DNA vaccines and bacterial DNA in immunity

Abstract

This thesis describes DNA-based vaccination and the importance of bacterial DNA in different immunological perspectives. Intranasal (i.n.) DNA vaccination utilizing a plasmid encoding the chlamydial heat shock protein 60 (p-hsp-60) generated lower bacterial burden and reduced pathology in the lungs of mice after subsequent infection with C. pneumoniae. This DNA vaccine- induced protection was dependent on T cells and induction of IFN-gamma. Co-administration of a plasmid encoding IL-12 ftirther enhanced the protective effect of the DNA vaccine. Interestingly, in the absence of CD8 T cells, CD4 T cells induced by the p-hsp-60 DNA vaccine were involved in deteriorating the outcome of infection. This detrimental response was linked to a shift towards a Th2 cytokine profile in the lungs.

In. DNA vaccination using the chlamydial outer membrane protein 2 (OMP- 2) combined with a CTLA-4 expression system also generated reduction of bacterial load in mouse lung after C. pneumoniae infection compared to controls. In contrast, subcutaneous (s.c.) OMP-2 protein-based immunization in Freund's complete adjuvant (FCA) generated increased lung bacterial load, with increased disease severity. This pathological outcome of disease was dependent on T cells and associated with a Th2 immune response. When oligodeoxynucleotides containing CpG motifs mixed with Freund's incomplete adjuvant (FIA- CpG ODN) were used as adjuvant this detrimental response was shifted towards partial protection against infection. This partial protection was linked to a Th1 type of immune response.

In an approach to identify protein(s) that are included in CpG DNA stimulation, an isolation procedure was developed for a crude protein extract prepared from peripheral blood mononuclear cells (PBMC). The binding between the protein(s) and CpG ODN in the extract was followed by electromobility shift assay (EMSA) analysis. Using a Series of different chromatographic purification steps and protein sequence analysis, heat shock protein 90 (hsp90) was identified as the most likely candidate. A direct binding of hsp90 to CpG ODN was confirmed by using commercially available hsp90 in EMSA analysis. Moreover, microscopic analysis revealed cellular redistribution of hsp90 from the cytoplasm into specific nuclear compartments upon CpG ODN treatment. Thus hsp90 may be a central protein in CpG DNA recognition and signaling.

Initially, the expression of the antimicrobial peptides LL-37 and HBD-1 were shown to be downregulated early during Shigella infection both in vivo and in vitro. The influence of different bacterial components involved in this downregulation was further investigated. Various bacterial components were utilized, including bacterial lysate, LPS and plasmid DNA prepared from different Shigella spp. Indeed, plasmid DNA was found to downregulate the expression of the antimicrobial peptide LL-37 in PBMC. Such a downregulation of antimicrobial peptides, in which plasmid DNA may act as one mediator, might represent an effective strategy in combating peptide attack during the initial phase of infection.