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Document Type

Dissertation - University Access Only

Award Date

2016

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

First Advisor

Radhey Shyam Kaushik

Abstract

Influenza viruses infect the epithelial cells of swine respiratory tract. We characterized MK1-OSU cells, a clonally derived cell line from tracheal and bronchial epithelial cells of 5 week old piglet.We also found that these cells were susceptible to Influenza A, B, and C virus infection. Hence, this cell line could serve as a model for studying the innate immune responses and pathogenesis of various influenza viruses. We also determined the innate immune responses of these cells against influenza A and B viruses. These responses were compared to that of SD-PJEC cells, which was derived from swine intestinal epithelium.TLR7, RIG1, and MDA5 protein expressions of MK1-OSU cells 24h post-infection with Influenza A strains decreased compared to non-infected control cells. Protein expression for TLR7 in SD-PJEC cells infected with Swine/MN/08 H1N1 (MN08) showed a similar decrease in expression, while.RIG1 and MDA5 protein expressions was not affected by infection in SDPJEC cells.Better understanding of differences in immune response of these cell lines could provide further insight to higher susceptibility of respiratory tract epithelium to influenza infection compared to digestive tract. In addition to studying innate immune response against influenza, we also investigated various approaches to develop vaccines against swine influenza. We conducted three different experiments using pigs as model. The first study was aimed to evaluate the protective efficacy of an intranasal Poly I:C adjuvanted UV inactivated bivalent swine influenza vaccine consisting of Swine/OH/24366/07 H1N1 and Swine/CO/99 H3N2, referred as PAV, in maternal antibody positive pigs against an antigenic variant and a heterologous swine influenza virus challenge. Groups of three-week-old commercial-grade pigs were immunized intranasally with PAV or a commercial vaccine (CV) twice at 2 weeks intervals. Three weeks after the second immunization, pigs were challenged with the antigenic variant MN08 and the heterologous Swine/NC/10 H1N2 (NC10) influenza virus. Intranasal administration of PAV induced challenge viruses specific IgG antibodies in the serum and IgA and IgG antibodies in the respiratory tract. Importantly, intranasal administration of PAV provided protection against the antigenic variant MN08 and the heterologous NC10 swine influenza viruses. These results indicated that Poly I:C or its homologues may be effective as vaccine adjuvants capable of generating cross-protective immunity against antigenic variants/heterologous swine influenza viruses in pigs. The second experiment was aimed to develop gnotobiotic pig model for influenza vaccine studies and also evaluate a universal peptide vaccine against Influenza. We used conserved epitopes of Influenza A as vaccine. Vaccines were administered intranasally to 10 d old gnotobiotic piglets along with heat labile toxin double mutant (dmLT) of Escherichia coli as adjuvant. A booster dose was administered intranasally 2 wk after initial vaccination. Mock administered piglets received only the adjuvant. Three weeks later, all pigs were infected with MN08 and were euthanized on 5d after virus challenge. All the gnotobiotic piglets developed macroscopic lung lesions following virus challenge. Vaccination using epitopes failed to induce antibody production against the HA epitopes. Also T cell epitopes used in the study did not offer protection against influenza, but we successfully developed gnotobiotic pig models for influenza vaccine studies. The objective of third experiment was to develop universal influenza vaccine by using highly conserved extracellular domain of Matrix protein 2 (M2e) of influenza virus as candidate. Four repeats of M2e gene were inserted into recombinant adenovector (Adeno-M2e) that could transiently express the protein. Twenty-five gnotobiotic pigs were divided in to 5 groups: 1) Mock-vaccination group receiveddmLT as adjuvant and recombinant adenovector containing the lac-Z gene 2) Vaccinated with dmLT and Adeno-M2e and challenged with MN08 3) Vaccinated with dmLT and Adeno-M2e and challenged with swine/IA//2007 H3N2 (IA07)4) Challenged with MN08 and 5) Challenged with IA07. Vaccines were delivered intranasally on 7 d and 28 d. Animals were challenged with SIV on 42 d and euthanized on 5 d post-challenge. Animals vaccinated with Adeno-M2e were protected against challenge withMN08.However, vaccine did not offer any protection against IA07. We also quantified the mRNA expression of various inflammatory genes in lungs of these animals using qRT-PCR. Vaccine protected group of animals had significantly higher expression of Interferons α and β, Interleukins 1α and 1β, and Interleukin 6 in lungs compared to mock-vaccinated animals. These results suggested that Adeno-M2e vaccine elicited immune response and protection against MN08 infection but failed to yield protection against IA07 infection.

Library of Congress Subject Headings

Swine influenza
Swine -- Virus diseases
Immune response
Influenza vaccines

Description

Includes bibliographical references

Format

application/pdf

Number of Pages

170

Publisher

South Dakota State University

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Rights Statement

In Copyright