Background: Members of the p47 (immunity-related GTPases (IRG) family) GTPases are essential, interferon-inducible resistance factors in mice that are active against a broad spectrum of important intracellular pathogens. Surprisingly, there are no reports of p47 function in humans.

Staphylococcus aureus is an important human pathogen with increasing clinical impact due to the extensive spread of antibiotic-resistant strains. Therefore, development of a protective polyvalent vaccine is of great clinical interest. We employed an intravenous immunoglobulin (IVIG) preparation as a source of antibodies directed against anchorless S. aureus surface proteins for identification of novel vaccine candidates. In order to identify such proteins, subtractive proteome analysis (SUPRA) of S. aureus anchorless cell wall proteins was performed. Proteins reacting with IVIG but not with IVIG depleted of S. aureus-specific opsonizing antibodies were considered vaccine candidates. Nearly 40 proteins were identified by this preselection method using matrix-assisted laser desorption ionization-time of flight analysis. Three of these candidate proteins, enolase (Eno), oxoacyl reductase (Oxo), and hypothetical protein hp2160, were expressed as glutathione S-transferase fusion proteins, purified, and used for enrichment of corresponding immunoglobulin Gs from IVIG by affinity chromatography. Use of affinity-purified anti-Eno, anti-Oxo, and anti-hp2160 antibodies resulted in opsonization, phagocytosis, and killing of S. aureus by human neutrophils. High specific antibody titers were detected in mice immunized with recombinant antigens. In mice challenged with bioluminescent S. aureus, reduced staphylococcal spread was measured by in vivo imaging. The recovery of S. aureus CFU from organs of immunized mice was diminished 10-to 100-fold. Finally, mice immunized with hp2160 displayed statistically significant higher survival rates after lethal challenge with clinically relevant S. aureus strains. Taken together, our data suggest that anchorless cell wall proteins might be promising vaccine candidates and that SUPRA is a valuable tool for their identification.

The enormous capacity of Staphylococcus aureus to acquire antibiotic resistance makes it a permanent task to search for and to develop new anti-infectives. One of the possible approaches is the early active immunization of risk patients and animal stocks to prevent S. aureus infections. Based on a S. aureus proteome screen with S. aureus-specific human antiserum, we have previously identified several anchorless cell wall proteins to be used as novel vaccine candidates. To develop an efficient anti-S. aureus vaccine, the supplemented adjuvants Montanide TM ISA 71 VG and ISA 206 were compared to Freund's adjuvant in terms of handling, induction of cytokine profile, triggering antigen-specific immunoglobulin production of different IgG subclasses and provision of increased survival rates in our S. aureus sepsis mouse model. Immunization with ISA 71 VG in comparison with Freund's adjuvant induced slightly delayed but comparably strong increase of antigen-specific antibody titres and conferred protective effect against S. aureus challenge. In contrast using ISA 206 as adjuvant, significantly lower IgG titres and consequently, no protective effect against S. aureus infection were observed. Handling and tolerability of the Montanide is superior to Freund's adjuvant. Montanide TM ISA 71 VG can serve as an effective adjuvant replacement for Freund's adjuvant in research with a prospective usage in animal and human vaccines against bacterial pathogens.