Akbari, Omid; (1999) Analysis of The Basis for Induction and Maintenance of T Cell Responses in DNA Vaccination. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This study is aimed at finding the role of dendritic cells in induction and maintenance of immune responses following DNA vaccination. For this purpose, two DNA vaccines were generated, one expressing full C5 cDNA (fifth complement of mice) and the other expressing just 1.6 kb of the 5' end of cDNA. Both vaccines express the epitope for recognition of the A18 T cell hybridoma and both induced C5 specific immune responses following i.m. inoculation of A/J mice. Three routes of entry i.e., (i.m.), abrasion (Scarification) of leg skin and abrasion of ear skin were chosen for inoculation with the DNA vaccine encoding the full C5 protein. Deep cervical lymph nodes were found to be the best targets following DNA inoculation of the ear. Kinetic analysis of these lymph nodes showed the highest T cell immune response 10 to 12 days following DNA vaccination. While all routes of vaccination led to C5 specific T cell responses with time, an early response was detected in cervical lymph nodes following vaccination in the ear. Isolation of dendritic cells from cervical lymph nodes following DNA vaccination allowed detection of C5 expression. C5 was actively expressed in a DC enriched population while the other cells in lymph node showed no expression. That suggested direct transfection of DCs following DNA vaccination. However, while DNA vaccination resulted in transfection of a small proportion of dendritic cells only, it led to general activation of all dendritic cells, providing optimal conditions for effective T cell activation. The sites, kinetics and extent of T cell activation following DNA vaccination was investigated in a transgenic model. It was demonstrated that T cell activation is initiated in the cervical draining lymph nodes and persisted for longer than 40 weeks although antigen expression was not demonstrable more than 12 weeks for keratinocytes and 2 weeks for dendritic cells. Crosspriming of dendritic cells by C5 expressed in keratinocytes did not occur unless keratinocyte death was induced by irradiation. About 2% of dendritic cells present in the draining lymph nodes were estimated to be transfected, both by confocal analysis of GFP expression and by functional titration assay. Notably, the construct used for the series of experiment described so far was shown to result in intra-cellular expression in mammalian cells, but not in secretion. A secretory C5 construct was constructed and it was shown that C5 specific antibodies have been detected only in the case of the secretory construct while the other constructs were unable to generate any antibodies.

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