The Role of NS1 in Flavivirus Immunity and Pathogenesis

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

Human monoclonal antibodies against NS1 protein protect against lethal West Nile virus infection

Envelope protein-targeted vaccines for flaviviruses are limited by concerns of antibody-dependent enhancement (ADE) of infections. Nonstructural protein 1 (NS1) provides an alternative vaccine target that avoids this risk since this protein is absent from the virion. Beyond its intracellular role in virus replication, extracellular forms of NS1 function in immune modulation and are recognized by host-derived antibodies. The rational design of NS1-based vaccines requires an extensive understanding of the antigenic sites on NS1, especially those targeted by protective antibodies. Here, we isolated human monoclonal antibodies (MAbs) from individuals previously naturally infected with WNV, mapped their epitopes using structure-guided mutagenesis, and evaluated their efficac

A simplified quantitative real-time PCR assay for monitoring SARS-CoV-2 growth in cell culture

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growt

Antibodies targeting epitopes on the cell-surface form of NS1 protect against Zika virus infection during pregnancy

Zika virus is an arthropod-transmitted flavivirus that can cause microcephaly and other fetal abnormalities during pregnancy. Here Wessel et al. develop antibodies against the Zika virus nonstructural protein 1 that protect non-pregnant and pregnant mice against infection, and define particular antibody epitopes and mechanisms underlying this protection

Privacy-preserving dataset combination and Lasso regression for healthcare predictions

Background: Recent developments in machine learning have shown its potential impact for clinical use such as risk prediction, prognosis, and treatment selection. However, relevant data are often scattered across different stakeholders and their use is regulated, e.g. by GDPR or HIPAA. As a concrete use-case, hospital Erasmus MC and health insurance company Achmea have data on individuals in the city of Rotterdam, which would in theory enable them to train a regression model in order to identify high-impact lifestyle factors for heart failure. However, privacy and confdentiality concerns make it unfeasible to exchange these data. Methods: This article describes a solution where vertically-partitioned synthetic data of Achmea and of Erasmus MC are combined using Secure Multi-Party Computation. First, a secure inner join protocol takes place to securely determine the identifiers of the patients that are represented in both datasets. Then, a secure Lasso Regression model is trained on the securely combined data. The involved parties thus obtain the prediction model but no further information on the input data of the other parties. Results: We implement our secure solution and describe its performance and scalability: we can train a prediction model on two datasets with 5000 records each and a total of 30 features in less than one hour, with a minimal difference from the results of standard (non-secure) methods. Conclusions: This article shows that it is possible to combine datasets and train a Lasso regression model on this combination in a secure way. Such a solution thus further expands the potential of privacy-preserving data analysis in the medical domain

Insights Into the Biogeochemical Cycling of Iron, Nitrate, and Phosphate Across a 5,300 km South Pacific Zonal Section (153°E–150°W)

Iron, phosphate and nitrate are essential nutrients for phytoplankton growth and hence their supply into the surface ocean controls oceanic primary production. Here, we present a GEOTRACES zonal section (GP13; 30-33oS, 153oE-150oW) extending eastwards from Australia to the oligotrophic South Pacific Ocean gyre outlining the concentrations of these key nutrients. Surface dissolved iron concentrations are elevated at >0.4 nmol L-1 near continental Australia (west of 165°E) and decreased eastward to ≤0.2 nmol L-1 (170oW-150oW). The supply of dissolved iron into the upper ocean (<100m) from the atmosphere and vertical diffusivity averaged 11 ±10 nmol m-2 d-1. In the remote South Pacific Ocean (170oW-150oW) atmospherically sourced iron is a significant contributor to the surface dissolved iron pool with average supply contribution of 23 ± 17% (range 3% to 55%). Surface-water nitrate concentrations averaged 5 ±4 nmol L-1 between 170oW and 150oW whilst surface-water phosphate concentrations averaged 58 ±30 nmol L-1. The supply of nitrogen into the upper ocean is primarily from deeper waters (24-1647 μmol m-2 d-1) with atmospheric deposition and nitrogen fixation contributing <1% to the overall flux, in remote South Pacific waters. The deep water N:P ratio averaged 16 ±3 but declined to <1 above the deep chlorophyll maximum (DCM) indicating a high N:P assimilation ratio by phytoplankton leading to almost quantitative removal of nitrate. The supply stoichiometry for iron and nitrogen relative to phosphate at and above the DCM declines eastward leading to two biogeographical provinces: one with diazotroph production and the other without diazotroph production

Rare and low-frequency coding variants alter human adult height

Height is a highly heritable, classic polygenic trait with ~700 common associated variants identified so far through genome - wide association studies . Here , we report 83 height - associated coding variants with lower minor allele frequenc ies ( range of 0.1 - 4.8% ) and effects of up to 2 16 cm /allele ( e.g. in IHH , STC2 , AR and CRISPLD2 ) , >10 times the average effect of common variants . In functional follow - up studies, rare height - increasing alleles of STC2 (+1 - 2 cm/allele) compromise d proteolytic inhibition of PAPP - A and increased cleavage of IGFBP - 4 in vitro , resulting in higher bioavailability of insulin - like growth factors . The se 83 height - associated variants overlap genes mutated in monogenic growth disorders and highlight new biological candidates ( e.g. ADAMTS3, IL11RA, NOX4 ) and pathways ( e.g . proteoglycan/ glycosaminoglycan synthesis ) involved in growth . Our results demonstrate that sufficiently large sample sizes can uncover rare and low - frequency variants of moderate to large effect associated with polygenic human phenotypes , and that these variants implicate relevant genes and pathways