Exploring the antigenic and immunostimulatory nature of dengue virus non-structural protein 1

Abstract

The medical burden of dengue virus (DENV) and other members of thenflavivirusngenus, including Japanese encephalitis virus, West Nile virus, yellow fever virus and Zika virus is significant. DENV is endemic in over 100 countries, responsible for an estimated 390 million infections, of which 100 million are associated with clinical diseasen(1). Though usually presenting as dengue fever, a mild, self-limiting febrile illness, in a small proportion of individuals infection can manifest with severe and often fatal haemorrhagic symptoms. This severe disease is clinically defined as dengue haemorrhagic fever (DHF) and consists of the febrile disease observed in DF, with the addition of haemorrhage of varying severity, thrombocytopenia and increased vascular permeabilityn(2). Patients lapseninto dengue shock syndrome (DSS) when plasma loss reaches a critical point.nDespite decades of research, the exact mechanism underlying the serious and often fatal pathologies observed during clinically severe DENV infections remains relatively unknown. It is understood that the vascular leak and associated systemic shock associated with the haemorrhagic symptoms of severe dengue infection are induced by interplay between viral factors and the hostrs immune response against the pathogen. It has long been theorised that the virally encoded protein non-structural protein 1 (NS1) plays a role in DHF/DSS, due to the correlation between high levels of circulating NS1 and disease severityn(3-8). Itnwas recently found the NS1 directly disrupts endothelial cell barrier function and indirectly via that activation of the innate immune receptor toll-like receptor 4 (TLR4), inducing thenproduction of pro-inflammatory cytokinesn(7).In light of the knowledge that DENV2 NS1 induces endothelial cell leak during severe disease, this candidature first aimed to develop a panel of NS1-specific recombinant monoclonal antibodies, which were antigenically validated prior to functional assessment using annin vitronendothelial cell leak assay. The use of this panel of monoclonal antibodies increases the understanding of the protein interactions that underpin the mechanism of NS1-mediated endothelial cell leak and identified key epitopes to inform the rational design of new therapies and vaccines.Recognition of NS1 as a critical component in severe disease has renewed interest in NS1 as a vaccine target. Extending upon this possibility, this thesis also investigated the potential for NS1 to repurposed as a TLR4-agonising adjuvant, with the aim of shaping the immune response towards a robust, more protective, Th1-like immune response. Testing alongside the established saponin adjuvant Quil-A successfully demonstrated the self-adjuvating properties of NS1 and suggests that NS1 acts synergistically with Quil-A when administered jointly. These findings further highlight the immunomodulatory nature of NS1 and also point to ways in which this effect can be managed to favour increased protection against severe dengue disease.nnFinally, this candidature aimed to establish a high yield mammalian NS1 expression system, in attempt to produce proteins representative of those produced during human infection and at biologically relevant concentrations. Codon optimisation of the NS1 gene sequence to codons preferentially utilised by Chinese hamster ovary (CHO) cells successfully enhanced the express of flavivirus NS1 to biologically relevant levels within the transfected cell supernatant. Additionally, this thesis showednnnovel expression of NS1 in CHO cells.nThe flavivirus NS1 proteins, though sharing a high degree of homology, are antigenically distinct, therefore limiting the ability for protein-specific antibody quantification. This thesis successfully demonstrated the development of a pan-flavivirus NS1 capture ELISA, shown to detect native NS1 derived from multiple DENV serotypes (DENV2-4), WNV and ZIKV. Additionally, a peptide tag was added to the recombinant NS1 protein, producing a means of antigenicity-independent detection and quantification.nThis thesis incorporated multiple research aims to better understand the mechanism of NS1-mediated dengue disease. The new reagents, expression systems and assays described within this thesis provide a platform to probe NS1 protein function at the molecular level. The identification of epitopes critical to NS1 induced leak and the proof of principle demonstration of NS1 as a TLR4 adjuvant provide new pathways to develop therapies and preventative measures against DENV. Together these results, contribute to better understanding of the interaction between NS1 and the immune system, and the role this enigmatic protein plays in pathogenesis, ultimately opening new avenues for further discovery.n