Novel diagnostic and therapeutic approaches for oropouche virus infection

Abstract

The present invention reveals MESD as a critical host dependency factor for Oropouche virus (OROV) infection. Initial identification was achieved through a CRISPR-Cas9 loss-of-function screen, which highlighted MESD amongst other potential factors. The ensuing research demonstrated that MESD knockout in HEK293T cells resulted in a significant reduction in OROV infection rates. Complementing MESDKO cells with FLAG-MESD re-established susceptibility, thus confirming MESD's essential role in the infection process. Interestingly, MESD specifically facilitates OROV infection without enhancing the infectivity of other viruses such as LACV, TOSCV, and WNV. The specificity of MESD for OROV was further validated by its interaction with OROV envelope glycoproteins, as shown through immunoprecipitation assays. These findings suggest that targeting MESD could be a novel and effective approach for diagnosing and treating OROV infections, potentially paving the way for innovative therapeutic strategies against this virus.

Description

[0001] NOVEL DIAGNOSTIC AND THERAPEUTIC APPROACHES FOR OROPOUCHE VIRUS INFECTION

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular infectious diseases and virology.

[0004] BACKGROUND OF THE INVENTION:

[0005] The Oropouche virus (OROV) is an arbovirus responsible for causing Oropouche fever, characterized by high fever, vomiting, photophobia, and in rare instances, aseptic meningitis or meningoencephalitis. Recent cases of OROV infection during pregnancy have been associated with stillbirth (Garcia Filho, Carlos, et al. "A Case of Vertical Transmission of Oropouche Virus in Brazil. " New England Journal of Medicine (2024)) and risk of birth defects (das Neves Martins, Fernanda Eduarda, et al. "Newborns with microcephaly in Brazil and potential vertical transmission of Oropouche virus: a case series. " The Lancet Infectious Diseases (2024)). Furthermore, first cases of fatal oropouche infections were also reported (Bandeira, Antonio Carlos, et al. "Fatal Oropouche virus infections in nonendemic region, Brazil, 2024. " Emerging Infectious Diseases 30.11 (2024): 2370)). Since its first isolation in 1955, OROV was predominantly endemo-epidemic in the pan-Amazonian regions of Latin America, with over 30 epidemics reported, but the virus has lately spread to non-endemic regions of Central and South America and the Caribbean Basin (Tilston-Lunel, Natasha L. "Oropouche Virus: An Emerging Orthobunyavirus. " Journal of General Virology 105.10 (2024): 002027). Diagnosing Oropouche fever presents challenges due to symptom similarities with other arboviral diseases such as Dengue, Zika, and Chikungunya viruses. Currently, there are no specific antiviral treatments or effective vaccines available for OROV infection. The zoonotic origin, history of human spillover, trisegmented RNA genome capable of reassortment, along with increased human-wildlife interaction due to deforestation, recent dissemination to non-endemic areas and increase in disease severity render OROV a significant epidemic threat.

[0006] OROV is classified under the genus Orthobunyavirus within the family Peribunyaviridae, one of the fourteen families that make up the order Bunyavirales. Orthobunyaviruses produce spherical, enveloped virus particles ranging from 100 to 120 nm in diameter. The genome of orthobunyaviruses consists of three negative-sense single-stranded RNA segments. The small segment (S) encodes the nucleocapsid N protein (25 to 30 kDa), which oligomerizes and encapsulates the viral genome, as well as the nonstructural protein NSs. The medium segment (M) encodes a polyprotein that is cotranslationally cleaved by host proteases to generate the viral surface glycoproteins (Gc and Gn) and a nonstructural protein NSm. The large segment (L) encodes the viral RNA-dependent RNA polymerase (RdRp), which catalyzes viral replication and transcription. The viral RNA segments are encapsulated by the N protein to form a ribonucleoprotein complex that interacts with both the RdRp and surface glycoproteins to enable virus particle assembly. The Gn (~32 kDa) and Gc (-110 kDa) glycoproteins are integral membrane proteins with N-terminal ectodomains. They associate within the host endoplasmic reticulum (ER) before being transported to the Golgi complex, where virion assembly predominantly occurs.

[0007] SUMMARY OF THE INVENTION:

[0008] The present invention is defined by the claims. In particular, the present invention relates to novel diagnostic and therapeutic approaches for oropouche virus infection.

[0009] DETAILED DESCRIPTION OF THE INVENTION:

[0010] Main definitions:

[0011] 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.

[0012] As used herein, the term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”).

[0013] As used herein, the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide that is different from the first one). 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.

[0014] 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.

[0015] 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 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

[0016] As used herein, the term “oropouche virus” or “OROV” has its general meaning in the art and refers to an arbovirus of the genus Orthobunyavirus that is known to cause febrile illness in humans. The virus is primarily transmitted through the bite of infected midges (Culicoides paraensis) and mosquitoes, leading to outbreaks in tropical regions, particularly in South and Central America. Oropouche virus infection can result in symptoms such as fever, headache, myalgia, and in some cases, encephalitis.

[0017] As used herein, the term "Gc" refers to the glycoprotein component of the viral envelope of the Oropouche virus. The Gc glycoprotein is an integral membrane protein that plays a critical role in the virus's ability to infect host cells. It is responsible for the attachment and entry of the virus into the host cell by interacting with specific receptors on the cell surface. Gc also acts as a class II fusion protein mediating low-pH-triggered fusion between viral and endosomal membranes. The Gc glycoprotein, along with the Gn glycoprotein, is synthesized in the host cell's endoplasmic reticulum and transported to the Golgi complex, where it undergoes further processing and assembly into mature virions. An exemplary amino acid sequence for Gc is shown as SEQ ID NO: 1.

[0018] SEQ ID NO: 1> Gc DEDCLSKDIKITYQELHNCIGPKIMGDTCVSKSELYSDLFSKNLVTEYDKKYFEPDTVNDQFNKIEFAQ DAHRMILLERILYKTECEMLSLKKNSGPYNVAWRTYFKNHNIDLCSRHNYKMICQCINTHSMCKNTDID YNKEIETYYKSNAAAYRADLNTIMDTLKTAFRGLTKVLIENYIEKDDSDALKALFSNITDSVQDNYQMI GILKFASKLLDINLGRSTRSAHHSIMTNEIPKSNPFTDYSYSNLNIKECMSPESLKCFKKRDSTPHTNH LLCKIDNKYKAFDWPEIETIQKGQKLCLGDSHCNLEFTAITADKIMSLTNCYKESFTAQPADMQAGIKK CSADEIGECTTLEDKTWPIIFCGGKYYYSDSKEHAKDGSINNYCLTNKCSEQRFPIHENWFKKCNWDKT HKEFSTMRQINYNDITSYRKAIESEIGTDLMTHHYKPTKNLPHWPRYHSIDVQGTESTEGIINGFIQN TI PAI SGLGVGYHLNFKGNQLFDI VI FVKKAVYKAQYQKAYTTGPS I S INI EHNERCTGHCPEKI PARE GWLTFSKEHTSSWGCEEYGCLAIDTGCLYGSCQDVIRPELDVYKKIGSEVSLIEICITLPHETYCNDMD ILEPIIGDKLSASFQNTQTNQLPTLIAYKKGKIYTGQINDIGNTALQCGSIQVINGSTIGTGSPKFDYI CHAMRRKDVIVRKCFNDNYQSCTRLEKRNDLIPYRKGDVIEVSKTGSNMGQMTFKIELGDINYKIFTKS IDLQMSGICAGCIDCAEGISCSINAEVPAETVCHCKTNCEDFINNIVFSPQIKNYNIKVHCKSKVEKIT AHICGRDIDLQLTIKPYNQKIDLSQLDESNYIREEDLQCGTWLCKVQKEGIDIIFKGLFSGLGRYWTIL IYSIIGWIIVILVYILLPIGRLLKAFLIRHEIEYAMEQKIK

[0019] As used herein, the term “Gn” refers to the glycoprotein component of the viral envelope of the Oropouche virus. The Gn glycoprotein is an integral membrane protein that plays a crucial role in the virus's infectious cycle. It facilitates the initial stages of viral attachment to host cells and aids in the viral entry process. Along with the Gc glycoprotein, the Gn glycoprotein is synthesized in the host cell's endoplasmic reticulum and transported to the Golgi complex for further processing and assembly into mature virions. An exemplary amino acid sequence for Gc is shown as SEQ ID NO:2.

[0020] SEQ ID NO: 2> Gn HPLSTSQIGDRCFAGGNLFKEMNLSVGLGEICVKDDISIVKSTTVFSKNKPALEATTKFYRSFIVKDWS ECNPVLDKFGNFMVLSVDDNGHIIPKMYTCRAACDIRLNKDNAEIILSSTKLNHFEIVGTTSTSGWFKN TITNNLEHTCEHVTVNCGQKSVKFHACFRQHRGCIRFFKGTYMPYSMIEAMCVNIELIILTLYIFAAII FALIITKSYVAYLLLPLFYPVTWFYGKVYKKINSCPNCLLASHPFTSCPKICICGSRFSCTEALKVHRM GKDCLGYKSLSKARQMCKSKSWSFTAAILTGLILMEFISPIAG

[0021] As used herein, the term “MESD” has its general meaning in the art and relates to a chaperone specifically assisting the folding of beta-propeller / EGF modules within the family of low-density lipoprotein receptors (LDLRs). This chaperone function is critical for the proper insertion of these receptors into the plasma membrane, where they play a key role in mediating cellular uptake of molecules such as cholesterol and fat-soluble vitamins. The correct folding and trafficking of LDLR family members, facilitated by MESD, is essential for maintaining cellular homeostasis and metabolic functions. An exemplary amino acid sequence for MESD is shown as SEQ ID NO:3.

[0022] SEQ ID NO: 3 >sp | Q14696 | MESD_HUMAN LRP chaperone MESD OS=Homo sapiens OX=9606 GN=MESD PE=1 SV=2 MAASRWARKAWLLCASDLLLLLLLLPPPGSCAAEGSPGTPDESTPPPRKKKKDIRDYNDADMARLLEQ WEKDDDIEEGDLPEHKRPSAPVDFSKIDPSKPESILKMTKKGKTLMMFVTVSGSPTEKETEEITSLWQG SLFNANYDVQRFIVGSDRAIFMLRDGSYAWEIKDFLVGQDRCADVTLEGQVYPGKGGGSKEKNKTKQDK GKKKKEGDLKSRSSKEENRAGNKREDL

[0023] 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.

[0024] 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 OROV 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.

[0025] 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.]).

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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. 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 OROV that provides at least partial protective immunity against a disease, condition, or disorder.

[0031] 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).

[0032] Screening methods:

[0033] The first object of the present invention relates to a method for identifying a substance useful for inhibiting the replication capacity of oropouche virus (OROV) comprising the steps of (a) contacting a polypeptide (Pl) containing an amino acid sequence of the human MESD protein with a polypeptide (P2) having an amino acid sequence of the OROV Gc or Gn glycoprotein, under conditions and for a time sufficient to permit binding and the formation of a complex between the two polypeptides (Pl) and (P2), in the presence of a test substance, and (b) detecting the formation of the complex, in which the ability of the test substance to inhibit the interaction between the two polypeptides (Pl) and (P2) is indicated by a decrease in complex formation as compared to the amount of complex formed in the absence of the test substance and (c) selecting the substance that inhibits the interaction.

[0034] In some embodiments, the polypeptide (Pl) comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:3.

[0035] In one embodiment, the polypeptide (Pl) comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 3 wherein the amino acid W in position 136 and the amino acid I in position 158 are conserved.

[0036] As used herein “amino acid conserved” refers to an amino acid residue that is functionally essential within a polypeptide or protein sequence, and which is not substituted by another amino acid in homologous sequences across different organisms or variants. The absence of substitution indicates that the residue plays a critical role in the biological activity, structural integrity, or molecular interactions of the protein, and that any change at this position would likely impair its function.

[0037] In some embodiments, the polypeptide (P2) comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO:2.

[0038] In some embodiments, the polypeptide (Pl) and / or (P2) is labelled with a detectable molecule. According to the invention, said detectable molecule may consist of any substance or substance that is detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful detectable molecules include radioactive substance (including those comprising32P,25S,3H, or125I), fluorescent dyes (including 5-bromodesosyrudin, fluorescein, acetylaminofluorene or digoxigenin), fluorescent proteins (including GFPs and YFPs), or detectable proteins or peptides (including biotin, polyhistidine tails or other antigen tags like the HA antigen, the FLAG antigen, the c-myc antigen and the DNP antigen).

[0039] According to the invention, the detectable molecule is located at, or bound to, an amino acid residue located outside the binding sites of the polypeptides, in order to minimise or prevent any artefact for the binding between said polypeptides or between the test substance and or any of said polypeptides.

[0040] In some embodiments, the polypeptides of the invention are fused with a tag. Tag that are routinely used in the art can be used. For instance, the polypeptide may be fused to a GST tag (Glutathione S-transferase) or polyhistidine tag. In said embodiments, the tag moiety of the said fusion protein may be used as detectable molecule. In the said fusion protein, the tag may be located either at the N-terminal end or at the C-terminal end. The tag detectable molecule may be detected when it is subsequently brought into contact with an anti-tag antibody, including with a labelled anti-tag antibody. Anti-tal antibodies labelled with various detectable molecules are easily commercially available.

[0041] In some embodiments, the polypeptides are fused with a portion of a transcription factor. The term “portion” when used herein for transcription factor, encompass complete proteins involved in multi protein transcription factors, as well as specific functional protein domains of a complete transcription factor protein. In some embodiments, the portion consists of either the DNA binding domain or the activator domain of a transcription factor. In some embodiments, the DNA binding domain and the activator domain both originate from the same naturally occurring transcription factor. In some embodiments, the DNA binding domain and the activator domain originate from distinct naturally occurring factors, while, when bound together, these two portions form an active transcription factor. Said protein moiety domain of transcription may be located either at the N-terminal end or at the C-terminal end. Such a DNA binding domain may consist of the well-known DNA binding domain of LexA protein originating form E. Colt. Moreover, said activator domain of a transcription factor may consist of the activator domain of the well-known Gal4 protein originating from yeast.

[0042] As used herein, the expression “inhibiting the interaction” means that the substance reduces 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% the interaction between the two polypeptides (Pl) and (P2).

[0043] In some embodiments the step (b) consists in generating physical values which illustrate or not the ability of said test substance to inhibit the interaction between the polypeptides (Pl) and (P2) 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 binding between polypeptides (Pl) and (P2), then the candidate is positively selected at step (c).

[0044] The substances that inhibit the interaction between the OROV glycoprotein and MESD protein encompass those substances that bind either to polypeptide (Pl) or polypeptide (P2), provided that the binding of the said substances of interest then prevents the interaction between said polypeptides.

[0045] Different assays that are routinely used in the art can be used for detecting the formation of the complex formed by the polypeptides (Pl) and (p2). In some embodiments, a two-hybrid assay may be used wherein a first polypeptide is fused or conjugated to a first portion of a transcription factor (e.g. a DNA binding portion) and the second polypeptide is fused the second portion of the transcription factor (e.g. activator domain of a transcription factor), wherein the binding together of the first and second portions generates a functional transcription factor that binds to a specific regulatory DNA sequence, which in turn induces expression of a reporter DNA sequence, said expression being further detected and / or measured. A positive detection of the expression of said reporter DNA sequence means that an active transcription factor is formed, due to the binding together of said polypeptides.

[0046] Therefore, in some embodiments of the invention, the assay of the invention comprises the following steps:

[0047] (1) providing a host cell expressing:

[0048] a first fusion polypeptide between (i) a first polypeptide (Pl) or (P2) and (ii) a first protein portion of transcription factor

[0049] a second fusion polypeptide between (i) a second polypeptide (Pl) or (P2) and (ii) a second portion of a transcription factor

[0050] said transcription factor being active on DNA target regulatory sequence when the first and second protein portion are bound together and

[0051] said host cell also containing a nucleic acid comprising (i) a regulatory DNA sequence that may be activated by said active transcription factor and (ii) a DNA report sequence that is operatively linked to said regulatory sequence

[0052] (2) bringing said host cell provided at step 1) into contact with a test substance to be tested (3) determining the expression level of said DNA reporter sequence

[0053] The expression level of said DNA reporter sequence that is determined at step (3) above is compared with the expression of said DNA reporter sequence when step (2) is omitted. A reduced expression level of said DNA reporter sequence in the presence of the test substance means that the said test substance effectively inhibits the binding between OROV glycoprotein and MESD protein and that said test substance may be positively selected.

[0054] Suitable host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). However preferred host cell are yeast cells and more preferably a Saccharomyces cerevisiae cell or a Schizosaccharomyces pombe cell. Similar systems of two-hybrid assays are well known in the art and therefore can be used to perform the assay according to the invention (see. Fields et al. 1989; Vasavada et al. 1991; Fearon et al. 1992; Dang et al., 1991, Chien et al. 1991, US 5,283,173, US 5,667,973, US 5,468,614, US 5,525,490 and US 5,637,463). For instance, as described in these documents, the Gal4 activator domain can be used for performing the assay according to the invention. Gal4 consists of two physically discrete modular domains, one acting as the DNA binding domain, the other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing documents takes advantage of this property. The expression of a Gall-LacZ reporter gene under the control of a Gal4-activated promoter depends on the reconstitution of Gal4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for p-galactosidase. A compete kit (MATCHMAKER, TM) for identifying protein-protein interactions is commercially available from Clontech.

[0055] The expression of said detectable marker gene may be assessed by quantifying the amount of the corresponding specific mRNA produced. However, usually the detectable marker gene sequence encodes for detectable protein, so that the expression level of the said detectable marker gene is assessed by quantifying the amount of the corresponding protein produced. Techniques for quantifying the amount of mRNA or protein are well known in the art. For example, the detectable marker gene placed under the control of regulatory sequence may consist of the P-galactosidase as above described.

[0056] In some embodiments, the assay comprises a step of subjecting to a gel migration assay the mixture of the first polypeptide (Pl) and the second polypeptide (P2) as above defined, with or without the test substance to be tested and then measuring the binding of the said polypeptides altogether by performing a detection of the complexes formed between said polypeptides. The gel migration assay can be carried out as known by the one skilled in the art.

[0057] Therefore, in some embodiments of the invention, the assay of the invention comprises the following steps:

[0058] (1) providing the polypeptides (Pl) and (P2) as defined above

[0059] (2) bringing into contact the test substance to be tested with said polypeptides

[0060] (3) performing a gel migration assay a suitable migration substrate with said polypeptides and said test substance as obtained at step (2) (4) detecting and quantifying the complexes formed between said polypeptides on the migration assay as performed at step (3).

[0061] The presence or the amount of the complexes formed between the proteins are then compared with the results obtained when the assay is performed in the absence of the test substance to be tested. Therefore, when no complexes between the proteins is detected or, alternatively when those complexes are present in a lower amount compared to the amount obtained in the absence of the test substance, means that the test substance may be selected as an inhibitor of the specific interaction between said host protein and said viral protein.

[0062] The detection of the complexes formed between the said two proteins may be easily performed by staining the migration gel with a suitable dye and then determining the protein bands corresponding to the protein analysed since the complexes formed between the first and the second proteins possess a specific apparent molecular weight. Staining of proteins in gels may be done using the standard Coomassie brilliant blue (or PAGE blue), Amido Black, or silver stain reagents of different kinds. Suitable gels are well known in the art such as sodium dodecyl (lauryl) sulfate-polyacrylamide gel. In a general manner, western blotting assays are well known in the art and have been widely described (Rybicki et al., 1982; Towbin et al. 1979; Kurien et al. 2006).

[0063] In some embodiments, the protein bands corresponding to the proteins submitted to the gel migration assay can be detected by specific antibodies. It may use both antibodies directed against polypeptide (Pl) and antibodies specifically directed against polypeptide (P2).

[0064] In some embodiments, both polypeptides are labelled with a detectable antigen as above described. Therefore, the proteins bands can be detected by specific antibodies directed against said detectable antigen. Preferably, the detectable antigen conjugates to the polypeptide (Pl) is different from the antigen conjugated to the polypeptide (P2). For instance, the first polypeptide (Pl) can be fused to a GST detectable antigen and the second polypeptide (P2) can be fused with the HA antigen. Then the protein complexes formed between the two proteins may be quantified and determined with antibodies directed against the GST and HA antigens respectively.

[0065] In some embodiments, the assay includes the use of an optical biosensor such as described by Edwards et al. (1997) or also by Szabo et al. (1995). This technique allows the detection of interactions between molecules in real time, without the need of labelled molecules. This technique is indeed bases on the surface plasmon resonance (SPR) phenomenon. Briefly, a first protein partner is attached to a surface (such as a carboxymethyl dextran matrix). Then the second protein partner is incubated with the previously immobilised first partner, in the presence or absence of the test substance to be tested. Then the binding including the binding level or the absence of binding between said protein partners is detected. For this purpose, a light beam is directed towards the side of the surface area of the substrate that does not contain the sample to be tested and is reflected by said surface. The SPR phenomenon causes a decrease in the intensity of the reflected light with a combination of angle and wavelength. The binding of the first and second protein partner causes a change in the refraction index on the substrate surface, which change is detected as a change in the SPR signal.

[0066] In some embodiments, the assay includes the use of affinity chromatography. Test substances for use in the assay above can also be selected by any immunoaffinity chromatography technique using any chromatographic substrate onto which the polypeptide (Pl) and / or (P2) as above defined, has previously been immobilised, according to techniques well known from the one skilled in the art. Briefly, the polypeptide may be attached to a column using conventional techniques including chemical coupling to a suitable column matrix such as agarose, Affi Gel®, or other matrices familiar to those of skill in the art. In some embodiments, the affinity column contains chimeric polypeptides in which the polypeptide (Pl) or (P2) is fused to a tag such as glutathion-s-transferase (GST). Then a test substance is brought into contact with the chromatographic substrate of the affinity column previously, simultaneously or subsequently to the other protein among the said first and second protein. The after washing, the chromatography substrate is eluted and the collected elution liquid is analysed by detection and / or quantification of the said later applied first or second protein, so as to determine if, and / or to which extent, the test substance has impaired or not the binding between both polypeptides (Pl) and (P2).

[0067] In some embodiments, the assay involves detection of a fluorescence signal. In some embodiments, the first polypeptide (Pl) and the second polypeptide (P2) as above defined are labelled with a fluorescent molecule or substrate. Therefore, the potential alteration effect of the test substance to be tested on the binding between the first polypeptide (Pl) and the second polypeptide (P2) as above defined is determined by fluorescence quantification. For example, the first polypeptide (Pl) and the second polypeptide (P2) as above defined may be fused with auto-fluorescent polypeptides, as GFP or YFPs as above described. The first polypeptide (Pl) and the second polypeptide (P2) as above defined may also be labelled with fluorescent molecules that are suitable for performing fluorescence detection and / or quantification for the binding between said proteins using fluorescence energy transfer (FRET) assay. The first polypeptide (Pl) and the second polypeptide (P2) as above defined may be directly labelled with fluorescent molecules, by covalent chemical linkage with the fluorescent molecule as GFP or YFP. The first polypeptide (Pl) and the second polypeptide (P2) as above defined may also be indirectly labelled with fluorescent molecules, for example, by non covalent linkage between said polypeptides and said fluorescent molecule. A suitable receptor / ligand couple may be the biotin / streptavidin paired member or may be selected among an antigen / antibody paired member. For example, a protein according to the invention may be fused to a poly-histidine tail and the fluorescent molecule may be fused with an antibody directed against the poly-histidine tail.

[0068] In some embodiments, a first polypeptide is labelled with a first fluorophore substance and the second polypeptide is labelled with a second fluorophore substance. The first fluorophore substance may have a wavelength value that is substantially equal to the excitation wavelength value of the second fluorophore, whereby the bind of said first and second proteins is detected by measuring the fluorescence signal intensity emitted at the emission wavelength of the second fluorophore substance. Alternatively, the second fluorophore substance may also have an emission wavelength value of the first fluorophore, whereby the binding of said and second proteins is detected by measuring the fluorescence signal intensity emitted at the wavelength of the first fluorophore substance.

[0069] The fluorophores used may be of various suitable kinds, such as the well-known lanthanide chelates. These chelates have been described as having chemical stability, long-lived fluorescence (greater than 0.1 ms lifetime) after bioconjugation and significant energy -transfer in specificity bioaffmity assay. Document US 5,162,508 discloses bipyridine cryptates. Polycarboxylate chelators with TEKES type photosensitizers (EP0203047A1) and terpyridine type photosensitizers (EP0649020A1) are known. Document W096 / 00901 discloses diethylenetriaminepentaacetic acid (DPTA) chelates which used carbostyril as sensitizer. Additional DPT chelates with other sensitizer and other tracer metal are known for diagnostic or imaging uses (e.g., EP0450742A1). In some embodiments, the fluorescence assay consists of a Homogeneous Time Resolved Fluorescence (HTRF) assay, such as described in document WO 00 / 01663 or US6,740,756, the entire content of both documents being herein incorporated by reference. HTRF is a TR-FRET based technology that uses the principles of both TRF (time-resolved fluorescence) and FRET. More specifically, the one skilled in the art may use a HTRF assay based on the time-resolved amplified cryptate emission (TRACE) technology as described in Leblanc et al. (2002). The HTRF donor fluorophore is Europium Cryptate, which has the long-lived emissions of lanthanides coupled with the stability of cryptate encapsulation. XL665, a modified allophycocyanin purified from red algae, is the HTRF primary acceptor fluorophore. When these two fluorophores are brought together by a biomolecular interaction, a portion of the energy captured by the Cryptate during excitation is released through fluorescence emission at 620nm, while the remaining energy is transferred to XL665. This energy is then released by XL665 as specific fluorescence at 665 nm. Light at 665nm is emitted only through FRET with Europium. Because Europium Cryptate is always present in the assay, light at 620nm is detected even when the biomolecular interaction does not bring XL665 within close proximity.

[0070] The test substance of the invention may be selected from a library of substances previously synthesized, a library of substances for which the structure is determined in a database, or from a library of substances that have been synthesized de novo. These substances can be classified into several categories:

[0071] (a) proteins or peptides, which can be naturally occurring or synthetically produced to interact specifically with certain molecular targets within the cell;

[0072] (b) nucleic acids, which include DNA, RNA, or their analogs and derivatives. Among these, antisense oligonucleotides are particularly noteworthy. These short, synthetic strands of nucleic acids are designed to bind to specific messenger RNA (mRNA) molecules, thereby blocking the expression of certain genes. This method can effectively reduce the production of proteins that are necessary for virus replication, providing a novel approach to antiviral therapy;

[0073] (c) organic or chemical substances, which encompass a wide range of small molecules capable of modulating biological processes. These substances can be identified through high-throughput screening methods and possess diverse chemical structures that enable them to interact with specific cellular targets.

[0074] In the context of this invention, the selection of antisense oligonucleotides as test substances is highly advantageous. These molecules are tailored to bind complementary sequences of mRNA, preventing the translation of viral proteins essential for the replication of the virus. The design and synthesis of antisense oligonucleotides can be precisely controlled, allowing for the development of highly specific inhibitors that minimize off-target effects. Furthermore, advancements in delivery methods enhance the stability and cellular uptake of antisense oligonucleotides, increasing their therapeutic potential. In some embodiments, the antisense is specific for MESD. For example, anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of MESD mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of MESD, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding MESD can be synthesized, e.g., by conventional phosphodiester techniques.

[0075] In some embodiments, the method of the present invention further comprises the step (d) consisting in determining whether the substance selected at step (c) inhibits the replication of OROV in a host cell and a step (e) that consists in positively selecting the test substance capable of inhibiting the replication of said OROV in said host cell.

[0076] In some embodiments, the method comprises the steps consisting of i) infecting said host cell with said OROV 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.

[0077] 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 OROV 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 OROV grown in the absence of said substance. Said replication capacity may be typically determined by any routine technique well known in the art. According to the present invention, any OROV strain can be used. Preferably, said OROV strain corresponds to a clinical isolate of at least one circulating strain of OROV.

[0078] 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.

[0079] 60 / 638,166, filed Dec. 23, 2004, and in U. S. Provisional Application No. 60 / 641,139, filed Jan.

[0080] 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.

[0081] In some embodiments, the infection of the cells with OROV 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.

[0082] 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.

[0083] 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 OROV infections as described infra. In some embodiments, the substances selected by the above-mentioned screening method may be used in the treatment of OROV infection. For example, therapeutic treatments include the reduction or amelioration of the progression, severity and / or duration of OROV infections, or the amelioration of one or more symptoms (specifically, one or more discernible symptoms) of OROV 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 OROV infection. In some embodiments, the therapeutic treatment includes the inhibition of the progression of an OROV 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 OROV infections.

[0084] In some embodiments, the substances selected by the above-mentioned screening method may be used in a prophylactic treatment. 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 OROV or not considered at high risk for complications, in order to reduce the chances of getting infected with the OROV and passing it on to a high-risk person in close contact with him (for instance, healthcare workers, nursing home workers, etc).

[0085] 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 OROV, to reduce the amount of OROV or to reduce or ameliorate the severity, duration, progression, or onset of a OROV infection, prevent the advancement of an OROV infection, prevent the recurrence, development, onset or progression of a symptom associated with an OROV infection, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy used against OROV 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 co-administered with other antiviral agents, e.g., when coadministered with an anti-OROV 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.

[0086] 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.

[0087] 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.

[0088] Attenuated viruses of the present invention:

[0089] A further object of the present invention relates to a live attenuated oropouche virus that is engineered to express a mutein of the glycoprotein Gc and / or Gn that comprises one or more mutations so that said glycoprotein mutein is no capable to bind to MESD in a host cell.

[0090] In some embodiments, the live attenuated OROV of the present invention is engineered to express a Gc and / or Gn mutein(s) that comprises one or more deletion so that said mutein is no capable to bind to MESD in a host cell. The deletion(s) may be continuous, or may comprise a plurality of sections of sequence. The deletion should remove a sufficient amount of the domain that is involved in the interaction with the MESD protein so that that said mutein is no capable to bind to MESD in a host cell. The deletion may, for example, remove at least 50, 60, 70, 80 or 90% of the domain that is involved in the interaction with MESD. In some embodiments, the deletion may be total, in which case 100% of said domain is absent, when compared to the corresponding amino acid sequence of the wild-type isolate.

[0091] 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.

[0092] 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.

[0093] The resulting attenuated OROV 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 oropouche 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.

[0094] The attenuation of the OROV can be confirmed by measuring its reduced replication and virulence in vitro and in vivo compared to the wild-type virus. The live attenuated OROV can also be tested for its immunogenicity and protective efficacy against oropouche infection in animal models.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] The live attenuated OROV 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 OROV-related vaccines and methods of the invention include (i) patients in areas in which OROV 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 a 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). 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 OROV can be performed following immunization.

[0100] Diagnostic methods:

[0101] A further object of the present invention relates to a method of testing whether a subject is predisposed a OROV infection comprising the steps consisting of i) measuring the expression level of MESD in a sample obtained from the subject and ii) comparing the expression level measured at step i) with a predetermined reference value and iii) concluding that the subject is predisposed to a OROV infection when differential between the measured expression level and the predetermined reference value is detected.

[0102] The method of the present invention is thus particularly suitable for discriminating subjects having a high risk of having a OROV infection from subjects having a low risk of having a OROV infection. The method the present invention is thus particularly suitable for carrying prophylactic behaviours including prophylactic treatments and / or isolations during a OROV outbreak.

[0103] As used herein, the term “risk" relates to the probability that an event will occur over a specific time period, as in the conversion to a OROV infection, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p / (l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. "Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to a OROV infection or to one at risk of developing a OROV infection. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of a OROV infection, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to a OROV infection, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for a OROV infection. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for a OROV infection. In some embodiments, the present invention may be used so as to discriminate those at risk for developing a OROV infection from normal.

[0104] The expression level may be measured by routine technique well known in the art. For instance, assays for measuring the expression level comprises quantifying the protein and thus typically invlove use of standard immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin / avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation. Use of a binding partner that is specific for HMESD is typically involved. The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal. As used herein, the term "labelled", with regard to the antibody, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy 5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. For example, radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Ini 11, Rel86, Rel88. The aforementioned assays generally involve the bounding of the binding partner (ie. Antibody or aptamer) in a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

[0105] In some embodiments, the assay consists in quantifying the amount of the mRNA. Methods for determining the quantity of mRNA are well known in the art. For example, the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and / or amplification (e.g., RT-PCR). Preferably quantitative or semi -quantitative RT-PCR is preferred. Real-time quantitative or semi -quantitative RT-PCR is particularly advantageous. Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

[0106] In some embodiments, the predetermined reference value is a threshold value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the total iron content in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured levels of the immune marker in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER. SAS, DESIGNROC. FOR, MULTIREADER POWER. SAS, CREATE-ROC. SAS, GB STAT VIO. O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

[0107] In some embodiments, the higher the expression level of MESD is, the higher the risk of having a OROV infection is.

[0108] Accordingly, in a particular embodiment, the invention relates to a method of testing a subject thought to have or be predisposed to having a OROV infection, which comprises the step of analysing a sample of interest obtained from said subject for detecting the presence of a genetic variant in the gene encoding for MESD protein.

[0109] As used herein, the term "genetic variant" has its general meaning in the art and denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. The alteration typically consists in a substitution, an insertion, and / or a deletion, at one or more (e.g., several) positions in the gene. Genetic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term is also known as “polymorphism”.

[0110] In some embodiments, the genetic variant is located in the promoter.

[0111] In some embodiments, the genetic variant is located in an intron.

[0112] In some embodiments, the genetic variant is located in an exon.

[0113] In some embodiments, the genetic variant is present is heterozygous (i.e. present in only one allele) or homozygous (i.e. present in the 2 alleles). In some embodiments, the method of the present invention comprises detecting one or more single nucleotide polymorphisms (SNP).

[0114] In some embodiments, the genetic variant is a single nucleotide polymorphism. As used herein, the term "single nucleotide polymorphism" or "SNP" has its general meaning in the art and refers to a single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the population.

[0115] Detecting the genetic variant may be determined according to any genotyping method known in the art. Typically, common genotyping methods include, but are not limited to, TaqMan assays, molecular beacon assays, nucleic acid arrays, allele-specific primer extension, allelespecific PCR, arrayed primer extension, homogeneous primer extension assays, primer extension with detection by mass spectrometry, sequencing, multiplex primer extension sorted on genetic arrays, ligation with rolling circle amplification, homogeneous ligation, OLA, multiplex ligation reaction sorted on genetic arrays, restriction-fragment length polymorphism, single base extension-tag assays, and the Invader assay. Such methods may be used in combination with detection mechanisms such as, for example, luminescence or chemiluminescence detection, fluorescence detection, time-resolved fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, and electrical detection. Various methods for detecting polymorphisms include, but are not limited to, methods in which protection from cleavage agents is used to detect mismatched bases in RNA / RNA or RNA / DNA, comparison of the electrophoretic mobility of variant and wild type nucleic acid molecules, and assaying the movement of polymorphic or wild-type fragments in polyacrylamide gels containing a gradient of denaturant using denaturing gradient gel electrophoresis. Sequence variations at specific locations can also be assessed by nuclease protection assays such as RNase and SI protection or chemical cleavage methods. Detecting the genetic variant may also be performed by sequencing. A variety of automated sequencing procedures can be used, including sequencing by mass spectrometry. The nucleic acid sequences of the present invention enable one of ordinary skill in the art to readily design sequencing primers for such automated sequencing procedures. Commercial instrumentation, such as the Applied Biosystems 377, 3100, 3700, 3730, and 3730x1 DNA Analyzers (Foster City, Calif.), is commonly used in the art for automated sequencing. Nucleic acid sequences can also be determined by employing a high throughput mutation screening system, such as the SpectruMedix system. A further object of the present invention relates to a method of testing a subject thought to have or be predisposed to having a OROV infection, which comprises the step of analysing a sample of interest obtained from said subject for detecting post-translational modifications of MESD protein.

[0116] The post-translational modifications MESD protein include but are not limited to phosphorylation, acetylation, glycosylation, and the like. Detecting the post-translational modifications of the MESD protein may be assessed by using a binding partner specific for a post-translational form of MESD protein. As described above, the binding partner may be an antibody (e.g., a radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein / ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to a specific form of the MESD protein. Said analysis can be assessed by a variety of techniques well known from one of skill in the art including, but not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (RIA).

[0117] 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.

[0118] FIGURES:

[0119] Figure 1. Genome-wide CRISPR-Cas9 identifies MESD as host dependency factors for OROV infection. A, B, Results of CRISPR-Cas9 loss-of-function OROV infection screen analyzed by MAGeCK RRA (A) and MAGeck MLE (B). Each circle represents individual gene. Y-axis represents the significance of sgRNA enrichment of genes in the selected population compared to the non-selected control population. X-axis represents a random distribution of the genes. All genes with FDR<0.05 were represented with enlarged circles (Benjamini -Hochberg procedure). Figure 2. MESD specifically promotes infection of OROV. A, MESDKOHEK293T cells were transduced with a lentiviral vector encoding FLAG-MESD or a control vector. Left panel shows an immunoblot of MESD in control and trans-complemented MESDKOHEK293T cells. Control, MESDKOand trans-complemented cells were inoculated with OROV (MOI 0.04) for 48 h, and infection was assessed by flow cytometry using an anti-OROVN antibody. Data are presented as mean value ± SD (n = 3 independent experiments performed in duplicate) and adjusted P-values are calculated by one-way ANOVA with a Dunnett’s multiple comparisons test (ns not significant; ****P<0.0001). B, Control and MESDKOHEK293T cells were inoculated with the OROV FG 2020 (MOI 0.04). At indicated time point after infection virus titers in supernatant were determined using titration of VeroE6 cells. Data are presented as mean ± SEM (n=2 independent experiments performed in duplicate) and adjusted P-values are calculated by two-way ANOVA with a Dunnett’s multiple comparisons test (ns not significant; * p< 0.05; ** p< 0.01). C, Control and MESDKOHEK293T cells were inoculated with OROV strain FG 2020 (MOI 0.04) or OROV strain BeAnl9991 (MOI 0.04) for 48 h, and infection was assessed by flow cytometry using an OROV anti-N antibody. D, Control and MESDKOHEK293T cells were inoculated with OROV strain FG 2020 (MOI 0.04), LACV (MOI 0.0016), TOSCV (MOI 5) and WNV (MOI 0.04) for 48 h, and infection was assessed by flow cytometry using an anti-OROV N antibody, anti-LACV Gc antibody, anti-whole TOSCV viral particle antibody and anti-WNV E antibody. C, D Data are presented as mean value ± SD (n = 3 independent experiments performed in duplicate) and adjusted P-values are calculated by oneway ANOVA with a Dunnett’s multiple comparisons test (ns not significant, ****P<0.0001). For panel C and D, the average number of infected cells in control duplicate, for each replicate, was taken as 100 %.

[0120] Figure 3. OROV envelope glycoproteins specifically interact with MESD. a, MESDKOHEK293T cells complemented with FLAG-MESD or an empty vector were transfected with a plasmid encoding the OROV M segment polyprotein cDNA with a N terminal HA tag (OROV-HA-M) or a plasmid encoding DENV C-prM-E. Cellular lysates were subject to immunoprecipitation with anti -FLAG beads followed by immunoblot analysis with anti -FLAG, anti-HA, anti-OROV Gc and anti-DENV E antibodies. Data shown are representative from 2 independent experiments, b, Control and MESDKOHEK293T cells complemented with FLAG-MESD or an empty vector were inoculated with OROV FG 2020 (MOI of 0.2) for 48 h. Cellular lysates were subject to immunoprecipitation with anti -FLAG beads followed by immunoblot analysis with anti-FLAG and anti-Gc antibodies, and anti-N (pan-OROV) mouse ascites. Data shown are representative from 2 independent experiments.

[0121] Figure 4. MESD regulate OROV dissemination at a post entry step, (a) Control and MESDKOHEK293T cells were resuspended with PBS-EDTA and incubated with OROV (MOI 5) at 4°C for 90 min. After extensive washing, vRNA from bound viral particles were quantified by RT-qPCR and normalized to the control cells. As a positive control, controls cells were also treated with proteolytic enzyme trypsin prior incubation with OROV. Data are presented as mean ± SD (n=2 independent experiments performed in triplicate) and adjusted P-values are calculated by one-way ANOVA with a Dunnett’s multiple comparisons test (* p< 0.05; *** p< 0.001; **** p<0.0001). (b) Control and MESDKOHEK293T cells were inoculated with OROV FG 2020 (MOI of 0.2). Inoculum was removed 2 hours after infection and replace with prewarm medium. At the indicated time points, cells were treated with trypsin to remove cell surface bound virus and viral RNA was quantified by qRT-PCR. Data are presented as mean ± SD (n=3 independent experiments performed in triplicate) and adjusted P-values are calculated by two-way ANOVA with a Dunnett’s multiple comparisons test (ns not significant; * p< 0.05; ** p< 0.01; *** p< 0.001; **** p<0.0001).

[0122] Figure 5. MESD mutants devoid of chaperone activity could not restore OROV infection in MEDKOcells, a, Immunoblot assessing the expression of FLAG-MESD in MESDKOHEK293T cells complemented with WT or point mutants of MESD using an anti-FLAG mAb. Data shown are representative from 2 independent experiments, b, MESDKOHEK293T cells complemented with an empty vector (E. V), MESD WT or point mutants were transfected with either a pcDNA3.1 vector backbone (control) or a plasmid encoding LRP6. LRP6 cell surface expression was quantified 48 hours after transfection by flow cytometry using an anti-LRP6 antibody. Data are presented as mean ± SD of the mean fluorescent intensity (MFI) (n=2 independent experiments performed in duplicate) and adjusted P-values are calculated by oneway ANOVA with a Dunnett’s multiple comparisons test (ns not significant; **** p<0.0001). c, MESDKOHEK293T cells complemented with an empty vector (E. V), MESD WT or point mutants were inoculated with OROV FG 2020 (MOI of 0.04) for 48 h, and infection was assessed by flow cytometry using an OROV anti-N antibody. The average number of infected cells in WT complemented cells duplicate, for each replicate, was taken as 100 %. Data are presented as mean value ± SD (n = 3 independent experiments performed in duplicate) and adjusted P-values are calculated by one-way ANOVA with a Dunnett’s multiple comparisons test, test (**** p<0.0001).

[0123] EXAMPLE:

[0124] The inventors reveal MESD as a critical host dependency factor for Oropouche virus (OROV) infection. Initial identification was achieved through a CRISPR-Cas9 loss-of-function screen, which highlighted MESD amongst other potential factors (Figure 1). The ensuing research demonstrated that MESD knockout in HEK293T cells resulted in a significant reduction in OROV infection rates (Figure 2). Complementing MESDKO cells with FLAG-MESD re-established susceptibility, thus confirming MESD's essential role in the infection process (Figure 2). Interestingly, MESD specifically facilitates OROV infection without affecting the infectivity of other viruses such as LACV, TOSCV, and WNV (Figure 2). The specificity of MESD for OROV was further validated by its interaction with OROV envelope glycoproteins, as shown through immunoprecipitation assays (Figure 3).

[0125] The OROV M segment encodes the viral glycoprotein Gn and Gc that require proper folding to traffic as a heterodimer from the ER to the Golgi apparatus where virus assembly occurs18. As MESD is an ER-resident chaperone and Gn / Gc are the only viral proteins expressed in the ER, the inventors hypothesized that MESD interacts with one or both of the viral glycoprotein. To test this, FLAG-MESD was immunoprecipitated from the lysates of HEK293T MESDKOcells complemented with MESD and transfected with a cDNA encoding the OROV M segment polyprotein (OROV-M cDNA) containing a HA epitope tag upstream Gn. To control for the specificity of this interaction, cells were also transfected with a plasmid encoding the dengue virus (DENV) E envelope glycoprotein. Both OROV glycoproteins coimmunoprecipitated with FLAG-MESD (Figure 3A), whereas no co-immunoprecipitation was detected with DENV E protein, confirming the specificity of this interaction. Because Gn / Gc form heterodimers, they next sought to investigate which of the viral glycoprotein interacts with MESD. HEK293T-MESDKOcells complemented with an empty vector or MESD were transfected with plasmids encoding HA-tagged Gn or HA-tagged Gc proteins, either individually or together, and cell lysates were subjected to immunoprecipitation with an anti-FLAG. They observed that both Gn and Gc individually co-immunoprecipitated with MESD (Data not shown). Finally, to assess whether MESD interacts with other viral proteins, HEK293T-MESDKOcells complemented with an empty vector or MESD were infected with OROV, and 48 hours after infection cell lysates were subjected to immunoprecipitation with an anti-FLAG. Immunoblotting with antibodies against OROV Gc and N proteins revealed that MESD co-immunoprecipitated with Gc but not with N (Figure 3B). However, due to the lack of suitable antibodies, they could not evaluate potential interaction with the viral L protein. Collectively, these findings confirmed that MESD specifically interact with both Gn and Gc.

[0126] Given that LRP1 has been identified as an entry factor for OROV, and that MESD is known to promote surface expression of members of the lipoprotein receptor-related protein (LRPs)34,37,38, they investigated whether MESD is required for OROV attachment to host cells. To address this, they performed a viral binding assay using control and MESDKOHEK293T cells. Cells were detached with PBS-EDTA, incubated in suspension with OROV at MOI of 5 for 1 hour at 4 °C and bound viral particles were quantified by RT-qPCR. As a positive control for loss-of-binding, control HEK293T cells were treated with trypsin to remove cell surface proteins prior incubation with OROV. Trypsin treatment significantly lowered OROV binding compared to untreated control cells, confirming the assay’s sensitivity. In contrast, MESD depletion did not affect OROV binding (Figure 4A), suggesting that MESD is not required for viral particle binding. To determine at which step of the OROV life cycle MESD acts, they challenged control and MESDKOHEK293T cells with OROV and quantified the viral RNA (vRNA) at different time point after infection (Figure 4B). At 4 h post-infection, vRNA levels were slightly, but non-significantly, reduced in MESDKOcells compared to controls. By 8 hours after infection, OROV vRNA levels were 7-fold lower in MESDKOcells, a reduction that remained approximatively constant at 24 hours after infection (9-fold reduction). At later time point, MESD depletion had a pronounced effect, with 153-fold and 199-fold decrease of the OROV vRNA levels at 48 and 72 hours after infection, respectively. These findings indicate that MESD functions at a post-entry stage of the viral replication cycle, likely by facilitating viral dissemination.

[0127] AlphaFold 3 modeling of the MESD-Gn / Gc multimeric complex identified several hydrophobic residues within the conserved core domain of MESD at the interface with Gc. Notably, substitution of these residues with positively charged arginine residues has been reported to impair MESD chaperone activity41. Therefore, the inventors hypothesized that the chaperone activity of MESD is required for its proviral function. To test this, single point mutations (W103R and I125R based on human MESD sequence without the 33 aa signal peptide) were introduced in the FLAG-MESD WT plasmid, and the corresponding cDNA were stably expressed in MESDKO293T cells via lentiviral transduction. Immunoblot analysis confirmed that FLAG-MESD W103R and I125R were expressed at levels comparable to the WT protein (Figure 5A). To verify that these substitutions abolish the chaperone activity of MESD, they performed a cell culture-based LRP6 maturation assay. This assay, measures the ability of MESD to promote proper folding and surface expression of LRP6, a receptor that is otherwise retained in the ER34HEK293T MESDKOcells complemented with wild-type or mutant MESD were transfected with an LRP6 cDNA, and surface expression of the receptor was quantified by flow cytometry (Figure 5B). In absence of MESD no expression of LRP6 on the cell surface was detected. In contrast, WT MESD efficiently promoted surface expression of the receptor, whereas complementation with mutated MESD did not promote LRP6 cell surface expression, confirming that these mutations abolish MESD chaperone activity. The inventors next examined the effect of these MESD substitutions on OROV infection. Cells expressing wild-type and mutated MESD were infected with OROV and infection was quantified 48 hours after infection by flow cytometry. Both W103R and I125R mutation significantly impaired the ability of exogeneous MESD to restore infection compared to WT protein (Figure 5C). Together, these results demonstrate that the proviral function of MESD depends on hydrophobic amino acids within its core domain that are essential for the chaperone activity, suggesting that MESD chaperone function is required for efficient OROV infection.

[0128] These findings suggest that targeting MESD could be a novel and effective approach for diagnosing and treating OROV infections, potentially paving the way for innovative therapeutic strategies against this virus.

[0129] REFERENCES:

[0130] 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 method for identifying a substance useful for inhibiting the replication capacity of oropouche virus (OROV) comprising the steps of (a) contacting a polypeptide (Pl) containing an amino acid sequence of the human MESD protein with a polypeptide (P2) having an amino acid sequence of the OROV Gc or Gn glycoprotein, under conditions and for a time sufficient to permit binding and the formation of a complex between the two polypeptides (Pl) and (P2), in the presence of a test substance, and (b) detecting the formation of the complex, in which the ability of the test substance to inhibit the interaction between the two polypeptides (Pl) and (P2) is indicated by a decrease in complex formation as compared to the amount of complex formed in the absence of the test substance and (c) selecting the substance that inhibits the interaction.

2. The method according to claim 1 that further comprises the step (d) consisting in determining whether the substance selected at step (c) inhibits the replication of OROV in a host cell and a step (e) that consists in positively selecting the test substance capable of inhibiting the replication of said OROV in said host cell.

3. A live attenuated oropouche virus that is engineered to express a mutein of the glycoprotein Gc and / or Gn that comprises one or more mutations so that said glycoprotein mutein is no capable to bind to MESD in a host cell.

4. The live attenuated OROV according to claim 3 that is engineered to express a Gc and / or Gn mutein(s) that comprises one or more deletion so that said mutein is no capable to bind to MESD in a host cell.

5. A vaccine composition comprising the live attenuated OROV according to claim 3 or 4.

6. The vaccine composition according to claim 5 that further comprises one or more adjuvants.

7. A method of vaccinating a patient in need of such treatment, comprising the steps of administering the vaccine composition according to claim 5 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.

8. A method of testing whether a subject is predisposed a OROV infection comprising the steps consisting of i) measuring the expression level of MESD in a sample obtained from the subject and ii) comparing the expression level measured at step i) with a predetermined reference value and iii) concluding that the subject is predisposed to a OROV infection when differential between the measured expression level and the predetermined reference value is detected.

9. A method of testing a subject thought to have or be predisposed to having a OROV infection, which comprises the step of analyzing a sample of interest obtained from said subject for detecting the presence of a genetic variant in the gene encoding for MESD protein.

10. The method of claim 11 that comprises detecting one or more single nucleotide polymorphisms (SNP).

11. A method of testing a subject thought to have or be predisposed to having a OROV infection, which comprises the step of analyzing a sample of interest obtained from said subject for detecting post-translational modifications of MESD protein.

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