Novel Structurally Designed Vaccine for S. aureus α-Hemolysin: Protection against Bacteremia and Pneumonia

et al. (2012) Novel Structurally Designed Vaccine for S. aureus a-Hemolysin: Protection against Bacteremia and Pneumonia. PLoS ONE 7(6): e38567. doi:10.1371/journal.pone.0038567 Novel Structurally Designed Vaccine for S. aureus a- Hemolysin: Protection against Bacteremia and Pneumonia Rajan P. Adhikari. 0 Hatice Karauzum. 0 Jawad Sarwar 0 Laura Abaandou 0 Mahta Mahmoudieh 0 Atefeh R. Boroun 0 Hong Vu 0 Tam Nguyen 0 V. Sathya Devi 0 Sergey Shulenin 0 Kelly L. Warfield 0 M. 0 Javad Aman 0 Michael Otto, National Institutes of Health, United States of America 0 Integrated Biotherapeutics Inc. , Gaithersburg, Maryland , United States of America Staphylococcus aureus (S. aureus) is a human pathogen associated with skin and soft tissue infections (SSTI) and life threatening sepsis and pneumonia. Efforts to develop effective vaccines against S. aureus have been largely unsuccessful, in part due to the variety of virulence factors produced by this organism. S. aureus alpha-hemolysin (Hla) is a pore-forming toxin expressed by most S. aureus strains and reported to play a key role in the pathogenesis of SSTI and pneumonia. Here we report a novel recombinant subunit vaccine candidate for Hla, rationally designed based on the heptameric crystal structure. This vaccine candidate, denoted AT-62aa, was tested in pneumonia and bacteremia infection models using S. aureus strain Newman and the pandemic strain USA300 (LAC). Significant protection from lethal bacteremia/sepsis and pneumonia was observed upon vaccination with AT-62aa along with a Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE) that is currently in clinical trials. Passive transfer of rabbit immunoglobulin against AT-62aa (AT62-IgG) protected mice against intraperitoneal and intranasal challenge with USA300 and produced significant reduction in bacterial burden in blood, spleen, kidney, and lungs. Our Hla-based vaccine is the first to be reported to reduce bacterial dissemination and to provide protection in a sepsis model of S. aureus infection. AT62-IgG and sera from vaccinated mice effectively neutralized the toxin in vitro and AT62-IgG inhibited the formation of Hla heptamers, suggesting antibody-mediated neutralization as the primary mechanism of action. This remarkable efficacy makes this Hla-based vaccine a prime candidate for inclusion in future multivalent S. aureus vaccine. Furthermore, identification of protective epitopes within AT-62aa could lead to novel immunotherapy for S. aureus infection. - Competing Interests: The authors have the following interests to declare. All authors, except for Tam Nguyen, are employed by Integrated Bio Therapeutics (IBT), who funded this study. Tam Nguyen served as a consultant to IBT. M. Javad Aman and Kelly L. Warfield are shareholders of Integrated BioTherapeutics. There are no products in development or marketed products to declare. The authors declare a patent application related to the vaccine described in this manuscript (PCT/US2012/024031; Filed on Feb 8, 2012, Title: IMMUNOGENIC COMPOSITION COMPRISING ALPHA-HEMOLYSIN OLIGOPEPTIDES). This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors. . These authors contributed equally to this work. Staphylococcus aureus (S. aureus) is a ubiquitous, formidable Grampositive pathogen associated with skin and soft tissue infections (SSTI), as well as life threatening sepsis and pneumonia [1]. Since its first emergence in the 1960s methicillin-resistant S. aureus (MRSA) has become endemic in hospitals and healthcare settings worldwide [2]. Since the 1990s, several community associated MRSA strains (CA-MRSA) have emerged and are spreading worldwide, posing a major global challenge [3,4,5]. There are currently no vaccines available for the prevention of S. aureus infections. The pathogenicity of S. aureus is dependent on numerous virulence factors, including cell surface proteins, polysaccharides, and secreted toxins. The latter cause tissue damage, promote bacterial dissemination and metastatic growth in distant organs, and allow the pathogen to evade the host innate immune response [6,7]. The pore-forming a-hemolysin (Hla), also known as a-toxin (AT), is produced by nearly all virulent strains and is implicated in several S. aureus diseases including SSTI [8] and pneumonia [9]. Several lines of evidence validate Hla as an important vaccine target for prevention of S. aureus infection or complications of disease: i) hla is encoded by a chromosomal determinant [10], and its production has been detected in most S. aureus isolates [11,12,13,14]; ii) a partially attenuated Hla mutant (H35L) or a truncated Hla protect mice against S. aureus pneumonia and skin infections [8,9,15]; and iii) passive immunization with antibodies raised against H35L protect mice from lethal toxin challenge and partially protect against bacterial challenge in pneumonia and skin infection models [16]. While the H35 mutation largely abrogates the lytic activity of Hla, a single point mutation is not considered sufficiently safe to be developed as vaccine for human use. Importantly, Panchal et al reported that several reverting point mutations can be identified that restore the lytic activity of HlaH35 mutants [17]. Furthermore, removal of 30 or 99 amino acids at the C terminus of the H35A mutant of Hla reactivated its hemolytic activity [18]. Therefore, there is a need to identify subdomain mutants of Hla with an increased safety profile capable of inducing protective immune responses. In this study, using a rational, structure-based approach, we designed several truncation mutants of Hla as vaccine candidates and examined their efficacy in two models of S. aureus infection. Importantly, this study demonstrates, for the first time, efficacy of a Hla based vaccine candidate against S. aureus bacteremia and distant organ bacterial seeding. Materials and Methods Bacteria S. aureus strain USA300 (Los Angeles County clone, LAC) was obtained from the NARSA repository and S. aureus strain Newman was kindly provided by Dr. Tim Foster (Trinity College Dublin, Ireland). Preparation of inoculation seeds for pneumonia model Newman or USA300 strains were grown overnight (ON) in a volume of 20 ml in brain heart infusion (BHI) medium at 37uC, shaking at 230 rpm using a 50 ml culture tube. Multiples of 20 ml cultures were prepared. ON cultures were centrifuged at 3000 rpm and washed twice in PBS using the original volume (20 ml) before pellet was re-suspended in 1 ml phosphate buffered saline (PBS). Multiples of re-suspended pellets were combined and mixed thoroughly on a vortex and further re-suspended with a 28 Gauge needle to keep chain formation of bacterial cells to a minimum. Subsequently 1ml aliquots of seed culture were prepared and stored at 280uC. Three aliquots were streaked out at different dilutions and different time points (to test stability of the seed) to e (...truncated)