Novel structurally designed vaccine for S. aureus α-hemolysin: protection against bacteremia and pneumonia.
ABSTRACT: 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.
Project description:Staphylococcus aureus - a commensal of the human skin, nares and gastrointestinal tract - is also a leading cause of bacterial skin and soft tissue infection (SSTIs), bacteremia, sepsis, peritonitis, pneumonia and endocarditis. Antibiotic-resistant strains, designated MRSA (methicillin-resistant S. aureus), are common and represent a therapeutic challenge. Current research and development efforts seek to address the challenge of MRSA infections through vaccines and immune therapeutics. Mice have been used as experimental models for S. aureus SSTI, bacteremia, sepsis, peritonitis and endocarditis. This work led to the identification of key virulence factors, candidate vaccine antigens or immune-therapeutics that still require human clinical testing to establish efficacy. Past failures of human clinical trials raised skepticism whether the mouse is an appropriate model for S. aureus disease in humans. S. aureus causes chronic-persistent infections that, even with antibiotic or surgical intervention, reoccur in humans and in mice. Determinants of S. aureus evasion from human innate and adaptive immune responses have been identified, however only some of these are relevant in mice. Future research must integrate these insights and refine the experimental mouse models for specific S. aureus diseases to accurately predict the failure or success for candidate vaccines and immune-therapeutics.
Project description:Recurrent Staphylococcus aureus skin and soft tissue infections (SSTIs) are common despite detectable antibody responses, leading to the belief that the immune response elicited by these infections is not protective. We recently reported that S. aureus USA300 SSTI elicits antibodies that protect against recurrent SSTI in BALB/c but not C57BL/6 mice, and in this study, we aimed to uncover the specificity of the protective antibodies. Using a proteomic approach, we found that S. aureus SSTI elicited broad polyclonal antibody responses in both BALB/c and C57BL/6 mice and identified 10 S. aureus antigens against which antibody levels were significantly higher in immune BALB/c serum. Four of the 10 antigens identified are regulated by the saeRS operon, suggesting a dominant role for saeRS in protection. Indeed, infection with USA300?sae failed to protect against secondary SSTI with USA300, despite eliciting a strong polyclonal antibody response against antigens whose expression is not regulated by saeRS. Moreover, the antibody repertoire after infection with USA300?sae lacked antibodies specific for 10 saeRS-regulated antigens, suggesting that all or a subset of these antigens are necessary to elicit protective immunity. Infection with USA300?hla elicited modest protection against secondary SSTI, and complementation of USA300?sae with hla restored protection but incompletely. Together, these findings support a role for both Hla and other saeRS-regulated antigens in eliciting protection and suggest that host differences in immune responses to saeRS-regulated antigens may determine whether S. aureus infection elicits protective or nonprotective immunity against recurrent infection.
Project description:Due to the emergence of highly virulent community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, S. aureus has become a major threat to public health. A majority of CA-MRSA skin and soft tissue infections in the United States are caused by S. aureus USA300 strains that are known to produce high levels of alpha hemolysin (Hla). Therefore, vaccines that contain inactivated forms of this toxin are currently being developed. In this study, we sought to determine the immune mechanisms of protection for this antigen using a vaccine composed of a genetically inactivated form of Hla (HlaH35L). Using a murine model of skin and soft tissue infections (SSTI), we found that BALB/c mice were protected by vaccination with HlaH35L; however, Jh mice, which are deficient in mature B lymphocytes and lack IgM and IgG in their serum, were not protected. Passive immunization with anti-HlaH35L antibodies conferred protection against bacterial colonization. Moreover, we found a positive correlation between the total antibody concentration induced by active vaccination and reduced bacterial levels. Animals that developed detectable neutralizing antibody titers after active vaccination were significantly protected from infection. These data demonstrate that antibodies to Hla represent the major mechanism of protection afforded by active vaccination with inactivated Hla in this murine model of SSTI, and in this disease model, antibody levels correlate with protection. These results provide important information for the future development and evaluation of S. aureus vaccines.
Project description:Phenol-soluble modulins (PSMs) are amphipathic, pro-inflammatory proteins secreted by most Staphylococcus aureus isolates. This study tested the hypothesis that in vitro PSM production levels are associated with specific clinical phenotypes.177 methicillin-resistant S. aureus (MRSA) isolates from infective endocarditis (IE), skin and soft tissue infection (SSTI), and hospital-acquired/ventilator-associated pneumonia (HAP) were matched by geographic origin, then genotyped using spa-typing. In vitro PSM production was measured by high performance liquid chromatography/mass spectrometry. Statistical analysis was performed using Chi-squared or Kruskal-Wallis tests as appropriate.Spa type 1 was significantly more common in SSTI isolates (62.7% SSTI; 1.7% IE; 16.9% HAP; p < 0.0001) while HAP and IE isolates were more commonly spa type 2 (0% SSTI; 37.3% IE; 40.7% HAP; p < 0.0001). USA300 isolates produced the highest levels of PSMs in vitro. SSTI isolates produced significantly higher quantities of PSM?1-4, PSM?1, and ?-toxin than other isolates (p < 0.001). These findings persisted when USA300 isolates were excluded from analysis.Increased in vitro production of PSMs is associated with an SSTI clinical source. This significant association persisted after exclusion of USA300 genotype isolates from analysis, suggesting that PSMs play a particularly important role in the pathogenesis of SSTI as compared to other infection types.
Project description:Staphylococcus aureus is a leading cause of superficial and invasive human disease that is often refractory to antimicrobial therapy. Vaccines have the potential to reduce the morbidity, mortality, and economic impact associated with staphylococcal infections. However, single-component vaccines targeting S. aureus have failed to show efficacy in clinical trials.A novel glycoengineering technology for creation of a multicomponent staphylococcal vaccine is described. Genes encoding S. aureus capsular polysaccharide (CP) biosynthesis, PglB (a Campylobacter oligosaccharyl transferase), and a protein carrier (detoxified Pseudomonas aeruginosa exoprotein A or S. aureus ? toxin [Hla]) were coexpressed in Escherichia coli. Recombinant proteins N-glycosylated with S. aureus serotype 5 or 8 CPs were purified from E. coli.Rabbits and mice immunized with the glycoprotein vaccines produced antibodies that were active in vitro in functional assays. Active and passive immunization strategies targeting the CPs protected mice against bacteremia, and vaccines targeting Hla protected against lethal pneumonia. The CP-Hla bioconjugate vaccine protected against both bacteremia and lethal pneumonia, providing broad-spectrum efficacy against staphylococcal invasive disease.Glycoengineering technology, whereby polysaccharide and protein antigens are enzymatically linked in a simple E. coli production system, has broad applicability for use in vaccine development against encapsulated microbial pathogens.
Project description:Staphylococcus aureus is an opportunistic pathogen that causes a range of serious infections associated with significant morbidity, by strains increasingly resistant to antibiotics. However, to date all candidate vaccines have failed to induce protective immune responses in humans. We need a more comprehensive understanding of the antigenic targets important in the context of human infection. To investigate infection-associated immune responses, patients were sampled at initial presentation and during convalescence from three types of clinical infection; skin and soft tissue infection (SSTI), prosthetic joint infection (PJI) and pediatric hematogenous osteomyelitis (PHO). Reactivity of serum IgG was tested with an array of recombinant proteins, representing over 2,652 in-vitro-translated open reading frames (ORFs) from a community-acquired methicillin-resistant S. aureus USA300 strain. High-level reactivity was demonstrated for 104 proteins with serum IgG in all patient samples. Overall, high-level IgG-reactivity was most commonly directed against a subset of secreted proteins. Although based on limited surveys, we found subsets of S. aureus proteins with differential reactivity with serum samples from patients with different clinical syndromes. Together, our studies have revealed a hierarchy within the diverse proteins of the S. aureus "immunome", which will help to advance efforts to develop protective immunotherapeutic agents.
Project description:Determine the prevalence and relatedness of Staphylococcus aureus anterior nares colonization in individuals with community-associated staphylococcal skin and soft-tissue infection (SSTI).Observational cohort.US Army soldiers undergoing infantry training.Trainees who developed SSTI from May 2010 to January 2012.Participants underwent anterior nares culture at the time of presentation for purulent SSTI. We determined the prevalence of S. aureus nasal colonization and strain relatedness between colonizing and clinical isolates with pulsed-field gel electrophoresis (PFGE).We enrolled 1,203 SSTI participants, of whom 508 had culture-confirmed S. aureus SSTI. Overall, 70% (357/508) were colonized with S. aureus. Phenotypically, concordant colonization was more common with methicillin-susceptible S. aureus (MSSA; 56%; 122/218) than methicillin-resistant S. aureus (MRSA) SSTI (41%; 118/290; P < .01). With PFGE, 48% (121 of 254) of clinical-colonizing pairs were indistinguishable, and concordant colonization was more common with MRSA (53%; 92/173) than MSSA SSTI (36%; 29/81; P < .01). Restricting analysis to concomitant MRSA-MRSA or MSSA-MSSA pairs, 92% (92/100) of MRSA SSTI were indistinguishable, and 40% (29/72) MSSA SSTI were indistinguishable (P < .01). All 92 MRSA pairs were USA300.On the phenotypic level, concordant anterior nares colonization with incident staphylococcal SSTI is more common in MSSA than MRSA; however, the opposite is observed when accounting for molecular typing, and MRSA SSTI displays greater concordance. USA300 was responsible for strain concordance with MRSA SSTI. Studies are needed to examine the roles of nasal and extra-nasal carriage, colonization preceding infection, and increased virulence in the pathogenesis of MRSA SSTI.ClinicalTrials.gov identifier: NCT01105767.
Project description:BACKGROUND:Microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) facilitate Staphylococcus aureus adherence to host tissue. We hypothesized that S. aureus isolates from implant-associated infections (IAIs) would differ in MSCRAMM profile and biofilm formation in vitro compared to skin and soft tissue infection (SSTI) isolates. METHODS:Pediatric patients and their isolates were identified retrospectively. IAI and SSTI isolates were matched (1:4). Pulsed field gel electrophoresis was performed to group isolates as USA300 vs. non-USA300. Whole genome sequencing was performed and raw sequence data were interrogated for presence of MSCRAMMs (clfA, clfB, cna, ebh, efb, fnbpA, fnbpB, isdA, isdB, sdrC, sdrD, sdrE), biofilm-associated (icaA,D,B,C), and Panton-Valentine leukocidin (lukSF-PV) genes, accessory gene regulator group, and multilocus sequence types. In vitro biofilm formation was assessed for 47 IAI and 47 SSTI isolates using a microtiter plate assay. Conditional logistic regression was performed for analysis of matched data (STATA11, College Station, TX). RESULTS:Forty-seven IAI and 188 SSTI isolates were studied. IAI isolates were more often methicillin susceptible S. aureus and non-USA300 vs. SSTI isolates [34 (72%) vs. 79 (42%), p = 0.001 and 38 (81%) vs. 57 (30%) p <0.001, respectively]. Greater than 98% of isolates carried clfA, clfB, efb, isdA, isdB, and icaA,D,B,C while cna was more frequently found among IAI vs. SSTI isolates (p = 0.003). Most isolates were strong biofilm producers. CONCLUSIONS:S. aureus IAI isolates were significantly more likely to be MSSA and non-USA300 than SSTI isolates. Carriage of MSCRAMMs and biofilm formation did not differ significantly between isolates. Evaluation of genetic polymorphisms and gene expression profiles are needed to further delineate the role of adhesins in the pathogenesis of IAIs.
Project description:Staphylococcus aureus causes serious sepsis and necrotic pneumonia worldwide. Due to the spread of multidrug-resistant strains, developing an effective vaccine is the most promising method for combating S. aureus infection. In this study, based on the immune-dominant areas of the iron surface determinant B (IsdB) and clumping factor A (ClfA), we designed the novel chimeric vaccine IsdB151-277ClfA33-213 (IC). IC formulated with the AlPO4 adjuvant induced higher protection in an S. aureus sepsis model compared with the single components alone and showed broad immune protection against several clinical S. aureus isolates. Immunisation with IC induced strong antibody responses. The protective effect of antibodies was demonstrated through the opsonophagocytic assay (OPA) and passive immunisation experiment. Moreover, this new chimeric vaccine induced Th1/Th17-skewed cellular immune responses based on cytokine profiles and CD4(+) T cell stimulation tests. Neutralisation of IL-17A alone (but not IFN-?) resulted in a significant decrease in vaccine immune protection. Finally, we found that IC showed protective efficacy in a pneumonia model. Taken together, these data provide evidence that IC is a potentially promising vaccine candidate for combating S. aureus sepsis and pneumonia.
Project description:UNLABELLED:Methicillin-resistant Staphylococcus aureus (MRSA) USA300 is a successful S. aureus clone in the United States and a common cause of skin and soft tissue infections (SSTIs). We performed whole-genome sequencing (WGS) of 146 USA300 MRSA isolates from SSTIs and colonization cultures obtained from an investigation conducted from 2008 to 2010 in Chicago and Los Angeles households that included an index case with an S. aureus SSTI. Identifying unique single nucleotide polymorphisms (SNPs) and analyzing whole-genome phylogeny, we characterized isolates to understand transmission dynamics, genetic relatedness, and microevolution of USA300 MRSA within the households. We also compared the 146 USA300 MRSA isolates from our study with the previously published genome sequences of the USA300 MRSA isolates from San Diego (n = 35) and New York City (n = 277). We found little genetic variation within the USA300 MRSA household isolates from Los Angeles (mean number of SNPs ± standard deviation, 17.6 ± 35; π nucleotide diversity, 3.1 × 10(-5)) or from Chicago (mean number of SNPs ± standard deviation, 12 ± 19; π nucleotide diversity, 3.1 × 10(-5)). The isolates within a household clustered into closely related monophyletic groups, suggesting the introduction into and transmission within each household of a single common USA300 ancestral strain. From a Bayesian evolutionary reconstruction, we inferred that USA300 persisted within households for 2.33 to 8.35 years prior to sampling. We also noted that fluoroquinolone-resistant USA300 clones emerged around 1995 and were more widespread in Los Angeles and New York City than in Chicago. Our findings strongly suggest that unique USA300 MRSA isolates are transmitted within households that contain an individual with an SSTI. Decolonization of household members may be a critical component of prevention programs to control USA300 MRSA spread in the United States. IMPORTANCE:USA300, a virulent and easily transmissible strain of methicillin-resistant Staphylococcus aureus (MRSA), is the predominant community-associated MRSA clone in the United States. It most commonly causes skin infections but also causes necrotizing pneumonia and endocarditis. Strategies to limit the spread of MRSA in the community can only be effective if we understand the most common sources of transmission and the microevolutionary processes that provide a fitness advantage to MRSA. We performed a whole-genome sequence comparison of 146 USA300 MRSA isolates from Chicago and Los Angeles. We show that households represent a frequent site of transmission and a long-term reservoir of USA300 strains; individuals within households transmit the same USA300 strain among themselves. Our study also reveals that a large proportion of the USA300 isolates sequenced are resistant to fluoroquinolone antibiotics. The significance of this study is that if households serve as long-term reservoirs of USA300, household MRSA eradication programs may result in a uniquely effective control method.