Date of Award

12-31-2023

Document Type

Doctoral Dissertation - Open Access

Degree Name

Doctor of Philosophy

Department

Basic Medical Sciences

First Advisor

Doris Bucher

Second Advisor

Christopher Whitehurst

Third Advisor

Tetyana Cheairs

Abstract

There is a continual public threat of influenza virus infections. This is related to viruses evolving in multiple animal reservoirs, making infection prevention in humans difficult to achieve and leading to seasonal epidemics and the semi-regular emergence of pandemics. Novel antibody-based therapies are of great interest to researchers because of a dramatic increase in drug resistant strains of influenza viruses. The identification of cross-reactive and broadly protective monoclonal antibodies targeting the influenza proteins presents a promising prospect for the development of a universal therapy. Although the research related to the monoclonal antibodies targeting the viral internal proteins is limited, multiple epitopes for broad protection have been identified in the stem and head domains of the viral hemagglutinin, neuraminidase and matrix protein 2 external domain. The hemagglutinin stem-based approaches have shown promising results of broader protection in animal studies, and their feasibility in humans is being evaluated in clinical trials. This research focuses on mouse monoclonal antibodies produced via the hybridoma technology that target the most abundant internal protein matrix 1 sharing the same 9 amino acid residues at the carboxyl terminal as the surface ectodomain of the matrix 2 protein of influenza A virus, and the surface exposed proteins hemagglutinin and neuraminidase of the influenza A and influenza B viruses. We have identified the binding spectrum and functional characteristics of four novel anti-influenza monoclonal antibodies. The novel monoclonal antibody 2B-B10-G9 targeting the matrix 1 of A/Puerto Rico/8/1934 virus showed heterosubtypic binding to the H1N1, H1N1pandemic, and H3N2 currently co-circulating subtypes of influenza A virus at 30 kDa region in Western blots that were quantified by image analysis with the Image J algorithm. The other three novel monoclonal antibodies 7-1E7-1G3, 28-1D6-1E4, and 28-1B12-1G4 showed a broad binding spectrum to the hemagglutinin xiv glycoproteins of co-circulating subtypes H1N1, H1N1 pandemic and H3N2 of influenza A viruses and the prototype B/Lee/1940 and current circulating Victoria lineage of influenza B viruses between 60-80 kDa, region detected with the same method. None of the novel monoclonal antibodies showed inhibition of hemagglutination of chicken erythrocytes with the A/Puerto Rico/8/1934 virus suggesting that they recognized the conserved epitopes distal to the receptor binding site of head domain of the hemagglutinin glycoprotein. Epitope mapping, using small molecular weight synthetic peptides representing the conserved epitopes of the head and stem domains of hemagglutinin protein of A/Puerto Rico/8/1934 via tricine-SDS-PAGE and western botting, revealed no binding signals for the novel monoclonal antibodies 7-1E7-1G3, 28- 1D6-1E4, and 28-1B12-1G4 targeting the surface glycoproteins hemagglutinin and neuraminidase of A/Puerto Rico/8/1934 and B/Lee/1940. They all demonstrated in vitro neutralization of A/Puerto Rico/ 8/1934 in the plaque reduction neutralization assay which was most likely related to inhibition of the fusion protein function via the stem-binding characteristics of antibodies which is further supported by their broad spectrum of binding, and lack of hemagglutination inhibition. In vitro low-dose quadruple combination therapy at 20 µg/ml (5 µg/ml of each monoclonal antibody) showed significant reduction of the plaque forming units with respect to the low-dose mono, dual, and triple therapies. All the antibodies at the dose of 150 µg/egg (15 µg/ml) suppressed in ovo expressions of the hemagglutinin, neuraminidase, and polymerase basic protein 1 genes of A/Puerto Rico /8/1934 with respect to the isotype control. 28-1D6-1E4 showed most significant suppression of the hemagglutinin, neuraminidase, and polymerase basic protein 1 genes of A/Puerto Rico/8/1934 with respect to the isotype treatment control at the same dose. The pooled in ovo treatment data of the antibodies showed the most significant suppression of the polymerase basic protein 1 genes of A/Puerto xv Rico /8/1934 with respect to the pooled data of isotype treatment controls. The quadruple combination therapy with the novel broadly neutralizing anti-influenza monoclonal antibodies targeting both the surface exposed glycoproteins hemagglutinin, neuraminidase, and the most abundant internal protein matrix 1 sharing the common amino acid residues as the ectodomain of matrix 2 protein were considered to have potential for use in the prophylaxis and treatment of influenza infections.

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