Patent Publication Number: US-2021163544-A1

Title: Stable Cell Line Secreting Chikungunya Virus (CHIKV) Virus Like Particles (VLP) for Vaccines

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. Provisional Application Serial No. 62/737,190, filed Sep. 27, 2018, the entire contents of which are incorporated herein by reference. 
     STATEMENT OF FEDERALLY FUNDED RESEARCH 
     Not applicable. 
    
    
     INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC 
     The present application includes a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 25, 2019, is named TECH2130WO_SeqList.txt and is 16, kilo bytes in size. 
     TECHNICAL FIELD OF THE INVENTION 
     The present invention relates in general to the field of stable cell line secreting Chikungunya Virus (CHIKV) Virus Like Particles (VLP) for use as vaccines. 
     BACKGROUND OF THE INVENTION 
     Without limiting the scope of the invention, its background is described in connection with Chikungunya Virus (CHIKV) Virus Like Particles (VLP). 
     Chikungunya is a viral disease caused by Chikungunya virus (CHIKV) and transmitted to humans by infected mosquitoes. The symptoms of Chikungunya include fever and joint pain. Other symptoms include headache, nausea, muscle pain, fatigue and rash. According to the World Health Organization (WHO) website, cince 2005, India, Indonesia, Maldives, Myanmar and Thailand have reported over 1.9 million cases. 
     Currently, there is no specific treatment or vaccine for CHIKV. Virus Like Particles (VLP) provides a safe, economical and effective vaccine platform for many viral diseases. The success of the VLP vaccine against Papilloma virus (HPV) exemplifies the success of this platform. Currently there are no VLP vaccines against CHIKV approved for human use. 
     One example of a VLP vaccine for CHIKV developed by the National Institutes of Health (NIH) relies on generating VLPs by transfecting 293T cells and collecting VLPs in the supernatant. This requires repeated transfection of cells making the platform expensive for use in developing countries where this problem persists. 
     SUMMARY OF THE INVENTION 
     In one embodiment, the present invention includes a nucleic acid encoding a consensus Chikungunya virus (CHIKV) nucleic acid sequence comprising the nucleic acid of SEQ ID NO:2. In one aspect, the nucleic acid is inserted into the lentiviral vector. In another aspect, the nucleic acid expresses one or more proteins of SEQ ID NO:1. In another aspect, the nucleic acid is transfected into a cell line. In another aspect, the nucleic acid is transfected into a human cell line. In another aspect, the nucleic acid is transfected into a 293T cell line. In another aspect, the nucleic acid is stably transfected in a cell line. 
     In another embodiment, the present invention includes a method of making a Chikungunya virus (CHIKV) Virus Like Particles (VLP) comprising: inserting one or more nucleic acids into a lentiviral backbone, wherein the nucleic acid encodes one or more Chikungunya virus (CHIKV) proteins; transfecting the one or more nucleic acids into the lentiviral backbone into a cell line; culturing the transfected cell line under conditions in which the Chikungunya virus (CHIKV) Virus Like Particles (VLP) are released from the cell line; and isolating the Chikungunya virus (CHIKV) Virus Like Particles (VLP) from a culture supernatant. In one aspect, the nucleic acid expresses one or more proteins of SEQ ID NO:1. In another aspect, the nucleic acid is transfected into a cell line. In another aspect, the nucleic acid is transfected into a human cell line. In another aspect, the nucleic acid is transfected into a 293T cell line. In another aspect, the nucleic acid is stably transfected in a cell line. 
     In another embodiment, the present invention includes a vaccine comprising an isolated and purified Chikungunya virus (CHIKV) Virus Like Particles (VLP) that comprises at least one CHIKV structural protein. 
     In another embodiment, the present invention includes a cell line transformed with a nucleic acid vector comprising a nucleic acid sequence that encodes comprising a lentiviral backbone and one or more Chikungunya virus (CHIKV) proteins. In one aspect, the one or more Chikungunya virus (CHIKV) proteins are expressed by a nucleic acid sequence codon optimized for expression in human cells. In another aspect, the one or more Chikungunya virus (CHIKV) proteins are expressed by a nucleic acid sequence codon optimized for expression in human cells of SEQ ID NO:2. In another aspect, the one or more Chikungunya virus (CHIKV) proteins have the amino acid sequence of SEQ ID NO:1. In another aspect, the cell line is stably transfected with the nucleic acid vector. 
     In another embodiment, the present invention includes a purified Chikungunya virus (CHIKV) Virus Like Particles (VLP) comprising the amino acid sequence of SEQ ID NO:1. In another aspect, the CHIKV VLP are produced in a stably transformed cell line. In another aspect, the CHIKV VLP are produced in a stably transformed human cell line. In another aspect, the CHIKV VLP are produced in a stably transformed 293T cell line. In another aspect, the CHIKV VLP are isolated from at least one of: a culture supernatant, or the cell lines. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which: 
         FIG. 1A  is a schematic of a lentiviral expression vector with CHKV structural proteins for production of capsid containing virus like particles. 
         FIG. 1B  is a Western Blot showing the culture supernatants harvested from 293T cells stably transfected and expressing the CHKV structural proteins (293T-CHKV-Lenti) and analyzed for CHKV E1-E2 protein expression. Lanes 1, 2 and 3 represent supernatants harvested from different days and C represents control supernatants from 293T cells. M-molecular weight markers. 
         FIGS. 2A and 2B  show the establishment of a single cell clones expressing CHKV Structural proteins. In  FIG. 2A , the CHKV structural proteins were cloned into the lentiviral vector pLenti6/5-D-Topo and used to produce lentiviral particles containing the CHKV-E1/E2. 293T cells were then transduced with the above lentiviral particles and cells selected by culturing in the presence of blasticidin. Bulk selected cells were confirmed for E1/E2 protein expression via western blotting. Subsequently, cells were plated in 96 well plates using limiting dilution and clones arising from single viable cells selected. In  FIG. 2B , the culture supernatants were harvested from 293T single cell clones and concentrated by ultracentrifugation. Expression of the E1/E2 proteins in the supernatants was determined by western blotting. 
         FIG. 3  is a flow cytometry analysis of single cell clones expressing the CHKV-E1/E2. Six different single cell clones of 293T cells expressing the CHKV-E1/E2 proteins were stained using the CHKV E protein antibody followed by flow cytometry analysis. The bulk selected cell line was used as control. The CH-6, CH-3 and CF-5 cell lines show good CHKV E protein expression. The flow cytometry is in the same order as the table below. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention. 
     To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims. 
     The present invention includes a CHIKV vaccine candidate that forms Virus Like Particles (VLPs) in a cell line that stably produces the CHIKV VLPs. The CHIKV virus genome was cleaved to express the capsid (C), pre-membrane (PrM), envelope (E), and other non-structural proteins, none of which are capable of generating host infections, but are still able to elicit an immune response. These CHIKV VLPs were then coupled with the backbone of a West Nile virus (WNV) reporter gene, to generate reporter virus-like particles, which can be detected by luciferase assays, and when used as a vaccine, were able to trigger the production of a robust immune response in animals. The antibodies elicited were further shown to be neutralizing antibodies against CHIKV vaccine. 
     As used throughout the present specification the following abbreviations are used: TF, transcription factor; ORF, open reading frame; kb, kilobase (pairs); UTR, untranslated region; kD, kilodalton; PCR, polymerase chain reaction; RT, reverse transcriptase. 
     The term “gene” is used to refer to a functional protein, polypeptide or peptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences, or fragments or combinations thereof, as well as gene products, including those that may have been altered by the hand of man. Purified genes, nucleic acids, protein and the like are used to refer to these entities when identified and separated from at least one contaminating nucleic acid or protein with which it is ordinarily associated. 
     As used herein, the term “vector” is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The vector may be further defined as one designed to propagate Chikungunya Virus (CHIKV) Virus Like Particle sequences, or as an expression vector that includes a promoter operatively linked to the Chikungunya Virus (CHIKV) Virus Like Particle sequence, or one designed to cause such a promoter to be introduced. The vector may exist in a state independent of the host cell chromosome, or may be integrated into the host cell chromosome. 
     The term “host cell” refers to cells that have been engineered to contain nucleic acid segment that encodes a Chikungunya Virus (CHIKV) Virus Like Particle, or altered segments, whether archeal, prokaryotic, or eukaryotic. Thus, engineered, or recombinant cells, are distinguishable from naturally occurring cells that do not contain recombinantly introduced genes through the hand of man. 
     As used herein, the terms “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or “nucleic acid fragment” are used interchangeably and is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5′-monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide. 
     As used herein, the term “isolated” refers to materials, such as nucleic acid molecules and/or proteins that are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. 
     The Chikungunya Virus (CHIKV) Virus Like Particles variants of the present invention may contain alterations in the coding regions, non-coding regions, or both. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as  E. coli ), as is the case in certain embodiments of the present invention and which are known to those of skill in the art following, e.g., Sambrook and Russell, Molecular Cloning 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. 5 (2001) and by Ausubel et al., Current Protocols In Molecular Biology, John Wiley and Sons, Inc. (1998), and updates thereof. 
     Stable cell line secreting Chikungunya Virus (CHIKV) Virus Like Particles for vaccine use. Chikungunya is a viral disease caused by Chikungunya virus (CHIKV) and transmitted to humans by infected mosquitoes. Symptoms of Chikungunya include fever and joint pain. Other symptoms include headache, nausea, muscle pain, fatigue and rash. Since 2005, India, Indonesia, Maldives, Myanmar and Thailand have reported over 1.9 million cases (WHO website). 
     Currently there is no specific treatment or vaccine for CHIKV. Virus Like Particles (VLP) provide a safe, economical and effective vaccine platform for many viral diseases. The success of the VLP vaccine against Papilloma virus (HPV) exemplifies the success of this platform. 
     Currently there are no VLP vaccines against CHIKV approved for human use. A VLP vaccine for CHIKV developed by NIH relies on generating VLPs by transfecting 293T cells and collecting VLPs in the supernatant. This requires repeated transfection of cells making the platform expensive for use in developing countries where this problem persists. 
     The present inventors have developed a VLP platform for the related arbovirus, Zika virus, using stable cell lines that constitutively secrete VLPs and demonstrated that this platform can provide an economical, safe and highly effective vaccine especially for use in humans. A similar stable cell line method was used to generate CHIKV VLP secreting cell line. The present invention provides three substantial improvements and advantages over the prior art. 
     1. The inventors generated a consensus sequence of 478 CHIKV sequences from year 2006 onwards to represent the most current CHIKV isolates. The artificial consensus sequence and translation was codon optimized to drive high expression of the proteins. The use of the consensus sequence provides a vaccine that is most relevant to current outbreaks. 
     2. The inventors used a lentiviral system to generate stable cell lines that constitutively express CHIKV structural proteins and secrete the VLPs in the supernatant. 
     3. Finally, the inventors optimized production and purification of the VLPs from these stable cell lines. 
     Chikungunya Virus Consensus Sequences used in the vaccine: 
     
       
         
           
               
               
            
               
                 Amino acid sequence: SEQ ID NO: 1. 
                   
               
               
                 MEFIPTQTFYNRRYQPRPWTPRPTIQVIRPRPRPQRKAGQLAQLISAVNKLTMRV 
               
               
                   
               
               
                 VPQQKPRKNRKNKKQKQKQQAPRNNTNQKKQPPKKKPVQKKKKPGRRERMCMKIEN 
               
               
                   
               
               
                 DCIFEVKHEGKVTGYACLVGDKVMKPAHVKGTIDNADLAKLAFKRSSKYDLECAQIPV 
               
               
                   
               
               
                 HMKSDASKFTHEKPEGYYNWHHGAVQYSGGRFTIPTGAGKPGDSGRPIFDNKGRVVAI 
               
               
                   
               
               
                 VLGGANEGARTALSVVTWNKDIVTKITPEGAEEWSLAIPVMCLLANTTFPCSRPPCTPC 
               
               
                   
               
               
                 CYEKEPEKTLRMLEDNVMSPGYYQLLQASLTCSPRRQRRSIKDHFNVYKATRPYLAHC 
               
               
                   
               
               
                 PDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPAD 
               
               
                   
               
               
                 AERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDGRKISHSCTHPFHHDPPVIGR 
               
               
                   
               
               
                 EKFHSRPQHGRELPCSTYAQSTAATAEEIEVHMPPDTPDRTLMSQQSGNVKITVNSQTV 
               
               
                   
               
               
                 RYKCNCGDSSEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAEFGDRKGK 
               
               
                   
               
               
                 VHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVT 
               
               
                   
               
               
                 HKKEIRLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTAVV 
               
               
                   
               
               
                 LSVASFILLSMVGVAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAATYQEAA 
               
               
                   
               
               
                 VYLWNEQQPLFWMQALIPLAALIVLCNCLRLLPCCCKMLTFLAVLSVGAHTVSAYEHV 
               
               
                   
               
               
                 TVIPNTVGVPYKTLVNRPGYSPMVLEMELLSVTLEPTLSLDYITCEYKTVIPSPYVKCCG 
               
               
                   
               
               
                 TAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDTENTQLSEAHVEKSESCKTEFASAY 
               
               
                   
               
               
                 RAHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVV 
               
               
                   
               
               
                 YKGDVYNMDYPPFGAGRPGQFGDIQSRTPESEDVYANTQLVLQRPSAGTVHVPYSQAP 
               
               
                   
               
               
                 SGFKYWLKERGASLQHTAPFGCQIATNPVRAMNCAVGNMPISIDIPDAAFTRVVDAPSL 
               
               
                   
               
               
                 TDMSCEVSACTHSSDFGGVAIIKYAASKKGKCAVHSMTNAVTIREAEIEVEGNSQLQISF 
               
               
                   
               
               
                 STALASAEFRVQVCSTQVHCAAECHPPKDHIVNYPASHTTLGVQDISATAMSWVQKITG 
               
               
                   
               
               
                 GVGLVVAVAALILIVVLCVSFSRH 
               
               
                   
               
               
                 DNA sequence SEQ ID NO: 2 
               
               
                 ATGGAGTTCATCCCCACACAGACCTTTTATAACCGGAGATACCAGCCCAGGC 
               
               
                   
               
               
                 CTTGGACCCCACGCCCAACAATCCAGGTCATCAGGCCTCGGCCAAGACCACAGAGG 
               
               
                   
               
               
                 AAGGCAGGACAGCTGGCACAGCTGATCAGCGCCGTGAATAAGCTGACCATGCGCGT 
               
               
                   
               
               
                 GGTGCCCCAGCAGAAGCCTCGGAAGAACAGAAAGAATAAGAAGCAGAAGCAGAAG 
               
               
                   
               
               
                 CAGCAGGCCCCAAGGAACAATACCAACCAGAAGAAGCAGCCCCCCAAGAAGAAGC 
               
               
                   
               
               
                 CTGTGCAGAAGAAGAAGAAGCCAGGCAGGCGCGAGCGCATGTGCATGAAGATCGA 
               
               
                   
               
               
                 GAATGATTGCATCTTCGAGGTGAAGCACGAGGGCAAGGTGACCGGCTACGCCTGTC 
               
               
                   
               
               
                 TGGTGGGCGACAAAGTGATGAAGCCCGCCCACGTGAAGGGCACAATCGACAACGC 
               
               
                   
               
               
                 CGATCTGGCCAAGCTGGCCTTCAAGAGGAGCTCCAAGTATGATCTGGAGTGCGCCC 
               
               
                   
               
               
                 AGATCCCCGTGCACATGAAGAGCGACGCCTCCAAGTTTACCCACGAGAAGCCTGAG 
               
               
                   
               
               
                 GGCTACTATAATTGGCACCACGGAGCAGTGCAGTACTCTGGAGGCAGGTTCACCAT 
               
               
                   
               
               
                 CCCTACAGGAGCAGGCAAGCCAGGCGACAGCGGCAGACCCATCTTTGATAATAAGG 
               
               
                   
               
               
                 GAAGAGTGGTGGCAATCGTGCTGGGAGGAGCAAACGAGGGCGCCAGAACCGCCCT 
               
               
                   
               
               
                 GAGCGTGGTGACATGGAATAAGGATATCGTGACCAAGATCACACCTGAGGGAGCA 
               
               
                   
               
               
                 GAGGAGTGGTCTCTGGCAATCCCAGTGATGTGCCTGCTGGCCAACACCACATTCCCA 
               
               
                   
               
               
                 TGTAGCCGGCCACCATGCACCCCATGCTGTTACGAGAAAGAGCCTGAGAAGACACT 
               
               
                   
               
               
                 GAGAATGCTGGAGGACAATGTGATGTCCCCTGGCTACTATCAGCTGCTGCAGGCCT 
               
               
                   
               
               
                 CTCTGACCTGTAGCCCACGGAGACAGAGGCGCTCTATCAAGGATCACTTTAACGTGT 
               
               
                   
               
               
                 ATAAGGCCACAAGGCCTTACCTGGCACACTGTCCAGACTGCGGAGAGGGACACTCT 
               
               
                   
               
               
                 TGCCACAGCCCAGTGGCCCTGGAGCGGATCAGAAATGAGGCCACCGATGGCACACT 
               
               
                   
               
               
                 GAAGATCCAGGTGAGCCTGCAGATCGGCATCAAGACCGACGATTCCCACGACTGGA 
               
               
                   
               
               
                 CAAAGCTGCGCTACATGGACAACCACATGCCAGCCGATGCAGAGAGGGCAGGACT 
               
               
                   
               
               
                 GTTCGTGAGAACCAGCGCCCCCTGTACAATCACCGGCACAATGGGCCACTTCATCCT 
               
               
                   
               
               
                 GGCAAGGTGCCCAAAGGGAGAGACCCTGACAGTGGGCTTTACCGATGGCCGCAAG 
               
               
                   
               
               
                 ATCTCTCACAGCTGTACACACCCTTTCCACCACGACCCTCCAGTGATCGGCCGCGAG 
               
               
                   
               
               
                 AAGTTTCACTCCCGGCCACAGCACGGAAGAGAGCTGCCCTGCTCTACCTATGCACA 
               
               
                   
               
               
                 GAGCACCGCCGCCACAGCCGAGGAGATCGAGGTGCACATGCCCCCTGACACCCCCG 
               
               
                   
               
               
                 ATCGGACACTGATGTCCCAGCAGTCTGGCAACGTGAAGATCACCGTGAATAGCCAG 
               
               
                   
               
               
                 ACAGTGAGATACAAGTGTAACTGCGGCGACTCTAGCGAGGGCCTGACCACAACCGA 
               
               
                   
               
               
                 TAAAGTGATCAACAATTGTAAGGTGGACCAGTGCCACGCCGCCGTGACCAACCACA 
               
               
                   
               
               
                 AGAAGTGGCAGTATAATTCCCCACTGGTGCCCAGGAACGCCGAGTTCGGCGATCGC 
               
               
                   
               
               
                 AAGGGCAAGGTGCACATCCCTTTTCCACTGGCCAATGTGACCTGCAGGGTGCCTAA 
               
               
                   
               
               
                 GGCCCGCAATCCAACCGTGACATACGGCAAGAACCAGGTCATCATGCTGCTGTATC 
               
               
                   
               
               
                 CTGACCACCCAACACTGCTGAGCTACAGGAACATGGGCGAGGAGCCTAATTATCAG 
               
               
                   
               
               
                 GAGGAGTGGGTGACCCACAAGAAGGAGATCCGCCTGACCGTGCCAACAGAGGGCC 
               
               
                   
               
               
                 TGGAGGTGACATGGGGCAACAATGAGCCCTATAAGTACTGGCCTCAGCTGTCCACC 
               
               
                   
               
               
                 AACGGAACAGCACACGGACACCCACACGAGATCATCCTGTACTATTACGAGCTGTA 
               
               
                   
               
               
                 CCCTACCATGACAGCCGTGGTGCTGAGCGTGGCCTCCTTCATCCTGCTGTCCATGGT 
               
               
                   
               
               
                 GGGAGTGGCAGTGGGAATGTGCATGTGCGCACGGAGAAGGTGCATCACCCCATATG 
               
               
                   
               
               
                 AGCTGACCCCCGGCGCCACAGTGCCTTTTCTGCTGTCTCTGATCTGCTGTATCCGGA 
               
               
                   
               
               
                 CCGCCAAGGCCGCCACATATCAGGAGGCCGCCGTGTACCTGTGGAACGAGCAGCAG 
               
               
                   
               
               
                 CCCCTGTTCTGGATGCAGGCCCTGATCCCTCTGGCCGCCCTGATCGTGCTGTGCAAT 
               
               
                   
               
               
                 TGCCTGAGACTGCTGCCTTGCTGTTGCAAGATGCTGACCTTTCTGGCCGTGCTGTCC 
               
               
                   
               
               
                 GTGGGCGCCCACACAGTGTCTGCCTACGAGCACGTGACCGTGATCCCCAATACAGT 
               
               
                   
               
               
                 GGGCGTGCCTTACAAGACCCTGGTGAACCGGCCAGGCTATTCTCCCATGGTGCTGG 
               
               
                   
               
               
                 AGATGGAGCTGCTGAGCGTGACCCTGGAGCCAACACTGTCCCTGGATTATATCACCT 
               
               
                   
               
               
                 GTGAGTACAAGACAGTGATCCCCAGCCCTTACGTGAAGTGTTGCGGCACCGCCGAG 
               
               
                   
               
               
                 TGTAAGGACAAGTCCCTGCCAGATTATTCTTGCAAGGTGTTCACAGGCGTGTATCCC 
               
               
                   
               
               
                 TTTATGTGGGGCGGCGCCTACTGTTTCTGCGACACCGAGAACACACAGCTGTCCGAG 
               
               
                   
               
               
                 GCCCACGTGGAGAAGTCCGAGTCTTGCAAGACCGAGTTTGCCTCTGCCTACAGAGC 
               
               
                   
               
               
                 CCACACAGCAAGCGCCTCCGCCAAGCTGAGAGTGCTGTACCAGGGCAACAATATCA 
               
               
                   
               
               
                 CCGTGGCCGCCTATGCCAATGGCGACCACGCCGTGACAGTGAAGGATGCCAAGTTC 
               
               
                   
               
               
                 ATCGTGGGACCCATGTCCTCTGCCTGGACCCCATTTGACAATAAGATCGTGGTGTAC 
               
               
                   
               
               
                 AAGGGCGACGTGTATAACATGGATTACCCACCCTTCGGCGCAGGCAGGCCTGGACA 
               
               
                   
               
               
                 GTTTGGCGATATCCAGAGCCGCACCCCAGAGTCCGAGGACGTGTATGCCAACACAC 
               
               
                   
               
               
                 AGCTGGTGCTGCAGAGGCCAAGCGCCGGCACCGTGCACGTGCCATACTCCCAGGCC 
               
               
                   
               
               
                 CCCTCTGGCTTCAAGTATTGGCTGAAGGAGAGGGGAGCATCCCTGCAGCACACCGC 
               
               
                   
               
               
                 ACCATTTGGCTGTCAGATCGCCACAAATCCCGTGAGAGCCATGAACTGCGCCGTGG 
               
               
                   
               
               
                 GCAATATGCCAATCAGCATCGACATCCCCGATGCCGCCTTCACCAGAGTGGTGGAC 
               
               
                   
               
               
                 GCCCCTTCCCTGACAGATATGAGCTGTGAGGTGTCCGCCTGCACCCACAGCTCCGAC 
               
               
                   
               
               
                 TTTGGCGGCGTGGCCATCATCAAGTACGCCGCCTCTAAGAAGGGCAAGTGTGCCGT 
               
               
                   
               
               
                 GCACAGCATGACCAACGCCGTGACAATCCGGGAGGCCGAGATCGAGGTGGAGGGC 
               
               
                   
               
               
                 AATAGCCAGCTGCAGATCTCTTTCAGCACCGCCCTGGCCTCCGCCGAGTTTAGAGTG 
               
               
                   
               
               
                 CAGGTGTGCTCTACACAGGTGCACTGTGCCGCCGAGTGCCACCCTCCAAAGGATCA 
               
               
                   
               
               
                 CATCGTGAACTATCCAGCATCCCACACAACCCTGGGAGTGCAGGACATCTCTGCCA 
               
               
                   
               
               
                 CCGCCATGAGCTGGGTGCAGAAGATCACAGGAGGAGTGGGACTGGTGGTGGCAGT 
               
               
                   
               
               
                 GGCCGCCCTGATCCTGATCGTGGTGCTGTGCGTGTCCTTCTCTAGACAC 
               
            
           
         
       
     
       FIG. 1A  is a schematic of a lentiviral vector expression that includes CHKV structural proteins for production of capsid containing virus like particles.  FIG. 1B  is a Western Blot showing the culture supernatants harvested from 293T cells stably transfected and expressing the CHKV structural proteins (293T-CHKV-Lenti) and analyzed for CHKV E1-E2 protein expression. Lanes 1, 2 and 3 represent supernatants harvested from different days and C represents control supernatants from 293T cells. M-molecular weight markers. 
       FIGS. 2A and 2B  show the establishment of a single cell clones expressing CHKV Structural proteins. In  FIG. 2A , the CHKV structural proteins were cloned into the lentiviral vector pLenti6/5-D-Topo and used to produce lentiviral particles containing the CHKV-E1/E2. 293T cells were then transduced with the above lentiviral particles and cells selected by culturing in the presence of blasticidin. Bulk selected cells were confirmed for E1/E2 protein expression via western blotting. Subsequently, cells were plated in 96 well plates using limiting dilution and clones arising from single viable cells selected. In  FIG. 2B , the culture supernatants were harvested from 293T single cell clones and concentrated by ultracentrifugation. Expression of the E1/E2 proteins in the supernatants was determined by western blotting. 
       FIG. 3  is a flow cytometry analysis of single cell clones expressing the CHKV-E1/E2. Six different single cell clones of 293T cells expressing the CHKV-E1/E2 proteins were stained using the CHKV E protein antibody followed by flow cytometry analysis. The bulk selected cell line was used as control. The CH-6, CH-3 and CF-5 cell lines show good CHKV E protein expression. The flow cytometry is in the same order as the table below the graph. 
     It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention. 
     It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. 
     All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. 
     The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. 
     As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. As used herein, the phrase “consisting essentially of” requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only. 
     The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. 
     As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%. 
     All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. 
     To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (0, or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim. 
     For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.