Application of mosquito saliva protein specific IgE as pathogenic target of severe dengue fever

Abstract

The application discloses application of mosquito saliva protein specific IgE as a pathogenic target point of severe dengue fever, and provides any one of the following (i)-(iii) applications: (i) application of AaISP specific IgE as a target point in preparation of a medicine for preventing and / or treating diseases caused by insect-borne virus infection; (ii) application of a reagent targeting AaISP specific IgE in preparation of a medicine for preventing and / or treating diseases caused by insect-borne virus infection; (iii) application of a reagent for inhibiting AaISP specific IgE sensitized effector cells in preparation of a medicine for preventing and / or treating diseases caused by insect-borne virus infection. The application further provides that the IgE antibody is administered to a host infected with dengue virus, so that the disease caused by the dengue virus can be relieved, and the severity of the dengue virus infection of the host can be reduced.

Description

Technical Field

[0001] This invention relates to the application of a mosquito salivary protein-specific IgE as a pathogenic target for severe dengue fever, belonging to the field of biomedicine. Background Technology

[0002] Dengue fever is a human disease caused by different serotypes of dengue virus (DENV) (DENV1 to DENV4), which are mainly transmitted by Aedes aegypti and Aedes albopictus mosquitoes. 1,2 The clinical manifestations of dengue fever are highly heterogeneous, ranging from asymptomatic infection and fever to severe hemorrhagic fever and even death. 1,3 It is noteworthy that dengue fever outbreaks have shown a rapid upward trend over the past decade. Currently, dengue fever is endemic or outbreak-prevalent in more than 100 countries and regions, putting approximately 2.5 billion people at risk of infection annually, with tens of millions experiencing clinical symptoms of varying severity. 3,4 Therefore, dengue fever, especially severe dengue fever (such as dengue hemorrhagic fever and dengue shock syndrome), has become a significant public health problem causing disease and economic burden in tropical regions worldwide, including Southeast Asia, the Indian subcontinent, and South America. 3,4 Under natural conditions, Aedes mosquitoes are the primary vector for DENV transmission; their blood-sucking habits enable the virus to spread efficiently to humans. 5 During the blood-feeding process, Aedes mosquitoes carrying DENV release large amounts of salivary effector molecules into the skin while searching for blood vessels, and simultaneously inject a large number of infectious DENV particles. 6 However, before being bitten by DENV-carrying mosquitoes, humans are usually already frequently and for a long period of time exposed to the bites of uninfected mosquitoes in the natural environment. 7,8 This is especially true for populations living in tropical regions. Such prolonged and repeated exposure can lead to persistent immunosensitization to mosquito saliva antigens. However, the role of this mosquito saliva-mediated immunosensitization in the transmission of DENV and the pathogenesis of dengue fever remains unclear.

[0003] Severe dengue fever is a life-threatening complication following dengue virus infection. However, due to insufficient understanding of its pathogenesis, effective prevention and treatment methods are currently lacking. It is generally believed that secondary infections caused by different serotypes of DENV can induce cross-reactive antibodies and T cells, thus making it more likely to cause severe dengue fever than the primary infection. 9-11 However, multiple clinical studies have found that the incidence of severe dengue fever is actually similar in patients with primary infection and those with secondary infection, suggesting that disease severity is not necessarily related to secondary infection. 12,13It is worth noting that initial DENV infection can also lead to severe illness and even death with a relatively high frequency. 14,15 This suggests that there may be other, yet unclear, pathogenic mechanisms in the process of primary DENV infection leading to severe dengue fever.

[0004] Immunoglobulin E (IgE) plays a dual role in human humoral immunity; it can participate in the body's defense against pathogens and also mediate pathological hypersensitivity reactions. 16,17 Antigen-specific IgE plays a central role in immediate inflammatory responses. In this process, effector cells sensitized by IgE, such as mast cells and basophils, are activated upon antigen cross-linking stimulation. 18,19 In this invention, it was discovered that naturally acquired IgE-mediated immunosensitization induced by mosquito bites can exacerbate the severity of DENV infection transmitted by Aedes mosquitoes and promote the progression of primary dengue virus infection to severe dengue fever.

[0005] References

[0006] 1.Diamond, MS, and Pierson, TC (2015). Molecular Insight into Dengue Virus Pathogenesis and Its Implications for Disease Control. Cell 162,488-492. 10.1016 / j.cell.2015.07.005.

[0007] 2. Simmons, CP, Farrar, JJ, Nguyen v, V., and Wills, B. (2012). Dengue. N Engl J Med 366, 1423-1432. 10.1056 / NEJMra1110265.

[0008] 3.Bhatt, S., Gething, P.W., Brady, O.J., Messina, J.P., Farlow, A.W.,Moyes, C.L., Drake, J.M., Brownstein, J.S., Hoen, A.G., Sankoh, O., et al.(2013). The global distribution and burden of dengue. Nature 496, 504-507.10.1038 / nature12060.

[0009] 4.Paz-Bailey, G., Adams, L.E., Deen, J., Anderson, K.B., andKatzelnick, L.C. (2024). Dengue. Lancet 403, 667-682. 10.1016 / s0140-6736(23)02576-x.

[0010] 5.Brady, O.J., and Hay, S.I. (2020). The Global Expansion of Dengue:How Aedes aegypti Mosquitoes Enabled the First Pandemic Arbovirus. Annu RevEntomol 65, 191-208. 10.1146 / annurev-ento-011019-024918.

[0011] 6.Wang, Z.Y., Nie, K.X., Niu, J.C., and Cheng, G. (2024). Researchprogress toward the influence of mosquito salivary proteins on thetransmission of mosquito-borne viruses. Insect Sci 31, 663-673. 10.1111 / 1744-7917.13193.

[0012] 7.Smith, D.L., Battle, K.E., Hay, S.I., Barker, C.M., Scott, T.W.,and McKenzie, F.E. (2012). Ross, macdonald, and a theory for the dynamics andcontrol of mosquito-transmitted pathogens. PLoS Pathog 8, e1002588. 10.1371 / journal.ppat.1002588.

[0013] 8.Scott, T.W., and Takken, W. (2012). Feeding strategies ofanthropophilic mosquitoes result in increased risk of pathogen transmission.Trends Parasitol 28, 114-121. 10.1016 / j.pt.2012.01.001.

[0014] 9.Halstead, S.B., and O'Rourke, E.J. (1977). Antibody-enhanced denguevirus infection in primate leukocytes. Nature 265, 739-741. 10.1038 / 265739a0.

[0015] 10.Morier, L., Kouri, G., Guzman, G., and Soler, M. (1987). Antibody-dependent enhancement of dengue 2 virus in people of white descent in Cuba.Lancet 1, 1028-1029. 10.1016 / s0140-6736(87)92289-6.

[0016] 11.Bournazos, S., Gupta, A., and Ravetch, JV (2020). The role ofIgG Fc receptors in antibody-dependent enhancement. Nat Rev Immunol 20, 633–643. 10.1038 / s41577-020-00410-0

[0017] 12.Aggarwal, C., Ahmed, H., Sharma, P., Reddy, ES, Nayak, K.,Singla, M., Maheshwari, D., Chawla, YM, Panda, H., Rai, RC, et al.(2024). Severe disease during both primary and secondary dengue virus infections in pediatric populations. Night Med 30, 670–674. 10.1038 / s41591-024-02798-x.

[0018] 13.Cardenas-Perea, ME, Flores-Mendoza, LK, Perez-Contreras, I., Diaz-Orea, MA, Gomez-Conde, E., Cortes-Hernandez, P., Reyes-Leyva, J.,Santos-Lopez, G., and Sosa-Jurado, F. (2020). Primary Dengue Infection inPatients Requiring Hospitalization During an Outbreak in a Low IncidenceMexican Region. Vector Borne Zoonotic Dis 20, 380–386. doi: 10.1089 / vbz.

[0019] 14.Sondo, AK, Diendere, EA, Meda, BI, Diallo, I., Zoungrana, J., Poda, A., Manga, NM, Bicaba, B., Gnamou, A., Kagone, CJ, et al.(2021). Severe dengue in adults and children, Ouagadougou (Burkina Faso),West Africa, October 2015-January 2017. IJID Reg 1, 53-59. 10.1016 / j.ijregi.2021.09.010.

[0020] 15.Ngwe Tun, MM, Muthugala, R, Nabeshima, T, Rajamanthri, L,Jayawardana, D, Attanayake, S, Soe, AM, Dumre, SP, Ando, ​​T, Hayasaka, D, et al. (2020). Unusual, neurological and severe dengue manifestations during the outbreak in Sri Lanka.

[0021] 16.Mukai, K., Tsai, M., Starkl, P., Marichal, T., and Galli, SJ(2016). IgE and mast cells in host defense against parasites and venoms.Semin Immunopathol 38, 581-603. 10.1007 / s00281-016-0565-1

[0022] 17.Gould, HJ, and Sutton, BJ (2008). IgE in allergy and asthma. Nat Rev Immunol 8, 205–217. 10.1038 / nri2

[0023] 18. Galli, SJ, and Tsai, M. (2012). IgE and mast cells in allergicdisease. Nat Med 18, 693-704. 10.1038 / nm.2755.

[0024] 19. Shamji, MH, Valenta, R., Jardetzky, T., Verhasselt, V., Durham, SR, Würtzen, PA, and van Neerven, RJJ (2021). The role of allergen-specific IgE, IgG and IgA in allergic disease. Allergy 76, 3627-3641.10.1111 / all.14908. Summary of the Invention

[0025] The problem the invention aims to solve

[0026] There is an urgent need in this field to provide a method that can alleviate arbovirus infection, reduce arbovirus-induced damage, and inhibit arbovirus replication within the host. Under natural conditions, proteins in mosquito saliva enhance viral infection in the host during mosquito bites. Currently, there is a lack of research on products and methods to inhibit such arbovirus infections.

[0027] Solution for solving the problem

[0028] [1]. Any of the following uses (i) to (iii):

[0029] (i) The use of AaISP-specific IgE as a target in the preparation of drugs for the prevention and / or treatment of diseases caused by arbovirus infection;

[0030] (ii) The use of agents targeting AaISP-specific IgE in the preparation of medicaments for the prevention and / or treatment of diseases caused by arbovirus infection;

[0031] (iii) The use of reagents that inhibit AaISP-specific IgE-sensitized effector cells in the preparation of medicaments for the prevention and / or treatment of diseases caused by arbovirus infection.

[0032] [2]. According to the use described in [1], wherein the disease caused by the vector virus infection is a disease in the host of the vector virus, the host of the vector virus being exposed to the bite of a transmission vector that does not carry the vector virus before being infected with the vector virus, or the host of the vector virus being sensitized by the salivary protein of the transmission vector before being infected with the vector virus.

[0033] [3]. According to the use described in [2], wherein the transmission vector that does not carry arbovirus contains salivary protein AaISP, or the salivary protein of the transmission vector contains AaISP.

[0034] [4]. According to the use described in [2] or [3], wherein the AaISP-specific IgE sensitized effector cells are exposed to the bite of an arbovirus that does not carry the arbovirus, or to the host of an arbovirus that is sensitized by the salivary protein of the arbovirus.

[0035] Preferably, the AaISP-specific IgE sensitizing effector cells include activated skin mast cells and / or myeloid cells that aggregate at the bite or sensitization site.

[0036] [5]. According to any one of [1]-[4], wherein the arbovirus includes one or more of Bunyaviridae, Flaviviridae, Reoviridae and Togaviridae;

[0037] Preferably, the arbovirus includes one or more of the genera Flavivirus, Alphavirus, and Orthobunyavirus.

[0038] More preferably, the arbovirus includes Flaviviridae;

[0039] More preferably, the vector-borne virus includes dengue virus.

[0040] [6]. The use according to any one of [1]-[5], wherein the disease caused by the vector-borne virus infection includes dengue fever;

[0041] Optionally, the dengue fever includes severe dengue fever.

[0042] [7]. The use according to any one of [1]-[6], wherein the drug reduces the severity of arbovirus infection, reduces viremia, reduces vascular leakage, and / or improves survival.

[0043] [8]. The use according to any one of [1]-[7], wherein the vector of the arbovirus includes one or more of mosquitoes, ticks or sandflies;

[0044] Preferably, the vector for the vector-borne virus is a mosquito;

[0045] More preferably, the mosquitoes include one or more of Culex, Culiseta, Anopheles, and Aedes;

[0046] Optionally, the mosquito is an Aedes mosquito, including one or more of Aedes aegypti, Aedes albopictus, Aedes polynesiensis, Aedes australis, Aedes cinereus, Aedes rusticus, and Aedes vexans.

[0047] [9]. The use according to any one of [1]-[8], wherein the host of the arbovirus is a mammal;

[0048] Preferably, the host of the arbovirus is a primate and / or a rodent.

[0049]

[10] . A pharmaceutical composition for the prevention and / or treatment of diseases caused by arbovirus infection, comprising the agents described in [1] that target AaISP-specific IgE and / or agents that inhibit the binding of AaISP-specific IgE to effector cells, and,

[0050] Optional, pharmaceutically acceptable carrier.

[0051] The effects of the invention

[0052] This invention provides an IgE antibody administered to a dengue virus-infected host, which can alleviate dengue virus-induced disease, reduce damage caused by dengue virus infection, inhibit dengue virus proliferation in the host, and reduce the severity of dengue virus infection in the host. The IgE antibody can inhibit specific IgE produced by the AaISP protein, further inhibiting the mast cell pathway-mediated viral infection enhancement effect. The antibody described in this invention can be formulated into a prophylactic product and is also suitable for early treatment after mosquito exposure prior to infection. Attached Figure Description

[0053] Figure 1 This is dengue virus infection that is spread naturally through prolonged exposure to mosquito bites.

[0054] Figure 1 A in the diagram is a schematic diagram of a long-term mosquito exposure experiment; Figure 1 B in the text represents AG6 after DENV2 infection. Control Group and AG6 Pre-AaWT DENV2 load in peripheral blood of mice in the group; Figure 1 C in the figure represents day 3 post-DENV2 infection, AG6 Control Group and AG6 Pre-AaWT DENV2 load in the footpads, draining lymph nodes, and spleen of mice in the group; Figure 1 In this context, D represents AG6 after DENV2 infection. Control Group and AG6 Pre-AaWT Survival curves of the mice group.

[0055] Figure 2 AaISP-mediated immune sensitization promotes the natural transmission of dengue virus infection and pathogenicity.

[0056] Figure 2 A in the figure represents the purity verification of recombinant mosquito salivary protein, which was verified by SDS-PAGE and Coomassie brilliant blue staining. Figure 2 B in the figure represents the immunoblotting method used to verify recombinant mosquito salivary proteins; Figure 2 C–D in the text refers to viremia in AG6 mice after they were immunized with different recombinant mosquito salivary proteins and then bitten by DENV2-infected mosquitoes. Figure 2 E in AG6 Control Group and AG6 AAEL000749-imm DENV2 load in peripheral blood of mice in the group; Figure 2 F in the text represents AG6 after DENV2 infection. Control Group and AG6 AAEL000749-imm DENV2 load in the footpads, draining lymph nodes, and spleen of mice in the group; Figure 2 In this context, G represents AG6 after DENV2 infection. Control Group and AG6 AAEL000749-imm Survival curves of the mice group.

[0057] Figure 3 AaISP cannot directly promote dengue virus infection.

[0058] Figure 3 In this context, A represents the DENV2 load in the supernatant 24 h after infection of primary macrophages. AaISP (0, 0.5, 1, 2, 4 μg / mL) or salivary gland extract (positive control SGE, 1, 2 μg / mL) were used to infect mouse macrophages for 1 h at an MOI of 1. Figure 3In this figure, B represents the DENV2 load in the peripheral blood of mice. Four-week-old female AG6 mice were injected intradermally with AaISP (100 ng or 1 μg) or AaNRP (positive control, 100 ng) + DENV2 (10 μg) via the footpad. 4 PFU).

[0059] Figure 4 AaISP is a key salivary protein that mediates repeated bites to promote dengue virus infection.

[0060] Figure 4 In the figure, A represents the expression of AaISP in the salivary glands of wild-type (AaWT) and AaISP knockout (AaISP-KO) mosquitoes. Scale bar: 50 μm; Figure 4 B in the diagram is a schematic diagram of the AaISP verification experiment; Figure 4 C in the text represents AG6 after DENV2 infection. Control / AaWT Group, AG6 Pre-AaWT / AaWT Group, AG6 Pre-AaISP-KO / AaWT Group and AG6 Pre-AaWT / AaISP-KO DENV2 load in peripheral blood of mice in the group; Figure 4 D in the figure represents day 3 post-DENV2 infection, AG6 Control / AaWT Group, AG6 Pre-AaWT / AaWT Group, AG6 Pre -AaISP-KO / AaWT Group and AG6 Pre-AaWT / AaISP-KO DENV2 load in the footpads, draining lymph nodes, and spleen of mice in the group; Figure 4 In this context, E represents AG6 after DENV2 infection. Control / AaWT Group, AG6 Pre-AaWT / AaWT Group, AG6 Pre-AaISP-KO / AaWT Group and AG6 Pre-AaWT / AaISP-KO Survival curves of mice in the group.

[0061] Figure 5 AaISP-specific T cells do not affect DENV2 infection.

[0062] Figure 5 A in the diagram represents a T cell adoptive transfer experiment. Figure 5 B in the text refers to the adopted child B6. Control T cells, B6 AaISP-imm DENV2 load in peripheral blood of AG6 mice infected with T cells or PBS (blank control); Figure 5 C in the text represents adoption B6 Control T cells, B6 AaISP-imm Survival curves of DENV2-infected AG6 mice with T cells or PBS (blank control).

[0063] Figure 6AaISP-specific IgE enhances dengue virus infection and disease severity.

[0064] Figure 6 In the diagram, A represents a schematic representation of a serum transfer experiment. Figure 6 The B in the text refers to intraperitoneal injection of B6. Control Serum, B6 AaISP-imm Serum or B6 AaISP-imm Serum ΔIgG / IgM / IgE / IgA DENV2 load in the peripheral blood of DENV2-infected AG6 mice; Figure 6 C in the text refers to intraperitoneal injection of B6. Control Serum, B6 AaISP-imm Serum or B6 AaISP-imm Serum ΔIgG / IgM / IgE / IgA Survival curves of DENV2-infected AG6 mice; Figure 6 D in the diagram represents a schematic representation of a serum IgE transfer assay. Figure 6 In this context, E represents the DENV2 load in the peripheral blood of AG6 mice infected with DENV2 after receiving IgE transfer from different sources. Figure 6 F in the figure represents the survival curves of DENV2-infected AG6 mice after receiving IgE transfer from different sources.

[0065] Figure 7 Repeated bites sensitize skin mast cells with AaISP-specific IgE.

[0066] Figure 7 In the diagram, A represents a skin RNA sequencing experiment. Figure 7 B in the figure represents the cluster analysis of genes related to mast cells in the skin from mosquito bites; Figure 7 C in the diagram is a flow cytometry experiment used to detect skin mast cells that bind to AaISP; Figure 7 In this context, D represents the proportion of skin mast cells that bind AaISP to all living skin cells.

[0067] Figure 8 AaISP-specific IgE mediates skin mast cell activation to recruit virus-susceptible myeloid cells.

[0068] Figure 8 A in the figure represents toluidine blue staining, which showed degranulation of skin mast cells 30 minutes after the bite. Figure 8 In the figure, B represents the degranulation of skin mast cells 30 minutes after the bite, as shown by immunofluorescence detection. Figure 8 C in the figure represents toluidine blue staining, which shows degranulation of skin mast cells 30 minutes after AaISP injection. Figure 8 D in the figure represents the change in the proportion of activated mast cells in the skin of the mouse paw pads over time after mosquito bites. Figure 8E in the figure represents the change over time in the proportion of neutrophils in the infiltrative myeloid cells of the mouse paw pad skin after mosquito bites. Figure 8 F in the figure represents the change over time in the proportion of monocytes among infiltrative myeloid cells in the skin of mouse paw pads after mosquito bites. Figure 8 In G, the proportion of dendritic cells in infiltrative myeloid cells in mouse paw pad skin changes over time after mosquito bites. Figure 8 In the figure, H represents the change over time in the proportion of macrophages among infiltrative myeloid cells in the skin of mouse paw pads after a mosquito bite.

[0069] Figure 9 Serum AaISP-specific IgE levels were positively correlated with the severity of dengue virus infection.

[0070] Figure 9 In this context, A represents the total IgE content in the serum of patients with mild dengue (DF, n=69) and severe dengue (SD, n=17). Figure 9 B in the table represents the AaISP-specific IgE level in the serum of patients with mild primary dengue (DF, n=69) and severe dengue (SD, n=17). Figure 9 C in the figure represents the average mosquito population density in Xishuangbanna region during April, June, and August from 2023 to 2025. Figure 9 In this context, D represents the total IgE content in serum samples from patients in the recovery period of primary DF (n=30) and SD (n=30) at 4, 6, and 8 months. Figure 9 E in the figure represents the AaISP-specific IgE level in serum samples from patients in the recovery phase of primary DF (n=30) and SD (n=30) at 4, 6 and 8 months.

[0071] Figure 10 Serum AaISP-specific IgE levels were positively correlated with the severity of dengue virus infection.

[0072] Figure 10 A in the diagram is a schematic diagram of a human IgE adoptive transfer experiment; Figure 10 In this context, B represents the serum IgE from different dengue patients transferred to the peripheral blood of FCER1A-KI AG6 mice after infection, along with the DENV2 load. Figure 10 In this context, C represents the viral load in the footpad skin, draining lymph nodes, and spleen of FCER1A-KI AG6 mice 3 days after the mice were infected with serum IgE from different dengue patients. Figure 10 D and Figure 10 E in the figure represents serum IgE from different dengue patients adopted into plasma exudates from different tissues and organs of FCEER1A-KI AG6 mice 10 days after infection; Figure 10F in the figure represents the survival curve of FCER1A-KI AG6 mice infected with DENV2 after adopting serum IgE from different dengue patients.

[0073] Figure 11 Anti-IgE antibodies can inhibit the production of AaISP-specific IgE to reduce the severity of dengue infection.

[0074] Figure 11 In this context, A represents the AaISP-specific IgE level in the serum of B6 and 129 mice after treatment with anti-IgE antibody. Figure 11 In this context, B represents the DENV2 load in the peripheral blood of DENV2-infected B6 and 129 mice after treatment with anti-IgE antibody. Figure 11 In the figure, C represents the vascular permeability of different tissues in DENV2-infected B6 and 129 mice on day 10 post-infection after treatment with anti-IgE antibody. Figure 11 D in the figure represents the survival curve of DENV2-infected B6 and 129 mice after treatment with anti-IgE antibody. Detailed Implementation

[0075] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0076] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0077] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0078] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0079] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., mean that a specific element (e.g., feature, structure, property, and / or characteristic) related to that embodiment is included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0080] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0081] In this specification, the term "treatment" means reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject before the onset of symptoms (e.g., based on a history of symptoms). Treatment may also continue after symptom relief, for example, to delay and / or prevent recurrence of the disease or disorder.

[0082] In this specification, the term "prevention" refers to preventive treatment for subjects who currently do not have or have not had any disease but are at risk of developing it, or who have had a disease in the past but are currently not at risk of relapse. In some implementations, subjects have a higher risk of developing a disease or a higher risk of disease relapse compared to the average healthy member of the subject population.

[0083] In this specification, "effective amount" means an amount sufficient to elicit the desired biological response. Effective amount can vary depending on factors such as the desired biological endpoint, pharmacokinetics, the condition being treated, the route of administration, and the age and health status of the subject. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is a prophylactic effective amount. In some embodiments, the effective amount is a single dose. In some embodiments, the effective amount is a combination of multiple doses.

[0084] In this specification, "therapeutic effective amount" is an amount sufficient to provide therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with a condition. A therapeutic effective amount refers to the amount of a therapeutic agent, alone or in combination with other therapies, that provides therapeutic benefit in the treatment of a condition. The term "therapeutic effective amount" may include amounts that improve overall therapy; reduce or avoid symptoms, signs, or causes of a condition; and / or enhance the therapeutic efficacy of another therapeutic agent.

[0085] In this specification, "preventive effective amount" is an amount sufficient to prevent the condition or one or more symptoms associated with the condition, or to prevent its recurrence. A preventive effective amount refers to the amount of a therapeutic agent, alone or in combination with other agents, that provides preventive benefit in preventing the condition. The term "preventive effective amount" may also include amounts that improve overall prevention or enhance the preventive efficacy of another preventive agent.

[0086] In this specification, "administration" means the method of giving a subject (e.g., a host) a dose of a compound or composition (e.g., a pharmaceutical composition). The compounds or compositions (e.g., pharmaceutical compositions) used herein may be administered via the following routes: intravenous (e.g., via intravenous infusion), subcutaneously, intramuscularly, intradermally, percutaneously, intra-arterially, intraperitoneally, intralesionally, intra-articularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, locally, intratumorally, intraperitoneally, subconjunctivally, intracysticly, intramucosally, intraperitoneally, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion directly immersing target cells, by catheter, by irrigation, as a cream, or as a lipid composition. The method of administration can vary depending on a variety of factors.

[0087] In this specification, the term "pharmaceutical acceptable" (or "pharmacologically acceptable") means a molecular entity and composition that, when appropriate, does not produce an adverse reaction, allergic reaction, or other adverse reaction when administered to animals or humans. As used herein, the term "pharmaceutical acceptable carrier" includes any and all solvents, dispersion media, coatings, antimicrobial agents, isotonic agents and absorption delay agents, buffers, excipients, binders, lubricants, gels, surfactants, etc., that can be used as a medium for pharmaceutically acceptable substances.

[0088] This invention provides for any of the following uses (i) to (iii):

[0089] (i) The use of AaISP-specific IgE as a target in the preparation of drugs for the prevention and / or treatment of diseases caused by arbovirus infection;

[0090] (ii) The use of agents targeting AaISP-specific IgE in the preparation of medicaments for the prevention and / or treatment of diseases caused by arbovirus infection;

[0091] (iii) The use of reagents that inhibit AaISP-specific IgE-sensitized effector cells in the preparation of medicaments for the prevention and / or treatment of diseases caused by arbovirus infection.

[0092] In some aspects of the present invention, reagents that target AaISP-specific IgE or inhibit AaISP-specific IgE-sensitized effector cells are provided for the prevention and / or treatment of diseases caused by arbovirus infection, the reduction of the severity of arbovirus infection and / or the inhibition of arbovirus proliferation in the host.

[0093] In some aspects of the present invention, a method for preventing and / or treating diseases caused by arbovirus infection is provided, comprising administering to a subject (host) an effective amount of a reagent targeting AaISP-specific IgE or a reagent inhibiting AaISP-specific IgE-sensitized effector cells.

[0094] (Reagents targeting AaISP-specific IgE, reagents inhibiting AaISP-specific IgE-sensitized effector cells)

[0095] In some implementations, agents targeting AaISP-specific IgE or agents inhibiting AaISP-specific IgE-sensitized effector cells may be present in the host to reduce the expression and / or production of AaISP-specific IgE or to inhibit AaISP-specific IgE-sensitized effector cells. These agents may exert their effects through various mechanisms, such as neutralizing free AaISP-specific IgE, blocking the binding of AaISP to the high-affinity receptor FcεRI on the surface of effector cells, clearing or inhibiting AaISP-specific IgE-producing B cells / plasma cells, inducing immune tolerance to AaISP, or inhibiting sensitized effector cells (e.g., mast cells, neutrophils, myeloid cells).

[0096] In some embodiments, the reagents targeting AaISP-specific IgE and the reagents inhibiting AaISP-specific IgE-sensitized effector cells are anti-IgE antibodies (targeting the IgE constant region): for example, monoclonal antibodies that can bind to the CH3 or CH4 domains of free IgE, thereby rapidly reducing the level of free IgE by blocking the binding of IgE to the high-affinity receptor FcεRI on the surface of effector cells (such as mast cells, neutrophils, and myeloid cells), and promoting the downregulation of FcεRI on the surface of sensitized effector cells, thereby inhibiting AaISP-specific IgE-induced sensitization and degranulation.

[0097] In some embodiments, the reagents targeting AaISP-specific IgE and the reagents inhibiting AaISP-specific IgE-sensitized effector cells are receptors or antagonists that block the binding of IgE to effector cells: such as soluble FcεRI or its Fc fusion protein, which can competitively bind to AaISP-specific IgE, preventing it from binding to receptors on effector cells, or anti-FcεRI antagonistic antibodies or fragments thereof, which directly occupy receptor binding sites and block the binding of IgE to effector cells.

[0098] In some implementations, the reagents targeting AaISP-specific IgE and the reagents inhibiting AaISP-specific IgE-sensitized effector cells are antibodies targeting sensitized effector cells: antibodies against sensitized effector cells (such as mast cells, myeloid cells) (such as CD123, KIT, Siglec-8, CD300a, etc.) clear or inhibit effector cells sensitized by AaISP-specific IgE through ADCC, CDC, or apoptosis induction.

[0099] In some implementations, the reagents targeting AaISP-specific IgE and the reagents inhibiting AaISP-specific IgE-sensitized effector cells are small molecule signal inhibitors: used to inhibit downstream signal transduction of IgE / FcεRI, thereby preventing the activation and degranulation of sensitized effector cells, and effectively blocking the release of IgE-mediated allergic mediators.

[0100] In some embodiments, the reagents targeting AaISP-specific IgE and the reagents inhibiting AaISP-specific IgE-sensitized effector cells are immunomodulatory molecules or vaccines: such as immunogenic peptide fragments of AaISP-specific IgE or anti-idiotype antibodies, which induce the host to produce an immune response against AaISP-specific IgE (e.g., produce anti-IgE antibodies) and reduce the titer of AaISP-specific IgE in the host.

[0101] In some preferred embodiments, the reagents for targeting AaISP-specific IgE and for inhibiting AaISP-specific IgE-sensitized effector cells described in this invention are anti-IgE antibodies.

[0102] In some preferred embodiments, the use of anti-IgE antibodies can effectively inhibit the activation of skin mast cells and block the subsequent recruitment of neutrophils and myeloid monocytes to the bite site.

[0103] In some exemplary embodiments, the present invention has found that treatment with anti-AaISP-specific IgE antibodies can significantly reduce viremia, alleviate vascular leakage, and significantly improve survival rates.

[0104] In some exemplary embodiments, the present invention has found that naturally acquired IgE-mediated immunosensitization induced in the host by the salivary protein AaISP from the bite of the vector can exacerbate the severity of vector-borne virus (e.g., dengue virus) infection and may promote the progression of primary vector-borne virus (e.g., dengue virus) infection to severe illness (e.g., severe dengue fever).

[0105] In some implementations, agents targeting AaISP-specific IgE or agents inhibiting AaISP-specific IgE-sensitized effector cells can reduce the level or activity of the AaISP gene or its expression product in the transmission vector. This reduces AaISP-specific IgE production in the host and inhibits AaISP-specific IgE-sensitized effector cells after the vector bites.

[0106] (AaISP gene)

[0107] In the above aspects of the present invention, the protein encoded by AAEL000749 was named Aedes aegypti immune-sensitizing salivary protein (AaISP) by screening Aedes aegypti salivary proteins.

[0108] In some exemplary implementations, the AAEL000749 gene is derived from the database name: VectorBase (https: / / vectorbase.org / ).

[0109] In some exemplary embodiments, the amino acid sequence of the protein encoded by AAEL000749 (SEQ ID NO: 1) is: MILQFWFVTFSVLFAARADENHSILIKLNDLDHRCFLPFSFTQMFSQQFYRHTREVTDRVSALKASIDTNLLELDQQIQQALDGIQSNESSSSTSATKSSGLTTIPIGSEPRVPALYERERYGGDWLVVMHRYDGSVKFDRTWAEYRDGFGMVGQEFWYGLERLHQLTKEKSYELMVEMEDFNGNLKYAWYDKFVVGPEEQRYALVELGTFNGTTDGDSLKPHKGSGSSTYDNDDFGCSNKYAKGGWWYYSGKCYGSSLTGIWKNELAYSSIVWVKFSDVSNTPLKLVRMMIRPKN

[0110] (Gene expression products)

[0111] In the above aspects of the present invention, gene expression products refer to molecules of various forms of genes at various stages, such as, but not limited to, molecules produced during gene amplification, replication, transcription, splicing, processing, translation, and modification, such as cDNA, mRNA, precursor proteins, mature proteins, and fragments thereof.

[0112] (Reagents targeting the AaISP gene or its expression product)

[0113] In some embodiments of the invention, the reagent targeting the AaISP gene or its expression product can recognize and bind to the AaISP gene or its expression product. In some embodiments, the reagent targeting the AaISP gene or its expression product can regulate the level or activity of the AaISP gene or its expression product. In some specific embodiments, the reagent targeting the AaISP gene or its expression product can reduce the level or activity of the AaISP gene or its expression product. In some specific embodiments, the reagent targeting the AaISP gene or its expression product can silence the AaISP gene or its expression product.

[0114] In other embodiments, the agent targeting the AaISP gene or its expression product causes a reduction or elimination of the expression and / or function of the AaISP gene (e.g., in the vector / cell). Exemplarily, the vector / cell does not contain the AaISP gene, or the biological function of the HRD1 gene expression product in the vector / cell is inhibited. In some specific embodiments, the expression or function of the AaISP gene in vector / cell treated with the agent targeting the AaISP gene or its expression product is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% compared to vector / cells not treated with the agent targeting the AaISP gene or its expression product.

[0115] In this invention, the reagent targeting the AaISP gene or its expression product can be a reagent used in at least one of gene knockout technology, gene silencing technology, inactivation mutation technology, and PROTAC technology, or it can be a small molecule inhibitor.

[0116] In some embodiments, the agent targeting the AaISP gene or its expression product is selected from, but not limited to, at least one of: nucleic acids, peptides, ribonucleoprotein complexes (RNPs), or small molecule inhibitors.

[0117] Nucleic acid

[0118] In some embodiments of the invention described above, the nucleic acid is selected from DNA, RNA, or DNA / RNA.

[0119] In some specific implementations, the nucleic acid includes at least one of antisense RNA molecules and RNA interference molecules.

[0120] polypeptide

[0121] In some embodiments of the invention described above, the polypeptide is selected from at least one of: antibodies or antigen-binding fragments thereof, proteins containing one or more zinc finger binding domains and enzyme domains (zinc finger system, or artificial zinc finger nucleases (ZFN)), and proteins containing transcription activator-like effector (TALE) nuclease domains and enzyme domains (TALEN system).

[0122] Ribonuclear protein complex (RNP)

[0123] In some embodiments of the invention described above, the RNP is selected from the CRISPR / CAS system.

[0124] In some specific implementations, the CRISPR / Cas system comprises nucleic acid molecules and enzyme proteins, wherein the nucleic acid molecules are guide RNA (gRNA) molecules, and the enzyme proteins are Cas proteins or Cas orthologs.

[0125] In some alternative embodiments, the enzyme protein is selected from Cas9, Cas12a, Cas12b, Cas13a, Cas13b, Cas13c, Cas13e, or Cas13f proteins or their orthologs.

[0126] Small molecule inhibitors

[0127] In some specific embodiments of the invention described above, the reagent targeting the AaISP gene or its expression product comprises a small molecule inhibitor that can reduce or silence the level or activity of the AaISP gene or its expression product.

[0128] In this invention, the term "small molecule" refers to a low molecular weight compound that may be artificially synthesized or obtained from a natural source and has a molecular weight of less than 2000 Daltons (Da), less than 1500 Da, less than 1000 Da, less than 900 Da, less than 800 Da, less than 700 Da, less than 600 Da, or less than 500 Da.

[0129] In some embodiments, small molecule inhibitors can be organic compounds, inorganic compounds, or combinations of organic and / or inorganic compounds. In some specific embodiments, small molecule inhibitors are chemically prepared active substances or compounds. Typically, these compounds are synthesized in a classical manner through chemical reactions between different organic and / or inorganic compounds.

[0130] In some implementations, the small molecule inhibitor can exert its activity in the administered form, or the small molecule inhibitor can be a prodrug. Therefore, "small molecule inhibitor" encompasses both the active form and the prodrug.

[0131] The term "prodrug" refers to a compound or substance that is converted into a therapeutically active agent under physiological conditions. In some embodiments, a prodrug is a compound or substance that, after administration, is metabolized in the subject's body into a pharmaceutically active form (e.g., through enzymatic activity in the subject's body).

[0132] In some preferred embodiments, the present invention uses the CRISPR-Cas9 system to knock out the AaISP gene, which is a vector for transmission.

[0133] In some exemplary embodiments, the present invention has not detected the protein in the salivary glands of AaISP gene knockout vectors.

[0134] In some exemplary embodiments, the present invention found that knocking out the AaISP gene of a vector using a CRISPR-Cas9 system significantly reduced vector-borne virus (e.g., dengue virus) infection caused by vector bites compared to not knocking out the AaISP gene.

[0135] In some exemplary embodiments, the present invention has found that knocking out the AaISP gene of a vector using the CRISPR-Cas9 system does not exacerbate subsequent infection by vector-borne viruses (e.g., dengue virus) transmitted by vectors with the AaISP gene not knocked out, compared to not knocking out the AaISP gene.

[0136] (Arbovirus)

[0137] In some implementations, the disease caused by the arbovirus infection is a disease in the host of the arbovirus, wherein the host of the arbovirus was exposed to the bite of a vector that does not carry the arbovirus before infection, or the host of the arbovirus was sensitized by the salivary proteins of the vector before infection.

[0138] In some embodiments, the non-arbovirus-carrying transmission vector contains the salivary protein AaISP, or the salivary protein of the transmission vector contains AaISP.

[0139] In some implementations, the AaISP-specific IgE-sensitized effector cells are either exposed to a host with an arbovirus that is not carried by a vector or to a host with an arbovirus that is sensitized by the salivary protein of the vector.

[0140] In some preferred embodiments, the AaISP-specific IgE sensitizing effector cells include activated skin mast cells and / or myeloid cells that aggregate toward the bite or sensitization site.

[0141] In the above aspects of the invention, a fundamental characteristic of arboviruses is that they replicate in mammalian and insect cells. Arboviruses are transmitted to mammals via mosquitoes, ticks, or sandflies.

[0142] In some implementations, arboviruses include one or more of the families Bunyaviridae, Flaviviridae, Reoviridae, and Togaviridae. In some specific implementations, arboviruses include one or more of the genera Flavivirus, Alphavirus, and Orthobunyavirus.

[0143] In some more specific implementations, examples of arboviruses include African Swine Fever virus, Tick-borne Encephalitis virus, Rift Valley Fever virus, Colorado Tick Fever virus, Equine Encephalosis virus, Chikungunya virus, Dengue virus (DV), Zika virus (ZV), and West Nile virus.

[0144] In some preferred embodiments, the arbovirus originates from the Flaviviridae family and the Flavivir genus.

[0145] In some preferred embodiments, the vector virus is dengue virus (e.g., dengue virus type 1, 2 or 3).

[0146] In some exemplary embodiments, the vector virus is dengue virus type-2 (hereinafter referred to as dengue virus or DENV2).

[0147] In some implementations, the diseases caused by arbovirus infection include dengue fever.

[0148] In some alternative implementations, the dengue fever includes severe dengue fever.

[0149] In some exemplary embodiments, the present invention has found that after a host is sensitized by the salivary protein AaISP from the bite of a vector, the host experiences a significant increase in viremia, increased viral load in tissues, and a significantly increased mortality rate after infection with vector-borne viruses (such as dengue virus).

[0150] In some exemplary embodiments, the present invention has found that AaISP neither directly infects vector-borne viruses (such as dengue virus) nor promotes viral transmission by modulating the innate antiviral immune response at the site of vector bites.

[0151] In some exemplary embodiments, the present invention has found that AaISP plays a key role in the immune sensitization phase induced by vector bites and in the subsequent transmission of vector-borne viruses (such as dengue virus) through vector bites.

[0152] In some exemplary embodiments, the present invention has found that AaISP-specific T cells do not exacerbate arbovirus (e.g., dengue virus) infection.

[0153] In some exemplary embodiments, the present invention has found that the AaISP of the transmission medium can bind to AaISP-specific IgE on the surface of sensitized effector cells (such as mast cells in the skin of sensitized animals), thereby inducing cross-linking and activating mast cells.

[0154] In some exemplary embodiments, the present invention has found that AaISP may induce skin mast cell activation in mosquito-bite-sensitized hosts.

[0155] In some exemplary embodiments, the present invention has found that AaISP-mediated IgE crosslinking plays a key role in the sensitization of effector cells (such as mast cells of sensitized animal skin) and further promotes the aggregation of a large number of sensitized effector cells (such as myeloid cells) highly susceptible to arboviruses (such as dengue virus) to the mosquito bite site, thereby promoting the establishment and amplification of arbovirus (such as dengue virus) infection.

[0156] In some implementations, the drug reduces the severity of arbovirus infection, reduces viremia, alleviates vascular leakage, and / or improves survival.

[0157] (Media of Communication)

[0158] In the above aspects of the present invention, the vector for the vector-borne virus can be a mosquito, tick, or sandfly. In some embodiments, the vector for the vector-borne virus is a mosquito (a mosquito-like insect), and correspondingly, the vector-borne virus can be a mosquito-borne virus.

[0159] In some embodiments of the invention, "mosquito" or "mosquito" refers to insects of the family Culicidae. The mosquitoes of the invention may include adult mosquitoes, mosquito larvae, pupae, or their eggs. Typically, the mosquito life cycle comprises four distinct stages: egg, larva, pupa, and adult. Thus, the mosquito life cycle begins when eggs are placed on the surface of water (e.g., Culex, Culiseta, and Anopheles) or on damp, flooded soil (e.g., Aedes). Most eggs hatch into larvae within 48 hours. The larvae live in water, feeding on microorganisms and organic matter, and then surface to breathe. They molt four times, growing larger after each molt, and on the fourth molt, the larva becomes a pupa. The pupal stage is a resting stage, a non-feeding period of approximately two days. During this time, the mosquito reaches adulthood. When development is complete, the pupal skin ruptures and the adult mosquito emerges.

[0160] In some embodiments, the mosquitoes belong to one or more of the subfamilies Anophelinae and Culicinae. In some specific embodiments of the invention, the mosquitoes include one or more of Culex, Culiseta, Anopheles, and Aedes.

[0161] In some more specific embodiments, the mosquitoes include, but are not limited to, Aedes, such as one or more of the following: Aedes aegypti, Aedes albopictus, Aedes polynesiensis, Aedes australis, Aedes cinereus, Aedes rusticus, and Aedes vexans.

[0162] In some preferred embodiments, the mosquitoes are Aedes aegypti and / or Aedes albopictus.

[0163] In some exemplary embodiments, the mosquito is Aedes aegypti.

[0164] (Host)

[0165] In the above aspects of the invention, "host" refers to a single-celled or multi-celled organism in which arboviruses can replicate, including cell lines and animals.

[0166] In some implementations, the host can be any mammal that can serve as a host for an arbovirus, such as a primate, preferably a human.

[0167] In other implementations, the host may also be a rodent, such as a mouse or rat.

[0168] In some implementations, the host is exposed to the bite of a vector that does not carry the vector virus before being infected with the vector virus, or the host is sensitized by the salivary proteins of the vector before being infected with the vector virus.

[0169] In some embodiments, the non-arbovirus-carrying transmission vector contains the salivary protein AaISP, or the salivary protein of the transmission vector contains AaISP.

[0170] In some exemplary embodiments, the present invention has found that the AaISP-specific IgE titer in the host is positively correlated with the severity of arbovirus (e.g., dengue virus) infection.

[0171] In some exemplary embodiments, the present invention has found a positive correlation between the level of AaISP-specific IgE in host serum and the severity of arbovirus (e.g., dengue virus) infection.

[0172] In some exemplary embodiments, the present invention has found that the higher the level of AaISP-specific IgE in the host serum, the stronger its ability to promote arboviruses (e.g., dengue virus) and pathogenicity.

[0173] In some exemplary embodiments, the present invention has found that host exposure to AaISP-specific IgE induced by uninfected vectors can promote infection with vector-borne viruses (e.g., dengue virus).

[0174] In some exemplary embodiments, the present invention has found that inhibiting the production of AaISP-specific IgE can effectively alleviate severe infection with arboviruses (e.g., dengue virus).

[0175] In some aspects of the present invention, a pharmaceutical composition for the prevention and / or treatment of diseases caused by arbovirus infection is provided, comprising the above-described reagents that target AaISP-specific IgE and / or reagents that inhibit the binding of AaISP-specific IgE to effector cells.

[0176] In some optional embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

[0177] Example

[0178] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0179] Experimental methods and materials used in the examples:

[0180] 1. Mice, mosquitoes, cells, and viruses

[0181] C57BL / 6J (B6; Cat# 219; Charles River) and 129 (Cat# 217; Charles River) mice were purchased from Charles River Laboratories. A / J (Cat# N000018; GemPharmatech) mice were purchased from GemPharmatech. B6. Cg-Fcer1a tm1Knt Tg(FCER1A)1Bhk / J(Fcer1α - / - FCER1A + / + FCER1A-KIB6 mice (Cat# Jax 010506; The Jackson Laboratory) were purchased from The Jackson Laboratory. Type I and Type II interferon receptor-deficient B6 mice (ifnagr...) were also used. - / - The AG6 mouse (FCER1A-KI AG6) was a gift from the Shanghai Pasteur Institute, Chinese Academy of Sciences. The humanized AG6 mouse carrying the human FCER1A gene knock-in and lacking endogenous mouse Fcer1a (FCER1A-KI AG6) was obtained by crossing AG6 mice with FCER1A-KI B6 mice. F1 offspring and subsequent generations carrying the target allele were intercrossed to obtain homozygous mice. Screening for FCER1A-KI AG6 mice was based on the following genotypes: homozygous deletion of mouse Fcer1a, stable integration of the human FCER1A knock-in allele, and double deficiency of Ifnar1 and Ifngr1. All genotypes were confirmed by PCR using primers and genotyping criteria provided by the manufacturer. The mice were housed and maintained in a specific pathogen-free animal facility at Tsinghua University.

[0182] Aedes aegypti (Rockefeller strain) were reared in a specially designed incubator (Cat# Model 818; Thermo Fisher) at 28°C and 80% humidity according to standard rearing procedures. C6 / 36 cells (Aedes albopictus larvae cells; Cat# CRL-1660; ATCC) were maintained at 28°C in RPMI 1640 medium (Cat#22400089; Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Cat# 16000-044; Gibco) and 1% antibiotic-antifungal solution (Cat# 15240-062; Invitrogen). Drosophila melanogaster S2 cells (Cat# CRL-1963; ATCC) were maintained at 28°C in Schneider medium (Cat# 21720024; Gibco) supplemented with 10% heat-inactivated FBS and 1% antibiotic-antifungal solution. Dengue virus type 2 (DENV2, New Guinea C strain, AF038403.1) was passaged in C6 / 36 cells cultured in serum-free VP-SFM (Cat# 11681-020; Gibco). Viral titers were determined using the widely used plaque formation assay.

[0183] 2. Constructing AaISP knockout Aedes aegypti mosquito strains via CRISPR-Cas9 gene editing

[0184] A CRISPR-Cas9-based Aedes aegypti mosquito strain with AAEL000749 knockout (AaISP-KO) was generated. Potential off-target effects of single-guide RNAs (sgRNAs) targeting AAEL000749 were designed and evaluated using the CHOPCHOP online tool (https: / / chopchop.cbu.uib.no). Five candidate sgRNA target sites were selected for experimental validation. Site-specific forward primers and universal reverse primers were used for these sites. Double-stranded DNA templates for sgRNA synthesis were generated using template-dependent PCR with overlapping oligonucleotides. Site-specific sgRNAs were generated in vitro using the MEGAscript T7 transcription kit (Cat# AM1334; Life Technologies) and purified using the MEGAclear transcription cleaning kit (Cat# AM1908; Life Technologies). Among the tested sgRNAs, a target sequence located in exon 2 of AAEL000749 (TAAGTTTGTGGTGGGCCCCG) (SEQ ID NO: 2) exhibited high cleavage activity and was selected for subsequent experiments.

[0185] To obtain the germline mutation, a CRISPR-Cas9 ribonucleoprotein complex was microinjected into the posterior end of the preblastocyst embryo before melanization. Genotyping analysis was performed on G0 generation adults, and the target region was amplified using flanking primers of AAEL000749. Adults exhibiting targeted cleavage were hybridized with wild-type (AaWT) Aedes aegypti. Similar genotyping was performed on G1 generation offspring to assess heritability. G1 generation male and female adults with the expected genotype predicted by the ICE analysis platform (https: / / ice.editco.bio) were paired to produce G2 generation offspring. Offspring homozygous for a one-base-pair deletion-frameshift mutation from the G2 generation were selected for co-culture to expand the mutant population. The knockout of the AaISP protein encoded by AAEL000749 was further validated by immunofluorescence staining and Western blotting using a specific self-made antibody.

[0186] 3. Expression and purification of recombinant Aedes aegypti salivary proteins

[0187] Recombinant Aedes aegypti salivary proteins were generated using a previously reported method. In short, the gene encoding the Aedes aegypti salivary protein was amplified using mosquito salivary gland cDNA as a template and then inserted into the pMT / Bip / V5-His A vector (Cat# V4130-20; Invitrogen) to construct a salivary protein plasmid. The mosquito salivary protein expression plasmid and hygromycin resistance plasmid were co-transfected into Drosophila melanogaster S2 cells at a 9:1 ratio to establish a cell line stably expressing the recombinant mosquito salivary protein. Expression of the recombinant salivary protein was induced by culturing cells in ExpressFive serum-free medium (Cat# 10486-025; Gibco) supplemented with 500 μM copper sulfate (Cat# HY-Y1878C; MCE). The recombinant protein was C-terminally labeled with a V5 tag and a 6x His tag, and subsequently purified using a cobalt column (Cat# 635515; Clontech). The purified recombinant salivary protein was validated by V5-tagged Western blotting analysis and its purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue staining.

[0188] 4. SDS-PAGE and Western blot for protein immunoblotting

[0189] Protein samples were diluted to appropriate concentrations with 1× loading buffer (Cat# 87787; ThermoFisher) containing SDS and β-mercaptoethanol, followed by denaturation at 100°C for 5 minutes. Samples were loaded onto 12% SDS-PAGE gels, with a concentration phase at 80 V and a separation phase at 120 V. Protein purity was assessed directly by Coomassie Brilliant Blue staining, or transferred to a polyvinylidene fluoride (PVDF) membrane (Cat# 1620177; Bio-Rad) for subsequent immunoblotting analysis. PVDF membranes were blocked with 5% skim milk at room temperature for 30 minutes, then incubated overnight at 4°C with primary antibody. After washing, the membranes were incubated at room temperature with horseradish peroxidase (HRP)-labeled secondary antibody for 1 hour. Protein bands were visualized using a chemiluminescent substrate (Cat# WBKLS0100; Millipore). The primary antibodies used included anti-V5 tag antibody (Cat# M167-3; MBL) and anti-β-actin antibody (Cat# HY-P81254; MCE), and the secondary antibodies included anti-rabbit IgG (H+L; Cat# 458; MBL) and anti-mouse IgG (H+L; Cat# 330; MBL).

[0190] 5. Mosquito infestation

[0191] Female Aedes aegypti or Aedes albopictus mosquitoes were anesthetized on a cold platform (Cat# 1431; BioQuip) and then intrathoracically inoculated with 20 PFU of DENV2. The inoculated mosquitoes were maintained at 28°C for 8 days and then used to bite AG6 mice for virus transmission.

[0192] 6. Mouse infection

[0193] For intradermal infection, 10 4PFU of DENV2 was injected intradermally into the left hind paw pad of AG6 mice. For mosquito bite infection, AG6 mice were first anesthetized with Avertin (Cat# HY-B1372; MCE), and then bitten by DENV2-infected Aedes aegypti mosquitoes for 30 minutes. Notably, immunocompetent mice (including strains 129, B6, and C3H) were infected in the same manner as AG6 mice. However, to increase susceptibility to DENV2 in these mice, type I interferon receptor blocking antibody (anti-IFNAR1; Cat# I-1188; Leinco; 1 mg / mouse) and type II interferon neutralizing antibody (anti-IFN-γ; Cat# I-1119; Leinco; 1 mg / mouse) were injected into the mice 24 hours before infection. Typically, each mouse was bitten by 5 DENV2-infected mosquitoes, unless otherwise stated. After infection, the weight and survival status of AG6 mice were monitored daily, and tail blood was collected from day 1 to day 7 post-infection to measure dynamic viremia by RT-qPCR.

[0194] 7. Mice exposed to mosquito bites

[0195] 2-week-old AG6 mice (AG6 Pre-AaWT AG6 mice were bitten twice a week by uninfected Aedes aegypti mosquitoes for four consecutive weeks. During each mosquito bite, AG6 mice were sedated and bitten by 10 uninfected Aedes aegypti mosquitoes per mouse. The corresponding unbitten mice (AG6) were... Control (A mosquito was used as a negative control.) The presence of blood on the mosquito's abdomen was used to confirm a successful bite. The mosquito exposure procedure for other inbred mouse strains was the same as described above. The same mosquito exposure protocol was also applied to Aedes albopictus or other inbred mouse strains. In experiments involving alternating mosquito species exposure, 2-week-old AG6 mice were exposed to uninfected female Aedes aegypti mosquito bites at weeks 1 and 3, and to Aedes albopictus mosquito bites at weeks 2 and 4. All other experimental conditions were the same as those used for single-species exposure.

[0196] 8. Screening for Aedes aegypti salivary proteins that enhance DENV2 transmission

[0197] To screen for mosquito salivary proteins that mediate enhanced DENV2 infection, 2-week-old AG6 mice were immunized twice at 2-week intervals, each time subcutaneously injected with a mixture of 2 μg of a single recombinant salivary protein and aluminum adjuvant (Cat# vac-alu-250; InvivoGen). Control AG6 mice were immunized with PBS or a mixture of 2 μg of bovine serum albumin (BSA; Cat# HY-D0842; MCE) and aluminum adjuvant. One week after the second immunization, AG6 mice immunized with mosquito salivary protein, BSA, or PBS were infected by the bites of 5 DENV2-infected Aedes aegypti mosquitoes per mouse.

[0198] 9. RNA extraction, reverse transcription, and RT-qPCR quantification

[0199] Total RNA was extracted using the AxyPrep Multisource Total RNA Miniprep Kit (Cat# AP-MN-MS-RNA-250; Axygen). Total RNA was reverse transcribed into total cDNA using the iScript cDNA Synthesis Kit (Cat# 170-8890; Bio-Rad). RNA quantification was performed using the iTaq Universal SYBR Green Supermix Kit (Cat# 1725121; Bio-Rad) on a Bio-Rad CFX-96 Touch real-time detection system. Dengue virus load in mouse blood and tissues was determined by absolute quantification and expressed as DENV2 copies / mL (blood or supernatant) or DENV2 copies / μg total RNA (tissue). In summary, the full-length coding sequence of the DENV2 NGC strain envelope protein was cloned into the pMT / Bip / V5-HisA vector to generate a DENV2 standard. The concentration of DENV2 standards was measured using a NanoDrop 2000 spectrophotometer (Cat# E112352; Thermo Fisher Scientific), and the DENV2 copy number in the standards was calculated using SnapGene software (version 8.2.2). The standards were then serially diluted 10-fold, and a standard curve was generated using qPCR analysis to establish the correlation between DENV2 copy number and the cycle threshold (Ct) value, from which a regression equation was derived. The Ct value of the test samples was applied to this equation to calculate the DENV2 genome copy number in each sample. Blood and tissue from uninfected mice were simultaneously treated as negative controls. The DENV2 copy number of these negative control samples, calculated according to the regression equation, was defined as the limit of detection (LOD) for absolute quantification. The LOD was 10² DENV2 copies / mL for blood or supernatant samples and 10² DENV2 copies / μg total RNA for tissue samples. Gene expression was normalized to the expression level of mosquito β-actin (AAEL011197) using the 2-ΔCt method. The RT-qPCR primers used are listed in Supplementary Table 3.

[0200] 10. Isolation and viral infection of mouse peritoneal macrophages

[0201] 5 mL of RPMI 1640 medium was injected intraperitoneally into B6 mice. After abdominal massage for 5 minutes and subsequent rest for 20 minutes, the culture medium containing macrophages was collected and centrifuged at 1,500 rpm for 10 minutes. The supernatant was discarded, and the cell pellet was resuspended in RPMI 1640 medium supplemented with 10% FBS. Macrophages were seeded at 100 μL / well in 96-well plates and incubated at 37°C for 2 hours. Non-adherent cells were removed by discarding the supernatant and washing twice with PBS; the remaining adherent cells were the peritoneal macrophages. Macrophages were then infected with DENV2 (MOI = 1) for 1 hour with different concentrations of AaISP (0, 0.5, 1, 2, and 4 μg / mL) or salivary gland extract (1 and 2 μg / mL). After infection, the cells were washed with PBS and cultured for another 24 hours in RPMI 1640 medium supplemented with 2% FBS and the corresponding concentrations of AaISP or salivary gland extract. Supernatant was collected 24 hours after infection, and viral load was quantified by RT-qPCR.

[0202] 11. Adoptive transfer of Pan-T cells

[0203] Three-week-old B6 mice were immunized twice at two-week intervals, each time subcutaneously injected with a mixture of purified AaISP and aluminum adjuvant. Corresponding B6 mice were immunized as controls with a mixture of PBS and aluminum adjuvant. One week after the second immunization, mouse spleens were collected. The spleens were dissociated into a single-cell suspension in ice-cold RPMI 1640 medium, washed with PBS, and then red blood cells were removed using erythrocyte lysis buffer (Cat# TNB4300; TONBO). Pan-T cells were isolated and counted using the MojoSort Mouse CD3 Selection Kit (Cat# 480099; Biolegend) via negative selection, following the manufacturer's instructions. The purity of pan-T cells was verified by flow cytometry. Four-week-old AG6 mice were intravenously injected with 5 x 10⁵ AaISP / a ... 7 T cells were obtained from AaISP-immunized mice or control mice. Twelve hours later, these mice were infected by the bites of DENV2-infected Aedes aegypti mosquitoes (five mosquitoes per mouse).

[0204] 12. Adoptive serum transfer

[0205] To specifically remove IgG or IgE, serum was incubated with Protein A+G agarose (IgG removal; Cat# P2019; Beyotime) or CaptureSelect IgE affinity matrix (IgE removal; Cat# 2943542010; ThermoScientific) at 4°C for 6 hours on a vortex mixer. The serum was then loaded into a chromatography column (Cat# 7321010; Bio-Rad) and flow-through filtered by gravity. The IgG or IgE-removed flow-through serum was collected. To specifically remove IgM or IgA, serum was incubated with biotin-labeled anti-mouse IgM antibody (IgM removal; Cat# 406504; Biolegend) or biotin-labeled anti-mouse IgA antibody (IgA removal; Cat# 407004; Biolegend) at 4°C for 1 hour. Subsequently, MojoSort streptavidin nanobeads (Cat# 480016; Biolegend) were added to the serum-antibody mixture and incubated on ice for another 30 minutes. The sample was then placed in a magnetic separator (Cat# 480019; Biolegend) for 10 minutes. Clear serum, free of IgM or IgA, was poured off and collected. Successful removal of specific immunoglobulins was verified by Western blotting. For adoptive serum transfer, 4-week-old AG6 mice received an intraperitoneal injection of serum (500 μL per mouse). A second injection of the corresponding serum was administered 12 hours later. Twelve hours after the second serum transfer, AG6 mice were infected via the bite of DENV2-infected Aedes aegypti mosquitoes.

[0206] 13. Purification and adoptive transfer of serum IgE

[0207] Serum IgE was purified from mouse or human serum samples using CaptureSelect IgE affinity matrix (Cat# 1943542250; Thermo Fisher) according to the manufacturer's instructions. For IgE transfer, 2 μg of purified IgE was intravenously injected into AG6 (mouse IgE transfer) or FCERT1A-KI AG6 (human IgE transfer) mice. Twelve hours post-transfer, mice were bitten by DENV2-infected Aedes aegypti mosquitoes (5 infected mosquitoes per mouse) to complete the transfer.

[0208] 14. Transcriptome Sequencing Sample Preparation

[0209] Two-week-old B6 mice were bitten twice weekly by uninfected Aedes aegypti mosquitoes (AaWT or AaISP-KO, 10 mosquitoes per mouse per bite) for four consecutive weeks, with corresponding un-bitten mice serving as negative controls (n=3 per group). Seven days after the last bite, both hind paw pads of the mice were bitten by uninfected AaWT or AaISP-KO mosquitoes (10 mosquitoes per mouse per hind paw pad). Thirty minutes after the bite, the mice were euthanized, and their hearts were perfused with ice-cold PBS to remove residual blood cells. Subsequently, both hind paw pads of each mouse were collected and combined as a single biological sample. Total RNA was then extracted from the paw pad skin using TRIzol (Cat# 15596026CN; Invitrogen) and subjected to transcriptome sequencing analysis.

[0210] 15. Toluidine blue staining

[0211] To assess degranulation of skin mast cells, mouse hind paw pads were exposed to bites from 10 Aedes aegypti mosquitoes for 30 minutes. Skin samples were fixed in 4% paraformaldehyde, then embedded in paraffin and sectioned. Sections were dewaxed with xylene, hydrated sequentially with a gradient of ethanol, and rinsed with tap water. For toluidine blue staining, sections were incubated in toluidine blue solution (Cat# C0637; Beyotime) for 5 minutes, rinsed, briefly differentiated in 1% glacial acetic acid, and then the reaction was terminated by washing with tap water. The degree of differentiation was then monitored under a microscope. After rinsing, sections were oven-dried, cleared in xylene for 5 minutes, and mounted with neutral resin for microscopic analysis.

[0212] 16. Immunofluorescence staining

[0213] Mice were euthanized, and their hearts were perfused with PBS to completely remove residual blood cells from the skin tissue. Skin samples from the paw pads at bite or injection sites were collected, fixed with 4% paraformaldehyde, and then embedded in paraffin and sectioned. Tissue sections were first dewaxed and hydrated, followed by heat-mediated antigen retrieval. Sections were then blocked with 0.3% Triton (Cat# X100-5ML; Sigma Aldrich), permeabilized for 5 minutes (omit this permeabilization step for membrane protein staining), and then blocked with 1% BSA (Cat# HY-D0842; MCE) for 1 hour. Sections were then incubated overnight at 4°C with primary antibody (1:200 dilution), and then incubated at room temperature with secondary antibody (1:200 dilution) for 50 minutes. Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-HEXB (Cat# DF3074; Affinity), Armenian hamster anti-mouse FcεRIα (Cat# 14-5898-82; eBioscience), rabbit anti-c-Kit (Cat# ab317843; Abcam), and mouse anti-V5 tag (Cat# 740058M; Invitrogen). Secondary antibodies used included: Alexa Fluor 488-labeled goat anti-Armenian hamster IgG (Cat# A78963; Invitrogen), Alexa Fluor 488-labeled goat anti-mouse IgG (Cat# A-21202; Invitrogen), and Alexa Fluor 594-labeled goat anti-rabbit IgG (Cat# A-11012; Invitrogen).

[0214] 17. Flow cytometry detection of skin mast cells bound to AaISP

[0215] Mice were euthanized and perfused with ice-cold PBS to remove circulating blood. Footpads were dissected, and the tissues were combined and digested with collagenase I and IV (1 mg / mL each; Cat# 17100017 and 17104019; Gibco) at 37°C for 1.5 hours. The digested tissues were filtered through a 70 μm cell filter (Cat# 352350; BD Falcon), washed with PBS, and subjected to erythrocyte lysis (Cat# TNB4300; TONBO). Single-cell suspensions from the footpads were incubated with 100 ng AaISP at 4°C for 1 hour, followed by blocking with anti-CD16 / CD32 antibody (2.4G2; Cat# 70-0161-U100; TONBO) and staining with a suite of antibodies including FITC anti-CD45 (30-F11; Cat# 35-0451-U100; TONBO), APC-Cyanine7 anti-CD117 (ACK2; Cat# 25-1172-U100; TONBO), and PE anti-V5 tag (TCM5; Cat# 12-6796-42; eBioscience). Live and dead cells were distinguished using Ghost Dye UV 450 (Cat# 13-0868-T100; TONBO). Samples were analyzed after staining using a Sony Spectral Cell Analyzer ID 7000 system. Skin mast cells that bound to AaISP were identified as CD45+CD117+V5+ cells.

[0216] 18. Flow cytometry detection of skin mast cell activation

[0217] To detect mast cell activation in the skin, mouse paw pads were bitten by Aedes aegypti mosquitoes (5 mosquitoes per hind paw pad per mouse) for 30 minutes. Mice were euthanized at 0, 0.5, 2, 4, and 12 hours and perfused with ice-cold PBS to remove circulating blood cells. The bitten paw pads were excised, minced, and enzymatically digested using the previously described method to obtain single-cell suspensions. Footpad skin cells were blocked with anti-CD16 / CD32 antibody and stained with a suite of antibodies including FITC anti-CD45 (30-F11; Cat# 35-0451-U100; TONBO), APC-Cyanine7 anti-CD117 (ACK2; Cat# 25-1172-U100; TONBO), violetFluor 450 anti-FcεRIα (M1 / 70; Cat# 75-5898-U100; TONBO), PerCP / Cyanine5.5 anti-CD63 (NVG-2; Cat# 143911; BioLegend), PE anti-CD203c (NP4D6; Cat# 324605; BioLegend), and the above-mentioned Ghost Dye UV 450. After staining, the samples were analyzed using a Sony Spectral Cell Analyzer ID7000 system. Activated skin mast cells were identified by gated CD45+CD117+FcεRIα+CD63+CD203c+ cells.

[0218] 19. Flow cytometry detection of myeloid cells in the skin

[0219] The proportions of neutrophils and other myeloid cells in the skin at the mosquito bite site were determined by flow cytometry. Mouse paw pads were bitten by Aedes aegypti mosquitoes (5 mosquitoes per hind paw pad per mouse) for 30 minutes. Mice were euthanized and perfused with ice-cold PBS at 0, 0.5, 2, 4, and 12 hours to remove circulating blood cells. The paw pads were dissected, and single-cell suspensions were prepared using the same method described previously. The obtained footpad skin cells were blocked with anti-CD16 / CD32 antibody and stained with a set of antibodies including FITC anti-CD45 (30-F11; Cat# 35-0451-U100; TONBO), violetFluor 450 anti-CD11b (M1 / 70; Cat# 75-0112-U100; TONBO), redFluor 710 anti-Ly6G (1A8; Cat# 80-1276-U100; TONBO), APC anti-CD163 (TNKUPJ; Cat# 17-1631-82; eBioscience), PerCP-Cyanine 5.5 anti-Ly6C (HK1.4; Cat# 65-5932-U100; TONBO), and Brilliant Violet 605 anti-IA / IE (Cat# ). 107639; BioLegend), APC-Cyanine7 anti-CD11c (N418; Cat# 25-0114-U100; TONBO), and Ghost Dye UV 450. After staining, samples were analyzed using the Sony Spectral Cell Analyzer ID7000 system. Skin neutrophils were identified as CD45+CD11b+Ly6G+ cells, monocytes as CD45+CD11b+Ly6C+Ly6G- cells, macrophages as CD45+CD11b+CD163+ cells, and dendritic cells as CD45+IA / I-E+CD11c+ cells.

[0220] 20. Enzyme-linked immunosorbent assay (ELISA)

[0221] IgE capture ELISA was used to detect the specific IgE titers of AaISP, AAEL006347, and AAEL006485 in human or mouse serum samples. Human serum was diluted 1:5, and mouse serum was diluted 1:10. Total IgE levels in human and mouse serum were determined according to the manufacturer's instructions using a human IgE ELISA kit (Cat# E-EL-H6104; Elabscience) and a mouse IgE ELISA kit (Cat# E-EL-M3034; Elabscience).

[0222] 21. Evans Blue (EB) Method for Determining Vascular Permeability

[0223] Vascular permeability was determined using the previously reported EB method. Briefly, 10 days post-infection, mice were intravenously injected with 150 μL of 1% EB dissolved in PBS (Cat# E6135; Macklin). After 30 minutes of systemic circulation, mice were perfused with 30 mL of PBS via the heart to remove residual EB from the blood vessels. Liver, spleen, lung, kidney, and small intestine were then collected. Approximately 50 mg of tissue was homogenized in 500 μL of N,N-dimethylformamide (Cat# N807506; Macklin). The homogenate was incubated at 60°C for 12 hours to extract EB, followed by centrifugation at 12,000 x g for 10 minutes. 100 μL of the supernatant was then transferred to a 96-well plate, and absorbance was measured at 620 nm. The EB content in the tissue was calculated according to a standard curve, expressed as ng EB / mg tissue.

[0224] 22. Removal of IgE from the body

[0225] To remove circulating IgE, mice were administered an intraperitoneal injection of an anti-IgE antibody (Cat# ICL-GE-90A; ICL; 20 ug / mouse) weekly, starting before mosquito bites. Control mice were injected with an equal dose of an isotype control antibody (Cat# 31245; Invitrogen). The efficiency of IgE removal was confirmed by detecting serum total IgE levels before and after antibody administration using ELISA.

[0226] 23. Statistical Analysis

[0227] Quantitative data conforming to a normal distribution and homogeneous variance are expressed as mean + / - SEM; otherwise, data are expressed as median. Statistical analysis was performed using GraphPad Prism 10.1.2 software. Comparisons of quantitative data between two groups were performed using unpaired t-tests (parametric method) or Mann-Whitney tests (non-parametric method). Comparisons of three or more groups were performed using one-way ANOVA, two-way ANOVA (for time-series viremia, cell percentage, and IgE titer tests), and post-ANOVA multiple unpaired t-tests or multiple Mann-Whitney tests. Survival curves were compared using the log-rank test. Statistical significance was set at p < 0.05. ns indicates no significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0228] Example 1. Mosquito salivary protein AaISP-specific IgE promotes the occurrence of severe dengue fever.

[0229] In tropical regions, people are frequently exposed to mosquito bites. Before being bitten by mosquitoes carrying dengue virus (DENV), they are often repeatedly exposed to bites from uninfected mosquitoes in the natural environment, which induces a sustained immune response against mosquito saliva antigens. Therefore, this invention investigates whether prior exposure to Aedes aegypti mosquito saliva antigens can regulate the mosquito-borne DENV infection process. To this end, this invention uses DENV-susceptible C57BL / 6J mice (AG6 mice) with type I and type II interferon receptor deficiency, and exposes them to bites from uninfected wild-type Aedes aegypti mosquitoes (AG6) for four consecutive weeks. Pre-AaWT (Group). During this period, each mouse was simultaneously bitten by 10 mosquitoes every 3–4 days (twice a week). Mice that had not received prior mosquito bite exposure served as negative controls (AG6). Control Group). Subsequently, all mice were infected by bites from Aedes aegypti mosquitoes carrying DENV2 (group). Figure 1 (A) The results showed that long-term exposure to AG6 without mosquito bites... Pre-AaWT The mice in this group showed higher levels of viremia ( Figure 1 (B) Higher tissue viral load ( Figure 1 (C) and higher mortality rate ( Figure 1 (D in the text) This suggests that mosquito salivary proteins may induce a specific adaptive immune response against salivary proteins, thereby promoting natural DENV2 infection and its pathogenesis.

[0230] Subsequently, this invention further identified key salivary proteins involved in mosquito-induced immune sensitization. Previous research (Amosquito salivary protein promotes flavivirus transmission by activation of autophagy. Nat Commun. 2020 Jan 14;11(1):260) identified 71 salivary proteins in the saliva of female Aedes aegypti mosquitoes using mass spectrometry. This invention utilizes Drosophila S2 cells to express and purify the 34 most abundant salivary proteins among them. Figure 2 A in Figure 2 (B in the text). Subsequently, each purified salivary protein was subcutaneously immunized with AG6 mice. After two immunizations, the mice were infected by bites from Aedes aegypti mosquitoes carrying DENV2, and the level of DENV2 viremia was detected by RT-qPCR. Figure 2 C and Figure 2(D in the original text). Screening of Aedes aegypti salivary proteins revealed that the protein encoded by AAEL000749 (SEQ ID NO: 1) significantly promoted mosquito-borne DENV infection. Immunosensitization with the AAEL000749-encoded protein significantly increased viremia in mice after DENV2 infection. Figure 2 E), increased viral load in tissues ( Figure 2 (F in the text), and the mortality rate after infection is significantly higher ( Figure 2 (G in the original text). In subsequent studies, the protein encoded by AAEL000749 was named Aedes aegypti immune-sensitizing salivary protein (AaISP). The above results indicate that AaISP is a key mosquito salivary protein that mediates enhanced infection.

[0231] Subsequently, this invention evaluated whether AaISP could regulate DENV2 infection under in vitro conditions. Different concentrations of purified AaISP protein were co-incubated with DENV2, and then added to primary macrophages derived from AG6 mice for infection experiments. Aedes aegypti salivary gland extract (SGE) was used as a positive control. The results showed that, consistent with previous studies, SGE significantly promoted DENV2 replication in primary macrophages, while AaISP did not show a promoting effect. Figure 3 (A in the original text). Furthermore, this invention further investigated whether AaISP could directly regulate DENV2 infection in vivo. AaISP and DENV2 were mixed and subcutaneously injected into the footpads of AG6 mice. AaNRP, a mosquito salivary protein known to promote dengue virus infection, was used as a positive control. Results showed that during infection, the viremia level in the AaISP-treated group was not significantly different from that in the control group (A in the original text). Figure 3 (B in the original text) indicates that AaISP does not directly promote the transmission and infection of DENV2 during mosquito bites. In summary, these results suggest that AaISP neither directly enhances DENV infection nor promotes viral transmission by modulating the innate antiviral immune response at the mosquito bite site.

[0232] To further verify the role of AaISP in dengue virus transmission, this invention used the CRISPR-Cas9 system to knock out the AaISP gene in Aedes aegypti mosquitoes. AaISP is highly expressed in the salivary glands of wild-type (AaWT) female Aedes aegypti, while the protein was not detected in the salivary glands of AaISP gene knockout mosquitoes (AaISP-KO). Figure 4 (A) in the text. Subsequently, the invention repeated... Figure 1 The experimental procedure is shown in A. First, uninfected wild-type Aedes aegypti mosquitoes (AG6) were used. Pre-AaWT) or AaISP-KO Aedes aegypti (AG6) Pre-AaISP-KO AG6 mice were pre-sensitized to mosquito saliva antigens by being bitten; mice that did not receive mosquito bites (AG6 mice) were also pre-sensitized to mosquito saliva antigens. Control ) as a negative control. Subsequently, mice were infected by biting with wild-type or AaISP-KO Aedes aegypti mosquitoes carrying DENV2, forming the following 4 experimental groups: (1) AG6 Control / AaWT Group: Unsensitized mice were bitten by wild-type mosquitoes carrying DENV2; (2) AG6 Pre-AaWT / AaWT Group: After being pre-sensitized by wild-type mosquitoes, the participants received bites from wild-type mosquitoes carrying DENV2; (3) AG6 Pre-AaISP-KO / AaWT Group: After pre-sensitization with AaISP-KO mosquitoes, the patients received bites from wild-type mosquitoes carrying DENV2; (4) AG6 Pre-AaWT / AaISP-KO Group: After pre-sensitization with wild-type mosquitoes, participants received bites from AaISP-KO mosquitoes carrying DENV2. Figure 4 (B in the text). The results show that, compared with AG6 Control / AaWT Compared to the group, AG6 Pre-AaWT / AaWT The mice in the group exhibited a more severe dengue virus infection phenotype, including higher viral load and higher mortality. Figure 4 (C–E). However, prior exposure to AaISP-KO mosquito bites does not increase the risk of subsequent DENV infection transmitted by wild-type mosquitoes (C–E). Figure 4 (C–E in the text). Notably, even after mice had been pre-sensitized to wild-type mosquitoes, DENV infection induced by AaISP-KO mosquito bites was significantly reduced compared to DENV infection transmitted by wild-type mosquitoes. Figure 4 (C–E in the text). These results indicate that AaISP plays a crucial role in both the mosquito bite-induced immune sensitization phase and the subsequent dengue virus transmission phase.

[0233] Example 2. AaISP-specific IgE promotes mosquito-borne dengue virus infection.

[0234] Subsequently, this invention aims to identify key immune effector molecules that promote the transmission of DENV2 infection by Aedes aegypti mosquitoes during AaISP-induced immunosensitization. To study AaISP-induced immunosensitization under immunocompetent conditions, C57BL / 6J (B6) mice were exposed to mosquito bites and immunized with AaISP, while subsequent dengue virus infection experiments were conducted using AG6 mice susceptible to DENV infection. Since AaISP exposure may simultaneously induce humoral and cellular immune responses, this invention first assesses the role of AaISP-specific T cells in DENV2 infection. B6 mice (B66) immunized twice with purified AaISP protein... AaISP-immT cells were isolated from the mosquitoes and total T cells (Pan-T cells) were obtained through a magnetic bead negative selection method. These cells were then transferred into AG6 mice. Subsequently, the recipient mice were infected by bites from Aedes aegypti mosquitoes carrying DENV2. Figure 5 A in the text). B6 mice immunized with adjuvant (B6) Control AG6 mice with AaISP-derived T cell transplantation served as the control group. Results showed that adoptive transfer of AaISP-specific T cells did not affect the DENV2 infection level transmitted by Aedes aegypti in AG6 mice. Figure 5 B and Figure 5 The C in the figure indicates that AaISP-specific T cells do not exacerbate DENV2 infection.

[0235] Therefore, this invention further evaluates the role of AaISP-specific humoral immune response in mosquito-borne DENV2 infection. Immunization induces the production of multiple immunoglobulin isotypes, including immunoglobulin G (IgG), IgM, IgA, IgE, and IgD. The first four are secretory antibodies, widely present in blood, tissue fluid, and mucosal secretions, and are the main effector molecules of the humoral immune response; while IgD is mainly expressed on the surface of B cells in a membrane-bound form, with extremely low levels in serum and mucosal secretions, and is not a core antibody type mediating humoral immunity. Therefore, this invention collects B6... AaISP-imm Mouse serum was used, and various immunoglobulins were removed using specific antibodies or affinity purification matrices targeting different antibody isotypes to obtain serum lacking specific antibody isotypes (e.g., B6). AaISP-imm Serum ΔIg To ensure sufficient antibody transfer efficiency, the serum deprived of specific antibody isotypes was injected twice into AG6 mice before mosquito-borne DENV2 infection. Figure 6 (A) The results showed that, compared with the removal of other antibody isotypes, the removal of IgE alone could completely eliminate B6. AaISP-imm dengue virus infection exacerbation effect induced by mouse serum ( Figure 6 B and Figure 6 (C in the middle).

[0236] To further verify this effect, the pathogenic immune response induced by AaISP mainly depends on AaISP-specific IgE. In this invention, AaISP-immunized B6 mice (B6...) were used to... AaISP-imm Group) and exposure to wild-type Aedes aegypti (B6) Pre-AaWT Group) or AaISP knockout Aedes aegypti (B6) Pre-AaISP-KO IgE was isolated from B6 mice bitten by (group) and used as B6 ControlIgE from mice in the control group served as a negative control. Subsequently, the IgE was transferred into the initial AG6 mice, which were then infected by bites from Aedes aegypti mosquitoes carrying DENV2. Figure 6 (D in the middle). The results show that, compared with B6 Control Compared to IgE from group B6, IgE from group B6 is superior. AaISP-imm Group and B6 Pre-AaWT IgE in all groups of mice significantly enhanced DENV2 infection in AG6 mice, as evidenced by a significant increase in viremia levels. Figure 6 E in the middle) and a significant increase in mortality ( Figure 6 (F in the original text). In contrast, it originates from B6. Pre-AaISP-KO IgE in the group of mice did not affect viremia or mortality. Figure 6 E and Figure 6 (F in the text). The above results indicate that AaISP-specific IgE induced by prior exposure to uninfected mosquitoes can promote mosquito-borne DENV2 infection.

[0237] Example 3. AaISP–IgE cross-linking activates skin mast cells and recruits monocytes to promote dengue virus infection.

[0238] Subsequently, this invention further investigated the molecular mechanism by which AaISP-specific IgE mediates immune activation in sensitized animals. Mosquito salivary proteins mainly act on the dermis, which is rich in mast cells. IgE can sensitize mast cells by binding to the high-affinity IgE receptor FcεRI on the surface of mast cells. Therefore, this invention hypothesizes that AaISP in mosquito saliva can bind to AaISP-specific IgE on the surface of mast cells in the skin of sensitized animals, thereby inducing FcεRI cross-linking and activating mast cells. To verify this hypothesis, this invention first evaluated AaISP-mediated gene expression regulation. The experimental group included the following three groups: mice (B6) repeatedly bitten by Aedes WT mosquitoes and then bitten again by Aedes WT mosquitoes. Pre-AaWT / AaWT Mice (B6) that were repeatedly bitten by AaISP-KO mosquitoes and then bitten by WT mosquitoes Pre-AaISP-KO / AaWT ), and mice (B6) that were repeatedly bitten by Aedes WT mosquitoes and then bitten by AaISP-KO mosquitoes. Pre-AaWT / AaISP-KO Unbitten mice were bitten by Aedes WT mosquitoes (B6). Control / AaWT ( ) served as a control group. Footpad skin tissue was collected 30 minutes after mosquito bites for RNA sequencing analysis. Figure 7 (A) Subsequently, this invention performed cluster analysis on genes associated with FcεRI activation and mast cell degranulation. The results showed that, with B6... Control / AaWT Compared to group B6 Pre-AaWT / AaWT In group B6, the expression of FcεRI activation and mast cell degranulation-related genes was significantly increased. However, in group B6...Pre-AaISP-KO / AaWT Group and B6 Pre-AaWT / AaISP-KO In this group, the upregulation effect of the above genes was significantly weakened. Figure 7 (B in the text) suggests that AaISP may induce skin mast cell activation in mosquito-bite-sensitized hosts.

[0239] Subsequently, this invention further evaluated the role of AaISP-specific IgE in the activation of skin mast cells. First, the sensitization of skin mast cells to AaISP-specific IgE was detected. B6 was taken separately... Control B6 Pre-AaWT and B6 Pre-AaISP-KO Single-cell suspensions were prepared from the paw pad skin of mice via enzymatic digestion. The cell suspensions were incubated with 100 ng of purified AaISP protein at 4 °C for 1 hour. The proportion of mast cells binding AaISP was then detected by flow cytometry. Figure 7 (C in the middle). The results show that, compared with B6 Control Compared to group B6 Pre-AaWT The proportion of AaISP-binding positive cells in the skin mast cells of mice in this group was significantly increased ( Figure 7 (D in the middle); and B6 Pre-AaISP-KO The proportion of AaISP-binding positive cells in mast cells of mice in group B6 Control Group similarity ( Figure 7 (D) It is worth noting that in B6 Pre-AaWT Pre-injection of mice with anti-mouse IgE antibody significantly reduced the proportion of AaISP-binding positive cells. Figure 7 (D in the text). These results indicate that previous bites of Aedes aegypti mosquitoes can sensitize skin mast cells with AaISP-specific IgE.

[0240] It is well known that mast cell activation typically triggers a degranulation response, i.e., the rapid release of inflammatory mediators (such as histamine and trypsin) stored in intracellular granules. Therefore, this invention investigated the degranulation of skin mast cells 30 minutes after a mosquito bite. The results showed that B6... Pre-AaWT / AaWT The number of degranulated mast cells was significantly increased in the group. However, regardless of whether it was in B6 Pre-AaISP-KO / AaWT Group B6, still treated with anti-IgE antibody Pre-AaWT / AaWT No obvious degranulation of skin mast cells was observed in any of the groups. This result was confirmed by toluidine blue staining (…). Figure 8 A) and immunofluorescence staining ( Figure 8 B) in the above is confirmed. Similarly, in B6... Pre-AaWT Subcutaneous injection of AaISP into mice induced significant activation and degranulation of skin mast cells; while in B6... Pre-AaISP-KO Mice or B6 treated with IgE clearance Pre-AaWT In mice, this effect completely disappeared. Figure 8 (C in the text). In summary, these results indicate that AaISP-specific IgE plays a decisive role in the activation of skin mast cells in mice previously exposed to mosquito bites.

[0241] Monocyte lineage immune cells (such as immature dendritic cell subsets and macrophages) are highly susceptible to DENV infection. Previous studies have shown that neutrophil-driven inflammatory responses can promote the aggregation of monocyte lineage cells at mosquito bite sites, and these highly susceptible myeloid cells can serve as new target cells to promote viral amplification. Therefore, inflammation-driven myeloid cell infiltration is an important factor determining the severity of cutaneous DENV infection and subsequent disease. Notably, mast cell degranulation can induce inflammatory neutrophil recruitment. Therefore, this invention further investigates whether AaISP-specific IgE-mediated skin mast cell activation promotes the aggregation of neutrophils and myeloid cells at the bite site. To analyze the dynamic changes in skin mast cell activation and myeloid cell recruitment, footpad skin tissue was collected at different time points after mosquito bites and analyzed by flow cytometry. Consistent with previous results, B6 Pre-AaWT / AaWT Mast cells in the skin at the bite site were rapidly activated, with activation levels peaking 0.5 hours after the bite and continuing to rise until 4 hours later; while B6 Control / AaWT Group and B6 Pre-AaISP-KO / AaWT No significant changes were observed in the group. Figure 8 (D) in B6. Control / AaWT Compared to group B6 Pre-AaWT / AaWT Numerous neutrophil infiltrations were detected in the group at 0.5, 2, 4, and 12 hours post-bite. Figure 8 (E in the text); Subsequently, myeloid mononuclear cell lineages highly susceptible to DENV significantly accumulated at the bite site at 4 and 12 hours post-bite ( Figure 8 (F–H in B6). However, in B6 Pre-AaISP-KO / AaWT In the group, whether it is neutrophils ( Figure 8 E in the text) or mononuclear lineage myeloid cells ( Figure 8 No recruitment occurred for F–G in B6. It is noteworthy that in B6… Pre-AaWT / AaWT Pre-injection of anti-IgE antibodies into mice effectively inhibited the activation of skin mast cells. Figure 8 (in D), and block subsequent neutrophil ( Figure 8 E in the middle) and myeloid cells of the mononuclear cell lineage ( Figure 8 The recruitment of F–G cells from mosquito bite sites. In summary, these results indicate that AaISP-mediated IgE crosslinking plays a crucial role in the activation of skin mast cells and further promotes the aggregation of a large number of highly susceptible myeloid cells to mosquito bite sites, thereby facilitating the establishment and amplification of dengue virus infection.

[0242] Example 4. Serum AaISP-specific IgE levels in dengue fever patients were positively correlated with disease severity.

[0243] The clinical manifestations of primary dengue virus infection are highly heterogeneous, progressing from asymptomatic infection to severe hemorrhagic fever and even death. Furthermore, previous studies have shown that mosquito saliva-specific IgE is commonly detected in the serum of individuals naturally exposed to mosquito bites. However, IgE levels vary considerably among individuals. Therefore, this invention further evaluated the titer of AaISP-specific IgE in the serum of patients with primary dengue virus infection across different clinical outcomes. This study recruited acute dengue fever patients in Xishuangbanna Hospital, Yunnan Province, in Southwest China in 2024, including 147 patients with dengue fever (DF) without warning signs and 47 patients with severe dengue (SD). Following World Health Organization (WHO) guidelines, the OD ratio of DENV-specific IgM / IgG was measured using an enzyme-linked immunosorbent assay (ELISA) with IgM and IgG capture method to determine patients with primary DENV infection. Of these, 69 DF patients and 17 SD patients were confirmed to have primary DENV infection. Subsequently, the AaISP-specific IgE titer in the serum of patients with primary dengue fever was detected using the IgE capture ELISA method. Clinical results showed that, compared with DF patients, the total IgE level in the serum of SD patients ( Figure 9 A) and AaISP-specific IgE levels ( Figure 9 Both B) were significantly elevated.

[0244] To further investigate the long-term association between serum AaISP-specific IgE levels and the severity of primary dengue virus infection under natural mosquito exposure conditions, this invention established a cohort of recovered dengue fever patients. Enrollment included patients diagnosed with and recovered from primary DENV infection within the past 2–5 years, including DF and SD patients. All subjects resided in Xishuangbanna, a tropical region with high densities of Aedes aegypti and Aedes albopictus mosquitoes. Figure 9 Therefore, these dengue fever survivors were continuously and frequently exposed to mosquito bites in the natural environment. This invention dynamically monitored the AaISP-specific IgE levels in the serum of these survivors in April, June, and August 2025. The results showed that, compared with DF survivors, SD survivors exhibited higher levels of total IgE and AaISP-specific IgE (C) throughout the follow-up period. Figure 9 D and Figure 9These results further reinforce the association between AaISP-specific IgE titers and the severity of primary dengue fever, indicating that this difference exists not only during the acute infection phase but can also be maintained long-term after recovery from infection and under natural mosquito exposure conditions.

[0245] Example 5. IgE from severe dengue fever patients exacerbates dengue virus infection in the body.

[0246] Subsequently, this invention further explores the role of IgE derived from dengue fever patients in the pathogenesis of dengue fever. Numerous studies have shown that IgE can bind to the high-affinity IgE receptor FcεRI, which is mainly expressed on the surface of specialized granulocytes such as mast cells and basophils. FcεRI is a tetrameric receptor complex composed of FcεRIα, FcεRIβ, and two FcεRIγ subunits. FcεRIα is responsible for recognizing the Fc domain of IgE, while FcεRIβ and FcεRIγ participate in downstream signal transduction. Based on this, this invention constructs a humanized AG6 mouse model: human FCER1A gene knocked in and mouse Fcer1a gene knocked out (FCER1A-KI AG6). This model enables human IgE to bind to FcεRI, activating granulocytes such as mast cells and basophils through antigen-mediated receptor cross-linking.

[0247] Subsequently, this invention utilized this humanized mouse model to evaluate whether IgE derived from patients with different clinical types of dengue fever could regulate the severity of infection in vivo. To this end, IgE was isolated and purified from the serum of patients with primary DF and SD, respectively. Each mouse was administered 2 μg of human IgE intravenously. FCERE1A-KI AG6 mice that did not receive human IgE transfer served as a negative control. Twelve hours after IgE transfer, mice were infected using bites from Aedes aegypti carrying DENV2 (…). Figure 10 (A in the text). The results showed that mice receiving IgE from SD patients exhibited the highest viremia compared to other groups. Figure 10 B) viral load in tissues ( Figure 10 C) Multi-organ vascular leakage ( Figure 10 (D in the middle) and mortality rate ( Figure 10 The E in the text. Compared with the negative control group, IgE from DF patients also enhanced DENV2 infection, but its promoting effect was significantly weaker than that from SD patients (E). Figure 10 (B–E in the text). In summary, these results indicate that the level of AaISP-specific IgE in human serum is positively correlated with the severity of primary dengue virus infection. The higher the level of AaISP-specific IgE, the stronger its ability to promote dengue virus infection and pathogenicity.

[0248] Example 6: Inhibiting IgE production can reduce the severity of dengue virus infection.

[0249] Mouse strains with strong AaISP-specific IgE production capabilities, such as B6 and 129 mice, are prone to severe dengue infection after mosquito bites. Therefore, this invention further evaluates whether inhibiting IgE production in these mouse strains can alleviate dengue severity. B6 and 129 mice were exposed to Aedes aegypti mosquito bites (B6) for four consecutive weeks. Pre-AaWT Group and 129 Pre-AaWT (Group). Subsequently, as Figure 11 As shown in Figure A, intraperitoneal injection of anti-IgE antibody (Cat# ICL-GE-90A; ICL; 20 ug / mouse) significantly reduced AaISP-specific IgE in mice.

[0250] Subsequently, mice were intraperitoneally injected with anti-IFN-α receptor blocking antibody (Cat# I-1188; Leinco; 1 mg / mouse) and anti-IFN-γ neutralizing antibody (Cat# I-1119; Leinco; 1 mg / mouse), and then infected by bites from Aedes aegypti mosquitoes carrying DENV2. Figure 11 The BD results showed that IgE clearance significantly reduced B6 Pre-AaWT Group and 129 Pre-AaWT In the study of the severity of DENV2 infection in mice, treatment with anti-IgE antibodies significantly reduced viremia, alleviated vascular leakage, and significantly improved mouse survival rate, indicating that inhibiting IgE production can effectively alleviate severe dengue symptoms. Furthermore, the enhanced inhibitory effect of IgE blockade was also observed in mice with different genetic backgrounds.

[0251] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0252] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. Any of the following uses (i) to (iii): (i) The use of AaISP-specific IgE as a target in the preparation of drugs for the prevention and / or treatment of diseases caused by arbovirus infection; (ii) The use of agents targeting AaISP-specific IgE in the preparation of medicaments for the prevention and / or treatment of diseases caused by arbovirus infection; (iii) The use of reagents that inhibit AaISP-specific IgE-sensitized effector cells in the preparation of medicaments for the prevention and / or treatment of diseases caused by arbovirus infection.

2. The use according to claim 1, wherein, The disease caused by arbovirus infection is a disease in the host of the arbovirus, wherein the host of the arbovirus is exposed to the bite of a vector that does not carry the arbovirus before infection, or the host of the arbovirus is sensitized by the salivary proteins of the vector before infection.

3. The use according to claim 2, wherein, The transmission vector that does not carry arboviruses contains the salivary protein AaISP, or the salivary protein of the transmission vector contains AaISP.

4. The use according to claim 2 or 3, wherein, AaISP-specific IgE-sensitized effector cells in hosts exposed to arboviruses from bites of vectors that do not carry the arbovirus, or in hosts sensitized by arboviruses from salivary proteins of vectors. Preferably, the AaISP-specific IgE sensitizing effector cells include activated skin mast cells and / or myeloid cells that aggregate at the bite or sensitization site.

5. The use according to any one of claims 1-4, wherein, The arboviruses include one or more of the following families: Bunyaviridae, Flaviviridae, Reoviridae, and Togaviridae; Preferably, the arbovirus includes one or more of the genera Flavivirus, Alphavirus, and Orthobunyavirus. More preferably, the arbovirus includes Flaviviridae; More preferably, the vector-borne virus includes dengue virus.

6. The use according to any one of claims 1-5, wherein, Diseases caused by vector-borne viral infections include dengue fever; Optionally, the dengue fever includes severe dengue fever.

7. The use according to any one of claims 1-6, wherein, The drug reduces the severity of arbovirus infection, reduces viremia, alleviates vascular leakage, and / or improves survival.

8. The use according to any one of claims 1-7, wherein, The vectors of the arboviruses include one or more of mosquitoes, ticks, or sandflies; Preferably, the vector for the vector-borne virus is a mosquito; More preferably, the mosquitoes include one or more of Culex, Culiseta, Anopheles, and Aedes; Optionally, the mosquito is an Aedes mosquito, including one or more of Aedes aegypti, Aedes albopictus, Aedes polynesiensis, Aedes australis, Aedes cinereus, Aedes rusticus, and Aedes vexans.

9. The use according to any one of claims 1-8, wherein, The host of the arbovirus is a mammal; Preferably, the host of the arbovirus is a primate and / or a rodent.

10. A pharmaceutical composition for the prevention and / or treatment of diseases caused by arbovirus infection, comprising the agent targeting AaISP-specific IgE as described in claim 1 and / or the agent inhibiting the binding of AaISP-specific IgE to effector cells, and, Optional, pharmaceutically acceptable carrier.

Images