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The activation of cellular immunity by DNA vaccination
Cellular immunity refers to the activation of both CD4+ T helper cells upon recognition of antigen presented on MHC class II molecules and CD8+ T cells (or CTL) that are activated by MHC class I presentation. All cell types present samples of self antigen (endogenous antigen) on MHC class I. Cells that present antigen that is not foreign to the CTL will be tolerated, but if antigen of unknown origin is presented to the CTL, it will destroy the infected host cell by apoptosis (reviewed by Van den Eynde and Morel, 2001). The unknown antigen normally originates from oncogenic genes, viral infection or intracellular bacteria (reviewed by Lehner and Cresswell, 1996; Whitton et al., 1999 and by Melief, 2003).
The expression of proteins from plasmid DNA in host cells is regarded to be equivalent to protein expression mediated by viral DNA or RNA. Therefore DNA vaccination could activate CTL mediated immunity through internal MHC class I antigen presentation (Corr et al., 1996). In order to activate these cells, antigen derived from the plasmid DNA must first be introduced into the MHC class I antigenpresentation pathway via the ubiquitination of proteins. Ubiquitination directs proteins to a proteosome complex where peptide fragments are generated (reviewed by Rechsteiner et al., 2000; Van den Eynde and Morel, 2001; Weissman, 2001 and by Yewdell and Bennink, 2001). These fragments are then transported to the endoplasmic reticulum where they associate with MHC class I molecules (reviewed by Hassett and Whitton, 1996 and by Yewdell and Bennink, 2001). Using an expression vector that contains the ubiquitin sequence will result in fusion proteins that are covalently linked to ubiquitin, consequently favouring a cytotoxic T cell response (Sykes and Johnston, 1999). Similarly heat shock protein 70 (HSP70) can also be used to direct recombinant protein towards the proteosome (Qazi et al., 2005; Li et al., 2006).
The activation of a memory CTL response by repeated boosting with plasmid DNA is not always successful. Therefore alternative vaccination strategies have been developed to improve the CTL response. So far the best results have been obtained by using a prime-boost immunisation strategy. This is achieved by priming the immune response using DNA vaccination, followed by either a modified viral or recombinant protein boost. Protection against several diseases that are controlled primarily by a cellular immune response has been demonstrated using this strategy (Hammond et al., 2001; Gilbert et al., 2002; Gonzalo et al., 2002; Moorthy et al., 2003; Stambas et al., 2005).
The role of CD4+ cells in the development of CTL responses still has to be elucidated. On the one hand, some scientists believe that CD4+ T cell help is required for the activation of both effector and memory CTL (mCTL) (Bennett et al., 1997; López-Días de Cerio et al., 1999; Behrens et al., 2004 and reviewed by Keene and Forman, 1982 and by Heath and Carbone, 1999). CD4+ and CD8+ specific epitopes derived from the one antigen must be presented on the same APC. This will facilitate interaction between CD4+ T cells and CD8+ T cells, thus the CD4+ T cells provide direct help.
1. CHAPTER 1: LITERATURE REVIEW
1.1. INTRODUCTION.
1.2. OBJECTIVES OF THIS STUDY.
1.3. REFERENCES
2. CHAPTER 2: DNA VACCINATION
2.1. INTRODUCTION.
2.2. MATERIALS AND METHODS
2.3. RESULTS.
2.4. DISCUSSION
2.5. REFERENCES
3. CHAPTER 3: HETEROLOGOUS NEEDLE AND NATURAL TICK CHALLENGE.
3.1. INTRODUCTION
3.2. MATERIALS AND METHODS
3.3. RESULTS.
3.3.1. Sequence analysis of selected 1H12 ORFs from different E. ruminantium isolates.
3.3.2. 1H12 vaccine tested against heterologous E. ruminantium challenge.
3.3.3 Field challenge on the farm Springbokfontein
3.4. DISCUSSION
3.5. REFERENCES
4. CHAPTER 4: HETEROLOGOUS PRIME-BOOST IMMUNISATION
4.1. Introduction
4.2. Materials And Methods
4.2.1. Materials.
4.2.2. Method8
4.3. RESULTS.
4.3.1. Preparation of recombinant LSDV.
4.3.2. Expression of the 1H12 recombinant proteins
4.3.3. Immunisation of sheep using the prime-boost strategy..
4.3.4. Prime-boost immunisation of sheep using the i.m. method of DNA inoculation
only.
4.3.4. Field challenge of sheep immunised using the prime-boost immunisation
strategy compared to DNA immunisation
4.5. REFERENCES
5. CHAPTER 5: CONCLUDING DISCUSSION.
5.1. REFERENCES
6 .APPENDIX A: MATERIALS, BUFFERS, MEDIA AND SOLUTIONS
7. APPENDIX B: SEQUENCE ALIGNMENTS.
8. APPENDIX C: PUBLICATIONS AND ETHICS
