Structure and protective efficacy of the Staphylococcus aureus autocleaving protease EpiP.

Despite the global medical needs associated with Staphylococcus aureus infections, no licensed vaccines are currently available. We identified and characterized a protein annotated as an epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. In addition, we determined the structure of the recombinant protein (rEpiP) by X-ray crystallography. The crystal structure revealed that rEpiP was cleaved somewhere between residues 95 and 100, and we found that the cleavage occurs through an autocatalytic intramolecular mechanism. The protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine whether the protein acts as a serine protease, we mutated the hypothesized catalytic serine 393 residue to alanine, generating rEpiP-S393A. The crystal structure of this mutant protein showed that the polypeptide chain was not cleaved and was not interacting stably with the active site. Indeed, rEpiP-S393A was shown to be impaired in its protease activity. Mice vaccinated with rEpiP were protected from S. aureus infection (34% survival, P=0.0054). Moreover, the protective efficacy generated by rEpiP and rEpiP-S393A was comparable, implying that the noncleaving mutant could be used for vaccination purposes.

Protective efficacy induced by recombinant Clostridium difficile toxin fragments.

Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.

MF59- and Al(OH)3-Adjuvanted Staphylococcus aureus (4C-Staph) Vaccines Induce Sustained Protective Humoral and Cellular Immune Responses, with a Critical Role for Effector CD4 T Cells at Low Antibody Titers.

Staphylococcus aureus (S. aureus) is an important opportunistic pathogen that may cause invasive life-threatening infections, like sepsis and pneumonia. Due to the increasing antibiotic resistance, the development of an effective vaccine against S. aureus is needed. Although a correlate of protection against staphylococcal diseases is not yet established, several findings suggest that both antibodies and CD4 T cells might contribute to optimal immunity. In this study, we show that adjuvanting a multivalent vaccine (4C-Staph) with MF59, an oil-in-water emulsion licensed in human vaccines, further potentiated antigen-specific IgG titers and CD4 T-cell responses compared to alum and conferred protection in the peritonitis model of S. aureus infection. Moreover, we showed that MF59- and alum-adjuvanted 4C-Staph vaccines induced persistent antigen-specific humoral and T-cell responses, and protected mice from infection up to 4 months after immunization. Furthermore, 4C-Staph formulated with MF59 was used to investigate which immune compartment is involved in vaccine-induced protection. Using CD4 T cell-depleted mice or B cell-deficient mice, we demonstrated that both T and B-cell responses contributed to 4C-Staph vaccine-mediated protective immunity. However, the role of CD4 T cells seemed more evident in the presence of low-antibody responses. This study provides preclinical data further supporting the use of the adjuvanted 4C-Staph vaccines against S. aureus diseases, and provides critical insights on the correlates of protective immunity necessary to combat this pathogen.