P.yoelii: Infection And Treatment Vaccine Induced Cross-Stage Immune Responses Associated With Sterile Protection Against Both Sporozoites And IRBCs Challenge

BackgroudMalaria is one of the most three infectious diseases all over the world. Effectivemalaria vaccine is urgently needed for eradicating this devastating disease. Sterileprotection against malaria infection has been achieved by the inoculation of whole parasitesattenuated to prevent symptoms that caused by blood stage infection. Sterile protection canbe achieved by~1,000bites of irradiated (irr) mosquito inoculation in human beings. Incomparison, using infection-treatment-vaccination (ITV) strategy, sterile protection can beachieved with less sporozoites (SPZ). For example, wild type Plasmodia sporozoites underthe treatment of anti-blood stage parasite drug chloroquine (CQ) can induce sterileprotection only with3doses of15mosquito bites. It suggesting that irr-SPZ is not asimmunogenic as ITV-SPZ. Since chloroquine resistance is widely spread around the world,which not only restricted its use for malaria control but also reduce the potential use forprophylaxis. Artemisinin derivatives such as artesunate (AS) shows the promises formalaria control with its effective and rapid inhibition of all forms of blood stage (BS)parasites. Moreover, the resistance to artemisinin of Plasmodium strains is limited. NeitherAS nor CQ affects the liver stages of malaria parasites, but CQ or AS kills different bloodstages of malaria parasites. CQ kills parasites at the trophozoite stage, AS acts on allintraerythrocytic stages of BS development. It promotes us to investigate if AS could be acandidate prophylactic drug in infection and treatment vaccine strategy, and the immunedifferences between CQ-ITV and AS-ITV.ObjectiveIn this study, we first assessed the possibility of using AS as potential drug for ITV. Then, we compared the immune differences between AS-ITV and CQ-ITV. Finally, themechanism of the difference between protections induced by these two drugs wasinvestigated.MethodsBALB/c mice were infected with10k wt P. yoelii SPZ by i.v. injection for3times at4week intervals. To control the blood stage infection, infected mice were treatedconsecutively with AS or CQ starting from day1to day10. Immunized mice werechallenged with10k SPZ or20k infected red blood cells1month post last vaccination.Blood smear checking was carried out at day2after each immunization and challenge, andreal-time qPCR (RT-qPCR) was used for confirming blood stage infection afterimmunization. After that, serum collected from immunized mice at day9post eachimmunization was used for detecting antibody expression by IFA and ELISA. Thenantibody function was investigated by inhibition of sporozoites invasion assay and serumadoptive transfer. Finally, the dynamics of T cells phenotype during immunization andCD8+T cells function were inspected by flow cytometry or inhibition of liver stagedevelopment assay respectively.ResultsWe observed that mice became parasitaemic four days post withdraw of CQ coverageand maintained in a low level of parasitemia, whereas AS treatment completely preventedblood stage infection during the vaccination phases. Meanwhile, RT-qPCR showed a closecorrespondence between the parasitemia levels determined by blood smear and thedetection of P. yoelii18S rRNA on blood samples taken on days11,14,18, and24postimmunization, as compared with that of mouse GAPDH. Moreover, complete protectionwas achieved against wild type P. yoelii sporozoite challenges after three doses of SPZinfections either both AS or CQ treatments and lasted at least6months. The infection andtreatment with CQ but not AS induced protection against blood stage challenge. We thenexamined the stage-specific immune responses associated with protection induced afterinfections under the drug treatments. We demonstrated that infection under the treatmentwith both drugs developed antibodies against circumsporozoite surface protein, recognizedsporozoite and liver stage parasites by IFA, inhibited sporozoite invasion to hepatocytes.The sera from mice received artesunate recognized only early blood stage parasites, whereas the sera from chloroquine treated mice recognized late blood stage of parasites.Moreover, both liver CD4+and CD8+Tem cell populations were significantly increased andin vitro cultured liver CD8+T cells significantly inhibited liver stage development from bothdrug-treated mice.ConclusionOur study detailed the inductions of stage-specific protective immunity under differentdrug prophylactic strategies against malaria. The SPZ infection covered with AS inducedpre-erythrocytic stage specific immunity, whereas the infection under the CQ coveragedeveloped immunity against both pre-erythrocytic and erythrocytic stage immunity. To ourknowledge, this is the first time that these ITV strategies showed the ability to induceprotection against both liver stage and BS malaria parasites depends on the stage of themalaria parasite affected by different drugs.Previous studies from other groups have shown that sterile protection induced byCQ-ITV in the mouse model depended on immune responses against both pre-erythrocyticand BS antigens and antibodies. CD8+T cells have been shown to play core role in malarialiver stage protection. In agreement with this, we observed that repeated exposure of miceunder either CQ-ITV or AS-ITV resulted in induction of total and malaria specific liverCD8+Tems. Meanwhile, the protection is also associated with antibodies. Both CQ-ITV andAS-ITV induced functional antibodies that were capable of blocking P. yoelii SPZ invasionof hepatocytes in vitro.This will inform us how choose specific drug or combination therapy that will promotethe naturally acquired immunity to develop against both liver pre-erythrocytic anderythrocytic stage-specific immunity. Finally, the immunity against only liver stagedevelopment under AS controlled infection may provide a unique model for the selection ofoptimal later liver stage vaccine candidates.