Application of scavenger receptor A antibody in preparation of medicine for inhibiting plasmodium transmission

Abstract

The invention relates to application of a scavenger receptor A antibody in preparation of a medicine for inhibiting plasmodium transmission, and belongs to the technical field of malaria prevention and treatment. Aiming at the technical problem that in the prior art, the drug resistance of plasmodium to drugs and the resistance of mosquito vector to insecticides are increasingly serious, so that the malaria propagation control effect is reduced, the invention provides a scavenger receptor A antibody which is specifically combined with a scavenger receptor A on the surface of macrophage to inhibit the phagocytic function of the scavenger receptor A, so that the control effect of malaria propagation is improved. Therefore, the proliferation of antimalarial active bacteria (such as Elizabeth, shigella sonnei or klebsiella oxytoca) in the stomach of the anopheles is promoted, and the development of plasmodium in the midgut of the anopheles is indirectly blocked. The antibody can be used alone or in combination with a Pfs25 antibody. The anopheles infection rate and the number of oocysts are remarkably reduced (for example, the infection rate is reduced from 81.7% to 6%), an eco-friendly biological control mode which is not prone to drug resistance is provided, and a new strategy is provided for malaria transmission blocking.

Description

Technical Field

[0001] This invention belongs to the field of malaria prevention and control technology, and relates to the application of scavenger receptor A antibody in the preparation of drugs that inhibit the spread of malaria parasites. Background Technology

[0002] Nearly half the world's population faces the threat of malaria infection. However, in recent years, malaria cases have increased rather than decreased. This is because previous malaria control tools have become less effective due to both mosquito resistance and the drug resistance of the malaria parasite. Malaria is unique in that it requires transmission via mosquitoes, so much research currently focuses on how to control malaria by reducing mosquito-borne infection rates. First, addressing drug resistance: The malaria parasite has developed resistance to drugs such as artemisinin, and mosquitoes are increasingly resistant to insecticides. New strategies targeting the mosquito-borne malaria parasite can bypass the limitations of traditional control methods and provide a more sustainable solution. Second, eco-friendly and precise control: Compared to large-scale insecticide use, intervention targeting the mosquito-borne malaria parasite reduces environmental damage and achieves precise control by specifically killing the parasite rather than the mosquito population. This research not only provides new tools for global malaria elimination but also offers insights for the control of other mosquito-borne diseases.

[0003] The mosquito stage of Plasmodium development is a natural bottleneck period, during which the parasite population is low, making it an ideal time to block its transmission. When an Anopheles mosquito bites an infected patient, the Plasmodium parasite and blood are inhaled into the mosquito's stomach. Simultaneously, the parasite begins its sexual development, undergoing stages such as gametophyte activation, silkening, fertilization, development into a zygote and then a motor zygote, before forming an oocyst on the basement membrane of the mosquito's stomach. Traditional methods of suppressing malaria transmission by killing Anopheles mosquitoes with antimalarial drugs and insecticides have significant drawbacks. Firstly, Plasmodium develops drug resistance over long-term evolution; secondly, Anopheles mosquitoes also develop resistance to insecticides. Therefore, new methods and approaches are urgently needed to reduce malaria transmission. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide the application of a scavenger receptor A antibody in the preparation of a drug for inhibiting the spread of malaria parasites; another objective is to provide the application of a combination of a scavenger receptor A antibody and a Pfs25 antibody in the preparation of a drug for inhibiting the spread of malaria parasites.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of a scavenger receptor A antibody in the preparation of a drug to inhibit the spread of Plasmodium; Preferably, the antigen amino acid sequence for preparing the scavenger receptor A antibody is shown in SEQ ID NO:1; Preferably, the scavenger receptor A antibody is a monoclonal antibody or a polyclonal antibody; Furthermore, the present invention also provides the application of a composition of scavenger receptor A antibody and Pfs25 antibody in the preparation of drugs to inhibit the spread of Plasmodium; Preferably, the antigenic amino acid sequence of the scavenger receptor A antibody is shown in SEQ ID NO:1; Preferably, the heavy chain sequence of the Pfs25 antibody is shown in SEQ ID NO:2, and the light chain sequence of the Pfs25 antibody is shown in SEQ ID NO:3.

[0006] The beneficial effects of this invention are as follows: In this invention, the scavenger receptor A antibody specifically binds to scavenger receptor A on the surface of macrophages, thereby inhibiting macrophage phagocytosis mediated by scavenger receptor A. The scavenger receptor A antibody can affect the phagocytosis of mosquito stomach bacteria by macrophages entering the mosquito stomach, thus enabling the rapid proliferation of antimalarial bacteria in the mosquito stomach after the mosquito's blood meal, thereby inhibiting the development of Plasmodium in the mosquito stage. This reduced the mosquito infection rate from 81.7% to 48.6%, a decrease of 33.1%. When Anti-Pfs25 antibody was used alone, the mosquito infection rate was 34.1%, and the infection rate was further reduced to 6% when combined with the scavenger receptor A antibody, indicating that the combined use of the two antibodies can significantly reduce mosquito infection with Plasmodium.

[0007] The key feature of this invention is that it does not directly affect the survival of Plasmodium and Anopheles mosquitoes. It reduces Plasmodium infection in Anopheles mosquitoes through biological control, thereby inhibiting transmission. By using scavenger receptor A antibody, administered via tail vein injection to mice two days after infection, the infection rate in Anopheles mosquitoes was significantly reduced. This invention significantly reduced the number of intestinal oocysts in infected Anopheles mosquitoes, decreasing the infection rate from 100% to 78.8%. The mechanism of this invention lies in the increased presence of Elizabethan bacteria (with antimalarial activity) in the Anopheles mosquito gut after using scavenger receptor A antibody. Elizabethkingia anophelis , Ea Shigella sonnei ( Shigella sonnei , Ss ) and Klebsiella pneumoniae ( Klebsiella oxytoca , Ko This invention indirectly affects the development of Plasmodium in the mosquito's stomach. It provides a new approach and strategy for reducing the spread of malaria.

[0008] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0009] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 for Plasmodium yoelii Three days after infecting mice with BY265-RFP, 50 μg of scavenger receptor A antibody was injected via the tail vein. On the fourth day post-infection, the mice were fed with Anopheles blood. Figure 1 A), 7 days after feeding on Anopheles blood, the midgut of the mosquitoes was dissected and the number of oocysts was counted, while the infection rate was calculated. Figure 1 B). (Parasites: parasite - Plasmodium; Anti-SR-A: scavenger receptor A antibody; Blood meal: blood meal; Oocyst count: oocyst count; Oocysts per midgut: number of oocysts per mosquito midgut; Control: solvent control group; Prevalence: infection rate) Figure 2 for Plasmodium berghei Three days after Pfs25 infection, mice were injected with 50 μg of scavenger receptor A antibody via the tail vein, followed by an injection of 20 μg of Pfs25 antibody via the tail vein one hour before feeding mosquito blood. Figure 2 A, B), nine days after feeding on Anopheles blood, the midgut of the mosquitoes was dissected and the number of oocysts was counted, while the infection rate was calculated. Figure 2 C). (Parasites: parasite - Plasmodium; Anti-SR-A: scavenger receptor A antibody; Anti-Pfs25: Pfs25 antibody; Blood meal: blood meal; Oocyst count: oocyst count; Oocysts per midgut: number of oocysts per mosquito midgut; Control: solvent control group; Prevalence: infection rate) Figure 3 for Plasmodium berghei Three days after Pfs25 infection, mice were injected with 50 μg of scavenger receptor A antibody via the tail vein. One hour before feeding mosquito blood, mice were injected with 20 μg of Pfs25 antibody via the tail vein. Twelve hours later, the midgut was dissected for quantitative PCR detection of intestinal bacteria. Figure 3 AC). (Control: solvent control group; Anti-Pfs25: Pfs25 antibody treatment group; Anti-SR-A: scavenger receptor A treatment group; Anti-Pfs25+Anti-SR-A: scavenger receptor A and Pfs25 antibody combined treatment group; Relative expression: relative expression level; Ea: Elizabethan bacillus; Ss: Shigella sonnei; Ko: Klebsiella pneumoniae) Figure 4 To pre-feed Anopheles mosquitoes, they are given sugar water containing antibiotics to remove their original gut flora, followed by feeding them a solution containing antibiotics. Ko , Ss or Ea The sugar water will infect Plasmodium yoelii Mice infected with BY265-RFP malaria parasites were fed Anopheles blood meals 4 days after infection. Seven days later, the mice were dissected to count the number of oocysts in the midgut of the Anopheles mosquitoes and calculate the infection rate. Figure 4 (Control: control group;) Ea Elizabethan colonization group; Ss : Shigella sonnei colonization group; Ko Klebsiella pneumoniae colonization group; Oocysts per midgut: number of oocysts per mosquito midgut; Prevalence %: infection rate Detailed Implementation The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0010] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0011] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0012] Materials and reagents used in this invention (1) Materials The Plasmodium johnsonii strain used in the experiment ( Plasmodium yoelii BY265-RFP), Plasmodium berghei strain ( Plasmodium berghei -Pfs25) and Anopheles stearensis ( Anopheles stephensi All of these are from the Malaria Research and Reference Reagent Resource Center (MR4); Female C57 BL / 6J mice, aged 6-8 weeks, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Elizabethan bacteria ( Elizabethkingia anophelis, Ea Shigella sonnei ( Shigella sonnei, Ss ) and Klebsiella pneumoniae ( Klebsiella oxytoca, Ko It was isolated from the midgut of Anopheles squarrosa raised in our laboratory. (2) Reagents: The anti-SR-A antibody was purchased from R&D Company in the United States; The Omega DNA extraction kit was purchased from Omega in the United States. PBS (Phosphate Buffered Saline) was purchased from Service Bio, China. Anhydrous diethyl ether was purchased from China Cloning Chemical Co., Ltd. Penicillin, streptomycin, and gentamicin were purchased from Gibco, Inc., USA. TB Green® Premix Ex TaqTMII was purchased from Takara Corporation, Japan; Anti-Pfs25 was expressed by GenScript China (see appendix for the complete sequence); Mercury was purchased from MedChemicalExpers, Inc. in the United States.

[0013] Example 1 Thawing in a 37°C water bath followed by liquid nitrogen cryopreservation P.yoelii BY265-RFP strain, 200 μl intraperitoneally injected, smear examination approximately three days later. When the protozoan rate is 5%–10%, blood is collected from the tail tip and prepared with PBS. 6 The malaria parasite-infected red blood cell attack fluid was injected into C57 mice via the tail vein.

[0014] 3-6 day old Anopheles streptococci were starved for 24 hours beforehand. Mice infected with Plasmodium malariae for 4 days were then fed with the mosquitoes' blood. After 30 minutes, the mice were removed, and the uneaten mosquitoes were removed. The mice were then placed in an artificial climate chamber at 23 ℃ and 70% ± 10 RH. Plasmodium yoeliiBY265-RFP) Infect P.yoelii Three days later, BY265-RFP mice were injected via tail vein with 200 μl of 50 μg of Anti-SR-A antibody or a solvent control. Figure 1 A). Plasmodium yoelii Three days after infecting mice with BY265-RFP, 50 μg of scavenger receptor A antibody was injected via the tail vein. On the fourth day post-infection, the mice were fed with Anopheles blood. Figure 1 A), 7 days after feeding on Anopheles blood, the midgut of the mosquitoes was dissected and the number of oocysts was counted, while the infection rate was calculated. Figure 1 B). (Parasites: parasite - Plasmodium; Anti-SR-A: scavenger receptor A antibody; Blood meal: blood meal; Oocyst count: oocyst count; Oocysts per midgut: number of oocysts per mosquito midgut; Control: solvent control group; Prevalence: infection rate) Example 2: Combined administration of Anti-SR-A and Anti-Pfs25 Thawing in a 37°C water bath followed by liquid nitrogen cryopreservation Plasmodium berghei -Pfs25 strain, intraperitoneal injection of 200 μl, smear examination approximately three days later. When the protozoan rate is 5%–10%, blood is collected from the tail tip and prepared with PBS to 10 μL. 6 The malaria parasite-infected red blood cell attack fluid was injected into C57 mice via the tail vein.

[0015] 3-6 day old Anopheles streptococci were starved for 24 hours beforehand. Mice infected with Plasmodium malariae for 4 days were then fed with the mosquitoes' blood. After 30 minutes, the mice were removed, and the uneaten mosquitoes were removed. The mice were then placed in an artificial climate chamber at 20°C and 70% ± 10 RH. Plasmodium berghei -Pfs25).

[0016] Infect Plasmodium berghei Three days later, Pfs25 mice were injected via tail vein with 200 μl of 50 μg of Anti-SR-A antibody or a solvent control. Subsequently, one hour before receiving mosquito blood, they were injected via tail vein with 20 μg of Anti-Pfs25 antibody or a solvent control. Figure 2 ). Plasmodium berghei Three days after Pfs25 infection, mice were injected with 50 μg of scavenger receptor A antibody via the tail vein, followed by an injection of 20 μg of Pfs25 antibody via the tail vein one hour before feeding mosquito blood. Figure 2 A, B), nine days after feeding on Anopheles blood, the midgut of the mosquitoes was dissected and the number of oocysts was counted, while the infection rate was calculated. Figure 2C). (Parasites: parasite - Plasmodium; Anti-SR-A: scavenger receptor A antibody; Anti-Pfs25: Pfs25 antibody; Blood meal: blood meal; Oocyst count: oocyst count; Oocysts per midgut: number of oocysts per mosquito midgut; Control: solvent control group; Prevalence: infection rate) Example 3 1. Evaluation of transmission inhibition effect 7 days after Anopheles infection ( Plasmodium yoelii BY265-RFP) or 9 days Plasmodium berghei -Pfs25) followed by dissection of the midgut of the Anopheles mosquito. The specific method is as follows: First, the Anopheles mosquito to be dissected is sucked out with a mosquito aspirator and anesthetized with anhydrous ether. Then, the mosquito is placed on a glass slide and its head is removed under a stereomicroscope. An appropriate amount of PBS is added to the glass slide. Dissection is performed using two 1 ml syringes. The left syringe is used to gently press on the thoracic cavity of the Anopheles mosquito, and then the blunt side of the right syringe is used to press down on the second-to-last segment of the abdomen of the Anopheles mosquito to remove the foregut and the Malpighian tubules attached to the tail of the midgut, thus obtaining the complete midgut of the Anopheles mosquito. Infected Plasmodium yoelii The intestines of BY265-RFP Anopheles mosquitoes were directly compressed and photographed under a fluorescence microscope. Oocysts were counted using ImageJ. Infection... Plasmodium berghei -Pfs25 Anopheles mosquito gut cells were stained with 1% mercuric chloride and counted under an optical microscope. The transmission inhibition effect was reflected by comparing the number of oocysts produced in the midgut of Anopheles mosquitoes and the percentage of infected oocysts in the experimental and control groups. The number of oocysts was determined by unpaired Mann-Whitney mosquito smears. U The ovarian cyst infection rate was analyzed using Fisher's exact test (two-sided);* p <0.05; ** p <0.01;*** p <0.001; **** p <0.0001.

[0017] 2. Quantitative PCR detection of Anopheles mosquito gut microbiota Infect Plasmodium bergheiPfs25 mice were fed mosquito blood, and the midgut of the mosquitoes was dissected 12 hours later for sampling. Each biological replicate consisted of 20 midgut samples, with at least three replicates. Total DNA was extracted using the Omega DNA extraction kit, following the manufacturer's instructions. DNA quality and concentration were then analyzed using a NanoDrop One Spectrophotometer. 100 ng of DNA was used as a template for quantitative PCR using a TB Green® PremixEx Taq™ II. Universal 16S rDNA primers were used to detect intestinal bacterial load, with the Anopheles S7 gene serving as an internal control. E. anopheles Specific primers were used to detect the expression level, with the Anopheles S7 gene used as an internal reference gene. Primers were designed using the NCBI primer blast tool. All experiments were performed using a Bio-Rad CFX-96 instrument. Relative expression levels were determined by 2... -ΔΔt Formula calculation.

[0018] 3. Anopheles mosquito gut microbiota clearance and specific bacterial colonization Female mosquitoes that had emerged from their molts three days prior were fed a 10% sugar solution supplemented with 10 U / ml penicillin, 15 μg / ml streptomycin, and 15 μg / ml gentamicin. This was then replaced with a solution containing 1×10 U / ml penicillin. 6 / mL of Elizabethan bacteria isolated ( Elizabethkingia anopheles, Ea Shigella sonnei ( Shigella sonnei, Ss ) or Klebsiella pneumoniae ( Klebsiella oxytoca, Ko A sugar solution was applied for 2 days to establish *Enterobacter anopheles* colonization in the midgut of mosquitoes. Detection was performed by quantitative PCR (primer information is shown in Appendix 1).

[0019] Appendix 1

[0020] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of scavenger receptor A antibody in the preparation of drugs to inhibit the spread of Plasmodium.

2. The application of the scavenger receptor A antibody according to claim 1 in the preparation of drugs to inhibit the transmission of Plasmodium, characterized in that: The antigenic amino acid sequence for preparing the scavenger receptor A antibody is shown in SEQ ID NO:

1.

3. The application of the scavenger receptor A antibody according to claim 2 in the preparation of drugs to inhibit the spread of Plasmodium, wherein the scavenger receptor A antibody is a monoclonal antibody or a polyclonal antibody.

4. Application of the combination of scavenger receptor A antibody and Pfs25 antibody in the preparation of drugs to inhibit the spread of Plasmodium.

5. The application according to claim 4, characterized in that, The antigenic amino acid sequence of the scavenger receptor A antibody is shown in SEQ ID NO:

1.

6. The application according to claim 4, characterized in that, The heavy chain sequence of the Pfs25 antibody is shown in SEQ ID NO:2, and the light chain sequence of the Pfs25 antibody is shown in SEQ ID NO:3.

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