Document Type

Dissertation - Open Access

Award Date

2018

Degree Name

Doctor of Philosophy (PhD)

Department

Biology and Microbiology

First Advisor

Radhey Kaushik

Second Advisor

Feng Li

Abstract

Swine influenza A viruses pose a serious concern for global health and worldwide swine industry considering their evasion from vaccine-induced immune responses. Formulation of effective influenza vaccines can be complicated by both antigenic shift to a different subtype of hemagglutinin (HAs) and antigenic drift within a particular HA subtype. To address these concerns, we employed a systematic approach to determine the antigenic determinants of swine influenza A viruses by focusing on H1 and H3 subtypes. In parallel, we used a cross-disciplinary approach including structural biology and modeling, virology, and immunology to identify swine influenza A virus-derived HA fusion intermediates broadly reactive to antibodies elicited from various strains or subtypes of swine influenza A viruses. Finally, we conducted a swine influenza vaccine study to evaluate the protective efficacy of a newly identified HA fusion intermediate. The vaccine candidate, when administered into pigs, protected swine against infections by swine influenza H1N1 and H3N2 viruses. For the antigenicity study, we performed a detailed analysis of the antigenic determinants of the Hemagglutinin (HA) protein of pdm09 strain (subtype H1N1) by the HA peptide array in Enzyme-linked immunosorbent assay with immune serum from experimental infected pigs by swine influenza A viruses (SIVs). The peptide array contains 139 peptides spanning the HA protein of H1N1pdm09 and peptides are 14- or 15-mers with 11 amino acid overlaps. A panel of swine antisera against SIV H1 clusters α, β, γ, δ-A, δ-B, H1N1pdm09, and H3 cluster was used in this study and SIVs used for swine hyperimmune sera production are representative subtypes and clusters of SIV circulating in North America. The results of these studies identified three immunodominant peptides, one located in the HA1 (amino acids 57-71) and two in the HA2 (amino acids 481-495 and 553-566), reactive to its homologous antisera (H1N1pdm09). Analysis of peptide reactivity with heterologous serum samples revealed that antibody responses to two HA2 peptides (amino acids 481-495 and 553-566) are completely conserved among antisera of all H1 clusters. In addition to H1N1pdm09, HA1 peptide (amino acids 57-71) was reactive to SIV H1 γ antisera Interestingly, none of peptides in HA peptide array of H1N1pdm09 were reactive to SIV H3 antisera, despite that this antisera has a HI antibody titer similar to those of H1 clusters antisera. Structural modeling localized two conserved immunedominant determinants to the membrane proximal external region (MPER) (amino acids 481-495) and the intracytoplasmic tail (amino acids 553-566) of the HA molecule. For the HA fusion intermediate project, through a structure-guided sitespecific mutagenesis targeting HA2 A and CD helical domains, we identified a mutation, named N439G, that attenuated viral replication but the resultant virus was still recognized by antiviral antibodies. The attenuation was mediated probably through the disruption of triple CD helix formation in the viruses. In light of the fact that N439G virus is highly attenuated in that it may prolong the exposure of HA fusion intermediate to host B cells toward antibody production, we next evaluated its vaccination efficacy in pigs. Significantly, pigs receiving N439G immunizations were substantially protected from both swine influenza A H1N1 and H3N2. In summary, the results of our antigenicity study provide an important foundation for further analyzing the immune response against these B cell epitopes residing in the HA protein during natural SIV infection and also provide potential peptide substrates for diagnostic assays and for vaccine strategies. Our universal vaccine candidate identified from this work should pave the way for further studies toward developing a universal swine influenza vaccine and defining a protective mechanism.

Description

Includes bibliographical references (pages 94-104)

Format

application/pdf

Number of Pages

122

Publisher

South Dakota State University

Rights

In Copyright - Educational Use Permitted
http://rightsstatements.org/vocab/InC-EDU/1.0/

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