Abstract

Antigenic similarities between the four distinct dengue virus (DENV) serotypes underlie disease enhancement observed in secondary DENV infection. Antibodies generated in the first infection weakly cross-react with the new infecting serotype to facilitate (enhance) viral replication through Fc receptor-mediated viral entry. This phenomenon is called antibody-dependent enhancement (ADE), and it is the largest obstacle to developing safe and broadly protective vaccines for DENV . The antibody repertoire after primary DENV infection is quite restricted, comprising predominantly of weakly cross-reactive antibodies targeting the fusion loop of the surface envelope (E) protein of the virus, while a small portion are serotype-specific antibodies responsible for conveying protective immunity against homotypic infection. Secondary DENV infection induces cross-serotype protection, in part mediated by a broadening and strengthening of antibody responses after heterotypic infection. However, it is not completely understood whether the post-secondary antibody response to the new serotype results from an extension and diversification of the DENV cross-reactive B cells generated during primary infection or is derived from de novo B cell responses. To evaluate how antibody diversity evolves across sequential infection, I first conducted a comprehensive analysis of the DENV -memory B cell receptor (BCR) repertoire of a highly exposed individual. This revealed that the majority of DENV -specific BCRs interacted with the E protein fusion loop and contained BCR physicochemical features opposite to those observed for DENV broadly neutralising antibodies. Despite their target, I identified fusion loop epitope antibodies with greater neutralisation activity than typically described for standard fusion loop epitope antibodies. The BCR signatures identified suggest an underlying selection for specific BCR features drives the clonal maturation trajectory away from enhancing, weakly cross-reactive fusion loop epitope antibodies toward protective and more specific antibodies. One of the greatest challenges to directly mapping this antibody diversification during infection has been our inability to sample and measure human germinal centre responses. To study this, we established a blinded Phase I trial to model sequential DENV infection in 45 individuals across different DENV immune histories (naïve, primary heterotypic, and polytypic) using a live-attenuated DENV3 vaccine. We successfully implemented a minimally invasive, ultrasound-guided, fine needle aspirate sampling of draining lymph nodes to capture DENV -responsive B cells at the site of their activation and affinity maturation. Flow cytometric analyses of circulating and lymph node-resident B cells revealed that naïve participants’ germinal centre responses were predominantly DENV3 -specific, whereas individuals with prior immunity demonstrated cross-reactive lymph node activity associated with a broadening and strengthening of DENV antibody responses as far as 57 days after vaccination. These data provide valuable insight into germinal centre activities that inform our basic understanding of DENV -specific B cell and antibody responses, as well as greater patterns of B cell imprinting, that will lay the foundation for the development of next-generation DENV vaccine strategies and therapeutic interventions.