Enhancement of bovine CD4(+) T cell-dependent immune responses induced by Anaplasma marginale MSP-1a DNA vaccine

The goal of this study was to enhance DNA vaccine induced bovine CD4 + T cell dependent immune responses. The hypothesis tested was that, incorporation of fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte macrophage colony-stimulating factor (GM-CSF), into a DNA vaccine would enhance dendritic cell (DC) recruitment and immune responses. In the first experiment, the gene encoding bovine Flt3L was cloned and characterized. Two conserved bovine Flt3L isoforms which differ in a defined region within the extracellular domain, were identified and shown to be uniformly transcribed in individuals with diverse MHC haplotypes. Bovine Flt3L isoform 1, but not 2, bound the human flt3 receptor and stimulated murine pro B cells transfected with the murine flt3 receptor. This retention of binding and function allowed definition of key residues by identifying sequences conserved among species. We have shown that a highly conserved, 18 aa sequence within the Flt3L extracellular domain is required for flt3 receptor binding and function. This definition of the required Flt3L structure will facilitate development of agonists to enhance DC recruitment for vaccines and immunotherapy. In the second experiment, DNA encoded bovine Flt3L and GM-CSF were used to determine whether co-administration of Flt3L and GM-CSF in a DNA vaccine vector would enhance DC recruitment and immune responses.; Pretreatment of a cutaneous immunization site with DNA encoded Flt3L and GM-CSF followed, 9 days later, by inoculation of a DNA vaccine expressing extracellular domain of Anaplasma marginale MSP1a antigen, enhanced CD4+ T cell responses. The dominant proliferative response by MSP1a-specific CD4+ T cells from the cytokine pretreated calves was mirrored by a strong MSP1a-specific IFN-γ response. However, vaccine-elicited antibody responses were not significantly changed by the cytokine treatment. The enhanced CD4+ T cell responses in calves treated with Flt3L and GMCSF could be attributed to the increased DC recruitment at the immunization site. Intradermal co-administration of the DNA encoded Flt3L and GM-CSF resulted in a statistically significant (P < 0.05) DC accumulation. Results indicate that DNA encoded Flt3L and GM-CSF recruits DC and augments antigen-specific CD4+ T cell responses associated with high IFN-γ secretion.