The role of antibodies in Dengue virus infection: Understanding protection and pathogenesis
Profound vascular leakage in conjunction with elevated viremia is the hallmark of Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS). Antibody (Ab)-dependent enhancement (ADE), in which pre-existing, cross-reactive Abs enhance virus infectivity, is thought to be responsible for increased viremia, while loss of endothelial cell (EC) barrier integrity is the precursor to plasma leakage. However, the relationship between viremia and vascular leak has not been established. The objective of this dissertation project was to determine the involvement of antibodies in the pathogenesis of vascular leak syndrome associated with DHF/DSS by establishing a relationship between Ab-mediated increase in viremia and changes in vascular permeability, the hallmark of DHF/DSS. Our approach focused on characterization of human monoclonal antibodies (hMAbs) from a previously dengue virus (DENV)-infected patient for their ability to both neutralize and enhance infection and increase vascular permeability in vitro. Our results revealed that the human antibody response to DENV E protein elicited by natural infection is predominantly comprised of broadly cross-reactive antibodies targeting domain II epitopes. Using a multiplex cytokine immunoassay, qRT-PCR, and plaque assay, we demonstrated an association between viral load and cytokine production in DENV-infected FcÎ³R-bearing K562 cells, and determined that DENV infection of K562 cells in the presence of hMAb resulted in a modulated inflammatory cytokine response with an overall pro-inflammatory profile. Using human microvascular ECs (HMEC-1), we further demonstrated an association between viral load, cytokine production, and the onset of permeability changes via an indirect mechanism in which inflammatory mediators released by DENV-infected K562 cells altered HMEC-1 barrier function and observed a synergistic effect between active DENV infection and release of inflammatory mediators by both K562 and HMEC-1 that increased permeability. Collectively, our results support the multifactorial nature of the pathogenesis underlying vascular leak, involving a complex interaction between ECs and FcÎ³R-bearing cells, and a synergistic relationship between enhanced viremia and inflammatory mediators leading to increased permeability. Our use of hMAbs provided a novel approach to understanding how Abs impact the vasculature during DENV infection and enable identification of Ab characteristics that may trigger vascular leak, a crucial concern for DENV vaccine design.