| Although malaria control programs have led to an extensive reduction in malaria incidence and mortality, it remains one of the most threatening diseases worldwide. The malaria blood-stage vaccine is regarded as the most cost-effective strategy to prevent malaria infection. However, there is no licensed vaccine available, although over 40 malaria vaccines have undergone clinical trials in the past ten years. Antigenic variation and polymorphism are proving to be a significant hurdle with blood-stage vaccines. Recently, malaria infection-treatment-vaccination(ITV), which involves infection with live malaria parasites under curative anti-malarial drug coverage, has been reported to induce antibodies specific for the merozoite surface antigens conserved between heterologous strains but not for the variant surface antigens. This may provide a new idea for designing the blood-stage subunit vaccines. However, the exact mechanism of Plasmodium specific B cell activation in ITV is still very unclear.As the important negative regulatory moleculars in innate immune system, PD-1/PD-L signaling has been confirmed in recent studies that it plays a very important role in regulation of TFH cells, then prompting the antibody-secreting of GC B cells. Meanwhile, the deficiency of PD-1/PD-L signaling will enhance the ability of B cells for antibody secretion, through the prompting TFH cells function in malaria infection. However, it is still unclear about how PD-1/PD-L signaling inducing the GC B cell activation in ITV vaccination.In the present study, we first confirmed the role of PD-1/PD-L signaling in ITV. Subsequently, Plasmodium specific antibody or CD4+ T cell, which was involved in the immune protection process, was confirmed by adoptive transfer or in vivo blocking experiments. Secondly, the titers of Plasmodium specific antibody and the frequency of GC B cells were detected in the immunized mice. Then, we tried to find the regulation mechanisms of GC B cells in PD-1 deficiency immunized mice, by detecting the concentrations of inflammatory cytokines and using the reverse antagonistic experiment. Finally, we will proceed a preliminary study of the regulation mechanism of TFH cell in ITV, by detecting the frequency of TFR cells and the maturation of CXCR5+- DC.1. The absence of PD-1 in mice can significantly enhance the protective immunity of ITV.1) Construction of ITV model of P.y 17XL: Mice were i.v. injected with 1×106 P.y 17 XL iRBC or an equivalent amount of normal RBC with or without 100 μL of 8 mg/mL CQ daily for 15 days starting from the day of iRBC injection. The mice were maintained for 21 days and challenged i.p. with 1×106 iRBC. The results showed that, compared with non-immunized WT mice, all immunized mice survived, although the parasitemia has reached the peak in sixth day after the challenge.2) The protective effect enhances in PD-1-/- mice following ITV immunization: The parasitemia and survival rate have been determined after the challenge of all immunized mice. Compared with the immunized WT mice, PD-1-deficiency could largely augment the protective efficacy in ITV, which indicated that PD-1/PD-L signaling plays the important role in Plasmodium blood-stage ITV.2. The lack of PD-1 can influence the capacity of GC B cells to secretion of Plasmodium specific antibody, leading to the enhanced protective effect in ITV immunized PD-1-/- mice.1) The enhanced protective efficacy of immunized WT mice was a result of increasing titers of Plasmodium specific antibody, but not due to the CD4+ T cell response: Three weeks after the immunization, the immunized serum was isolated and adoptively transferred to na?ve mice. After the P.y 17 XL challenge, the parasitemia and survival rate were observed. The results show that the mice that received sera from the ITV-immunized mice cleared the parasites at day 18 post-challenge, and all of the mice survived. Meanwhile, the CD4+ T cells of the immunized WT mice were depleted, and the mice were challenged with P.y 17 XL. It is found that no significant difference in the parasitemia or survival rate was found between the ITV-immunized mice injected with control Ab and those injected with anti-CD4 Ab. These results demonstrated that antibodies are capable of mediating protection of ITV-immunized mice against malaria parasite challenge.2) The increased titer of Plasmodium specific antibody results in the enhanced protection in PD-1-/- immunized mice: The levels of Plasmodium specific IgG were compared between the ITV-immunized WT mice and PD-1-/- mice. The results show that the levels of Plasmodium specific total IgG and isotype IgG2 a in the immunized WT mice were much lower than those in the immunized PD-1-/- mice, although no significant difference in the IgG1 levels was found between the two types of immunized mice. Meanwhile, sera from the ITV-immunized WT mice or PD-1-/- mice were adoptively transferred to the na?ve mice, and the recipient mice were then challenged with P.y 17 XL. As a result, the parasite in the blood of the mice that received the immunized PD-1-deficient mice sera was much lower than that of mice received the immunized WT mice sera before the eighth day after challenge. These results indicated that PD-1-/- ITV immunized mice can enhance immune protection by increasing the Plasmodium antibody secretion.3) The enhanced protection of PD-1-/- immunized mice mainly relied on the increasing frequency and number of GC B cells: The frequency and number of GC B cells in the spleen were detected at days 7, 9, 11, 14 and 21 after the last immunization. The results showed that the frequency and number of GC B cells in both the ITV-immunized WT mice and PD-1-/- mice gradually increased overtime. However, the the GC B frequency and number of the ITV-immunized PD-1-/- mice was much higher than that of the ITV-immunized mice at days 14 and 21.These results suggested that PD-1-/- could promote the expansion of GC B cells in the spleen in ITV immunized mice. This is consistent with the elevated level of Ab in the sera3. The lack of PD-1 can enhance the GC B cell function, by increasing the secretion of inflammatory cytokines and enhancing the activities of TFH cells.1) The frequency, number of TFH cells in spleen and the concentration of IFN-γ、IL-10、MCP-1 in serum have increased significantly in PD-1-/- ITV immunized mice: The frequency and number of TFH cells in both the WT immunized mice and PD-1-/- immunized mice gradually reduced over time. The highest TFH cells frequency and number were observed at day 7 after the last immunization and the frequency and number were reduced to the baseline level at day 21. However, the frequency and number of the TFH cells from the immunized PD-1-/- mice were more than 3-fold greater than that of the immunized WT mice at days 7 after the last immunization. Meanwhile, the concentrations of inflammatory cytokines in serum were detected at days 21 after the last immunization. The results have found the concentrations of IFN-γ、IL-10、MCP-1 of the ITV-immunized PD-1-/- mice were much higher than that of the ITV-immunized mice.Thus, the increased Plasmodium specific TFH cells and cytokines were closely associated with the expansion of GC B cells and elevated Ab in the sera.2) The frequency, number of GC B cells have been decreased significantly, when IFN-γ、IL-10 and MCP-1 have been blocked separately in PD-1-/- immunized mice: Blocking antibodies against IFN-γ、IL-10、MCP-1 have been separately i.p. injected into the PD-1-/- immunized mice at days 1, 3, 5, or days 3, 5,7,9, 11 after the last immunization. The total numbers of lymphocyte, GC B cells’ number and frequency in the spleen have been detected at days 7 or 14 after the last immunization. The results have found there is no difference between the WT immunized mice and PD-1-/- immunized mice at days 7 after the last immunization. However, comparing with the normal PD-1-/- immunized mice, all blocking groups have lower numbers of total lymphocyte, GC B cells in the spleen at days 14 after the last immunization. These results suggested that IFN-γ、IL-10 and MCP-1 may promote the GC B cells survival in PD-1-/- ITV immunized mice.4. The mechanisms of TFH cells activation in PD-1-/- ITV immunized mice.1) IFN-γ、IL-10 and MCP-1 may not be involved in TFH cells activation in PD-1-/- immunized mice: The cytokines blocking and TFH cells’ detection were consist with the above. The results have found there is no difference between the normal immunized group and cytokines blocking group both at the days 7 and 14 after the last immunization. These results suggested IFN-γ、IL-10 and MCP-1 may not be involve in TFH cells development.2) The function of TFH cells does not depend on the spleen DC maturation in PD-1-/- immunized mice: Spleen lymphocytes were isolated at days 2, 4, 6 after single immunization and the maturation of CXCR5+ DC has been detected by FACS. We found that the expression levels of co-stimulatory molecules(CD86、MHC-II、CD40) have been increased at different degrees in all immunized mice and there is no difference between the WT and PD-1-/- immunized mice. These results indicated that the increased number of TFH cells may not be associated with the activation of DC.3) TFR cells may not be involved in the regulation of TFH cells’ function in PD-1-/- immunized mice: Spleen lymphocytes were isolated at days 7, 14, 21 after the last immunization and the frequency of TFR cells have been detected by FACS. The results have found PD-1-/- mice have higher frequency of TFR cells than WT, although it has decreased significantly in all immunized mice. These results suggested that TFH cells’ function may not be regulated by TFR cells in PD-1-/- immunized mice.In conclusion, we are succeeding in establishing the model of P.y 17 XL ITV. By using this model, we found that PD-1-/- can enhance the protection efficiency by regulating the GC B cells’ secretion of Plasmodium specific antibody in ITV immunized mice. In addition, we also found that the function of GC B cells has close relationship with the cytokines in serum and TFH cells in spleen. Further researches indicated that DC maturation, inflammatory cytokines in serum and TFR cells’ function may not be involved in the regulation of TFH cells in PD-1-/- ITV immunized mice. These findings have implications not only in rational design of effective blood-stage subunit vaccines against malaria parasites, but also would further our understanding of the mechanisms of immune protection induced by the whole blood stage vaccine. |
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