MURINE MODELS TO STUDY IMMUNITY AND IMMUNISATION AGAINST RESPIRATORY VIRAL PATHOGENS

Abstract

This thesis investigates two key concepts (vaccination and elicitation of host immune responses) in prevention of respiratory virus infections, focusing on SARS coronavirus (SARS-CoV) and influenza virus (InfV). A novel approach to increasing DNA vaccine efficacy was investigated. The integrin-binding motif, Arg-Gly-Asp (RGD) was incorporated into a mammalian expression vector expressing codon-optimized extra-cellular domain of SARS-CoV spike protein, and tested by immunizing C57BL/6 mice. Immune responses were characterized by 51Cr-release assay and IFN-gamma secretion ELISPOT assay against RMA/S target lines presenting predicted MHC class I H2-Kb epitopes (including residues 884-891 and 116-1123 within the S2 subunit). Immunization with the Spike-RGD/His DNA vaccine generated robust cell-mediated immune responses. The role of interferon regulatory factor-4 (IRF-4) as a direct transducer of influenza virus-mediated signaling is unclear. IRF-4 -/- mice in C57BL/6 background infected with a lethal dose of InfV A/Puerto Rico/8/1934 H1N1 had the highest lung viral titres and were unable to mount a detectable antibody response in virus neutralization test. Lung homogenates revealed a dysregulation of pro-inflammatory cytokines (IL-1ß and IL-6 levels: suppressed; GM-CSF and TNF-a levels: augmented). Th-1 cytokines (IL-2, IFN-¿) levels decreased, whereas Th2 cytokines (IL-4, IL-10) were increased. Chemokine CXCL1 indicated a dysfunction in neutrophil attractant activity. IFN-a and -ß mRNA levels remained unchanged. However, IFN-¿ mRNA levels were undetectable due to dysfunctional T lymphocytes. Microarray analysis revealed that besides being involved in signaling pathways of both innate and adaptive immune responses, the IRF-4 gene also played multiple roles in other previously unknown pathways. The absence of IRF-4 which has critical function in the development of lymphoid and myeloid cells appears to be detrimental to InfV-infected mice, resulting in the failure to arm the mice with the necessary protective immunity to mount an efficient adaptive immune response.