Project Summary RNA viruses comprise some of the most deadly human pathogens described to date, with new agents constantly emerging as humans encroach on once untouched ecosystems. Two such viruses, Hendra (HeV) and Nipah (NiV), were first described in 1994 and 1998, respectively, and have causes sporadic outbreaks of disease in humans in Australia and Southeast Asia. Recently, though, Nipah virus has begun spreading geographically into new regions of India, causing disease with mortality rates reaching close to 100%. Because of the ability to cause such high morbidity and mortality, coupled with the fact that human-to-human transmission has been observed with Nipah virus, the WHO designated these agents as priority diseases in 2018, with urgent needs for accelerated research and development of vaccines and therapeutics. Despite this urgency, no treatments or vaccines have been licensed to date to combat these viruses, and major gaps in knowledge surrounding the human immune response to Hendra and Nipah exist. The overarching goal of this proposal is to elucidate the molecular and structural mechanisms of neutralization by human monoclonal antibodies targeting the attachment glycoproteins of Hendra and Nipah viruses. I have isolated a large panel of monoclonal antibodies from a human subject inoculated with the Hendra equine vaccine and have shown that these antibodies bind to the attachment glycoproteins or Hendra and/or Nipah viruses. A subset of antibodies potently neutralize both Hendra and Nipah virus isolates. My central hypothesis is that human antibodies targeting the henipavirus attachment glycoprotein neutralize by blocking receptor attachment and/or preventing fusion. In Aim 1, I will use a flow cytometric assay and pre- and post-attachment neutralization tests with chimeric Cedar virus bearing Hendra or Nipah virus glycoproteins to determine the molecular mechanisms by which antibodies neutralize virus. I will elaborate on these findings in Aim 2 by using a variety of structural and biochemical techniques to map the antigenic sites on the henipavirus attachment glycoprotein, with an emphasis on antigenic sites that elicit potently neutralizing, cross-reactive antibodies. My studies to date suggest the most potent antibodies able to bind both Hendra and Nipah virus attachment glycoproteins function by blocking viral attachment to the host receptor ephrin-B2, but that at least one other distinct antigenic site elicits neutralizing antibodies that use a distinct mechanism to neutralize virus. This proposal will provide insight into the human humoral response to henipavirus infection and will help to rationally guide the design of vaccines and antibody therapeutics.