Potent neutralizing antibodies elicited by dengue vaccine in rhesus macaque target diverse epitopes

There is still no safe and effective vaccine against dengue virus infection. Epidemics of dengue virus infection are increasingly a threat to human health around the world. Antibodies generated in response to dengue infection have been shown to impact disease development and effectiveness of dengue vaccine. In this study, we investigated monoclonal antibody responses to an experimental dengue vaccine in rhesus macaques. Variable regions of both heavy chain (VH) and light chain (VL) were cloned from single antibody-secreting B cells. A total of 780 monoclonal antibodies (mAbs) composed of paired VH and VL were characterized. Results show that the vaccination induces mAbs with diverse germline sequences and a wide range of binding affinities. Six potent neutralizing mAbs were identified among 130 dengue envelope protein binders. Critical amino acids for each neutralizing antibody binding to the dengue envelope protein were identified by alanine scanning of mutant libraries. Diverse epitopes were identified, including epitopes on the lateral ridge of DIII, the I-III hinge, the bc loop adjacent to the fusion loop of DII, and the beta-strands and loops of DI. Significantly, one of the neutralizing mAbs has a previously unknown epitope in DII at the interface of the envelope and membrane protein and is capable of neutralizing all four dengue serotypes. Taken together, the results of this study not only provide preclinical validation for the tested experimental vaccine, but also shed light on a potential application of the rhesus macaque model for better dengue vaccine evaluation and design of vaccines and immunization strategies. Author summary Dengue virus (DENV) is a leading cause of human illness in the tropics and subtropics, with about 40% of the world's population living in areas at risk for infection. There are four DENV serotypes. Patients who have previously been infected by one dengue serotype may develop more severe symptoms such as bleeding and endothelial leakage upon secondary infection with another dengue serotype. This study reports the extensive cloning and analysis of 780 monoclonal antibodies (mAbs) from single B cells of rhesus macaques after immunization with an experimental dengue vaccine. We identified a panel of potent neutralizing mAbs with diverse epitopes on the DENV envelope protein. Antibodies in this panel were found to bind to the lateral ridge of DIII, the I-III hinge, the bc loop adjacent to the fusion loop of DII, and the beta-strands and the loops of DI. We also isolated one mAb (d448) that can neutralize all four dengue serotypes and binds to a novel epitope at the interface of the DENV envelope and membrane proteins. Further investigation of these neutralizing monoclonal antibodies is warranted for better vaccine efficacy evaluation and vaccine design.