Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors

To test the effect of co-expression of immunomodulatory molecules, together with target antigen, two recombinant Streptococcus gordonii strains were constructed which secreted either murine interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) in addition to a surface anchored test antigen (the conserved C-repeat region (CRR) of the M6 protein of Streptococcus pyogenes). The secretion of functional cytokines by S. gordonii was achieved by in-frame fusion of sequences encoding mature IL-2 or IFN-gamma to the sequences encoding the leader signal of the M6 protein. Expression of the M protein CRR region from a separate chromosomal site produced double recombinants expressing a secreted cytokine and the M protein CRR region anchored to the surface. Protein expression was verified by streak blot, immunoblot, and ELISA on both the single and double recombinants. A cytokine bioassay using HT-2 cells verified biological activity of recombinant IL-2 secreted from S. gordonii. When mice were immunized subcutaneously with the different S. gordonii expression strains, cytokine co-expression apparently modulated the systemic immune response. These results show that streptococci can deliver biologically active molecules such as cytokines along with antigens to the immune system. These results demonstrate that a cytokine-secreting, noninvasive, bacterial vaccine vector can be used to modulate immune responses to a co-expressed antigen.