Fusion protein for preventing plasmodium infection, immunogenic composition, and use

Abstract

Provided are a fusion protein for preventing Plasmodium infection, an immunogenic composition, and a recombinant vaccine. Also provided is a novel fusion molecular architecture, comprising a fusion protein containing ADF1, Pfj4, 14-3-3 protein and SUI1, or a fusion protein containing the N-terminal domain of a TRAP antigen and the C-terminal domain of a CSP antigen. Further provided is a composition of the two fusion proteins, which can be used in the research and development of a nucleic acid vaccine or a subunit vaccine. The fusion protein exhibits good immunogenicity, is capable of providing an effective immune protection effect for Plasmodium infection, and has good prospects for application.

Description

A fusion protein and immunogenic composition for preventing Plasmodium infection and their applications Technical Field

[0001] This invention belongs to the field of biomedical technology, particularly the field of immunotherapeutic drugs, and specifically relates to a fusion protein for preventing malaria parasite infection, an immunogenic composition, and its applications. Background Technology

[0002] Malaria is an infectious disease caused by Plasmodium parasites, primarily transmitted by Anopheles mosquitoes. Plasmodium is a single-celled parasite widely distributed in tropical and subtropical regions, especially sub-Saharan Africa, South Asia, and South America. Currently, the main pathogenic Plasmodium species include: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Among them, Plasmodium falciparum is the deadliest, causing severe malaria symptoms such as high fever, anemia, cerebral malaria, and multiple organ failure, resulting in hundreds of thousands of deaths annually, especially among children and pregnant women.

[0003] The pathogenicity of Plasmodium primarily stems from its complex life cycle and multiple virulence factors within the host. After entering the human body through mosquito bites, Plasmodium first multiplies in the liver, then invades red blood cells, triggering rupture and an immune response, leading to the typical symptoms of malaria. The virulence factors of Plasmodium include surface proteins (such as merozoite surface protein MSP1), antigenic variant proteins (such as PfEMP1), and immune evasion mechanisms. These factors enable Plasmodium to evade the host's immune system and survive long-term within the host.

[0004] Traditional treatments for malaria primarily rely on antimalarial drugs such as chloroquine, artemisinin, and their derivatives. However, the malaria parasite has developed widespread resistance to these drugs, particularly the artemisinin resistance of *Plasmodium falciparum*, which has emerged in Southeast Asia and is gradually spreading to other regions, posing a serious challenge to global malaria control. Vaccination is a more effective solution for malaria prevention. Compared to drug treatment, vaccines can activate the host's immune system, producing specific antibodies and immune cells against the malaria parasite, thus providing long-term protection against infection. Furthermore, effective vaccines can reduce reliance on antimalarial drugs, suppress the emergence and spread of drug-resistant malaria parasites, and alleviate the pressure on global malaria control. However, due to the complex life cycle and strong immune evasion mechanisms of the malaria parasite, developing an effective malaria vaccine faces significant challenges. Currently, the only approved malaria vaccine is RTS,S / AS01 (trade name Mosquirix), but its protective effect is limited and its duration of protection is short. RTS,S / AS01 is not available in China, and there is currently a lack of commercially available malaria vaccines in China.

[0005] Currently, several vaccines based on the major antigens of Plasmodium falciparum have entered clinical trials. For example, the RTS,S / AS01 vaccine, based on the circomyosporin (CSP) of Plasmodium falciparum, has been widely used in several African countries, but its protective efficacy is limited and its duration is short. The PfSPZ vaccine, based on live attenuated Plasmodium falciparum, is currently undergoing clinical trials, and preliminary results show that it has a high protective efficacy; however, the production cost of live vaccines is high, the risks are significant, and it is difficult to promote its widespread use. CIS43LS and L9LS are two monoclonal antibodies targeting the circomyosporin (CSP) of Plasmodium falciparum, capable of blocking Plasmodium infection in the liver. CIS43LS showed highly effective protection in Phase I clinical trials, with protection lasting for several months after a single injection; L9LS is an improved version of CIS43LS with stronger neutralizing capacity and a longer half-life, and is currently undergoing Phase II clinical trials. Preliminary results show that L9LS has a protective efficacy of over 80% against Plasmodium falciparum infection in malaria-endemic areas. However, the protection effect of CIS43LS and L9LS is relatively short-lived and the production cost is high, which also makes it difficult to promote them on a large scale.

[0006] Although several malaria vaccines have entered clinical trials, most still face challenges such as the inability to provide long-term protection and the lack of cross-immunity. Studies have shown that long-term protection after Plasmodium infection depends on T-cell-mediated immune responses. However, traditional vaccines (such as subunit vaccines RTS, S / AS01 and antibody vaccines CIS43LS, L9LS) primarily rely on inducing humoral immune responses and struggle to effectively activate T-cell immunity, thus failing to address the issue of long-term protection. Furthermore, since current research largely focuses on vaccine design based on the CSP antigen of Plasmodium falciparum, and CSP proteins vary significantly among different Plasmodium species, these vaccines, while providing specific protection against Plasmodium falciparum, cannot prevent or treat infections caused by other Plasmodium species such as Plasmodium vivax and Plasmodium malariae.

[0007] In summary, although several malaria vaccines have entered the research and clinical trial stages, there is still an urgent need in the field for a malaria vaccine with cross-immunogenicity that can provide long-term protection. Nucleic acid vaccines, through precise molecular design, can fuse and express multiple effective antigens, overcoming the challenge of cross-immune protection. Simultaneously, by expressing antigen proteins intracellularly, nucleic acid vaccines can simultaneously activate humoral and T-cell immune responses, providing a new direction for malaria vaccine development. Therefore, as an emerging technology, nucleic acid vaccines hold the promise of overcoming the limitations of traditional vaccines and providing a new solution for malaria control. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a novel fusion protein, immunogenic composition, recombinant vaccine, molecular architecture design, and applications for the prevention of Plasmodium infection. The present invention provides a novel fusion molecular architecture comprising fusion proteins encoded by four genes: ADF1, Pfj4, 14-3-3 protein, and SUI1; or fusion proteins encoded by two genes: the N-terminal domain of the TRAP antigen and the C-terminal domain of the CSP antigen; and combinations of the above two fusion proteins, which can be used in the development of nucleic acid vaccines or subunit vaccines. The present invention has found that the novel fusion molecule exhibits good immunogenicity and can provide effective immune protection against Plasmodium infection, almost completely inhibiting Plasmodium amplification in some individuals. The present invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, and uses.

[0009] One aspect of the present invention provides a fusion protein, characterized in that it is a fusion protein selected from any of the following:

[0010] (1) Fusion protein A, wherein fusion protein A comprises actin-depolymerizing factor 1 (ADF1) antigen, heat shock protein DNAJ-like Pfj4 (Pfj4) antigen, 14-3-3 protein antigen, and translation initiation factor SUI1 (SUI1) antigen;

[0011] (2) Fusion protein B, wherein fusion protein B contains thrombospondin related anonymous protein (TRAP) antigen and circumsporozoite protein (CSP) antigen.

[0012] Furthermore, the fusion protein also comprises a combination of the fusion protein A and the fusion protein B.

[0013] Furthermore, the antigen is derived from Plasmodium.

[0014] Furthermore, the antigen is derived from one or more of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

[0015] Furthermore, the antigen is derived from Plasmodium falciparum.

[0016] Furthermore, the fusion protein A or fusion protein B has functional elements added at the N-terminus or C-terminus to promote fusion protein expression or epitope presentation.

[0017] Furthermore, the fusion protein A comprises, from the N-terminus to the C-terminus, the actin depolymerization factor 1 antigen, the DNAJ-like heat shock protein Pfj4 antigen, the 14-3-3 protein antigen, and the translation initiation factor SUI1 antigen; optionally, the antigens are linked together by a linker.

[0018] Furthermore, for fusion protein A, the amino acid sequence of the actin depolymerization factor 1 antigen is shown in SEQ ID NO:1, the amino acid sequence of the DNAJ-like heat shock protein Pfj4 antigen is shown in SEQ ID NO:2, the amino acid sequence of the 14-3-3 protein antigen is shown in SEQ ID NO:3, the amino acid sequence of the translation initiation factor SUI1 antigen is shown in SEQ ID NO:4, and the amino acid sequence of the linker is shown in SEQ ID NO:12.

[0019] Furthermore, for fusion protein B, the TRAP antigen is the full-length sequence or the N-terminal domain of TRAP, and the CSP antigen is the full-length sequence or the C-terminal domain of CSP.

[0020] Furthermore, the N-terminus of the fusion protein B further comprises a signal peptide and / or an Fc domain; more preferably, the N-terminus of the fusion protein B further comprises a signal peptide and an Fc domain in sequence.

[0021] Further, the signal peptide is the signal peptide of human cyanidin protein, and the Fc domain is the Fc domain of human IGHG1 protein; optionally, the TRAP antigen and the Fc domain are connected by a spacer sequence.

[0022] Further, for fusion protein B, the TRAP antigen is the N-terminal domain of TRAP, and the amino acid sequence of the N-terminal domain of TRAP is shown in SEQ ID NO:5; the CSP antigen is the C-terminal domain of CSP, and the amino acid sequence of the C-terminal domain of CSP is shown in SEQ ID NO:6; the amino acid sequence of the signal peptide is shown in SEQ ID NO:9; the amino acid sequence of the Fc domain is shown in SEQ ID NO:10; and the amino acid sequence of the spacer sequence is shown in SEQ ID NO:11.

[0023] Furthermore, the amino acid sequence of the fusion protein A is shown in SEQ ID NO:7, and the amino acid sequence of the fusion protein B is shown in SEQ ID NO:8.

[0024] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it comprises a nucleic acid encoding a fusion protein according to any one of the present invention.

[0025] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention.

[0026] Furthermore, the recombinant gene expression cassette also includes one or more of a promoter, a terminator, and a regulatory sequence.

[0027] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention or the recombinant gene expression cassette described in the present invention.

[0028] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0029] Furthermore, the prokaryotic vector comprises an Escherichia coli vector.

[0030] Furthermore, the Escherichia coli vector includes, but is not limited to, pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, and pBR vector.

[0031] Furthermore, the eukaryotic vector includes, but is not limited to, yeast expression vectors, insect expression vectors, and mammalian cell expression vectors.

[0032] Furthermore, the yeast expression vector includes, but is not limited to, pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector.

[0033] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention, or the recombinant gene expression cassette described in the present invention, or the recombinant vector described in the present invention.

[0034] Furthermore, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0035] Furthermore, the eukaryotic cells include mammalian cells, insect cells, and yeast cells.

[0036] Furthermore, the yeast cells include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0037] Furthermore, the prokaryotic cells include, but are not limited to, Escherichia coli cells, Bacillus subtilis cells, and Pseudomonas cells.

[0038] Furthermore, the *E. coli* cells include, but are not limited to, BL21(DE3), DH5α, TOP10, and Rosetta.

[0039] Another aspect of the present invention provides an immunogenic composition or pharmaceutical composition, characterized in that it comprises one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention.

[0040] Furthermore, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0041] Furthermore, the immunogenic composition or pharmaceutical composition comprises the fusion protein A and the fusion protein B.

[0042] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present invention.

[0043] Furthermore, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine.

[0044] Furthermore, the recombinant vaccine is a nucleic acid vaccine.

[0045] Another aspect of the present invention provides the use of one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present invention, and / or one or more recombinant vaccines according to the present invention in the preparation of vaccines or pharmaceuticals for prevention, treatment and / or inoculation.

[0046] Furthermore, the drug is used for the prevention and / or treatment of Plasmodium infection.

[0047] Furthermore, the malaria parasite is one or more of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

[0048] Furthermore, the malaria parasite is Plasmodium falciparum.

[0049] Another aspect of the present invention provides a method for the prevention and / or treatment of a disease, characterized in that it comprises administering to a subject one or more fusion proteins according to any one of the present inventions, and / or one or more recombinant nucleic acid molecules according to the present inventions, and / or one or more recombinant gene expression cassettes according to the present inventions, and / or one or more recombinant vectors according to the present inventions, and / or one or more recombinant host cells according to the present inventions, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present inventions, and / or one or more recombinant vaccines according to the present inventions.

[0050] Furthermore, the disease is a Plasmodium infection.

[0051] Furthermore, the malaria parasite is one or more of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

[0052] Furthermore, the malaria parasite is Plasmodium falciparum.

[0053] The fusion protein, immunogenic composition, recombinant vaccine, etc. of the present invention have the following beneficial technical effects:

[0054] 1. This invention, from the perspective of reverse vaccinology, gradually screens and eliminates candidates, and finally obtains four antigens: ADF1, Pfj4, 14-3-3 protein, and SUI1, which are used to construct new fusion proteins.

[0055] 2. Existing technologies show that simply selecting the RTS domain of CSP cannot provide sufficient protective efficacy. Based on the design strategy for CSP antigen in the RTS,S / AS01 vaccine, this invention further optimizes the design by re-selecting the C-terminal domain of CSP and adding the N-terminal domain of TRAP antigen to form another new fusion protein, which provides more effective epitopes while ensuring molecular conformational stability.

[0056] 3. This invention discovers that the novel fusion molecule possesses excellent immunogenicity and can provide immune protection, making it suitable for the development of nucleic acid vaccines or subunit vaccines. The immunogenic composition of this invention can provide effective immune protection against Plasmodium infection.

[0057] 4. This invention also discovers that the new fusion molecule can be expressed at high levels in eukaryotic cells, providing correct and sufficient immune epitopes and good immune protection, thus providing a new technology for the design of immunotherapeutic drugs for multivalent malaria vaccines.

[0058] 5. Example 6 of the present invention shows that both vaccine A and vaccine B designed based on the present invention can be correctly expressed in eukaryotic cells, and the expressed protein structure is correct and stable, which is beneficial to the presentation of immune epitopes.

[0059] 6. Example 7 of the present invention shows that, in a mouse infection model, vaccines A, B, and combinations of vaccines A and B based on the present invention can all provide good immune protection, and can significantly inhibit the proliferation of Plasmodium in the liver stage, whether it is cellular immunity or humoral immunity.

[0060] 7. Embodiment 7 of the present invention also demonstrates that the combined use of vaccine A and vaccine B can provide a superimposed immune protective effect, achieving unexpected technical results. This result also indicates that the development of malaria vaccines needs to simultaneously consider humoral immunity and cellular immunity.

[0061] 8. Examples 6 and 7 of this invention jointly demonstrate that the vaccine based on this invention can induce effective immunity in mouse model animals and has a good preventive effect against Plasmodium infection. This invention can be applied to the production and research and development of immunotherapeutic drugs for humans and animals, filling a gap in the current field of malaria vaccine research and development, and has extremely high commercial value and broad application prospects. Attached Figure Description

[0062] Figures 1A-1D show the homology analysis of the four cell immune targets screened in this invention in different Plasmodium species; Figure 1A shows the homology analysis of the ADF1 cell immune target in different Plasmodium species; Figure 1B shows the homology analysis of the Pfj4 cell immune target in different Plasmodium species; Figure 1C shows the homology analysis of the 14-3-3 cell immune target in different Plasmodium species; Figure 1D shows the homology analysis of the SUI1 cell immune target in different Plasmodium species.

[0063] Figure 2 is a schematic diagram of the interception strategy of the N-terminal domain of the TRAP antigen and the C-terminal domain of the CSP antigen in this invention.

[0064] Figure 3 is a non-restrictive molecular diagram of vaccines A and B of the present invention.

[0065] Figures 4A and 4B are the quality control results of vaccines A and B of the present invention, respectively.

[0066] Figures 5A and 5B show the expression of vaccines A and B of the present invention after in vitro transfection into HEK293 cells.

[0067] Figure 6 is a schematic diagram of the schedule for immunizing mice with the vaccine of the present invention.

[0068] Figure 7 shows the qPCR relative quantitative detection results of parasite load in liver tissue of mice immunized with the vaccine of this invention 42 hours after challenge.

[0069] Figure 8 shows the ELISA results of CSP-specific IgG antibodies in the serum of mice immunized with the vaccine of this invention, 42 hours after challenge. Detailed Implementation

[0070] Terms and Definitions

[0071] The term "malaria parasite" refers to single-celled parasites belonging to the genus *Plasmodium*, primarily including *Plasmodium falciparum*, *Plasmodium vivax*, *Plasmodium ovale*, *Plasmodium malariae*, and *Plasmodium knowlesi*. Malaria parasites are transmitted by mosquitoes and infect the red blood cells and liver cells of mammals (including humans), causing malaria.

[0072] The term "malaria infection" refers to an infection caused by parasites of the genus Plasmodium, including species such as Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium norotri. Malaria infection is typically transmitted through the bite of infected Anopheles mosquitoes and is characterized by periodic fever, anemia, hepatosplenomegaly, and in severe cases, organ failure or death.

[0073] The term "ADF1" refers to the actin-depolymerizing factor 1 (ADF1) antigen from Plasmodium. It comprises isolated wild-type ADF1 polypeptides and segments of proteins from Plasmodium, as well as variants that stimulate an immune response against the Plasmodium ADF1 protein. Preferably, the amino acid sequence of ADF1 is shown in SEQ ID NO:1.

[0074] The term "Pfj4" refers to the Pfj4 antigen, a heat shock protein DNAJ-like Pfj4, derived from Plasmodium. It comprises isolated wild-type Pfj4 polypeptides and segments of proteins from Plasmodium, as well as variants that stimulate an immune response against the Plasmodium Pfj4 protein. Preferably, the amino acid sequence of Pfj4 is as shown in SEQ ID NO:2.

[0075] The term "14-3-3 protein" refers to the 14-3-3 protein antigen derived from Plasmodium, also known as "14-3-3". It comprises proteins containing isolated wild-type 14-3-3 polypeptides and segments thereof from Plasmodium, and variants capable of stimulating an immune response against the Plasmodium 14-3-3 protein. Preferably, the amino acid sequence of the 14-3-3 protein is as shown in SEQ ID NO:3.

[0076] The term "SUI1" refers to the translation initiation factor SUI1 antigen from Plasmodium. It comprises isolated wild-type SUI1 polypeptides and segments of proteins from Plasmodium, as well as variants that stimulate an immune response against the Plasmodium SUI1 protein. Preferably, the amino acid sequence of SUI1 is as shown in SEQ ID NO:4.

[0077] The term "N-terminal domain of TRAP" refers to the N-terminal domain of the thrombospondin-related anonymous protein (TRAP) antigen from Plasmodium. Proteins comprising the isolated wild-type TRAP peptide and its segments from Plasmodium, and variants capable of stimulating an immune response against the Plasmodium TRAP protein. Preferably, the amino acid sequence of the N-terminal domain of TRAP is as shown in SEQ ID NO:5.

[0078] The term "C-terminal domain of CSP" refers to the C-terminal domain of the circumsporozoite protein (CSP) antigen from Plasmodium. The protein comprises an isolated wild-type CSP C-terminal polypeptide and its segments from Plasmodium, and variants capable of stimulating an immune response against Plasmodium CSP proteins. Preferably, the amino acid sequence of the C-terminal domain of CSP is as shown in SEQ ID NO:6.

[0079] The term "immune response" refers to a humoral response, a cellular response, or both in an organism. Immunity should be measurable by assays, including but not limited to assays measuring the presence or amount of antibodies that specifically recognize proteins or cell surface proteins, assays measuring T cell activation or proliferation, and / or assays measuring the regulation of the activity or expression of one or more cytokines.

[0080] The terms "administration" or "inoculation" refer to the administration of the nucleic acid vaccine or vaccine composition based on the present invention, preferably via intramuscular or subcutaneous routes, although other routes of administration may also be used, such as oral, intranasal (e.g., aerosol or other non-injectable), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or by combination of routes. Intramuscular administration in the neck muscles of animals is preferred. Boosting regimens can be used to adjust the administration regimen to provide optimal immunization.

[0081] The term “expression” includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0082] The term "recombinant nucleic acid molecule" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides can be contained in a suitable vector, which can then be transformed into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide," a "recombinant protein," or a "fusion protein."

[0083] The term "recombinant expression vector" refers to a DNA structure containing a polynucleotide encoding, for example, a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence transcribed into mRNA and translated into protein; and (3) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner and any vector, including plasmids, viruses, bacteriophages, and transposons, may be used. Possible vectors used in this disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, from viruses such as lentiviruses, retroviruses, vaccinia virus, adenovirus, fowlpox virus, baculovirus, SV40, and pseudorabies virus. Self-replicating vectors and non-self-replicating vectors are included.

[0084] The term "mRNA" refers to messenger RNA, which is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template. It carries genetic information and can guide protein synthesis.

[0085] The term "5'-UTR" refers to the "5' untranslated region" or "5'UTR," which is a portion of a gene transcribed into a primary RNA transcript (precursor mRNA) and located upstream of the coding sequence. Primary transcripts are the initial RNA products, containing introns and exons, produced by DNA transcription. Many primary transcripts must undergo RNA processing to form physiologically active RNA. The processing to form mature mRNA includes end modification, intron removal, capping, and / or cleavage of individual rRNA molecules from the precursor RNA. Therefore, the 5'UTR of mRNA is a portion of mRNA that is not translated into protein and is located upstream of the coding sequence. In a genome sequence, the 5'UTR is generally defined as the region between the transcription start site and the start codon. The length of the 5' untranslated region (5'UTR) of vertebrate mRNA can range from tens to hundreds of bases.

[0086] The term "3'-UTR" refers to the "3'-untranslated region" or "3'UTR," which refers to the region located at the 3' end of a gene, downstream of the stop codon in a protein-coding region, and which is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule. The 3'-UTR typically extends from the stop codon of the translation product to a poly(A) sequence that usually attaches after transcription. The 3'-UTR of mammalian mRNA typically has a homologous region known as the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-UTR may contain one or more inverted repeats that can fold to create stem-loop structures that act as barriers to ribonucleases or interact with proteins known to enhance RNA stability (e.g., RNA-binding proteins).

[0087] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce recombinant vaccines based on the present invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells.

[0088] The terms “individual,” “patient,” or “subject” include mammals. Mammals include, but are not limited to, domesticated animals (e.g., pigs, cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats).

[0089] The terms “transformation,” “transfection,” and “transduction” have the meanings generally understood by those skilled in the art: the process of introducing exogenous DNA or RNA into a host.

[0090] The term "pharmaceutical combination" or "pharmaceutical composition" refers to excipients widely used in the pharmaceutical manufacturing industry. The primary purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature, and / or has specific functionalities, and also to provide a method for its effective absorption in a subject. Pharmaceutically acceptable carriers can be inert fillers or active ingredients that provide a function to the pharmaceutical combination (e.g., stabilizing the overall pH of the composition or preventing degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, flow aids, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0091] The term "treatment" refers to exposing a subject to (e.g., administering medication) a recombinant vaccine, composition, or the like based on the present invention after contracting a disease, thereby reducing the symptoms of the disease compared to when not exposed, without implying the necessity of completely suppressing the symptoms. Contracting a disease means the appearance of disease symptoms in the body.

[0092] The term "prevention" refers to the reduction of symptoms after contracting a disease by exposing (e.g., administering medication) a subject to a recombinant vaccine, composition, etc. based on the present invention before contracting the disease, compared to the absence of exposure, and does not imply the necessity of completely suppressing the disease.

[0093] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art described herein.

[0094] This invention discloses a fusion molecular architecture for preventing Plasmodium infection, a method for preparing a recombinant vaccine based on this architecture, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0095] The fusion protein and encoding nucleic acid and their elements provided by this invention, as well as the preparation method and application, all utilize commercially available raw materials and reagents. Based on conventional knowledge in molecular cloning, expression construction, vaccine preparation, and immunization, those skilled in the art can implement the methods and embodiments of this invention.

[0096] The present invention will be further illustrated below with reference to the embodiments. Preferably, a nucleic acid vaccine architecture is selected for the preparation of the recombinant vaccine.

[0097] Example 1: Screening of Cellular Immunogens and Construction of Fusion Proteins

[0098] To screen antigens suitable for cellular immunization, this invention adopts a reverse vaccinology approach. Transcriptome data for *Plasmodium falciparum*, *Plasmodium berghei*, *Plasmodium malariae*, *Plasmodium vivax*, and *Plasmodium ovale* were downloaded from the NCBI database. Through technical analysis, a series of candidate antigens with high transcriptional levels were obtained. These candidate antigens share the following characteristics across different *Plasmodium* species: high transcriptional levels and sequence homology exceeding 80% across different *Plasmodium* sources. Since malaria vaccines are ultimately used for human immunization, antigens with high homology to human proteins need to be excluded. Furthermore, considering that proteins with very small molecular weights provide limited immunotopes during vaccine design, while proteins with very large molecular weights present difficulties in expression and production, a step-by-step screening and elimination process is necessary. Ultimately, four antigens were preferably obtained: ADF1 (actin-depolymerizing factor 1), Pfj4 (heat shock protein DNAJ-like Pfj4), 14-3-3 protein, and SUI1 (translation initiation factor SUI1).

[0099] As shown in Figure 1A, the ADF1 antigen showed >86% homology among the five Plasmodium species; as shown in Figure 1B, the Pfj4 antigen showed >88% homology among the five Plasmodium species; as shown in Figure 1C, the 14-3-3 protein (also known as 14-3-3) antigen showed >98% homology among the five Plasmodium species; and as shown in Figure 1D, the SUI1 antigen showed >93% homology among the five Plasmodium species. Furthermore, the amino acid lengths of the four preferred antigens ranged from 100 to 300, which is the preferred length for constructing fusion proteins.

[0100] Example 2: Selection of TRAP antigen and CSP antigen and construction of fusion protein

[0101] Existing research has shown that antibodies specifically binding to the CSP antigen of Plasmodium falciparum have been proven effective in blocking Plasmodium infection, with a protection rate of approximately 80%. However, due to the short duration of protection and the inability to induce long-term immune protection, their application is limited and difficult to promote widely. From the perspective of preventive vaccines, humoral immunity vaccines remain indispensable. Currently, the RTS,S / AS01 vaccine uses the CSP antigen as the immunogen. Clinical results show that after four doses, the protection rate is around 30%. This demonstrates that simply selecting the RTS domain of CSP cannot provide sufficient protective efficacy; additional humoral immune antigens are needed to improve the protective effect.

[0102] This invention further optimizes the design strategy for the CSP antigen in the RTS,S / AS01 vaccine. As shown in Figure 2, this invention re-selects the C-terminal domain of the CSP antigen and adds the N-terminal domain of the TRAP antigen to form a fusion protein, providing more effective epitopes while ensuring molecular conformational stability.

[0103] Example 3: Construction of the recombinant nucleic acid vaccine of the present invention

[0104] To prepare a recombinant nucleic acid vaccine containing the antigen of the present invention, a non-limiting structural diagram of the nucleic acid vaccine of the present invention is shown in Figure 3. Figure 3 is an exemplary schematic diagram of the molecular structure of the fusion protein expressed by vaccines A and B of the present invention. To prepare a recombinant nucleic acid vaccine capable of producing the molecular structure shown in Figure 3, a gene expression cassette is first constructed to express the antigen sequence described in the present invention. The expression cassette, from the 5' end to the 3' end, sequentially includes: a 5' UTR, a CDS region, a 3' UTR, and PolyA, wherein the CDS region contains the fusion molecular structure described in the present invention. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained through gene synthesis (commissioned by GenScript). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector that can be used for in vitro RNA transcription to obtain a vector plasmid for preparing the recombinant nucleic acid vaccine.

[0105] According to the above method, a carrier for use in subsequent embodiments is prepared:

[0106] (1) Preparation vector of recombinant nucleic acid vaccine A based on the present invention

[0107] Step a: Synthesize the fusion fragment of the four genes “ADF1-Pfj4-[14-3-3]-SUI1”. The genes are linked by a linker sequence as shown in SEQ ID NO:12. The amino acid sequence of the fusion protein encoded by the ADF1-Pfj4-[14-3-3]-SUI1 fusion gene is shown in SEQ ID NO:7. Specifically, the amino acid sequence of the ADF1 antigen is shown in SEQ ID NO:1, the amino acid sequence of the Pfj4 antigen is shown in SEQ ID NO:2, the amino acid sequence of the 14-3-3 antigen is shown in SEQ ID NO:3, and the amino acid sequence of the SUI1 antigen is shown in SEQ ID NO:4.

[0108] Step b: Construct a nucleic acid vaccine architecture carrier.

[0109] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0110] Step c: Prepare recombinant plasmids.

[0111] The gene synthesized in step a is inserted into the vector structure in step b to obtain the recombinant nucleic acid vaccine A preparation vector based on the present invention.

[0112] (2) Preparation vector of recombinant nucleic acid vaccine B based on the present invention

[0113] Step a: Synthesize the gene fragment “human signal peptide-human IGHG1 Fc domain-TRAP antigen N-terminal domain-CSP antigen C-terminal domain”. The human signal peptide is the signal peptide of human Azurocidin protein, and its amino acid sequence is shown in SEQ ID NO:9. The amino acid sequence of the human IGHG1 Fc domain is shown in SEQ ID NO:10. The amino acid sequence of the TRAP antigen N-terminal domain is shown in SEQ ID NO:5. The amino acid sequence of the CSP antigen C-terminal domain is shown in SEQ ID NO:6. The human IGHG1 Fc domain and the TRAP antigen N-terminal domain are connected by a spacer sequence as shown in SEQ ID NO:11. The amino acid sequence of the fusion protein encoded by the “TRAP antigen N-terminal domain-CSP antigen C-terminal domain” fusion gene is shown in SEQ ID NO:8.

[0114] Step b: Construct a nucleic acid vaccine architecture carrier.

[0115] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0116] Step c: Prepare recombinant plasmids.

[0117] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine preparation vector B based on the present invention.

[0118] Table 1. Protein amino acid sequences of the architectural elements involved in this invention.

[0119] Example 4: Preparation of the recombinant nucleic acid vaccine of the present invention

[0120] (1) Preparation of capped mRNA vaccines

[0121] Step a: Linearize the vector plasmid used in Example 1 for producing capped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0122] Step b: The linearized plasmid was subjected to an in vitro co-transcriptional capping reaction to add a 7-methylguanylate cap structure to the 5' end of the transcribed mRNA and the template DNA was degraded.

[0123] (2) Preparation of non-capped mRNA vaccines

[0124] Step a: Linearize the vector plasmid used in Example 1 for producing uncapped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0125] Step b: Perform an in vitro uncapped transcription reaction on the linearized plasmid and degrade the template DNA.

[0126] (3) DNA vaccine preparation

[0127] Step a: Amplify the vector plasmid used in Example 1 for producing DNA vaccines to obtain a large number of target plasmids for purification.

[0128] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0129] Example 5: Quality control of recombinant nucleic acid in vitro transcription and vaccine preparation according to the present invention

[0130] Vaccine A (recombinant nucleic acid vaccine A based on the present invention) and vaccine B (recombinant nucleic acid vaccine B based on the present invention) were prepared using the method for preparing capped mRNA vaccines in Example 4. The purity of the produced recombinant nucleic acids was tested, and the purity of the recombinant nucleic acids used in the experiments was greater than 80%. The quality control peak diagrams of the recombinant nucleic acids based on the present invention are shown in Figures 4A and 4B. Specifically, (1) Recombinant nucleic acid vaccine A based on the present invention has a purity of 83.9%; (2) Recombinant nucleic acid vaccine B based on the present invention has a purity of 86.8%. The above purities all meet the quality requirements for cell transfection experiments and vaccine production.

[0131] Example 6: In vitro expression effect of the recombinant nucleic acid of the present invention

[0132] Using cell transfection reagents, vaccines A and B from Example 5 were transfected into HEK293T cells, and proteins were collected for Western blot analysis. Figures 5A and 5B show the in vitro expression WB (Western blot) results of vaccines A and B transfected into HEK293 cells. The antigen expressed by vaccine A is a cellular immune antigen, which theoretically should be significantly expressed in cell lysates; the antigen expressed by vaccine B is a humoral immune antigen, which theoretically should be significantly expressed in the supernatant. Vaccines containing multiple cellular immune antigens showed significant protein expression in cell lysates; vaccines containing multiple humoral immune antigens showed significant protein expression in the supernatant.

[0133] The molecular weights of the proteins in vaccines A and B are shown in Table 2. Vaccine A is significantly expressed intracellularly, demonstrating that the molecular architecture for promoting antigen fusion expression provided by this invention enables multiple antigens to be successfully translated and correctly folded in eukaryotic cells, exhibiting structural stability and a long half-life. Vaccine B is significantly expressed in the supernatant, demonstrating that the molecular architecture for promoting the secretion of fusion proteins provided by this invention enables multiple antigens to be successfully translated, correctly folded, and secreted extracellularly in eukaryotic cells. Therefore, vaccines designed based on this invention, whether cellular immune antigen fusion proteins or humoral immune antigen fusion proteins, can be correctly expressed in eukaryotic cells, and the expressed protein structures are correct and stable, which is beneficial for immunotope presentation.

[0134] Table 2 Protein molecular weights of vaccines A and B

[0135] Example 7: Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse infection model.

[0136] To verify whether cell-mediated immune antigen fusion molecules and humoral immune antigen fusion molecules have immune protective effects, and to verify whether their combined immunity has an additive and synergistic protective effect, this embodiment uses vaccine A, vaccine B, and a combination of vaccine A and vaccine B to conduct an immunotherapy experiment.

[0137] Since the Plasmodium berghei used in this experiment is a species that rarely infects humans, this invention employs a genetically modified mutant strain of Plasmodium berghei that expresses the circomyosporin protein (CSP) of Plasmodium falciparum. CSP is the main pathogenic protein of Plasmodium falciparum and a key target for vaccine design; therefore, this mutant strain provides an ideal experimental model for verifying the efficacy of malaria vaccines.

[0138] Furthermore, malaria parasites transition from the hepatic stage to the intraerythrocytic stage (hemocyte stage) 48 hours after infection, making accurate quantitative detection of the hepatic stage parasites impossible at this time. Therefore, this invention involves dissection and sample collection 42 hours after challenge to ensure quantitative detection is completed before the parasites fully enter the intraerythrocytic stage. This timing more accurately reflects the vaccine's inhibitory effect on the hepatic stage parasites, thus providing a more realistic assessment of the vaccine's protective efficacy.

[0139] Twenty 6-week-old Balb / c mice were used in the experiment. The specific immunization grouping and treatment procedures are shown in Table 3. In this table and below, the dosage refers to the amount of active ingredient (i.e., the recombinant nucleic acid vaccine of this invention). The immunization and sampling procedures are shown in Figure 6.

[0140] Table 3. Immunization and challenge schedule for experimental animals in Example 7. Note: PBS refers to the use of PBS solution instead of vaccine, serving as the control group (non-treatment group) in the immunotherapy experiment.

[0141] Mice in each group underwent two immunizations according to the immunization protocol shown in Table 3, and were challenged on day 35. Mice were euthanized by cervical dislocation 42 hours after challenge, and whole liver tissue was collected and mechanically ground in Trizol to a paste. Total RNA was extracted from the liver tissue using the Trizol method, and its concentration was measured using Nanodrop. The total RNA concentration was adjusted to 500 ng / μl with RNase-free water, and 1 μg of total RNA was reverse transcribed to obtain cDNA. 1 μl of cDNA was used for qPCR to detect the relative expression level of Plasmodium 18S in the liver tissue, which was used to assess the Plasmodium load in the liver tissue. Primers used in the qPCR experiment are shown in Table 4.

[0142] Table 4 Primers used in Example 7 qPCR experiment Note: The internal reference gene in Table 4 is GapDH.

[0143] The test results are shown in Figure 7. Immunization with vaccine A or vaccine B alone, or with a combination of vaccine A and vaccine B, all provided significant protective effects. Among them, immunization with vaccine B alone was more effective than immunization with vaccine A alone, while immunization with a combination of vaccine A and vaccine B was the most effective, achieving almost complete suppression in 2 / 5 of the individuals.

[0144] Forty-two hours after challenge, mouse serum was collected to detect the titer of CSP-specific IgG antibodies. The full-length CSP protein of Plasmodium falciparum expressed in vitro was used as the antigen to coat ELISA plates, which were then blocked with 1% casein. The primary antibody was mouse serum after challenge (1% casein, 1:100 dilution), with two replicates per sample. The secondary antibody was goat anti-mouse IgG-HRP (1% casein, 1:100 dilution). The results are shown in Figure 8. Vaccine A, a cell-mediated immunization vaccine, did not contain the CSP antigen and therefore induced the lowest CSP antibody titer. Vaccine B, a humoral immunization vaccine, contained the C-terminal domain of the CSP antigen and induced high levels of specific IgG antibodies. Mixed immunization with vaccines A and B also induced high antibody titers.

[0145] The results of the two experiments above show that the humoral immune vaccine B of the present invention can induce mice to produce high levels of specific antibodies, thereby playing a significant protective role in the early stage of Plasmodium infection; while the cell immune vaccine A of the present invention can effectively activate the cell immune response in mice. Although it cannot completely inhibit the proliferation of Plasmodium in the early stage of infection, it can produce a significant synergistic effect when used in combination with the humoral immune vaccine B, achieving the superposition of protective efficacy.

[0146] Furthermore, since the antigen sequence used in the vaccine design is entirely derived from Plasmodium falciparum, and the challenge experiment used a genetically modified Plasmodium berghei (expressing the Plasmodium falciparum CSP protein), the inhibitory effect exhibited by the cell-mediated immunotherapy vaccine further demonstrates that the vaccine of this invention can provide good cross-immune protection. This characteristic lays an important foundation for developing a broad-spectrum malaria vaccine with "one vaccine for multiple protections," and has significant reference value and application prospects.

[0147] In summary, the antigen and fusion molecular architecture provided by this invention can induce effective immunity in mouse model animals, demonstrating good preventive effects against Plasmodium infection. Therefore, this invention can be applied to the production and development of animal immunotherapies, filling a gap in the current field of malaria vaccine development, and possesses extremely high commercial value and broad application prospects.

[0148] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A fusion protein, characterized in that, It is a fusion protein selected from any of the following: (1) Fusion protein A, wherein fusion protein A comprises actin-depolymerizing factor 1 (ADF1) antigen, heat shock protein DNAJ-like Pfj4 (Pfj4) antigen, 14-3-3 protein antigen, and translation initiation factor SUI1 (SUI1) antigen; (2) Fusion protein B, wherein fusion protein B comprises hemagglutinin-related anonymous protein (TRAP) antigen and circumsporozoite protein (CSP) antigen; preferably, the fusion protein further comprises a combination of fusion protein A and fusion protein B.

2. The fusion protein according to claim 1, characterized in that, The antigen is derived from Plasmodium; preferably, the antigen is derived from one or more of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi; more preferably, the antigen is derived from Plasmodium falciparum.

3. The fusion protein according to claim 1 or 2, characterized in that, Optionally, the fusion protein A or fusion protein B may have functional elements added to its N-terminus and / or C-terminus to promote fusion protein expression or epitope presentation.

4. The fusion protein according to claim 1 or 2, characterized in that, The fusion protein A comprises, from N-terminus to C-terminus, the actin depolymerization factor 1 antigen, the DNAJ-like heat shock protein Pfj4 antigen, the 14-3-3 protein antigen, and the translation initiation factor SUI1 antigen; optionally, the antigens are linked together by a linker.

5. The fusion protein according to claim 4, characterized in that, For fusion protein A, the amino acid sequence of the actin depolymerization factor 1 antigen is shown in SEQ ID NO:1, the amino acid sequence of the DNAJ-like heat shock protein Pfj4 antigen is shown in SEQ ID NO:2, the amino acid sequence of the 14-3-3 protein antigen is shown in SEQ ID NO:3, the amino acid sequence of the translation initiation factor SUI1 antigen is shown in SEQ ID NO:4, and the amino acid sequence of the linker is shown in SEQ ID NO:

12.

6. The fusion protein according to claim 1, characterized in that, For fusion protein B, the TRAP antigen is a full-length sequence or the N-terminal domain of TRAP, and the CSP antigen is a full-length sequence or the C-terminal domain of CSP; preferably, the N-terminus of fusion protein B further comprises a signal peptide and / or an Fc domain; more preferably, the N-terminus of fusion protein B further comprises a signal peptide and an Fc domain in sequence; most preferably, the signal peptide is the signal peptide of human cyanidin protein, and the Fc domain is the Fc domain of human IGHG1 protein; optionally, the TRAP antigen and the Fc domain are connected by a spacer sequence.

7. The fusion protein according to claim 6, characterized in that, For fusion protein B, the TRAP antigen is the N-terminal domain of TRAP, and the amino acid sequence of the N-terminal domain of TRAP is shown in SEQ ID NO:5; the CSP antigen is the C-terminal domain of CSP, and the amino acid sequence of the C-terminal domain of CSP is shown in SEQ ID NO:6; the amino acid sequence of the signal peptide is shown in SEQ ID NO:9; the amino acid sequence of the Fc domain is shown in SEQ ID NO:10; and the amino acid sequence of the spacer sequence is shown in SEQ ID NO:

11.

8. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein A is shown in SEQ ID NO:7, and the amino acid sequence of the fusion protein B is shown in SEQ ID NO:

8.

9. A recombinant nucleic acid molecule, characterized in that, The nucleic acid comprising encoding the fusion protein of any one of claims 1-8.

10. A recombinant gene expression cassette, characterized in that, It includes the recombinant nucleic acid molecule of claim 9.

11. A recombinant vector, characterized in that, It comprises the recombinant nucleic acid molecule of claim 9 or the recombinant gene expression cassette of claim 10.

12. A recombinant host cell, characterized in that, It comprises the recombinant nucleic acid molecule of claim 9, the recombinant gene expression cassette of claim 10, or the recombinant vector of claim 11.

13. An immunogenic composition or pharmaceutical composition, characterized in that, The composition comprises one or more of the fusion proteins of any one of claims 1-8, and / or one or more of the recombinant nucleic acid molecules of claim 9, and / or one or more of the recombinant gene expression cassettes of claim 10, and / or one or more of the recombinant vectors of claim 11, and / or one or more of the recombinant host cells of claim 12; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable vector.

14. The immunogenic composition or pharmaceutical composition according to claim 13, characterized in that, The immunogenic composition or pharmaceutical composition contains the fusion protein A and the fusion protein B.

15. A recombinant vaccine, characterized in that, The recombinant vaccine comprises one or more fusion proteins according to any one of claims 1-8, and / or one or more recombinant nucleic acid molecules according to claim 9, and / or one or more recombinant gene expression cassettes according to claim 10, and / or one or more recombinant vectors according to claim 11, and / or one or more recombinant host cells according to claim 12, and / or one or more immunogenic compositions or pharmaceutical compositions according to claim 13 or 14; preferably, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine; more preferably, the recombinant vaccine is a nucleic acid vaccine.

16. Use of one or more fusion proteins according to any one of claims 1-8, and / or one or more recombinant nucleic acid molecules according to claim 9, and / or one or more recombinant gene expression cassettes according to claim 10, and / or one or more recombinant vectors according to claim 11, and / or one or more recombinant host cells according to claim 12, and / or one or more immunogenic compositions or pharmaceutical compositions according to claim 13 or 14, and / or one or more recombinant vaccines according to claim 15 in the preparation of vaccines or pharmaceuticals for prevention, treatment and / or inoculation.

17. The use according to claim 16, characterized in that, The drug is used to prevent and / or treat Plasmodium infection; preferably, the Plasmodium is one or more of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi; more preferably, the Plasmodium is Plasmodium falciparum.

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