ORCID

http://orcid.org/0000-0002-4452-8152

Date of Award

Winter 12-15-2022

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

The presence of neutralizing antibodies typically correlates strongly with protection from infection, particularly for RNA viruses; elicitation of neutralizing antibody responses is thus the primary goal of most vaccines, and monoclonal antibodies (mAbs) can be leveraged as potent therapeutics. Thus, understanding the properties of individual antibodies that comprise polyclonal antibody responses and their epitopes is essential for countermeasure design. In this dissertation, I explore the epitopes and functional properties of monoclonal antibodies targeting the structural proteins of two families of RNA viruses, flaviviruses and coronaviruses.

Flaviviruses are globally distributed arthropod-borne positive-sense RNA viruses transmitted primarily by either mosquitos or ticks. Symptomatic infection can range from mild febrile illness to potentially fatal neuroinvasive or hemorrhagic disease, depending on the virus. Virions are 50 nm diameter spheres comprised of envelope protein (E), a three-domain class II viral fusion protein, which forms an icosahedral, proteinaceous outer shell encapsulating the viral envelope and genome. Given its exposure on the surface of the virus, E is the most significant target of neutralizing antibodies. Here, we characterize the properties of mAbs raised against Powassan virus (POWV) and a phylogenetically distinct strain of dengue virus (DENV), DKE-121.

POWV is the sole tick-borne flavivirus circulating in North America. In cases of severe neuroinvasive disease, for which no approved prophylactic or therapeutic countermeasures exist, approximately 10% die, and nearly 50% of survivors suffer from long-term neurological sequelae. We identified 20 neutralizing monoclonal antibodies which cluster into 6 competition groups based on binding to recombinant POWV E. The most potently neutralizing mAbs specifically bind domain III (DIII) of POWV E, a subset of which also cross-react to other tick-borne flaviviruses. Crystallographic analysis of the cross-neutralizing DIII-specific mAb POWV-80 in complex with POWV DIII showed binding to the cryptic C-C′ loop epitope via conserved residues exemplified by tick-borne encephalitis virus (TBEV). Furthermore, docking of POWV-80 into the cryo-EM structure of TBEV revealed a binding orientation unlike other C-C′ loop antibodies but reminiscent of highly neutralizing lateral ridge antibodies, suggesting a mechanism by which POWV-80 neutralizes more potently than C-C′ loop antibodies to other flaviviruses.

Alongside our investigation of POWV, we also generated a panel of monoclonal antibodies against a highly divergent strain of DENV, DKE-121. Dengue viruses are a group of serologically distinct, globally-distributed mosquito-borne flaviviruses which cause hundreds of millions of infections per year. DKE-121 displays approximately 12% sequence divergence in E from its closest relative, DENV-4, much of which is localized to neutralizing epitopes on E. One of our mAbs, DKE-121 E77, showed potent, type-specific neutralization of DKE-121. To understand the basis of this type specificity, we solved cryo-EM structures of both the unbound DKE-121 virus and DKE-121 virions bound by E77, revealing that E77 binds the lateral ridge epitope on DIII, a frequent target of highly neutralizing, type-specific antibodies among flaviviruses. Further investigation will focus on dissecting the specific residues involved in type-specific binding to DKE-121 DIII by E77 and how often these residues occur in circulating DENV strains worldwide.

Lastly, we explore the features of two SARS-CoV-2 neutralizing antibodies, 2B04 and 2H04. SARS-CoV-2, the etiologic agent of COVID-19, is responsible for a global pandemic that has caused hundreds of millions of infections and millions of deaths since its emergence in late 2019. Unlike flaviviruses, SARS-CoV-2 employs a class I viral fusion protein, Spike (S), which binds angiotensin-converting enzyme II (ACE2) on host cell surfaces and drives membrane fusion, enabling viral entry. Cryo-EM analysis of SARS-CoV-2 spike bound by the potently neutralizing 2B04 reveals engagement of the receptor-binding motif (RBM) on the receptor-binding domain (RBD) of S. In contrast, 2H04 bound an epitope adjacent to the RBM and did not block direct ACE2 binding, despite potent neutralization. Mechanistic studies revealed that 2H04 can block SARS-CoV-2 attachment to host cells and neutralizes most effectively prior to viral attachment, indicating possible alternative mechanisms of neutralization. Additionally, we show that SARS-CoV-2 can escape 2B04 and 2H04 neutralization in-vitro by epitope residue substitutions, some of which have since emerged in prominent SARS-CoV-2 variants of concern.In summary, these studies reveal how nuance in epitope sequence and structure plays a key role in determining the functional properties of cognate neutralizing antibodies. Overall, this work expands our understanding of the critically important features of antibodies in the defense against pathogens, strengthening the foundation upon which countermeasure design efforts depend for success.

Language

English (en)

Chair and Committee

Daved H Fremont

Committee Members

Michael S Diamond

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