Studies in DNA immunization

This thesis describes investigations and developments of DNA immunization techniques, which were applied in the study of two different pathogens; HIV-1 and Chlamydia pneumoniae. DNA vaccines, i.e. naked plasmid DNA encoding an antigen, represent a novel strategy for generating protective immunity against several infectious diseases. This technique may eventually provide the means to develop safe and efficient vaccines without the need to use infectious agents.

When developing an HIV-1 vaccine, evoking an immune response against early expressed HIV-1 proteins e.g. those that have regulatory function, may be crucial since this would potentially enable elimination of infected cells, before release of new viral particles. Our aim was to investigate the DNA immunization approach in the induction of immune responses against such early HIV-1 proteins. Mice were immunized with plasmid DNA encoding Tat, Rev or Nef respectively, either by intramuscular injection or by epidermal gold particle delivery. The mice developed both humoral and cellular immune responses against all three proteins. IL-2 secretion from T cells in vitro and DTH responses in vivo suggest a Th1-like response. The T cell responses were stronger and longer lasting when plasmids were given epidermally, coated on gold particles.

A vector encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), coinoculated with Nef plasmid, dramatically increased Nef-specific humoral and cellular immune responses. The immuno-stimulatory activity of GM-CSF DNA was locally restricted and was only observed if both plasmids were simultaneously injected at the same site. The T cell response was of Th I type as judged by IL-2 and IFN-[gamma] secretion.

The influence of a secretion signal proceeding the gene encoding the antigen was investigated. Two sets of plasmids were constructed, encoding secreted or intracellular forms of HIV-1 Nef, HIV-1 Tat or C. pneumoniae outer membrane protein (OMP2) proteins respectively. Upon intradermal immunization with such plasmids, dramatically raised antibody titers were obtained, provided that the antigen was exported out from the transfected cell. Further, specific T cell responses were analyzed after Nef-DNA immunization, these were of Th1 -like phenotype regardless of whether the Nef protein was secreted or not.

The DNA vaccine approach was also evaluated in a C pneumoniae mouse model. We investigated the efficacy of DNA vaccination using the heat shock protein (hsp60) gene of C. pneumoniae, for protection of mice against infection with the bacteria. Intranasal but not intradermal administration of plasmids encoding hsp60 (pHSP-60) generated a 5-20 fold reduction of bacteria in lungs of mice, which coincided with diminished severity of disease. Co-inoculation with IFN-[gamma] or IL-12 genes, but not with GM-CSF, further increased the protection. The DNA vaccine-induced protection was dependent on T cells and induction of IFN-[gamma]. Interestingly, CD4+ T cells induced by the vaccine, in absence of CD8+ T cells, were involved in deteriorating the outcome of infection. This deterioration was linked with a shift towards a Th2 cytokine pattern.