Differential dengue tropism & neutralization : potential mechanisms of pathogenesis /

by Martin, Nicole C; Couillard, Nicole

Nicole C. Martin Emerging Infectious Diseases Graduate Program Department of Microbiology and Immunology Uniformed Services University Doctor of Philosophy 2006 Major Advisor: LCDR Timothy H. Burgess, M.D., Department of Virology, Infectious Diseases Directorate, Naval Medical Research Center Title of Dissertation: Differential Dengue Tropism & Neutralization: Potential Mechanisms of Pathogenesis The mechanisms underlying the pathogenesis of dengue hemorrhagic fever (DHF) remain poorly understood, but a clear correlation appears to exist between dengue viral load and disease severity. While antibody-dependent enhancement and T-cell-mediated pathogenesis theories suggest an immunopathologic basis for the development of severe disease, intriguing evidence suggest a role for viral strain differences. Consistent genetic differences exist in the envelope glycoproteins of dengue 2 strains associated with DHF epidemics (Asian genotype) and dengue 2 strains only associated with DF (American genotype). It has also been established that dengue virus infection can be mediated by C- iii type lectins DC-SIGN and L-SIGN. DC-SIGN is found on dendritic cells, a presumed target in human infection. L-SIGN is found on liver sinusoidal endothelial cells. Such cells have been shown to tolerize naïve T cells. To avoid the pitfalls associated with current measures of infection and neutralization, we developed an assay that uses cells expressing these relevant lectin receptors and low-passage viral isolates that yields results within 24 hours. Using this assay, we examined whether Asian and American genotype dengue 2 viruses exhibit differences in utilization of these two receptors. Our results showed that American strains infect DC-SIGN bearing cells to a greater extent than L- SIGN bearing cells while Asian strains preferentially infect L-SIGN bearing cells. A single mutation in the envelope glycoprotein of an American strain at E390 from aspartic acid (American) to asparagine converted the C-type lectin binding phenotype from an American strain to an Asian strain by the observation that the E390 amino acid (aa) in the Asian strain is also asparagine. Furthermore, Asian and American strains differed in their sensitivity to antibody neutralization. The neutralizing capacity of mAbs 3H5 and 4G2 for Asian virus was significantly decreased when infection was measured in L-SIGN bearing cells compared to DC-SIGN bearing cells. We also found that serum from Venezuelan DF patients had much greater neutralizing capacity for Asian virus in L- SIGN cells than serum from patients who progressed to DHF. Further, the magnitude of neutralization of L-SIGN-mediated Asian virus infection was inversely associated with disease severity. Taken together, our studies suggest that differences in receptor utilization and neutralization sensitivity between Asian and American dengue 2 strains may contribute to our understanding of the role that viral strain differences play in dengue pathogenesis. iv