Human B Cell Responses to Malaria Vaccination and Infect

防治疟疾的疫苗是降低非洲儿童发病率和死亡率的一种非常理想的公共卫生工具。为了实现这一目标，重要的是详细了解对目前候选疫苗的免疫反应的性质以及生活在非洲疟疾流行地区的个人的免疫状况。在本报告所述期间，该项目是皮尔斯博士与疟疾疫苗开发股的路易斯·米勒博士及其同事之间的合作努力。在细胞水平上的免疫反应在美国的个体中进行了评估，这些个体参加了两项恶性疟原虫候选疫苗的临床试验，明矾上的AMA-1，明矾加TLR-9配体CpG的一项试验，以及在马里参加平行研究的非洲成年人中。此外，在马里开展了一项研究，以确定疟疾感染对记忆B细胞的产生和维持的影响。为了评估这些个体的免疫状态，利用了有关免疫应答中淋巴细胞离散亚群功能的新信息和血清学试剂的可用性来识别这些信息。B细胞抗体对疟疾的反应被认为是控制寄生虫感染的核心，因此我们最初的研究集中在美国和非洲参加1期疫苗临床试验的个体外周血中B细胞的特征。外周血细胞用流式细胞术检测B细胞标志物CD19、CD27和CD38。荧光标记的AMA-1用于鉴定抗原特异性B细胞。此外，通过体外对tlr9配体CpG的反应，用ELISPOT和记忆B细胞鉴定抗原特异性抗体分泌浆细胞。在接种疫苗前以及一次免疫和二次免疫（一次免疫后28天或56天）后的第3、7和14天对细胞进行分析。到目前为止获得的结果表明，CD19+ B细胞的百分比在接种疫苗后没有变化。然而，定义为CD27+和CD38+以及CD19+上CD19-的浆细胞数量在第一次接种疫苗后7 - 14天和第二次接种疫苗后3天出现增加。疫苗接种的效果也反映在一次和二次免疫后3至7天记忆B细胞（定义为CD27+细胞）数量的增加上。初步结果表明，初次免疫后外周抗原特异性记忆B细胞数量增加，而二次免疫后则没有增加。对参加类似的AMA-1或明矾I期研究的非洲人B细胞的平行分析显示，接种疫苗后B细胞的总体反应相似。然而，记忆B细胞较早出现在外周血中，在初次接种疫苗后3天，这表明生活在流行地区的个体已经对AMA-1免疫。类似的分析正在进行中，以分析记忆B细胞对含有AMA-1明矾和CpG的疫苗在美国的I期试验中的反应。类似的分析也计划在马里进行AMA-1明矾和CpG的I期试验。这些结果令人鼓舞，表明相关B细胞亚群的离散变化可以在疫苗接种反应中检测到。这些变化可能最终为监测疫苗效力和指导未来的疫苗战略提供重要的新参数。

A vaccine to combat malaria is a highly desirable public health tool to reduce morbidity and mortality in African children. In order to achieve this goal it will be important to gain a detailed understanding of both the nature of the immune response to the current vaccine candidates as well as the immunological status of individuals living in areas in African where malaria is endemic. Over the reporting period this project represented a collaborative effort between Dr. Pierce and Dr. Louis Miller and his colleagues in the Malaria Vaccine Development Unit (MVDU). The immune response at the cellular level was evaluated in individuals in the U.S. enrolled in two clinical trials of the Plasmodium falciparum vaccine candidate, AMA-1 on alum, and one trial of AMA-1 on alum plus the TLR-9 ligand CpG, and in African adults enrolled in parallel studies in Mali. In addition, a study was initiated in Mali to determine the effect of malaria infection on the generation and maintenance of memory B cells. To evaluate the immune status of these individuals, advantage was taken of new and emerging information concerning the function of discrete subsets of lymphocytes in immune responses and the availability of serological reagents to identify these. The B cell antibody response to malaria is believed to be central to the control of parasite infections and thus our initial studies focused on a characterization of B cells in the peripheral blood of individuals enrolled in phase 1 vaccine clinical trials in the U.S. and in Africa. Peripheral blood cells were analyzed by flow cytometry for the B cell markers CD19, CD27, and CD38. Fluorescently labeled AMA-1 was used to identify antigen-specific B cells. In addition, antigen-specific antibody secreting plasma cells were identified by ELISPOT and memory B cells by their response in vitro to the TLR 9 ligand CpG. Cells were analyzed prior to vaccination and at days 3, 7 and 14 following the primary immunization and the secondary immunization (given either 28 or 56 days after the primary). The results acquired thus far showed that the percentage of CD19+ B cells did not change in response to vaccination. However, the number of plasma cells defined as CD27+ and CD38+ and either CD19+ on CD19-, showed an increase between 7 and 14 days after the first vaccination and 3 days after the second vaccination. The effect of vaccination was also mirrored in an increase in the number of memory B cells, defined as CD27+ cells, 3 to 7 days after both the primary and secondary immunization. Preliminary results indicate that the number of antigen-specific memory B cells increased in the periphery after the primary but not after the secondary immunization. Parallel analyses of the B cells in Africans enrolled in a similar Phase I study of AMA-1 or alum showed similar overall B cell response to vaccination. However, memory B cells appeared in the peripheral blood early, at three days post primary vaccination suggesting that individuals living in endemic areas are already immune to AMA-1. Similar analyses are in progress to analyze the memory B cell response to a vaccine containing AMA-1 on alum and CpG in a Phase I trial in the U.S. A similar analyses is planned for both AMA-1 on alum plus CpG Phase I trial in Mali. These results are encouraging indicating that discrete changes in relevant B cell subpopulations can be detected in response to vaccination. These changes may ultimately provide important new parameters to monitor the efficacy of vaccines and guide future vaccine strategies. At present, there is little known about the immunological status at the cellular level of the target population for the vaccine, namely children, in Africa chronically infected with malaria. A detailed analysis of the immune cells in the peripheral blood of chronically infected individuals and a comparison of their profiles with that of nonimmune and vaccinated individuals should provide important new information concerning the repercussion of malaria infection on the immune system and the impact of those effects on the potential to respond to current vaccine candidates. In collaboration with scientists at the MRTC in Mali, we initiated in May 2006, a study to characterize the peripheral blood cells of children and adults in areas in Africa where malaria is endemic. The analyses is a longitudinal study correlating the levels of parasitemia and the immune cell profile in individuals. Lastly, studies will be initiated to determine the effect of malaria infection on both the generation and maintenance of immunological memory. Several anecdotal observations suggest that immunological memory is difficult to establish and only short-lived in individuals living in malaria endemic regions. A clinical protocol will be developed to enroll recent immigrants to the U.S. from West Africa who have malaria infections. These individuals will be treated with anti-malaria drugs and vaccinated for polio and the memory B cell and plasma cells response to polio and to a panel of malaria antigens will be followed with time. The results of this study will hopefully provide new information concerning the generation and maintenance of immunological memory during and following malaria infections.