Nearly all licensed vaccines protect the host through antibodies rather than cell-mediated immunity. In addition to being directed against the right pathogen antigen and having the right affinity and immunoglobulin class, a critical aspect of vaccine-induced serological protection is the duration of antibody titer post-boost. Our basic understanding of how T-cell dependent antibody responses develop after infection or vaccination involves presentation of antigens by dendritic cells to specific CD4+ T cells, which then proliferate and differentiate into help T cells (Tfh) that begin to support responses of antigen-engaged B cells that have moved to the T-B border of secondary lymphoid tissues (spleen or lymph node). These interactions can generate a population of short-lived, high-rate antibody secreting cells (the extrafollicluar antibody response), which contributes to acute host defense but not to long-lived protection. The latter derives from additional events that involve migration of antigen-activated T cells and the associated antigen-specific B cells into the B cell follicle where they continue to interact in an antigen-dependent manner to set up the germinal center reaction. This leads to somatic hypermutation and isotype class switching, producing antibodies of higher affinity and different effector class, while generating both memory B cells and also plasmablasts that have the capacity to become long-lived plasma cells if they reach the proper supportive niche, which is mainly located in the bone marrow. While this general outline is well-established, the molecular signals that guide the engagement of T and B cells to generate a maximally productive response, the role of Tfh in determining the choice between memory B cells and long-lived plasma cells, and what determines how plasmablasts become long-lived plasma cells remain unclear. This project is a collaborative effort among three laboratories with expertise in vaccine development, adjuvant function, and the cellular immune reactions involved in antibody responses that aims to examine how adjuvants affect each of these key steps in humoral immune responses and how variations in the quality and quantity of Tfh, stimuli for B cells, and niche space for plasma cells affects the persistence of antibody responses post-vaccination. During the past year, progress has been made in developing a strategy for conducting mouse and follow-on NHP experiments to address these fundamental issues. A schema has been developed to systematically test a variety of adjuvants with candidate antigens selected for a transmission-blocking malarial vaccine with respect to their capacity to produce durable antibody responses of adequate titer. Preliminary data have been gathered about how to measure Tfh responses and hence, to correlate these responses with robust, long-lived antibody responses in the context of a single antigen formulated with each of a diverse set of adjuvants. In this work, we used a model system in which mice were immunized with a peptide epitope from Toxoplasma emulsified in Complete Freunds Adjuvant and then measured T cell responses using a specific Class II MHC tetramer at day 14 post-injection. With this protocol we found that approximately 7% of the CD4+, Foxp3- cells in draining lymph nodes from immunized mice were Tfh as defined by co-staining with CXCR5 and BCL-6;of these roughly 15% were tetramer positive. A more stringent analysis using a combination of 4 Tfh canonical markers revealed that approximately 1 % of the CD4+, Foxp3- cells in immunized mice were CXCR5+, BCL-6 +, ICOShi, PD1hi versus 0.1 % in sham immunized mice. These responses were substantially reduced in immunized mice deficient in the TLR/IL-1R adaptor MyD88. This work is currently being extended with kinetic analyses and to studies with Incomplete Freunds adjuvant to evaluate the contribution of the mycobacterial component in CFA to Tfh induction. A full-time fellow has been recruited to specifically investigate regulation of the bone marrow plasma cell niche, a well as to develop methods for direct and quantitative analysis of Tfh formation via dendritic cell and antigen-specific B cell interactions, for assessment of B cell activation by antigen and innate stimuli, for examination of the outcome of Tfh-B cell interactions in germinal centers, and for the determination of the proportion of memory B cells vs. plasmablasts formed by this latter response. We anticipate collecting valuable data on the relationship between adjuvant and antibody response persistence within the coming few months, while advancing the cellular immunological studies of how adjuvants influence the various aspects of the T-cell dependent B cell response during the same interval. The data generated by these rodent experiments will be used to plan NHP studies focused on determining if the top candidate adjuvants produce the same types of responses in these primates at the macro (i.e., antibody titer) level and with respect to the cellular events involved, such as bone marrow niche regulation. A key aim of this project is the identification of vaccine formulations that will extend the duration of the antibody response against malaria vaccine candidates. Our prototype target malaria antigen is Pfs25, which is undergoing Phase 1 trials in humans as a Pichia-expressed recombinant protein conjugated to the carrier protein ExoProtein A (EPA) expressed in E. coli with a molar ratio of 3:1, and formulated with the commercially available adjuvant Alhydrogel: Pfs25-EPA/Alhydrogel. Pfs25-EPA/Alhydrogel is undergoing Phase 1 trials in malaria-nave volunteers in the US (dose-escalating trial) and malaria-experienced volunteers in Mali (dose-escalating;double-blinded;placebo-controlled trial). Sera collected from volunteers in the US and Mali are being assessed for seroreactivity by standardized ELISA, and transmission-blocking antibody activity is measured in membrane feeding assays. Serum antibody levels in either assay will be measured before and after each vaccine dose, and then periodically thereafter to assess the duration of the antibody response, including the functional antibody response. In Mali, mosquitoes are fed directly on vaccinees to determine their infectivity/malaria transmission potential, and this will be related to the antibody measurements. The Pfs25-EPA/Alhydrogel product will be a benchmark against which we will compare novel Pfs25 products and formulations in our animal studies.

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