Title

Modulating immune responses with DNA vaccine constructs expressing differing levels of antigen

Date of Completion

January 2001

Keywords

Biology, Molecular|Health Sciences, Immunology

Degree

Ph.D.

Abstract

To test the hypothesis that the amount of expressed foreign antigen produced by DNA vaccines may improve the overall intensity, duration and effectiveness of the ensuing immune response, we explored two approaches for controlling antigen expression. The first approach employed a DNA vaccine in which antigen expression was driven by an inducible promoter. Although this approach was ultimately unsuccessful due to the inability to adequately control levels of gene expression, an alternative approach utilizing differing 3 polyadenylation sequences (polyA) was more promising. Three identical plasmid constructs, differing only in their polyA sequences (HBVpA, BGHpA, PpA) were prepared using traditional cloning techniques. It was hypothesized that increased levels of HBs expression, afforded by the HBVpA enhancer sequence would result in higher antibody titers and T-cell activation. In addition, the ability to bias the phenotype (Th1or Th2) and quality (antibody affinity) of the immune response was examined. In vitro studies utilizing these three constructs showed that the highest level of secreted HBs antigen was produced by the HBVpA > BGHpA > PpA. Following immunization, total IgG titers were highest in mice receiving the “enhanced” vaccine, with both HBVpA and BGHpA constructs yielding a Th1 biased immune response as evidenced by high IgG2a titers and IFN-γ levels. In addition, the HBVpA construct resulted in seroconversion in 100% of vaccinated mice, in contrast to 40–50% in BGHpA mice and 0% in the βpA group. The quality of the antibody response, as measured by antibody affinities did not significantly differ. Although mice immunized with the βpA construct yielded no antibody responses, they did elicit the highest levels of IFN-γ. Taken together, these findings suggest that differing levels of gene expression not only affects the overall seroconversion rates, but also may play a role in modulating the Th1or Th2 bias with respect to a particular antigen. ^