Immunological responses to emerging phleboviruses

Emerging viruses of the Phenuiviridae family threaten human and animal health across the globe. The mosquito-borne phlebovirus, Rift Valley fever virus (RVFV) is widespread across the African continent wherein regular deadly outbreaks amongst livestock constitute a significant economic burden. In 2009, a tick-borne banyangvirus, severe fever with thrombocytopaenia syndrome virus (SFTSV), emerged in China. Since emergence, SFTSV has spread rapidly and become a serious public health concern in China, Korea, and Japan, with human fatality rates reaching 30%. The growing health burden of phenuiviruses is further highlighted by the emergent tick-borne pathogen, Heartland banyangvirus (HRTV), a genetic relative of SFTSV that is responsible for sporadic human disease outbreaks in the USA. Targeting of the glycoprotein capsomers displayed on the phenuivirus surface by the antibody-mediated immune response is key to controlling infection. To enhance understanding of the molecular basis for immune recognition, we raised monoclonal antibodies (mAbs) against RVFV Gn, one of which exhibited protective efficacy in a mouse infection model. Structural characterisation, using X-ray crystallography, revealed that neutralising antibodies (nAbs) were directed to the membrane-distal domain B of RVFV Gn and likely prevented virus entry into a host cell by blocking the required rearrangements within viral capsomers necessary for membrane fusion. Additionally, we structurally characterised non-neutralising mAbs that target RVFV Gn. These data revealed a previously unreported domain repositioning of RVFV Gn and uncover a potential intermediate that precedes fusogenic rearrangements of the Gn-Gc lattice. These data form a model for antibody-mediated neutralisation of RVFV and provide a blueprint for the rational design of immunotherapeutics and vaccines capable of preventing RVFV infection. Finally, we generated additional libraries of immunoglobulins that target phlebo- and banyangviruses. A nanobody library was generated by vaccination with a leading RVFV vaccine candidate and recombinant RVFV Gn and Gc, which revealed a neutralising response directed to RVFV Gc. This thesis reports the expression and purification of recombinant banyangvirus Gn glycoproteins from HRTV and SFTSV. A heterologous prime-boost regime with the recombinant Gn glycoproteins was implemented and cross-reactive mAbs binding both SFTSV and HRTV were isolated. These nanobodies and mAbs may constitute key reagents for future structural studies, viral detection, and neutralisation efforts. Furthermore, it is likely that they will provide additional insight into the fundamental biology of the immune response to phenuiviruses. The work described herein provides a molecular appraisal of key effectors of the immune response to emerging phenuiviruses. Isolation of novel immunoglobulins and elucidation of their modes of action inform aspects of basic phenuiviral biology and enable future rationalised vaccination and treatment strategies.