Cytotoxic T lymphocytes (CTL) have been implicated as important effectors of recovery from viral infections in experimental murine models including influenza. However, while the cellular and soluble requirements for induction of CTL have been studied extensively, areas remain where understanding of the mechanisms and effects of induction are unclear. The work presented in this thesis aimed to provide novel insight into the biology of the CTL response and to demonstrate the practical application of these biological phenomena in vaccine design.
An experimental murine peptide vaccination and viral infectious challenge model was designed using influenza virus as both the biological source from which the peptide vaccines were derived and against which their efficacy was tested. The model allowed the quantitative measurement of a primary CD8+ CTL response whose protective effect could be measured without the confounding effect of a simultaneously induced B cell response. It also allowed the induction of the a CD4+ T helper response. In a lipopeptide form the peptides could be administered via both systemic and mucosal routes.
Chapter 3 describes experiments which aimed to determine whether a concomitant CD4+ T cell response is required for CD8+ CTL induction. Groups of BALB/c mice were vaccinated with formulations containing varying doses and combinations of the minimal influenza peptide epitopes, H-2Kd-restricted nucleoprotein residues 147-155 (NP147-155) and the I-Ed-restricted hemagglutinin light chain residues 166-180 (HA2166-180) emulsified in the protein-free adjuvant Montanide ISA 720. Induction of CTL by NP147-155 occurred over a restricted dose range that was increased by inclusion of HA166-180 mixed or covalently bound to NP147-155. None of the formulations induced a specific antibody response against influenza A Mem/71 which expresses both epitopes. The unexpected observation was made that, depending on dose, inclusion of a class II-restricted peptide either augmented or inhibited the induction of CTL. Assessment of the relationship between CTL induction and viral clearance revealed that the degree of protection against viral challenge correlated to the strength of the primary CTL response.
Chapter 4 describes experiments using a lipopeptide which was assembled to deliver the NP147-155 -HA166-180 peptide construct directly to the respiratory tract, the target organ for influenza infection, or via the conventional subcutaneous route. Subcutaneous and intranasal lipopeptide vaccination induced a strong CTL response in local lymphoid tissue. Subcutaneous lipopeptide vaccination also induced a disseminated response with measurable CTL responses in distant lymphoid tissue and the primary CTL dose response revealed an optimal mid-dose range similar to that described in Chapter 3. Dose-dependent induction of CTL activity and viral clearance after subcutaneous lipopeptide vaccination compared favourably with that induced by the peptide construct emulsified in M720. Paradoxically, although intranasal lipopeptide immunization induced a strong dose-dependent primary CTLp response, it did not protect significantly against live influenza challenge.
Chapter 5 describes experiments which test the hypothesis that the effect of varying the dose of either the MHC class II-restricted HA2166-180 or the MHC class I-restricted NP147-155 was to alter the relative quantities of peptide-specific cytokine responses. Dose responses for peptide specific cytokine gene expression by CD4+ and CD8+ T cells were correlated with CD8+ CTL activity using a quantitative competitive PCR technique. The data are consistent with the hypothesis that the dose-dependant patterns of cytotoxic activity correlate with predominant type 1 cytokine gene expression by CD4+ and CD8+ T cells. They confirm peptide-specific cytokine gene expression by CD4+ T cells can be deviated toward type 1 or type 2 profiles by altering the dose of peptide. They also illustrate that cytokine gene responses of both the CD4+ and CD8+ T cell populations is altered by the inclusion of another peptide in the vaccination model. Finally, they support the hypothesis that the mechanism underlying the CD4+ -mediated dose dependent augmentation or inhibition of CTL activity is a change not simply in the magnitude of the CD4+ cytokine response but in the relative quantities of type 1 and 2 cytokines.
In summary, the data are consistent with the hypothesis that the induction requirement of CTL for T cell help is conditional on the dose of antigen. The dose effect is not linear but consistent with the mechanism of cytokine-mediated immune deviation. The importance of these findings to efficient vaccine design are demonstrated by the close quantitative correlation between CTL induction and viral clearance.