Avanish K. Varshney

Diverse Enterotoxin Gene Profiles among Clonal Complexes of Staphylococcus aureus Isolates from the Bronx, New York

ABSTRACT Staphylococcal enterotoxins (SE) can cause toxin-mediated disease, and those that function as superantigens are implicated in the pathogenesis of allergic diseases. The prevalence of 19 enterotoxin genes was determined by PCR in clinical S. aureus strains derived from wounds (108) and blood (99). We performed spa typing and multilocus sequence typing (MLST) to determine clonal origin, and for selected strains staphylococcal enterotoxin B (SEB) production was measured by enzyme-linked immunosorbent assay. Strains carried a median of five SE genes. For most SE genes, the prevalence rates among methicillin-resistant and methicillin-sensitive S. aureus isolates, as well as wound- and blood-derived isolates, did not differ. At least one SE gene was detected in all except two S. aureus isolates (>99%). Complete egc clusters were found in only 11% of S. aureus isolates, whereas the combination of sed, sej, and ser was detected in 24% of clinical strains. S. aureus strains exhibited distinct combinations of SE genes, even if their pulsed-field gel electrophoresis and MLST patterns demonstrated clonality. USA300 strains also showed considerable variability in SE content, although they contained a lower number of SE genes (mean, 3). By contrast, SE content was unchanged in five pairs of serial isolates. SEB production by individual strains varied up to 200-fold, and even up to 15-fold in a pair of serial isolates. In conclusion, our results illustrate the genetic diversity of S. aureus strains with respect to enterotoxin genes and suggest that horizontal transfer of mobile genetic elements encoding virulence genes occurs frequently.

Generation, Characterization, and Epitope Mapping of Neutralizing and Protective Monoclonal Antibodies against Staphylococcal Enterotoxin B-induced Lethal Shock

T-cell stimulating activity of Staphylococcal enterotoxin B (SEB) is an important factor in the pathogenesis of certain staphylococcal diseases including SEB mediated shock. SEB is one of the most potent superantigens known and treatment of SEB induced shock remains a challenge. We generated and characterized murine monoclonal antibodies (mAbs) to SEB in mice. We tested mAbs neutralize mitogenic effects of SEB in vitro and in vivo with T-cell proliferation assays and 2 murine models for SEB induced lethal shock (SEBILS). Epitope mapping suggests that all these mAbs recognize conformational epitopes that are destroyed by deleting the C terminus of the protein. Further site-directed mutagenesis identified potential residues involved in binding to SEB that differ between Methicillin resistant and sensitive Staphylococcus aureus strains. Only mAb 20B1 was effective as a monotherapy in treating SEBILS in HLA DR3 transgenic mice, which exhibit enhanced sensitivity to SEB. It is noteworthy that mAbs, 14G8 and 6D3 were not protective when given alone in the HLA DR3 mice but their efficacy of protection could be greatly enhanced when mAbs were co-administered simultaneously. Our data suggest combinations of defined mAbs may constitute a better treatment strategy and provide a new insight for the development of passive immunotherapy.

Augmented Production of Panton-Valentine Leukocidin Toxin in Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Is Associated with Worse Outcome in a Murine Skin Infection Model

The role of Panton-Valentine leukocidin (PVL) in Staphylococcus aureus infections is controversial. We used a mouse model of skin infection to compare the virulence of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains with different levels of PVL production. Differences in PVL production were not associated with mutations in the genes lukS-PV and lukF-PV. However, MSSA and MRSA strains that produced high levels of PVL caused larger skin abscesses, higher bacterial burdens, and more tissue inflammation than did low-PVL-producing strains. Together, these data suggest that (1) the effect of PVL on the pathogenesis of staphylococcal infection may depend on the level of toxin produced and (2) many strains of MSSA that cause soft-tissue infections produce higher levels of PVL than do MRSA strains.

Staphylococcal Enterotoxin B–Specific Monoclonal Antibody 20B1 Successfully Treats Diverse Staphylococcus aureus Infections

BACKGROUND Methicillin-resistant Staphylococcus aureus (MRSA) has become a major health threat in the United States. Staphylococcal enterotoxin B (SEB) is a potent superantigen that contributes to its virulence. High mortality and frequent failure of therapy despite available antibiotics have stimulated research efforts to develop adjunctive therapies. METHODS Treatment benefits of SEB-specific monoclonal antibody (mAb) 20B1 were investigated in mice in sepsis, superficial skin, and deep-tissue infection models. RESULTS Mice challenged with a SEB-producing MRSA strain developed fatal sepsis, extensive tissue skin infection, and abscess-forming deep-seeded thigh muscle infection. Animals preimmunized against SEB or treated passively with mAb 20B1 exhibited enhanced survival in the sepsis model, whereas decrease of bacterial burden was observed in the superficial skin and deep-tissue models. mAb 20B1 bound to SEB in the infected tissue and decreased abscess formation and proinflammatory cytokine levels, lymphocyte proliferation, and neutrophil recruitment. CONCLUSIONS mAb 20B1, an SEB-neutralizing mAb, is effective against MRSA infection. mAb 20B1 protects against lethal sepsis and reduces skin tissue invasion and deep-abscess formation. The mAb penetrates well into the abscess and binds to SEB. It affects the outcome of S. aureus infection by modulating the hosts proinflammatory immune response.

Isotype Switching Increases Efficacy of Antibody Protection against Staphylococcal Enterotoxin B-Induced Lethal Shock and Staphylococcus aureus Sepsis in Mice

ABSTRACT Staphylococcal enterotoxin B (SEB) is a potent toxin that is produced by Staphylococcus aureus strains and is classified as a category B select agent. We have previously shown that monoclonal antibody (MAb) 20B1, a murine anti-SEB IgG1, successfully treats SEB-induced lethal shock (SEBILS) and bacteremia that is caused by SEB-producing S. aureus. In this study, we have generated two isotype switch variants of the original IgG1 MAb 20B1, an IgG2a and IgG2b, both bearing the same variable region sequence, and compared their neutralizing and protective activity in in vitro and in vivo assays, respectively. All 3 isotypes demonstrated comparable affinity to SEB and comparable 50% inhibitory concentrations (IC50s) in T cell proliferation assays. In vivo, however, the IgG2a isotype variant of 20B1 exhibited significantly greater protection than IgG1 or IgG2b in murine SEB intoxication and S. aureus sepsis models. Protection was associated with downmodulation of inflammatory host response. Our data demonstrate that changing the isotype of already protective MAbs, without affecting their antigen specificity or sensitivity, can result in an enhancement of their protective ability. Isotype selection, therefore, should be carefully considered in the development of toxin-neutralizing MAbs and the design of antibody therapeutics. IMPORTANCE The purpose of this study was to enhance the protective efficacy of an existing, protective monoclonal antibody against staphylococcal enterotoxin B. Using two in vivo mouse models, our study demonstrates that the protective efficacy of a monoclonal antibody may be improved by inducing an isotype switch at the Fc region of an antibody, without altering the antigen specificity or sensitivity of the antibody. The development of therapeutic MAbs with higher efficacy may allow for the achievement of equal therapeutic benefit with a lower dosage. In turn, the use of lower doses may reduce the cost of these therapies, while reducing the potential for adverse side effects. The purpose of this study was to enhance the protective efficacy of an existing, protective monoclonal antibody against staphylococcal enterotoxin B. Using two in vivo mouse models, our study demonstrates that the protective efficacy of a monoclonal antibody may be improved by inducing an isotype switch at the Fc region of an antibody, without altering the antigen specificity or sensitivity of the antibody. The development of therapeutic MAbs with higher efficacy may allow for the achievement of equal therapeutic benefit with a lower dosage. In turn, the use of lower doses may reduce the cost of these therapies, while reducing the potential for adverse side effects.

Optimal Irrigation and Debridement of Infected Joint Implants An In Vitro Methicillin-Resistant Staphylococcus aureus Biofilm Model

Acute postoperative and acute, late hematogenous prosthetic joint infections have been treated with 1-stage irrigation and debridement with polyethylene exchange. Success rates, however, are highly variable. Reported studies demonstrate that detergents are effective at decreasing bacterial colony counts on orthopedic implants. Our hypothesis is that the combination of a detergent and an antiseptic would be more effective than using a detergent alone to decrease colony counts from a methicillin-resistant Staphylococcus aureus biofilm-coated titanium alloy disk simulating an orthopedic implant. In our study of various agents tested, chlorhexidine gluconate scrub (antiseptic and detergent) was the most effective at decreasing bacterial colony counts both prereincubation and postreincubation of the disks; pulse lavage and scrubbing were not more effective than pulse lavage alone.

Humanized Staphylococcal Enterotoxin B (SEB)–Specific Monoclonal Antibodies Protect From SEB Intoxication and Staphylococcus aureus Infections Alone or as Adjunctive Therapy With Vancomycin

BACKGROUND Staphylococcal enterotoxin B (SEB), a potential biological warfare agent, is a potent superantigen that contributes to the virulence of methicillin-resistant Staphylococcus aureus (MRSA), which is a major health threat in the United States. Efforts to develop toxin-neutralizing antibodies as adjunctive therapies are justified, given the high mortality and frequent failure of therapy despite available antibiotics. METHODS Murine SEB-specific mAb 20B1 was humanized, and treatment benefits of Hu-1.6/1.1 and Hu-1.4/1.1 variants were investigated in mice in an SEB intoxication model, as well as in sepsis and deep-tissue infection models. RESULTS Hu-1.6/1.1 and Hu-1.4/1.1 protected mice against SEB-induced lethal shock. Hu-1.6/1.1 also enhanced survival of mice that developed fatal sepsis after challenge with a SEB-producing MRSA strain. Combined treatment of Hu-1.6/1.1 with vancomycin further increased survival and altered cytokine responses, compared with monotherapy with either monoclonal antibody or vancomycin alone. Efficacy was also demonstrated in the deep-tissue infection model, where Hu-1.4/1.1 bound to SEB in vivo and decreased abscess formation, as well as proinflammatory cytokine levels. CONCLUSIONS SEB-neutralizing mAb 20B1 was successfully humanized. The mAb affects outcome by modulating the proinflammatory host response in both the sepsis and the intoxication models, which justifies further development.

Mechanisms mediating enhanced neutralization efficacy of staphylococcal enterotoxin B by combinations of monoclonal antibodies.

Background: Staphylococcal enterotoxin B is a potent superantigen that causes lethal toxic shock syndrome. Results: Ternary and binary complex of SEB with SEB specific mAbs identified three distinct epitopes. Conclusion: Two different mechanisms illustrate how cocktails of mAbs enhance neutralization efficacy. Significance: SEB neutralization via combination of mAbs is superior to monotherapy and can include non-neutralizing mAbs. Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.

Frequency of Panton-Valentine Leukocidin-Producing Methicillin-Sensitive Staphylococcus Strains in Patients with Complicated Skin and Soft Tissue Infection in Bronx, New York

ABSTRACT lukF-PV was present in 36% of skin and soft tissue infection (SSTI)-derived methicillin-susceptible Staphylococcus aureus (MSSA) strains and comprised six distinct clones, which contained fewer enterotoxin genes than strains without lukF-PV. Clinical presentations and outcomes of lukF-PV + methicillin-resistant S. aureus (MRSA) and MSSA SSTIs were comparable. In multivariable analysis, the presence of lukF-PV remained a significant predictor for incision and drainage among MSSA strains.

Detection and Measurement of Staphylococcal Enterotoxin-Like K (SEl-K) Secretion by Staphylococcus aureus Clinical Isolates

ABSTRACT Staphylococcal enterotoxin-like K (SEl-K) is a potent mitogen that elicits T-cell proliferation and cytokine production at very low concentrations. However, unlike the classical enterotoxins SEB and toxic shock syndrome toxin 1 (TSST-1), the gene for SEl-K is commonly present in more than half of all Staphylococcus aureus clinical isolates and is present in almost all USA300 community-acquired methicillin-resistant S. aureus (CA-MRSA) isolates. Sequencing of the sel-k gene in over 20 clinical isolates and comparative analysis with all 14 published sel-k sequences indicate that there are at least 6 variants of the sel-k gene, including one that is conserved among all examined USA300 strains. Additionally, we have developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) that specifically detects and measures SEl-K protein in culture supernatants and biological fluids. Quantification of in vitro SEl-K secretion by various S. aureus isolates using this novel capture ELISA revealed detectable amounts of SEl-K secretion by all isolates, with the highest secretion levels being exhibited by MRSA strains that coexpress SEB. In vivo secretion was measured in a murine thigh abscess model, where similar levels of SEl-K accumulation were noted regardless of whether the infecting strain exhibited high or low secretion of SEl-K in vitro. We conclude that SEl-K is commonly expressed in the setting of staphylococcal infection, in significant amounts. SEl-K should be further explored as a target for passive immunotherapy against complicated S. aureus infection.