Methods for the prophylactic treatment of chikungunya virus infections

Abstract

The present invention provides live attenuated vaccines for the prophylactic treatment of Chikungunya virus infections. The vaccines comprise a genetically modified Chikungunya virus that has a reduced replication capacity due to a mutation or deletion in the R5 region of the hypervariable domain of nsP3, a nonstructural protein that interacts with host factors. The inventors have indeed discovered that the R5 region of the hypervariable domain of nsP3, a nonstructural protein that interacts with host factors, is critical for CHIKV replication and pathogenesis. Specifically, the inventors have shown that the R5 region is required for the interaction of nsP3 with FHL1, BIN1 and CD2AP, three host proteins that are involved in the regulation of actin cytoskeleton and membrane trafficking. These host proteins are expressed in muscle and joint tissues, which are the main targets of CHIKV infection and inflammation. By mutating or deleting the R5 region of nsP3, the inventors have generated a genetically modified CHIKV (CHIKV-ΔR5) that has a reduced replication capacity and virulence compared to the WT virus. Moreover, the inventors have demonstrated that CHIKV-ΔR5 is able to elicit a strong neutralizing antibody response and protect mice from lethal challenge with WT virus. The present invention also provides screening methods for identifying test substances that are capable of inhibiting the interaction between nsP3 and FHL1, CD2AP and BIN1, wherein the selected test substances would be suitable for the treatment of CHIKV infections.

Description

[0001] METHODS FOR THE PROPHYLACTIC TREATMENT OF CHIKUNGUNYA

[0002] VIRUS INFECTIONS

[0003] FIELD OF THE INVENTION:

[0004] The present invention is in the field of medicine, in particular virology and vaccinology.

[0005] BACKGROUND OF THE INVENTION:

[0006] Chikungunya virus (CHIKV) is a member of the Alphavirus genus in the Togaviridae family, which consists of more than 30 arthropod-borne viruses that cause human and animal diseases. Chikungunya virus was first isolated in 1953 from a patient with fever and rash in Tanzania, and the name derives from a Makonde word meaning "that which bends up", referring to the characteristic stooped posture of patients with joint pain. Chikungunya virus is transmitted by the bite of infected mosquitoes, mainly of the Aedes genus, such as 4. aegypti and A. albopictus. These mosquitoes are widely distributed in tropical and subtropical regions, and can also transmit other viruses, such as dengue, yellow fever, and Zika.

[0007] Chikungunya virus infection is a re-emerging global health problem, with sporadic and unpredictable outbreaks occurring in various parts of the world. Since 2004, there have been several large epidemics of chikungunya virus infection, affecting millions of people in Africa, Asia, Europe, and the Americas. The most recent outbreak started in 2013 in the Caribbean islands and spread to North, Central, and South America, causing more than 2 million cases. According to the World Health Organization, at least 5 million chikungunya virus infections were reported over the past 15 years, underscoring it as a major public health threat. Moreover, with increasing international travel and climate change, the potential range and incidence of chikungunya virus infection are expected to expand further.

[0008] The clinical manifestations of chikungunya virus infection vary from asymptomatic or mild illness to severe and debilitating disease. The typical signs and symptoms include sudden onset of high fever, viremia, intense joint pain, recurring mild joint pain, and maculopapular rash, which can affect all sex and age groups. The joint pain usually affects multiple joints, especially the hands, wrists, ankles, and feet, and can persist for weeks or months, causing significant morbidity and reduced quality of life. In some cases, chronic inflammatory arthritis can develop, resembling rheumatoid arthritis. Other complications of chikungunya virus infection include neurological disorders, such as encephalitis, meningitis, and Guillain-Barre syndrome; ocular diseases, such as uveitis and retinitis; cardiovascular problems, such as myocarditis and pericarditis; and renal, hepatic, and hematological abnormalities. The case-fatality ratio of chikungunya virus infection is estimated to be 0.3-1 per 1000, with most deaths occurring in neonates, adults with underlying conditions, and older people.

[0009] Currently, there is no specific antiviral treatment or licensed vaccine for chikungunya virus infection. The management of patients relies on symptomatic and supportive care, such as analgesics, anti-inflammatory drugs, and fluid replacement. Therefore, there is an urgent medical need for effective and safe prophylaxis against chikungunya virus infection, especially for high-risk populations, such as travelers, military personnel, and health-care workers. Several vaccine candidates have been developed and tested in preclinical and clinical studies, but none of them has reached the market yet. Among the different types of vaccines, live attenuated vaccines have shown promising results in terms of inducing robust and durable immune responses, with a single dose or a short immunization schedule. Live attenuated vaccines are derived from natural or engineered strains of chikungunya virus that have reduced virulence and replication capacity, but retain their antigenicity and immunogenicity. Live attenuated vaccines can stimulate both humoral and cellular immunity, as well as mucosal and memory responses, providing protection against chikungunya virus infection and disease. One example of a live attenuated vaccine for chikungunya virus infection is VLA1553, developed by Valneva, a biotechnology company based in France and Austria. VLA1553 is derived from the East / Central / South African genotype of chikungunya virus, which is responsible for most of the recent outbreaks. VLA1553 has been genetically modified to delete a segment of the non- structural protein 1 (nsPl), which is essential for viral RNA replication. This deletion renders the virus unable to replicate in human cells, but still capable of expressing the structural proteins that elicit an immune response. VLA1553 is administered as a single intramuscular injection, with the aim of conferring long-lasting immunity against chikungunya virus infection and is currently evaluated in clinical trial (Schneider, Martina, et al. "Safety and immunogenicity of a single-shot live-attenuated chikungunya vaccine: a double-blind, multicentre, randomised, placebo-controlled, phase 3 trial." The Lancet 401.10394 (2023): 2138-2147). Another example of a live attenuated vaccine for chikungunya virus infection is CHIKV-AFHL1, developed by researchers from the National University of Singapore and Duke-NUS Medical School (Ng, Wern Hann, et al. "FHL1 promotes chikungunya and o ’nyong-nyong virus infection and pathogenesis with implications for alphavirus vaccine design. " Nature Communications 14.1 (2023): 6605). CHIKV-AFHL1 is based on an Asian genotype of chikungunya virus, which was isolated from a patient in Singapore during the 2008 outbreak. CHIKV-AFHL1 has been engineered to remove the binding site of a host protein called factor H-like protein 1 (FHL1). By deleting this binding site, the virus loses its ability to interact with FHL1 and becomes less pathogenic and more immunogenic. CHIKV-AFHL1 is administered as a single subcutaneous injection, with the aim of inducing protective immunity against chikungunya virus infection and preventing chronic joint pain.

[0010] SUMMARY OF THE INVENTION:

[0011] The present invention is defined by the claims. In particular, the present invention relates to live attenuated vaccines for the prophylactic treatment of Chikungunya virus infections.

[0012] DETAILED DESCRIPTION OF THE INVENTION:

[0013] The present invention provides live attenuated vaccines for the prophylactic treatment of Chikungunya virus infections. The vaccines comprise a genetically modified Chikungunya virus that has a reduced replication capacity due to a mutation or deletion in the R5 region of the hypervariable domain of nsP3, a nonstructural protein that interacts with host factors. In particular, the inventors showed that the R5 region of the hypervariable domain of nsP3 is required for the interaction of nsP3 with FHL, BINI and CD2AP, but dispensable for G3BP1 interaction. They also showed that CHIKV-AR5 exhibited a reduced replication capacity compared to CHIKV-WT in BHK21, U2OS and VeroE6 cells. These results thus suggest that the R5 domain of nsP3 is important for efficient viral replication and host factor recruitment. One of the objectives of the present invention is to provide a live attenuated vaccine that can induce a protective immune response against CHIKV infection, while avoiding the adverse effects of wild-type (WT) virus replication in host cells. The inventors have discovered that the R5 region of the hypervariable domain of nsP3, a nonstructural protein that interacts with host factors, is critical for CHIKV replication and pathogenesis. Specifically, the inventors have shown that the R5 region is required for the interaction of nsP3 with FHL1, BINI and CD2AP, three host proteins that are involved in the regulation of actin cytoskeleton and membrane trafficking. These host proteins are expressed in muscle and joint tissues, which are the main targets of CHIKV infection and inflammation. By mutating or deleting the R5 region of nsP3, the inventors have generated a genetically modified CHIKV (CHIKV-AR5) that has a reduced replication capacity and virulence compared to the WT virus. Moreover, the inventors have demonstrated that CHIKV-AR5 is able to elicit a strong neutralizing antibody response and protect mice from lethal challenge with WT virus.

[0014] The inventors have thus disclosed a novel mechanism of CHIKV attenuation that is based on blocking the interaction between the virus and FHL1, and simultaneously CD2AP and BINI, which is tantamount to interfering with the virus' ability to replicate in muscle and joint tissues. This strategy effectively lowers the viral load to a level that will induce an immune response while preventing the pathology associated with infection. Therefore, the inventors claim that their invention covers not only the specific CHIKV-AR5 vaccine, but also any other interventions or strategies that would prevent virus replication in these two tissues and lead to virus attenuation. Such interventions or strategies could include, but are not limited to, the use of small molecules, antibodies, RNA interference. . . that target the R5 region of nsP3 or its host interactors. The inventors contend that their invention provides a broad and novel approach to combat CHIKV infection and its devastating consequences.

[0015] Main definitions:

[0016] As used herein, the term “chikungunya virus” or "CHIKV" refers to a virus that belongs to the genus Alphavirus and causes Chikungunya fever, a disease characterized by fever, rash, and joint pain. CHIKV has a single-stranded positive-sense RNA genome that encodes four nonstructural proteins (Nspl-4) and five structural proteins (C, E3, E2, 6K, and El). CHIKV is transmitted by mosquitoes of the Aedes genus and can infect both humans and animals.

[0017] As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein.

[0018] As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification. According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

[0019] As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. As used herein, the term "deletion" refers to a type of mutation that involves the removal of one or more amino acids from a polypeptide sequence. Deletions can affect the function and structure of proteins, depending on the size and location of the deletion. Within the specification, the mutation are references according to the standard mutation nomenclature.

[0020] As used herein, the term “nsP3” refers to nonstructural protein 3, which is found in alphaviruses. nsP3 is a multifunctional protein that plays a crucial role in the replication and assembly of alphaviruses. It is a component of the viral replication complex and is involved in various steps of the viral life cycle. nsP3 is typically produced as part of a polyprotein precursor that is processed into individual nonstructural proteins, including nsp3, by viral proteases. nsP3 consists of multiple domains with distinct functions. Exemplary amino acid sequences for nsP3 are represented by SEQ ID NO:1.

[0021] SEQ ID NO : 1 NSP3 of CHIKV (the R5 domain is underlined)

[0022] APSYRVKRMDIAKNDEECWNAANPRGLPGDGVCKAVYKKWPESFKNSATPVGTAKTVMCGTYPVIHAVG PNFSNYSESEGDRELAAAYREVAKEVTRLGVNSVAI PLLSTGVYSGGKDRLTQSLNHLFTAMDSTDADW IYCRDKEWEKKI SEAIQMRTQVELLDEHI SIDCDIVRVHPDSSLAGRKGYSTTEGALYSYLEGTRFHQTA VDMAEIHTMWPKQTEANEQVCLYALGESIESIRQKCPVDDADASSPPKTVPCLCRYAMTPERVTRLRMNH VTSI IVCSSFPLPKYKIEGVQKVKCSKVMLFDHNVPSRVSPREYRSSQESAQEASTITSLTHSQFDLSVD GEILPVPSDL DADAP AL E PAL D D GAT HT L P S T T GN LAAVSDWVI STVPVAPPRRRRGRNLTVTCDEREGN I TPMASVRFFRAELCPWQETAETRDTAMSLQAPPSTATE PNH P PI S FGAS S ET FP I T FGD FNEGE I E S L SSELLTFGDFLPGEVDDLTDSDWSTCSDTDDEL

[0023] As used herein, the term "R5 domain" refers to a specific domain of nsP3 that is located between amino acids 391 and 460. The R5 region is essential for the interaction of nsP3 with host proteins, such as FHL1, CD2AP, and BINI, and for the modulation of the replication of Chikungunya virus in the host cell. The R5 region is represented by the underlined sequence in SEQ ID NO: 1.

[0024] As used herein, the term "FHL1" refers to a protein that is encoded by the FHL1 gene and belongs to the four-and-a-half LIM domain family of proteins. FHL1 is expressed in various tissues, such as skeletal muscle, heart, and kidney, and plays a role in muscle development, differentiation, and regeneration. FHL1 interacts with several viral proteins, including the nsP3 protein of Chikungunya virus, and modulates the innate immune response against viral infection.

[0025] As used herein, the term "CD2AP" refers to a protein that is encoded by the CD2AP gene and belongs to the adaptor protein family. CD2AP is expressed in various tissues, such as lymphoid organs, kidney, lung, and brain, and plays a role in cell adhesion, cytoskeleton organization, endocytosis, and signal transduction. CD2AP interacts with several viral proteins, including the nsP3 protein of Chikungunya virus, and modulates the innate immune response against viral infection.

[0026] As used herein, the term "BINI" refers to a protein that is encoded by the BINI gene and belongs to the BAR (Bin / Amphiphysin / Rvs) domain family of proteins. BINI is expressed in various tissues, such as skeletal muscle, brain, and immune cells, and plays a role in membrane remodeling, endocytosis, and signal transduction. BINI interacts with several viral proteins, including the nsP3 protein of Chikungunya virus, and modulates the innate immune response against viral infection. As used herein, the term "mutein" refers to a variant of a protein that has one or more amino acid substitutions, deletions, or insertions compared to the original protein. A mutein can have altered biological properties, such as affinity, stability, activity, or interactions with other molecules. A mutein can be produced by genetic engineering, mutagenesis, or other methods. For example, a mutein of the nsP3 protein of Chikungunya virus can have different binding capacities to host proteins, such as FHL1, CD2AP, and BINI, and affect the viral replication and pathogenesis.

[0027] As used herein, the term "host cell" refers to a cell that is infected by a virus and supports its replication. Depending on the type of virus, a host cell may have specific receptors, enzymes, or factors that facilitate viral entry, transcription, translation, assembly, or release.

[0028] As used herein, the term “engineered” refers to an aspect of having been manipulated and altered by the hand of man. In particular, the term “engineered virus” refers to a virus that has been subjected to manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference virus such as otherwise identical virus that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation. In some embodiments, a genetic manipulation is or comprises one or more of (i) introduction of a polynucleotide not present in the virus prior to the manipulation (i.e., of a heterologous polynucleotide); (ii) removal of a polynucleotide, or portion thereof, present in the virus prior to the manipulation; and / or (iii) alteration (e.g., by sequence substitution) of a polynucleotide, or portion thereof, present in the virus prior to the manipulation. Those of ordinary skill in the art will appreciate that reference to an “engineered virus” herein may, in some embodiments, encompass both the particular virus to which the manipulation was applied and also any progeny of such virus.

[0029] As used herein, the term “patient” is interchangeable with the term “individual” or “subject”, and may refer to a subject to be treated by the methods disclosed herein. Typically, the patient is affected or likely to suffer from a CHIKV infection. In some embodiments, the patient is a mammal. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bushbabies, monkeys, apes, and humans), rabbits, dogs (e.g., companion dogs, service dogs, or work dogs such as police dogs, military dogs, race dogs, or show dogs), horses (such as race horses and work horses), cats (e.g., domesticated cats), livestock (such as pigs, bovines, donkeys, mules, bison, goats, camels, and sheep), and deer. In some embodiments, the mammal is a human. In some embodiments, the patient is a human infant. In some embodiments, the patient is a human child. In some embodiments, the patient is a human adult.

[0030] As used herein, the term "treatment" or "treat" refers to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of patients at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during the treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0031] As used herein, the terms "prophylaxis" or "prophylactic use" and "prophylactic treatment" refer to any medical or public health procedure whose purpose is to prevent, rather than treat or cure a disease. As used herein, the terms "prevent", "prevention" and "preventing" refer to the reduction in the risk of acquiring or developing a given condition, or the reduction or inhibition of the recurrence or said condition in a subject who is not ill, but who has been or may be near a subject with the disease.

[0032] As used herein, the term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.

[0033] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

[0034] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[0035] As used herein, the term "vaccine composition" is intended to mean a composition which can be administered to humans or to animals in order to induce an immune system response; this immune system response can result in the activation of certain cells, in particular antigen- presenting cells (APCs), T lymphocytes and B lymphocytes.

[0036] As used herein, the terms “live vaccine composition”, “live vaccine”, “live bacterial vaccine”, and similar terms refer to a composition comprising a strain of live CHIKV that provides at least partial protective immunity against a disease, condition, or disorder.

[0037] As used herein, the term “adjuvant” refers to a compound that can induce and / or enhance the immune response against an antigen when administered to a patient or an animal. It is also intended to mean a substance that acts generally to accelerate, prolong, or enhance the quality of specific immune responses to a specific antigen. In the context of the present invention, the term "adjuvant" means a compound, which enhances both the innate immune response by affecting the transient reaction of the innate immune response and the more long-lived effects of the adaptive immune response by activation and maturation of the antigen-presenting cells (APCs) especially Dendritic cells (DCs).

[0038] Attenuated viruses of the present invention:

[0039] The first object of the present invention relates to a live attenuated chikungunya virus that is engineered to express a mutein of the nonstructural protein 3 (nsP3) that comprises one or more mutations in the R5 domain so that said nsP3 mutein is no capable to bind to FHL1, CD2AP and BINI in a host cell.

[0040] The live attenuated CHIKV of the present invention can be derived from any genotype of chikungunya virus, such as the African or Asian genotype, that has been genetically modified to introduce one or more mutations in the nsP3 protein. In some embodiments, The live attenuated CHIKV of the present derives from a strain isolated in the He de la Reunion (France).

[0041] In some embodiments, The live attenuated CHIKV of the present invention is engineered to express a nsP3 mutein that comprises one or more deletion in the R5 domain that said nsP3 mutein is no capable to bind to FHL1, CD2AP and BINI in a host cell.

[0042] The deletion(s) may be continuous, or may comprise a plurality of sections of sequence. The deletion should remove a sufficient amount of the R5 domain so that that said nsP3 mutein is no capable to bind to FHL1, CD2AP and BINI in a host cell. The deletion may, for example, remove at least 50, 60, 70, 80 or 90% of the R5 domain. In some embodiments, the deletion may be total, in which case 100% of the R5 domain is absent, when compared to the corresponding amino acid sequence of the wild-type isolate.

[0043] In some embodiments, The live attenuated CHIKV of the present invention is engineered to express a nsP3 mutein wherein the R5 domain is deleted.

[0044] In some embodiments, The live attenuated CHIKV of the present invention is engineered to express a nsP3 that consists of an amino acid sequence having a least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:1 wherein the domain that ranges from the amino acid residue at position 391 to the amino acid residue at position 460 is deleted. In some embodiments, The live attenuated CHIKV of the present invention is engineered to express a nsP3 that consists of the amino acid sequence having as set forth in SEQ ID NO: 1 wherein the domain that ranges from the amino acid residue at position 391 to the amino acid residue at position 460 is deleted.

[0045] The mutation(s) can be introduced by any suitable method, such as site-directed mutagenesis, or reverse genetics. Methods for introducing deletion in the viral genome are well known in the art. For example, homologous recombination may be used, in which a transfer vector is created in which the relevant gene(s) are missing and used to transfect virus-infected cells. Recombinant viruses expressing the new portion of sequence may then be selected.

[0046] Thus, certain aspects of the present invention are drawn to a method of producing a vaccine comprising the steps of introducing the engineered virus into host cells and allowing the virus to replicate in the host cells to produce a viral vaccine.

[0047] The resulting attenuated CHIKV can be propagated in cell culture and purified by standard techniques. The attenuated viruses of the present invention can be propagated in various cell lines that are permissive for chikungunya virus infection. Examples of such cell lines include, but are not limited to, Vero cells, C6 / 36 cells, BHK-21 cells, HeLa cells, 293T cells, CHO cells, COS cells, and SF9 cells. The choice of the cell line may depend on the efficiency of virus replication, the yield of virus production, and the safety of the cell culture. The cell line may also be modified to express certain viral proteins or factors that can enhance the virus propagation. For the cell line may also be genetically engineered to have reduced expression or activity of host factors that can restrict the virus infection, such as interferons, PKR, or OAS. The cell line may also be treated with chemicals or drugs that can modulate the cellular environment or metabolism to favor the virus propagation. For example, the cell line may be treated with cycloheximide, puromycin, or actinomycin D, which can inhibit the host protein synthesis and reduce the antiviral response. Alternatively, the cell line may be treated with sodium butyrate, trichostatin A, or valproic acid, which can induce histone acetylation and increase the viral gene expression.

[0048] The attenuation of the CHIKV can be confirmed by measuring its reduced replication and virulence in vitro and in vivo compared to the wild-type virus. The live attenuated CHIKV can also be tested for its immunogenicity and protective efficacy against chikungunya infection in animal models.

[0049] In addition, certain aspects of the present invention provide a method of vaccinating a patient in need of such treatment, comprising the steps of administering the viral vaccine of the present invention to the patient and allowing the vaccine to produce viral proteins for immune surveillance and / or to stimulate the immune system for antibody production in the patient.

[0050] It is contemplated that the vaccine composition of the invention may be combined with one or more additional components to form a more effective vaccine. Non-limiting examples of additional components include, for example, one or more additional antigens, immunomodulators or adjuvants to stimulate an immune response to the vaccine composition of the present invention and / or the additional component(s). For example, it is contemplated that immunomodulators can be included in the vaccine to augment a cell or a patient's (e.g., an animal's) response. Immunomodulators can be included as purified proteins, nucleic acids encoding immunomodulators, and / or cells that express immunomodulators in the vaccine composition. Adjuvants that are known to those skilled in the art can be used in the administration of the viruses of the invention. Adjuvants that can be used to enhance the immunogenicity of the viruses include, for example, liposomal formulations, synthetic adjuvants, such as (e.g., QS21), muramyl dipeptide, monophosphoryl lipid A, or polyphosphazine. Although these adjuvants are typically used to enhance immune responses to inactivated vaccines, they can also be used with live vaccines. In the case of a virus delivered via a mucosal route (for example, orally) mucosal adjuvants such as the heat-labile toxin of E. coli (LT) or mutant derivations of LT can be used as adjuvants.

[0051] The vaccine composition of the present invention may also be mixed with one or more additional components (e.g., excipients, salts, etc.) that are pharmaceutically acceptable and compatible with at least one active ingredient (e.g., antigen). Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and combinations thereof.

[0052] The vaccine composition of the present invention may be formulated into a neutral or salt form. A pharmaceutically acceptable salt, includes the acid addition salts (formed with the free amino groups of the peptide) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. A salt formed with a free carboxyl group also may be derived from an inorganic base such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and such organic bases as isopropylamine, trimethylamine, 2 ethylamino ethanol, histidine, procaine, and combinations thereof. In addition, if desired, the vaccine composition may comprise minor amounts of one or more auxiliary substances such as for example wetting or emulsifying agents, pH buffering agents, etc. that enhance the effectiveness of the vaccine composition or vaccine.

[0053] The live attenuated CHIKV viruses of the present invention can be administered as primary prophylactic agents in patients at risk of infection, or can be used as secondary agents for treating infected patients. Examples of patients who can be treated using the CHIKV-related vaccines and methods of the invention include (i) patients in areas in which CHIKV is endemic, such as Asia, and Africa, (ii) foreign travelers, or (iii) military personnel,. Moreover, inhabitants of regions where the disease has been observed to be expanding (e.g., Southern Europe), or regions where it may be observed to expand in the future (e.g., regions infested with Aedes aegypti or Aedes albopictus) can be treated according to the invention. The vaccine compositions of the present invention can be administered using methods that are well known in the art, and appropriate amounts of the vaccines administered can readily be determined by those of skill in the art. For example, the viruses of the invention can be formulated as sterile aqueous solutions containing between 102and 107infectious units (e.g., plaque-forming units or tissue culture infectious doses) in a dose volume of 0.1 to 1.0 ml, to be administered by, for example, intramuscular, subcutaneous, or intradermal routes. Further, the vaccine compositions of the present invention can be administered in a single dose or, optionally, administration can involve the use of a priming dose followed by a booster dose that is administered, e.g., 2-6 months later, as determined to be appropriate by those of skill in the art. The manner of administration of an vaccine compositions of the present invention may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. For example, a vaccine may be conventionally administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, intrapericardially, orally, rectally, nasally, or topically. The vaccination schedule and dosages may be varied on a patient-by-patient basis, taking into account, for example, factors such as the weight and age of the patient, the type of disease being treated, the severity of the disease condition, previous or concurrent therapeutic interventions, the manner of administration and the like, which can be readily determined by one of ordinary skill in the art. For instance a suitable dosage range may be, for example, of the order of several hundred micrograms active ingredient per vaccination. In other non-limiting examples, a dose may also comprise from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per vaccination, and any range derivable therein. In nonlimiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above. A suitable regime for initial administration and booster administrations (e.g., inoculations) are also variable, but are typified by an initial administration followed by subsequent inoculation(s) or other administration(s).

[0054] The course of the immunization may be followed by assays for antibodies for the supernatant antigens. The assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescents, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays. Other immune assays can be performed — and assays of protection from challenge with the CHIKV can be performed following immunization.

[0055] Screening methods:

[0056] A further object of the present invention relates to a method for identifying a substance useful for inhibiting the replication capacity of chikungunya virus (CHIKV) in a subject comprising the steps of (a) determining whether a test substance is capable of inhibiting the interaction between nsP3 and FHL1, CD2AP and BINI and (b) selecting the substance that inhibits the interaction. In some embodiments the step (a) consists in generating physical values which illustrate or not the ability of said test substance to inhibit the interaction between nsP3 and FHL1, CD2AP and BINI and comparing said values with standard physical values obtained in the same assay performed in the absence of the said test substance. The “physical values” that are referred to above may be of various kinds depending of the binding assay that is performed, but notably encompass light absorbance values, radioactive signals and intensity value of fluorescence signal. If after the comparison of the physical values with the standard physical values, it is determined that the said test substance inhibits the interaction between nsP3 and FHL1, CD2AP and BINI, then the candidate is positively selected at step (b).

[0057] In some embodiments, the test substances encompass the substances that bind either to nsP3, FHL1, CD2AP or BINI, provided that the binding of the said substances of interest then prevents the interaction between said polypeptides.

[0058] Different assays that are routinely used in the art can be used for determining whether the test substance can inhibit the interaction between nsP3 and FHL1, CD2AP and BINI. For instance, one method that could be used for determining whether the test substance can inhibit the interaction between nsP3 and FHL1, CD2AP and BINI is an immunoprecipitation assay. In this method, a first polypeptide (e.g. nsP3 R5 region) is tagged with a molecule that can be recognized by a specific antibody (e.g. FLAG tag). The second polypeptide (e.g. FHL1, CD2AP and / or BINI) is tagged with a different molecule that can be detected by another means (e.g. GFP tag). The test substance is added to a cell lysate or a recombinant protein mixture containing both tagged polypeptides, and then the antibody against the first tag is used to pull down the first polypeptide and any bound molecules. The precipitated complex is then analyzed by western blotting or fluorescence microscopy to see if the second polypeptide is coprecipitated with the first one. If the test substance inhibits the interaction between the two polypeptides, then the second polypeptide will not be present in the complex. Alternatively, a surface plasmon resonance (SPR) assay could be used to measure the binding affinity and kinetics of the interaction between nsP3 and FHL1, CD2AP and BINI in the presence or absence of the test substance. In this method, one of the polypeptides (e.g. nsP3 R5 region) is immobilized on a sensor chip, while the other polypeptide (e.g. FHL1, CD2AP and / or BINI) is injected as a solution over the sensor chip. The binding of the two polypeptides causes a change in the refractive index at the sensor surface, which is measured by a detector as a function of time. The test substance is added to the solution of the second polypeptide before or after the injection, and the effect of the test substance on the binding curve is monitored. If the test substance inhibits the interaction between the two polypeptides, then the binding curve will show a reduced response or a faster dissociation rate. A third method that could be used for determining whether the test substance can inhibit the interaction between nsP3 and FHL1, CD2AP and BINI is a fluorescence resonance energy transfer (FRET) assay. In this method, the two polypeptides are fused to fluorescent proteins that have overlapping emission and excitation spectra (e.g. CFP and YFP). When the two polypeptides are in close proximity, the energy from the excited donor protein (e.g. CFP) is transferred to the acceptor protein (e.g. YFP), which emits a different wavelength of light. The test substance is added to a cell expressing the fusion proteins or a recombinant protein mixture containing the fusion proteins, and then the fluorescence intensity and ratio of the donor and acceptor proteins are measured by a fluorometer or a microscope. If the test substance inhibits the interaction between the two polypeptides, then the FRET signal will decrease or disappear.

[0059] The test substances that could be used in the screening method of the present invention may include, but are not limited to, small organic molecules, oligonucleotides, peptides, antibodies, or natural products. In some embodiments, the test substances are small organic molecules that have a molecular weight of less than 1000 Da. In some embodiments, the test substances are oligonucleotides, such as antisense, siRNA, miRNA, or aptamers, that can bind to the mRNA or the protein of nsP3, FHL1, CD2AP, or BINI and modulate their expression or function. In some embodiments, the test substances are peptides that mimic or interfere with the binding domains of nsP3, FHL1, CD2AP, or BINI and disrupt their interaction. In some embodiments, the test substances are antibodies that recognize and block the binding sites of nsP3, FHL1, CD2AP, or BINI and prevent their association. In some embodiments, the test substances are natural products, such as plant extracts, fungal metabolites, or marine compounds, that can affect the activity or stability of nsP3, FHL1, CD2AP, or BINI.

[0060] In some embodiments, the screening method of the present invention further comprises the step (c) consisting in determining whether the substance selected at step (b) inhibits the replication of CHIKV in a host cell and a step (d) that consists in positively selecting the test substance capable of inhibiting the replication of said CHIKV in said host cell.

[0061] In some embodiments, the screening method thus comprises the steps consisting of i) infecting said host cell with said CHIKV and ii) culturing said infected cell in presence of the test substance, iii) comparing the replicating capacity of the virus with the replication capacity determined in the absence of the test substance and iv) positively selecting the test substance that provides a decrease in the replication capacity of the virus.

[0062] The expression "inhibiting the replication capacity," as used herein with reference to a viral phenotype, means that the virus grows to a lower titer in the presence of a substance as above described relative to the virus grown in the absence of said substance. In some embodiments, the presence of said substance which will inhibit the ability of an CHIKV to replicate in a host cell by at least about 10%, or by at least about 20%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 60%, or by at least about 70%, or by at least about 80%, or by at least about 90%, or by at least about 100%, or by at least about 200%, or by at least about 300%, or by at least about 400%, or by at least about 500% when compared to said CHIKV grown in the absence of said substance. Said replication capacity may be typically determined by any routine technique well known in the art.

[0063] According to the present invention, any CHIKV strain can be used. Preferably, said CHIKV strain corresponds to a clinical isolate of at least one circulating strain of CHIKV.

[0064] According to the invention, any eukaryotic cell may be used in the screening method of the invention. In some embodiments the cell is a human cell. In some embodiments, the cell is a cell line. Non-limiting examples of cell lines that can be suitable for the invention include but are not limited to BS-C-1, CV-1, Vero, Vero 76, Vero C1008, Vero 76, Cos-1, Cos-7, Huh7, FR11K-4, LLC-MK2 original, LLC-MK2 derivative, MDCK, RD, A549, MRC-5, KB, PER.C6, HEK-293 and CaCo-2 cells. Typically, cells are cultured in a standard commercial culture medium, such as Dulbecco's modified Eagle's medium supplemented with serum (e.g., 10% fetal bovine serum), or in serum free medium, under controlled humidity and C02 concentration suitable for maintaining neutral buffered pH (e.g., at pH between 7.0 and 7.2). Suitable serum free media are described, for example, in U.S. Provisional Application No. 60 / 638,166, filed Dec. 23, 2004, and in U.S. Provisional Application No. 60 / 641,139, filed Jan. 5, 2005, each of which is hereby incorporated by reference in its entirety. Optionally, the medium contains antibiotics to prevent bacterial growth, e.g., penicillin, streptomycin, etc., and / or additional nutrients, such as L-glutamine, sodium pyruvate, nonessential amino acids, additional supplements to promote favorable growth characteristics, e.g., trypsin, (3- mercaptoethanol, and the like. In some embodiments, the infection of the cells with CHIKV is carried out at an m.o.i. (multiplicity of infection) of about 0.0001 to 10, preferably of 0.002 to 0.5. Typically, the MOI is 0.1, 0.01 or 0.001 for Vero cells or 0.1, 0.05, 0.01 or 0.001 for Huh7 cells while preferably an MOI is used of 0.001 for Vero cells or 0.05 for Huh7 cells.

[0065] Typically, the cells can be grown in culture under conditions permissive for replication and assembly of viruses. In some embodiments, cells can be cultured at a temperature below about 37° C, preferably at a temperature equal to, or less than, about 35° C. Typically, the cells are cultured at a temperature between about 32° C. and about 35° C. In some embodiments, the cells are cultured at a temperature between about 32° C. and 34° C, e.g., at about 33° C.

[0066] As described above, the methods of the present invention are particularly useful for screening a plurality of substances that may be used for the treatment or prevention of CHIKV infections. In particular , the selected test substances are particularly suitable for interfering with the virus' ability to replicate in muscle and joint tissues. Thus the selected test substance are thus particularly suitable for lowering the viral load to a level that will induce an immune response while preventing the pathology associated with infection. Therefore, the selected test substances can thus prevent virus replication in muscle and joint tissues and lead to virus attenuation. Thus the test substances can be used in the treatment of CHIKV infection. For example, therapeutic treatments includes the reduction or amelioration of the progression, severity and / or duration of CHIKV infections, or the amelioration of one or more symptoms (specifically, one or more discernible symptoms) of CHIKV infections, resulting from the administration of at least one substance selected by the above mentioned screening method. In some embodiments, the therapeutic treatment includes the amelioration of at least one measurable physical parameter of a CHIKV infection. In some embodiments, the therapeutic treatment includes the inhibition of the progression of an CHIKV infection, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In some embodiments, the therapeutic treatment includes the reduction or stabilization of CHIKV infections.

[0067] In some embodiments, the substances selected by the above mentioned screening method may be used in a prophylactic treatment. In particular, the prophylactic use includes the use in situations in which an outbreak has been detected, to prevent contagion or spread of the infection in places where a lot of people that are at high risk of serious complications live in close contact with each other (e.g. in a hospital ward, daycare center, prison, nursing home, etc). Prophylactic use may also include treating a person who is not ill with the CHIKV or not considered at high risk for complications, in order to reduce the chances of getting infected with the CHIKV and passing it on to a high-risk person in close contact with him (for instance, healthcare workers, nursing home workers, etc).

[0068] Typically, the substances selected by the above mentioned screening method are administered to the subject in an effective amount. As used herein, an "effective amount" refers to an amount sufficient to elicit the desired biological response. In the present invention the desired biological response is to inhibit the replication of CHIKV, to reduce the amount of CHIKV or to reduce or ameliorate the severity, duration, progression, or onset of a CHIKV infection, prevent the advancement of an CHIKV infection, prevent the recurrence, development, onset or progression of a symptom associated with an CHIKV infection, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy used against CHIKV infections. The precise amount of compound administered to a subject will depend on the mode of administration, the type and severity of the infection and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. When coadministered with other anti-viral agents, e.g., when co-administered with an anti-CHIKV medication, an "effective amount" of the second agent will depend on the type of drug used. Suitable dosages are known for approved agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of condition(s) being treated and the amount of a compound described herein being used. In cases where no amount is expressly noted, an effective amount should be assumed. For example, compounds described herein can be administered to a subject in a dosage range from between approximately 0.01 to 100 mg / kg body weight / day for therapeutic or prophylactic treatment.

[0069] In some embodiments the substances selected by the above mentioned screening method are used in combination with an additional suitable therapeutic agent, for example, an antiviral agent or a vaccine. When "combination therapy" is employed, an effective amount can be achieved using a first amount of a substance selected by the above mentioned screening method and a second amount of an additional suitable therapeutic agent (e.g. an antiviral agent). As used herein, the terms "in combination" or "co-administration" can be used interchangeably to refer to the use of more than one therapy (e.g., one or more prophylactic and / or therapeutic agents). The use of the terms does not restrict the order in which therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject. Specific examples that can be coadministered with a substance selected by the above mentioned screening method include nonsteroidal anti-inflammatory drugs (NSAIDS). Examples of Aspirin, Naproxen, Sulindac, Ibuprofen, Indomethacin, Valproic acid, Fenamic acid, Flurbiprofen, Diclofenac, Diflunisal, Salsalate, Choline Magnesium Trisalicylate, Dexibuprofen, Fenoprofen, Detoprofen, Dexketoprofen, Oxaprozin, Loxoprofen, Tolmetin, Etodolac, Ketorolac, Aceclofenac, Nabum etone, Piroxicam, Meloxicam, Tenoxicam, Droxicam, Lornoxicam, Isoxicam, Mefenamic acid, Meclofenamic acid, Flufenamic acid, Tolfenamic acid, Selective COX-2 inhibitors, and Licofelone.

[0070] The substances selected by the above mentioned screening method can be formulated into pharmaceutical compositions that further comprise a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In some embodiments, the present invention relates to a pharmaceutical composition comprising a substance selected by the above mentioned screening method described above, and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. In some embodiments, the present invention is a pharmaceutical composition comprising an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices.

[0071] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0072] FIGURES:

[0073] Figure 1. RMN studies identified the R5 region of CHIKV nsP3 HVD that is involved in the interaction with FHL1. (A) Schematic of full-length CHIKV nsP3 highlighted the R5 region within the HVD domain. (B) Amino acid sequence alignment of the nsP3 region containing the R5 sequence between WT CHIKV strain and viruses (SEQ ID NO:2) either deleted for the R5 sequence (AR5) (SEQ ID NO:3) or carrying the 6 amino acid substitutions (CHIKV-AFHL1) (SEQ ID NO:4) published by Ng et al. (doi.org / l 0.1038 / s4 l 467-023-42330- 2). The domains of interaction with FHL1 LIM domains identified by RMN structural studies are mapped to the R5 sequence. R4 minimal sequence of interaction with FHL1 is underlined. Boxes indicate the domain of interaction with CD2AP and BINI.

[0074] Figure 2. The R5 region of nsP3 HVD is required for interaction with FHL1 and CD2AP, but dispensable for G3BP1 interaction. 293T cells were co-transfected with plasmids encoding HA-tagged FHL1A and FLAG-tagged CHIKV nsP3 WT or CHIKV lacking the amino acid region R5 (AR5) or carrying the corresponding randomized sequence (R5_Scr). Cellular lysates were subject to immunoprecipitation with anti -FLAG beads followed by immunoblot analysis with anti-FLAG, anti-FHLl, anti-HA, anti-G3BPl or anti-CD2AP mAbs. Images from one out of two reproducible experiments are shown.

[0075] Figure 3. The nsP3 R5 region is important for CHIKV infection. BHK21, U2OS and VeroE6 cells were transfected with in iv' / m-tran scribed viral RNA from CHIKV21-WT or CHIKV21-AR5. Supernatants from transfected cells were collected at 48 hours after transfection and viral titers were determined on Vero E6 cells by plaque assays. PFU, plaque forming units. Data shown are mean + / - SD.

[0076] EXAMPLE:

[0077] Methods:

[0078] Co-immunoprecipitation assay. HEK-293T cells were plated in 10 cm dishes (5.106cells / dish). Twenty -four hours later, the cells were transfected with a total of 15 pg of DNA expression plasmids (7.5 pg of each plasmid). Twenty -four hours post-transfection, the cells washed once with PBS and collected with a cell scrapper. After 5 min centrifugation (400 x g for 5 min), cells pellets were lysed for 30 min in cold IP lysis buffer supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail, and then cleared by centrifugation for 15 min at 6,000 x g. Supernatants were incubated overnight at 4°C, with either anti-FLAG magnetic beads. Beads were washed three times with BO 15 buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgC12, 10% Glycerol, 0.5 mM EDTA, 0.05% Triton, 0.1% Tween-20). The retained complexes were eluted twice with either 3xFLAG-peptide (200 pg / ml; SIGMA F4799- 4MG) for 30 min at room temperature. Samples were prepared and subjected to immunoblot. For input, 1% of whole cell lysate were loaded on the gel.

[0079] Genomic viral RNA transfection and kinetic of viral amplification. To assess CHIKV RNA replication within the cells, we transfected cells with capped genomic viral RNA generated from pCHIKV-WT and pCHIKV-AR5. Cells were plated on 48 well plate (3 x 104cells) and transfected with 500 ng of purified RNA using the Lipofectamine MessengerMax reagent according to the manufacturer’s instruction (Thermo Fisher Scientific). At 48 hours after transfection, supernatants were collected and viral particles released from the transfected cells were quantified on Vero E6 cells by plaque assay and expressed as PFU (plaque forming unit) per mL.

[0080] Results:

[0081] Figures 1 to 3 showed that the R5 region of the hypervariable domain of nsP3 is required for the interaction of nsP3 with FHL, BINI and CD2AP, but dispensable for G3BP1 interaction. The results also showed that CHIKV-AR5 exhibited a reduced replication capacity compared to CHIKV-WT in BHK21, U2OS and VeroE6 cells. These results thus suggest that the R5 domain of nsP3 is important for efficient viral replication and host factor recruitment. By mutating or deleting the R5 domain, one could produce attenuated viruses that could be used for vaccinating patients.

[0082] REFERENCES:

[0083] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A live attenuated chikungunya virus (CHIKV) that is engineered to express a mutein of the nonstructural protein 3 (nsP3) that comprises one or more mutations in the R5 domain so that said nsP3 mutein is no capable to bind to FHL1, CD2AP and BINI in a host cell.

2. The live attenuated CHIKV according to claim 1 that is engineered to express a nsP3 mutein that comprises one or more deletion in the R5 domain that said nsP3 mutein is no capable to bind to FHL1, CD2AP and BINI in a host cell.

3. The live attenuated CHIKV according to claim 2 that is engineered to express a nsP3 mutein wherein the R5 domain is deleted4. The live attenuated CHIKV according to claim 3 that is engineered to express a nsP3 that consists of an amino acid sequence having a least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:1 wherein the domain that ranges from the amino acid residue at position 391 to the amino acid residue at position 460 is deleted5. The live attenuated CHIKV according to claim 4 that is engineered to express a nsP3 that consists of the amino acid sequence having as set forth in SEQ ID NO: 1 wherein the domain that ranges from the amino acid residue at position 391 to the amino acid residue at position 460 is deleted.

6. A method of producing a vaccine comprising the steps of introducing the live attenuated CHIKV according to any one of claims 1 to 5 into host cells and allowing the virus to replicate in the host cells to produce a viral vaccine.

7. A vaccine composition comprising the live attenuated CHIKV according to any one of claims 1 to 5.

8. The vaccine composition according to claim 8 that further comprises one or more adjuvants.

9. A method of vaccinating a patient in need of such treatment, comprising the steps of administering the vaccine composition according to claim 1 to the patient and allowingthe vaccine to produce viral proteins for immune surveillance and / or to stimulate the immune system for antibody production in the patient.

10. A method for identifying a substance useful for inhibiting the replication capacity of chikungunya virus (CHIKV) in a subject comprising the steps of (a) determining whether a test substance is capable of inhibiting the interaction between nsP3 and FHL 1 ,CD2AP and BINI and (b) selecting the substance that inhibits the interaction.

11. The method according to claim 10 which further comprises the step (c) consisting in determining whether the substance selected at step (b) inhibits the replication of CHIKV in a host cell and a step (d) that consists in positively selecting the test substance capable of inhibiting the replication of said CHIKV in said host cell.

12. The method according to claim 11 that comprises the steps consisting of i) infecting said host cell with said CHIKV and ii) culturing said infected cell in presence of the test substance, iii) comparing the replicating capacity of the virus with the replication capacity determined in the absence of the test substance and iv) positively selecting the test substance that provides a decrease in the replication capacity of the virus.

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