Study On The Effect Of Exposure To Plasmodium Antigen PfCSP During Pregnancy On Vaccine Immunity In Offspring Mice

Objective:Establishing an animal model of pregnant mice to analyze the effect of embryonic exposure to Plasmodium antigen on the immune characteristics of offspring mice against PfCSP.Investigate the effect of exposure to the PfCSP antigen during pregnancy on the protective effect of offspring mice vaccines.Provide an immunological basis for developing a more effective malaria vaccine in the future.Methods:(1)PfCSP recombinant plasmid was constructed,the corresponding recombinant protein was expressed and purified in vitro,and preparing PfCSP recombinant protein as a vaccine;(2)Let adult BALB/c mice mating and check the pregnancy status of females.Pregnant mice were inoculated with 100 μg PfCSP recombinant protein or the same volume of PBS in the tail vein on the 7th day of gestation and fed until natural delivery.A certain number of neonatal mice were taken for the corresponding experiments,and feeding the rest offspring mice until adulthood for the rest experiments.The newborn mice’s thymus,spleen,and liver were dissected and separated 3-5 days after birth to prepare the single-cell suspension.The changes in CD4/CD8+T cell subsets in the thymus;CD4/CD8+T,Treg,B,NK cell subsets in the spleen;NK cell subsets in the liver;were all detected by flow cytometry.(3)4-week-old offspring mice were injected with the transgenic Plasmodium strain P.y-PfCSP-PfVAR2CSA(1×104 parasites/mouse)through the tail vein,and the parasitemia and survival time of offspring mice were observed;(4)The peripheral blood of newborn mice was taken 3-5 days after birth,and the changes of serum cytokines(TNF-α,IFN-γ,IL-2,IL-4,IL-10 and TGF-β)of the offspring mice in the experimental group and the control group were detected by ELISA;(5)After the newborn offspring mice were raised to 4 weeks of age,the offspring mice of the PfCSP experimental group and PBS control group were subcutanouslly immunized with PfCSP antigen,and after 3 times immunization and one boost,the thymus,spleen and liver were dissected and isolated,single-cell suspension was prepared,and the changes of CD4/CD8+T cell subsets in the thymus;T,CD4/CD8+T,Treg,B,NK cell subsets in the spleen;NK cells in the liver;were measured by flow cytometry;(6)The peripheral blood of adult offspring mice was taken and the serum IgG level mice was detected by ELISA;(7)Adult offspring mice were inoculated with 1 × 104 transgenic parasite P.y-PfCSP-PfVAR2CSA via the tail vein,and the proliferation of spleen cells after PfCSP stimulation,parasitemia and survival time of offspring mice were observed.Results:(1)CD4+T cells were significantly increased(P<0.01),and CD4+CD8+T cells were significantly decreased(P<0.05)in the thymus of neonatal mice exposed to PfCSP during pregnancy.The percentage of CD4+T and NK cells in the spleen of neonatal mice exposed to PfCSP during pregnancy was significantly increased(P<0.05);(2)Peripheral blood IL-10 in the PfCSP exposure group was decreased(P>0.05);(3)After inoculation of P.y-PfCSP-PJVAR2CSA parasite in 4-week-old mice,the parasitemia in mice exposed to PfCSP in the embryonic stage was higher than that in the unexposed group(P<0.05).Compared with the PfCSP exposed group,the overall survival time of mice in the PfCSP unexposed group was longer;however,the difference in survival curve between the two groups was not statistically significant.(4)The 4-week-old mice exposed to PfCSP during pregnancy were immunized by subcutaneous injection of PfCSP recombinant antigen.In the thymus of adult offspring mice immunized with the PfCSP vaccine,the proportion of CD4+T cells was significantly increased(P<0.05),and the proportion of CD4+CD8+T cells was significantly decreased(P<0.05)in the PfCSP exposure group during pregnancy;Compared with PBS+PBS group,the proportion of T cells in the spleen of PBS+PfCSP group was significantly increased(P<0.05);(5)Compared with the PfCSP exposed group,the level of IgG in the PfCSP unexposed group had an upward trend,but there was no significant difference in the level of IgG between the four groups of mice(P>0.05);(6)4-week-old offspring mice were injected with PfCSP recombinant protein subcutaneously for vaccine immunization,and inoculating transgenic Plasmodium P.y-PfCSP-PfVAR2CSA through the tail vein.There was no significant difference in parasitemia between the two groups inoculated with transgenic Plasmodium through the tail vein;however,the survival time of the mice exposed to PfCSP during pregnancy decreased significantly.Conclusion1.Exposure to PfCSP during pregnancy significantly increased the proportions of CD4+T in the thymus,CD4+T,and NK cell subgroups in the spleen.It significantly decreased the proportions of CD4+CD8+T cells in the thymus of neonatal rats.2.Maternal exposure to PfCSP during pregnancy affected the proportion of CD4+T and CD4+CD8+T cells in the thymus of adult offspring mice.This effect was retained from neonatal to adulthood,forming an imprinting effect.3.Maternal exposure to Plasmodium antigen PfCSP during pregnancy decreased the survival time and increased parasitemia of offspring infected with transgenic parasite P.y-PfCSP-PfVAR2CSA.It weakened the protective effect of the vaccine.