Degree Type

Thesis

Date of Award

2009

Degree Name

Master of Science

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Marcus E. Kehrli

Second Advisor

James A. Roth

Abstract

Current swine influenza vaccines fail to protect against the broad range of strains circulating within the United States. To induce effective broad cross-protection against influenza, strong humoral and cell-mediated immune responses are needed. However, animals are often vaccinated at a young age when they exhibit a host of dysregulated or insufficient immune responses including biased T cell polarization and immature inflammatory responses. Porcine neonates have diminished expression of IL-18 in their respiratory tract, a major site of infection in young animals. Initially named interferon-g inducing factor (IGIF), IL-18 is most widely known for its potent ability to up-regulate expression of interferon-g (IFN-g), resulting in a phenotypic TH1 driven T cell polarization. For many infections, such as influenza, deficiencies in IL-18 expression have been shown to result in deleterious effects on the development of protective immune responses. Insufficient expression of IL-18 during neonatal development in swine may result in a reduced expansion of a TH1 immune response and may result in improper immunological polarization in response to vaccination. Based on findings by Kim et al. (2001), two amino acid conversions were introduced (E41A & K88A) to porcine IL-18 by PCR primer site directed mutagenesis. Synonymous mutations to human IL-18 (huIL-18) by Kim et al. (2001, 2002) resulted in 4-fold increases in biological activity and extended half-life. Replication-defective adenoviruses expressing either the wild type porcine IL-18 or the mutated form was generated. Additionally, to create a succinct swine influenza modified live virus (MLV) vaccine expression rIL-18 protein, the mutated form of IL-18 (mutIL-18) was inserted into the truncated region of the NS1126 gene of an attenuated H3N2 modified live swine influenza virus. The properties of biological expression and biological activity of both wild type IL-18 and mutated IL-18 were assessed in vitro and in vivo. Replication-defective adenovirus expression vectors were confirmed to express high levels (>10ng/mL) of wild type and mutated forms of IL-18. The rIL-18 expressing constructs (MLV+mutIL-18, MLV+Ad5wtIL-18, and MLV+Ad5mutIL-18) were administered as single dose influenza vaccines. The immunomodulatory effects of wild type and mutated forms of IL-18 were evaluated for their efficacy to enhance the immune protection afforded by the MLV vaccine alone in response to heterosubtypic influenza challenge (H1N1). The MLV+Ad5mutIL-18 vaccination group resulted in significantly higher antibody titers post challenge compared to the MLV alone. Additionally, both groups receiving either mutIL-18 construct (MLV+mutIL-18, MLV+Ad5mutIL-18) exhibited significantly lower viral shedding post challenge, lower viral replication in the lungs, and reduced microscopic lung lesion scores at time of necropsy. Flow cytometric analysis of circulating lymphocyte populations revealed significant differences in cell populations between IL-18/non-IL-18 groups and adenovirus and non-adenovirus groups. CD4+CD8+ double positive (DP) and gamma/delta T cells were the main cell populations activated upon influenza vaccination and heterosubtypic challenge. Results indicate that, along with rIL-18 administration, the type of expression vector (adenovirus or influenza) plays a significant role in determining cytokine expression and responding T cell populations

Copyright Owner

Matthew Allan Kappes

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

182 pages

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