Study On The Mechanism Of The Effects Of Immunization Dose On The Long-term Protection Of Malaria ITV

Background:Malaria is one of the three most serious infectious diseases in the world.The increasing antimalarial drug resistance and limited malaria control methods have made the development of malaria vaccine become more urgent.The blood-stage of plasmodium is the key period of inducing clinical symptoms.And the purpose of blood-stage malaria vaccine is to inhibit the proliferation of Plasmodium in red blood cells,so as to control symptoms and prevent the disease.Although several blood-stage subunit vaccines have entered the clinical trial stage,the results were not satisfactory because of their limited efficacy,short-term protection and difficulty in resisting high antigenic polymorphism.Therefore,the whole parasite vaccine in the blood stage of malaria has been paid more attention because it contains a large number of antigens and can produce multi-antigen protection.Related research shows that Infection treatment vaccine?ITV?can lead to sterile protection against malaria infection in mice and humans.However,this protection is difficult to maintain for a long time.Memory B cell?MBC?plays an important role in maintaining the long-term protection of malaria blood stage vaccine.Related studies have shown that ITV immunization with an extremely low dose of the parasite can induce stronger protection than high immune dose.This indicates that low-dose immunization may confer better protection.B10 cells play a vital role in maintaining immune tolerance and inhibiting inflammation via releasing interleukin-10?IL-10?.Thus,we explore the role of MBCs and B10 cells in the immune dose-mediated long-term protection decline of malaria blood stage ITV.Objective:1.To explore whether immune dose can mediate the long-term protection difference of malaria blood stage ITV.2.To explore the role of MBCs in the immune dose-mediated malaria blood stage ITV long-term protection difference.3.To explore the function of B10 cells and related inflammatory factors in the immune dose-mediated malaria blood stage ITV long-term protection difference,and explore its mechanism.Methods:C57BL/6 mice and BALB/c mice were immunized thrice by intravenous injection?i.v.?10⁵ P.yoelii265-infected red blood cells?Py-i RBCs?at two-week intervals and challenged with 10³ homogeneous i RBC at 1,3,and 6 months after last immunization.The result showed that C57BL/6 mice are more sensitive to P.yoelii than other mouse strains.Herein,C57BL/6 mice were immunized with 10³,10⁵,or 10⁷ ITV thrice in a 14-day interval.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.Cytometric bead array?CBA?was used to detect the level of cytokines in serum.Results:The results showed that the long-term protection difference of ITV mediated by immune dose was correlated with the IL-10 secreted by B10 cells.In addition,the decrease of long-term protection of ITV may be related to the decrease of MBCs in spleen of mice with the increase of time.The experimental results are as follows:1.The ITV protection of C57BL/6 mice decreased faster than that of BALB/c mice,so C57BL/6 mice were selected as the animal model of this experiment.2.The 10⁷group revealed the highest peak parasitemia compared to the other two lower dose groups.In addition,the 10³group gained sterile protection in the 1-month challenge,and maintained more than 60%protection for at least 6 months.However,the 10⁵ and 10⁷immunized groups only exhibited 16.7%protection after6 months.3.The number of MBCs in all three groups at 6 months was significantly lower than that at 3 months?10³:p=0.0023;10⁵:p=0.0007;10⁷:p=0.001?,but no significant difference was found in PD-1 expression on MBCs in the three immunized groups at different times.4.At 1 month,the number of B10cells of ITV-immunized mice in 10³ and 10⁵ groups was significantly lower than that in the control group?10³:p=0.0497,10⁵:p=0.0232?,and that in the 10⁷group was significantly higher than that in the 10³?p=0.0471?and 10⁵?p=0.0128?groups.This was in accordance with the protection assay at 1 month.At 3months,the number of B10 cells in 10⁵ group was significantly lower than that in control?p=0.0275?,10³?p=0.0075?and 10⁷groups?p=0.0071?.5.Pearson correlation analysis showed that the level of IL-10 was proportional to the number of B10 cells?r=0.41,p=0.015?,the amount of IL-6?r=-0.626,p<0.001?and IFN-??r=-0.638,p<0.001?was inversely proportional to the amount of IL-10.Conclusions:Taken together,immunization dose could mediate the long-term protection of Plasmodium blood stage ITV in C57BL/6 mice.Higher immunization dose could not produce better long-term protection.CD19⁺CD1d^hiCD5^hi B cells can downregulate ITV protection against malaria via IL-10 secretion.In addition,the decrease of long-term protection of ITV may be related to the decline of the number of MBCs in the spleen of mice.These results could facilitate the design of an effective long-lasting malaria vaccine with the aim of maintaining MBCs function.