ORCID

http://orcid.org/0000-0002-0438-7842

Date of Award

Spring 5-15-2023

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Immunology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

ABSTRACT OF THE DISSERTATIONThe Role of NS1 in Flavivirus Immunity and Pathogenesis by Alex W. Wessel Doctor of Philosophy in Biology and Biomedical Sciences Immunology Washington University in St. Louis, 2023 Professor Michael Diamond, Chair Flaviviruses are a genus of enveloped, arthropod-transmitted RNA viruses that include clinically relevant pathogens such as yellow fever, dengue, Zika, and West Nile viruses. These viruses cause a spectrum of potentially life-threatening diseases including hepatitis, vascular shock, congenital abnormalities, and encephalitis. Dengue virus, alone, infects up to 390 million individuals in any given year, and its endemic regions continue to expand along with the geographical spread of its mosquito vectors. Flaviviruses also have a history of emergence and reemergence, such as the recent dissemination of Zika virus (ZIKV) to Oceania and the Americas in 2015. During this epidemic, the virus caused an estimated 1.5 million infections in Brazil, drawing global attention due to new clinical manifestations of microcephaly and other congenital malformations. The dissemination of West Nile virus (WNV) to North America in 1999 also resulted in thousands of infections, along with considerable morbidity and mortality due to neuroinvasive disease. Notwithstanding this epidemiology, no approved vaccine or antiviral therapeutic exists for many flaviviruses such as ZIKV and WNV. The approved vaccines for dengue, Japanese encephalitis, and yellow fever viruses have demonstrated efficacy by targeting the flavivirus envelope protein, generating neutralizing

antibody responses. However, cross-reactive, sub-neutralizing antibodies reportedly can enhanceinfection of homologous and heterologous flaviviruses, particularly for dengue virus and ZIKV. This phenomenon, termed antibody-dependent enhancement (ADE), remains a concern for envelope-targeted vaccines and antibody therapeutics. An alternative approach in the field has been to target the flavivirus nonstructural protein 1 (NS1), a membrane-associated and secreted glycoprotein also involved in virus replication. Prior reports indicate that anti-NS1 monoclonal antibodies (mAbs) can confer protection against certain flaviviruses, but the mechanisms and epitopes associated with protection are poorly characterized. Herein, I describe correlates of protection by anti-NS1 monoclonal antibodies (mAbs) for ZIKV and WNV. Panels of anti-ZIKV and anti-WNV NS1 human and murine mAbs were generated to study their efficacy in vivo. Administration of anti-ZIKV NS1 human and murine mAbs to non-pregnant mice conferred protection against lethal ZIKV challenge. Additionally, several mAbs conferred protection to the developing fetus upon infection of pregnant mice by limiting viral burden in the fetal head and placenta. Protection correlated with the avidity of mAb binding to cell-surface-expressed NS1 and with Fc effector function engagement, suggesting that anti-NS1 mAbs may protect through clearance of virus-infected cells. The protective mAbs mapped to exposed epitopes on the cell-surface form of NS1, including the outer, electrostatic surface of the wing domain and loop face of the β-platform domain. Administration of anti-WNV NS1 human mAbs protected mice against lethal WNV challenge. For most mAbs, protection also correlated with binding to cell-surface-expressed NS1. Epitope mapping through mutagenesis revealed most of the protective mAbs to bind the outer surface of the wing domain or loop face of the β-platform. Unlike the anti-ZIKV NS1 mAbs, however, one protective mAb was identified that mapped to the flexible loop of the wing domain. Additionally, one mAb conferred protection

against WNV despite lacking binding to cell-surface NS1 and mapping to a poorly exposed epitopeon cell-surface NS1. These data suggest that NS1-specific mAbs likely protect through Fcmediated clearance of infected cells expressing NS1 on the surface. However, other mechanisms potentially exist through binding to secreted NS1 and blocking its pathogenic functions. The protective epitopes defined in these studies can be informative for the design of NS1-targeted vaccine immunogens and antibody therapeutics that avoid ADE. Cell-surface-expressed and secreted NS1 are thought to contribute to viral pathogenesis through interaction with host factors such as complement, toll-like receptors, and endothelial cells. Despite these ascribed functions, their relevance during in vivo infection has not been clearly established for many flaviviruses, such as WNV. Mutagenesis studies are often complicated by the inability to uncouple the role of NS1 in replication from its accessory roles in pathogenesis. A proline to lysine substitution at residue 101 (P101K) of WNV NS1 did not affect WNV replication yet resulted in decreased lethality in mice, accompanied by lower levels of virus spreading to the brain. The NS1-P101K substitution led to lower levels of secreted NS1 in circulation in mice, and exogenous reconstitution of the NS1 levels restored virus levels in the brain. These studies suggest that the levels of NS1 in circulation during infection can contribute to WNV dissemination to the brain, and support the general notion that NS1 contributes to flavivirus disease.

Language

English (en)

Chair and Committee

Michael Diamond

Committee Members

Adrianus Boon, Daved Fremont, Michael Gross, Robyn Klein,

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