Vaccine against mosquito extracellular vesicles for protecting against flaviviruses

Abstract

The present invention relates to an isolated mosquito extracellular vesicle for use as an immunogenic agent in the prevention of at least one vector-borne disease. The present invention also relates to a vaccine composition comprising at least one mosquito extracellular vesicle. The present invention further relates to the use of a mosquito extracellular vesicle in identifying at least one immunogenic peptide capable of reducing, in a vertebrate, the transmission of a vector-borne disease when the vertebrate is bitten by the vector, and to a method for identifying at least one immunogenic peptide capable of blocking, in a vertebrate, the transmission of a vector-borne disease when the vertebrate is bitten by the vector, the method comprising a step of identifying in vitro at least one target antibody present in an animal serum sample immunised against mosquito extracellular vesicles.

Description

[0001] MOSQUITO EXTRACELLULAR VESICLE VACCINE TO PROTECT AGAINST FLAVIVIRUSES

[0002] DESCRIPTION

[0003] Technical field

[0004] The present invention relates to a product for its use as an immunogenic agent in the prevention of at least one vector-borne disease, as well as to a vaccine composition comprising this product, and to the use of this product for identifying at least one immunogenic peptide capable of reducing, in a vertebrate, the transmission of a vector-borne disease during a bite of the vertebrate by the vector.

[0005] The present invention finds application in the pharmaceutical field, particularly in the field of vaccines.

[0006] State of the art

[0007] Mosquito-borne flaviviruses include, but are not limited to, dengue, Zika, yellow fever, Japanese encephalitis, and West Nile fever viruses. These flaviviruses alone are responsible for nearly half a billion infections annually, causing the deaths of approximately 250,000 people and costing affected societies more than €10 billion. Furthermore, due to the geographical distribution of mosquito vectors, almost all of humanity lives in an area at risk of infection. Despite this alarming situation, there are no effective means of control: there is no curative treatment, vector control has moderate efficacy, and the vaccination strategy is at an impasse because current vaccines targeting flaviviruses increase the severity of symptoms under certain conditions. Indeed, there are currently several limitations to vaccine strategies against flaviviruses.On the one hand, there is a growing demand for vaccines against several flaviviruses to address the growing diversity of viral risk. Indeed, several flaviviruses currently threaten human populations and the emergence of new flaviviruses as important pathogens is expected in the near future due to global changes. However, there are only a few flavivirus vaccines that target yellow fever, Japanese encephalitis and dengue fever. Thus, humanity will be faced with an increasing need for vaccines against a growing number of flaviviruses. On the other hand, vaccines targeting flaviviral proteins can carry health risks. The recent example of the dengue vaccine, DENGVAXIA® (SANOFI-Pasteur) is an illustration. This vaccine, initially deployed in the Philippines, revealed that vaccination increases the risk of severe forms of dengue fever, under certain conditions.Indeed, children vaccinated against dengue had an increased risk of mortality when they had never been infected with the virus before immunization. This phenomenon is linked to the amplification of infection by non-neutralizing antibodies generated during vaccination. These antibodies facilitate the internalization of the virus into macrophage-like cells, increasing infection and the severity of symptoms. Even more alarmingly, this amplification of infection by non-neutralizing antibodies could also operate between flavivirus species because antibodies against a first flavivirus infection increase secondary infection by another flavivirus species. These data strongly suggest that an immune response induced by a vaccine against one flavivirus species could aggravate infection by another flavivirus species and seriously calls into question the vaccine strategy of targeting flaviviral proteins.

[0008] There is therefore a real need for a new tool for preventing flaviviruses, overcoming these defects, drawbacks and obstacles of the prior art. Description of the invention

[0009] Following extensive research, the inventors have developed a broad-spectrum vaccine against several flaviviruses, targeting mosquito extracellular vesicles.

[0010] The inventors of the present invention have surprisingly demonstrated that extracellular vesicles (EVs) in mosquito saliva promote infection by several flaviviruses.

[0011] Indeed, the inventors unexpectedly showed that lipids contained in mosquito EVs amplify skin infection and viral transmission. They further demonstrated that supplementation with EV lipids increases infection by several flaviviruses in several cell models relevant for transmission, including primary skin fibroblast cells and primary immune cells primarily infected by viruses upon biting. The inventors further validated the effect of EV lipids with a mouse model by showing that injection of the virus supplemented with mosquito EV lipids increases infection and symptoms in an animal model. Thus, the inventors identified an amplification factor for pan-flaviviral transmission.

[0012] Based on this information, the inventors developed a novel vaccine strategy that involves immunizing against these mosquito extracellular vesicles to impair the initiation of skin infection and thus reduce viral pathogenesis and / or symptoms of infection.

[0013] The inventors of this invention are thus the very first to have demonstrated, in a completely unexpected manner, that immunization with non-infectious mosquito extracellular vesicles protects against infection by flaviviruses.

[0014] The inventors have notably demonstrated that immunizing mice with mosquito vesicles protects against infection by West Nile virus through mosquito bites, with an almost total reduction in mortality.

[0015] Thus, the invention provides a technical solution with the following advantages:

[0016] - Pan-flaviviral protection. Indeed, by targeting a factor necessary for the infection of several flaviviruses, the invention protects against several flaviviruses with a single vaccine.

[0017] - Increased health safety. By targeting mosquito salivary proteins, the invention circumvents the health risks associated with the vaccine strategy targeting flaviviral proteins.

[0018] - By immunizing with vesicles, an immune response against several antigens is generated and therefore a stronger protective effect.

[0019] Thus, a first subject of the invention relates to an extracellular vesicle isolated from a mosquito for its use as an immunogenic agent in the prevention of at least one vector-borne disease.

[0020] For the purposes of the present invention, the term "extracellular vesicle", also referred to by the acronym "EV", means any non-replicating vesicle secreted by at least one cell of at least one mosquito, in particular by the cells of the salivary glands. EVs are delimited by a lipid bilayer maintained by transmembrane proteins, which have a part in the lumen and a part external to the EV. There are 2 types of EVs, which are differentiated according to their biogenesis: exosomes which are produced by release of intraluminal vesicles by fusion with the plasma membrane, microvesicles which are secreted by budding of the plasma membrane. These EVs are different from VME (extracellular membrane vesicles) or OMV in English, which are produced by bacteria by budding of the external membrane.EVs can be internalized by receptor cells and thus transfer their material from the secreting cell to a recipient cell.

[0021] Notably, EVs have a lipid composition including ceramides; sphingomyelins; phosphatidylserines; phosphatidylcholines; phosphatidylethanolamines; phosphatidylinositols; cholesterol; cholesterol esters; fatty acids, including saturated fatty acids (SAFA); monounsaturated fatty acids (PUFA) and polyunsaturated fatty acids; triacylglycerides; diacylglycerides; fatty acids and N-acylethanolamines. Without wishing to be bound by a particular mechanism of action, it appears that EV sphingomyelins are the lipids responsible for increasing / facilitating mosquito EV infection. Advantageously, immunization against EVs neutralizes the function of sphingomyelins in increasing / facilitating infection.

[0022] The extracellular vesicles may be isolated from at least one biological fluid secreted by at least one mosquito, such as saliva, hemolymph, or excreta. Alternatively, the extracellular vesicles may be isolated from at least one mosquito cell extract, such as a cell culture, salivary gland extract, egg homogenate, intestinal homogenate, larval homogenate, mosquito body homogenate, or ex vivo mosquito tissue or organ culture. In the case of cell culture, the cells can be chosen from the cell lines Aedes aegypti Aag2, RML-12 and CCL125, Aedes albopictus C6 / 36, C7-10 and U4.4, Culex quinquefasciatus Hsu, Culex tritaeniorhynchus TRA-171 and Anopheles gambiae Mos.55, Suai B or 4a-3B. Advantageously, these cell models make it possible to overcome the limited quantities of saliva produced by mosquitoes.

[0023] The size of the extracellular vesicles is not limiting for the implementation of the invention. Thus, this size may depend on the biological fluid in which they are secreted, and / or the cells secreting them. For example, the diameter of the extracellular vesicles may vary between approximately 20 and approximately 800 nm, for example between approximately 20 and approximately 400 nm. In particular, within a population of EVs secreted by a given cell type, the diameters of the EVs are not all identical, and may vary between 20 and 800 nm, for example between approximately 20 and approximately 400 nm. This heterogeneity in size may be due to differences in their formation mechanisms, their functions or their biological contents. Thus, within the same population of EVs, there may be different subpopulations of EVs, such as exosomes, for example with a diameter between 30 and 150 nm, and microvesicles, for example with a diameter greater than 150 nm.

[0024] The aforementioned biological fluids or cell extracts may be derived from infected mosquitoes or infected cells, or alternatively from uninfected mosquitoes or cells. Whether derived from infected or uninfected mosquitoes or cells, the EVs may contain viruses in the case where they are derived from infected mosquitoes, liquids, extracts or cells, or may not contain them in the opposite case. However, in all cases, the EVs are not infectious, which may be defined by any method known to those skilled in the art, for example by viral titration on mammalian or invertebrate cells, as described for example by Pompon et al. ([2]). Preferably, when dealing with infected cells, the method for preparing mosquito EVs comprises a step of separating the EVs and the viral material, in order to avoid any transfer of viral material during immunization.Advantageously, the use of biological fluid, extract or uninfected cells makes it possible not to introduce viral material during immunization and / or not to have to isolate the EVs from the viruses before immunization. Indeed, the presence of viral protein would trigger immunization against these viral proteins, in addition to the immunization created by the EVs and this immunization against the viral proteins would protect against infection, thus masking the protective effect of the EVs. Furthermore, in the context of the invention, it has been shown that it is the immunization against the EVs which reduces the infection by inhibiting the effect of facilitating infection by the mosquito EVs at the bite, without there being any need for a transfer of viral material to the host. Indeed, as explained above, the EVs used are not infectious, even if they may contain viral material.

[0025] Extracellular vesicles may be isolated by any purification method known to those skilled in the art, for example by differential ultracentrifugation, density gradient separation, size filtration, immunoaffinity, affinity for certain lipids, affinity by surface charges, size exclusion chromatography, precipitation kits, molecular weight cut-off centrifugal filters, tangential flow filtration, membrane affinity column, flow cytometry, this list not being limiting.

[0026] The mosquito can be any species of mosquito that carries viruses, including flaviviruses. It can be, for example, a mosquito of the genus Aedes, for example Aedes aegypti, Aedes albopictus or Aedes japonicus, or of the genus Culex, for example Culex pipiens, Culex quinquefasciatus, Culex tarsalis, Culex molestus, Culex tritaeniorhynchus, or of the genus Anopheles, such as Anopheles gambiae, Anopheles coluzii, or Anopheles arabiensis.

[0027] The vector-borne disease may be, for example, at least one disease selected from dengue fever, Zika, yellow fever, Japanese encephalitis, West Nile fever, St. Louis encephalitis, Tembusu virus disease, and Usutu virus disease.

[0028] Extracellular vesicles are used as an "immunogenic agent" within the meaning of the present invention for their ability to trigger an immune response in an organism. This immune response is directed against all or part of the extracellular vesicles. "Prevention" within the meaning of the present invention means reducing the risk of contracting a disease or mitigating its effects in an organism, compared to an organism in which the preventive act, namely the administration of EVs, has not taken place. Advantageously, prevention is the consequence of an immune response in the organism following the administration of EVs. Without wishing to be bound by a theory or mechanism of action, the administration of EVs would trigger the production of antibodies and activate the specific immune cells capable of recognizing EVs and preventing infection of the skin, necessary for systemic infection and transmission.Indeed, the initiation stage of skin infection is a weak point in transmission because only a few hundred infectious viruses are injected with mosquito saliva. Furthermore, blocking skin infection prevents transmission because skin infection is necessary for systemic infection and transmission. Consequently, the prevention according to the invention would reduce or prevent disease transmission during a bite.

[0029] Prevention can occur by administering isolated EVs to a human or non-human vertebrate. Examples of non-human vertebrates include, but are not limited to, mammals such as monkeys, cats, dogs, cows, sheep, rabbits, pigs, goats, horses, or donkeys; birds such as ducks, turkeys, or chickens; or fish.

[0030] Another subject of the invention relates to a vaccine composition comprising at least one mosquito extracellular vesicle as defined above. The vaccine composition has the effects of preventing at least one vector-borne disease as described above.

[0031] The vaccine composition of the invention may further comprise any additional ingredient conventionally used in this type of specific formulation, in particular to ensure its efficacy and safety depending on the vertebrate to be vaccinated. This may be at least one substance chosen from vehicles, excipients, adjuvants, buffers, preservatives, immune regulators and stabilizers. This may be, for example, sugars, such as sucrose, sorbitol, proteins, such as human albumin or bovine serum, polyols, such as glycerin, amino acids, phenol, phenoxyethanol, immune agonists, salts, such as aluminum salts, aluminum phosphates, emulsions, such as squalene-based emulsions, or liposomes, this list not being limiting.

[0032] The composition may be in any form conventionally used in the field of vaccines. It may be, for example, a form chosen from injectable form, oral form, intranasal form, intradermal form, transdermal form, intramuscular form or oropharyngeal route. Preferably, it is an injectable form.

[0033] According to the invention, the composition may comprise any pharmaceutically acceptable and effective dose of extracellular vesicle. The dose may be adapted to the vertebrate to be vaccinated by a person skilled in the art, depending on his general knowledge. It may be, for example, a dose allowing the administration of EV between 35 and 100 nmol of protein equivalent in humans, or between 5 and 20 nmol in mice, or between 20 and 50 nmol in rabbits.

[0034] Another subject of the invention relates to a method for preparing mosquito extracellular vesicle, comprising the following steps:

[0035] 1) culture of mosquito cells, uninfected or infected by a virus,

[0036] 2) separation of the culture supernatant,

[0037] 3) purification of extracellular vesicles from the culture supernatant, for example by ultracentrifugation, 4) optionally, in the case of a culture of virus-infected cells, separation of EVs and viral particles or viral material.

[0038] The mosquito cells are as defined above. Preferably, they may be the Aedes aegypti Aag2 cell line (Zhang, Rudian et al. ([3]).

[0039] The in vitro conditions and the culture medium used are those known to those skilled in the art, in particular depending on the cell line chosen, to allow the cells to divide and secrete the extracellular vesicles.

[0040] The step of separating the culture supernatant, which contains the extracellular vesicles, can be carried out by any technique known to those skilled in the art, such as, for example, centrifugation, filtration, sedimentation, chromatography or ultrafiltration.

[0041] The step of separating the extracellular vesicles from the culture supernatant can be carried out by any technique known to those skilled in the art, such as for example by differential ultracentrifugation, density gradient separation, size filtration, immunoaffinity, affinity for certain lipids, affinity by surface charges, size exclusion chromatography, precipitation kits, molecular weight cut-off centrifugal filters, tangential flow filtration, membrane affinity column, flow cytometry, this list not being limiting. Preferably, it can be ultracentrifugation.

[0042] The optional step of separating EVs from viral particles or viral material may be carried out by any technique known to the person skilled in the art, such as differential centrifugation and ultracentrifugation, density gradient, for example on sucrose or iodixanol, membrane filtration, size exclusion chromatography, immunoaffinity or flow cytometry analysis, this list not being exhaustive. The extracellular vesicles obtained are in particular intact vesicles, or non-intact vesicles allowing the immunization of a vertebrate. The term "intact vesicle" means a vesicle whose lipid bilayer membrane is not altered. This allows the EVs to retain a content inside the lipid bilayer membrane and to present the antigens on the surface. The term "non-intact vesicle" means a vesicle whose lipid bilayer membrane is altered.It may be, for example, a fragment of a vesicle, or a vesicle whose membrane is denatured, or interrupted in one or more places.

[0043] Another subject of the invention relates to the use of a mosquito extracellular vesicle to identify, in vitro, at least one immunogenic peptide capable of reducing, in a vertebrate, the transmission of a vector-borne disease during a bite of the vertebrate by the vector.

[0044] Another subject of the invention relates to a method for identifying at least one immunogenic peptide capable of blocking or reducing, in a vertebrate, the transmission of a vector-borne disease during a bite of the vertebrate by the vector, comprising a step of in vitro identification of at least one antibody target present in a sample of serum from an animal immunized against mosquito extracellular vesicles.

[0045] Advantageously, the identified immunogenic peptides may correspond to all or part of the external part, or to all or part of the internal part, or to all or part of the transmembrane part, of a transmembrane protein of the mosquito extracellular vesicle.

[0046] The step of identifying the targets of the antibodies present in the serum sample can be carried out by any technique known to those skilled in the art. This may be, for example, Enzyme-Linked Immunosorbent Assay (ELISA), immunoprecipitation, Western blot, immunofluorescence, yeast display screening, immunohistochemistry or protein chips. Other advantages may also become apparent to those skilled in the art upon reading the examples below, illustrated by the attached figures, given for illustrative purposes.

[0047] Brief description of the figures

[0048] Figure 1 represents: A) The normalized number of viral genomic RNA copies in human liver cells (Huh7) infected with Dengue virus (DENV), Zika virus (ZIKV) and West Nile virus (WNV), supplemented with lipids from extracellular vesicles (VE-lipids; volume = 0.01 or 0.1 μl of lipid extract) or in the control group without supplementation (CTRL). * indicates a statistical p value < 0.05; ** < 0.01; and *** < 0.001; according to a T test. B) The severity of symptoms in a mouse infected by injection of West Nile virus (WNV) supplemented with mosquito EV lipids (VE-lipids) or not (CTRL), as a function of the number of days post-injection.C) Survival of mice infected by injection of West Nile fever virus (WNV) supplemented with VE lipids (virus + VE lipids), or not (CTRL virus), or without infection (No infection), as a function of the number of days post-injection.

[0049] - Figure 2 shows A) The severity of symptoms after mosquito bites infected with West Nile virus for non-immunized control mice (CTRL) or mice immunized with mosquito EVs (Immunized) as a function of the number of days after bite infection. B) The percentage of survival for the control group (Non-immunized) and for the group immunized with mosquito EVs (Immunized) as a function of the number of days after bite infection.

[0050] - Figure 3 represents the size distribution of mosquito EVs within EVs isolated from mosquito cells. A) Cryo electron microscopy photo. Arrows indicate lipid bilayer vesicular structures corresponding to EVs. B) Distribution of the proportion (%) of EVs for the different size ranges, ranging from 20 to 400 nm in diameter, within EVs isolated from mosquito cells.

[0051] - Figure 4 represents the identification of sphingomyelins as the lipids that amplify infection by Aag2 mosquito EVs from Aedes aegypti. A) Lipid composition of mosquito EVs. Cer, Ceramide; SM, Sphingomyelin; PS, Phosphatidylserine; PC, Phosphatidylcholine; PE, Phosphatidylethanolamine; PI, Phosphatidylinositol; Choi, Cholesterol; CholE, Cholesterol esters; SAFA, Saturated fatty acids; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; TAG, triacylglycerides; DAG, diacylglycerides; FA, Fatty acids; NAE, N-acylethanolamine. B) Effect of sphingomyelins removal from VE-lipids on infection of human cells (Huh7) by West Nile virus.Human cells were infected with the virus in the presence of lipid vehicle (CTRL), lipids extracted from mosquito EVs (EV-lipids), or extracted lipids from which sphingomyelins had been removed by pretreatment with sphigomyelinase (EV-lipids + SMase). * indicates a statistical p-value < 0.05; ** < 0.01; according to a T-test.

[0052] EXAMPLES

[0053] Example 1: Preparation of isolated extracellular vesicles

[0054] EVs are purified from the Aag2 cell line (Aedes aegypti) (Peleg, J. “Growth of Arboviruses in Primary Tissue Culture of Aedes Aegypti Embryos.” American Journal of Tropical Medicine and Hygiene 17, no. 2 (1968): 219-, ([1])). It should be noted that the Aag2 cells used for EV purification are not infected with a virus, especially not with a flavivirus. The use of uninfected cells makes it possible to avoid introducing viral material during immunization. Indeed, the presence of viral proteins would have triggered immunization against these viral proteins and the effect of EVs could not have been evaluated as such.

[0055] Material

[0056] • Aag2 cells

[0057] • Roswell Park Memorial Institute (RPMI) medium 1640, GlutaMAX™ supplement

[0058] • Penicillin-Streptomycin (PS) (10,000 U / mL)

[0059] • Non-Essential Amino Acids (NEAA) Solution (100X)

[0060] • Fetal calf serum (FCS)

[0061] • Dulbecco's phosphate buffered saline (DPBS), devoid of calcium and magnesium (1X)

[0062] • RIPA buffer (1X)

[0063] Method

[0064] • Incubate Aag2 cells at 28°C / 5% CO2 in culture medium (e.g., RPMI + 1% PS + 1X NEAA) supplemented with 10% decomplemented FCS.

[0065] • When the cells reach 80-90% confluence, remove the culture medium and replace it with fresh medium without FBS.

[0066] • Incubate at 28°C / 5% CO2 for 48 hours.

[0067] • Collect the supernatant without detaching the cell layer.

[0068] • Centrifuge the supernatant at 300g for 10 minutes at 4°C.

[0069] • Collect the supernatant.

[0070] • Centrifuge the supernatant at 2000g for 10 minutes at 4°C.

[0071] • Collect the supernatant. Centrifuge the supernatant at 10,000g for 30 minutes at 4°C.

[0072] Collect the supernatant.

[0073] Centrifuge the supernatant at 100,000g for 155 minutes at 4°C.

[0074] Discard the supernatant.

[0075] Wash the pellet containing EVs with ice-cold 1X DPBS.

[0076] Centrifuge the supernatant at 100,000g for 155 minutes at 4°C.

[0077] Discard the supernatant.

[0078] Resuspend the pellet in ice-cold 1X DPBS.

[0079] Take a fraction of the resuspended pellet to quantify protein content: o Mix 1:1 in RIPA buffer. o Quantify protein content using Qubit.

[0080] • Store the VE solution at -80°C.

[0081] Advantageously, the use of this cell line makes it possible to obtain quantities of EVs suitable for the immunization of animals.

[0082] Example 2: Amplification of infection and transmission by lipids contained in mosquito EVs

[0083] Material

[0084] • EVs isolated from Aag2 cells

[0085] • HuH7 cells (clone JTC-39)

[0086] • Dulbecco's modified Eagle medium (DMEM, Gibco), supplemented with 10% fetal bovine serum

[0087] • Dengue virus type 2 (DENV2) NGC strain, West virus

[0088] Nile (WNV) strain IS98, and Zika virus (ZIKV) strain PF-251013-18

[0089] • C57BI6 / J mice, males, 4-5 weeks old (Charles River Laboratories)

[0090] • Methanol:Chlorform:Nuclease-Free Water (2:1:1 volume) Methanokchloroform (1:1 volume)

[0091] DMSO (dimethyl sulfoxide)

[0092] Lipid extraction method

[0093] • Aag2 EVs are mixed with nuclease-free Methanol:Chlorform:Water

[0094] • Vortex and centrifuge the mixture

[0095] • Collect the single-phase liquid phase

[0096] • Dry the liquid under nitrogen

[0097] • Resuspend the pellet in DMSO

[0098] • Add the methanol and chloroform solution

[0099] • Mix using a vortex and centrifuge the mixture

[0100] • Dry the liquid under nitrogen

[0101] • Resuspend the pellet in DMSO

[0102] Method for infecting cells with lipids contained in EVs

[0103] • Plate the Huh7 cells

[0104] • Infect with DENV, ZIKV or WNV at a multiplicity of infection (MOI = 0.1)

[0105] • Supplement the inoculum with lipid extracts contained in EVs

[0106] • Control cells receive the same volume of DMSO

[0107] • Incubate cells for 72 hours after infection

[0108] • Collect cells and quantify viral genomes (gRNA) by RT-qPCR

[0109] Method of infecting mice with lipids contained in EVs

[0110] Upon receipt of the mice, they are placed in cages in groups of 4 to 5 with food and water available at will. • Wait a week for the mice to acclimatize to their new environment.

[0111] • Anesthetize the mice

[0112] • Inject intradermally into each mouse 10 μL of a solution containing 1000 particles forming units of West Nile virus, extracts of lipids contained in EVs and PBS.

[0113] • Control mice were injected with 10 μl of West Nile virus, an equal volume of DMSO and PBS.

[0114] • A second group of control mice received an equal volume of DMSO and PBS, without West Nile virus.

[0115] • The mice are returned to their cages.

[0116] • Clinical signs and survival are monitored daily for up to 12 days after injection.

[0117] In the context of the invention, it has been shown that lipids contained in mosquito EVs amplify infection in human cells and viral transmission in a mouse model (Fig. 1A, B and C). In particular, it has been shown that EV lipids increase infection by several flaviviruses (dengue virus (DENV), Zika virus (ZIKV) and West Nile virus (WNV)) (Fig. 1 A), that EV sphyngomyelins increase the translation of viral proteins and that, as a result, viral transmission is increased.

[0118] Furthermore, it has been shown in the context of the invention that supplementation with EV lipids increases infection by several flaviviruses, including dengue virus, Zika virus and West Nile virus, on several cellular models relevant for transmission, such as primary skin fibroblast cells and immune cells primarily infected by viruses during the bite. In addition, the effect of EV lipids was validated with a mouse model by showing that injection of lipids with virus increases infection of the animal (Fig 1 B and C). In conclusion, our results identified an amplification factor for pan-flaviviral transmission.

[0119] Example 3: Immunization of wild-type mice against extracellular vesicles

[0120] Purified EVs as described in Example 1 are used to immunize wild-type mice (without genetic modification) to study the immune response under conditions where immunity is complete.

[0121] 100g of purified and intact EV (diluted in PBS) in combination with Alum adjuvant were injected into wild-type mice twice at two-week intervals intramuscularly (Fig. 2A).

[0122] Control mice received no injection.

[0123] To assess the effect of EV immunization on biting transmission, Aedes aegypti mosquitoes were injected by intrathoracic inoculation with West Nile virus, in order to homogenize the inoculum received by each mosquito and therefore their level of infection.

[0124] EV-immunized mice and control mice were bitten by 3 infected mosquitoes, for which infection was validated. Symptoms and viremia were quantified in mice for two weeks after the last immunization. Previously, it was established that, under the same conditions without immunization, symptoms appear before 7 days post-bite.

[0125] The results show that immunized mice have fewer symptoms of infection (assessed by symptom severity; Fig. 2A) and better survival (Fig. 2B) compared to control mice.

[0126] These results show that immunization of mice attenuated viral transmission and pathology associated with systemic infection. Thus, vaccination against mosquito EVs protects against bite-borne infection by mosquito-borne flaviviruses.

[0127] Material

[0128] • EVs isolated from Aag2 cells as described in Example 1

[0129] • Dulbecco's phosphate buffered saline (DPBS), devoid of calcium and magnesium (1X)

[0130] • Alhydrogel® adjuvant 2% (InvivoGen)

[0131] • C57BI6 / J mouse, male, 4-5 weeks old (Charles River Laboratories)

[0132] Method

[0133] • Upon arrival, the mice are placed in cages in groups of 4 to 5 and provided with enrichment, food and water at will.

[0134] • One-week acclimatization of the mice to their new environment.

[0135] • Preparation of the immunization solution for the required number of mice. For 1 mouse: 50pL of Alhydrogel 2% adjuvant + 50pL VE (10pg of protein equivalent) diluted in PBS.

[0136] • Inject 100 µl of immunization solution into each mouse intramuscularly.

[0137] • Replacing the mice in their cages.

[0138] • After 2 weeks, inject the mice with the same immunization solution (booster).

[0139] • Replacing the mice in their cages.

[0140] • Wait 2 weeks before the viral challenge by mosquito bite.

[0141] • Control mice do not receive any injection. Example 4: Characterization of mosquito EV size.

[0142] Mosquito EVs were isolated as previously reported and analyzed by cryo-electron microscopy and NTA (Nanoparticle Tracking Assay).

[0143] Cryomicroscopy identified spherical vesicular structures with a lipid bilayer. These structures allowed the identification of EVs and thus demonstrated the existence of EVs produced by mosquito cells (Fig. 3A).

[0144] NTA allowed defining the size of the EV population (Fig. 3B). This size varies from 20 to 400 nm.

[0145] Material

[0146] • EVs isolated from Aag2 cells as described in Example 1

[0147] • Dulbecco's phosphate buffered saline (DPBS), devoid of calcium and magnesium (1X)

[0148] • NanoSight NS300 (Malvern)

[0149] • JEOL 2200FS (JEOL, EUROPE) electron microscope.

[0150] Method

[0151] • 3pl of VE solution are deposited on polarized Lacey carbon-supported copper grids.

[0152] • The grids are left to dry at 95% humidity for 3 seconds on blotting paper, frozen in ethanol and stored in liquid nitrogen

[0153] • The grid was observed under a transmission electron microscope.

[0154] • Isolated EVs are diluted in PBS to a concentration of 10 8 particles per ml before analysis with NTA.

[0155] • The size class distribution was determined. Example 5: Identification of sphingomyelins as the lipids responsible for increasing / facilitating infection by mosquito EVs.

[0156] To identify the lipid class responsible for facilitating mosquito EV infection, we described the lipid composition of mosquito EVs using targeted lipidomics. Among others, we identified lipids of the sphingomyelin class (Fig. 4A).

[0157] To determine the function of sphingomyelin in enhancing mosquito EV infection, we observed the effect of sphingomyelin-depleted EV lipids on West Nile virus infection. Briefly, EVs were isolated as described in Example 1 and used to isolate lipids as in Example 2. The extracted lipids were treated with a sphingomyelinase that degrades sphingomyelin. The sphingomyelinase-treated lipids were then heated to 60°C to degrade the enzyme and inhibit its effect on infection. The resulting lipids containing all EV lipids except sphingomyelin were used to supplement human cells during West Nile virus infection. Control cells were supplemented with the same volume of DMSO (the lipid-containing diluent, CTRL) and EV-extracted lipids (EV-lipids).

[0158] We then assessed infection by quantifying gRNA by RT-qPCR at 72h post-infection. We observed that supplementation with EV lipids increased infection, as shown in Example 2. In contrast, removal of sphingomyelins abolished the increase in infection (Fig. 4B). These results demonstrate that sphingomyelins in EVs are responsible for the increase in infection. Thus, without wishing to be bound by a particular mechanism of action, the immunization object of the present invention could neutralize the function of sphingomyelins in the increase / facilitation of infection. REFERENCES Peleg, J. “Growth of Arboviruses in Primary Tissue Culture of Aedes Aegypti Embryos.” American Journal of Tropical Medicine and Hygiene 17, no. 2 (1968): 219-. Pompon, Julien, Menchie Manuel, Geok Kee Ng, Benjamin Wong, Chao Shan, Gayathri Manokaran, Ruben Soto-Acosta, et al.“Dengue Subgenomic Flaviviral RNA Disrupts Immunity in Mosquito Salivary Glands to Increase Virus Transmission.” PLoS Pathogens 13, no. 7 (2017): e1006535. https: / / doi.org / 10.1371 / journal.ppat.1006535. Zhang, Rudian, Yibin Zhu, Xiaojing Pang, Xiaoping Xiao, Renli Zhang, and Gong Cheng. “Regulation of Antimicrobial Peptides in Aedes Aegypti Aag2 Cells.” Frontiers in Cellular and Infection Microbiology 7 (2017). https: / / www.frontiersin.org / articles / 10.3389 / fcimb.2017.00022.

Claims

CLAIMS 1. Isolated mosquito extracellular vesicle for use as an immunogenic agent in the prevention of at least one vector-borne disease.

2. Extracellular vesicle for use according to claim 1, in a human or non-human vertebrate.

3. Extracellular vesicle for use according to claim 1 or 2, the mosquito being chosen from the genus Aedes, for example Aedes aegypti, Aedes albopictus, or Aedes Japonicus, from the genus Culex, for example Culex pipiens, Culex quinquefasciatus, Culex tarsalis, Culex molestus or Culex tritaeniorhynchus, or from the genus Anopheles, for example Anopheles gambiae, Anopheles coluzii, or Anopheles arabiensis.

4. Extracellular vesicle for use according to any one of the preceding claims, said vesicle being isolated from at least one biological fluid secreted by said mosquito, or from at least one mosquito cell extract.

5. Extracellular vesicle for use according to claim 4, said biological liquid being chosen from saliva, hemolymph or excreta, and said cellular extract being chosen from a cell culture, a salivary gland extract, an egg homogenate, an intestinal homogenate, a larval homogenate, or a homogenate of the mosquito body, or an ex vivo culture of mosquito tissues or organs.

6. Extracellular vesicle for use according to any one of the preceding claims, the virus being transmitted by a vector, said virus being at least one virus chosen from flaviviruses.

7. Extracellular vesicle for use according to any one of the preceding claims, said disease being at least one disease selected from dengue fever, Zika, yellow fever, Japanese encephalitis, West Nile fever, Saint Louis encephalitis, disease caused by the Tembusu virus and disease caused by the Usutu virus.

8. Vaccine composition comprising at least one mosquito extracellular vesicle as defined in any one of claims 1 to 7.

9. Vaccine composition according to claim 8, further comprising at least one substance selected from vehicles, excipients, adjuvants, buffers, preservatives, immune regulators and stabilizers.

10. Vaccine composition according to claim 8 or 9, said composition being in injectable form.

11. A method for preparing mosquito extracellular vesicle, comprising the following steps: 1) culture of mosquito cells, uninfected or infected by a virus, 2) separation of the culture supernatant, 3) purification of extracellular vesicles from the culture supernatant, for example by ultracentrifugation.

12. Method according to claim 11, wherein said cells are selected from the cell lines Aedes aegypti Aag2, RML-12 and CCL125, Aedes albopictus C6 / 36, C7-10 and U4.4, Culex quinquefasciatus Hsu, Culex tritaeniorhynchus TRA-171 and Anopheles gambiae Mos.55, Sua1 B or 4a-3B.

13. Use of a mosquito extracellular vesicle to identify at least one immunogenic peptide capable of reducing, in a vertebrate, the transmission of a vector-borne disease during a bite of the vertebrate by the vector.

14. Method for identifying at least one immunogenic peptide capable of blocking, in a vertebrate, the transmission of a vector-borne disease during a bite of the vertebrate by the vector, comprising a step of in vitro identification of at least one antibody target present in a sample of serum from an animal immunized against mosquito extracellular vesicles.

15. Use according to claim 13 or method according to claim 14, wherein said at least one immunogenic peptide corresponds to all or part of the external part, or to all or part of the internal part, or to all or part of the transmembrane part, of a transmembrane protein of said at least one mosquito extracellular vesicle.

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