(CORE B) Research in this P01 will depend on unique antibody reagents that are developed and implemented by the Immunology Core B. Monoclonal antibodies (MAbs) generated by Core B from memory B cells (MBCs) will be essential to address critical questions about the epitopes and lineage of neutralizing antibodies produced after natural dengue virus (DENV) infection or immunization with a tetravalent live attenuated dengue vaccine. Core B will meet these needs and provide infrastructure support for Projects 1 and 2 of the P01. Specifically, the Core B facility will receive frozen PBMCs from individuals who have experienced documented repeat DENV infections from Nicaragua, as has previously been done to successfully isolate human MAbs. Core B also will receive PBMCs from DENV-nave and DENV-exposed recipients of the DENVax tetravalent vaccine candidate from Phase 2 clinical trials. MAbs will be generated from MBCs after EBV transformation or DENV-specific MBC sorting and identification of wells containing DENV-specific antibodies, followed by electrofusion with a myeloma partner cell to create stable hybridomas or recombinant cloning and expression of heavy and chain variable regions. During this process, Core B will produce recombinant DENV proteins and purified viruses for limited initial characerization that will be performed by the Core (e.g., single dilution neutralization, type- specificity vs cross-reactivity, E vs prM protein specificity) to facilitate prioritization of clones for fusion and large-scale MAb production. Purified MAbs then will be sent to the laboratories of Project 1 (natural infections) and Project 2 (vaccinees) for more detailed characterization of type specificity, epitope mapping, and functional anaysis. Core B will also provide Projects 1 and 2 with recombinant DENV proteins from Nicaraguan or vaccine strains, respectively. Based on characterization by Projects 1 and 2, the Core will sequence the antibody genes of all MAbs of functional interest and then perform high throughput sequence analysis of total B cell repertoires from PBMCs collected from prior natural infections in the same individuals (Project 1) or PBMCs collected prior to vaccination in DENV-exposed recipients (Project 2) in order to identify ?siblings? of neutralizing antibodies isolated at later time-points. We hypothesize that highly neutralizing cross-reactive MAbs isolated at later time-points are derived from pre-existing MBCs from a prior DENV exposure that have undergone further somatic hypermutation and affinity maturation. Overall, Core B will provide platforms for generating human MAbs from naturally-infected or vaccinated individuals and sequencing antibody lineages. The functional analysis of these antibody repertoires by Projects 1 and 2 will help to elucidate correlates of humoral immune protection after natural infection and immunization.
(CORE B) By using a common core facility to generate monoclonal antibodies for distribution to Projects 1 and 2 linked to sequencing data, collaborative experimentation can be performed to define the specificity of the humoral response after natural infection (primary versus secondary) or immunization in dengue-nave vs dengue- exposed vaccinees. This program should help define the complex interplay between host and virus in different human populations and improve efforts to successfully develop an effective tetravalent dengue vaccine that confers long-term protection to all four DENV serotypes.
Showing the most recent 10 out of 54 publications
