Malaria is a life-threatening global epidemic disease and has caused more than 400,000 deaths in 2019. To control and prevent malaria, the development of a vaccine is a potential method. An effective malaria vaccine should either combine antigens from all stages of the malaria parasite\'s life cycle, or epitopes of multiple key antigens due to the complexity of the Plasmodium parasite. Malaria\'s random constructed antigen-1 (M.RCAg-1) is one of the recombinant vaccines, which was selected from a DNA library containing thousands of diverse multi-epitope chimeric antigen genes. Moreover, besides selecting an antigen, using an adjuvant is another important procedure for most vaccine development procedures. Freund\'s adjuvant is considered an effective vaccine adjuvant for malaria vaccine, but it cannot be used in clinical settings because of its serious side effects. Traditional adjuvants, such as alum adjuvant, are limited by their unsatisfactory immune effects in malaria vaccines, hence there is an urgent need to develop a novel, safe and efficient adjuvant. In recent years, Pickering emulsions have attracted increasing attention as novel adjuvant. In contrast to classical emulsions, Pickering emulsions are stabilized by solid particles instead of surfactant, having pliability and lateral mobility. In this study, we selected aluminum hydroxide gel (termed as \"alum\") as a stabilizer to prepare alum-stabilized Pickering emulsions (ALPE) as a malaria vaccine adjuvant. In addition, monophosphoryl lipid A (MPLA) as an immunostimulant was incorporated into the Pickering emulsion (ALMPE) to further enhance the immune response. In vitro tests showed that, compared with alum, ALPE and ALMPE showed higher antigen load rates and could be effectively endocytosed by J774a.1 cells. In vivo studies indicated that ALMPE could induce as high antibody titers as Freund\'s adjuvant. The biocompatibility study also proved ALMPE with excellent biocompatibility. These results suggest that ALMPE is a potential adjuvant for a malaria vaccine.

Infection treatment vaccine (ITV) can lead to sterile protection against malaria infection in mice and humans. However, parasite breakthrough is frequently observed post-challenge. The mechanism of rapid decline in protection after the last immunization is unclear. Herein, C57BL/6 mice were immunized with 103, 105, or 107 ITV thice at 14-day intervals. Mice were challenged with 103 parasites at 1, 3, and 6 months after last immunization and the protection was checked using blood smear. The phenotypes of B cells were analyzed by flow cytometry. The levels of serum cytokines were quantified using cytometric bead array. The 103 ITV vaccination group exhibited 100% protection at 1 month after last immunization, and the 105 group showed sterile protection at 3 months after last immunization. However, the 107 group showed only partial protection. Further, the protection declined to 16.7% at 6 months after last immunization in 105 and 107 groups, whereas it maintained for more than 60% in 103 group. The number of memory B cells (MBC) decreased along with the decline in protection. However, programmed cell death protein 1 (PD-1) expressed on MBCs did not show significant variation among the three groups. Interestingly, CD19+CD1dhiCD5hi B cells, defined as B10 cells, exhibited negative regulation with respect to protection. The numbers of CD19+CD1dhiCD5hi B cells in the 103 group at 1 months and in the 105 group at 3 months post-immunization were the lowest compared to those in the other groups. Moreover, the serum levels of interleukin 10 (IL-10) in these two groups were also significantly lower than those in other groups. We conclude that higher immunization dose may not lead to better protection with the malaria vaccine as CD19+CD1dhiCD5hi B cells can downregulate ITV protection against malaria via IL-10 secretion. These results could facilitate the design of an effective long-lasting malaria vaccine with the aim of maintaining MBC function.

Many malaria antigens contain multiple disulphide bonds involved in the formation of inhibitory B-cell epitopes. Producing properly folded malaria antigens in sufficient quantities for vaccination is often a challenge. The 42-kDa fragment of Plasmodium falciparum merozoite surface protein 1 (MSP142 ) is such a kind of malaria antigen. In this study, we investigated the expression of MSP142 in a rice system (9522, a cultivar of Oryza sativa ssp. japonica), which was used as a bioreactor for protein production. The MSP142 gene was synthesized according to rice-preferred codons and transformed into rice plants via an Agrobacterium-mediated method. The recombinant antigen was efficiently expressed in rice seeds with a level up to 1.56% of total soluble protein and was recognized by both the conformational monoclonal antibody 5.2 (mAb5.2) and the pooled sera of P. falciparum malaria patients. Rabbits were immunized intramuscularly with the purified MSP142 formulated with Freund\'s adjuvant. High antibody titres against MSP142 were elicited. The rabbit immune sera reacted well with the native protein of P. falciparum parasite and strongly inhibited the in vitro growth of blood-stage P. falciparum parasites, demonstrating that transgenic rice can become an efficient bioreactor for the production of malaria vaccine antigens.

OBJECTIVE: To explore the effect of immunogenicity and immunizing protection of GAMA gene DNA vaccine, which was related with merozoite, ookinete and sporozoite invasion.
METHODS: Gene fragments were obtained using PCR technique and eukaryotic expression vector (containing immunostimulatory sequence) was built. BALB/c mice were divided into PBS control group, empty vector control group and study group and were immunized at week 0, 3 and 6 respectively. Blood was collected 2 weeks after each immunization and serum was separated to detect the IgG, IgG1 and IgG2a levels. Spleen of mice was obtained for preparation of splenic mononuclear cell and the cytokine IL-4 and IFN-γ levels were detected. Indirect immunofluorescence and western blot were employed to verify the specificity of antiserum. Sporozoite and merozoite invasion were used respectively to detect the immune protective effect 2 weeks after the third immunization. Ookinete conversion rate in vitro and oocyst numbers of mosquito stomach were observed to evaluate the transmission-blocking levels.
RESULTS: In GAMA DNA vaccine group: antiserum could be combined with recombinant protein specifically and green fluorescence signals of merozoite, ookinete and sporozoite were observable, while specific fragments and fluorescence signals were not observable in empty vector group. Compared with control group, specific IgG in DNA vaccine immunity group significantly increased (P < 0.01), and IgG1 and IgG2a all increased (P < 0.01). IL-4, IFN-γ content in study group significantly increased, compared with control group (P < 0.01). GAMA DNA vaccine immunity could not obviously block the erythrocyte-stage infection (caused by sporozoite invasion); compared with control group, liver worm load was slightly reduced (P < 0.05), and antiserum ookinete numbers (cultured in vitro) had no significant difference with oocyst numbers of mosquito stomach in DNA vaccine group.
CONCLUSIONS: GAMA has good antigenicity, which could stimulate the body to produce specific immune responses; while DNA vaccine immunity could not play a good protective effect, the effect of which is only limited to the slight reduction of liver worm load, and has no obvious erythrocyte-stage protective effect and transmission-blocking effect. Therefore, trying other immunization strategies for further research on the value of GAMA (as multi-stage antigen vaccine and multi-stage combined vaccine components of the life-cycle of plasmodium) is necessary.

Malaria is a severe, life-threatening infectious disease that endangers human health. However, there are no vaccines or immune strategy of vaccines succeeding in both erythrocytic and pre-erythrocytic stage. During the liver stage of the Plasmodium life cycle, sporozoites invade the host liver cells. The sporozoites, then, induce a cellular immune response via the major histocompatibility complex (MHC) molecules on their surfaces. The cytotoxic T lymphocytes (CTLs) then recognize the corresponding antigen-MHC complex on the surfaces of these infected liver cells and kill them. However, dominant epitopes with high MHC affinity are prone to mutation due to immune selection pressure. CTLs evoked by the original dominant epitopes cannot recognize the mutated epitopes, leading to immune evasion. In this study, we have modified the cryptic epitopes of different antigens in the sporozoite and liver stages of Plasmodium falciparum to increase their immunogenicity without changing T cell antigen receptor (TCR)-peptide binding specificity. In addition, we have also added an important erythrocytic phase protective antigen, named apical membrane antigen 1 (AMA-1), to this process with the goal of constructing a complex multi-stage, multi-epitope recombinant DNA vaccine against P. falciparum. The vaccine was tested in HHD-2 mice. The method involved multiple stages of the P. falciparum life cycle as well as elucidation both humoral and cellular immunity. The conclusion drawn from the study was that the vaccine might provide an important theoretical and practical basis for generating effective preventative or therapeutic vaccine against P. falciparum.

Since PvTARAg55 protein (PvTARAg55) of Plasmodium vivax (P. vivax) is expressed during the parasite\'s sporozoite stage, it was strongly suggested, as a potential candidate for the development of a vaccine against malaria. PvTARAg55 polymorphisms were examined among isolates from various locations in Asian countries mainly; thus the current study could set the valuable baseline data for the development of a vaccine and clinical trials. A total of 59 samples were collected from Asian countries and one isolate from Africa. PvTARAg55 gene from 59 isolates was amplified, sequenced, and analyzed. PvTARAg55 contained a highly conserved tryptophan-rich domain (TRD) and a variable alanine-rich domain (ARD). In comparison to the Sal-1 strain, 10 allelic types of PvTARAg55 were found among 59 isolates. The main observed variations were the insertions and deletions of repeated sequences in the Ala-rich domain. Four types of GGVAAAP repeats were found at codon 324. Interestingly, GGVAAAP was found to be majority of Sal-1 type in the world. Two repeats (x2) were found in isolates from Korea, China, and India. Type of total deletion of GGVAAAP and three repeat (x3) were found from Indonesia isolates. Furthermore, \"second insertion repeats\"—with one or two repeats—were found with AFGAPSGFAPRP amino acid sequences at codon 338. Two repeats (x2) of AFGAPSGFAPRP were found in Indonesia, and PNG isolates. Finally, a \"third repeat\" was present with TTVNPEA amino acid sequences at codon 429 (the Indonesian isolates had three TTVNPEA sequences at that position). Isolates from ROK revealed \"conserved sequences\" in tryptophan-rich domain of PvTARAg55 with single amino acid substitutions (M180I). Hence, the extensive antigenic diversity of PvTARAg55 should be taken in account during the vaccine development.

Although great efforts have been undertaken for the development of malaria vaccines, no completely effective malaria vaccines are available yet. Despite being clinically silent, the pre-erythrocytic stage is considered an ideal target for the development of malaria vaccines. Sporozoite asparagine-rich protein 1 (SAP1) is a sporozoite-localized protein that regulates the expression of UIS (upregulated in infectious sporozoites) genes, which are essential for the infectivity of sporozoites. In this study, a recombinant DNA vaccine encoding a predicted antigenic determinant region of Plasmodium yoelii SAP1 (PySAP1) was constructed. Immunization of mice with this DNA vaccine construct resulted in significant elevation of cytokines such as IFN-γ, IL-2, IL-4 and IL-10, and total IgG as compared with control groups immunized with either the empty DNA vector or saline. After challenge with sporozoites, the group receiving the DNA vaccine showed delayed development of parasitemia and prolonged survival time compared with the control group. The DNA vaccine provided partial protection against P. yoelii 17XL infection, with an overall protection rate of 20%. In addition, the DNA vaccine did not show integration into the host genome. Further studies of SAP1 are needed to test whether it can be used as subunit vaccine candidate.