Anti-plasmodium vivax propagation blocking composition

Abstract

The present disclosure relates to a composition for inhibiting cross-species infection of malaria caused by Plasmodium vivax in a subject. Preferably, the disclosed compositions are prepared in the form of a vaccine comprising: a plurality of polynucleotides, each of the plurality of polynucleotides comprising a sequence as set forth in SEQ ID No.1 or SEQ ID No.2, expressed in the subject for inducing an immune response responsive to a malaria infection thereof; a liquid phase of lipid nanoparticles, the liquid phase of lipid nanoparticles configured to form a protective layer, the protective layer encapsulating a plurality of polynucleotides within the protective layer; and a pharmaceutically acceptable adjuvant.

Description

Technical Field

[0001] This disclosure relates to a vaccine against the transmission of malaria infection. More specifically, the disclosed vaccine is an mRNA-based vaccine that effectively blocks the transmission or spread of malaria infection between two species, such as between a human host and a mosquito. Background Technology

[0002] Malaria is a global public health problem. The pathogen of this disease is the Plasmodium parasite, which is transmitted by female Anopheles mosquitoes. Plasmodium falciparum and Plasmodium vivax are the main parasitic species of malaria in humans. Currently, 3.5 billion people are at risk of Plasmodium vivax infection[1]. A growing body of research suggests that Plasmodium vivax can lead to complications and even death, particularly in pregnant women and young children[2]. In particular, Plasmodium vivax infection is associated with chronic anemia and can lead to maternal anemia, miscarriage, low birth weight, and congenital malaria[3]. Compared to Plasmodium falciparum, Plasmodium vivax infection is more complex to manage due to the parasite’s ability to remain dormant in the liver of an infected individual for months to years before being reactivated[4]. These reservoirs of dormant parasites, known as dormants, not only prolong clinical onset but also sustain transmission, leading to widespread epidemics worldwide[4]. Furthermore, Plasmodium vivax is more effectively transmitted to mosquitoes[5,6]. Currently, drugs suitable for killing dormant cells are limited to the 8-aminoquinoline class drugs primaquine and tafenoxane, which are known to cause acute hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. In addition, primaquine and tafenoxane are contraindicated during pregnancy and lactation. These drugs are not recommended for children under six months of age. Therefore, alternative methods are needed for controlling, curing, or even containing the spread of vivax malaria[7].

[0003] Vaccines are among the most successful and cost-effective public health tools for eradicating, containing, and reducing infectious diseases. Thus, malaria vaccines designed to reduce the transmission of the mosquito-specific stage or mosquito-specific parasite from humans to mosquitoes, known as transmission blocking vaccines (TBVs), are considered an important tool for eliminating Plasmodium vivax[8]. As an effective TBV is expected to reduce overall malaria transmission, it will also have an impact on the intensity of mosquito-to-human transmission and the size of the dormant reservoir in the population. Pvs25, a protein antigen expressed on the surface of the kinetid zygote of Plasmodium vivax, has been proposed as a leading TBV candidate due to its potent transmission blocking efficacy[12,13][9-11]. Studies evaluating the transmission of Plasmodium vivax malaria using recombinant Pvs25 formulated with dehydrated gels have revealed that the vaccine significantly reduced the number of parasites in mosquitoes, but the titer was insufficient to be considered an effective vaccine

[14] . Another study to test a transmission blocking vaccine based on Pvs25 / Montanide ISA 51 observed a highly functional antibody response. Nevertheless, this study encountered problems with local reactions, which forced the termination of further development of the formulation

[15] . In view of this, there is an urgent need for a new vaccination platform, particularly a Pvs25-based transmission blocking vaccine that can induce potent and durable transmission blocking immunity while being safe for clinical use.

[0004] As mentioned above, conventional vaccine approaches used in early studies to develop vaccines against Plasmodium vivax, such as live attenuated pathogen, inactivated pathogen, and protein subunit vaccines, have encountered serious setbacks. Nevertheless, in recent years, various forms of mRNA-based vaccines have been shown to be highly effective against cancer and infectious diseases compared to conventional approaches.

[16] One of the most promising vaccine platforms involves nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs). Nucleoside-modified mRNA-LNP vaccines developed by Pfizer / BioNTech and Moderna have been shown to be effective in containing Covid-19.

[17] A single dose of nucleoside-modified mRNA-LNP vaccine elicited potent and sustained protective neutralizing antibody responses against Zika virus and influenza virus in mice and nonhuman primates.[18-20] Furthermore, comparative studies have confirmed that nucleoside-modified mRNA-LNP vaccines are superior to conventional vaccine forms, such as protein subunit vaccines with added MF59 adjuvant and inactivated pathogen vaccines.

[18] Nucleoside-modified mRNA-LNP vaccines have a unique ability to effectively induce follicular helper T cells

[18] , which are key drivers of antibody affinity maturation and the production of protective neutralizing antibodies

[21] . Recently, nucleoside-modified mRNA vaccines encoding Plasmodium falciparum CSP have been found to be immunogenic in mice and protective in both homologous and heterologous transgenic rodent models, making them a powerful platform for further development of malaria vaccines

[22] , particularly effective against Plasmodium vivax infection. Summary of the Invention

[0005] One object of this disclosure is to provide a composition that, upon administration of the disclosed composition to a subject via a predetermined route, induces an immune response in the subject against malaria infection, particularly malaria infection caused by Plasmodium vivax.

[0006] Another object of this disclosure is to provide a vaccine composition that effectively inhibits, prohibits, and / or reduces the likelihood of cross-species transmission of malaria, the pathogen of which includes, but is not limited to, Plasmodium vivax. In particular, the disclosed composition is capable of inducing an immune response in a subject receiving the vaccine to delay, prohibit, and / or prevent the development of merozoites and / or dormant cells in the subject and / or mosquitoes that feed on the subject, through one or more antibodies produced by the subject that are associated with the received vaccine composition. The subject may not yet be infected with malaria.

[0007] Further objectives of this disclosure relate to an mRNA-based vaccine composition designed to prevent the development of merozoites and / or dormant cells in mammalian subjects after administration of an effective dose of one or more drugs over a period of time.

[0008] Another object of this disclosure is to provide a method for inducing an immune response in a subject by administering an mRNA-based vaccine composition prepared using a polynucleotide sequence of a Plasmodium vivax kinetic surface protein antigen (i.e., the Pvs25 protein).

[0009] This disclosure satisfies at least one of the above-mentioned objectives in whole or in part, wherein one embodiment of this disclosure is a vaccine composition for preventing cross-species infection of malaria caused by Plasmodium vivax in a subject, and even for preventing cross-species infection of malaria caused by Plasmodium vivax in mosquitoes that may carry Plasmodium vivax merozoites and / or dormant zoites and feed on the subject. Preferably, the vaccine composition comprises: a plurality of polynucleotides, each of the plurality of polynucleotides comprising the sequence as described in SEQ ID No. 1 or SEQ ID No. 2, the polynucleotides being expressed in the subject to induce an immune response in response to malaria infection; a liquid phase of lipid nanoparticles configured to form a protective layer encapsulating the plurality of polynucleotides within the protective layer; and a pharmaceutically acceptable adjuvant.

[0010] In some embodiments, the polynucleotide of the disclosed composition has uridine, which is substituted or replaced by any one or a combination of pseudouridine and 1-methyl-pseudouridine.

[0011] In many other embodiments, the lipid nanoparticles comprise cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and PEG. More preferably, the molar ratio of the cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and PEG is 40-60:5-15:30-50:1-5.

[0012] In some embodiments, the polynucleotide further comprises a region encoding at least one signal peptide, which is incorporated upstream of the polynucleotide sequence SEQ ID No. 1 or SEQ ID No. 2 in the expression construct carrying the polynucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2. In some embodiments, the incorporation of the signal peptide may facilitate better recognition of the expressed peptide by the host or subject receiving the vaccine. According to several preferred embodiments, the signal peptide is derived from any of the MHC class II signal peptides. Some examples of the signal peptides used are detailed in SEQ ID No. 5 or SEQ ID No. 6.

[0013] In other embodiments, the sequences of SEQ ID No. 1 and / or SEQ ID No. 2 are derived from the Sal I strain of Plasmodium vivax.

[0014] Another aspect of this disclosure relates to a method for inducing an immune response in a subject to malaria infection caused by Plasmodium vivax. Essentially, the disclosed method comprises administering a vaccine composition to the subject via an intramuscular or subcutaneous route, the vaccine composition comprising: a plurality of polynucleotides, each of which comprises a sequence as described in SEQ ID No. 1 or SEQ ID No. 2, the polynucleotides being expressed in the subject to induce an immune response to malaria infection; a liquid phase of lipid nanoparticles configured to form a protective layer encapsulating the plurality of polynucleotides within the protective layer; and a pharmaceutically acceptable adjuvant. Preferably, the polynucleotides have uridine, which is substituted with any one or a combination of pseudouridine and 1-methyl-pseudouridine.

[0015] According to various embodiments of the disclosed method, the lipid nanoparticles comprise cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and PEG. More preferably, the molar ratio of the cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and PEG is 40-60:5-15:30-50:1-5. Attached Figure Description

[0016] Figure 1 These are gel images, which show that various constructs with different modifications as described in Example 1 were successfully expressed for further experimental study. Figure 2 This is a graph showing the results of antigen-specific antibody responses in mice immunized with a nucleoside-modified construct incorporating the polynucleotide sequence of Pvs25; Figure 3 These are photomicrographs showing the results of testing the surface of the natural antigen on the surface of the Plasmodium vivax kinetus using mixed plasma from immunized mice. The top subplot shows nuclear staining with DAPI, followed by staining with anti-mouse IgG Alexa Fluor 488 secondary antibody in the second row, the third row showing the merged image, and the bottom row showing the differential interference contrast (DIC) image (dots represent individual mice, horizontal lines represent the geometric mean of the 95% CI, and vertical lines represent error bars).

[0017] Figure 4 This is a graph showing the functional activity of antiserum induced by Pvs25 mRNA-LNP vaccine, where TRA represents the percentage reduction in mean oocyst density relative to non-immune serum in the presence of each specific immune serum (using one-way ANOVA with Bonferroni correction, *p<0.05). Figure 5This is a graph showing the detection of Pvs25-specific antibody responses in mice using ELISA (n=17 per group) one month after the initial vaccination (month 0) and one month after the booster vaccination (month 1). Figure 6a The graph shows the activity index of anti-Pvs25. Figure 6b A graph showing the IgG2a / IgG1 ratio against Pvs25 (using one-way ANOVA with Bonferroni correction, *p<0.05). Figure 7a The graph shows the frequencies at which CD4-T cells from mice immunized using different constructed modalities produced different amounts of interleukins and interferons. Figure 7b A graph showing the frequency of different interleukins and interferons produced by CD8-T cells of mice immunized using different constructs prepared; Figure 8 This is a graph showing the immune responses induced in mice immunized with different constructs prepared during a 7-month period; Figure 9 Includes a graph showing the transmission reduction activity (TRA) induced by different immunization regimens in the first and seventh months; Figure 10 It shows TRA (from Figure 9 A graph showing the relationship between () and total IgG produced; and Figure 11 This is a sequence listing showing SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.15 and SEQ ID No.6. Detailed Implementation

[0018] In the following description, this disclosure will be presented in accordance with preferred embodiments and with reference to the accompanying drawings. However, it should be understood that the description of preferred embodiments and drawings is merely for the purpose of discussing various disclosed embodiments, and it is foreseeable that those skilled in the art can devise various modifications without departing from the scope of the appended claims.

[0019] As used in this article, the terms "polynucleotide" or "nucleic acid" refer to mRNA, RNA, cRNA, cDNA, or DNA. The term typically refers to oligonucleotides that are longer than 30 nucleotide residues.

[0020] As used herein, the term "gene" can refer to a functionally significant DNA sequence. It can be a naturally occurring nucleic acid sequence or a recombinant nucleic acid sequence derived from a natural source or a synthetic construct. The term "gene" can also refer to, for example, but not limited to, cDNA and / or mRNA that are directly or indirectly encoded by a genomic DNA sequence or directly or indirectly derived from a genomic DNA sequence.

[0021] According to one aspect of this disclosure, a composition is provided for preventing cross-species infection of malaria caused by Plasmodium vivax in a subject. Preferably, the composition is configured to inhibit, prevent, prohibit, and / or stop the development of merozoites and / or dormant cells that may be carried by a human subject and / or a vector (such as a female mosquito of the Anopheles genus). The disclosed vaccine composition typically comprises: a plurality of polynucleotides, each of which comprises a sequence as described in SEQ ID No. 1 or SEQ ID No. 2, the polynucleotides being expressed in the subject to induce an immune response responsive to malaria infection; a liquid phase of lipid nanoparticles configured to form a protective layer encapsulating the plurality of polynucleotides within the protective layer; and a pharmaceutically acceptable adjuvant.

[0022] Referring to the sequence listing of SEQ ID No. 1, the inventors of this disclosure use the polynucleotide sequence of the Plasmodium vivax kinesi surface protein antigen, namely the Pvs25 protein, as a template or basic template to prepare the polynucleotide sequence in mRNA form of SEQ ID No. 1. Using mRNA instead of conventional protein-based vaccines, the disclosed composition is relatively safer to use, considering that mRNA is a non-infectious and non-integrative platform that does not expose subjects to the risk of infection or insertional mutations. Furthermore, mRNA is the smallest genetic vector; therefore, anti-vector immunization can be avoided, and mRNA vaccines can be administered repeatedly. In addition, the mRNA platform of this disclosure can be degraded through normal cellular processes, and its in vivo half-life can be regulated by various modifications introduced and the delivery methods used to further reduce the possibility of any undesirable outcomes. It is noteworthy that the polynucleotides used in the preparation of the vaccine composition in this disclosure can be of natural origin (preferably further processed through one or more purification steps) or synthetic origin. The Pvs25 sequence used may be modified or not modified, depending on the specified result to be achieved, as will be further elaborated below. For example, in some embodiments, the polynucleotide contains uridine, which is present in a natural or universal mRNA template, and is substituted by any one or a combination of pseudouridine and 1-methyl-pseudouridine. The use of pseudouridine and / or 1-methyl-pseudouridine incorporated into the disclosed composition ensures higher levels of protein expression in subjects. The substitution of uridine with pseudouridine and / or 1-methyl-pseudouridine also reduces the risk of severe inflammatory responses in subjects after receiving the disclosed vaccine composition.

[0023] According to further embodiments of the disclosed compositions, the polynucleotides used may contain an I130T mutation as an additional modification to the polynucleotide sequence for improving the overall performance of the disclosed compositions. Figure 11 The sequence of the polynucleotide with the expected mutation is disclosed in SEQ ID No. 2. Preferably, the included mutation enhances the transmission blocking activity against other variants of the Plasmodium parasite, particularly the Plasmodium vivax variant carrying the aforementioned mutation. Furthermore, the sequences of the peptides encoded by the corresponding peptides in SEQ ID No. 1 and SEQ ID No. 2 are shown in SEQ ID No. 3 and SEQ ID No. 4.

[0024] In some embodiments, the polynucleotide further comprises a region encoding at least one signal peptide, which is incorporated upstream of the SEQ ID No. 1 or SEQ ID No. 2 sequence in the expression construct carrying the polynucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2. In some embodiments, the incorporation of the signal peptide may facilitate better recognition of the expressed peptide by the host or subject receiving the vaccine. According to several preferred embodiments, the signal peptide is derived from any of the MHC class II signal peptides. Some examples of the signal peptides used are detailed in SEQ ID No. 5 or SEQ ID No. 6. In particular, in some embodiments, each of the plurality of polynucleotides further comprises SEQ ID No. 5 and / or SEQ ID No. 6 located upstream or downstream of SEQ ID No. 1 or SEQ ID No. 2.

[0025] As described above, a liquid phase composed of LNPs is used to form a protective layer to encapsulate, encapsulate, or package various polynucleotides. By mixing LNPs with polynucleotides under regulated and controlled conditions, the LNPs self-assemble into particles of 80-100 nm in size. The protective layer established by the LNPs creates a neutral surface relative to the cellular environment, thereby preventing the encapsulated polynucleotides from extensively binding to serum proteins before expression. More specifically, LNPs loaded with mRNA are taken up via endocytosis, then destroyed by endosomal acidification via the endosomal pathway, and a portion of the RNA escapes from the endosome to enter the cytosol, where protein production from the mRNA occurs [35-37]. In some embodiments, the lipid nanoparticles comprise cationic lipids, distearylphosphatidylcholine (DSPC), cholesterol, and PEG. More preferably, the molar ratio of the cationic lipids, distearylphosphatidylcholine (DSPC), cholesterol, and PEG is 40-60:5-15:30-50:1-5.

[0026] For various embodiments, the adjuvant can be any one or a combination of aqueous solution, saline solution, Ringer's solution, isotonic sodium chloride, synthetic monoglyceride, synthetic diglyceride, polyethylene glycol, glycerol, propylene glycol, antibacterial agent, antioxidant, chelating agent, buffer, tension regulator, and cryoprotectant. Preferably, the antibacterial agent can be benzyl alcohol, methylparaben, etc. Preferably, the antioxidant can be ascorbic acid, sodium bisulfite, etc. Preferably, the buffer can be acetate, citrate, or phosphate, etc. Preferably, the tension regulator can be sodium chloride, glucose, etc. Preferably, the cryoprotectant can be sucrose, trehalose, etc. In addition, chelating agents such as ethylenediaminetetraacetic acid can also be used.

[0027] Another aspect of this disclosure relates to a method for inducing an immune response in a subject to malaria infection caused by Plasmodium vivax. The induced immune response results in the production of antibodies responsive to the Pvs25 antigen, which in turn prevents or inhibits the development of Plasmodium vivax (particularly merozoites and / or dormant cells) in the subject. It is important to note that the produced antibodies can be transferred to malaria vectors, such as female Anopheles mosquitoes that happen to feed on the blood of a subject carrying the antibodies. These antibodies are configured to achieve the same result in mosquitoes, preventing or inhibiting the development of Plasmodium vivax merozoites and / or dormant cells. Therefore, mosquitoes carrying these antibodies will be unable to transmit malaria, thereby reducing the number of available malaria vectors. Through this inhibitory mechanism, the disclosed method is able to reduce or prevent cross-species transmission of malaria, particularly caused by Plasmodium vivax. The disclosed method preferably includes the step of administering a vaccine composition to the subject via an intramuscular or subcutaneous route. More specifically, the vaccine composition used in the disclosed method comprises: a plurality of polynucleotides, each of which comprises a sequence as described in SEQ ID No. 1, the polynucleotides being expressed in the subject to induce an immune response in response to malaria infection; a liquid phase of lipid nanoparticles configured to form a protective layer encapsulating the plurality of polynucleotides within the protective layer; and a pharmaceutically acceptable adjuvant.

[0028] Corresponding to the vaccine compositions mentioned above, the polynucleotides of the vaccine compositions used in the disclosed methods have uridine, which is replaced by any one or a combination of pseudouridine and 1-methyl-pseudouridine to improve the expression of antigen proteins in subjects.

[0029] Similarly, the lipid nanoparticles present in the vaccine composition used in the disclosed method comprise cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and PEG. More preferably, the molar ratio of the cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and PEG is 40-60:5-15:30-50:1-5.

[0030] The following examples are intended to further illustrate this disclosure, but are not intended to limit this disclosure to the specific implementations described herein.

[0031] Example 1 Western blotting using a Pvs25-specific antibody confirmed the expression of Pvs25 in all mRNA-LNPs of human monocyte-derived dendritic cells (mo-Des). Results are presented in... Figure 1All eight constructs were designed based on the Pvs25 gene sequence of the reference Plasmodium vivax Sal I strain. Four constructs expressed Pvs25 without a C-terminal GPI anchor. Two of them (Pvs25A and Pvs25A I130T) had wild-type signal peptide sequences; one of these (Pvs25A I130T) contained the dominant I130T substitution found in Asian Plasmodium vivax isolates. Two other constructs (Pvs25A IL-2 SP and Pvs25A HLA-DR SP) had exogenous signal peptide sequences (human IL-2 and HLA-DR), known to enhance protein expression. The other four constructs encoded Pvs25 with a C-terminal GPI anchor. Pvs25F encoded the full-length sequence of the Pvs25 gene from Sal I. The other three constructs (Pvs25F I130T, Pvs25F IL-2 SP, and Pvs25F HLA-DR SP) contain the full-length sequence of Pvs25 and have a) an I130T mutation, b) an IL-2 signal peptide, or c) an HLA-DR signal peptide, respectively. Cells transfected with polycytidine (PolyC) RNA-LNP were used as a negative control. Recombinant protein (Pvs25A) produced from a cell-free wheat germ system was used as a positive control.

[0032] Example 2 Following administration of the various constructs prepared in Example 1 to mice, experiments were performed to assess the immune response. Mice were immunized using the various prepared Pvs25 mRNA-LNP constructs. Specifically, BALB / C mice were immunized via intramuscular injection of 3 µg, 10 µg, or 30 µg of Pvs25 mRNA-LNP, with a priming-boost regimen (N=6 per group) at 4-week intervals. In the control group, mice received 30 µg of PolyC RNA-LNP (N=6). Results are presented in... Figure 2 middle.

[0033] Furthermore, after final immunization with 30 µg of Pvs25F mRNA-LNP (Pvs25F) or Poly C mRNA-LNP (PolyC), reactivity to the natural antigens on the surface of the Plasmodium vivax kinetus was assessed by immunofluorescence assay (IFA) using mixed plasma from immunized BALB / c mice. Results are shown in... Figure 3 middle.

[0034] Example 3 Direct membrane feeding assay (DMFA) was performed to evaluate the transmission reduction activity (TRA) of antiserum from immunized mice at different dilutions. Laboratory-raised uninfected mosquitoes were fed blood from Plasmodium vivax malaria patients (n=5) containing either immune serum (from mice that received 30 µg of each of the eight Pvs25 mRNA-LNPs) or non-immunized serum (from mice that received 30 µg of PolyC RNA-LNP serum). Seven days after feeding, the mosquitoes were dissected, and oocysts were counted under an optical microscope. The results are shown below. Figure 4 As shown.

[0035] Example 4 This disclosure compares the immunogenicity and immunodynamics of four different immunization regimens: a) homologous primary-boost vaccination with Pvs25F mRNA-LNP, b) homologous primary-boost vaccination with Pvs25 protein (with ISA-51 adjuvant), c) heterologous vaccination with Pvs25 mRNA-LNP / Pvs25 protein, and d) heterologous vaccination with Pvs25 protein / Pvs25F mRNA-LNP. Mice were randomly assigned to eight groups (n=17 mice per group). In four vaccination groups, mice were vaccinated with homologous or heterologous primary-boost regimens (10 µg mRNA or 10 µg recombinant protein vaccine, with a four-week interval between primary and booster vaccinations). In the other four groups, 10 µg of Poly(C)RNA-LNP or Montanide ISA-51 VG was administered to each vaccination group as a negative control.

[0036] ELISA testing showed no Pvs25-specific antibody response in the serum of the control group. Figure 5 The mRNA / mRNA homologous primary-boost vaccination produced the strongest Pvs25-specific antibody response, with a GMT of approximately 140,000. The protein / protein homologous vaccination had a GMT of approximately 40,000. The protein / mRNA vaccination had a GMT of approximately 80,000, and the mRNA / protein vaccination had a GMT of approximately 46,000.

[0037] This disclosure further characterizes the quality of the Pvs25-specific antibody. The IgG1 and IgG2a subclasses are expressed as the IgG2a / IgG1 ratio and antigen-antibody affinity index. Here, mixed sera are used to determine the IgG2a / IgG1 ratio and affinity index (…). Figure 6a and Figure 6b The IgG subclass patterns were similar across all vaccination regimens, with an IgG2a / IgG1 ratio of approximately 0.75. Affinity indices were also similar across groups.

[0038] Example 5 Experiments were conducted to assess the production of IFN-γ and IL-2 by CD4+ and CDS+ T cells. Specifically, spleen cells were obtained from immunized mice one month post-immunization and then stimulated with Pvs25 peptide to analyze IFN-γ and IL-2 production by CD4+ and CDS+ T cells, with controls (n=2) selected from four predetermined controls. Results are summarized in […]. Figure 7a and Figure 7b middle.

[0039] More specifically, the cellular immune responses of mouse spleen cells in homologous and heterologous primation-boost assays were evaluated. As measured by IFN-γ and IL-2 production, mRNA / mRNA homologous primation-boost vaccination induced the strongest Pvs25-specific CD4+ and CDS+ T cell responses, while protein / protein vaccination induced almost no T cell responses. The results are summarized in […]. Figure 7a and Figure 7b Similarly, mRNA / mRNA homologous vaccination elicited the strongest memory B cell response, which was almost absent in protein / protein homologous vaccination. Protein / mRNA heterologous vaccination produced a positive but moderate cellular response, while mRNA / protein vaccination elicited a very low cellular response, similar to protein / protein vaccination. The results are presented in Figure 7.

[0040] Example 6 This disclosure also uses ELISA to determine the Pvs25 antibody response in mice immunized with four different constructs prepared over a 7-month period, with measurements performed monthly. Nine mice in each group were maintained for 7 months following a booster dose at month 0. Results are summarized in Figure 8 In particular, Pvs25 antibody levels were tracked monthly for 7 months following the booster vaccination to assess the durability of the antibody response. Figure 8 In all vaccination groups, antibody levels peaked one month after the booster and declined over the following months. Consistent with previous results, GMT was highest in the mRNA / mRNA group and lowest in the protein / protein group. By month 7, antibody levels had GMTs of 36,000 for mRNA / mRNA, 2,300 for protein / protein, 25,000 for protein / mRNA, and 18,000 for mRNA / protein.

[0041] In addition, the transmission-reducing activity in immunized mice was investigated. Specifically, a direct membrane feeding assay (DMFA) was performed using mixed serum obtained from mice at months 1 and 7 against blood infected with *Plasmodium vivax* from four different patients. Transmission-reducing activity (TRA) was determined at serum dilutions of 1:2, 1:10, and 1:50, with the results shown in [Figure / Table / Insert Table ... Figure 9 In particular, following booster immunizations in both homologous and heterologous primate-boost assays, Pvs25 antibody levels in mice (n=8-9 per group) were monitored monthly for 7 months to assess antibody response persistence via ELISA. Results are presented in... Figure 9 In the middle. A similar pattern was observed when TRA was tracked over time; TRA peaked one month after intensification ( Figure 9 ).

[0042] At this point, all vaccination regimens demonstrated full efficacy (100% TRA) at a serum dilution of 1:2. However, at the same 1:2 dilution, the TRA of the protein / protein vaccine decreased to 63%, while the other three vaccination strategies maintained high efficacy (>99%) 7 months after the booster. Among the four vaccination regimens, the mRNA / mRNA group showed the most durable functional response, as the TRA in this group remained above 80% even at a 1:50 dilution. When the TRA data for all immunization regimens were pooled, a clear dose-response relationship between TRA and total IgG was observed, as expected. Figure 10 The half-maximal inhibitory concentration (IC50) of total IgG from Pvs25 was 781 (CI95: 564-1003) reciprocal titer units.

[0043] It should be understood that this disclosure may be embodied in other specific forms and is not limited to the only embodiments described above. However, modifications and equivalents of the disclosed concept (such as those readily apparent to those skilled in the art) are intended to be included within the scope of the appended claims.

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Claims

1. A vaccine composition for preventing cross-species infection of malaria caused by Plasmodium vivax in subjects, comprising: A plurality of polynucleotides, each of which comprises a sequence as described in SEQ ID No. 1 or SEQ ID No. 2, wherein the polynucleotides are expressed in the subject to induce an immune response in response to malaria infection; A liquid phase of lipid nanoparticles, wherein the liquid phase of the lipid nanoparticles is configured to form a protective layer, the protective layer encapsulating the plurality of polynucleotides within the protective layer; and Pharmaceutically acceptable adjuvants.

2. The vaccine composition according to claim 1, wherein the polynucleotide has uridine, and the uridine is substituted by any one or a combination of pseudouridine and 1-methyl-pseudouridine.

3. The vaccine composition according to claim 1, wherein the lipid nanoparticles comprise cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and PEG.

4. The vaccine composition according to claim 3, wherein the molar ratio of the cationic lipid, distearate phosphatidylcholine (DSPC), cholesterol and PEG is 40-60:5-15:30-50:1-5.

5. The vaccine composition according to claim 1, wherein the sequences of SEQ ID No. 1 and / or SEQ ID No. 2 are derived from the Sal I strain of Plasmodium vivax.

6. The vaccine composition according to claim 1, wherein each of the plurality of polynucleotides further comprises SEQ ID No. 5 and / or SEQ ID No. 6 located upstream or downstream of SEQ ID No. 1 or SEQ ID No.

2.

7. The vaccine composition according to claim 1, wherein the pharmaceutically acceptable adjuvant is any one or a combination of aqueous solution, saline solution, Ringer's solution, isotonic sodium chloride, synthetic monoglyceride, synthetic diglyceride, polyethylene glycol, glycerol, propylene glycol, antibacterial agent, antioxidant, chelating agent, buffer, tension modifier and cryoprotectant.

8. A method for inducing an immune response in a subject to malaria infection caused by Plasmodium vivax, the method comprising: The vaccine composition is administered to the subject via an intramuscular or subcutaneous route, the vaccine composition comprising: A plurality of polynucleotides, each of which comprises a sequence as described in SEQ ID No. 1 or SEQ ID No. 2, wherein the polynucleotides are expressed in the subject to induce an immune response in response to malaria infection; A liquid phase of lipid nanoparticles, wherein the liquid phase of the lipid nanoparticles is configured to form a protective layer, wherein the protective layer encapsulates the plurality of polynucleotides within the protective layer; and Pharmaceutically acceptable adjuvants.

9. The method of claim 8, wherein the polynucleotide has uridine, and the uridine is substituted by any one or a combination of pseudouridine and 1-methyl-pseudouridine.

10. The method of claim 8, wherein the lipid nanoparticles comprise cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and PEG.

11. The method according to claim 8, wherein the molar ratio of the cationic lipid, distearate phosphatidylcholine (DSPC), cholesterol and PEG is 40-60:5-15:30-50:1-5.

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