S. aureus α toxin promotes opportunistic co-infection, which can be neutralized with a pathogen-specific antibody.

Clinical severity of Staphylococcus aureus respiratory infection correlates with alpha toxin (AT) expression. AT activates the NLRP3 inflammasome; deletion of Nlrp3, or AT neutralization, protects mice from lethal S. aureus pneumonia. We tested the hypothesis that this protection is not due to a reduction in inflammasome-dependent cytokines (IL-1β/IL-18) but increased bactericidal function of macrophages. In vivo, neutralization of AT or NLRP3 improved bacterial clearance and survival, while blocking IL-1β/IL-18 did not. Primary human monocytes were used in vitro to determine the mechanism through which NLRP3 alters bacterial killing. In cells treated with small interfering RNA (siRNA) targeting NLRP3 or infected with AT-null S. aureus, mitochondria co-localize with bacterial-containing phagosomes. Mitochondrial engagement activates caspase-1, a process dependent on complex II of the electron transport chain, near the phagosome, promoting its acidification. These data demonstrate a mechanism utilized by S. aureus to sequester itself from antimicrobial processes within the cell.

c Alpha-toxin (AT) is a major virulence determinant in Staphylococcus aureus skin and soft tissue infection models. We previously demonstrated that prophylactic administration of 2A3, an AT-neutralizing monoclonal antibody (MAb), prevents S. aureus disease in a mouse dermonecrosis model by neutralizing AT-mediated tissue necrosis and immune evasion. In the present study, MEDI4893*, an affinity-optimized version of 2A3, was characterized for therapeutic activity in the dermonecrosis model as a single agent and in combination with two frontline antibiotics, vancomycin and linezolid. MEDI4893* postinfection therapy was found to exhibit a therapeutic treatment window similar to that for linezolid but longer than that for vancomycin. Additionally, when combined with either vancomycin or linezolid, MEDI4893* resulted in reduced tissue damage, increased neutrophil and macrophage infiltration and abscess formation, and accelerated healing relative to those with the antibiotic monotherapies. These data suggest that AT neutralization with a potent MAb holds promise for both prophylaxis and adjunctive therapy with antibiotics and may be a valuable addition to currently available options for the treatment of S. aureus skin and soft tissue infections.

Bacterial pneumonia, such as those caused by Staphylococcus aureus, is associated with an influx of inflammatory neutrophils into the lung tissue and airways. Regulation and clearance of recruited neutrophils is essential for preventing tissue damage by “friendly fire”, a responsibility of macrophages in a process called efferocytosis. We hypothesized that S. aureus impairs efferocytosis by alveolar macrophages (AMs) through the activity of the secreted virulence factor alpha toxin (AT), which has been implicated in altering the antimicrobial function of AMs. Infection of mice lacking AMs resulted in significantly increased numbers of neutrophils in the lung, while clearance of neutrophils delivered intranasally into uninfected mice was reduced in AM depleted animals. In vitro, sublytic levels of AT impaired uptake of apoptotic neutrophils by purified AMs. In vivo, the presence of AT reduced uptake of neutrophils by AMs. Differential uptake of neutrophils was not due to changes in either the CD47/CD172 axis or CD36 levels. AT significantly reduced lung expression of CCN1 and altered AM surface localization of DD1α, two proteins known to influence efferocytosis. We conclude that AT may contribute to tissue damage during S. aureus pneumonia by inhibiting the ability of AM to clear neutrophils at the site of infection.

Secreted alpha-toxin and surface-localized clumping factor A (ClfA) are key virulence determinants in Staphylococcus aureus bloodstream infections. We previously demonstrated that prophylaxis with a multimechanistic monoclonal antibody (MAb) combination against alpha-toxin (MEDI4893*) and ClfA (11H10) provided greater strain coverage and improved efficacy in an S. aureus lethal bacteremia model. Subsequently, 11H10 was found to exhibit reduced affinity and impaired inhibition of fibrinogen binding to ClfA002 expressed by members of a predominant hospital-associated methicillin-resistant S. aureus (MRSA) clone, ST5. Consequently, we identified another anti-ClfA MAb (SAR114) from human tonsillar B cells with >100-fold increased affinity for three prominent ClfA variants, including ClfA002, and potent inhibition of bacterial agglutination by 112 diverse clinical isolates. We next constructed bispecific Abs (BiSAbs) comprised of 11H10 or SAR114 as IgG scaffolds and grafted anti-alpha-toxin (MEDI4893*) single-chain variable fragment to the amino or carboxy terminus of the anti-ClfA heavy chains. Although the BiSAbs exhibited in vitro potencies similar to those of the parental MAbs, only 11H10-BiSAb, but not SAR114-BiSAb, showed protective activity in murine infection models comparable to the respective MAb combination. In vivo activity with SAR114-BiSAb was observed in infection models with S. aureus lacking ClfA. Our data suggest that high-affinity binding to ClfA sequesters the SAR114-BiSAb to the bacterial surface, thereby reducing both alpha-toxin neutralization and protection in vivo. These results indicate that a MAb combination targeting ClfA and alpha-toxin is more promising for future development than the corresponding BiSAb.