A multi-stage dual-target tandem malaria mRNA vaccine combining prevention and treatment

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a multi-stage dual-target tandem malaria mRNA vaccine for combined prevention and treatment; the multi-stage dual-target tandem malaria mRNA vaccine comprises a coding RNA with a nucleotide sequence as shown in SEQ ID NO:5; the present invention solves the problem of difficulty in expressing Plasmodium antigen protein through an LNP mRNA vaccine technology platform; the present invention creatively selects essential proteins of the liver stage and the erythrocytic stage after Plasmodium infects the human body as candidate antigens, and develops a "multi-stage-multivalent" tandem malaria mRNA sequence; the present invention solves the problems of low expression of effective antigen amount, low antibody titer produced by the vaccine, low protection rate, and short protection time through sequence optimization, linker selection and antigen combination order; at the same time, the vaccine has the dual effects of transmission blocking and post-infection treatment, and serves as a double insurance for the prevention and treatment of malaria.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a multi-stage dual-target tandem malaria mRNA vaccine combining prevention and treatment. Background Art

[0002] Malaria is a major global infectious disease that seriously endangers human health. With the increasing clinical resistance of various antimalarial drugs such as artemisinin, nearly half of the world's population is still at risk of malaria infection, which is an important obstacle to the sustainable development of today's society and the realization of the goal of a "human health community". How to effectively curb the prevalence and spread of malaria in tropical and subtropical countries and regions is a scientific problem that malaria researchers around the world urgently need to solve. Vaccines are the most effective strategy and tool for the prevention and control of infectious diseases. However, due to the complex life history of Plasmodium and the high variability of Plasmodium antigen proteins, research on malaria vaccines has been slow.

[0003] Malaria vaccine research mainly focuses on attenuated vaccines, inactivated vaccines, subunit vaccines, and nucleic acid vaccines. Malaria vaccines can be divided into the following categories based on the differences in the target stage of Plasmodium and the function of the vaccine: pre-erythrocytic vaccines, erythrocytic vaccines, transmission blocking vaccines, placental malaria vaccines, multi-stage multi-target vaccines, and whole-worm vaccines. Currently, the only malaria vaccine approved for marketing is the protein vaccine RTS,S that targets the Plasmodium liver antigen PfCSP, but it is limited in the population and has a low protective effect. RTS,S, the only malaria vaccine approved by the WHO for use in children in medium- and high-risk areas for malaria transmission, has been shown to have disadvantages such as high side effects and low protective efficiency.

[0004] Therefore, the development of new malaria vaccines is urgent. Summary of the invention

[0005] Currently, common malaria vaccines include Plasmodium falciparum liver-stage vaccine (RTS,S / AS01), erythrocytic stage recombinant protein vaccine and whole-worm attenuated vaccine candidate.

[0006] (1) Plasmodium falciparum liver stage vaccine (RTS,S / AS01)

[0007] RTS,S / AS01 (RTS,S) is the world's first malaria vaccine approved by the World Health Organization (WHO). It is a subunit vaccine that fuses the C'-terminal sequence of the circumsporozoite protein CSP of Plasmodium falciparum (including NANP antigen repeat sequence and T cell epitope sequence) with the surface antigen of hepatitis B virus (HBsAg) and assembles it into a virus-like particle structure, and enhances the immunogenicity of the vaccine through the new liposome immune adjuvant AS01. Since October 2021, RTS,S has been approved for use in children over 5 months old in medium- and high-risk areas of malaria transmission in Africa. However, according to data from phase 3 and 4 clinical studies in Africa, the RTS,S vaccine can only produce a protective efficiency of about 30% for specific children after four immunizations, and the effectiveness gradually fades after several months of vaccination. It is undeniable that RTS,S does not meet the official WHO standards for malaria vaccines (protection rate>50%, protection time>1 year).

[0008] (2) Erythrocytic recombinant protein vaccine

[0009] Apical membrane antigen-1 (AMA1), a membrane protein present in Plasmodium falciparum, is essential for the parasite's merozoites to complete the invasion process. Important progress has been made in the study of vaccines for AMA1. In one experiment, the AMA1-3D7 protein from Plasmodium falciparum was recombined with the adjuvant system AS02A (i.e., FMP2.1 / AS02A). This AMA1-based vaccine enhanced the growth inhibition of Plasmodium, but this effect was not related to the allele specificity of the malaria epidemic season. It was confirmed in field studies that although the AMA1 vaccine alone can produce an immune response to some homologous parasites, it failed to provide a protective effect in the homologous CHMI experiment, indicating that the vaccine has poor broad-spectrum protection.

[0010] (3) Whole-worm attenuated vaccine candidate

[0011] The principle of the whole-worm attenuated sporozoite vaccine is that irradiated attenuated sporozoites or genetically attenuated sporozoites can still invade liver cells normally, but their development in liver cells is blocked to varying degrees, and no erythrocytic stage malarial parasites will appear; the blocked erythrocytic stage malarial parasites can continue to stimulate the body's immune system, but can eliminate the inhibitory effect of erythrocytic stage malarial parasites on the body's immune response against erythrocytic stage malarial parasites. Attenuated sporozoites are directly inoculated into the human body intravenously, and have shown very good protection in clinical trials. Tests have shown that the whole-worm sporozoite vaccine can induce very high anti-CSP antibodies in the human body. However, the common problems of the above-mentioned attenuated sporozoite vaccines are: a. Since sporozoites cannot be cultured in vitro and can only be obtained by infecting mosquitoes, their sources are obviously limited; b. Sporozoites must be separated under sterile conditions and transported through a cold chain, which will affect their immunogenicity due to the reduced ability of sporozoites to infect liver cells during transportation; c. After one year, the protective efficiency of the attenuated sporozoite candidate vaccine dropped to below 60%, and field trials could not achieve an ideal protective immune response.

[0012] In addition, existing malaria vaccines have the following problems: (1) Since the beginning of this century, nearly 10 malaria vaccine research applications have been registered for clinical trials each year (data from the global clinical research database: https: / / clinicaltrials.gov / ), of which about 150 vaccine projects have been proven to have failed. Currently, only one vaccine based on the malarial parasite liver stage protein PfCSP (RTS,S / AS01, trade name MosquirixtM) has been approved for use. However, data from Phase III clinical studies in Africa showed that the RTS,S vaccine can only produce about 30% protection efficiency for specific children after four immunizations, and the effectiveness gradually fades after several months of vaccination. (2) Plasmodium antigens and pathogenic proteins are highly variable, and malarial parasites themselves have strong host immune escape. Traditional malaria vaccines designed based on a single target lack broad-spectrum effectiveness and long-term protection. (3) Plasmodium proteins often have long redundant sequences or repeated sequences, which are difficult to express in vitro, and the quality and immunogenicity of antigenic proteins are low. (4) Currently available multi-antigen combination vaccines are mostly a combination of mosquito-stage antigens and liver-stage antigens. The antibodies produced by the mosquito-stage antigens cannot play a good neutralizing role, nor can they simultaneously play the dual role of transmission blocking and post-infection treatment.

[0013] In order to solve the problems existing in the above-mentioned malaria vaccines, the present invention provides a multi-period dual-target tandem malaria mRNA vaccine that combines prevention and treatment. A technical solution close to the present invention is to develop an mRNA vaccine using the LNP system with PfCSP as the target. The other party used different peptide segments of PfCSP to design mRNA vaccines. Among the many types of vaccines, mRNA vaccines are undoubtedly the most beautiful dark horse. The development and application of the new crown mRNA vaccine marks the emergence of a new generation of biotechnology platforms. mRNA vaccines have broad application prospects. mRNA not only has the technical advantage of rapid response, but also can provide a more efficient and longer-lasting protection period.

[0014] The present invention provides a coding RNA for a vaccine for preventing and treating Plasmodium infection, wherein the coding RNA is RH5-linker-CSP;

[0015] The RH5-linker-CSP further comprises at least one heterologous 5' untranslated region and / or at least one heterologous 3' untranslated region; and at least one coding sequence operably linked to the 3' untranslated region and / or the 5' untranslated region, wherein the coding sequence encodes a coding sequence obtained by combining a tandem combination of a reticulocyte binding protein 5 derived from Plasmodium and one or more circumsporozoite proteins or other antigenic proteins;

[0016] Wherein, the RH5 is the reticulocyte binding protein 5 of Plasmodium, and the nucleotide sequence of the RH5 is shown in SEQ ID NO: 8;

[0017] The CSP is circumsporozoite protein, and the nucleotide sequence of the CSP is shown in SEQ ID NO:7.

[0018] Preferably, the linker in the RH5-linker-CSP is EAAAK3, GGGGS3 or T2A;

[0019] Or, the linker in the RH5-linker-CSP is a rigid, flexible and shearable linker of the same type as the EAAAK3, GGGGS3 or T2A;

[0020] Wherein, the nucleotide sequence of RH5-EAAAK3-CSP is shown in SEQ ID NO: 4;

[0021] The nucleotide sequence of RH5-T2A-CSP is shown in SEQ ID NO: 5;

[0022] The nucleotide sequence of RH5-GGGGS3-CSP is shown in SEQ ID NO:6.

[0023] Preferably, the malarial parasite is selected from Plasmodium falciparum, Plasmodium knowlesi, Plasmodium ovale, Plasmodium paraovale, Plasmodium vivax or Plasmodium cynomolgus.

[0024] Preferably, the Plasmodium falciparum is Plasmodium falciparum 3D7 strain.

[0025] The present invention also provides an RNA encoding a vaccine for preventing and treating Plasmodium infection, wherein the RNA encoding the vaccine for preventing and treating Plasmodium infection is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of the RH5-linker-CSP;

[0026] or a fragment or variant of any of these sequences.

[0027] The present invention also provides an RNA encoding a vaccine for preventing and treating Plasmodium infection, wherein the RNA encoding the vaccine for preventing and treating Plasmodium infection is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence expressed by the RH5-linker-CSP;

[0028] or a fragment or variant of any of these sequences.

[0029] The present invention also provides a vaccine encoding RNA for preventing and treating Plasmodium infection. The vaccine encoding RNA comprises RH5-linker-CSP, 5'-Cap and 3'-poly A tail.

[0030] Preferably, the 5'-Cap is m7G, cap0, cap1, cap2, modified cap0 or modified cap1 structure;

[0031] The 3'-poly A tail is a polyadenylic acid sequence containing 30-150 adenosine nucleotides.

[0032] The present invention also provides an mRNA vaccine, which comprises any one of the coding RNAs and a shell.

[0033] Preferably, the shell is a lipid nanolayer.

[0034] The present invention also provides a nucleotide sequence, which is used to transcribe any of the coding RNAs.

[0035] The present invention also provides a biological material for expressing any one of the coding RNA or the nucleotide sequence.

[0036] The present invention also proposes a multi-stage dual-target tandem malaria mRNA vaccine for combined prevention and treatment, wherein the multi-stage dual-target tandem malaria mRNA vaccine comprises the coding RNA according to claim 1, a 5'-Cap and a 3'-polyA tail; wherein the 5'-Cap is m7G, cap0, cap1, cap2, a modified cap0 or a modified cap1 structure;

[0037] The 3'-poly A tail is a polyadenylic acid sequence containing 30-150 adenosine nucleotides.

[0038] Preferably, the preparation method of the multi-stage dual-target tandem malaria mRNA vaccine is:

[0039] Inserting the RH5-linker-CSP and the DNA expression element into an expression plasmid to obtain a recombinant plasmid;

[0040] Using the recombinant plasmid to express and obtain tandem mRNA;

[0041] The tandem mRNA is packaged to obtain the multi-stage dual-target tandem malaria mRNA vaccine combining prevention and treatment.

[0042] Preferably, when packaging the tandem mRNA to obtain the multi-stage dual-target tandem malaria mRNA vaccine combining prevention and treatment, the method comprises:

[0043] The tandem mRNA is packaged using lipid nanoparticles to obtain the multi-stage dual-target tandem malaria mRNA vaccine that combines prevention and treatment.

[0044] Compared with the prior art, the invention has the following beneficial effects:

[0045] The present invention intends to develop a "multi-stage-multivalent" tandem malaria mRNA sequence that can simultaneously target the liver stage and the erythrocytic stage for candidate antigens at different stages of Plasmodium infection.

[0046] The present invention mainly solves the following difficult problems in the research and development of malaria vaccines:

[0047] (1) Due to the peculiarities of malarial parasite evolution, the virulence-related proteins of malarial parasites usually contain many redundant sequences and highly repetitive sequences, which makes it very difficult to express malarial parasite proteins in vitro, making it impossible to obtain high-quality antigen proteins for immunization. mRNA vaccine technology introduces nucleic acid sequences into cells and expresses proteins through intracellular elements, making the antigen proteins closer to the original state in terms of structure and modification, thereby providing higher immunogenicity.

[0048] (2) At present, malaria vaccines are mostly targeted at single-stage antigens, or multiple antigens at the transmission blocking stage. Due to the characteristics of malarial parasites such as immune escape and high antigen variability, vaccines targeting single antigens are difficult to have good protective effects. For multiple antigens at the transmission stage, it is usually a combination of mosquito stage antigens and liver stage antigens. The antibodies produced by mosquito stage antigen immunity can only be inhaled into the mosquito body through the process of mosquito bites to produce effects. As time goes by, the antibody content in the human body will become less and less, and the transmission blocking effect will become lower and lower. Therefore, vaccines with multiple antigen combinations at the transmission stage are also difficult to obtain efficient and lasting immune protection effects. As the most important liver stage and erythrocyte stage in malaria infection, there is currently no vaccine targeting these two stages of antigen combination. The present invention creatively combines the antigens of the two most critical stages of the human body during malarial parasite infection (liver stage is the initial stage of infection, and erythrocyte stage is the stage of malaria onset). The vaccine will take into account the two effects of transmission blocking and post-infection treatment. The antigens PfCSP and PfRH5 selected for the two stages are essential proteins for liver stage infection and erythrocyte infection, respectively.

[0049] (3) The present invention also finds the optimal combination order and linker sequence for the tandem expression of multiple antigens by optimizing the tandem sequence between multiple antigens and the linker sequence connecting multiple antigens, thereby ensuring that the mRNA vaccine can efficiently express multiple antigens in vivo and efficiently induce the body to simultaneously produce RH5 and CSP specific antibodies, thereby exerting an immune protection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a schematic diagram of the structure of the malaria mRNA vaccine in Example 1;

[0051] Figure 2 The results of detecting protein expression in 293T cells 12h, 24h and 48h after transfection of eight malaria mRNA vaccines in Example 2;

[0052] Figure 3The detection process and results of antibody titer after immunization with single-target RH5 and CSP mRNA vaccines in Example 3; wherein a is a schematic diagram of the immunization procedure and sampling time, and b is the absorbance value of the serum sample at a wavelength of 450nm when diluted 10,000 times;

[0053] Figure 4 The results of the persistence test of neutralizing antibodies in mice after immunization with the single-target RH5 and CSP mRNA vaccines in Example 4; wherein a is a schematic diagram of the immunization procedure and sampling time, and b is the titer test results of the RH5 and CSP mRNA immunization groups after immunization;

[0054] Figure 5 This is one of the antibody titer test results after immunization with the dual-target RH5 and CSP tandem mRNA vaccine in Example 5;

[0055] Figure 6 This is the second result of the antibody titer test after immunization with the dual-target RH5 and CSP tandem mRNA vaccine in Example 5;

[0056] Figure 7 The evaluation process and results of the inhibitory effect of RH5-specific antibodies in malaria mRNA vaccines on the growth of erythrocytic Plasmodium in Example 6; wherein, a is a schematic diagram of an experiment evaluating the inhibition of erythrocytic growth of Plasmodium falciparum by specific RH5 antibodies in malaria mRNA vaccines, b is a result of an experiment evaluating the inhibition of erythrocytic growth of Plasmodium falciparum by specific RH5 antibodies in malaria mRNA vaccines, and c is a result of evaluating the high inhibitory effect of specific RH5 antibodies on Plasmodium falciparum from different clinical sources after the third immunization with RH5-EAAAK3-CSP, RH5-T2A-CSP and RH5-GGGGS3-CSP mRNA vaccines;

[0057] Figure 8 The result diagram of the successful construction of the PbPfCSP-GFPLuc gene-modified insect strain in Example 7; wherein, a is a schematic diagram of the construction of the PbPfCSP-GFPLuc gene-modified insect strain, PCR identification primers and product size; b is the PCR identification result of 7 mice electroporated using F1 / R2, F2 / R1 and F1 / P3 primers; c is the bioluminescence imaging result of mice infected with the PbPfCSP-GFPLuc gene-modified insect strain and the Pb WT insect strain using a small animal living imager;

[0058] Fig. 9Schematic diagram and results of the experiment on the inhibition of liver stage growth of PbPfCSP-GFPLuc transgenic malarial parasite sporozoites by specific CSP antibodies in malaria mRNA vaccines in Example 8; wherein, a is a schematic diagram of the experimental operation of evaluating the specific CSP antibodies in malaria mRNA vaccines against the infection of PbPfCSP-GFPLuc gene-modified parasite sporozoites in human hepatocytes HepG2, and b is the inhibitory effect of specific CSP antibodies on the invasion of PbPfCSP-GFPLuc parasite sporozoites into human hepatocytes after the third immunization with seven vaccines;

[0059] Fig.10 The results of antibody subtype analysis after immunization with the malaria mRNA vaccine in Example 9; wherein, a is the antibody subtype analysis of serum against the liver stage antigen CSP after the third immunization with RH5-EAAAK3-CSP, RH5-T2A-CSP, RH5-GGGGS3-CSP and CSP mRNA vaccines, and b is the antibody subtype analysis of serum against the erythrocytic stage antigen RH5 after the third immunization with RH5-EAAAK3-CSP, RH5-T2A-CSP, RH5-GGGGS3-CSP and RH5 mRNA vaccines;

[0060] Fig.11 These are the results of the protective experiment of mice immunized with malaria mRNA vaccine in Example 10; wherein, a is a schematic diagram of the animal immunization procedure, sampling and infection time, and b is the survival curve of mice twelve days after infection with PbPfCSP-GFPLuc gene-modified insect strain sporozoites for RH5-EAAAK3-CSP, RH5-T2A-CSP, RH5-GGGGS3-CSP, CSP mRNA vaccine and PBS control group. DETAILED DESCRIPTION

[0061] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] Example 1 Construction, in vitro preparation and packaging of six dual-target tandem and two single-target malaria mRNA vaccines

[0063] 1. In vitro transcription and capping of mRNA vaccines

[0064] Schematic diagram of the structures of RH5-EAAAK3-CSP, CSP-EAAAK3-RH5, RH5-GGGGS3-CSP, CSP-GGGGS3-RH5, RH5-T2A-CSP, CSP-T2A-RH5, CSP and RH5 malaria mRNA vaccines are shown in Figure 1 The malaria mRNA vaccine sequence information, nucleotide length and delivery system details are shown in Table 1.

[0065] Table 1 Malaria mRNA vaccine sequence information, nucleotide length and delivery system details

[0066]

[0067] In this embodiment, the basic plasmid used for in vitro transcription of mRNA vaccine is pUC57, which is provided by Nanjing GenScript Biotechnology Co., Ltd.

[0068] The DNA expression elements of the mRNA vaccine are inserted into the basic plasmid pUC57 by molecular biological methods, including: (1) T7 promoter; (2) 5' UTR sequence upstream of the coding region; (3) DNA coding region, including RH5 and CSP DNA coding RNA sequences of different orders and different linkers (specific DNA coding RNA sequences are shown in SEQ ID NO: 1-8); (4) downstream 3' UTR sequence; (5) poly-A tail.

[0069] First, eight in vitro transcription plasmids were digested with restriction endonuclease BspQ1 to obtain linearized plasmids. Conventional DNA purification methods were used to purify the plasmids to obtain templates for in vitro transcription. T7 High Yield RNA Transcription Kit with CAP1 GAG (3'OMe) (E139, Suzhou Jinan Protein Technology Co., Ltd.) was used to transcribe the DNA template containing T7 promoter, AG start sequence and poly A tail coding region, and CAP1 GAG (3'OMe) cap analogs were incorporated during the transcription process to obtain mRNA with 5'-Cap and 3'-poly A tail. Lithium chloride was then used to purify the mRNA, and RiboGreen dye was used for RNA quantification.

[0070] SEQ ID NO: 1: CSP-EAAAK3-RH5 nucleotide sequence

[0071]

[0072] SEQ ID NO: 9: CSP-EAAAK3-RH5 amino acid sequence

[0073] 。

[0074] SEQ ID NO: 2: CSP-GGGGS3-RH5

[0075] ATGATGCGGAAGCTGGCCATCCTGAGCGGTTCCAGCTTCCTGTTTGTGGAAGCCCTGTTCCAAGAGTACCATCGCTACGGCAGCAGCAGCAACACCAGAGTGCTGAACGAGCTGAACTACGACAACGCCGGCACCAACCTGTACAACGAGCTGGAAATGAACTACTACGGCAAGCAAGAGAACTGGTACAGCCTGAAGAAGAACAGCAGAAGCCTGGGCGAGAACGACGACGGCAACAAGCAAGAGAGAACT. CGAGGACAACGAGAAGCTGCGGAAGCCCAAGCACAAGAAGCTGAAGCAGCCCGCCGACGGCAATCCCGATCCTAACGCCAATCTCTAACGTGGACCCCAACGCTAACCCAATGTGGACCCAAATGCCAATCCAAATGTCGATCCAATGCAAACCCTAACGCAAACCCGAATGCTAACCCAAACGCTAATCCGAACGCAAATCCCAATGCCAATCTAATGCTAATCCCAACGCCAATC CAAACGCTAACCCTAATGCAAATCCTAACGCTAACCCCAACGCAAACCCGAACGCCAATCCCTAACGCCAATCCGAATGCAAACCCCAATGCTAACCCCAATGGCAAATCCCAATGTGGACCCCTAACCGCAAACCCAAACCGCAAATGCAAACCCAATCCAAATGCAAATCCCAACGCTAACCCTAACCGCAAACCCAAACCGCAAACCCAAATGCTAATCCTAACAAGAACAATCAAGGCAACGGCCAGGGCCACAACATGCCCAACGATCCCAACAGAAACGTGGACGACGCCAACGCCAACGCGCGTGAAGAAACAACAACAACAATAGGAACCCAGCGACAACAAGCACATCAAAGGA

[0076] CCTGAACAAGATCCAGAACAGCCTGAGCACCGAGTGGTCCCCATGTAGCGTGACATGCGG

[0077] CAACGGAATCCAAGTGCGGATCAAGCCTGGCAGCGCCAACAAGCCTAAGGATGAGCTGGA

[0078] CTACGCCAATGACATCGAGAAGAAAATCTGCAAGATGGAAAAGTGCAGCAGCGTGTTCAA

[0079] CGTGGTCAACAGCAGCATCGGCCTGATTATGGTGCTGTCCTTCCTGTTCCTGAACGGCGG

[0080] AGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCCATGATCAGAATCAAGAAGAA

[0081] ACTGATCCTGACCATCATCTACATCCACCTGTTCATCCTGAACCGGCTGAGCTTCGAGAA

[0082] TGCCATCAAAAAGACCAAAAATCAAGAGAACAACCTGACGCTGCTCCCCATCAAGTCCAC

[0083] CGAGGAAGAGAAGGACGACATCAAGAACGGCAAGGATATCAAGAAAGAGATCGACAACGA

[0084] CAAAGAGAACATCAAGACCAACAACGCCAAGGACCACAGCACCTACATCAAGAGCTATCT

[0085] CAACACCAACGTGAACGACGGCCTGAAGTACCTGTTTATCCCCAGCCACAACAGCTTTAT

[0086] CAAAAAGTACTCCGTGTTCAATCAGATCAACGACGGCATGCTGCTGAATGAGAAGAACGA

[0087] CGTGAAAAACAACGAAGATTACAAGAACGTGGACTACAAGAATGTGAACTTCCTGCAGTA

[0088] CCACTTCAAAGAGCTGAGCAACTACAATATCGCCAACTCCATCGACATCCTGCAAGAGAA

[0089] AGAGGGCCACCTGGACTTCGTGATCCATCCTCACTACACCTTTCTGGACTACTACAAGCA

[0090] CCTGAGCTACAACAGCATCTACCACAAGTCCAGCACATACGGCAAGTGATCGCCGTGGA

[0091] CGCCTTCATTAAGAAGATTAACGAGACTTACGACAAAGTGAAGTCCAAGTGCAACGACAT

[0092] TAAGAATGACCTGATCGCCACCATCAAGAAGCTCGAGCACCCCTACGATATCAACAACAA

[0093] GAACGATGACAGCTACCGCTACGACATCAGCGAGAAAATCGACGACAAGTCCGAGGAAAC

[0094] AGACGACGAGACAGAGAGAGGGTGGAAGATAGCATCCAGGACACCGACAGCAATCACACCC

[0095] TAGCAACAAGAAAAAGAACGACCTCATGAACCGGACCTTCAAGAAAATGATGGACGAGTA

[0096] CAACACGAAGAAGAAAAAAGCTGATCAAGTGCATCAAGAATCACGAGAACGATTTCAACAA

[0097] GATCTGCATGGACATGAAGAACTACGGGACCAATCTGTTCGAGCAGCTGTCCTGCTATAA

[0098] CAACAACTTCTGCAACACGAACGGCATCCGCTACCACTACGATGAGTACATCCACAAGCT

[0099] GATCCTGTCCGTCAAGTCCAAGAATCTGAACAAGGACCTGTCCGACATGACCAATATCCT

[0100] GCAGCAGAGCGAACTGCTGCTGACCAACCTCAACAAAAAGATGGGCAGCTACATCTATAT

[0101] CGATACCATCAAGTTCATCCACAAAGAGATGAAGCACATCTTCAACCGGATCGAGTACCA

[0102] CACCAAGATCATCAACGACAAGACGAAGATCATCCAGGATAAGATCAAGCTGAACATCTG

[0103] GCGCACCTTCCAGAAAGACGAGCTGCTGAAGCGGATCCTGGACATGAGCAACGAGTACAG

[0104] TCTGTTCATCACCAGCGACCACCTGAGACAGATGCTGTACAACACCTTCTACAGCAAAGA

[0105] GAAGCACCTTAACAACATCTTTCACCACCTGATCTACGTGCTGCAGATGAAGTTCAACGA

[0106] CGTGCCCATTAAGATGGAATACTTCCAGACCTACAAAAAGAACAAGCCCCTGACACAGTGA。

[0107] SEQ ID NO: 10: Amino acid sequence of CSP-GGGGS3-RH5

[0108] 。

[0109] SEQ ID NO: 3: CSP-T2A-RH5 sequence

[0110] ATGATGCGGAAGCTGGCCATCCTGAGCGTGTCCAGCTTCCTGTTTGTGGAAGCCCTGTTCCAAGAGTACCAGTGCTACGGCAGCAGCAGCAACACCAGAGTGCTGAACGAGCTGAACTACGACAACGCCGGCACCAACCTGTACAACGAGCTGGAAATGAACTACTACGGCAAGCAAGAGAACTGGTACAGCCTGAAGAAGAACAGCAGAAGCCTGGGCGAGAACGACGACGGCAACAACGAGGACAACGAGAAGCTGCGGAAGCCCAAGCACAAGAAGCTGAAGCAGCCCGCCGACGGCAATCCCGATCCTAACGCCAATCCTAACGTGGACCCCAACGCTAACCCCAATGTGGA

[0111] CCCAAATGCCAATCCAAATGTCGATCCCAATGCAAACCCTAACGCAAACCCGAATGCTAA

[0112] CCCAAACGCTAATCCGAACGCAAATCCCAATGCCAATCCTAATGCTAATCCCAACGCCAA

[0113] TCCAAACGCTAACCCTAATGCAAATCCTAACGCTAACCCCAACGCAAACCCGAACGCCAA

[0114] TCCTAACGCCAATCCGAATGCAAACCCCAATGCTAACCCCAATGCAAATCCCAATGTGGA

[0115] CCCTAACGCAAATCCTAATGCAAATCCGAACGCAAATCCTAACGCAAACCCAAACGCCAA

[0116] TCCAAATGCAAACCCTAACGCTAATCCAAATGCTAATCCCAATGCAAACCCGAATGCAAA

[0117] TCCCAACGCTAACCCTAACGCTAACCCCAATGCCAATCCAAACGCAAATCCAAATGCTAA

[0118] CCCGAACGCTAACCCAAACGCAAACCCAAATGCTAATCCTAACAAGAACAATCAAGGCAA

[0119] CGGCCAGGGCCACAACATGCCCAACGATCCCAACAGAAACGTGGACGAGAACGCCAACGC

[0120] CAACAGCGCCGTGAAGAACAACAACAATGAGGAACCCAGCGACAAGCACATCAAAGAGTA

[0121] CCTGAACAAGATCCAGAACAGCCTGAGCACCGAGTGGTCCCCATGTAGCGTGACATGCGG

[0122] CAACGGAATCCAAGTGCGGATCAAGCCTGGCAGCGCCAACAAGCCTAAGGATGAGCTGGA

[0123] CTACGCCAATGACATCGAGAAGAAAATCTGCAAGATGGAAAAGTGCAGCAGCGTGTTCAA

[0124] CGTGGTCAACAGCAGCATCGGCCTGATTATGGTGCTGTCCTTCCTGTTCCTGAACGGCAG

[0125] CGGAGAAGGCAGAGGCAGCCTTCTGACTTGTGGCGACGTGGAAGAGAATCCCGGACCTAT

[0126] GATCCGCATCAAGAAGAAACTGATCCTGACCATCATCTACATCCACCTGTTCATCCTGAA

[0127] CCGGCTGAGCTTCGAGAATGCCATCAAAAAGACCAAGAATCAAGAGAACAACCTGACACT

[0128] GCTGCCCATCAAGTCCACCGAGGAAGAGAAGGACGACATCAAGAACGGCAAGGATATCAA

[0129] GAAAGAGATCGACAACGACAAAGAGAACATCAAGACCAACAACGCCAAGGACCACAGCAC

[0130] CTACATCAAGAGCTATCTCAACACCAACGTGAACGACGGCCTGAAGTACCTGTTTATCCC

[0131] CAGCCACAACAGCTTTATCAAAAAGTACTCCGTGTTCAATCAGATCAACGACGGCATGCT

[0132] GCTGAATGAGAAGAACGACGTGAAAAACAACGAAGATTACAAGAACGTGGACTACAAGAA

[0133] TGTGAACTTCCTGCAGTACCACTTCAAAGAGCTGAGCAACTACAATATCGCCAACTCCAT

[0134] CGACATCCTGCAAGAGAAAGAGGGCCACCTGGACTTCGTGATCATCCCTCACTACACCTT

[0135] TCTGGACTACTACAAGCACCTGAGCTACAACAGCATCTACCACAAGTCCAGCACATACGG

[0136] CAAGTGTATCGCCGTGGACGCCTTCATTAAGAAGATTAACGAGACTTACGACAAAGTGAA

[0137] GTCCAAGTGCAACGACATTAAGAATGACCTGATCGCCACGATCAAGAAGCTCGAGCACCC

[0138] CTACGATATCAACAACAAGAACGATGACAGCTACCGCTACGACATCAGCGAGGAAATCGACGACAAGTCCGAGGAAACAGACGACGAGACAGAGGAAGTGGAAGATAGCATCCAGGACACCGACAGCAATCACACCCCTAGCAACAAGAAAAAGAACGACCTCATGAACCGGACCTTCAAGAAAATGATGGACGAGTACAACACGAAGAAGAAAAAGCTGATCAAGTGCATTAAGAACCACGAGAACGATTTCAACAAGATCTGCATGGACATGAAGAACTACGGGACCAATCTGTTCGAGCAGCTGTCCTGCTATAACAACAACTTCTGCAACACGAACGGCATCCGCTACCACTACGATGAGTACATCCACAAGCTGATCCTGTCCGTCAAGTCCAAGAATCTGAACAAGGACCTGTCCGACATGACCAATATCCTGCAGCAGAGCGAACTGCTGCTGACCAACCTCAACAAAAAGATGGGCTCCTACATCTATATCGACACCATCAAGTTCATCCACAAAGAGATGAAGCACATCTTCAACCGGATCGAGTACCACACCAAGATCATCAACGACAAGACGAAGATCATCCAGGATAAGATCAAGCTGAACATCTGGCGCACCTTCCAGAAAGACGAGCTGCTGAAGCGGATCCTGGACATGAGCAACGAGTACAGTCTGTTCATCACCAGCGACCACCTGAGACAGATGCTGTACAACACCTTCTACAGCAAAGAGAAGCACCTTAACAACATCTTCCACCACCTGATCTACGTGCTGCAGATGAAGTTCAACGACGTCCCCATTAAGATGGAATACTTCCAGACCTACAAAAAGAACAAGCCCCTGACACAGTGA。

[0139] SEQ ID NO: 11: Amino acid sequence of CSP-T2A-RH5

[0140] 。

[0141] SEQ ID NO: 4: RH5-EAAAK3-CSP nucleotide sequence

[0142]

[0143] SEQ ID NO: 12: RH5-EAAAK3-CSP amino acid sequence

[0144] 。

[0145] SEQ ID NO: 5: RH5-T2A-CSP sequence

[0146] ATGATACGAATTAAGAAAAAACTCATACTGACAATCATATACATTCACCTTTTCATTTTGAACCGACTCTCCTTTGAGAATGCAATTAAAAAAACCAAAAATCAAGAGAACAACCTGACGTTGCTTCCCATCAAGAGCACAGAGGAGGAGAAGGACGACATAAAAAATGGAAAAGATATCAAGAAGGAAATCGATAACGATAAAGAAAACATAAAAACCAATAATGCCAAAGACCATTCTACGTATATCAAAAGTTACTTGAATACGAACGTGAATGACGGTTTGAAGTACCTGTTTATACCATCTCATAACAGCTTCATCAAAAAATATAGTGTGTTTAACCAGATTAACGACGGCATGTTGTTGAATGAGAAGAACGACGTGAAAAATAACGAAGATTACAAAAACGTGGATTATAAGAACGTAAACTTCCTGCAATACCATTTTAAGGAGCTTAGTAACTACAACATTGCCAACAGTATCGATATACTGCAAGAGAAAGAAGGGCATCTCGATTTTGTAATCATACCTCATTACACCTTCCTCGACTATTACAAGCATCTCAGTTATAATAGTATCTATCACAAGAGTAGTACTTATGGCAAATGCATAGCCGTTGACGCCTTCATTAAAAAGATAAACGAAACTTATGACAAAGTCAAATCCAAATGTAACGACATTAAGAATGACCTTATTGCGACAATCAAAAAGTTGGAGCATCCTTATGACATTAATAATAAAAATGACGACTCTTATAGGTACGACATATCAGAGGAAATTGACGACAAATCAGAAGAGACCGATGACGAGACTGAAGAAGTAGAGGACAGCATCCAAGAC

[0147] ACAGACAGTAACCATACACCCTCTAACAAGAAAAAAAACGATTTGATGAATCGGACGTTT

[0148] AAAAAGATGATGGATGAATATAACACAAAGAAGAAGAAGCTGATCAAATGCATTAAGAAC

[0149] CACGAAAATTCAATAAAATCTGCATGGACATGAAGAACTATGGCACTAATCTGTTC

[0150] GAACAACTGAGCTGTTACAATAATAATTTCTGCAACACAAATGGAATCCGCTACCATTAC

[0151] GATGAGTACATTCATAAACTTATACTCAGCGTGAAGTCCAAGAACCTCAATAAGGATCTT

[0152] AGTGATATGACCAATATACTCCAACAATCAGAGCTCCTGTTGACCAACCTTAACAAGAAA

[0153] ATGGGTTCTTACATCTACATCGACACCATCAAGTTCATCCACAAAGAGATGAAACACATA

[0154] TTCAACCGAATTGAATATCATACTAAAATCATTAACGACAAGACAAAGATTATCCAAGAC

[0155] AAAATCAAACTTAACATATGGAGAACCTTCCAGAAAGACGAGTTGCTCAAACGCATATTG

[0156] GACATGTCCAACGAATACTCCCTGTTTATTACTAGTGATCATCTGAGGCAGATGCTCTAT

[0157] AACACGTTCTATTCAAAAGAAAACACCTTAACAATATTTTTCCACCACCCTTATTTACGTA

[0158] TTGCAGATGAAATTCAATGATGTACCCATCAATTGGAGTATTTCCAGACGTACAAGAAG

[0159] AATAAACCACTTACACAGGGATCTGGCGAAGGACGCGGCTCTCTGCTGACATGTGGGGAC

[0160] GTTGAAGAAAACCCCGGGCCCATGATGAGAAAGCTCGCTATACTGAGTGTCTCATCCTTC

[0161] CTGTTCGTTGAGGCGCTGTTCCAAGAATATCAATGCTATGGATCATCAAGCAATACTCGA

[0162] GTACTGAACGAGTTGAATTACGACAACGCCGGTACGAACCTCTATAACGAGCTGGAGATG

[0163] AACTACTATGGAAAGCAGGAAAACTGGTACAGTCTTAAAAAAAATAGCAGATCTCTTGGT

[0164] GAAAACGATGATGGCAACAATGAGGACAACGAGAAACTGCGCAAACCTAAGCATAAAAAG

[0165] CTGAAGCAGCCGGCTGATGGTAATCCAGATCCCAATGCTAACCCTAACGTAGACCCTAAC

[0166] GCTAACCCAAATGTGGATCCAAACGCGAACCCAAACGTTGACCCAAACGCGAATCCCAAC

[0167] GCCAATCCAAATGCTAACCCTAATGCTAATCCAAACGCAAATCCCAATGCGAACCCGAAC

[0168] GCTAATCCCAATGCCAATCCCAACGCAAACCCTAACGCGAACCCGAACGCAAATCCTAAT

[0169] GCTAATCCAAACGCTAACCCAAACGCTAATCCCAACGCGAATCCGAATGCGAACCCAAAT

[0170] GCCAACCCCAACGTGGACCCTAATGCAAATCCTAACGCGAATCCGAACGCCAATCCTAAT

[0171] GCCAACCCAAATGCGAATCCTAACGCTAACCCAAACGCCAATCCAAATGCGAACCCCAAT

[0172] GCGAACCCAAATGCTAACCCTAATGCGAACCCAAACGCGAACCCTAACGCGAACCCTAAC

[0173] GCCAACCCCAACGCCAATCCGAACGCTAATCCAAATGCAAACCCAAACGCTAATCCAAAT

[0174] AAAAATAACCAAGGTAATGGGCAGGGGCATAACATGCCGAACGATCCTAATCGGAATGTCGACGAGAACGCGAATGCGAATTCTGCTGTGAAAAATAATAATAATGAAGAGCCCTCCGATAAGCATATTAAAGAATATCTTAATAAGATCCAGAATTCCCTGTCCACCGAGTGGTCCCCGTGTTCCGTAACTTGCGGAAACGGCATTCAGGTGCGCATCAAGCCTGGTAGCGCGAACAAACCCAAAGATGAGCTTGATTACGCTAATGACATTGAGAAAAAGATTTGTAAAATGGAAAAGTGTTCTAGCGTGTTCAATGTAGTGAATTCCTCCATCGGCTTGATAATGGTCTTGTCATTTCTTTTTTTGAACTGA。

[0175] SEQ ID NO: 13: Amino acid sequence of RH5-T2A-CSP

[0176] 。

[0177] SEQ ID NO: 6: Nucleotide sequence of RH5-GGGGS3-CSP

[0178] ATGATCCGCATCAAGAAGAAGCTGATCCTGACCATCATCTACATCCACCTGTTCATCCTGAACCGGCTGAGCTTCGAGAACGCCATCAAAAAGACCAAGAATCAAGAGAACAACCTGACGCTGCTGCCCATCAAGAGCACCGAGGAAGAGAAGGACGACATCAAGAACGGCAAGGATATCAAGAAAGAGATCGACAACGACAAAGAGAACATCAAGACCAACAACGCCAAGGACCA

[0179] CAGCACCTACATCAAGTCCTACCTGAACACCAACGTGAACGACGGCCTGAAGTACCTGTT

[0180] TATCCCCAGCCACAACAGCTTTATCAAAAAGTACAGCGTGTTCAACCAGATCAACGACGG

[0181] CATGCTGCTGAACGAGAAGAACGACGTGAAGAACAACGAGGACTACAAGAACGTCGACTA

[0182] TAAGAATGTGAACTTCCTGCAGTACCACTTCAAAGAGCTGAGCAACTACAATATCGCCAA

[0183] CAGCATCGACATCCTGCAAGAGAAAGAGGGCCACCTGGACTTCGTGATCATCCCTCACTA

[0184] CACCTTTCTGGACTACTACAAGCACCTGAGCTACAACTCCATCTACCACAAGTCCTCCAC

[0185] CTACGGCAAGTGTATCGCCGTGGACGCCTTCATTAAGAAGATTAACGAGACTTACGACAA

[0186] AGTGAAGTCCAAGTGCAACGACATTAAGAATGACCTGATCGCCACGATCAAGAAGCTGGA

[0187] ACACCCCTACGATATCAACAACAAGAACGATGACAGCTACCGCTACGACATCAGCGAGGA

[0188] AATCGACGACAAGTCCGAGGAAACAGACGACGAGACAGAGGAAGTGGAAGATAGCATCCA

[0189] GGACACCGACAGCAATCACACCCCTAGCAACAAGAAAAAGAACGACCTCATGAACCGGAC

[0190] CTTCAAGAAAATGATGGACGAGTACAACACCAAGAAGAAAAAGCTCATCAAGTGCATTAA

[0191] GAACCACGAGAACGACTTCAACAAGATCTGCATGGACATGAAGAACTACGGCACCAACCT

[0192] GTTCGAGCAGCTGTCCTGCTACAACAACAACTTCTGCAACACGAACGGCATCCGCTACCA

[0193] CTACGATGAGTACATCCACAAGCTGATTCTGAGCGTCAAGTCCAAGAACCTGAACAAGGA

[0194] CCTGTCCGACATGACCAATATCCTGCAGCAGAGCGAGCTGCTGCTGACCAATCTGAACAA

[0195] GAAGATGGGCTCCTACATCTATATCGACACCATCAAGTTCATCCACAAAGAGATGAAGCA

[0196] CATCTTCAACCGGATCGAGTACCACACGAAGATCATCAACGACAAGACTAAGATCATCCA

[0197] GGATAAGATCAAGCTGAACATCTGGCGCACCTTCCAGAAGGATGAGCTGCTGAAGCGGAT

[0198] CCTGGACATGAGCAACGAGTACTCCCTGTTCATTACCAGCGACCACCTGAGACAGATGCT

[0199] CTACAACACCTTCTACAGCAAAGAGAAGCACCTCAACAACATCTTCCACCACCTGATCTA

[0200] CGTGCTGCAGATGAAGTTCAACGACGTGCCGATCAAGATGGAATACTTCCAGACCTACAA

[0201] GAAGAACAAACCCCTGACACAAGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGG

[0202] AGGATCCATGATGCGGAAGCTGGCCATCCTGAGCGTGTCCAGCTTCCTGTTTGTGGAAGC

[0203] CCTGTTCCAAGAGTACCAGTGCTACGGCAGCTCCAGCAACACCAGAGTGCTGAATGAGCT

[0204] GAACTACGACAACGCCGGCACAAATCTGTACAACGAGCTGGAAATGAACTACTACGGGAA

[0205] GCAAGAGAACTGGTACAGCCTGAAGAAGAACTCCGAAGCCTGGGAGAGACGACGACGG

[0206] CAACAATGAGGACAACGAGAAGCTGCGGAAGCCCAAGCACAAGAAACTGAAGCAGCCCGCCGACGGCAATCCCGATCCTAACGCCAATCCTAACGTGGACCCCAACGCTAACCCCAATGTGGACCCAAATGCCAATCCAAATGTCGATCCCAATGCAAACCCTAACGCAAACCCGAATGCTAACCCAAACGCTAATCCGAACGCAAATCCCAATGCCAATCCTAATGCTAATCCCAACGCCAATCCAAACGCTAACCCTAATGCAAATCCTAACGCTAACCCCAACGCAAACCCGAACGCCAATCCTAACGCCAATCCGAATGCAAACCCCAATGCTAACCCCAATGCAAATCCCAATGTGGACCCTAACGCAAATCCTAATGCAAATCCGAACGCAAATCCTAACGCAAACCCAAACGCCAATCCAAATGCAAACCCTAACGCTAATCCAAATGCTAATCCCAATGCAAACCCGAATGCAAATCCCAACGCTAACCCTAACGCTAACCCCAATGCCAATCCAAACGCAAATCCAAATGCTAACCCGAACGCTAACCCAAACGCAAACCCAAATGCTAATCCTAACAAGAACAATCAAGGCAACGGCCAGGGCCACAACATGCCCAACGATCCCAACAGAAACGTGGACGAGAATGCCAACGCCAACAGCGCCGTGAAAAACAACAACAATGAAGAACCCAGCGACAAGCACATCAAAGAGTACCTTAACAAGATCCAGAACAGCCTGTCCACCGAGTGGTCCCCATGTAGCGTGACATGCGGCAATGGCATCCAAGTGCGGATCAAGCCTGGCAGCGCCAACAAGCCCAAGGATGAACTGGACTACGCCAACGACATCGAGAAGAAAATCTGCAAAATGGAAAAGTGCAGCTCCGTGTTCAATGTCGTGAACAGCAGCATCGGCCTGATTATGGTGCTGTCCTTCCTGTTCCTGAACTGA。

[0207] SEQ ID NO: 14: RH5-GGGGS3-CSP amino acid sequence

[0208] 。

[0209] SEQ ID NO: 7: CSP nucleotide sequence

[0210]

[0211] SEQ ID NO:15:CSP amino acid sequence

[0212] MMRKLAILSVSSFLFVEALFQEYQCYGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQENWYSLKKNSRSLGENDDGNNEDNEKLRKPKHKKLKQPADGNPDPNANPNVDPNANPNVDPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNAN PNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEKCSSVFNVVNSSIGLIMVLSFLFLN。

[0213] SEQ ID NO: 8: RH5

[0214]

[0215] SEQ ID NO: 16: RH5 amino acid sequence

[0216] .

[0217] 2. Lipid nanoparticle (LNP) packaging of mRNA

[0218] Cationic lipids, phosphatidylcholine, cholesterol and PEG lipids were mixed in a ratio of 50:10:38.5:1.5, and then mixed and packaged with the above-mentioned 5'-end capped mRNA using the NanoAssemblr Benchtop nanoliposome packaging instrument produced by Precision Nano Systems. After packaging, the buffer solution was replaced with PBS by centrifugation or dialysis. After packaging, the mRNA packaging efficiency was identified using the Quan-iTRibogreen RNAreagent kit from Thermo Fisher, and the packaging efficiency met the standards for mRNA vaccines.

[0219] Example 2 In vitro expression and identification of malaria mRNA vaccine

[0220] In this example, the malaria mRNA vaccines CSP-EAAAK3-RH5, CSP-GGGGS3-RH5, CSP-T2A-RH5, RH5-EAAAK3-CSP, RH5-GGGGS3-CSP, RH-T2A-CSP, RH5 and CSP were transfected into mammalian cells 293T at a transfection dose of 5 μg, and the negative control was transfected with an equal amount of LNPs.

[0221] Before transfection, it is necessary to observe that the cells have normal morphology, uniform size, basically no dead cells, and a cell density of 70-80%. Prepare 50μL mRNA reagent solution (5μg) + 450μL opti-MEM for each well of the six-well plate, a total of 500μL solution, mix well by pipetting, and let stand for 5 minutes. During the standing period, aspirate the cell supernatant, add 1mL PBS to wash away the residual culture medium, add the premixed opti-mRNA solution, 500μL per well, slowly add along the wall of the culture dish, and place it in a 37°C incubator for 8 hours. After 8 hours, change the liquid, aspirate the supernatant, add 2mL of DMEM complete culture medium containing 10% FBS, and culture at 37°C. After 12h, 24h, and 48h of transfection, collect cell samples and prepare protein samples, and detect the in vitro expression of malaria mRNA vaccine by protein immunoblotting (Western blot, WB) experiment. The specific method is as follows:

[0222] 1. Protein sample preparation:

[0223] 1. Aspirate the culture medium, add 1 mL of 4°C pre-cooled PBS (Gibco) to each well, lay it flat and shake it gently to wash the cells, then discard the washing solution, wash the cells three times in total, and finally use 1 mL of PBS to blow off the attached 293T cells and transfer them to a new 1.5 mL EP tube;

[0224] 2. The protein sample was disrupted using non-contact ultrasonication (3s on, 5s off) for 5 min;

[0225] 3. Add 5× protein loading to the protein sample to a final concentration of 1×, mix well, boil at 100°C for 10 min, and cool to room temperature for later use.

[0226] 2. Western Blotting:

[0227] 1. Sample loading: Add 10 μL of protein sample to each well, ensuring that the sample is completely added to the SDS-PAGE gel wells;

[0228] 2. Electrophoresis: 80V for 20min, 120V for 60min. Electrophoresis can be terminated when bromophenol blue just runs out, and transfer to membrane;

[0229] 3. Transfer: Use commercial fast transfer solution (20×) (NCM Biotech, catalog number: WB4600) for transfer; Preparation of 1× fast transfer solution (1000mL): Take 50mL fast transfer solution (20×) + 850mL deionized water + 100mL anhydrous ethanol; Make a transfer sandwich structure, then transfer to the electrophoresis tank, set the constant current to 400mA, and the electrophoresis time to 30min to complete the protein transfer;

[0230] 4. Blocking: Use 1×TBST to prepare 5% skim milk, ensure that the skim milk is mixed evenly without particles, put the PVDF membrane with transferred protein into the WB incubation box containing 5% skim milk blocking solution, shake and block at room temperature for 1 hour or incubate at 4°C overnight;

[0231] 5. Primary antibody: The specific antiserum obtained from the third immunization with malaria RH5 and CSP mRNA vaccines was used as the primary antibody for western blot experiments. The primary antibody dilution ratio was 1:1000 and incubated at 4°C with shaking overnight.

[0232] 6. Wash the membrane: wash four times with 1×TBST, 10 min each time;

[0233] 7. Secondary antibody: Use HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:5000 and incubate at room temperature with shaking for 1 hour;

[0234] 8. Wash the membrane: wash four times with 1×TBST, 10 min each time;

[0235] 9. Development: Use a commercial ECL kit for development and take photos to preserve the results.

[0236] The results are as follows Figure 2 As shown, eight malaria mRNA vaccines were transfected and the protein expression of transfected 293T cells was detected 12h, 24h and 48h after transfection. The results showed that the protein expression level of the mRNA vaccine was the highest 12h after transfection, and the expression level gradually decreased with time. It also showed that the expression levels of RH5-EAAAK3-CSP, RH5-GGGGS3-CSP and RH5-T2A-CSP in the malaria tandem mRNA vaccine were higher than those of CSP-EAAAK3-RH5, CSP-GGGGS3-RH5 and CSP-T2A-RH5.

[0237] Example 3 Detection of antibody titers after immunization with single-target RH5 and CSP mRNA vaccines

[0238] In this example, 6-8 week old female BALB / c mice (purchased from Vital River) were used for animal experiments. The animal immunization procedure and sampling time are shown in Figure 3a. The experimental group was immunized with 10 μg of single-target RH5 and CSP mRNA vaccines, and the control group was intramuscularly injected with an equal volume of PBS. Immunization was performed 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). The specific experimental method is as follows:

[0239] 1. Antigen coating: dilute RH5 or CSP antigen with carbonate buffer (0.05M, pH9.6) at 2μg / mL, 50μL / well, add to a 96-well polyvinyl chloride microplate, shake gently to mix, spread 50μL antigen coating solution evenly on the bottom of the well, and incubate at 4°C overnight;

[0240] 2. Blocking: Take out 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°C for 1 hour, wash three times with PBST, and pat dry;

[0241] 3. Incubate with primary antibody: dilute the serum sample in a gradient from 1:100, then add the diluent to each well, 50 μL / well, incubate at 37°C for 2 hours, wash three times with PBST, and pat dry;

[0242] 4. Incubate with secondary antibody: Add 1:5000 diluted horseradish peroxidase (HRP) labeled goat anti-mouse IgG to a 96-well plate, 50 μL / well, incubate at 37°C for 1 hour, wash three times with PBST, and pat dry;

[0243] 5. Color development: Add TMB color development solution to a 96-well plate, 50 μL / well, and incubate at room temperature away from light for 8-10 minutes;

[0244] 6. Stop reaction: add stop solution into 96-well plate, 50 μL / well;

[0245] 7. Measure OD value: Use an enzyme-labeled instrument to measure the absorbance (OD value) at a wavelength of 450nm; the endpoint titration is defined as the serum dilution factor corresponding to the absorbance of serum at 450nm being 2.1 times greater than that of negative serum.

[0246] The ELISA method was used to detect the antibody titers of the first, second, and third immunizations of the single-target RH5 and CSP mRNA vaccines. The results are as follows Figure 3 b shows that the antibody titer after the second immunization is basically the same as that after the third immunization, and the difference is not statistically significant; this indicates that the single-target RH5 and CSP mRNA vaccines can induce the body to produce high levels of antibody titers after the second immunization.

[0247] Example 4 Detection of persistence of neutralizing antibodies in mice after immunization with single-target RH5 and CSP mRNA vaccines

[0248] In this example, 6-8 week old female mice of BALB / c strain (purchased from Vital River) were used for animal experiments. The immunization experimental group was immunized with 10 μg of single-target RH5 and CSP mRNA vaccines, respectively, and the control group was intramuscularly injected with an equal volume of PBS. The immunization was performed once every three weeks, and blood was collected two weeks after each immunization to prepare serum. The serum of the RH5 and CSP mRNA vaccine immunization groups was collected at 39 weeks and 46.4 weeks after immunization, respectively. Figure 4 a), the persistence of antibody titer was detected by ELISA method; the specific experimental method was the same as that in Example 3.

[0249] The results are as follows Figure 4 As shown in b, the antibody titer of the serum of the CSP mRNA immunization group at 46.4 weeks after immunization and the antibody titer after three immunizations were both greater than 1×10 6 The antibody titer of the RH5 mRNA immunization group at 39 weeks after immunization and the antibody titer after three immunizations were both greater than 1×10 6 , indicating that single-target CSP and RH5 mRNA vaccines can establish long-term and sustained humoral immunity in mice after immunization.

[0250] Example 5 Detection of antibody titer after immunization with dual-target RH5 and CSP tandem mRNA vaccine

[0251] In this embodiment, female mice of BALB / c strain at 6-8 weeks of age (purchased from Weitong Lihua Company) were used for animal experiments. The experimental groups were CSP-EAAAK3-RH5, CSP-GGGGS3-RH5, CSP-T2A-RH5, RH5-EAAAK3-CSP, RH5-GGGGS3-CSP, RH-T2A-CSP, RH5 and CSP mRNA vaccine immunization groups, and the negative control group was 1×PBS. All mice in the mRNA vaccine immunization group were immunized in the zeroth week, the third week and the sixth week, wherein the dual-target tandem mRNA vaccine immunization dose was 20 μg, the RH5 and CSP immunization groups were 10 μg, and the mice in the negative control group were injected with the same volume of PBS at the same time. The vaccination method was intramuscular injection. Serum samples of each mouse were collected two weeks after each immunization for detection of antibody titer and subtype analysis of immune serum. The experimental method of detection is shown in Example 3.

[0252] The results are as follows Figure 5 and Figure 6 As shown, Figure 5 and Figure 6They were coated with CSP and RH5 antigens, and CSP-EAAAK3-RH5, CSP-GGGGS3-RH5, CSP-T2A-RH5, RH5-EAAAK3-CSP, RH5-GGGGS3-CSP, RH-T2A-CSP, RH5 and CSP were detected by ELISA. The antibody titer of the mRNA vaccine after the third immunization showed that the specific CSP antibody titer of the six dual-target tandem mRNA vaccine immunization groups was basically equivalent to that of the CSP immunization group, among which the specific CSP antibody titer uniformity of the five mice in the RH5-T2A-CSP immunization group was the best; the results of the RH5 antigen-coated ELISA antibody titer test showed that the specific RH5 antibody titer of the RH5-EAAAK3-CSP, RH5-T2A-CSP and RH5-GGGGS3-CSP immunization groups was basically equivalent to that of the RH5 immunization group. In summary, RH5-EAAAK3-CSP, RH5-T2A-CSP and RH5-GGGGS3-CSP can be used as preferred mRNA vaccines for subsequent studies.

[0253] Example 6 Evaluation of the inhibitory effect of RH5-specific antibodies in malaria mRNA vaccines on the growth of erythrocytic Plasmodium

[0254] In this example, the antiserum IgG two weeks after the third immunization was first purified using the NAb Protein G Spin Kit, 0.2mL (Thermo Scientific, Catalog No.: 89949), and the antibody buffer was replaced with RPMI 1640, and the concentration was measured for later use, and then the growth inhibition effect of the RH5-specific antibody in the malaria mRNA vaccine on the red stage malarial parasite was evaluated. The specific method is as follows:

[0255] 1. Freshly prepare 2× complete culture medium (CM) (containing 20% ​​human serum, 2× sodium bicarbonate, 2× gentamicin), which can be stored at 37°C for 6 hours (for example, prepare 50 mL 2×CM: 45 mL CM + 1.6 mL 7.5% sodium bicarbonate solution + 5 mL human serum + 50 μL 10 mg / mL gentamicin solution, and sterilize with a 0.22 μm filter);

[0256] 2. Synchronize Plasmodium falciparum by the Percoll / Sorbitol method to obtain highly synchronized late Plasmodium falciparum (40-44h) for growth inhibition assay (GIA); first synchronize most Plasmodium falciparum parasites over 38h with 40%-60% Percoll / Sorbitol, then synchronize the ring stage with 5% Sorbitol after 4-14h, and perform GIA experiment when Plasmodium falciparum grows to 40-44h;

[0257] 3. The GIA system is 40 μL / well, the blood pressure product is 1%, and the initial parasite rate is 0.3%±0.1%; the purified IgG is diluted according to the concentration to ensure that the volume added to the immune test well is ≥5 μL, the final concentration of IgG in the immune test well and the blank test well is 500 μg / mL, and RPMI 1640 is added to 20 μL; then 20 μL of Plasmodium falciparum suspension (formula: 2×CM with a parasite rate of 0.3%±0.1% and a blood pressure product of 2%) is added to each well; the immune test well is IgG purified from serum after immunization with malaria mRNA vaccine, the blank test well is IgG purified from serum after immunization with 1×PBS, and the control well is without any IgG, and three replicate wells are set;

[0258] 4. After 40-48 hours, centrifuge and discard the supernatant, use a 10μL pipette to draw the red blood cell precipitate onto a clean slide, prepare a single-layer blood smear, perform Giemsa staining, calculate the infection status of 5000 red blood cells, and perform statistical analysis to calculate the inhibition rate.

[0259] Figure 7 a is a schematic diagram of the experiment to evaluate the inhibition of the growth of Plasmodium falciparum erythrocytic stage by specific RH5 antibodies in malaria mRNA vaccines. Figure 7As shown in b, the results showed that the RH5-T2A-CSP immunization group had a growth inhibition effect on malarial parasites (inhibition rate as high as 90.69%) and the RH5 group (inhibition rate was 91.56%). Among them, the RH5-EAAAK3-CSP, RH5-T2A-CSP and RH5-GGGGS3-CSP immunization groups had a growth inhibition effect on the erythrocytic stage of malarial parasites that was significantly better than the neutralizing antibodies (mAb-RH5-Cell) produced by immunization with the recombinant protein vaccine based on RH5 that has entered the clinical research stage. 2019). According to the results of the erythrocytic growth inhibition experiment, the RH5-EAAAK3-CSP, RH5-T2A-CSP and RH5-GGGGS3-CSP tandem mRNA vaccines have good inhibitory effects and can all be used as candidate vaccines. It was also found that after the third immunization with RH5-EAAAK3-CSP, RH5-T2A-CSP and RH5-GGGGS3-CSP mRNA vaccines, specific RH5 antibodies had a high inhibitory effect on the erythrocytic stage of Plasmodium falciparum from different clinical sources ( Figure 7 c), indicating that the immune protection of RH5-EAAAK3-CSP, RH5-T2A-CSP and RH5-GGGGS3-CSP mRNA vaccines is broad-spectrum.

[0260] Example 7 Establishment of an in vivo evaluation system for immune protection of liver stage vaccine (PbPfCSP-GFPLuc gene modified insect strain)

[0261] In this embodiment, firstly, negative screening was performed on Plasmodium berghei stably expressing GFP-Luciferase (inserted into the P230p gene site) (fed with 5-fluorocytosine, the parasite rate was 0, i.e., negative screening was successful) to obtain a transgenic strain in which GFP-Luciferase was fully integrated into the genome, and CRISPR / Cas9 technology was used to successfully screen the GFP-Luciferase strain to obtain a transgenic strain (PbPfCSP-GFPLuc) in which the CSP gene of Plasmodium berghei was replaced with the CSP gene of Plasmodium falciparum. The specific method is as follows:

[0262] 1. According to Figure 8 a Schematic diagram of constructing PbPfCSP-GFPLuc gene modified plasmid;

[0263] 2. Resuscitate the previously frozen Plasmodium berghei stably expressing GFP-Luciferase (inserted into the P230p gene site) gene, and inject it intraperitoneally into ICR female mice; observe the smear 3 days later, and when the Plasmodium grows to the schizont stage, kill the mice and collect blood; wash once with 10mL RPMI 1640 culture medium, configure a 60% percoll column on the bottom layer and a 40% percoll column on the top layer, centrifuge at 3800rpm for 10min, take the middle and late stage Plasmodium on the liquid surface, wash once with 10mL RPMI 1640 culture medium, discard the supernatant, and keep the precipitate for later use;

[0264] 3. Dissolve 5 μg of PbPfCSP-GFPLuc plasmid in 100 μL Cytomix solution, add 5 μL of schizont-containing red blood cells, mix gently with a pipette tip, transfer all into the electroporation cup, and select the Bio-rad electroporator T-16 program for electroporation;

[0265] 4. After the electric shock, add 200 μL PBS to resuspend, mix gently, and use a 1 mL syringe to inject into the tail vein of ICR mice; 24 hours after the tail vein injection, take blood from the tail tip of the mouse, and smear to observe whether there is malarial parasite. If the parasite rate reaches 0.2-1%, add pyrimethamine (7 μg / mL, 0.1% DMSO) to the drinking water of the mice to screen for successfully transfected gene-modified Plasmodium berghei;

[0266] 5. After 5-7 days of drug administration, smear check whether there is malarial parasite. When the parasite rate reaches about 3-5%, extract DNA and use Figure 8 The three pairs of primers in a were used for PCR amplification, restriction digestion and sequencing of PCR products;

[0267] 6. The correctly sequenced gene-modified strains are screened for monoclonal strains, which are then used in subsequent in vivo evaluation experiments of the immune protection effect of liver stage vaccines.

[0268] The results are as follows Figure 8 As shown, Figure 8 a is a schematic diagram of constructing the PbPfCSP-GFPLuc gene modified insect strain, PCR identification primers and product size; Figure 8 b is to first use F1 / R2 and F2 / R1 primers to perform PCR identification on the 7 electroporated mice. The results show that mice No. 2, No. 3 and No. 7 can amplify the target band of the expected size, and the size of their restriction PCR products is correct. The full-length F1 / P3 large fragment was further amplified from mice No. 2, No. 3 and No. 7. Only mouse No. 3 could amplify the full-length fragment. Subsequently, the gene-modified insect strain of mouse No. 3 was monocloned for subsequent experiments; Figure 8c is the in vivo imaging of mice infected with PbPfCSP-GFPLuc and Pb WT strains. The results show that obvious biochemiluminescence signals can be observed in mice infected with PbPfCSP-GFPLuc strain, indicating that an in vivo evaluation system for the immune protection effect of liver stage vaccine has been successfully established.

[0269] Example 8 CSP-specific antibodies in malaria mRNA vaccines inhibit the infection of human hepatocytes by sporozoites of PbPfCSP-GFPLuc gene-modified worm strains

[0270] In this example, the antiserum IgG two weeks after the third immunization was first purified using the NAb ProteinG Spin Kit, 0.2mL (Thermo Scientific, Catalog No.: 89949), and the antibody buffer was replaced with RPMI 1640, and the concentration was measured for later use, and then the effect of CSP-specific antibodies in malaria mRNA vaccines on inhibiting the infection of human hepatocytes by sporozoites of PbPfCSP-GFPLuc gene-modified worm strains was evaluated. The specific method is as follows:

[0271] 1. 12 hours in advance, plate 96-well cell plates, with 2.5×10 4 HepG2 cells;

[0272] 2. The next day, prepare fresh four-antibody complete medium, commercial DMEM medium supplemented with fetal bovine serum (10%), penicillin-streptomycin (working concentration of 100 U / mL), amphotericin B solution (working concentration of 0.25 μg / mL) and gentamicin (working concentration of 50 μg / mL);

[0273] 3. Dissect the salivary glands of Anopheles stephensi. First, anesthetize the Anopheles stephensi with CO2, transfer to 70% ethanol for 30 seconds, then transfer to 1×PBS to wash off the ethanol, and finally wash once with RPMI 1640 medium containing 2% fetal bovine serum, transfer to a slide for dissection, dissect an appropriate amount of salivary glands and transfer to 200 μL RPMI 1640 medium containing 2% fetal bovine serum, use a syringe with a 29.5G needle to mechanically break and release the sporozoites in the salivary glands, and then use a white blood cell counting plate to count the sporozoites;

[0274] 4. Wash the counted sporozoites three times with 1×PBS to remove impurities as much as possible; then mix 50 μL of immune group IgG (final IgG concentration of 500 μg / mL) with 50 μL of sporozoite suspension (containing 1.25×10 4 Sporozoites) were incubated at room temperature for 20 min, transferred to a 96-well plate using a pipette, and the cell plate was centrifuged at 110 × g for 1 min to accelerate the interaction between sporozoites and hepatocytes, and the cell plate was cultured in a di-gas incubator;

[0275] 5. After 3 hours of culture, wash three times with 1× PBS medium preheated at 37°C, discard each time, and replace the medium with four-antibody complete medium, once every 12 hours and 24 hours;

[0276] 6. After culturing for 48 hours, discard the culture medium, wash twice with 1× PBS, extract RNA according to the instructions of the RNA extraction kit (RNeasy Micro Kit, catalog number: 74004) of Qiagen Company, and determine the RNA concentration using a UV spectrophotometer;

[0277] 7. Use TaKaRa's reverse transcription kit (PrimeScript RT reagent Kit with gDNAEraser, catalog number: RR047A) to remove gDNA and reverse transcribe RNA. For detailed steps, see the instructions;

[0278] 8. The probe qPCR method was used to detect the copy numbers of 18S rRNA of PbPfCSP-GFPLuc sporozoites and GAPDH gene of human hepatocytes, so as to calculate the inhibition rate of CSP-specific antigen blocking sporozoites from infecting human hepatocytes. Three replicate wells were set for each sample.

[0279] Fig. 9 a is a schematic diagram of the experiment to evaluate the growth inhibition of PbPfCSP-GFPLuc transgenic Plasmodium berghei liver stage by specific CSP antibodies in malaria mRNA vaccines. The results are shown in Fig. 9 As shown in b, the results showed that the RH5-EAAAK3-CSP, RH5-T2A-CSP and RH5-GGGGS3-CSP immunization groups had growth inhibition effects on the liver stage of Plasmodium (inhibition rates were 95.43%, 90.61% and 91.40%, respectively) which were basically equivalent to the CSP group (inhibition rate was 91.76%). According to the results of the liver stage growth inhibition experiment, RH5-EAAAK3-CSP, RH5-T2A-CSP and RH5-GGGGS3-CSP tandem mRNA vaccines can all be used as candidate vaccines.

[0280] Example 9 Analysis of antibody subtypes after malaria mRNA vaccine immunization

[0281] The serum of the immunization group and the control group was collected two weeks after the first, second and third immunization, and the specific antibody subtype was detected by ELISA. The specific experimental method is as follows:

[0282] 1. Antigen coating: dilute RH5 or CSP antigen with carbonate buffer (0.05M, pH9.6) at 2μg / mL, 50μL / well, add to a 96-well polyvinyl chloride microplate, shake gently to mix, spread 50μL antigen coating solution evenly on the bottom of the well, and incubate at 4°C overnight;

[0283] 2. Blocking: Take out 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°C for 1 hour, wash three times with PBST, and pat dry;

[0284] 3. Incubate with primary antibody: dilute the serum sample at 1:10000 (preliminary experimental results show that at this dilution concentration, its OD450 value is in the effective section of the four-parameter fitting curve, which is not shown), then add the diluted serum sample to each well, 50 μL / well, incubate at 37°C for 2 hours, wash three times with PBST, and pat dry;

[0285] 4. Incubate with secondary antibody: add 1:5000 diluted horseradish peroxidase (HRP)-labeled goat anti-mouse IgG1, IgG2a, IgG2b and IgG3 (Southern Biotech; catalog numbers: 1070-05, 1080-05, 1090-05, 1100-05, respectively) to a 96-well plate, 50 μL / well, incubate at 37°C for 1 h, wash three times with PBST, and pat dry;

[0286] 5) Color development: Add TMB color development solution to a 96-well plate, 50 μL / well, and incubate at room temperature in the dark for 8-10 minutes;

[0287] 6) Stop reaction: add stop solution into 96-well plate, 50 μL / well;

[0288] 7) OD value measurement: Use an enzyme-labeled instrument to measure the absorbance (OD value) at a wavelength of 450 nm.

[0289] The results are as follows Fig.10As shown, the CSP-specific antibody immune response induced by RH5-EAAAK3-CSP, RH5-T2A-CSP, RH5-GGGGS3-CSP and CSP mRNA vaccines was mainly Th1 (cellular immunity) and Th2 type immune response (humoral immunity); while the RH5-specific antibody immune response induced by RH5-T2A-CSP and RH5 mRNA vaccines was mainly Th2 type immune response, among which the RH5-specific antibody immune response induced by RH5-EAAAK3-CSP and RH5-GGGGS3-CSP mRNA vaccines was mainly Th1 and Th2 type; the antibody subtype IgG1 antibody titer of RH5-T2A-CSP after the third immunization was not significantly different from that of the CSP immunization group or the RH5 immunization group, indicating that the RH5-T2A-CSP tandem mRNA vaccine can efficiently activate Th1 and Th2 type immune responses, and has a double-layer immune protection effect of humoral and cellular immunity.

[0290] Example 10 Malaria mRNA vaccine challenge protection experiment in mice

[0291] In this example, 6-8 week old female BALB / c mice (purchased from Vital River) were used for animal experiments. The animal immunization procedure, sampling time and challenge time are shown in the figure. Fig.11 a, The experimental groups were divided into RH5-EAAAK3-CSP, RH5-T2A-CSP, RH5-GGGGS3-CSP, CSP mRNA vaccine immunization groups and 1×PBS control group, with the immunization doses of 20μg, 20μg, 20μg and 10μg, respectively, and the immunization volume of the 1×PBS control group was 200μL; immunization was performed once every three weeks, and the virus was challenged one month after the third immunization. 2000 PbPfCSP-GFPLuc sporozoites were injected into the tail vein of each mouse, and blood smears were prepared every day for Giemsa staining after infection. The mice were observed until the 12th day after infection, and the data were statistically analyzed to draw the survival curve.

[0292] The results are as follows Fig.11 As shown in b, on the sixth day after the tail vein injection of 2000 PbPfCSP-GFPLuc sporozoites, all five mice in the PBS control group were infected with the PbPfCSP-GFPLuc strain, while none of the mice in the CSP, RH5-EAAAK3-CSP, RH5-T2A-CSP and RH5-GGGGS3-CSP mRNA vaccine immunization groups were infected, indicating that the protective efficacy of the above four mRNA vaccines was as high as 100%.

[0293] In summary, the present invention solves the problem of difficult expression of Plasmodium antigen protein through the LNP mRNA vaccine technology platform. The present invention creatively selects essential proteins for the two most important periods (liver stage and erythrocytic stage) of Plasmodium infection in the human body as candidate antigens, and develops "multi-period-multivalent" tandem malaria mRNA sequences that can simultaneously have transmission blocking and post-infection treatment efficacy through sequence design, sequence optimization and other technical research and development. The present invention solves the problems of low expression of effective antigen amount, low antibody titer produced by vaccine, and low protection rate through sequence optimization, linker selection and antigen combination order. At the same time, this vaccine has the dual efficacy of transmission blocking and post-infection treatment, and plays a double insurance for the prevention and treatment of malaria.

[0294] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A coding RNA for a vaccine for preventing and treating Plasmodium infection, characterized in that The coding RNA is RH5-linker-CSP; Wherein, the RH5 is the reticulocyte binding protein 5 of Plasmodium, and the nucleotide sequence of the RH5 is shown in SEQ ID NO: 8; The CSP is circumsporozoite protein, and the nucleotide sequence of the CSP is shown in SEQ ID NO: 7; The linker in the RH5-linker-CSP is EAAAK3, GGGGS3 or T2A; Wherein, the nucleotide sequence of RH5-EAAAK3-CSP is shown in SEQ ID NO: 4; The nucleotide sequence of RH5-T2A-CSP is shown in SEQ ID NO: 5; The nucleotide sequence of RH5-GGGGS3-CSP is shown in SEQ ID NO:

6.

2. The RNA encoding the vaccine for preventing and treating Plasmodium infection according to claim 1, characterized in that The malarial parasite is selected from Plasmodium falciparum, Plasmodium knowlesi, Plasmodium ovale, Plasmodium paraovale, Plasmodium vivax or Plasmodium cynomolgus.

3. The RNA encoding the vaccine for preventing and treating Plasmodium infection according to claim 2, characterized in that: The Plasmodium falciparum is the Plasmodium falciparum 3D7 strain.

4. An mRNA vaccine, characterized in that: The mRNA vaccine comprises the coding RNA and the shell as described in any one of claims 1-3.

5. The mRNA vaccine according to claim 4, characterized in that The shell is a lipid nanolayer.

6. A DNA molecule, characterized in that The DNA molecule is used to transcribe the coding RNA according to any one of claims 1 to 3.

7. A biological material for expressing the coding RNA according to any one of claims 1 to 3 or the DNA molecule according to claim 6.

8. A multi-stage dual-target tandem malaria mRNA vaccine combining prevention and treatment, characterized in that: The multi-stage dual-target tandem malaria mRNA vaccine comprises the coding RNA, 5'-Cap and 3'-poly A tail of claim 1; wherein the 5'-Cap is m7G, cap0, cap1, cap2, modified cap0 or modified cap1 structure; The 3'-poly A tail is a polyadenylic acid sequence containing 30-150 adenosine nucleotides.

9. The multi-stage dual-target tandem malaria mRNA vaccine combining prevention and treatment according to claim 8, characterized in that: The preparation method of the multi-stage dual-target tandem malaria mRNA vaccine is: Inserting the RH5-linker-CSP and the DNA expression element into an expression plasmid to obtain a recombinant plasmid; Using the recombinant plasmid to express and obtain tandem mRNA; The tandem mRNA is packaged to obtain the multi-stage dual-target tandem malaria mRNA vaccine combining prevention and treatment.

10. The multi-stage dual-target tandem malaria mRNA vaccine combining prevention and treatment according to claim 9, characterized in that: When the tandem mRNA is packaged to obtain the multi-stage dual-target tandem malaria mRNA vaccine combining prevention and treatment, it includes: The tandem mRNA is packaged using lipid nanoparticles to obtain the multi-stage dual-target tandem malaria mRNA vaccine that combines prevention and treatment.

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