Influence Of Antimalarial Treatment On The Acquired Immunity In Mice Against Malaria

PrefaceMalaria is one of the three most life-threatening infectious diseases worldwide. World malaria report 2005 reported that there were about 500 million episodes of malaria and over 3 billion people lived under the threat of malaria. The phenomenon of reinfection is one of its characteristics. Incomplete insight into the mechanisms of host protective immunity and diverse parasitic immune evasion hamper the development of effective vaccines. Because there is still no safe and effective malaria vaccine, since 1930s chemotherapy has been the best a-vailable way for human to control malaria infection. Chloroquine (CQ) is conventional antimalarial, but as the emergence and spread of drug-resistant Plasmodium strains, in 2001 World Health Organization (WHO) recommended the therapy with a combination of CQ and artemisinin in endemic where drug-resistant Plasmodium exists.Although patients gain great benefits from antimalarial treatment, reinfection is still prevalent in endemic areas. This might be ascribed to difficulty in establishing long-lasting protective immunity against parasites, as others research showed the " boosting" of immunity was much less if longer intervals between attacks. Control of infection at low parasitemia levels, instead of eradication of parasites , referred to premunition, is a hallmark of malaria. This seems conflicting to the basic principle for antimalairal treatment which get rid of parasite radically in order to avoid drug-resistance. As mentioned in rodent models, infected mice those received radical antimalarial treatment from the start of infectioncould not resist homologous Plasmodium reinfection. And it was also shown that CQ and AR (artesunate, belongs to artemisinin) might interfere in some aspects of the immunity. Thus whether radical antimalarial treatment might influence the acquisition of protective immunity and the resistance against reinfection should be studied in detail.Our previous study showed that mice could survival Plasmodium yoelii without drug treatment. It mainly contributed to the protective immunity that obtained via the establishment of Thl cellular immune response in early phase and then antibody-mediated response to eradicate parasites in later phase. Interferon gamma (IFN-7) and specific IgG were the key molecules in this process. In this research, parasitemia of P. yoelii 17XNL infected BALB/c and P. yoelii 17XL infected DBA/2 mice were self-resloved or eridicated by different dose of CQ or AR, then mice were reinfected with homologous or heterologous Plasmodium . We investigated the effect of antimalarial treatment on the development and dynamic changes of host accquired immunity against primary infection and parasite reinfection. These findings would provide essential experimental data for scientific , rational clinical antimalarial usage and for the feasibility in combination of antimalarial and malaria vaccine.Materials and methodsBALB/c and DBA/2 mice were infected intraperitoneally with erythrocytes parasitized with P. yoelii 17XNL (non-lethal) or P. yoelii 17XL (lethal) , then treated by different dose of CQ or AR. On day 60 PI ( about 1 month interval after all mice cleared parasites) , mice of all groups were reinfected with homologous ( P. yoelii 17XNL or P. yoelii 17XL) or heterologous ( P. bergheiANKA) Plasmodium . We observed the levels of parasitemia during primary and challenged infection by microscopic detection on Giemsa-stained thin blood smears. We also- detected the levels of IFN-7 in cultured supernatants of spleen cells and the levels of IgG in sera by ELISA. Data were presented as means ± standard errors of the means (SEM ). Statistical significance of differences were analyzed by Student^ t test. A value of P <0. 05 was considered significant.ResultsSelf-resolved and different dose of CQ or AR treated BALB/c and DBA/2 mice all produced high levels of IFN-7 during early phase of primary infection. Subsequently the levels of P. yoelii specific antibody increased significantly, and were of no significant difference between self-resolved and antimalarial treated groups. Moreover, there were no significant differences in the proliferation ability of spleen cells among all groups in two kinds of mice. When challenged by homologous Plasmodium , most BALB/c and DBA/2 mice of all groups uniquely showed resisitant, extremely low parasitemia was only shown in a few mice after challenge and then soon disappeared. For all groups of BALB/c and DBA/2 mice the levels of primary infected P. yoelii specific IgG were high. BALB/c mice of all groups also resisted P. yoelii 17 XL challenge, but when challenged by P. berghei ANKA, all infected and died. :DiscussionCQ and AR are commonly most used and efficient antimalarials, but a few researches showed they might inhibit some aspects of the immune system of host, and the mean of antimalarial treatment was reported to interfere in the response of mice against P. berghei NK65 reinfection. In this study, two substrains ( non-lethal and lethal) of P. yoelii were introduced to infect BALB/c mice and DBA/ 2 mice, respectively, both of which caused self-resolving infections. And then we studied the effect of different dose of CQ or AR treatment on the profile of protective immunity during malaria primary infection and reinfection. Our results suggested that although there were significant differences in the time course and the ability in controlling levels of parasitemia in both of models, CQ and AR treated or not, or treated by different dose we used affected neither the acquisition of protective immunity against P. yoelii of different virulence nor the resistance to homologous Plasmodium reinfection. Moreover, it was parasite specific antibody response that responsible for the clearance of reinfected parasites.Stephens et al. and our previous study suggested that it was the Thl cellular immune response that IFN-'y secretion was predominant in the early phase of infection and subsequent effective antibody-mediated immune response in later phase that limited parasitemia and eventually cleared parasite. In this research, our results showed that even though different dose of CQ or AR treatments efficiently eliminated parasitic infection, they had less influence on the development of immunity during Plasmodium yoelii primary infection, since higher IFN-'y level in early phase and production profile of specific IgG in later phase were also observed in both CQ and AR treated groups, just as shown in untreated and self-resolved mice. What￡ more, there was no difference in proliferation ability of spleen cells from all groups.Research has shown that specific IgG was major effector, responsible for resistance against homologous Plasmodium reinfection. Transfer of immune serum from P. chabaudi AS infected mice completely protected the infected recipient mice against reinfection. Furthermore, depletion of IgG from immune serum significantly reduced the protective effect of the serum. The incapacity of CD28 ~ mice to acquire a full protective immunity to P. chabaudi correlated with an impaired production of IgG. Infected mice treated by combination of CQ and IL-12, induced high levels of anti-malaria antibodies as well as sterile immunity a-gainst reinfection. According to our results, each treated group and self-resolved groups of both kinds of mice all had high levels of P. yoelii specific IgG and showed significant protection against homologous Plasmodium reinfection. Mice either treated by different antimalarials or treated by different dose of antimalari-al showed similar resistance against reinfection compared with self-resolved mice. These results indicated different dose of CQ or AR treatment did not alter the protective response against P. yoelii reinfection and specific IgG was the major effector in this process. But whether the persistence period of protective immunity was affected needs further research.Research has shown production of IFN-7 by peripheral blood mononuclear cells (PBMC) from mild Plas modium falciparum patient in response to merozo-ite antigen peptides was associated with significantly delayed first reinfection. While, IFN-7 receptor deficient mice were equally protected upon reinfectionlike wild type mice, indicating less function for IFN-7 in the control of malaria reinfection. Elisa et al. recently reported the numbers of IFN-7-producing spleen cells were not increased in the first days after reinfection. In this research , both kinds of mice self-resolved or treated all had no elevated production profile of IFN-*y during reinfection. This phenomenon could be due to the fact that the high level of IgG had already controlled or eradicated the parasites. This suggested the immune mechanism agaist reinfection may be different from that a-gainst primary infection. Thus, the function of IFN-7 in resistance to reinfection needs to be clarified by more researches.Taken together, this work presented evidence that in P. yoelii 17XNL infected BALB/c mice and P. yoelii 17XL infected DBA/2 mice models, CQ or AR treatment didnt interfere in the development and maintenance of immunity during primary infection and specific immnunity against homologous Plasmodium reinfection. Consequently, these findings provided essential experimental data for rational usage of antimalarial and the feasibility in combination of antimalarial and malaria vaccine.Conclusion1. Different dose of CQ or AR treatment didnt interfere in the development of primary immune response in P. yoelii 17XNL infected BALB/c mice and P. yoelii 17XL infected DBA/2 mice.2. Different dose of CQ or AR treatment didnt interfere in the development of specific immunity to homologous Plasmodium reinfection in P. yoelii 17XNL infected BALB/c mice and P. yoelii 17XL infected DBA/2 mice.3. Specific IgG is major effect molecular responsible for resistance against homologous Plasmodium reinfection.