Document Type

Dissertation - Open Access

Award Date

2020

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

First Advisor

Feng Li

Keywords

Antibody-dependent cellular cytotoxicity (ADCC), Hemagglutinin (HA), Human antibody, Influenza, Transchromosomic cattle

Abstract

Influenza remains a global health risk and challenge. Currently, NA inhibitors are extensively used to treat influenza, but their efficacy is compromised by the emergence of drug resistant variants. Antibodies targeting influenza A virus surface glycoproteins are critical components of influenza therapeutic agents and may provide alternative strategies to the existing countermeasures. This study characterized both polyclonal and monoclonal antibodies produced from the transchromosomic (Tc) cattle platform. A polyclonal antibody, designated SAB-100, was generated from Tc cattle after immunized with H1N1,H3N2, and influenza B virus. The peptide-based Enzyme-linked immunosorbent assay shows that the polyclonal antibody recognizes three distinct epitopes, one out of three is located in HA2 and is highly conserved among different subtypes of HAs. This work shows the antigenic moieties of influenza HA, which may assist the design and development of vaccines and therapeutic agents. Next, one human monoclonal antibody (mAb), 53C10, also generated from the Tc cattle platform was characterized. This mAb was capable of neutralizing diverse clades of the H1 subtype. In vitro selection of antibody escape mutants reveals that 53C10 recognizes a novel non-continuous epitope that overlaps with the receptor binding site (RBS) and the introduction of three substitutions in hemagglutinin (HA) can completely abrogate antibody recognition. Further characterization showed that two substitutions in the escape mutant may not affect antibody binding but may serve as compensatory substitutions. Numerous studies confirmed the protective role of neutralizing antibodies. Contrarily, non-neutralizing antibodies and their therapeutic potential are less well defined. Our enzyme-linked immunosorbent assay (ELISA), western blot and flow cytometry assays demonstrated that one human mAb isolated from Tc cattle recognizes diverse influenza A HAs from subtype H1. Despite the broad binding to H1 HAs, 38C2 mAb demonstrated no detectable neutralizing activities against H1N1 virus in vitro. Further investigation of 38C2’s mechanism of action revealed that this monoclonal antibody can induce potent antibody-dependent cellular cytotoxicity (ADCC) in vitro. There is a correlation between the binding affinity of 38C2 to HA and the potency of ADCC induced by 38C2. Detailed epitope mapping and structural modeling demonstrate that 38C2 binding to a linear epitope which located in the HA trimer interface. Conservative analysis show that the epitope recognized by 38C2 is highly conserved in H1 HA. In summary, the results of our neutralizing antibody may indicate the potential role of 53C10 in the treatment of H1 influenza virus infection in humans. Our non-neutralizing antibody characterization emphasizes the crucial role of Fc-effector functions and may provide a plausible way to develop a universal influenza vaccine.

Library of Congress Subject Headings

Immunoglobulins.
Influenza viruses.
Cattle -- Physiology.
Antibody-dependent cell cytotoxicity.
Immunology.
Antigenic determinants.

Format

application/pdf

Number of Pages

132

Publisher

South Dakota State University

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Included in

Microbiology Commons

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Rights Statement

In Copyright