Monoclonal antibody based on plasmodium falciparum antigen PfRH5 and application of monoclonal antibody in malaria treatment

Abstract

The invention relates to the technical field of biological medicines, and discloses a monoclonal antibody based on a plasmodium falciparum antigen PfRH5 and application of the monoclonal antibody in malaria treatment. The preparation method of the monoclonal antibody based on the plasmodium falciparum antigen PfRH5 comprises the following steps: preparing an RH5 mRNA vaccine through an mRNA vaccine technology, and then screening through a hybridoma B cell technology to obtain the monoclonal antibody based on the plasmodium falciparum antigen PfRH5. According to the monoclonal antibody based on the plasmodium falciparum antigen PfRH5, the problem that RH5 is difficult to express is successfully solved by combining an mRNA vaccine technology, the RH5 with native conformation is successfully utilized for immunization, and a plurality of monoclonal antibodies with efficient inhibitory activity are successfully screened by combining a hybridoma B cell technology; the antibodies have brand-new epitopes, and a new scheme is provided for treatment of malaria in the red phase.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a monoclonal antibody based on Plasmodium falciparum antigen PfRH5 and its application in malaria treatment. BACKGROUND

[0002] Malaria is an infectious disease caused by Plasmodium, mainly transmitted through mosquito bites. According to the statistics of the World Health Organization, there were about 200 million cases of malaria worldwide in 2023, causing about 600,000 deaths, which caused a heavy disease burden. At present, although there are vaccines and antimalarial drugs, Plasmodium has begun to develop resistance to traditional treatment methods (artemisinin combination therapy, ACT), which has gradually lost its effectiveness. Therefore, the development of new antimalarial methods has become the focus of attention for those skilled in the art.

[0003] Monoclonal antibody drugs show great potential in the treatment of various diseases due to their high specificity and long-acting nature. In the field of malaria, monoclonal antibody drugs CIS43LS and L9LS are two preventive monoclonal antibodies targeting CSP antigens that have entered the clinical research stage. Among them, CIS43LS is an antibody targeting the junction region of Plasmodium falciparum circumsporozoite protein, which prolongs the serum half-life through LS mutation of the Fc region. Preliminary clinical trials show that CIS43LS has good safety and preventive efficacy in preventing malaria infection. L9LS is a second-generation anti-malaria monoclonal antibody, which is 3 times more potent than CIS43. L9LS also increases the half-life through LS mutation modification of the Fc region, which can target the small NVDP gene repeats on the Plasmodium falciparum circumsporozoite protein, achieve cytolysis of sporozoites and prevent liver cell infection. Clinical trial results show that L9LS has high efficiency in preventing malaria infection.

[0004] However, the monoclonal antibodies currently in clinical research are mainly antibodies targeting Plasmodium liver infection and mosquito infection stages, and their main functions are transmission blocking and prevention of infection. However, in areas with high population density and high mosquito prevalence, a large portion of the population cannot avoid Plasmodium infection and will develop to the red inner stage, showing clinical symptoms and death. Therefore, the development of therapeutic antibodies targeting the only pathogenic period of malaria infection is particularly important, especially in the case of Plasmodium resistance to traditional treatment methods. The development of highly effective therapeutic antibodies can effectively reduce the mortality rate of malaria.

[0005] The currently approved malaria vaccine RTS, S / AS01 (Mosquirix TM) and its iterative product R21, are a class of subunit vaccines based on CSP protein, which works by preventing P. falciparum infection in the liver; however, the RTS, S / AS01 vaccine only shows partial effect (about 30%~50%) in phase II / III clinical trials, and the protection effect of R21 is only 66%, both of which have not reached the minimum requirement of WHO; therefore, there is an urgent need for a vaccine that can protect the immunized from the infection of Plasmodium and the occurrence of clinical symptoms.

[0006] It has been proven in the prior art that antigen immunization can induce the production of effective antibodies that inhibit the growth and development of the blood stage of Plasmodium, among these antigens, the growth inhibition effect of antibodies produced by immunization of apical membrane antigen 1 (PfAMA1) is the best, therefore, PfAMA1 has always been the focus of research on blood stage malaria vaccine; however, further research has found that antibodies against PfAMA1 are only effective at very high concentrations; and due to the genetic diversity of AMA1 protein, PfAMA1 induces antibodies against specific Plasmodium strains, which have poor broad-spectrum neutralization (A.L. Goodman, S.J. Draper, Ann. Trop. Med. Parasitol. 104, 189 (2010)); in addition, in addition to the limitation of antigens, specific adjuvants are also needed to induce sufficient antibody response, so as to produce effective immune response in human trials, which makes the development of vaccine more complex and increases the risk of side effects; therefore, developing a new type of vaccine that can produce strong immune response without excessive dependence on adjuvants is a key challenge in current malaria vaccine research.

[0007] It has been found that reticulocyte binding protein homolog 5 (PfRH5) is a potential more effective candidate for malaria vaccine antigen (WO2012 / 114125); the reticulocyte binding protein homolog (PfRH) family contains six members, PfRH1, PfRH2a, PfRH2b, PfRH3, PfRH4 and PfRH5, each of which participates in the binding of Plasmodium to red blood cells except PfRH3; PfRH is an adhesin on the surface of the schizont form of Plasmodium, which binds to the receptors on the surface of red blood cells, thereby helping Plasmodium successfully invade red blood cells; antibodies induced by PfRH5 can very efficiently inhibit the growth and development of P. falciparum in the blood stage in vitro, which is better than the effect of antibodies induced by AMA1, and is still effective at a lower concentration, in addition, the antibodies induced by PfRH5 are also effective against genetic diversity strains of Plasmodium; therefore, PfRH5 is a promising candidate antigen for malaria vaccine.

[0008] Currently, monoclonal antibodies against RH5 use truncated RH5 expressed in vitro as antigens. These truncated RH5s contain the core structural regions from PfRH5, and the monoclonal antibodies selected after immunization have better expression characteristics and thermal stability, which is conducive to the industrial production of subunit vaccines.

[0009] However, recent research shows that natural malaria infection can induce rare but potent neutralizing antibodies against RH5. These antibodies target the same protein sites as those induced by potent vaccines, suggesting that full-length RH5 protein may be more effective as an antigen for immunization. However, due to the complexity of the protein structure, full-length RH5 is difficult to express in vitro, and truncated RH5 proteins may have some structural differences from the full-length protein, which will lead to differences in immunogenicity. Therefore, the development of vaccines against RH5 and the screening of monoclonal antibodies should focus on solving the problems of full-length protein expression and the screening of highly effective adjuvants, and using new adjuvants and full-length proteins for immunization to obtain more effective immune effects and screen for more effective monoclonal antibodies. Summary of the Invention

[0010] The purpose of this invention is to provide a monoclonal antibody based on the Plasmodium falciparum antigen PfRH5 and its application in the treatment of malaria.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] One of the technical solutions of this invention:

[0013] A method for preparing a monoclonal antibody based on the Plasmodium falciparum antigen PfRH5 includes the following steps:

[0014] An RH5 mRNA vaccine was prepared using mRNA vaccine technology, and then monoclonal antibodies based on the Plasmodium falciparum antigen PfRH5 were screened using hybridoma B cell technology.

[0015] Furthermore, the sequence of the RH5 mRNA vaccine is T7 promoter-5'UTR-RH5 CDS-3'UTR-polyA.

[0016] Furthermore, in the RH5 CDS, the amino acid sequence of RH5 is shown in SEQ ID NO:1; SEQ ID NO:1

[0017] MIRIKKKLILTIIYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKDDIKNGKDIKKEIDNDKENIKTNNAKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNNEDYKNVDYKNVNFLQYHFKELSNYNIANSIDILQEKEGHLDFVIIPHYTFLDYYKHLSYNSIYHKSSTYGKCIAVDAFIKKINETYDKVKSKCNDIKNDLIATIKKLEHPYDINNKNDDSYRYDISEEIDDKSEETDDETEEVEDSIQDTDSNHTPSNKKKNDLMNRTFKKMMDEYNTKKKKLIKCIKNHENDFNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRYHYDEYIHKLILSVKSKNLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHIFNRIEYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPLTQ.

[0018] Further, in the RH5 CDS, the nucleotide sequence of the CDS is shown as SEQ ID NO: 2; the nucleotide sequence of the RH5 CDS is shown as SEQ ID NO: 2

[0019]

[0020] Further, the preparation method of the RH5 mRNA vaccine comprises the following steps:

[0021] 1) Construction of in vitro transcription plasmid:

[0022] On the basis plasmid pUC57 for in vitro transcription mRNA vaccine, each DNA expression element of mRNA vaccine is inserted by molecular biology method to obtain an in vitro transcription plasmid;

[0023] 2) In vitro transcription and capping of RH5 mRNA vaccine:

[0024] The in vitro transcription plasmid obtained in step 1) is subjected to enzyme cutting using restriction endonuclease BspQ1 to obtain a linearized plasmid; purification is performed using a conventional DNA purification method to obtain an in vitro transcription template; the in vitro transcription template is subjected to transcription using a T7 High Yield RNA Transcription Kit with CAP1 GAG kit, and CAP1 GAG (3' OMe) cap analogs are incorporated during the transcription process to obtain 5' cap and 3' poly A tail modified mRNA; lithium chloride purification is performed;

[0025] 3) Lipid nanoparticle packaging mRNA:

[0026] Cationic lipids, phosphatidylcholine, cholesterol and PEG lipids are mixed in a molar ratio of 50:10:38.5:1.5, and the Nanoassemblr Benchtop nanoliposome packaging instrument is used to mix and package the 5' cap and 3' poly A tail modified mRNA obtained in step 2); after the packaging is completed, the buffer solution is replaced with PBS using centrifugation or dialysis method to obtain the RH5 mRNA vaccine.

[0027] Further, the hybridoma B cell technology comprises the following steps:

[0028] 1) Sp2 / 0 cell recovery culture:

[0029] ① Take out the sp2 / 0 cells in the liquid nitrogen, immediately place in a 37℃ water bath kettle, transfer to a 50mL centrifuge tube, take three times the volume of 10% 1640 culture medium, slowly drop and shake, centrifuge at 300g for 3min to remove the supernatant, resuspend with 10mL culture medium, transfer to a 10cm culture dish, and place in an incubator for culture;

[0030] ② Sp2 / 0 cell expansion culture: after 24 h of cell recovery, observe the cell state, the cell membrane is uniformly bright and the size is uniform, shake the culture dish simply, suspend the cells with poor state and dead cells, carefully aspirate the supernatant, add 10 mL of culture medium and incubate, observe and replace the liquid every 24 h, until the cells expand to 80% to 90% of the culture dish bottom, after replacing the liquid, blow gently with a pipette and transfer to a T75 culture medium, supplement 15 mL of 10% 1640 culture medium, incubate, and subculture after 2 to 3 days at a ratio of 1:3 to 1:4;

[0031] 2) Preparation of feeder layer cells:

[0032] ① One day before fusion, prepare 3 to 5 balb / c blank mice, soak in 75% alcohol for 5 min for surface disinfection, fix the mouse abdomen upwards, fix the limbs and tail on the foam board with a fixed needle, and cut the outermost abdominal skin with sterile dissection tools and tear it open with forceps and fix it on the foam board;

[0033] ② Take 10 mL of preheated serum-free 1640 culture medium with a 10 mL syringe, replace it with a 1 mL syringe needle, first slowly push 5 mL of culture medium into the mouse abdominal cavity;

[0034] ③ Take another new 10 mL syringe, replace it with a 1 mL syringe needle, avoid the fat layer of the abdominal cavity, and suck the culture medium in the mouse abdominal cavity;

[0035] ④ Repeat steps ② to ③ by injecting 5 mL of serum-free culture medium again;

[0036] ⑤ After removing the needle, transfer the abdominal cavity cell culture medium to a 50 mL centrifuge tube, centrifuge at 300 g for 5 min, resuspend by blowing after adding 30 mL of HAT 1640 culture medium, and culture in 3 pieces of 96-well plate culture dishes, and incubate overnight in an incubator;

[0037] 3) Preparation of mouse spleen cells:

[0038] ① 3 to 7 days before the cell fusion experiment, select mice with high neutralization titer, and inject RH5 protein plus immune into the tail vein;

[0039] ② On the day of fusion, dissect the mouse, disinfect, cut open the peritoneal membrane, remove the spleen, remove the connective tissue and fat layer around the spleen, grind the head end with a sterile syringe, add 5 mL of 1640 culture medium several times in the middle, aspirate the grinding product, filter it through a 100 μm cell screen into a 50 mL centrifuge tube, until the spleen tissue changes from red to white, only the connective tissue is left, the remaining tissue is filtered, the 1640 culture medium and residual cells are collected;

[0040] ③Centrifugal to supernatant after washing with 10 mL PBS, add 3 mL red blood cell lysis solution, room temperature for 2 min, then add 9 mL 10% 1640 medium to terminate the reaction; 500g 3min centrifugal to supernatant, washed with 10% 1640 medium twice, and through 40 μm mesh to remove the cell cluster, resuspended with 10 mL 10% 1640 medium, and counted with red blood cell counter;

[0041] 4) Mouse spleen-SP2 / 0 cell fusion:

[0042] ① Prepare 1L of sterile pure water at 37℃, keep constant temperature in water bath before fusion;

[0043] ② Shake T75 flask to make sp2 / 0 cells fall off, collect into 50mL centrifuge tube, 300g 3min centrifugal to supernatant, washed with PBS twice, then resuspended with 10 mL 10% 1640 medium, and counted;

[0044] ③ Mix all the number of mouse spleen cells and the corresponding number of sp2 / 0 cells, 500g 3min centrifugal to supernatant, and tap the sedimented cells;

[0045] ④ Take out the preheated 37℃ pure water, place the cell tube bottom under the 37℃ pure water surface, use pipette to suck 1 mL PEG, add to the cells at a constant speed, then continue to shake the cells, and add 19 mL pure 1640 medium to terminate the reaction;

[0046] ⑤ Centrifugal the cells with 300g for 3min to remove the supernatant, resuspend the cells with 30 mL HAT1640 medium and plate into 96-well plates, 200uL medium per well, incubate in the incubator for 9-10 days, without changing the liquid and supplementing the liquid, ensure the water tank is full of water to reduce evaporation of the medium;

[0047] 5) Detection after fusion:

[0048] ① 2-3 days after fusion, cell clusters can be observed under a microscope, and cells that have not fused successfully die, marked with marker pen to indicate whether there are cell clusters and the number of cell clusters;

[0049] ② 8 days after fusion, take 50uL cell supernatant for ELISA detection, and add 50uL HAT medium to the original well to prevent cell death;

[0050] ③ According to the ELISA detection results, transfer the cells with OD>1 and 0.4<OD<1 to 24-well plates, add 1 mL HT1640 medium, and incubate;

[0051] IV. After the hybridoma cells grow to 80% in the 24-well plate, the supernatant is removed, 500 uL HT1640 medium is added, and cell counting is performed, 100 cells are plated in a 96-well plate, and the rest of the cells are transferred to a 6-well plate supplemented with 2 mL of HT1640 medium, and after growing to 80%, the supernatant is removed, the cells are resuspended with 1 mL of medium, 100 cells are plated in a 96-well plate, and the rest of the cells are frozen in 2 tubes, which are labeled as positive or weakly positive parent clones;

[0052] IV. In this way, the positive clones are continuously diluted and monoclonally cultured by limited dilution method until ELISA detection of the whole page positive.

[0053] The second technical solution of the present application is:

[0054] The monoclonal antibody based on the Plasmodium falciparum antigen PfRH5 prepared by the preparation method of the monoclonal antibody based on the Plasmodium falciparum antigen PfRH5.

[0055] The third technical solution of the present application is:

[0056] The application of the monoclonal antibody based on the Plasmodium falciparum antigen PfRH5 in the treatment of malaria.

[0057] Compared with the prior art, the present application has the following beneficial effects:

[0058] The monoclonal antibody based on the Plasmodium falciparum antigen PfRH5 provided by the present application successfully solves the problem of RH5 expression difficulty by combining mRNA vaccine technology, and successfully uses RH5 with natural conformation for immunization, and combines hybridoma B cell technology to successfully screen a plurality of monoclonal antibodies with high inhibition activity, and these antibodies have a brand-new antigen epitope, which will provide a new scheme for the treatment of red inner stage malaria;

[0059] The present application combines mRNA vaccine technology and monoclonal antibody screening technology, introduces nucleic acid sequences into cells, expresses proteins through intracellular elements, makes the structure and function of Plasmodium protein (RH5) in the natural state closer to the natural state, can produce antibodies closer to natural infection, thereby providing higher immunogenicity and producing more effective antibodies;

[0060] The present application uses the natural adjuvant properties of LNP, and immunization by mRNA vaccine can more effectively stimulate the immune system and produce more antibodies;

[0061] The application screens a plurality of monoclonal antibodies through hybridoma B cell technology, determines that the antibodies have a brand-new epitope through structural biology means, confirms a brand-new RH5 antigen epitope, and comprehensively analyzes effective antigen epitopes of RH5 in combination with antibody in-vitro neutralization capacity, thereby providing a basis for subsequent vaccine design and antibody combination taking RH5 as a target. BRIEF DESCRIPTION OF DRAWINGS

[0062] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The detailed description is made with reference to the accompanying drawings.

[0063] Figure 1 Immunoblotting experiment result graph for RH5 mRNA vaccine protein;

[0064] Figure 2 Animal immunization program and sampling time schematic diagram;

[0065] Figure 3 Indirect enzyme-linked immunosorbent assay result graph for RH5 mRNA vaccine;

[0066] Figure 4 Total IgG growth inhibition effect evaluation result graph of RH5 mRNA vaccine on red blood stage plasmodium after immunization;

[0067] Figure 5 Positive monoclonal screening flow chart;

[0068] Figure 6 45 heavy-light chain combinations obtained from 28 positive monoclonal antibodies;

[0069] Figure 7 Growth inhibition verification result graph of 16 monoclonal antibodies;

[0070] Figure 8 Competitive Elisa experiment result graph of 10 representative monoclonal antibodies;

[0071] Figure 9 Group division result graph of 10 representative monoclonal antibodies;

[0072] Figure 10 P20-2 hydrogen-deuterium exchange experiment result graph. DETAILED DESCRIPTION

[0073] The following detailed description of various example embodiments of the application will not be considered limiting of the application, but rather as a description of certain aspects, features and embodiments of the application. It is understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application.

[0074] In addition, for numerical ranges recited herein, every integer value within the range is specifically included. For ranges recited in terms of "between," every intervening value between the recited values is specifically included. For ranges recited in terms of "between" it should be understood that every intervening value within the range is specifically included and that the endpoints are specifically contemplated. These are only examples of what is specifically recited herein and are not intended to limit the application in any way.

[0075] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in further detail the methods and / or materials associated with the documents. In the case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0076] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all such modifications and variations warrant the patentable subject matter under the patent laws. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0077] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0078] Example 1

[0079] Construction, in vitro preparation and packaging of RH5 mRNA vaccine

[0080] Construction, in vitro preparation and packaging of RH5 mRNA vaccine were performed according to the sequence: T7 promoter-5'UTR-RH5 CDS-3'UTR-polyA, which specifically included the following steps:

[0081] 1) Construction of in vitro transcription plasmid:

[0082] On the basis plasmid pUC57 (purchased from Nanjing Kingsri Biotechnology Co., Ltd.) for in vitro transcribed mRNA vaccine, the DNA expression elements of mRNA vaccine were inserted by molecular biology methods, including: T7 promoter, 5' end UTR sequence upstream of coding region (5' UTR), DNA coding region including RH5 and CDS (RH5 CDS, wherein the amino acid sequence of RH5 is shown as SEQ ID NO: 1, and the nucleotide sequence of CDS is shown as SEQ ID NO: 2), 3' end UTR sequence downstream (3' UTR), polyadenylic acid tail (polyA), to obtain an in vitro transcription plasmid;

[0083] SEQ ID NO: 1

[0084] MIRIKKKLILTIIYIHLFILNRLSFENAIKKTKNQENNLTLLPIKSTEEEKDDIKNGKDIKKEIDNDKENIKTNNAKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNEKNDVKNNEDYKNVDYKNVNFLQYHFKELSNYNIANSIDILQEKEGHLDFVIIPHYTFLDYYKHLSYNSIYHKSSTYGKCIAVDAFIKKINETYDKVKSKCNDIKNDLIATIKKLEHPYDINNKNDDSYRYDISEEIDDKSEETDDETEEVEDSIQDTDSNHTPSNKKKNDLMNRTFKKMMDEYNTKKKKLIKCIKNHENDFNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRYHYDEYIHKLILSVKSKNLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHIFNRIEYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDHLRQMLYNTFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPLTQ;

[0085] SEQ ID NO: 2

[0086]

[0087] 2) In vitro transcription and capping of RH5 mRNA vaccine:

[0088] The in vitro transcription plasmid obtained in step 1) was digested using restriction enzyme BspQ1 to obtain a linearized plasmid; purification was performed using a conventional DNA purification method to obtain an in vitro transcription template; the in vitro transcription template was transcribed using a T7 High Yield RNA Transcription Kit with CAP1 GAG(3’OMe) kit (E139, Suzhou Epiomics Technologies Co., Ltd.), and CAP1 GAG(3’OMe) cap analogs were incorporated during transcription to obtain 5’ cap and 3’ poly A tail modified mRNA; lithium chloride purification and RiboGreen dye RNA quantification were performed;

[0089] 3) Lipid nanoparticle (LNP) packaging of mRNA:

[0090] Cationic lipids, phosphatidylcholine, cholesterol, and PEG lipids were mixed in a molar ratio of 50:10:38.5:1.5, and the Nanoassemblr Benchtop nanoliposome packaging instrument (Precision Nano Systems) was used to mix and package the 5’ cap and 3’ poly A tail modified mRNA obtained in step 2); after packaging was complete, the buffer solution was changed to PBS using centrifugation or dialysis to obtain the RH5 mRNA vaccine.

[0091] The mRNA packaging efficiency was identified using a Quan-iT Ribogreen RNA reagent kit (Thermo Fisher), and the packaging efficiency met the standard of mRNA vaccine after identification;

[0092] The sequence information, length, and delivery system of the RH5 mRNA vaccine are shown in Table 1.

[0093] Table 1 Sequence information, length, and delivery system of RH5 mRNA vaccine

[0094]

[0095] Example 2

[0096] In vitro expression and identification of RH5 mRNA vaccine

[0097] The mammalian cell 293T is transfected with the RH5 mRNA vaccine, the transfection dose is 5 μg, and the same amount of LNP is transfected as a negative control; before transfection, the cell morphology is observed to be normal, the size is uniform, there are basically no dead cells, and the cell density is 70% to 80%; 50 μL of the RH5 mRNA vaccine (5 μg) + 450 μL of opti-MEM is prepared for each hole of the six-hole plate, a total of 500 μL of solution, which is mixed uniformly after being blown and then is left to stand for 5 min; during the standing, the cell supernatant is sucked off, 1 mL of PBS is added to wash away the residual culture medium, and the premixed opti-mRNA solution is added, 500 μL per hole, slowly along the wall of the dish, and is left to stand in a 37°C incubator for 8 h; after 8 h, the liquid is changed, 2 mL of DMEM complete culture medium containing 10% FBS is added after the supernatant is sucked off, and is left to stand for culture at 37°C; after 12 h, 24 h and 48 h of transfection, a protein sample is prepared, and the in-vitro expression of the malaria mRNA vaccine is detected by Western blot (WB) experiment; the specific method is as follows:

[0098] 1) Protein sample preparation:

[0099] ① The culture medium is sucked off, 1 mL of 4°C pre-cooled PBS (Gibco) is added to each hole, the cells are washed gently by shaking, and then the washing liquid is discarded, and the cells are washed three times, finally, 1 mL of PBS is used to blow down the adherent 293T cells and is transferred to a new 1.5 mL EP tube;

[0100] ② The protein sample is broken by non-contact ultrasonic crushing (3 s on, 5 s off) for 5 min;

[0101] ③ 5× protein loading is added to the protein sample to a final concentration of 1×, which is mixed and boiled at 100°C for 10 min, and is cooled to room temperature for standby.

[0102] 2) Western blot:

[0103] ① Loading: 10 μL of the protein sample is loaded per hole to ensure that the sample is completely added to the SDS-PAGE gel hole;

[0104] ② Electrophoresis: 80 V for 30 min, 120 V for 60 min, and the electrophoresis is terminated when the bromophenol blue just runs out, and then the membrane is transferred;

[0105] ③ Membrane transfer: the commercial rapid membrane transfer solution (20×) (NCM Biotech, product number: WB4600) is used for membrane transfer; 1× rapid membrane transfer solution (1000 mL) is prepared by adding 50 mL of rapid membrane transfer solution (20×) to 85 mL of deionized water and 100 mL of absolute ethanol; the transfer sandwich structure is prepared, and then is transferred to the electrophoresis tank, a constant current of 400 mA is set, and the electrophoresis time is 30 min to complete the protein membrane transfer;

[0106] IV. Blocking: 5% skim milk was prepared using 1x TBST, make sure the skim milk is mixed well without particles, put the PVDF membrane with transferred protein into the WB incubation box with 5% skim milk blocking solution, shake at room temperature for 1h;

[0107] V. Primary antibody: the specific antisera obtained from the third immunization of the pre-immune malaria RH5 and CSP mRNA vaccine were used as the primary antibody of the western blot experiment, the dilution ratio of the primary antibody was 1:1000, shake incubate at 4℃ overnight;

[0108] VI. Membrane washing: wash with 1x TBST for four times, each time for 10min;

[0109] VII. Secondary antibody: use HRP labeled goat anti-mouse IgG secondary antibody diluted at 1:5000, shake incubate at room temperature for 1h;

[0110] VIII. Membrane washing: wash with 1x TBST for four times, each time for 10min;

[0111] IX. Development: use commercial ECL kit for development, take pictures to save the results;

[0112] The results of the western blot experiment of the RH5 mRNA vaccine are shown in Figure 1 ;

[0113] As can be seen from Figure 1 , the protein expression amount of the RH5 mRNA vaccine is the highest at 12h after transfection, and gradually decreases with time.

[0114] Example 3

[0115] Detection of antibody titer after immunization of RH5 mRNA vaccine

[0116] BALB / c female mice of 6-8 weeks old (purchased from Vivotec Labs) were used for animal experiments of the RH5 mRNA vaccine, and the animal immunization program and sampling time are shown in Figure 2 ;

[0117] The experimental group was immunized with RH5 mRNA, and immunized once every three weeks, and blood was collected two weeks after each immunization to prepare serum; the specific antibody titer was detected by indirect enzyme-linked immunosorbent assay (ELISA), and the specific experimental method is as follows:

[0118] 1) Antigen coating: dilute RH5 antigen with carbonate buffer (0.05M, pH=9.6), 2μg / mL, 50μL / well, add to a polyvinyl chloride 96-well microplate, gently shake to mix, 50μL of antigen coating solution evenly covers the bottom of the well, 4℃ incubate overnight;

[0119] 2) Blocking: Remove the plate, discard the coating solution, wash three times with PBST (PBS containing 0.05% Tween-20), add 1% BSA (PBS preparation) to the 96-well plate, 50 μL / well, incubate at 37℃ for 1 h, wash three times with PBST, and pat dry;

[0120] 3) Incubation with primary antibody: Serum samples were serially diluted from 1:100, and then the diluent was added to each well at 50 μL / well. After incubation at 37°C for 2 h, the samples were washed three times with PBST and patted dry.

[0121] 4) Incubation with secondary antibody: Add 50 μL / well of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG diluted 1:5000 to a 96-well plate, incubate at 37°C for 1 h, wash three times with PBST, and pat dry.

[0122] 5) Color development: Add 50 μL of TMB color development solution to a 96-well plate and incubate at room temperature in the dark for 8–10 min.

[0123] 6) Termination of reaction: Add the stop solution to the 96-well plate, 50 μL / well;

[0124] 7) OD value measurement: The absorbance (OD value) at a wavelength of 450nm was measured using an ELISA reader; the endpoint titration was defined as the serum dilution factor corresponding to the serum absorbance at 450nm being greater than that of negative serum by 2.1 times.

[0125] Results of the indirect enzyme-linked immunosorbent assay (ELISA) for RH5 mRNA vaccine are as follows: Figure 3 As shown;

[0126] Depend on Figure 3 It can be seen that the RH5 mRNA vaccine highly activates the mouse immune system after the second and third immunizations (to better illustrate the antibody titers of the first, second, and third immunizations, Figure 4 The image shows the absorbance of a serum sample at a wavelength of 450 nm when diluted 10,000 times.

[0127] Example 4

[0128] Evaluation of the inhibitory effect of total IgG on the growth of intraerythrocytic stage Plasmodium after RH5 mRNA vaccine immunization

[0129] Antiserum IgG was purified two weeks after the third immunization using the NAb Protein GSpin Kit (0.2 mL, Thermo Scientific, catalog number: 89949). The antibody buffer was replaced with RPMI 1640, and the concentration was determined for later use. The inhibitory effect of RH5 mRNA vaccine-specific antibodies on the growth of Plasmodium erythrophagus was then evaluated. The specific method is as follows:

[0130] 1) Prepare fresh 2× complete culture medium (CM) (containing 20% ​​human serum, 2× sodium bicarbonate, and 2× gentamicin) and store it at 37℃ for later use. For example, to prepare 50mL of 2×CM: 45mL CM + 5mL human serum + 1.6mL 7.5% sodium bicarbonate solution + 50μL 10mg / mL gentamicin solution, and sterilize with a 0.22μm filter;

[0131] 2) Synchronize Plasmodium falciparum using the Percoll / Sorbitol method to obtain highly synchronized late-stage Plasmodium falciparum (40-44h) for growth inhibition assay (GIA); synchronize most of the Plasmodium falciparum that are older than 38h with 40%-60% Percoll / Sorbitol, and then synchronize with 5% Sorbitol for 4-14h. When the Plasmodium falciparum grows to 40-44h, perform the GIA experiment.

[0132] 3) The GIA system consisted of 40 μL / well, with a volumetric saturation of 1% and an initial parasite rate of 0.3% ± 0.1%. The purified IgG was diluted according to its concentration to ensure that the volume added to the immunization wells was ≥ 5 μL. The final concentration of IgG in the immunization wells and blank wells was 500 μg / mL. RPMI 1640 was added to 20 μL. Then, 20 μL of Plasmodium falciparum suspension (formulation: 2×CM with a parasite rate of 0.3% ± 0.1% and a volumetric saturation of 2%) was added to each well. The immunization wells contained IgG purified from serum after immunization with malaria mRNA vaccine, the blank wells contained IgG purified from serum after immunization with 1×PBS, and the control wells contained no IgG. Three replicate wells were set up.

[0133] 4) After 40–48 hours, centrifuge and discard the supernatant. Use a 10 μL pipette to aspirate the red blood cell precipitate onto a clean glass slide to prepare a monolayer blood smear. Perform Giemsa staining, calculate the infection status of 5000 red blood cells, and perform statistical analysis to calculate the inhibition rate.

[0134] The evaluation results of the inhibitory effect of total IgG on the growth of Plasmodium erythrocytic stage after RH5 mRNA vaccine immunization are as follows: Figure 4 As shown;

[0135] Depend on Figure 4 It can be seen that the growth inhibition rate of Plasmodium in the RH5 immunization group was 91.562%, which was significantly better than the neutralizing antibody (mAb-RA-Cell2019) produced by RH5-based recombinant protein vaccine immunization that has entered the clinical research stage. Moreover, after the third immunization, the specific RH5 antibody had a high inhibitory effect on Plasmodium falciparum from different clinical sources, which indicates that the RH5 antibody has a good broad spectrum.

[0136] Example 5

[0137] Hybridoma B cell preparation and positive monoclonal screening

[0138] Select the mouse with the highest inhibition of RH5 antibody titer and the strongest antiserum neutralization ability as the experimental object, prepare the spleen cells from the mouse spleen, use the peritoneal macrophages of blank mice as the feeder layer cells, fuse with sp2 / 0 tumor cells, and continuously perform subsequent positive screening, the specific method is as follows:

[0139] Main materials: myeloma cells sp2 / 0, spleen cells (immune completed mice), mouse peritoneal macrophages (blank mice), 1640 culture medium (gibco);

[0140] Other materials: mouse dissection tools, fixed needle, 40 or 100 μm cell sieve (corning), T75 culture bottle, 10 cm culture dish, 96 well cell culture plate, 37°C 5% CO2 incubator, fetal bovine serum (gibco), penicillin-streptomycin double antibody (gibco), polyethylene glycol 1500 (roche), 50xHAT (sigma), 50xHT (sigma), PSB (gibco);

[0141] Solution configuration:

[0142] 1640 culture medium: without other added ingredients;

[0143] 10% 1640 culture medium: 500 mL 1640 culture medium + 50 mL fetal bovine serum + 5 mL P / S double antibody, store at 4°C in the dark, and preheat at 37°C for 10 min before use;

[0144] 50xHAT solution: 50xHAT powder in one bottle, dissolved in 10 mL PBS and filtered through a 0.45 μm filter membrane;

[0145] 50xHT solution: 50xHT powder in one bottle, dissolved in 10 mL PBS and filtered through a 0.45 μm filter membrane;

[0146] HAT1604 culture medium: 500 mL 1640 culture medium + 100 mL fetal bovine serum + 5 mL P / S double antibody + 10 mL 50xHAT solution, store at 4°C in the dark, and preheat at 37°C for 10 min before use;

[0147] HT1604 culture medium: 500 mL 1640 culture medium + 50 mL fetal bovine serum + 5 mL P / S double antibody + 10 mL 50xHT solution, store at 4°C in the dark, and preheat at 37°C for 10 min before use;

[0148] Experimental steps:

[0149] 1) Sp2 / 0 cell resuscitation culture:

[0150] ① Take out the sp2 / 0 cells in liquid nitrogen, immediately placed in 37°C water bath, transfer to 50mL centrifuge tube, take three times the volume of 10% 1640 medium slowly drop, shake, 300g 3min centrifugation, after removing the supernatant, with 10mL medium resuspended, transfer to 10cm culture dish, put into incubator and culture;

[0151] ② Sp2 / 0 cell expansion culture: after 24h of cell resuscitation, observe the cell state, the cell membrane is uniform and the size is uniform, shake the culture dish, make the state of the cell suspension, carefully suck the supernatant, add 10mL medium and culture, observe and replace liquid every 24h, until the cell expansion is 80% to 90% of the culture dish bottom, replace liquid with pipette, and transfer to T75 medium, add 15mL 10% 1640 medium, and culture, 2-3 days later, subculture at 1:3 to 1:4 (pipette blow or directly shake, no need for trypsin digestion);

[0152] 2) Preparation of feeder layer cells:

[0153] ① The day before fusion, prepare 3-5 balb / c blank mice, soak in 75% alcohol for 5min for surface disinfection, fix the mouse abdomen upwards, four limbs and tail on the foam board, use sterile dissection tools to cut the outermost abdominal skin and tear it open with forceps and fix it on the foam board;

[0154] ② Take 10mL preheated serum-free 1640 medium with 10mL syringe, replace 1mL syringe needle, first push 5mL medium into the mouse abdominal cavity;

[0155] ③ Take another new 10mL syringe, replace 1mL syringe needle, avoid fat layer in abdominal cavity, suck the medium in the mouse abdominal cavity;

[0156] ④ Repeat steps ② to ③ by injecting 5mL serum-free medium again;

[0157] ⑤ After removing the needle, transfer the abdominal cavity cell culture medium to a 50mL centrifuge tube, centrifuge at 300g for 5min, add 30mL HAT1640 medium, resuspend and blow evenly, then culture in 3 pieces of 96-well plate culture dish, and culture in incubator overnight;

[0158] 3) Preparation of mouse spleen cells:

[0159] ① 3-7 days before cell fusion experiment, select mice with high neutralization titer, inject RH5 protein plus immune in tail vein;

[0160] ② Fusion the same day dissection of mice, disinfection, cut open the peritoneal membrane after taking out the spleen, remove the connective tissue and fat layer around the spleen, with a sterile syringe push the head end grinding, multiple times in the middle of 5 mL 1640 culture medium, suction grinding products, filtered to 50 mL centrifuge tube with 100 μm cell screen, until the spleen tissue by red white, only connective tissue, end grinding, filter the remaining tissue, medium, collect 1640 culture medium, residual cells;

[0161] ③ After centrifugation to remove the supernatant with 10 mL PBS wash, add 3 mL red blood cell lysis solution, room temperature for 2 min, then add 9 mL 10% 1640 culture medium to stop the reaction; 500g 3min centrifugation to remove the supernatant, washed with 10% 1640 culture medium twice, and remove the cell aggregation through 40 μm screen, resuspended the cells with 10 mL 10% 1640 culture medium, and use the red blood cell counter to count the cells;

[0162] 4) Mouse spleen-SP2 / 0 cell fusion:

[0163] ① Prepare 1L of sterile pure water at 37℃, keep constant temperature in the water bath before fusion;

[0164] ② Shake the T75 culture bottle to make the sp2 / 0 cells fall off, collect into a 50 mL centrifuge tube, 300g 3min centrifugation to remove the supernatant, wash with PBS for 2 times, then resuspend the cells with 10 mL 10% 1640 culture medium, and count the cells;

[0165] ③ Mix all the number of mouse spleen cells and the corresponding number of sp2 / 0 cells, 500g 3min centrifugation to remove the supernatant, and tap the sedimented cells;

[0166] ④ Take out the preheated 37℃ pure water, place the cell tube bottom below the 37℃ pure water surface, use a pipette to suck 1 mL PEG, add it to the cells at a constant speed, then continue to shake the cells, and then add 19 mL pure 1640 culture medium to stop the reaction;

[0167] ⑤ Centrifuge the cells at 300g for 3min to remove the supernatant, resuspend the cells with 30 mL HAT1640 culture medium and plate into a 96-well plate, 200uL medium per well, incubate in the incubator for 9-10 days, do not change the liquid and do not supplement the liquid, ensure the water tank is full of water in the incubator to reduce evaporation of the culture medium;

[0168] 5) Detection after fusion:

[0169] ① 2-3 days after fusion, cell clusters can be observed under a microscope, cells that have not fused successfully die, and marker pen is used to mark the presence or absence of cell clusters and the number of cell clusters;

[0170] ②On the 8th day after fusion, 50 uL of cell supernatant was taken for ELISA detection, and 50 uL of HAT medium was added to the original hole to avoid cell death;

[0171] ③According to the ELISA detection results, the positive (OD>1) and weak positive (0.4<OD<1) cells were transferred to a 24-well plate, 1 mL of HT1640 medium was added, and static culture was carried out;

[0172] ④After the hybridoma cells grew to 80% in the 24-well plate, the supernatant was removed, 500 uL of HT1640 medium was added, and cell counting was carried out, 100 cells were plated in a 96-well plate (plating density 0.5 cells per plate, a total of two plates, 200 uL per well), the rest of the cells were transferred to a 6-well plate and supplemented with 2 mL of HT1640 medium, and after growing to 80%, the supernatant was removed, the cells were resuspended with 1 mL of medium, 100 cells were plated in a 96-well plate, and the rest was frozen in 2 tubes, labeled as positive (weak positive) parent clones.

[0173] ⑤In this way, the positive clones were continuously diluted and monoclonal cultured by limited dilution method until the whole plate was positive in ELISA detection, indicating that the clones before plating were positive monoclonal.

[0174] The positive monoclonal screening process is shown in Figure 5 ;

[0175] The 35 positive parent clones obtained by screening are shown in Table 2;

[0176] Table 2 35 positive parent clones

[0177]

[0178]

[0179] On the basis of the 35 positive parent clones obtained by screening, 28 positive monoclonals were screened by limited dilution method, as shown in Table 3;

[0180] Table 3 28 positive monoclonals

[0181]

[0182] Example 6

[0183] Monoclonal antibody lineage analysis and preparation of representative antibodies and effectiveness determination

[0184] A large number of positive monoclonal hybridoma cells screened and extracted RNA, cDNA by reverse transcription, combined with heavy and light chain universal primers for PCR to obtain hybridoma B cell antibody heavy and light chain sequence, by sequence analysis of the antibody lineage analysis and classification, in each lineage to select a representative antibody expression, heavy and light chain nucleic acid sequence cloned into a humanized vector for large-scale expression, after obtaining the antibody, by in vitro growth inhibition test, test the neutralizing ability of the antibody, finally, screening out the candidate antibody with broad neutralization performance, the specific method is as follows:

[0185] 1) Resuscitation has screened all positive monoclonal and expanded culture to 6 hole plate;

[0186] 2) by trypsin digestion to collect cells, and using Trizol to dissolve the cells, and then using the commercial RNA extraction kit for mRNA extraction;

[0187] 3) take 1ug of mRNA using commercial reverse transcription kit for reverse transcription reaction, obtain cDNA;

[0188] 4) using the conserved universal primer of antibody heavy and light chain nucleic acid sequence for PCR reaction, obtain the antibody heavy and light chain in each hybridoma B cell;

[0189] 5) obtain all the antibody heavy and light chain sequence information, find the use of these antibodies by Igblast VDJ gene, and divide these antibodies into different lineages;

[0190] 6) select the representative antibody of each lineage for expression and purification, and construct it into the antibody humanized expression plasmid vector;

[0191] 7) the plasmid encoding antibody light chain or heavy chain is extracted according to the HP Plasmid DNA Maxi Kit(Omega, D6922-02) manual, and then the plasmid encoding antibody heavy chain and light chain is co-transfected into 293F cells according to the molar ratio of 1:1;

[0192] 8) after continuous culture for 6 days, the cell supernatant is collected and the secreted antibody in the cell supernatant is affinity purified by protein A(Cytiva, 7040201) antibody affinity column, and finally the antibody obtained by affinity purification is replaced into PBS buffer by gel filtration chromatography, and stored in-80℃ refrigerator for standby;

[0193] 9) freshly prepared 2x complete culture medium(Complete culture medium, CM)(containing 20% human serum, 2x sodium bicarbonate, 2x gentamicin), stored at 37℃ for 6h;

[0194] 10) Synchronization of P. falciparum by 40%~60% Percoll / Sorbitol and 5% Sorbitol method to obtain highly synchronized late trophozoites and early late P. falciparum (34~40h) for Growth Inhibition Assay (GIA); first, most of P. falciparum at 38h or more is synchronized by 40%~60% Percoll / Sorbitol, and then 5% Sorbitol is used to synchronize the ring stage after 4~14h, and GIA is performed when P. falciparum grows to 34~40h;

[0195] 11) The GIA system is 40μL / well, the hematocrit is 1%, and the initial parasite rate is 0.3%±0.1%; the purified monoclonal antibody is diluted according to the concentration, and the volume added to the immune experimental well is ≥5μL, and RPMI 1640 is added to 20μL; then P. falciparum suspension (formula: 2×CM with a parasite rate of 0.3%±0.1% and a hematocrit of 2%) is added to each well at 20μL, and three repeated wells are set;

[0196] 12) After 40~48h, 120μL 1×PBS is used to wash twice, centrifugation is performed to discard 120μL supernatant, 120μL LDH substrate is added for incubation in the dark, 20min later, the plate is read by an enzyme label instrument, and finally statistical analysis is performed to calculate the inhibition rate.

[0197] As shown in Figure 6 , 45 heavy-light chain combinations are obtained from 28 positive monoclonal antibodies, through lineage analysis, the 45 heavy-light chain combination antibodies are divided into 16 lineages, combined with the expression of these antibodies in hybridoma B cells and 293T cells, 16 monoclonal antibodies are selected, each monoclonal antibody represents a lineage, and 16 monoclonal antibodies are shown in Table 4, and growth inhibition verification is performed using these monoclonal antibodies, and the growth inhibition verification results of the 16 monoclonal antibodies are shown in Figure 7 .

[0198] Table 4 16 monoclonal antibodies

[0199]

[0200]

[0201] As Figure 7 can be seen, the growth inhibition effect of monoclonal antibody P20-2 is optimal, and the inhibition effect is better than that of RH5 monoclonal antibody R5.016 which has entered the clinical research stage.

[0202] The heavy chain amino acid sequence of the monoclonal antibody P20-2 is shown in SEQ ID NO: 3, wherein the CDRs are three, respectively located at the 29th-36th, 54th-60th and 99th-112th amino acid residues;

[0203] SEQ ID NO: 3

[0204] VHSQVQMKESGPGLVQPSQSLSITCTVSGFPLSGYGVHWVRQSPGKGLEWLGVIWSGGSTDYNAAFISRLSITKDNSRSQVLFKMNSLQPNDTAIYYCARNHYYEYDWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC;

[0205] The light chain amino acid sequence of the monoclonal antibody P20-2 is shown in SEQ ID NO: 4, wherein the CDRs are three, respectively located at the 30th-35th, 53rd-55th and 92nd-100th amino acid residues;

[0206] SEQ ID NO: 4

[0207] VHSDVQITQSSSSSSVSLGDRVTITCRASEDIFNRLAWYQQKPGNAPRLLISGASSLESGVLSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWIIPYTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0208] Example 7

[0209] Analysis and identification of candidate antibody epitopes

[0210] The epitope competition between antibodies was analyzed by competitive ELISA, and the antigen binding epitopes of the 10 representative monoclonal antibodies further screened were analyzed by hydrogen-deuterium exchange mass spectrometry;

[0211] The 10 representative monoclonal antibodies further screened are shown in Table 5;

[0212] Table 5. 10 representative monoclonal antibodies screened further

[0213]

[0214] The specific steps of the antigen binding epitope analysis are as follows:

[0215] 1) Biotin labeling of 10 monoclonal antibodies and two control antibodies R5.004 and R5.016, respectively, first incubating RH5 protein with unmarked protein, then adding each antibody with Biotin labeling, respectively, and developing color to analyze the epitope competition relationship between each antibody;

[0216] 2) 2 μL of pfRH5 protein sample (40 μM) or pfRH5-antibody complex (40 μM) was added to 18 μL of D2O buffer (25 mM Tris-HCl, pD = 8.5, 100 mM NaCl) for deuterium labeling, then 20 μL of termination buffer (8 M urea, 0.5 M TCEP, pH = 3.0) was added to the above system at different time points 0 s, 10 s, 100 s, 1000 s, 10000 s to terminate the hydrogen-deuterium exchange reaction, and finally 60 μL of dilution buffer (1.5% FA, 50 mM TCEP) was added to the above system and stored in a -80 °C freezer after quick freezing with liquid nitrogen;

[0217] 3) In the mass spectrometry experiment, the above sample was thawed on ice and quickly injected into the HPLC-MS system (Thermo ultimate 3000) connected in series with a protease enzyme column (NovabioAssays, NBA2014002), a Trap column (ThermoFisher, 2100100) and a BioBasic C8 analysis column (BIOBASIC 8, 03-051-676). The peptide segments after enzyme treatment were captured by the Trap column and then entered the BioBasic C8 analysis column for separation. Subsequently, they were directly analyzed by Orbitrap Fusion mass spectrometer (Thermo) through electrospray ionization. The sequence identification of the peptide segments was carried out by mass spectrometry analysis of the non-deuterium sample (0 s time point sample). The mass spectrometry analysis was carried out in tandem MS / MS mode (orbi / orbi). All MS / MS spectra were searched for peptide segments using the MASCOT algorithm, with FDR set to 1%, thereby obtaining sequence identification of all peptide segments. Subsequent mass spectrometry analysis of deuterium samples at other time points was carried out in primary MS mode;

[0218] 4) Mass spectrometry data of all samples are input into HD-Examiner v2.3 (Sierra Analytics) software for processing. The m / z range, charge state, peptide retention time, and peptide coverage of each peptide are manually checked. HD-Examiner software corrects the mass spectrometry results of the fully deuterated samples and calculates the deuteration level (%D) of each peptide at different time points, thereby determining the antigen-binding epitope of each antibody.

[0219] The results of a competitive ELISA experiment using 10 representative monoclonal antibodies are as follows: Figure 8 As shown;

[0220] exist Figure 8 In the ELISA test, the larger the value and the lighter the color, the less competition there is; the smaller the value and the darker the color, the more competition there is. Based on the results of the competition ELISA test, the 10 representative monoclonal antibodies can be preliminarily divided into several different groups.

[0221] The grouping results of 10 representative monoclonal antibodies are as follows: Figure 9 As shown;

[0222] Depend on Figure 9 It can be seen that P20-2 is a separate group.

[0223] The results of the hydrogen-deuterium exchange experiment of P20-2 are as follows: Figure 10 As shown;

[0224] Depend on Figure 10 It is known that P20-2 recognizes I328-E341, which is located near the natural receptor Basigin epitope on the erythrocyte surface of pfRH5.

[0225] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

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