Student Theses and Dissertations

Date of Award

2022

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Nussenzweig Laboratory

Abstract

The last decade has seen a number of viral epidemics and global pandemic. Increased global connectivity makes the likelihood of future mass outbreaks ever greater. Understanding the adaptive immune response to these pathogens will expand basic understanding of viral immunity mechanisms and can help inform vaccine and therapeutic development. This thesis describes the characterization of the human antibody response to several important viral pathogens, all of which are either emerging pathogens posing significant public health burdens or are already responsible for epidemic outbreaks. These include tick-borne encephalitis virus (TBEV), Powassan virus (POWV), Zika virus (ZIKV) and severe acute respiratory syndrome coronavirus (SARS-CoV-2). The first part of this thesis focuses on encephalitis-causing tick-borne flaviviruses, specifically TBEV and POWV. Both TBEV and POWV are emerging human pathogens that cause potentially fatal disease; there are no disease-specific treatments. Mouse monoclonal antibodies are protective against TBEV and POWV, but little is known about the human antibody response to active infection. Although 10,000 to 20,000 cases of TBEV infection are reported per year, study of the immune response to POWV is additionally complicated by the paucity of confirmed cases. Here I report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals, and to POWV in a cohort of infected individuals. Memory B cells expressing anti- TBEV envelope domain-III (EDIII) antibodies were isolated using a combination of single-cell sorting and nested polymerase chain reaction (PCR). Expanded clones of memory B cells expressing closely related anti-EDIII antibodies were found in both groups of volunteers. Antibodies representative of clonal and unique sequences found in TBEV-infected and -vaccinated donors were expressed as monoclonals and characterized functionally. In reporter virus particle (RVP) neutralization assays, the most potently neutralizing antibodies with IC50s below 1ng/mL were found only in convalescent donors. Neutralization assays using authentic TBEV in vitro confirmed potent neutralization activity in at least the four antibodies tested. Select antibodies also neutralized RVPs representative of other emerging tick-borne flaviviruses including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV. Shifting focus to the ongoing SARS-CoV-2 pandemic, this virus continues to be a global problem in part because of the emergence of variants of concern that evade neutralization by antibodies elicited by Wuhan-Hu-1 infection or vaccination. I report on the human neutralizing antibody and memory responses to the Gamma variant in a cohort of hospitalized individuals from Manaus, Brazil. Plasma from infected individuals potently neutralized viruses pseudotyped with the Gamma SARS-CoV-2 spike protein, but neutralizing activity against Wuhan-Hu-1, Beta, Delta, or Omicron was significantly lower. Memory B cells expressing anti-Gamma receptor binding domain (RBD) antibodies were again isolated using a combination of single-cell sorting and nested polymerase chain reaction (PCR). Monoclonal antibodies representative of clonal and unique antibody sequences were expressed and characterized functionally. In an in vitro neutralization assay using pseudovirus, antibodies from Gamma-infected individuals were shown to neutralize Gamma and Beta pseudoviruses more effectively than Wuhan-Hu-1. Sixty-nine % and 34% of Gamma-neutralizing antibodies failed to neutralize Delta or Wuhan-Hu-1. In contrast to class 1 and 2 antibodies that dominate the response to Wuhan-Hu-1 or Beta, 43% of antibodies elicited by Gamma infection recognized Class 3 epitopes. These results have implications for variant-specific vaccines, suggesting that these strategies would have limited efficacy. In the course of characterization, specific antibodies with unique properties were identified. In the final part of this thesis, I return to these antibodies to investigate them in more detail. They include antibody T025, which was shown to be efficacious at preventing disease in an in vivo model of infection. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Antibody T036, also from a TBEV-infected donor, was shown separately to enhance RVP and authentic virus infection in vitro in a Fc receptor-independent manner. Enhancement could be ablated by addition of 4G2, a mouse anti-fusion loop monoclonal antibody. T036 was shown to bind a cryptic epitope on the TBEV EDIII, suggesting that T036 binds the virion during or after conformational change of the E protein. Separately, Z039 and Z015 had been shown in previous studies of anti-ZIKV EDIII antibodies in ZIKV-exposed cohorts to exhibit unexpectedly broad cross-reactivity. Z039 and Z015 were confirmed to bind ZIKV EDIII, as well as Dengue virus 1-4 EDIIIs, and yellow fever virus and West Nile virus EDIIIs, respectively. in vitro neutralization assays using authentic virus confirmed corresponding patterns of antibody-mediated neutralization. Z039 was shown to recognize an epitope on the flavivirus EDIII similar to that of Z021, an anti-Zika antibody with comparatively limited cross-reactivity, although Z039 appears to make contacts with the EDIII backbone which may partially explain its breadth of activity. Z015, on the other hand, does not compete with Z021 or Z004 and recognizes a distinct but cryptic epitope on the yellow fever virus EDIII.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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