Analysis of Type II Secretion of Recombinant Pneumococcal PspA and PspC in a Salmonella enterica serovar Typhimurium Vaccine with Regulated Delayed Antigen Synthesis

Center for Infectious Diseases and Vaccinology, The Biodesign Institute and School of Life Science, Arizona State University, Tempe, Arizona 85287

^* To whom correspondence should be addressed. Email: rcurtiss{at}asu.edu.

	Abstract

Recombinant attenuated Salmonella vaccines (RASV) have been used extensively to express and deliver heterologous antigens to host mucosal tissues. Immune responses can be enhanced greatly when the antigen is secreted to the periplasm or extracellular compartment. The most common method for accomplishing this is by fusion of the antigen to a secretion signal sequence. Finding an optimal signal sequence is typically done empirically. To facilitate this process, we constructed a series of plasmid expression vectors, each containing a different type II signal sequence. We evaluated the utility of these vectors by fusing two different antigens, the -helix domain of pneumococcal surface protein A (PspA) or the pneumococcal surface protein C (PspC) to the signal sequences of -lactamase (bla SS), ompA, phoA, and the signal sequence and C-terminal peptide of -lactamase (bla SS+CT) on Asd⁺ plasmids under the control of the P_trc promoter. Strains were characterized for level of expression, subcellular antigen location, and the capacity to elicit antigen-specific immune responses and protection against challenge with Streptococcus pneumoniae in mice. The immune responses to each protein varied depending on the signal sequence used. Strains carrying the bla SS-pspA and bla SS+CT-pspC fusions yielded the highest amounts of secreted PspA and PspC, respectively, and induced the highest serum IgG titers, although all fusion proteins tested induced some level of antigen-specific IgG responses. Consistent with the serum antibody responses, RASV expressing the bla SS-pspA and bla SS+CT-pspC fusions induced the greatest protection against S. pneumoniae challenge.