Abstract
Previous efforts towards S. aureus vaccine development have largely focused on cell surface antigens to induce opsonophagocytic killing aimed at providing sterile immunity, a concept successfully applied to other Gram-positive pathogens such as Streptococcus pneumoniae. However, these approaches have largely failed, possibly in part due to the remarkable diversity of the staphylococcal virulence factors such as secreted immunosuppressive and tissue destructive toxins. S. aureus produces several pore-forming toxins including the single subunit alpha hemolysin as well as bicomponent leukotoxins such as Panton-Valentine leukocidin (PVL), gamma hemolysins (Hlg), and LukED. Here we report the generation of highly attenuated mutants of PVL subunits LukS-PV and LukF-PV that were rationally designed, based on an octameric structural model of the toxin, to be deficient in oligomerization. The attenuated subunit vaccines were highly immunogenic and showed significant protection in a mouse model of S. aureus USA300 sepsis. Protection against sepsis was also demonstrated by passive transfer of rabbit immunoglobulin raised against LukS-PV. Antibodies to LukS-PV inhibited the homologous oligomerization of LukS-PV with LukF-PV as well heterologous oligomerization with HlgB. Importantly, immune sera from mice vaccinated with the LukS mutant not only inhibited the PMN lytic activity produced by the PVL-positive USA300 but also blocked PMN lysis induced by supernatants of PVL-negative strains suggesting a broad protective activity towards other bicomponent toxins. These findings strongly support the novel concept of an anti-virulence, toxin-based vaccine intended for prevention of clinical S. aureus invasive disease, rather than achieving sterile immunity. Such a multivalent vaccine may include attenuated leukotoxins, alpha hemolysin, and superantigens.
Highlights
Staphylococcus aureus (SA) is a ubiquitous, formidable Grampositive pathogen associated with a wide range of pathologies from skin and soft tissue infections (SSTI) to life-threatening systemic infections
CA-methicillin resistant SA (MRSA) USA300 was grown for 18 hours in Tryptic soy broth (TSB, Difco Laboratories, Detroit, Mich.). 10 ml of TSB in 25 ml flask was inoculated with a single bead of S. aureus USA300 from 280uC stored bead stock and culture grown overnight at 37uC, with shaking at 230 rpm
We have recently generated an all-atom model of the pre-pore conformation of LukF-PV/LukSPV heterodimer and octamer [18] which was used in the current study for rational design of attenuated Panton-Valentine leukocidin (PVL) mutants
Summary
Staphylococcus aureus (SA) is a ubiquitous, formidable Grampositive pathogen associated with a wide range of pathologies from skin and soft tissue infections (SSTI) to life-threatening systemic infections. The range of pathologies reflects the diverse abilities of this microbe to escape the innate and adaptive immune responses using multiple virulence factors including coagulases, capsular polysaccharides, adhesins, proteases, exoproteins that inactivate the complement system, pore-forming toxins, superantigens and other innate response mediators [1,5]. Most previous approaches for vaccine development have focused on achieving sterile immunity and largely ignored the potential for an anti-virulence approach aimed at clinical protection against invasive disease. Toward this goal, key secreted toxins of S. aureus such as superantigens and pore-forming toxins represent excellent vaccine targets
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