NYMC Student Theses and Dissertations

Date of Award

5-22-2019

Document Type

Doctoral Dissertation - Open Access

Degree Name

Doctor of Philosophy

Department

Microbiology and Immunology

First Advisor

Doris Bucher

Abstract

Seasonal epidemics caused by influenza A viruses (IAV) result in an estimated 290,000- 650,000 deaths worldwide each year (17). While antivirals targeted to influenza exist, resistance to these drugs is increasing and regular vaccination remains the most effective way to prevent infection (26, 73, 99). However due to the persistence of antigenic drift and shift, influenza vaccines must be updated each season and antigenic mismatches can reduce efficacy (24, 118). Immunity to influenza either from vaccination or infection is principally mediated by antibodies generated to one of its major surface proteins, Hemagglutinin (HA). HA is a homotrimer, each monomer HA0 is composed of a globular head and stem (100, 111). Proteases present in the host cleave native HA0 into two subunits, HA1 and HA2. Residues in HA1 form the receptor binding pocket within the head and facilitate interaction with the host cell. The stem is encoded by both HA1 and HA2 and contains the fusion peptide required for cytosolic release (10, 112). Most antibodies isolated from patients following infection or vaccination are targeted to the receptor binding domain of the HA head. These antibodies are efficient neutralizers but highly strain specific, recognizing only antigenically similar strains of the same IAV group and consequently lose relevance rapidly as viruses undergo drift (68, 141). However, patients immunized with heterosubtypic HA can also develop antibodies that target the HA stem domain. These antibodies are often effective against multiple HA subtypes, reflecting strong structural constraints imposed on the HA stem epitope, and are referred to as “broadly neutralizing”. It is because of this strong conservation that there has been an interest in characterizing HA stem antibodies in the development of a universal flu vaccine (22, 115, 123). Since their discovery in 1991, dozens xi of broadly neutralizing stem antibodies have been isolated in humans and even more have been derived using targeted technologies, including hybridoma cell generation (69). We used two immunization protocols that each featured successive injection of influenza with antigenically distinct HA components, H1 then H3, followed by hybridoma cell development to generate candidate monoclonal antibodies (mAbs) targeted to the IAV surface glycoproteins. We screened antibody containing hybridoma supernatant for activity against influenza proteins using ELISA, Neuraminidase Inhibition (NI), and Hemagglutinin Inhibition (HAI) assays. Those antibodies which showed appreciable activity against the immunizing viruses from hybridoma generation were purified for further study. Of 114 antibodies, mAb 1G3 (IgG1) was found by Western blotting to successfully bind the HA protein of H1, H1pdm, and H3 influenza A viruses as well as influenza B viruses of both major lineages (Yamagata and Victoria). Significantly, mAb 1G3 was able to successfully neutralize both influenza A and B viruses in vitro as shown by plaque assay and indirect immunofluorescence of infected cells. 1G3 was also shown to be effective against H1, H3, and an influenza B virus in ovo, delaying onset of positive viral titer and preventing viral expansion overall. Although we believe mAb 1G3 to have a conformational epitope, linear epitope mapping revealed that 1G3 likely binds in the stalk domain near to the fusion peptide and across the C terminus of HA1 and N terminus of HA2. We found this locus to be strongly conserved among heterosubtypic influenza strains, including influenza B. Importantly, only one other antibody known as CR9114 has been identified that can neutralize both influenza A and B viruses in vivo (32). CR9114’s epitope appears to have significant overlap with the proposed epitope of our mAb 1G3, although they are not identical. Where CR9114 xii displayed no activity against B viruses in vitro, 1G3 is the only antibody to date that inhibits both A and B viruses in cells. This previously unseen neutralization profile may suggest the possible presence of an additional inhibition mechanism or mechanisms active in cell culture. While CR9114 prevents the fusogenic conformational change of HA within the host endosome and incites Antibody Dependent Cellular Cytotoxicity (ADCC) in vivo, we have demonstrated through Western blotting and neuraminidase inhibition that our mAb 1G3 likely also prevents extracellular HA cleavage and interferes with virion budding (11, 29, 122). Therefore, mAb 1G3 is a unique and previously uncharacterized antibody that is broadly neutralizing against Group 1 and Group 2 influenza A as well as both lineages of influenza B.

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