DNA immunization, inoculation with an antigen-expressing plasmid DNA, is a new method for generating an antigen-specific immune response. At the time these investigations began, very little was known about the immune response produced by DNA vaccines. Thus, the first aim of our studies was to perform a detailed examination of the antibody response generated by DNA immunization with an influenza hemagglutinin (HA)-expressing DNA in BALB/c mice. Using several different routes and methods of DNA immunization, we observed a number of findings. Although all three forms of DNA immunization elicited strong anti-HA antibody responses, i.m. and i.d. saline DNA immunization required approximately 100 times more DNA than a gene gun DNA immunization to raise an equivalent titer of anti-HA antibody. Indeed, as little as one inoculation and one boost by gene gun of 0.0004 μg of DNA produced a measurable antibody response in 50% of mice. Unexpectedly, we found the isotype of the antibody response differed among groups of mice immunized by different forms of DNA immunization. Intramuscular and i.d. saline DNA immunization produced predominantly an IgG2a anti-HA antibody response, whereas gene gun DNA immunization elicited mostly an IgG1 anti-HA antibody response. Considering that IgG2a and IgG1 antibody isotypes were known to correlate with Th1 and Th2 immune responses, respectively, we analyzed the type of immune responses produced by i.m., i.d., and gene gun DNA immunization. We found that i.m. and i.d. saline DNA immunization produced a Th1 predominant cellular immune response. In contrast, gene gun DNA immunization produced a Th2 cellular immune response. The differences in the type of immune responses were found to be due to the method of DNA immunization, and not due to the route of DNA inoculation. A gene gun DNA immunization of muscle produced the same IgG1, Th2 immune response as a gene gun DNA immunization of skin, while a saline DNA immunization of muscle and skin produced mostly an IgG2a, Th1 immune response. Each method of DNA immunization created good memory Th cell responses. The type of immune response created by an initial DNA immunization remained fixed even after multiple boosts with the identical method of DNA immunization, following a boost with the alternative method of DNA immunization, or after a viral challenge. The differentiation of naive Th cells into Th1 or Th2 cells depends on a variety of factors. We performed many experiments to elucidate which factors played a role in the generation of Th1 or Th2 immune responses following saline DNA immunization and gene gun DNA immunization. DNA dose response studies revealed the use of different doses of DNA between groups of saline DNA and gene gun DNA immunized mice did not account for the differentiation of distinct Th cell subsets. Cytokine production inducible by a number of factors inherently associated with either saline DNA or gene gun DNA immunization did not affect Th differentiation. For instance, contamination of plasmid DNA with lipopolysaccharide did not account for differences in the immune response. Immunostimulatory CpG sequences did not affect Th differentiation following DNA immunization, but they did enhance the IgG2a antibody response to coinoculated HA protein. Finally, cotransfection of IFNγ or IL-4 expressing plasmids with an HA-expressing plasmid by gene gun inoculation or as a saline DNA injection did not shift the type of immune response in a Th1 or Th2 direction, respectively. Thus, it appeared that increased cytokine stimulation was not responsible for selective Th subset differentiation. One factor related to the method of DNA immunization did seem to correlate with Th1 differentiation. Deposition of plasmid DNA extracellulary by saline DNA injections (as opposed to intracellular DNA delivery by gene gun) may have stimulated Th1 immune responses. Manipulating a gene gun DNA immunization to deliver DNA to the dermis (and thus extracellularly) shifted the immune response from that of a Th2 type to a mixed Th1/Th2 type. Furthermore, evidence was gathered demonstrating that pDNA can interact with cell surface molecules and that specific sequences in pDNA can act as a ligand and bind to molecules. Taken together, our data led us to propose a new model for Th1 differentiation following saline DNA immunization. We believe extracellular pDNA binds to an APC cell surface molecule which activates the cell. The activated APC preferentially stimulates naive Th cells to differentiate into Th1 cells. Finally, studies using a variety of mice differing in their genetic backgound and MHC genotype demonstrated the generality of our findings regarding i.m. saline DNA inoculations of an HA-expressing pDNA. Saline DNA immunization produced IgG2a, Th1-predominant immune responses independent of the genetic background and MHC genotype of the mice. In contrast, the type of immune response elicited by a gene gun DNA immunization was dependent on the MHC genotype of mice. Thus the type of immune response produced by gene gun DNA immunization probably depends on the specific antigen (and its effect on MHC-peptide/TcR interaction and signaling) and is less likely due to any inherent feature associated with the process of gene gun DNA delivery.

Yung Chung Ming Edmund. / Thesis submitted in: September 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 44-54). / Abstracts in English and Chinese. / Title Page / Contents / Abstract / 摘要 / List of Figures and tables / Abbreviations / Text / References / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- An overview to the study of CTLA-4 gene in childhood Graves' disease (GD) / Chapter 1.1.1 --- "Graves' disease 226}0ؤ features, incidence, aetiology and pathogenesis page" / Chapter 1.1.1.1 --- GD features 226}0ؤ from clinical to laboratory --- p.1 / Chapter 1.1.1.2 --- GD incidence - from adult to children --- p.2 / Chapter 1.1.1.3 --- GD aetiology - from environment to genes --- p.3 / Chapter 1.1.1.4 --- GD pathogenesis - from auto-antigen to autoantibody --- p.4 / Chapter 1.1.2 --- CTLA-4 gene study in Graves' disease --- p.5 / Chapter 1.1.3 --- Conclusion --- p.6 / Chapter 1.2 --- "Objectives, hypothesis and planning of the study" --- p.7 / Chapter 1.2.1 --- Objectives --- p.7 / Chapter 1.2.2 --- Hypothesis --- p.7 / Chapter 1.2.3 --- Planning --- p.7 / Chapter Chapter 2: --- Literature Review --- p.8 / Chapter 2.1 --- The CD28 / CTLA-4: B7 co-stimulatory pathway and Graves' disease --- p.8 / Chapter 2.1.1 --- Overview of co-stimulation and T cell activation --- p.8 / Chapter 2.1.2 --- Overview of the CD28 gene --- p.9 / Chapter 2.1.3 --- Overview of the CTLA-4 gene --- p.10 / Chapter 2.1.4 --- Co-stimulation and Graves' disease --- p.13 / Chapter 2.2 --- The study of CTLA-4 gene polymorphism in Graves' disease --- p.14 / Chapter Chapter 3: --- Methodology --- p.16 / Chapter 3.1 --- Recruitment of subjects --- p.16 / Chapter 3.1.1 --- Recruitment of cases --- p.16 / Chapter 3.1.2 --- Recruitment of controls --- p.16 / Chapter 3.1.3 --- Ethical approval --- p.17 / Chapter 3.2 --- Peripheral blood collection and genomic DNA preparation --- p.17 / Chapter 3.2.1 --- Peripheral blood collection --- p.17 / Chapter 3.2.2 --- White blood cell harvesting --- p.17 / Chapter 3.2.3 --- White blood cell digestion --- p.17 / Chapter 3.2.4 --- DNA extraction --- p.17 / Chapter 3.3 --- Polymerase Chain Reaction (PCR) amplification of CTLA-4 gene exon one --- p.18 / Chapter 3 .4 --- PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of CTLA-4 gene codon 17 A/G dimorphism --- p.19 / Chapter 3.5 --- PCR-Single Strand Conformational Polymorphism (PCR-SSCP) analysis of of CTLA-4 gene codon 17 A/G dimorphism --- p.21 / Chapter 3.5.1 --- Preparation of SSCP gel and buffer --- p.21 / Chapter 3.5.2 --- ´5ةend labelling of forward PCR primer --- p.21 / Chapter 3.5.3 --- Preparation of PCR fragment for SSCP analysis --- p.21 / Chapter 3.5.4 --- SSCP analysis --- p.22 / Chapter 3.5.5 --- Autoradiography --- p.22 / Chapter 3.6 --- Sequence confirmation of the SSCP fragment by PCR cycle sequencing --- p.22 / Chapter 3.6.1 --- Preparation of sequencing template from SSCP fragment --- p.22 / Chapter 3.6.2 --- PCR cycle sequencing --- p.23 / Chapter 3.6.3 --- Preparation of cycle sequencing products for electrophoresis --- p.23 / Chapter 3.6.4 --- Sequencing by capillary electrophoresis (CE) --- p.24 / Chapter 3.7 --- Statistical analysis --- p.24 / Chapter Chapter 4: --- Results and Data Analysis --- p.26 / Chapter 4.1 --- Results --- p.26 / Chapter 4.1.1 --- Demographic data of case and control subjects --- p.26 / Chapter 4.1.2 --- PCR amplification of CTLA-4 gene exon one --- p.26 / Chapter 4.1.3 --- PCR-RFLP analysis of CTLA-4 gene codon 17 A/G dimorphism locus --- p.26 / Chapter 4.1.4 --- PCR-SSCP analysis of CTLA-4 gene codon 17 A/G dimorphism locus --- p.29 / Chapter 4.1.5 --- PCR cycle sequencing of the SSCP fragments --- p.31 / Chapter 4.2 --- Data analysis --- p.32 / Chapter 4.2.1 --- Overview of data --- p.32 / Chapter 4.2.2 --- CTLA-4 exon one polymorphism analysed with respect to sex --- p.32 / Chapter 4.2.3 --- CTLA-4 exon one polymorphism in patients with Graves' disease and controls --- p.34 / Chapter Chapter 5: --- Discussion --- p.36 / Chapter Chapter 6: --- Summary and Conclusions --- p.42