The adaptive immune system encodes the ability to remember an initial encounter with an antigen and to respond to that antigen upon re-exposure in a rapid and robust fashion for the life time of the individual. This phenomenon of immunological memory is a fundamental property of the adaptive immune system and is the basis for all vaccine development. For most vaccines, neutralizing antibodies plays a critical role in protective immune responses, and thus the mechanisms that underlie the generation and maintenance of humoral memory are of considerable interest. Long-term humoral immunity is encoded in memory B cells (MBC) and long-lived plasma cells (LLPC) which are generated as a part of the primary immune response. LLPC are terminally differentiated cells that reside in the bone marrow and are responsible for the long-term maintenance of serum Ab levels which play a key role in the initial control of pathogens and their toxins upon reinfection. MBC are capable of mounting an antigen-induced response by proliferating and differentiating into plasma cells (PC) resulting in rapid, high titer secondary Ab responses upon re-exposure to pathogens. MBC may provide protection even in individuals with insufficient neutralizing Ab titers. Despite the central role of MBC in combating infections, our understanding of the cellular and molecular mechanisms that underlie the generation and maintenance of B cell memory is incomplete. Efforts to develop new vaccines would benefit from a more detailed knowledge of these processes, particularly vaccines against a pathogen such as Plasmodium falciparum in malaria which appear to subvert immunological memory. This project represented a collaborative effort between Dr. Pierce and Dr. Louis Miller and his colleagues in the Malaria Vaccine Development Branch (MVDB). Over the last year we have focused our efforts on gaining an understanding of the generation, maintenance and activation of B cell memory in nave individuals in response to vaccination. Of particular interest in this process is the role of TLR9, a pattern recognition receptor that initiates innate immune responses. TLR9 detects microbial DNA with hypomethylated CpG motifs and in humans is preferentially expressed by plasmacytoid dendritic cells (PDC) and B cells. TLR9 ligands have been indirectly implicated in the maintenance of B cell memory although at present the role of TLR9 in the generation of B cell memory has not been addressed. ? ? We have taken advantage of recent advances in the identification of antigen-specific human memory B cells in peripheral blood to describe the generation and maintenance of malaria-specific memory B cells in the U.S. in response to vaccines currently under development in the MVDB. Over the last year, in collaboration with the MVDB, we described the acquisition of antigen-specific memory B cells in the peripheral blood of volunteers enrolled in two trials of the malaria vaccines, one composed of P. falciparum apical membrane antigen 1 (AMA1) and one of merozoite surface protein 1 (MSP1), both on alum either alone or in combination with the TLR9 agonist, CpG. Volunteers received three doses of the vaccine 28 days apart and peripheral blood samples were collected 3, 7, 14 and 28 days after each vaccination and up to 200 days following the third and last vaccination. The longitudinal design of this study permitted a detailed characterization of the kinetics of MBC generation and maintenance in response to primary and secondary vaccination. The capacity for a detailed characterization was most apparent in the analysis of the AMA1-C1 vaccine trial in which PBMC samples were collected at several time points after each vaccination. We found that the acquisition of memory B cells is a dynamic process in which the antigen-specific memory B cell pool rapidly expands and then contracts following vaccination. In individuals who received CpG-containing vaccines, antigen-specific memory B cells appeared more rapidly, in greater numbers, and persisted for longer. The percentage of vaccine-specific memory B cells present at the time of re-immunization predicted antigen-specific antibody levels 14 days later and at steady state, there was a positive correlation between antigen-specific memory B cells and antibody levels. We also observed an antigen-independent decrease in the total IgG+ memory B cell pool in circulation 3 days after each vaccination, possibly the result of adjuvant-induced trafficking of memory B cells into tissues. Consistent with this possibility we observed a large increase in the total number of plasma cells in circulation, suggesting that memory B cells induced to leave the circulation gave rise to plasma cells. These are the first data describing the naive human memory B cell response to vaccination and will serve as a baseline for similar analyses in malaria endemic areas.
