Methods for studying memory B-cell immunity against malaria

Abstract: Plasmodium falciparum malaria remains one of the world’s deadliest infectious diseases and the search for an effective vaccine is highly warranted. Memory B cells (MBCs) and the antibodies they produce, once activated, is believed to play an important role in the protective immunity against malaria, but the mechanism of acquiring and maintaining these cells is poorly understood. New and sensitive tools able of gathering detailed information regarding the development and maintenance of antigen-specific MBCs could increase the understanding of protective immunity but also be used for the evaluation of new vaccines. In Study I, we developed the reversed B-cell FluoroSpot assay, a new assay format based on an established technique for single-cell analysis. Using hybridomas and splenocytes from immunized mice together with a tag/anti-tag approach for detection, we showed proof-of-principle that the assay could be used for multiplex analysis of single B cells specific to four different antigens simultaneously, as well as detecting B cells displaying cross-reactivity against antigen variants. In Study II, we adapted the assay for studies on humans and measured MBC responses against hepatitis B virus, tetanus toxoid and cytomegalovirus. We also measured MBC frequencies before and after vaccination against hepatitis B and used new FluoroSpot reader functions to assess spot volume. We showed that the assay could be used to detect B cells against all of the antigens simultaneously and also changes in MBC frequencies and spot volume before and after vaccination. In Study III, we adapted the multiplex assay further for studies on P. falciparum antigen-specific MBCs and used it to study the kinetics of MBC responses in primary infected and previously exposed travelers diagnosed with malaria in Sweden. We showed that primary infected individuals could acquire and maintain P. falciparum-antigen specific MBCs as efficiently as previously exposed individuals during a one year follow up period, but that the maintenance and magnitude of antibody levels in plasma were higher in the previously exposed individuals. In Study IV, we used the assay developed in Study III to analyze P. falciparum antigen-specific MBCs in children living in areas with endemic transmission of malaria in Kenya. We identified that high levels of MBCs against certain P. falciparum antigens were associated with a reduced risk of a subsequent clinical malaria episode, and that proportions of MBCs specific to some, but not all, P. falciparum antigens, increase with age, but also some decrease with cumulative number of infections. We conclude that the multiplex FluoroSpot method developed in this thesis provide insights towards the acquisition and maintenance of P. falciparum malaria-induced MBCs. We believe that the reversed B-cell FluoroSpot assay is a sensitive and highly adaptable method to assess MBC responses against multiple antigens and will be a powerful tool for future studies on protective immunity to malaria, but also other fields of research.

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