Elucidating mechanisms of protection against dengue severity through immunity to dengue virus nonstructural protein 1

Almost half of the world’s population is at risk of infection by dengue virus (DENV), a mosquito-borne flavivirus consisting of serotypes DENV1-4 that is the causative agent of dengue disease. Of the estimated 50-100 million dengue cases annually, 5% develop severe complications known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which are characterized by severe vascular leak of fluid from the blood into tissues. The triggers of pathogenic DENV-associated vascular leak are still under investigation, but recent work has identified secreted DENV non-structural protein 1 (NS1) as a direct mediator of endothelial cell hyperpermeability and vascular leak. Antibodies against NS1 have been shown to prevent morbidity and mortality in mouse models of DENV infection and can inhibit DENV NS1-induced endothelial dysfunction in vitro, highlighting their potential as a therapeutic against DENV and a target for vaccine design. The mechanistic basis by which anti-NS1 antibodies prevent endothelial dysfunction is largely unknown. DENV NS1 is a multifunctional viral protein that plays many roles in the viral life cycle. In addition to inducing endothelial dysfunction, it has been shown to participate in the formation of the viral replication complex, antagonize the complement cascade, and activate innate immune cells. The mechanisms underlying NS1-induced innate immune activation, in particular, are not well understood, and the contribution of this activation during DENV infection is unclear. This dissertation details the exploration of how both innate immunity and antibodies targeting NS1 inhibit DENV pathogenesis./nFirst, we identify inflammasomes, a class of cytosolic innate immune sensors in the nucleotide-binding oligomerization domain-like receptor (NLR) family, as sensors of DENV NS1. Inflammasomes can sense pathogenic stimuli through NLRs like NLRP3, which leads to activation of caspase-1 and release of IL-1 family cytokines such as IL-1β. We show that DENV NS1 activates inflammasomes in mouse and human macrophages in a caspase-1/11-dependent manner. In mice, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3 inflammasome and does not result in detectable inflammatory cell death. Using caspase-1/11- and NLRP3-deficient mice, we find that caspase-1/11 deficiency makes mice more susceptible to DENV compared to caspase-1/11-functional mice, whereas NLRP3 deficiency has no effect on morbidity and mortality during DENV infection, mirroring the pattern of NS1-induced inflammasome activation. Taken together, we demonstrate that NS1 can trigger inflammasome activation and identify a beneficial role for the inflammasome pathway in DENV infection, broadening our understanding of innate immune-mediated protective responses to DENV./nNext, we characterized the mechanisms behind the protective ability of an NS1-specific monoclonal antibody (mAb), 2B7. mAb 2B7 inhibits NS1 binding to endothelial cells and NS1-induced hyperpermeability in vitro and prevents vascular leak and mortality in vivo. We mutated amino acids within the 2B7 epitope guided by a recent crystal structure of the 2B7 antigen-binding fragment (F’ab) in complex with NS1 in order to define the basis of antibody-mediated inhibition of NS1-induced endothelial dysfunction. We found that several amino acids within the core of the 2B7 epitope are conserved across flaviviruses, imbuing 2B7 with pan-flavivirus NS1 cross-reactivity. We also identified several mutants that could still bind to endothelial cells but were unable to induce hyperpermeability, suggesting that amino acids within the 2B7 epitope are molecular determinants of NS1 pathogenesis. The structures and mutagenesis results suggest that 2B7 prevents NS1-induced endothelial dysfunction though two mechanisms: 1) indirect steric hinderance of the cell-binding domain of NS1, abrogating interaction with endothelial cells and 2) direct blockade of amino acids responsible for an additional step in NS1 pathogenesis downstream of binding. Lastly, we find that humans infected with DENV can generate 2B7-like antibody responses against DENV NS1, opening the possibility for rational vaccine design targeting this important epitope./nTaken together, these findings highlight the importance of immune responses to DENV NS1 in preventing disease and elucidate new aspects of DENV NS1’s protective and pathogenic roles during DENV infection.

Advisor: Harris, Eva