aqui dejo parte del documento oficial del invento del virus chinjunguya.. usado para generar millones y enfermar alos boricuas y algunos latinoamericanos .. tomen nota y EDUQUENA OTROS. Yucayeque Cerouno Description FIELD OF THE INVENTION [0001] The present invention relates to binding molecules against Chikungunya virus. Particularly, said binding molecules are capable of neutralizing Chikungunya virus infectivity. Said binding molecules can be used with therapeutic, diagnosis or research purposes, among others. The invention also relates to a pharmaceutical composition comprising said binding molecules and more particularly, to the use thereof in the treatment or prevention of Chikungunya fever. BACKGROUND OF THE INVENTION [0002] Chikungunya fever is an emerging, epidemic disease, caused by an arbovirus and transmited by the Aedes mosquitoes, of much significance for WHOs South-East Asia Region. The disease has been reported from countries of South and East Africa, South Asia and South-East Asia. In WHOs South-East Asia Region, outbreaks have been reported from India, Indonesia, Myanmar, Sri Lanka, Thailand and Maldives. Massive outbreaks of Chikungunya fever have occurred in recent years in India and in the island countries of the Indian Ocean. Similarly, Maldives reported outbreaks of Chikungunya fever for the first time in December 2006. Although not a killer disease, high morbidity rates and prolonged polyarthritis leading to considerable disability in a proportion of the affected population can cause substantial socio-economic impact in affected countries (WHO, guidelines for prevention&control of Chikungunya fever, 2009). [0003] The Chikungunya virus (CHIKV) is a member of the genus alphavirus and family Togaviridae (reviewed by Strauss and Strauss, 1994, Microbiol Rev 58, 491-562). The alphaviruses are small enveloped single-stranded positive RNA viruses exhibiting a large cell tropism. The viral surfaces are covered in membrane-anchored spikes composed of triplets of heterodimers of the envelope E1 and E2 glycoproteins. The viral spike proteins facilitate attachment to cell surfaces and viral entry. The E1 envelope glycoprotein is a class II fusion protein that mediates low pH-triggered membrane fusion during virus infection. E2 is a 50 kDa type I transmembrane glycoprotein: the first 260 amino acids constitute the ectodomain, followed by about 100 amino acids that form the stem region, a spanning region of 30 amino acids, and a short cytoplasmic endodomain of 30 amino acids (Plenetv, et al., 2001, Cell 105, 127-136; Mukhopadhyay, et al., 2006, Structure 14, 63-73). pE2 (the 62-kDa precursor to the E3 and E2 proteins) and E1 are assembled as heterodimers in the endoplasmic reticulum (Strauss and Strauss, 1994, Microbiol Rev 58, 491-562). After the cleavage of pE2 in the Golgi apparatus to form E3 and E2, the E1-E2 complexes are transported to the plasma membrane (PM). The interaction of the cytoplasmic E2 endodomain with the preassembled nucleocaspid is one of the initial steps in the process of virus envelopment at the PM. Integrity of virion is maintained by direct interactions between E1 and E2 (Strauss and Strauss, 1994, Microbiol Rev 58, 491-562). During the course of alphavirus life cycle, the E2 glycoprotein is responsible for receptor binding. In general, neutralizing antibodies against alphaviruses recognize epitopes in E2 rather than E1 (Roehrig, J. T. 1986. The use of monoclonal antibodies in studies of the structural proteins of togaviruses and flaviviruses, p.251-278. In S. Schlesinger and M. J. Schlesinger (ed.), The Togaviridae and Flaviviridae. Plenum Publishing Corp., New York). [0004] Biological diagnosis of CHIK virus infection is essentially based on quantitative real-time RT-PCR-based method during the initial viraemic phase (Edwards et al., 2007, J.Clin.Virol. 39, 271-275; Laurent et al., 2007, Clin. Chem. 53, 1408-1414; Parida et al., 2007, J. Clin. Microbiol. 45, 351-357). Serological methods detect anti-CHIK IgM early times after the first clinical manifestations and specific IgG after two weeks (Pialoux et al., 2007, Lancet Infect. Dis. 7, 319-327). However, ELISA and immunodetection assays are poorly specific and sensitive due the cross reactivity of Chikungunya virus with related members of the Semliki Forest (SF) antigenic complex (Greiser-Wilke et al., 1991, J. Clin. Microbiol. 29, 131-137). [0005] More recently, Brehin and colleagues (Brehin et al., 2008, Virology 371, 185-195) have developed some monoclonal antibodies (mAbs) reactive to CHIKV E2 glycoprotein for diagnosis and research purposes, which were also described in WO 2009/031045 . The three anti-CHIKV-E2 mAbs showed cross-reactivity with the Onyong-nyong viral strains Igbo-Ora and ONN-59. CHIKV, Igbo-Ora and ONN-59 are serologically classified in the SF antigenic complex (Strauss and Strauss, 1994, Microbiol Rev 58, 491-562). These monoclonal antibodies are of murine origin, and although they have demonstrated significant reactivity with CHIKV-associates E2 glycoprotein they failed to neutralize CHIKV infection of primate cells in vitro. Besides, a first commercial Chikungunya virus indirect immunofluorescense test (IIFT) is commercialized by Euroimmun AG, Germany for analyzing the CHIKV specific immune response. This test was evaluated by Litzba et al. (Litzba et al., 2008, Journal of virological methods, 149(1), 175-179). [0006] In the recent years, there has been an explosive re-emergence of Chikungunya fever. Thus, although some advances have been undertaken to provide a reliable test allowing the detection and monitoring of CHIKV specific antibodies, there is still a need to develop new anti-CHIKV monoclonal antibodies for diagnosis and research purposes on Chikungunya virus infection. [0007] On the other hand, a specific treatment is not available and there is no approved vaccine for the prevention of Chikungunya fever. Currently, vector control is the only way to prevent and control the outbreaks. Vector control is not an easy task and insecticide spraying is not always effective and desirable (WHO, guidelines for prevention&control of Chikungunya fever, 2009). [0008] Symptomatic treatment is recommended after excluding more serious conditions. Symptomatic or supportive treatment basically comprises rest and use of acetaminophen or paracetamol to relieve fever and ibuprofen, naproxen or other non-steroidal anti-inflammatory agent (NSAID) to relieve the arthritic component. Patients with persistent or chronic phase of arthritis who fail to respond to NSAID may show some response to chloroquine phosphate. The latter may act as a weak broad spectrum antiviral agent apart from being an anti-inflammatory agent. Use of corticosteroids in managing Chikungunya related arthropathy has in general been a contentious issue and has to be the last resort in a clinical decision (WHO, guidelines for prevention&control of Chikungunya fever, 2009). [0009] While there has been extensive work in vaccinology for several other alphaviruses (Rayner et al., 2002, Rev Med Virol 12, 279-296; Nalca et al., 2003, Antiviral Res 60, 153-174); Johnston & Davis, 2004, Arch Virol Suppl 18, 207-220), the history of vaccine development for CHIKV is short and none of these efforts have yet resulted in a licensed vaccine. Recently, a Phase II study was performed with a serially passaged live chikungunya virus (Edelman et al., 2000, Am J Trop Med Hyg. 62(6), 681-5) with good immunogenicity and tolerance results. However, this vaccine seems not to have reached market authorization and further efforts are being made to find a Chikungunya vaccine, see for example the in vivo study on the immunogenicity of consensus-based DNA vaccines against CHIKV performed by Muthumani et al. (Muthumani K. et al., 2008, Vaccine 26(40), 5128-34). [0010] With regards to the immunotherapy strategies against Chikungunya virus infection, the use of a concentrate of human immunoglobulins (IgA, IgM and IgG) has been previously described, as well as F(ab)2 and/or Fab fragments specific to an arbovirus (i.e. Chikungunya virus) for use as a medicament in the treatment of arbovirosis, see WO 2007/118986 . [0011] Furthermore, studies with neutralizing mAbs have been reported for several alphaviruses including Sindbis virus (SIN), Venezuelan equine encephalitis virus (VEE), Ross River virus (RR), Semliki Forest virus (SF), Eastern equine encephalitis virus (EEE) and Western equine encephalitis virus (WEE) (see, e.g. Roehrig, J. T. 1986. The use of monoclonal antibodies in studies of the structural proteins of togaviruses and flaviviruses, p.251-278. In S. Schlesinger and M. J. Schlesinger (ed.), The Togaviridae and Flaviviridae. Plenum Publishing Corp., New York). Recently, human polyvalent immunoglobulins were purified from plasma samples obtained from donors in the convalescent phase of CHIKV infection, and the preventive and curative effects of these immunoglobulins were investigated (Couderc et al., 2009, J Infect Dis. 2009, 200(4), 489-91). However, to our knowledge, at present no neutralizing monoclonal antibodies against Chikungunya virus have been described, let alone fully human neutralizing antibodies. [0012] Since at present there is no vaccine or specific treatment available on the market to combat the Chikungunya fever, several efforts are being undertaken to obtain a safe and active therapy to be administered to patients suffering from Chikungunya fever and also to obtain a protective therapy against the virus infection. Accordingly, there is a need for providing therapies that are useful in the prevention and/or treatment of the Chikungunya fever. SUMMARY OF THE INVENTION [0013] The invention provides new binding molecules against Chikungunya virus (CHIKV). In particular, two monoclonal antibodies specifically binding to an epitope located in an antigenic site of one of the Chikungunya virus envelope proteins are provided. Furthermore, these antibodies are fully human antibodies with CHIKV neutralizing properties which make them particularly useful for the treatment or prevention of Chikungunya fever, thus avoiding all the secondary effects or unwanted reactions associated to murine, chimeric or humanized antibodies in the treatment of human patients. [0014] In accordance with a first aspect of this invention, we provide an isolated binding protein that specifically binds to Chikungunya virus, comprising a heavy chain amino acid sequence comprising at least one of the CDRs selected from the group consisting of: (a) CDRH1s of SEQ ID NOs: 10 or 30, (b) CDRH2s of SEQ ID NOs: 12 or 32, and (c) CDRH3s of SEQ ID NOs: 14 or 34, and/or a light chain amino acid sequence comprising at least one of the CDRs selected from the group consisting of: (d) CDRL1s of SEQ ID NOs: 16 or 36, (e) CDRL2s of SEQ ID NOs: 18 or 38, and (f) CDRL3s of SEQ ID NOs: 20 or 40. [0015] A second aspect of the present invention relates to a functional variant of a binding protein of the invention characterized in that said functional variant binds to Chikungunya virus. [0016] A third aspect of the present invention refers to an immunoconjugate of a binding protein or a functional variant of the invention wherein said binding protein or functional variant is coupled to at least one labeling and/or effector group. [0017] A further aspect of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a binding protein, a functional variant or an immunoconjugate of the invention. [0018] Another aspect of the present invention pertains to a vector comprising a nucleic acid molecule of the invention. A further aspect of the invention relates to a host cell comprising the nucleic acid molecule or the vector of the invention. [0019] In accordance to an additional aspect of this invention, we provide a method for producing a binding protein, a functional variant or an immunoconjugate of the invention, comprising the step of producing said binding protein, functional variant or immunoconjugate in a host cell of the invention and optionally, isolating said binding protein, functional variant or immunoconjugate. [0020] In yet another aspect of the present invention, we provide a pharmaceutical composition comprising as an active agent at least one isolated binding protein, at least one functional variant or at least one immunoconjugate of the invention; and a pharmaceutically acceptable carrier, diluent or adjuvant. [0021] A related aspect of the present invention refers to an isolated binding protein, a functional variant or an immunoconjugate of the invention for use as a medicament. Moreover, the present invention relates in a further aspect to a isolated binding protein, a functional variant, an immunoconjugate and/or the pharmaceutical composition of the invention for use in the prevention or treatment of the infection of an arbovirus from the Togaviridae family, preferably from the genus alphavirus more preferably the Chikungunya virus. [0022] In even another aspect, the present invention relates to the use of a binding protein, a functional variant or an immunoconjugate of the invention for diagnostic or screening purposes. [0023] A further aspect of the present invention relates to a kit comprising at least one isolated binding protein, at least one functional variant, at least one immunoconjugate and/or at least one pharmaceutical composition of the invention. BRIEF DESCRIPTION OF THE FIGURES [0024] Figure 1A shows the nucleotide (SEQ ID N°1) and corresponding amino acid (SEQ ID N° 50) sequences of the heavy chain variable domain (VH) and a partial region of the Constant Heavy 1 (CH1) domain of the 5F10F175E2 antibody and Figure 1B shows the nucleotide (SEQ ID N° 3) and corresponding amino acid (SEQ ID N° 51) sequences of whole light chain of the 5F10F175E2 antibody. Leader sequences are indicated in light grey. Variable domains (VH and VL, respectively) are highlighted in dark grey. The Complementary Determining Regions (CDRs) are also indicated for each of the variable domains. Figure 2A shows the nucleotide (SEQ ID N° 21) and corresponding amino acid (SEQ ID N° 52) sequences of the heavy chain variable domain (VH) and a partial region of the Constant Heavy 1 (CH1) domain of the 8B10F8 antibody and Figure 2B shows the nucleotide (SEQ ID N° 23) and corresponding amino acid (SEQ ID N° 53) sequences of whole light chain of the 8B10F8 antibody. The leader sequence (5 Untranslated region; 5UTR) is indicated in light grey. Variable domains (VH and VL, respectively) are highlighted in dark grey. The Complementary Determining Regions (CDRs) are also indicated for each of the variable domains. Figure 3 shows the results of the indirect immunofluorescense assay performed to assess the infection of HEK 293T cells by Chikungunya virus, confirming the in vitro neutralizing properties of the recombinant antibodies (rec8B10F8 and rec5F10F175E2) and of the isolated antibodies (8B10F8 and 5F10F175E2) against the Chikungunya virus A226 and A226V strains. Images were obtained using a fluorescent microscope (x10 magnificence, NIKON ECLIPSE TS 100). Figure 4 shows the dose-response neutralization curves of the recombinant mAbs (rec8B10F8 and rec5F10F175E2) against Chikungunya virus A226 and A226V strains, as neutralization percentage (%) relative to an irrelevant IgG1. rec5F10F175E2 antibody is represented as a square (-■-),rec8B10F8 antibody is represented as a triangle (-▲-) and both antibodies used in combination as a dot (-•-). Figure 5 shows the results obtained by a western blot immunoassay on the recombinant antibodies (rec8B10F8 and rec5F10F175E2) against infected cell lysates and purified viral particles of both A226 and A226V Chikungunya virus strains. (E1/E2) represents the envelope glycoproteins E1 and E2 and (C) represents the viral capsid protein. Figure 6 shows the mouse signal peptide of Ig kappa chain (SEQ ID N° 48), followed by the first nucleotides/amino acids of the human IgG1 constant region sequence (SEQ ID N° 49). The Sal I and Nhe I sites are shown in bold case. DETAILED DESCRIPTION Binding proteins [0025] In a first aspect, the present invention encompasses binding proteins capable of specifically binding to Chikungunya virus (CHIKV). [0026] The term specifically binding, as used herein, in reference to the interaction of a binding protein, e. g. an antibody, and its binding partner, e. g. an antigen, means that the interaction is dependent upon the presence of a particular structure, e. g. an antigenic determinant or epitope, on the binding partner. In other words, the antibody preferentially binds or recognizes the binding partner even when the binding partner is present in a mixture of other molecules or organisms. The binding may be mediated by covalent or noncovalent interactions or a combination of both. In yet other words, the term specifically binding means immunospecifically binding to an antigen or a fragment thereof and not immunospecifically binding to other antigens. A binding protein that immunospecifically binds to an antigen may bind to other peptides or polypeptides with lower affinity as determined by, e. g., radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), BIACORE, or other assays known in the art. Binding proteins or fragments thereof that immunospecifically bind to an antigen may be cross-reactive with related antigens. Preferably, binding proteins or fragments thereof that immunospecifically bind to an antigen do not cross-react with other antigens. [0027] Chikungunya virus is part of the genus alphavirus. According to Strauss and Strauss (Strauss and Strauss, 1994, Microbiol Rev 58, 491-562), in total, the genus alphavirus have 26 recognized members (i.e. genotypes). Alphaviruses have been classified in 4 main groups according to its serological cross-reaction, i.e., the Venezuelan equine encephalitis (VEE/EEE) group, the Semliki Forest (SF) group, the Sindbis virus (SIN) group and the Western Equine Encephalitis (WEE) group. Table I below lists some of the currently recognized alphaviruses, together with their geographical distribution and their serological group. Besides binding to Chikungunya virus, the binding proteins of the invention may also be capable of binding to other genotypes of the genus alphavirus, including but not limited to those listed in Table I. Table I Group Virus Geographic distribution VEE/EEE Eastern equine encephalitis (EEE) America Venezuelan equine encephalitis (VEE) America Everglades America Mucambo America Pixuna America SF Semliki Forest (SF) Africa, Eurasia Middelburg Africa Chikungunya Africa, Asia ONyong Nyong Africa Ross River Australia Barma Forest Australia Getah Australia, Asia Sagiyama Japan Bebaru Malaysia Mayaro South America Una South America Ndumu Africa SIN Sindbis (SIN) Africa, Asia, Europe, Australia, Scandinavia Aura America Whataroa New Zeeland Babanki Africa Kyzylagash Russia WEE Western equine encephalitis America [0028] Furthermore, the binding proteins of the invention may even be capable of binding to viruses other than alphaviruses of the Togaviridae family, such as those belonging to the genus rubivirus or others. Further information on the Togaviridae family and its taxonomic structure and members can be found on the Index of Viruses - Togaviridae (2006). In: ICTVdB - The Universal Virus Database, version 4. Büchen-Osmond, C (Ed), Columbia University, New York, USA. [0029] The binding proteins of the invention may be capable of specifically binding to Chikungunya virus in its natural form or in its inactivated/attenuated form. General viral inactivation methods well known to the skilled artisan such as inter alia pasteurization (wet heat), dry heat treatment, vapor heat treatment, treatment with low pH, treatment with organic solvent/detergent, nanofiltration and/or UV light irradiation may be used. Preferably, the inactivation is performed by heat-treatment for 1 hour at 56 °C. [0030] The binding proteins of the invention may also be capable of specifically binding to one or more fragments of the Chikungunya virus such as inter alia a preparation of one or more proteins and/or (poly) peptides derived from Chikungunya virus or a cell transfected with a Chikungunya virus protein and/or (poly) peptide. For methods of treatment and/or prevention of Chikungunya virus the binding proteins of the invention are preferably capable of specifically binding to surface accessible proteins of Chikungunya virus such as the E1 or E2 glycoproteins (Strauss and Strauss, 1994, Microbiol Rev 58, 491-562). For diagnostic purposes the binding proteins of the invention may also be capable of specifically binding to proteins not present on the surface of Chikungunya virus. The amino acid sequence of surface accessible and internal proteins of various known strains of Chikungunya virus can be found in the EMBL-database and/or other databases. [0031] Preferably, the fragment at least comprises an antigenic determinant recognized by the binding proteins of the invention. An antigenic determinant as used herein is a moiety, such as a Chikungunya virus (poly) peptide, (glyco)protein, or analog or fragment thereof, that is capable of binding to a binding proteins of the invention with sufficiently high affinity to form a detectable antigen-binding protein complex. [0032] Typically, binding proteins according to the invention can bind to their binding partners, i. e. Chikungunya virus or fragments thereof such as Chikungunya virus proteins, with an affinity constant (Kd-value) that is lower than 0.2x10-4 M, 1.0x10-5 M, 1.0x10-6 M, 1.0x10-7 M, preferably lower than 1.0x10-8 M, more preferably lower than 1.0x10-9 M, more preferably lower than 1.0x10-10 M, even more preferably lower than 1.0x10-11 M, and in particular lower than 1.0x10-12 M. The affinity constants can vary for different antibody isotypes. Affinity constants can for instance be measured using surface plasmon resonance, i.e. an optical phenomenon that allows for the analysis of real- time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, in particular using the BIACORE system (GE Healthcare). [0033] The binding proteins according to the invention may bind to Chikungunya virus in purified/isolated or non-purified/non-isolated form. The binding proteins may bind to Chikungunya virus in soluble form such as for instance in a sample or may bind to Chikungunya virus bound or attached to a carrier or substrate, e. g., microtiter plates, membranes and beads, etc. Carriers or substrates may be made of glass, plastic (e. g., polystyrene), polysaccharides, nylon, nitrocellulose, or teflon, etc. The surface of such supports may be solid or porous and of any convenient shape. Alternatively, the binding proteins may also bind to fragments of Chikungunya virus such as proteins or (poly) peptides of the Chikungunya virus. In an embodiment the binding proteins are capable of specifically binding to the Chikungunya virus E2 or E1 protein or to a fragment thereof. The Chikungunya virus proteins or (poly) peptides may either be in soluble form or may bind to Chikungunya virus bound or attached to a carrier or substrate as described above. In another embodiment cells tranfected with Chikungunya virus proteins or (poly) peptides may be used as binding partner for the binding proteins. [0034] In one embodiment of the present invention, the isolated binding protein of the invention comprises a heavy chain amino acid sequence comprising at least one of the CDRs selected from the group consisting of: (a) CDRH1s of SEQ ID NOs: 10 or 30, (b) CDRH2s of SEQ ID NOs: 12 or 32, and (c) CDRH3s of SEQ ID NOs: 14 or 34, and/or a light chain amino acid sequence comprising at least one of the CDRs selected from the group consisting of: (d) CDRL1s of SEQ ID NOs: 16 or 36, (e) CDRL2s of SEQ ID NOs: 18 or 38, and (f) CDRL3s of SEQ ID NOs: 20 or 40. Preferably, the isolated binding protein comprises both the heavy chain amino acid sequence and the light chain amino acid sequence. [0035] The term complementarity determining regions (CDR) as used herein means sequences within the variable regions of binding proteins, such as immunoglobulins, that usually contribute to a large extent to the antigen binding site which is complementary in shape and charge distribution to the epitope recognized on the antigen. The CDR regions can be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of proteins or protein fragments, either as present on the protein in its native conformation or, in some cases, as present on the proteins as denatured, e. g., by solubilization in SDS. Epitopes may also consist of posttranslational modifications of proteins. [0036] The CDR3 region of the variable domain of the heavy chain (CDRH3) provides typically with the greatest source of molecular diversity within the antibody binding-site (Xu et al., 2000, Immunity, 13(1):37-45). Accordingly, preferably, the isolated binding protein of the invention comprises a heavy chain amino acid sequence comprising at least one of the CDRs selected from SEQ ID NO: 14 and SEQ ID NO: 34. [0037] In another embodiment of the present invention, the isolated binding protein of the invention comprises a heavy chain amino acid sequence that comprises a CDRH1 selected from SEQ ID NOs: 10 or 30, a CDRH2 selected from SEQ ID NOs: 12 or 32, and a CDRH3 selected from SEQ ID NOs: 14 or 34, and/or a light chain amino acid sequence that comprises a CDRL1 selected from SEQ ID NOs: 16 or 36, a CDRL2 selected from SEQ ID NOs: 18 or 38, and a CDRL3 selected from SEQ ID NOs: 20 or 40. Preferably, said isolated binding protein comprises both the heavy chain amino acid sequence and the light chain amino acid sequence. [0038] In a further embodiment, the isolated binding protein of the invention comprises a heavy chain amino acid sequence that comprises the CDRH1 of SEQ ID NO: 10, the CDRH2 of SEQ ID NO: 12, and the CDRH3 of SEQ ID NO: 14, and/or a light chain amino acid sequence that comprises the CDRL1 of SEQ ID NO: 16, the CDRL2 of SEQ ID NO: 18, and the CDRL3 of SEQ ID NO: 20. [0039] In yet another embodiment, the isolated binding protein of the invention comprises a heavy chain amino acid sequence that comprises the CDRH1 of SEQ ID NO:30, the CDRH2 of SEQ ID NO:32, and the CDRH3 of SEQ ID NO:34, and/or a light chain amino acid sequence that comprises the CDRL1 of SEQ ID NO:36, the CDRL2 of SEQ ID NO:38, and the CDRL3 of SEQ ID NO:40. [0040] In another embodiment, the isolated binding protein of the invention comprises a heavy chain amino acid sequence which comprises a VH domain amino acid sequence selected from the group consisting of SEQ ID Nos: 6 or 26, and/or a light chain amino acid sequence that comprises a VL domain amino acid sequence selected from the group consisting of SEQ ID NOs: 8 or 28. In a preferred embodiment, the isolated binding protein of the invention comprise the VH domain amino acid sequence of SEQ ID NO: 6 and the VL domain amino acid sequence of SEQ ID NO: 8. Alternatively, the isolated binding protein of the invention comprises the VH domain amino acid sequence of SEQ ID NO: 26 and the VL domain amino acid sequence of SEQ ID NO: 28. [0041] In further embodiment, the isolated binding protein of the invention comprises a heavy chain amino acid sequence which comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 22 and/or a light chain amino acid sequence that comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 24. In a preferred embodiment, the isolated binding protein of the invention comprises a heavy chain amino acid sequence which comprises the amino acid sequence of SEQ ID NO: 2 and a light chain amino acid sequence which comprises the amino acid sequence of SEQ ID NO:4. Alternatively, the isolated binding protein of the invention comprises a heavy chain amino acid sequence which comprises the amino acid sequence of SEQ ID NO: 22 and a light chain amino acid sequence which comprises the amino acid sequence of SEQ ID NO: 24. [0042] In a particular embodiment, the binding protein of the invention is a scaffold protein having an antibody like binding activity or an antibody, i.e. an anti-CHIKV antibody. [0043] Within the context of the present invention, the term scaffold protein, as used herein, means a polypeptide or protein with exposed surface areas in which amino acid insertions, substitutions or deletions are highly tolerable. Examples of scaffold proteins that can be used in accordance with the present invention are protein A from Staphylococcus aureus, the bilin binding protein from Pieris brassicae or other lipocalins, ankyrin repeat proteins, and human fibronectin (reviewed in Binz and Plückthun, 2005, Curr Opin Biotechnol 16, 459-69 and in Plückthun, A., 2009, Recombinant Antibodies for Immunotherapy: Alternative Scaffolds: Expanding the Options of Antibodies (Little, M., ed), pp. 243-271, Cambridge University Press, New York). Engineering of a scaffold protein can be regarded as grafting or integrating an affinity function onto or into the structural framework of a stably folded protein. Affinity function means a protein binding affinity according to the present invention. A scaffold can be structurally separable from the amino acid sequences conferring binding specificity. A scaffold protein having an antibody like binding activity can for instance be derived from an acceptor polypeptide containing the scaffold domain, which can be grafted with binding domains of a donor polypeptide to confer the binding specificity of the donor polypeptide onto the scaffold domain containing the acceptor polypeptide. Said inserted binding domains may be, for example, the complementarity determining region (CDR) of an antibody, in particular an anti-CHIKV antibody. In one embodiment of the present invention, at least one of said inserted binding domains is one of the CDRs of the antibodies identified in the Examples (i.e. 8B10F8, 5F10F175E2, rec8B10F8 and rec5F10F175E2 antibodies), as above described. Insertion can be accomplished by various methods known to those skilled in the art including, for example, polypeptide synthesis, nucleic acid synthesis of an encoding amino acid as well by various forms of recombinant methods well known to those skilled in the art. [0044] In a preferred embodiment, the binding protein of the invention is an antibody. The term antibody or anti-CHIKV antibody, as used herein, means a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody (Jones et al., 1986, Nature 321, 522-525; Riechmann et al., 1988, Nature 332, 323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2, 593-596), a chimeric antibody (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81, 6851-6855), a multispecific antibody (e.g. a bispecific antibody) formed from at least two antibodies, or an antibody fragment thereof. The term antibody fragment comprises any portion of the afore-mentioned antibodies, preferably their antigen binding or variable regions. Examples of antibody fragments include Fab fragments, Fab fragments, F(ab)2 fragments, Fv fragments, diabodies (Hollinger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90, 6444-6448), single chain antibody molecules (Plückthun, A., 1994, in The pharmacology of monoclonal antibodies: Antibodies from Escherichia coli (Rosenberg, M., and Moore, G. P., eds), Vol. 113, pp. 269-315, Springer Verlag, Berlin) and other fragments as long as they exhibit the desired capability of binding to Chikungunya virus. [0045] In addition, the term antibody or anti-CHIKV antibody, as used herein, may include antibody-like molecules that contain engineered sub-domains of antibodies or naturally occurring antibody variants. These antibody-like molecules may be single-domain antibodies such as VH-only or VL-only domains derived either from natural sources such as camelids (Muyldermans et al., 2001, J Biotechnol. 74(4), 277-302) or through in vitro display of libraries from humans, camelids or other species (Holt et al., 2003, Trends Biotechnol. 21, 484-90). [0046] In another preferred embodiment the binding protein of the invention is an antibody fragment. [0047] In accordance with the present invention, the Fv fragment is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen- binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind the antigen, although usually at a lower affinity than the entire binding site. Derivatives of Fv fragments, such as scFv (single-chain Fv) and dsFv (disulfide-stabilized Fv), which have been modified to increase stability of the recombinant Fv fragment are also included. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab fragment by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. The F(ab)2 fragment originally is produced as a pair of Fab fragments which have hinge cysteines between them. Methods of preparing such antibody fragments, such as papain or pepsin digestion, are known to those skilled in the art. [0048] In a further preferred embodiment of the present invention, the anti-CHIKV antibody of the invention is of the IgA-, IgD-, IgE-, IgG- or IgM-type, preferably of the IgG- or IgM-type including, but not limited to, the IgG1-, IgG2-, IgG3-, IgG4-, IgM1-and IgM2-type. In most preferred embodiments, the antibody is of the IgG1-, IgG2- or IgG4- type. More preferably, of the IgG1- type. [0049] In certain respects, e.g. in connection with the generation of antibodies as therapeutic candidates against Chikungunya virus, it may be desirable that the anti-CHIKV antibody of the invention is capable of fixing complement and participating in complement-dependent cytotoxicity (CDC). There are a number of isotypes of antibodies that are capable of the same including without limitations the following: murine IgM, murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgG1, human IgG3, and human IgA. It will be appreciated that antibodies that are generated need not initially possess such an isotype but, rather the antibody as generated can possess any isotype and the antibody can be isotype switched by appending the molecularly cloned V region genes or cDNA to molecularly cloned constant region genes or cDNAs in appropriate expression vectors using conventional molecular biology techniques that are well known in the art and then expressing the antibodies in host cells using techniques known in the art. The isotype-switched antibody may also possess an Fc region that has been molecularly engineered to possess superior CDC over naturally occurring variants (Idusogie et al., 2001, J Immunol. 166, 2571-2575) and expressed recombinantly in host cells using techniques known in the art. Such techniques include the use of direct recombinant techniques (see e.g. US 4,816,397 ), cell-cell fusion techniques (see e.g. US 5,916,771 and US 6,207,418 ), among others. In the cell-cell fusion technique, a myeloma or other cell line such as CHO is prepared that possesses a heavy chain with any desired isotype and another myeloma or other cell line such as CHO is prepared that possesses the light chain. Such cells can, thereafter, be fused and a cell line expressing an intact antibody can be isolated. By way of example, a human anti-CHIKV IgG4 antibody, that possesses the desired binding to the Chikungunya virus antigen, could be readily isotype switched to generate a human IgM, human IgG1 or human IgG3 isotype, while still possessing the same variable region. Such molecule might then be capable of fixing complement and participating in CDC. [0050] Moreover, it may also be desirable for the anti-CHIKV antibody of the invention to be capable of binding to Fc receptors on effector cells, such as monocytes and natural killer (NK) cells, and participate in antibody- dependent cellular cytotoxicity (ADCC). There are a number of isotypes of antibodies that are capable of the same, including without limitations the following: murine IgG2a, murine IgG2b, murine IgG3, human IgG1 and human IgG3. It will be appreciated that antibodies that are generated need not initially possess such an isotype but, rather the antibody as generated can possess any isotype and the antibody can be isotype switched by appending the molecularly cloned V region genes or cDNA to molecularly cloned constant region genes or cDNAs in appropriate expression vectors using conventional molecular biological techniques that are well known in the art and then expressing the antibodies in host cells using techniques known in the art. The isotype-switched antibody may also comprise an Fc region that has been molecularly engineered to possess superior ADCC over naturally occurring variants (Shields et al., 2001, J Biol Chem. 276, 6591- 6604) and expressed recombinantly in host cells using techniques known in the art. Such techniques include the use of direct recombinant techniques (see e.g. US 4,816,397 ), cell-cell fusion techniques (see e.g. US 5,916,771 and US 6,207,418 ), among others. Such molecule might then be capable of binding to FcyR on effectors cells and participating in ADCC. [0051] Antibodies with superior ADCC activity may be obtained by modifying the oligosaccharides profile of IgGs, by using different strategies such as for example, glycosylation inhibition, genetic modifications, amino acid changes in FcR, transgenesis or production in cell lines producing naturally unfucosilated antibodies. Examples of particular genetic engineering strategies are those used by companies such as GLYCART, KYOWA or LFB. GLYCART BIOTECHNOLOGY AG (Zurich, CH) has expressed N-acetyl-glucosaminyltransferase III (GnTIII) which catalyzes the addition of the bisecting GlcNac residue to the N-linked oligosaccharide, in a CHO cell line, and showed a greater ADCC of the IgG1 antibody produced ( WO 99/54342 ; WO 03/011878 ; WO 2005/044859 ). On the other hand, by removing or supplanting fucose from the Fc portion of the antibody, KYOWA HAKKO KOGYO (Tokyo, Japan) has enhanced Fc binding and improved ADCC, and thus the efficacy of the Mab ( US 6,946,292 ). More recently, Laboratoire Francais du Fractionnement et des Biotechnologies (LFB) (France) showed that the ratio Fuc/Gal in Mab oligosaccharide should be equal or lower than 0.6 to get antibodies with a high ADCC ( FR 2 861 080 ). Preferably, an antibody with a glycosylation profile providing enhanced cell-mediated effector functions is obtained by expressing said antibody in a cell line producing naturally unfucosilated antibodies, such as for example, an avian cell, preferably a duck cell. Particularly interesting is the production of an antibody of the invention in an avian embryonic derived stem cell line EBx® marketed by Vivalis (Nantes, France) as described in WO 2008/142124 , which will provide the antibody with an optimized ADCC activity. Furthermore, an antibody produced in the avian embryonic derived stem cell line EBx® will display a human-like glycosylation pattern. [0052] Thus, according to a preferred embodiment, an antibody of the invention has an optimized ADCC activity. Preferably, said antibody has been produced in an avian embryonic derived stem cell line EBx® and thus, has a glycosylation profile providing it with enhanced cell-mediated effector functions. More preferably, the avian embryonic derived stem cell line EBx® is from chicken or duck, and more preferably are chicken EB 14 cells, duck EB 66 or duck EB 24 cells, which may be genetically engineered to express recombinant proteins. [0053] The binding proteins of the invention can be obtained or derived by a variety of ways, specifically by using well known methods for obtaining antibodies or antibody fragments, such as generation of antibodies by the use of mouse hybridomas (see, for example, Köhler et al., 1975, Nature 256, 495-97), production of chimaeric (Hardman et al., 1989, Int J Cancer., 44(3), 424-33) or humanized (Winter and Harris, 1993, Immunology Today 14 (6), 243-246) antibodies or antibody fragments by using recombinant DNA techniques. Preferably, fully human antibodies are obtained using technologies for the production of mAbs derived from human immunoglobulin gene sequences, such as, genetically engineered animals (e.g. Xenomouse® strains (Abgenix, Inc., Fremont, Canada), by using recombinant library methods, such as phage display, yeast display, ribosome display, E.coli display, etc. (See, e.g., Clackson et al., 1991, Nature 352, 624-628 ; Marks et al., 1991, J. Mol. Biol. 222, 581-597; Feldhaus and Siegel, 2004, J Immunol Methods. 290, 69-80; Groves and Osbourn, 2005, Expert Opin Biol Ther. 5(1), 125-135; and Jostock and Dubel, 2005, Comb. Chem. High Throughput Screen. 8, 127-133). Preferably, native human antibodies are obtained from recently developed technologies, including the use of human B cells directly by Human-Human hybridoma (Karpas et al., 2001, Proc Natl Acad Sci U S A. 98(4), 1799-804), Hybrid hybridoma (Schmidt E, 2001, J Immunol Methods. 255(1-2), 93-102), B cell immortalization and cloning (Lanzavecchia et al., Curr Opin Biotech, 2007, 18, 523-8), genetic programming of immortalized B cells (Kwakkenbos et al., 2009, Nature) or Single-cell RT-PCR (Tiller et al., 2008, J Immunol Methods 329(1-2), 112-124; Wrammert et al., 2008, Nature 453(7195):667-71). [0054] Particularly preferred is the generation of native human antibodies from human B cells, such as described in the Examples of the invention [0055] Accordingly, the antibody of the invention could be an antibody of animal origin (e.g. murine), a chimaeric, humanized or fully human antibody. [0056] A problem with murine antibodies derives from the fact that there are many sequence differences between rodent immunoglobulins and human immunoglobulins (Kabat EA, Wu TT, Perry HM, et al. Sequences of proteins of immunological interest. US Department of Health and Human Services, U.S. Government Printing Office, 1991.). Consequently, use of rodent monoclonal antibodies into a human recipient usually results in an antiglobulin response, detectable at about 8-12 days with a peak at about 20-30 days (Isaacs JD. The antiglobulin response to therapeutic antibodies. Seminars in Immunology 1990; 2: 449-456). The presence of this immunological response will usually render the treatment inoperative after 10 days. Furthermore, later retreatment is not possible, due to the rapid onset of a secondary response. [0057] In order to avoid immune response from the patient, is thus important to use monoclonal antibodies that are as near as possible to human antibodies. Thus, by re-engineering and de-immunization techniques chimaeric antibodies and humanized antibodies may be obtained based on murine antibodies. In a chimaeric antibody, the whole of the variable regions of a mouse or rat (or of a non-human) antibody are expressed along with human constant regions. This will lead to an antibody with proper human effector functions, while decreasing the immunogenicity caused by the xenogeneic Fc region. In a humanized antibody, only the CDRs from the rodent antibody V-regions are combined with framework regions from human V-regions. It is expected that these antibodies should thus be less immunogenic than chimaeric antibodies. [0058] In a preferred embodiment, the antibody of the invention is fully human. Preferably, the antibody of the invention is a native human antibody or antibody fragment. [0059] A fully human antibody is an antibody containing exclusively human sequences. Thus, a fully human antibody shall not induce an immune response when administered to a human recipient. Preferably, a human antibody of the invention is a native human antibody, in which the antibody is naturally occurring in a human, as opposed to a human antibody in which the individual heavy and light chains are isolated from humans but are assembled randomly (i.e. by using library methods such as phage display) creating all forms of natural and unnatural antibodies. [0060] Specifically, native human antibodies are those that arise naturally as the result of the functioning of an intact human immune system. The utility of native antibodies for the treatment of human viral diseases has been established through experience with hyperimmune human globulins. Native antibodies, as a class, differ in some respects from those obtained by library methods (phage or transgenic mouse) and possess distinct properties that may make them ideal therapeutics for human diseases. (See Dessain et ah, Exploring the Native Human Antibody Repertoire to Create Antiviral Therapeutics in Current Topics in Microbiology and Immunology 317: 155-183 (2008), (c) Springer-Verlag New York). Specifically, there is a specific advantage of native antibodies expressed from human B cells over phage-derived antibodies, due to the limitations in a phage approach to recreate all of the original or native heavy chain: light chain pairings, thus preventing important antibody structures from being incorporated into a phage-generated library. The term native human antibodies includes native human antibody fragments as described herein and in particular, Fv fragments (including derivatives thereof such as scFv and dsFv), Fab fragments, Fab fragments or F(ab)2 fragments. Specifically, the binding site of these native human antibody fragments will correspond to that of a native human antibody (i.e., particular combination of heavy and light chain sequences naturally occurring in a human). [0061] Human antibodies of the invention, however, may contain residues or modifications (such as post-translational modifications) not found in a naturally occurring human antibody, including those modifications and variant sequences described herein. These modifications are typically made to further refine or enhance antibody desired properties, such as those providing a better performance, increased antibody life-time, increased estability, increased ADCC activity, etc. [0062] In addition, the binding proteins of the invention preferably neutralize Chikungunya virus infectivity. Accordingly, in a further preferred embodiment, the invention relates to a binding protein of the invention having Chikungunya virus neutralizing activity. This may be achieved by preventing the attachment of Chikungunya virus to its receptors on host cells or inhibition of the release of RNA into the cytoplasm of the cell or prevention of RNA transcription or translation. [0063] Preferably, the binding proteins of the invention may also be capable of neutralizing other genotypes of the genus alphavirus. Furthermore, the binding proteins of the invention may even be capable of neutralizing infectivity of viruses other than alphaviruses of the Togaviridae family, such as those belonging to the genus rubivirus or others. Further information on the Togaviridae family and its taxonomic structure and members can be found on the Index of Viruses - Togaviridae (2006). In: ICTVdB - The Universal Virus Database, version 4. Büchen-Osmond, C (Ed), Columbia University, New York, USA. [0064] The binding proteins of the invention may prevent Chikungunya virus from infecting host cells by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to infection of host cells by Chikungunya virus in the absence of said binding proteins. The neutralizing activity of the binding protein may be measured, for instance, by a standard plaque reduction neutralization test (PRNT) as shown in Example 4 and Figure 3 or by other in vitro neutralization assays, such as rapid fluorescent focus inhibition test (RFFIT), (Vene et al., 1998, Journal of Virological Methods 73(1), Pages 71-75). The neutralizing activity could also be investigated by analyzing the cell supernatant for the presence of Chikungunya viral genome by quantitative real time PCR (qRT-PCR). [0065] Alternatively, the binding protein may be characterized in that it has a Chikungunya virus neutralizing activity of at least 2500 IU/mg protein. More preferably, said binding protein has a Chikungunya virus neutralizing activity of at least 2800 IU/mg protein, at least 3000 IU/mg protein, at least 3200 IU/mg protein, at least 3400 IU/mg protein, at least 3600 IU/mg protein, at least 3800 IU/mg protein, at least 4000 IU/mg protein, at least 4200 IU/mg protein, at least 4400 IU/mg protein, at least 4600 IU/mg protein, at least 4800 IU/mg protein, at least 5000 IU/mg protein, at least 5200 IU/mg protein, at least 5400 IU/mg protein. The neutralizing activity of the binding protein may be measured, for instance, by as standard plaque reduction neutralization test (PRNT) as shown in Example 4 and Figure 3 or by other in vitro neutralization assays, such as rapid fluorescent focus inhibition test (RFFIT), (Vene et al., 1998, Journal of Virological Methods 73(1), Pages 71-75). The neutralizing activity could also be investigated by analyzing the cell supernatant for the presence of Chikungunya viral genome by quantitative real time PCR (qRT-PCR). [0066] In a preferred embodiment, the neutralizing protein of the invention is selected from the anti-CHIKV neutralizing fully human antibodies shown in the Examples (8B10F8, 5F10F175E2, rec8B10F8 and rec5F10F175E2). More preferably, from the IgG1 type recombinant antibodies rec8B10F8 and rec5F10F175E2 which are characterized in Example 4. Functional variants [0067] A second aspect of the invention includes functional variants of binding proteins as defined herein. [0068] The term functional variant, as used herein, refers to a binding molecule that comprises a nucleotide and/or amino acid sequence that is altered by one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acid sequences of the parent binding protein and that is still capable of competing for binding to the binding partner, e.g. Chikungunya virus or a fragment thereof, with the parent binding molecule. In other words, the modifications in the amino acid and/or nucleotide sequence of the parent binding molecule do not significantly affect or alter the binding characteristics of the binding molecule encoded by the nucleotide sequence or containing the amino acid sequence, i. e. the binding molecule is still able to recognize and bind its target. Whether a modification in the amino acid sequence results in a functional binding protein (i.e. in a binding protein that binds to Chikungunya virus or a fragment thereof), can readily be determined by assaying the specific activity of the resulting binding protein in ELISA or FACS for binding to Chikungunya virus or a fragment thereof or other in vitro or in vivo functional assay. Preferably, the functional variants should also have Chikungunya virus neutralizing activity. [0069] The functional variant may preferably have conservative sequence modifications including nucleotide and amino acid substitutions, additions and deletions. Furthermore, functional variants can comprise truncations of the amino acid sequence at either or both the amino or carboxy termini. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and may comprise natural as well as non-natural nucleotides and amino acids. [0070] Non-natural amino acids may include, for example, stereoisomers (e.g. D-amino acids) of the 20 conventional amino acids, unnatural amino acids such as a -, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids. Examples of unconventional amino acids, which may also be suitable components for the binding protein of the invention, include: 4- hydroxyproline, [gamma]-carboxyglutamate, [epsilon]-N,N,N-trimethyllysine, [epsilon]-N- acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, [sigma]-N-methylarginine, and other similar amino acids and imino acids, e.g. 4-hydroxyproline. [0071] Especially preferred variations in the nucleotide or amino acid sequences shown in SEQ ID NOs: 1-46 are those that lead to a reduced susceptibility to proteolysis or oxidation, alter glycosylation patterns or alter binding affinities or confer or modify other physicochemical or functional properties of the binding protein. In particular, conservative amino acid replacements are contemplated. Conservative amino acid substitutions include the ones in which the amino acid residue is replaced with an amino acid residue having similar structural or chemical properties. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e. g., lysine, arginine, histidine), acidic side chains (e. g., aspartic acid, glutamic acid), uncharged polar side chains (e. g., asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e. g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e. g., threonine, valine, isoleucine) and aromatic side chains (e. g., tyrosine, phenylalanine, tryptophan). It will be clear to the skilled artisan that other classification of amino acid residue families than the one used above can also be employed. Furthermore, a variant may have non-conservative amino acid substitutions, e.g., replacement of an amino acid with an amino acid residue having different structural or chemical properties. Similar minor variations, may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing immunological activity may be found using computer programs well known in the art. SEQ ID NO: 1, 21, 41 or 43. EL DOCUMENTO ES MAS LARgo, PERO AQUI ESTA LA PAGINA google/patents/EP2374816A1?cl=en
Posted on: Wed, 01 Oct 2014 03:23:18 +0000
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