Ala→Val)
G
C



06.27 (Genotype 3)
C
A
T
T (Ala→Val)
G
C



06.49 (Genotype 4)
C
A
C
T (Ala→Val)
G
T



05.209 (Genotype 5)
T
G (Gln→Arg)
T
C
A
C



S27 (a)
C
A
T
C
G
C







(a) Only sites that are variable among Indian Ocean outbreak isolates are represented; those sites that were distinct between Indian Ocean outbreak isolates and other viruses are given as Supplementary Information



(b) S27 numbering













TABLE 4







Primers used for RT-PCR and sequencing











Fragment
Gene
Primer (a)
Sequence (5′ to 3′)
SEQ ID NO.





FG1
5′NC
   18F
CACGTAGCCTACCAGTTTCTTA
35



nsP1
  871R
ATGGAACACCGATGGTAGGTG
36


 


FG2
nsP1
  616F
AACCCCGTTCATGTACAATGC
37



nsP1
 1435R
CGGTACCACAAAGCTGTCAAAC
38


 


FG3
nsP1
 1317F
CACTGACCTGCTGCTGTCTATG
39



nsP2
 2130R
AGTCCTGCAGCTTCTTCCTTC
40


 


FG4
nsP1
 1412F
CGAGTTTGACAGCTTTGTGGTA
41



nsP2
 2227R
ATGACTGCAATTTTGTATGGGC
42


 


FG5
nsP2
 1908F
CAATCTCGCCTGAAGACTTCC
43



nsP2
 2709R
TCCACTACAATCGGCTTGTTG
44


 


FG6
nsP2
 2530F
GTGCGGCTTCTTCAATATGATG
45



nsP2
 3343R
TCCAGGCCTATTATCCCAGTG
46


 


FG7
nsP2
 2577F
AACATCTGCACCCAAGTGTACC
47



nsP2
 3504R
GTCTCCTGTTGGCCGGTATAAT
48


 


FG8
nsP2
 3332F
TAATAGGCCTGGAGGGAAGATG
49



nsP3
 4134R
CTACGCACTCTTCATCGTTCTT
50


 


FG9
nsP2
 3885F
GAACGAGTCATCTGCGTATTGG
51



nsP3
 4725R
ATATCTCTGCCATATCCACTGC
52


 


FG10
nsP3
 4458F
TCTTTACAGCCATGGACTCGAC
53



nsP3
 5273R
CGACAGGTACGGTGCTCATTAC
54


 


FG11
nsP3
 5065F
TGTACAGGAAGCGAGTACGACC
55



nsP4
 5874R
TCTACTTTGCGCGACTGATACC
56


 


FG12
nsP4
 5630F
ACGGACGACGAGTTACGACTAG
57



nsP4
 6380R
CCCAGTATTCTTGGTTGCATG
58


 


FG13
nsP4
 6184F
AAAACAGCACGCTTACCACG
59



nsP4
 6936R
AACTTGAAGCGCGTACCTGTC
60


 


FG14
nsP4
 6732F
TCATAGCCGCACACTTTAAGC
61



nsP4
 7495R
AGGACCGCCGTACAAAGTTAC
62


 


FG15
nsP4
 7278F
GCAGGTGACGAACAAGATGAG
63



C
 8034R
CCGCTTAAAGGCCAATTTG
64


 


FG16
C
 7910F
TCGAAGTCAAGCACGAAGG
65



E2
 8670R
GTCTGTCGCTTCATTTCTGATG
66


 


FG17
E3
 8459F
TGCTTGAGGACAACGTCATGAG
67



E2
 9240R
TTTGTGATTGGTGACCGCG
68


 


FG18
E2
 9093F
AGTCCGGCAACGTAAAGATCAC
69



6K
 9861R
AAAGGTTGCTGCTCGTTCCAC
70


 


FG19
E2
 9648F
AGTTGTGTCAGTGGCCTCGTTC
71



E1
10403R
TAAAGGACGCGGAGCTTAGCTG
72


 


FG20
E1
10145F
ACAAAACCGTCATCCCGTCTC
73



E1
11158R
TGACTATGTGGTCCTTCGGAGG
74


 


FG21
E1
10959F
CAGCAAGAAAGGCAAGTGTGC
75



3′NC
11770R
TTTGCCAATTATGGTATTCA
76





(a) The primer name indicates their position and direction on the nucleotide sequence of the S27 genome.













TABLE 5





Amino-acid changes observed between strain S27 and Indian Ocean


outbreak strains in the non-structural proteins







nsP1 and nsP2




















Protein

























































Protein
172
234
301
383
384
481
488
507
 54
374
 642
 643
 793


position















Poly-
172
234
301
383
384
481
488
507
589
909
1177
1178
1328


peptide















position















S27
L
E
T
M
I
T
Q
L
S
H
C
S
A


05.115
V
K
T
L
L
I
R
R
N
Y
Y
N
V


05.61
V
K
T
L
L
I
R
R
N
Y
Y
N
V


06.21
V
K
T
L
L
I
R
R
N
Y
Y
N
V


 


06.27
V
K




L
L
I
R
R
N
Y




N
V


 


06.49
V
K
T
L
L
I
R
R
N
Y
Y
N
V


05.209
V
K
T
L
L
I
R
R
N
Y
Y
N
V










nsP3 and nsP4


















Protein
nsP3
nsP3
nsP3
nsP3
nsP3
nsP3
nsP3
nsP3
nsP3
nsP3
nsP3





Protein
 175
 217
 326
 331
 337
 352
 358
 376
 382
 460
 461


position













Poly-
1508
1550
1659
1664
1670
1685
1691
1709
1715
1793
1794


peptide













position













S27
V
Y
P
V
T
K
S
I
A
L
L


05.115
I
H
S
A
I
E
S
T
T
E
P


05.61
I
H
S
A
I
E
S
T
T
nd
nd


06.21
I
H
S
A
I
E
S
T
T
E
P


 


06.27
I
H
S
A
I
E
S
T
T




P


 


06.49
I
H
S
A
I
E
S
T
T
E
P


 


05.209
I
H
S
A
I
E




T
T
E
P












nsP3 and nsP4


















Protein
nsP3
nsP3
nsP3
nsP4
nsP4
nsP4
nsP4
nsP4
nsP4






Protein
 462
 471
 524
 75
 254
 500
 514
 555
 604



position












Polypeptide
1795
1804
1857
1938
2117
2363
2377
2418
2467



position












S27
S
P
R
T
T
Q
I
V
V



05.115
N
S
STOP
A
A
L
T
I
I



05.61
nd
nd
nd
A
A
L
T
I
I



06.21
N
S
STOP
A
A
L
T
I
I



06.27
N
S
STOP
A
A
L
T
I
I



06.49
N
S
STOP
A
A
L
T
I
I



05.209
N
S
STOP
A
A
L
T
I
I





Grayed cells correspond to aa that were variable among Indian Ocean outbreak isolates













TABLE 6





Amino-acid changes observed in the structural genes among S27, Ross and Indian Ocean outbreak strains.






























(a) When two nt positions were variable in the same codon, only the position of the upstream nt is given


Grayed cells correspond to amino acid changes among Indian Ocean outbreak isolates.













TABLE 7







Sequence used for the phylogenetic analysis of partial E1 sequences















Genomic

Isolation




Accession No.
Strain
domain
Strain Origin
Date
Phylogroup
Reference





AF192906
CAR 256
E1 partial
Central African Region
Unknown
Central Africa
1


AF192907
Ag41855
E1 partial
Uganda
1982
Central Africa
1


AY549583
ChikRCA
E1 partial
DRC (b)
1996
Central Africa
2


AF192903
AR 18211
E1 partial
South African Republic
1976
Central-East/South Africa
1


AF192904
SA H2123
E1 partial
South African Republic
1976
Central-East/South Africa
1


AF192905
Ross
E1 partial
Tanzanie
1953
Central-East/South Africa
1


AF490259
Ross
Complete
Tanzanie
1953
Central-East/South Africa
na




genome


AF369024
S27
Complete
Tanzanie
1952
Central-East/South Africa
3




genome


AY549576
DRC010
E1 partial
DRC (b)
2000
Central Africa
2


AY549577
DRC027
E1 partial
DRC (b)
2000
Central Africa
2


AY549579
DRC1719
E1 partial
DRC (b)
2000
Central Africa
2


AY549575
DRC007
E1 partial
DRC (b)
2000
Central Africa
2


AY549578
DRC1718
E1 partial
DRC (b)
2000
Central Africa
2


AY549581
DRC1725
E1 partial
DRC (b)
2000
Central Africa
2


AY549582
DRC1728
E1 partial
DRC (b)
2000
Central Africa
2


AY549580
DRC1720
E1 partial
DRC (b)
2000
Central Africa
2


AY549584
DRC1730
E1 partial
DRC (b)
2000
Central Africa
2


AF192896
644188
E1 partial
Thailand
1988
Asian
1


AF192899
3412/78
E1 partial
Thailand
1978
Asian
1


AF192894
RSU1
E1 partial
Indonesia
1985
Asian
1


AF192895
H15483
E1 partial
Philippines
1985
Asian
1


AF192898
1455/75
E1 partial
Thailand
1975
Asian
1


AF192901
Gibbs 63-263
E1 partial
India
1963
Asian
1


AF192902
PO731460
E1 partial
India
1973
Asian
1


AF192897
C-03295
E1 partial
Thailand
1995
Asian
1


AF192900
SV045196
E1 partial
Thailand
1996
Asian
1


L37661
Vaccine strain
polyprotein
Na
na
Asian
na




gene


AF192892
37997
E1 partial
Na
na
West African
1


AY726732
37997
Complete
Senegal
1983
West African
4




genome


AF192891
PM2951
E1 partial
Senegal
1966
West African
1


AF192893
IbH35 E1
E1 partial
Nigeria
1964
West African
1





References: (1) Powers et al., Pastorino et al., 2004; (3) Khan et al., 2002; (4) Vanlandingham et al., 2005.


(b) Democratic Republic of the Congo













TABLE 8







Sequence percent similarity based on amino acids and nucleotides (in parentheses) 


for the structural (SP) and non-structural (NSP) proteins of selected Alphaviruses.















05.115/06.49
05.115
06.49


Virus
Strain
Accession No.
NSP
SP
SP





CHIKV
05.115
To be submitted
 100 (100)
100 (100)
—



06.49
To be submitted
  100 (99.97)
99.91 (99.95)
100 (100)



S27
AF369024
98.79 (97.3)
98.47 (97.34)
98.38 (97.33)



37997
AY726732
95.88 (85.5)
95.82 (84.87)
95.74 (84.81)



Nagpur
AY424803
NA
97.18 (94.85)
97.10 (94.79)



Vaccine
L37661
NA
96.92 (94.24)
96.83 (94.19)


ONNV
Gulu
M20303
85.90
87.30
87.22


SFV
42S RNA genome
X04129
70.55
65.20
65.20


RRV
NB5092
M20162
69.66
64.40
64.40


SINV
HRSP
J02363
59.25
47.40
47.31





CHIKV: chikungunya virus;


ONNV: o'nyong-nyong virus;


SFV: Semliki Forest virus;


RRV: Ross River virus;


SINV: Sindbis virus


NA: Not Available.













TABLE 9





List of biological assays performed to validate the 


reactivity of anti-CHIK E2 MAbs


BIOLOGICAL ASSAYS
















 
ELISA on solubilized antigens from CHIK virions



ELISA on purified CHIK virions (La Réunion Isl.)



ELISA on purified CHIK virions (+ TX-100)



ELISA on purified CHIK virions (+ NP-40)



IF assay on CHIKV-infected VERO cells



FACS analysis on cell surface of CHIKV-infected VERO cells



Western blot on recombinant CHIK sE2 from S2 cells



IF assay on stable TRIP/CHIK.sE2-transduced 293A cell clone



Western blot on recombinant CHIK sE2 from TRIP/CHIK.sE2-



transduced 293A cell clone









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## Claims

1. A vector comprising a nucleotide sequence encoding a Chikungunya virus E2 protein and a heterologous nucleotide sequence regulating expression of the E2 protein, wherein the E2 protein comprises a threonine at amino acid position 489 of the Chikungunya virus E2 protein and a methionine at amino acid position 637 of the Chikungunya virus E2 protein.

2. The vector of claim 1, further comprising a nucleotide sequence encoding a Chikungunya E1 protein and a heterologous nucleotide sequence regulating expression of the E1 protein, wherein the E1 protein comprises a valine at amino acid position 1078 of the Chikungunya virus E1 protein, a glutamic acid at position 1093 of the Chikungunya virus E1 protein and an alanine at position 1131 of the Chikungunya virus E1 protein.

3. The vector of claim 1, further comprising a nucleotide sequence encoding a Chikungunya 6K protein and a heterologous nucleotide sequence regulating expression of the 6K protein, wherein the 6K protein comprises an isoleucine at amino acid position 756 of the Chikungunya virus 6K protein and a valine at position 802 of the Chikungunya virus 6K protein.

4. The vector of claim 1, wherein the E2 protein further comprises a lysine at amino acid position 382, a methionine at amino acid position 399, a glutamic acid at amino acid position 404, a threonine at amino acid position 485, a methionine at amino acid position 506, a threonine at amino acid position 536, an asparagine at amino acid position 624, a threonine at amino acid position 669, a threonine at amino acid position 700, and an alanine at amino acid position 711 of the Chikungunya virus E2 protein.

5. The vector of claim 4, wherein the vector encodes the amino acid sequence of SEQ ID NO:21.

6. The vector of claim 5, wherein the vector is a viral vector.

7. The vector of claim 6, wherein the vector is a recombinant lentiviral vector.

8. The vector of claim 5, wherein the vector is plasmid.

9. The vector of claim 5, wherein the vector comprises a synthetic soluble E2 gene that has been modified for optimal codon usage in mammalian cells.

10. A method for producing a Chikungunya virus E2 protein comprising introducing the vector of claim 5 into a host cell.

11. The vector of claim 1, wherein the vector encodes the amino acid sequence of SEQ ID NO:27.

12. A method for producing a Chikungunya virus E2 protein comprising introducing the vector of claim 11 into a host cell.

13. The vector of claim 1, wherein the vector is a viral vector.

14. The vector of claim 13, wherein the vector is a recombinant lentiviral vector.

15. The vector of claim 1, wherein the vector is plasmid.

16. The vector of claim 1, wherein the vector comprises a synthetic soluble E2 gene that has been modified for optimal codon usage in mammalian cells.

17. A method for producing a Chikungunya virus E2 protein comprising introducing the vector of claim 1 into a host cell.