| Malaria remains a fatal public health problem resulting in high morbidity and mortality in tropical and subtropical regions[2],it is initiated by the bite of an infected anopheline female mosquito with the inoculation of sporozoites[1].A high-efficiency vaccine is regarded as the most promising and cost-effective tool for malaria control.Although the scientists have made great efforts to develop subunit vaccines against malaria in the post fifty years,no high-efficiency subunit vaccine is licensed.The failure is greatly attributed to the only one or several antigens are involved in the subunit malaria vaccine designation,and high polymorphisms of the target antigens existing among the different strains of malaria parasites.In contrast,a whole-organism vaccine with maximal spectrum of antigens was appreciated recently.The whole-organism vaccines,such as irradiation-or genetically attenuated sporozoite and whole-killed blood-stage vaccine,have been demonstrated to induce the protective immunity in both human and mice.Of those,whole-killed blood-stage vaccine was prepared by repetitive freeze-thaw of the blood-stage infected RBCs,and immunized the mice along with alum.Our previously research have showed that immunization of whole-killed blood-stage vaccine could induce the protective immunity,but needed a high dose of p RBCs,which limits its wide application.Additionally,the protection efficacy needs to be greatly improved.A strong adjuvant has been demonstrated as a solution to this problem.Chloroquine,which was discovered in 1934,was used as an antimalarial drug since 1947[4].It shows its maximum efficiency when the parasites stays in RBCs while it is ineffective against sporozoites.Several studies reported that chloroquine can regulate host immune responses.However,whether it could inhibit or enhance host immune responses remains contradictory.Our previous study found that chloroquine alone could enhance the immune responses [5].Therefore,we investigated whether chloroquine could be a novel adjuvant,and enhance the protective efficacy of the whole-killed blood stage vaccine(WKV),and explored the underlying mechanisms.As cross-protection has been reported between the vaccine against pre-erythrocytic stage and vaccine against the blood stage,we also test whether chloroquine could also promote the cross-protection of the whole-killed blood stage vaccine against the liver stage.1.Chloroquine greatly enhances the protection efficacy of whole-killed blood stage vaccine.1.1.Chloroquine enhances the protective efficiency of whole-killed blood stage vaccinesAlthough whole-killed blood stage vaccine could induce the mice to generate protective immunity against the blood stage,the protective efficacy was not enough.However,the adding of chloroquine to the whole-killed blood stage could greatly enhance the protective efficacy of the immunized mice against blood stage.The parasite of the immunized mice was eliminated rapidly and all the mice survived after challenge.Additionally,injection of chloroquine alone every two weeks had no effect on the parasitemia of mice challenged with pRBCs.This indicated that the chloroquine can promote the protection efficacy of the whole-killed blood stage vaccine through acting on host immune system,but not dependent on its antimalaria effect.1.1.1.The effect of chloroquine on promoting the protection efficacy of the whole-killed blood stage vaccine is dose relevant.Two weeks after immunization with the whole-killed blood stage vaccine mixed with different concentration of chloroquine(10mg/kg,20mg/kg and 40mg/kg),the mice were then attacked with P.y NSM pRBCs.As a result,although the protectionefficacy increased when the mice were immunized with the whole-killed blood stage vaccinemixed with different concentration of chloroquine,but the effect of chloroquine was not dose dependent.The protectionefficacywas higher when the mice immunized with the whole-killed blood stage vaccinemixed with 20mg/kg of chloroquine.1.1.2.Alum was necessary for the chloroquine-enhanced immunogenicity of p RBCs lysate.Two weeks after the ultimate immunization with pRBCs lysate,alum plus pRBCs lysate,chloroquine plus pRBCs lysate,or pRBCs lysate plus chloroquine and alum,mice were attacked with P.y.NSM pRBCs.As a result,the parasitemia in mice immunized with the chloroquine plus pRBCs lysate was comparable to that of mice immunized with pRBCs lysate alone,indicating that chloroquine alone could not promote the immunogenicity of p RBCs lysate.However,the parasitemia was much lower in mice immunized with the p RBCs lysate plus chloroquine and alum than that of mice immunized with pRBCs lysate plus alum.This result indicated that alum was necessary for chloroquine to enhance the immunogenicity of pRBCs lysate.1.2.The underlying mechanisms of chloroquine to enhance the protection efficacy of the whole-killed blood stage vaccine.1.2.1.Antibody is essential for the chloroquine-enhanced protective immunity of the whole-killed blood stage vaccineTwo weeks post final immunization,both the frequency of spleenic GC B cells and the level of antibody in serum were compared when mice immunized with whole-killed blood stage vaccine with or without chloroquine.As a result,both the GC B frequency and the antibody level were much higher in mice immunized with whole-killed blood stage vaccine plus chloroquine than in mice immunized with whole-killed blood stage vaccine alone,indicating that chloroquine could promote the immunized mice to generate antibodies.To verify whether the increased antibodies contribute to the enhanced protection efficacy of mice induced by the whole-killed blood stage vaccine plus chloroquine,the serum from immunized mice was adoptive transferred to the na?ve mice,and then challenged with P.y.NSM p RBCs.The parasitemia was significantly lower in the mice received with serum from mice immunized with whole-killed blood stage vaccine plus chloroquine than that of mice received with serum from mice immunized with whole-killed blood stage vaccine alone.1.2.2.CD4+T cells were essential for the chloroquine-enhanced protection efficacy of mice induced by the whole-killed blood stage vaccineTwo weeks post the ultimate immunization,the splenic lymphocytes were isolated,and both the proliferation of CD4+ T cells and the secretion of IFN-γ were analysised by FACS.Both the proliferation capacity and IFN-γ secretion of CD4+ T cells were greatly increased in the mice immunized with whole-killed blood stage vaccine plus chloroquine,as compared to the mice immunized with whole-killed blood stage vaccine.To investigate the contribution of CD4+ T cells in the chloroquine-enhanced protective immunity of the whole-killed blood stage vaccine,CD4+ T cells in the immunized mice were depleted prior to pRBCs challenge.The parasitemia in the immunized mice significantly increased after depleted CD4+ T cells.This suggested that the enhanced protective immunity induced by the whole-killed blood stage vaccine plus chloroquine was closely associated with the increased CD4+T cell responses.2.Whole-killed blood stage vaccine resisted to sporozoites challenge.2.1.Whole-killed blood stage vaccine confers the protection against pre-erythrocytic stage.To investigate whether the immunization with the whole-killed blood stage vaccine could also confer the cross-protection against sporozoite,the liver parasite load of the immunized mice and the control mice was compared after chall enged with P.yoelii NSM sporozoite.As a result,the parasite load in the liver of the immunized mice is much lower than that of the control mice.Consistently,the appearance of parasites in the blood of the immunized mice is 2-day delay as compared to the control mice.Additionally,the parasitemia of the immunized mice is reduced at a much higher level as compared to that of the control mice at late infection stage.Although all the control mice died,the immunized mice finally survive and all the parasites are cleared.This indicates that the blood stage developed from the pre-erythrocytic stage is encountered with the blood stage-specific immune responses at the late infection stage.2.2.CD8+ but not CD4+ T cells were essential for pre-erythrocytic stage immunity.Two weeks post the ultimate immunization with vaccines,the CD4+ and CD8+ T cells of the immunized mice were exhausted,and then the mice were attacked with sporozoites.No significant change was observed in the liver parasite load of the immun ized mice depleted with or without CD4+T cells,but the parasite burden of the immunized mice greatly increased after CD8+T cells are depleted.Identical to the liver parasite burden,the parasitemia in the immunized mice is comparable to that of the contr ol na?ve mice after depleted CD8+T cells,while depletion of CD4+ T cells has no significant effect on the parasitemia at the early stage.This result demonstrated that the protective immunity induced by the whole-killed blood stage vaccine against the sporozoites is largely dependent on the parasite-specific CD8+ T cells but not CD4+ T cells.2.3.Chloroquine cannot promote the efficiency of whole-killed vaccine against sporozoite.Two weeks after the ultimate immunization,the mice were attacked with spor ozoites,and the liver parasite load was detected 42 h later.As a result,there is no significant difference when the mice immunized with whole-killed blood stage vaccine with or without chloroquine.This indicates that chloroquine cannot enhane the protec tion efficacy of P.y NSM whole-killed blood stage vaccine against sporozoites.In conclusion,an appropriate concentration of chloroquine can greatly enhance the protection efficacy of whole-killed blood stage vaccine,and might provide us with a safety and effective vaccine to control the malaria.Additionally,our result also indicates that chloroquine could be used as a novel adjuvant,which could promote the antibody generation and CD4+ T cell responses. |
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