Antibacterial Monoclonal Antibodies Do Not Disrupt the Intestinal Microbiome or Its Function.

Antibiotics revolutionized the treatment of infectious diseases; however, it is now clear that broad-spectrum antibiotics alter the composition and function of the host's microbiome. The microbiome plays a key role in human health, and its perturbation is increasingly recognized as contributing to many human diseases. Widespread broad-spectrum antibiotic use has also resulted in the emergence of multidrug-resistant pathogens, spurring the development of pathogen-specific strategies such as monoclonal antibodies (MAbs) to combat bacterial infection. Not only are pathogen-specific approaches not expected to induce resistance in nontargeted bacteria, but they are hypothesized to have minimal impact on the gut microbiome. Here, we compare the effects of antibiotics, pathogen-specific MAbs, and their controls (saline or control IgG [c-IgG]) on the gut microbiome of 7-week-old, female, C57BL/6 mice. The magnitude of change in taxonomic abundance, bacterial diversity, and bacterial metabolites, including short-chain fatty acids (SCFA) and bile acids in the fecal pellets from mice treated with pathogen-specific MAbs, was no different from that with animals treated with saline or an IgG control. Conversely, dramatic changes were observed in the relative abundance, as well as alpha and beta diversity, of the fecal microbiome and bacterial metabolites in the feces of all antibiotic-treated mice. Taken together, these results indicate that pathogen-specific MAbs do not alter the fecal microbiome like broad-spectrum antibiotics and may represent a safer, more-targeted approach to antibacterial therapy.

Mouse model of hematogenous implant-related Staphylococcus aureus biofilm infection reveals therapeutic targets.

Infection is a major complication of implantable medical devices, which provide a scaffold for biofilm formation, thereby reducing susceptibility to antibiotics and complicating treatment. Hematogenous implant-related infections following bacteremia are particularly problematic because they can occur at any time in a previously stable implant. Herein, we developed a model of hematogenous infection in which an orthopedic titanium implant was surgically placed in the legs of mice followed 3 wk later by an i.v. exposure to Staphylococcus aureus This procedure resulted in a marked propensity for a hematogenous implant-related infection comprised of septic arthritis, osteomyelitis, and biofilm formation on the implants in the surgical legs compared with sham-operated surgical legs without implant placement and with contralateral nonoperated normal legs. Neutralizing human monoclonal antibodies against α-toxin (AT) and clumping factor A (ClfA), especially in combination, inhibited biofilm formation in vitro and the hematogenous implant-related infection in vivo. Our findings suggest that AT and ClfA are pathogenic factors that could be therapeutically targeted against Saureus hematogenous implant-related infections.

Treatment Efficacy of MEDI3902 in Pseudomonas aeruginosa Bloodstream Infection and Acute Pneumonia Rabbit Models.

Pseudomonas aeruginosa is a challenge for clinicians due to increasing drug resistance and dwindling treatment options. We report on the activity of MEDI3902, an antibody targeting type 3 secretion protein PcrV and Psl exopolysaccharide, in rabbit bloodstream and lung infection models. MEDI3902 prophylaxis or treatment was protective in both acute models and exhibited enhanced activity when combined with a subtherapeutic dose of meropenem. These findings further support MEDI3902 for the prevention or treatment of serious P. aeruginosa infections.

Pseudomonas aeruginosa PcrV and Psl, the Molecular Targets of Bispecific Antibody MEDI3902, Are Conserved Among Diverse Global Clinical Isolates.

Background: Bispecific antibody MEDI3902, targeting the Pseudomonas aeruginosa type 3 secretion system (PcrV) and Psl exopolysaccharide, is currently in phase 2b development for prevention of nosocomial pneumonia in patients undergoing mechanical ventilation. We surveyed a diverse collection of isolates to study MEDI3902 epitope conservation and protective activity. Methods: P. aeruginosa clinical isolates (n = 913) were collected from diverse patients and geographic locations during 2003-2014. We conducted whole-genome sequencing; performed PcrV and Psl expression analyses via immunoblotting and enzyme-linked immunosorbent assay, respectively; performed crystallography to determine the MEDI3902 PcrV epitope, using anti-PcrV Fab and PcrV components (resolved at 2.8 Å); and evaluated MEDI3902 protective activity against select isolates in vitro and in vivo. Results: Intact psl operon and pcrV genes were present in 94% and 99% of isolates, respectively, and 99.9% of isolates contained at least one of the genetic elements. Anti-Psl binding was confirmed in tested isolates harboring a complete Psl operon or lacking nonessential psl genes. We identified 46 PcrV variant sequences, and MEDI3902-PcrV contact residues were preserved. MEDI3902 maintained potent in vivo activity against various strains, including strains expressing only a single target. Conclusions: Psl and PcrV are highly prevalent in global clinical isolates, suggesting MEDI3902 can mediate broad coverage against P. aeruginosa.

The Neutrophilic Response to Pseudomonas Damages the Airway Barrier, Promoting Infection by Klebsiella pneumoniae.

Pseudomonas aeruginosa and Klebsiella pneumoniae are two common gram-negative pathogens that are associated with bacterial pneumonia and can often be isolated from the same patient. We used a mixed-pathogen pneumonia infection model in which mice were infected with sublethal concentrations of P. aeruginosa and K. pneumoniae, resulting in significant lethality, outgrowth of both bacteria in the lung, and systemic dissemination of K. pneumoniae. Inflammation, induced by P. aeruginosa activation of Toll-like receptor 5, results in prolonged neutrophil recruitment to the lung and increased levels of neutrophil elastase in the airway, resulting in lung damage and epithelial barrier dysfunction. Live P. aeruginosa was not required to potentiate K. pneumoniae infection, and flagellin alone was sufficient to induce lethality when delivered along with Klebsiella. Prophylaxis with an anti-Toll-like receptor 5 antibody or Sivelestat, a neutrophil elastase inhibitor, reduced neutrophil influx, inflammation, and mortality. Furthermore, pathogen-specific monoclonal antibodies targeting P. aeruginosa or K. pneumoniae prevented the outgrowth of both bacteria and reduced host inflammation and lethality. These findings suggest that coinfection with P. aeruginosa may enable the outgrowth and dissemination of K. pneumoniae, and that a pathogen- or host-specific prophylactic approach targeting P. aeruginosa may prevent or limit the severity of such infections by reducing neutrophil-induced lung damage.

Alanine Scanning Mutagenesis of the MEDI4893 (Suvratoxumab) Epitope Reduces Alpha Toxin Lytic Activity In Vitro and Staphylococcus aureus Fitness in Infection Models.

Alpha toxin (AT) is a cytolytic pore-forming toxin that plays a key role in Staphylococcus aureus pathogenesis; consequently, extensive research was undertaken to understand the AT mechanism of action and its utility as a target for novel prophylaxis and treatment strategies against S. aureus infections. MEDI4893 (suvratoxumab) is a human anti-AT IgG1 monoclonal antibody (MAb) that targets AT and is currently in phase 2 clinical development. As shown previously, the MEDI4893-binding epitope on AT is comprised of the highly conserved amino acid regions 177 to 200 and 261 to 271, suggesting these amino acids are important for AT function. To test this hypothesis and gain insight into the effect of mutations in the epitope on AT neutralization by MEDI4893, nine MEDI4893 contact residues in AT were individually mutated to alanine. Consistent with our hypothesis, 8 out of 9 mutants exhibited >2-fold loss in lytic activity resulting from a defect in cell binding and pore formation. MEDI4893 binding affinity was reduced >2-fold (2- to 27-fold) for 7 out of 9 mutants, and no binding was detected for the W187A mutant. MEDI4893 effectively neutralized all of the lytic mutants in vitro and in vivo When the defective mutants were introduced into an S. aureus clinical isolate, the mutant-expressing strains exhibited less severe disease in mouse models and were effectively neutralized by MEDI4893. These results indicate the MEDI4893 epitope is highly conserved due in part to its role in AT pore formation and bacterial fitness, thereby decreasing the likelihood for the emergence of MAb-resistant variants.

Neutralizing Alpha-Toxin Accelerates Healing of Staphylococcus aureus-Infected Wounds in Nondiabetic and Diabetic Mice.

Staphylococcus aureus wound infections delay healing and result in invasive complications such as osteomyelitis, especially in the setting of diabetic foot ulcers. In preclinical animal models of S. aureus skin infection, antibody neutralization of alpha-toxin (AT), an S. aureus-secreted pore-forming cytolytic toxin, reduces disease severity by inhibiting skin necrosis and restoring effective host immune responses. However, whether therapeutic neutralization of alpha-toxin is effective against S. aureus-infected wounds is unclear. Herein, the efficacy of prophylactic treatment with a human neutralizing anti-AT monoclonal antibody (MAb) was evaluated in an S. aureus skin wound infection model in nondiabetic and diabetic mice. In both nondiabetic and diabetic mice, anti-AT MAb treatment decreased wound size and bacterial burden and enhanced reepithelialization and wound resolution compared to control MAb treatment. Anti-AT MAb had distinctive effects on the host immune response, including decreased neutrophil and increased monocyte and macrophage infiltrates in nondiabetic mice and decreased neutrophil extracellular traps (NETs) in diabetic mice. Similar therapeutic efficacy was achieved with an active vaccine targeting AT. Taken together, neutralization of AT had a therapeutic effect against S. aureus-infected wounds in both nondiabetic and diabetic mice that was associated with differential effects on the host immune response.

Multimechanistic Monoclonal Antibodies (MAbs) Targeting Staphylococcus aureus Alpha-Toxin and Clumping Factor A: Activity and Efficacy Comparisons of a MAb Combination and an Engineered Bispecific Antibody Approach.

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.

Targeting Alpha Toxin To Mitigate Its Lethal Toxicity in Ferret and Rabbit Models of Staphylococcus aureus Necrotizing Pneumonia.

The role broad-spectrum antibiotics play in the spread of antimicrobial resistance, coupled with their effect on the healthy microbiome, has led to advances in pathogen-specific approaches for the prevention or treatment of serious bacterial infections. One approach in clinical testing is passive immunization with a monoclonal antibody (MAb) targeting alpha toxin for the prevention or treatment of Staphylococcus aureus pneumonia. Passive immunization with the human anti-alpha toxin MAb, MEDI4893*, has been shown to improve disease outcome in murine S. aureus pneumonia models. The species specificity of some S. aureus toxins necessitates testing anti-S. aureus therapeutics in alternate species. We developed a necrotizing pneumonia model in ferrets and utilized an existing rabbit pneumonia model to characterize MEDI4893* protective activity in species other than mice. MEDI4893* prophylaxis reduced disease severity in ferret and rabbit pneumonia models against both community-associated methicillin-resistant S. aureus (MRSA) and hospital-associated MRSA strains. In addition, adjunctive treatment of MEDI4893* with either vancomycin or linezolid provided enhanced protection in rabbits relative to the antibiotics alone. These results confirm that MEDI4893 is a promising candidate for immunotherapy against S. aureus pneumonia.

Properdin deficiency protects from 5-fluorouracil-induced small intestinal mucositis in a complement activation-independent, interleukin-10-dependent mechanism.

Intestinal mucositis is a serious complication of chemotherapy that leads to significant morbidity that may require dose or drug adjustments. Specific mitigating strategies for mucositis are unavailable, due partly to an incomplete understanding of the pathogenic mechanisms. We have previously shown an effect of properdin, a positive regulator of complement activation, in models of colitis. Here we use properdin-deficient (PKO ) mice to interrogate the role of properdin and complement in small intestinal mucositis. Mucositis was induced by five daily injections of 5-fluorouracil (5-FU) in wild-type (WT), PKO , interleukin (IL)-10-/- and properdin/IL-10-/- double knock-out (DKO) mice. At the time of euthanasia their jejunum was collected for histology, immunohistochemistry and cytokine and complement activation measurements. Complement became activated in mice receiving 5-FU, indicated by increased intestinal levels of C3a and C5a. Compared to WT, PKO mice experienced significantly less mucositis, despite C3a levels as high as inflamed WT mice and slightly less C5a. Conversely, PKO mice had higher intestinal levels of IL-10. IL-10 expression was mainly by epithelial cells in both uninflamed and inflamed PKO mice. IL-10-/- mice proved to be highly susceptible to mucositis and DKO mice were equally susceptible, demonstrating that a lack of properdin does not protect mice lacking IL-10. We interpret our findings to indicate that, to a significant extent, the inflammation of mucositis is properdin-dependent but complement activation-independent. Additionally, the benefit achieved in the absence of properdin is associated with increased IL-10 levels, and IL-10 is important in limiting mucositis.

New Strategies Targeting Virulence Factors of Staphylococcus aureus and Pseudomonas aeruginosa.

Morbidity, mortality, and economic burden of nosocomial pneumonia caused by Staphylococcus aureus and Pseudomonas aeruginosa remain high in mechanically ventilated and hospitalized patients despite the use of empirical antibiotic therapy or antibiotics against specific classes of pathogens and procedures to reduce nosocomial infections in hospital settings. Newer agents that neutralize or inhibit specific S. aureus or P. aeruginosa virulence factors may eliminate or reduce the risk for developing pneumonia before or during mechanical ventilation and may improve patient outcomes through mechanisms that differ from those of antibiotics. In this article, we review the types, mechanisms of action, potential advantages, and stage of development of antivirulence agents (AVAs) that hold promise as alternative preventive or interventional therapies against S. aureus­ and P. aeruginosa­associated nosocomial pneumonias. We also present and discuss challenges to the effective utilization of AVAs separately from or in addition to antibiotics and the design of clinical trials and meaningful study end points.

Pseudomonas aeruginosa Bacteremic Patients Exhibit Nonprotective Antibody Titers Against Therapeutic Antibody Targets PcrV and Psl Exopolysaccharide.

BACKGROUND: The type 3 secretion protein PcrV and Psl exopolysaccharide are promising therapeutic antibody targets against Pseudomonas aeruginosa. We examined P. aeruginosa bloodstream infection (BSI) isolates for the ability to express PcrV and Psl and evaluated corresponding patient serum for active titers to these targets. METHODS: We identified 114 patients with acute P. aeruginosa BSI; 56 cases were accompanied by acute sera. Serum was evaluated for PcrV- and Psl-specific immunoglobulin G (IgG) and for cytotoxicity and opsonophagocytosis. Isolates were evaluated for susceptibility to antibiotics, expression of PcrV and Psl, and susceptibility to the anti-PcrV/Psl bispecific antibody and clinical candidate MEDI3902. RESULTS: In-hospital mortality for patients with P. aeruginosa BSI was 39%. A total of 26% of isolates were resistant to ≥3 antibiotic classes. Although PcrV and/or Psl were detected in 99% of isolates, a majority of patients lacked active titers to PcrV (100%) and Psl (98%). In addition, MEDI3902 was active against all tested isolates. CONCLUSIONS: A vast majority of P. aeruginosa BSI isolates express PcrV and Psl; however, patient sera most often lacked IgG and functionally active responses to these targets. These results suggest that therapies directed at PcrV and Psl could be a promising approach for combating P. aeruginosa bloodstream infections.

Target-Agnostic Identification of Functional Monoclonal Antibodies Against Klebsiella pneumoniae Multimeric MrkA Fimbrial Subunit.

The increasing incidence of Klebsiella pneumoniae infections refractory to treatment with current broad-spectrum antibiotic classes warrants the exploration of alternative approaches, such as antibody therapy and/or vaccines, for prevention and treatment. However, the lack of validated targets shared by spectrums of clinical strains poses a significant challenge. We adopted a target-agnostic approach to identify protective antibodies against K. pneumoniae Several monoclonal antibodies were isolated from phage display and hybridoma platforms by functional screening for opsonophagocytic killing activity. We further identified their common target antigen to be MrkA, a major protein in the type III fimbriae complex, and showed that these serotype-independent anti-MrkA antibodies reduced biofilm formation in vitro and conferred protection in multiple murine pneumonia models. Importantly, mice immunized with purified MrkA proteins also showed reduced bacterial burden following K. pneumoniae challenge. Taken together, these results support MrkA as a promising target for K. pneumoniae antibody therapeutics and vaccines.

Critical Role of Alpha-Toxin and Protective Effects of Its Neutralization by a Human Antibody in Acute Bacterial Skin and Skin Structure Infections.

Methicillin-resistant Staphylococcus aureus (MRSA) causes large-scale epidemics of acute bacterial skin and skin structure infections (ABSSSI) within communities across the United States. Animal models that reproduce ABSSSI as they occur in humans are urgently needed to test new therapeutic strategies. Alpha-toxin plays a critical role in a variety of staphylococcal infection models in mice, but its role in the pathogenesis of ABSSSI remains to be elucidated in rabbits, which are similar to humans in their susceptibility to S. aureus superantigens and certain bicomponent pore-forming leukocidins. We report here a new rabbit model of ABSSSI and show that those infected with a mutant deficient in expression of alpha-toxin (Δhla) developed a small dermonecrotic lesion, whereas those infected with isogenic USA300 MRSA wild-type or complemented Δhla strains developed ABSSSI that mimic the severe infections that occur in humans, including the large central dermonecrotic core surrounded by erythema, induration, and marked subcutaneous hemorrhage. More importantly, immunoprophylaxis with MEDI4893*, an anti-alpha-toxin human monoclonal antibody, significantly reduced the severity of disease caused by a USA300 wild-type strain to that caused by the Δhla mutant, indicating that this toxin could be completely neutralized during infection. Thus, this study illustrates a potential high standard for the development of new immunotherapeutic agents in which a toxin-neutralizing antibody provides protection to the same degree achieved with a toxin gene knockout. When MEDI4893* was administered as adjunctive therapy with a subtherapeutic dose of linezolid, the combination was significantly more efficacious than either agent alone in reducing the severity of ABSSSI.

MEDI4893* Promotes Survival and Extends the Antibiotic Treatment Window in a Staphylococcus aureus Immunocompromised Pneumonia Model.

Immunocompromised individuals are at increased risk of Staphylococcus aureus pneumonia. Neutralization of alpha-toxin (AT) with the monoclonal antibody (MAb) MEDI4893* protects normal mice from S. aureus pneumonia; however, the effects of the MAb in immunocompromised mice have not been reported. In this study, passive immunization with MEDI4893* increased survival rates and reduced bacterial numbers in the lungs in an immunocompromised murine S. aureus pneumonia model. Lungs from infected mice exhibited alveolar epithelial damage, protein leakage, and bacterial overgrowth, whereas lungs from mice passively immunized with MEDI4893* retained a healthy architecture, with an intact epithelial barrier. Adjunctive therapy or prophylaxis with a subtherapeutic MEDI4893* dose combined with subtherapeutic doses of vancomycin or linezolid improved survival rates, compared with the monotherapies. Furthermore, coadministration of MEDI4893* with vancomycin or linezolid extended the antibiotic treatment window. These data suggest that MAb-mediated neutralization of AT holds promise in strategies for prevention and adjunctive therapy among immunocompromised patients.

Anti-alpha-toxin monoclonal antibody and antibiotic combination therapy improves disease outcome and accelerates healing in a Staphylococcus aureus dermonecrosis model.

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.

Synthetic Enterobacterial Common Antigen (ECA) for the Development of a Universal Immunotherapy for Drug-Resistant Enterobacteriaceae.

All Enterobacteriaceae express a polysaccharide known as enterobacterial common antigen (ECA), which is an attractive target for the development of universally acting immunotherapies. The first chemical synthesis of ECA-derived oligosaccharides for the development of such therapies is described. A number of synthetic challenges had to be addressed, including the development of concise synthetic procedures for unusual monosaccharides, the selection of appropriate orthogonal protecting groups, the development of stereoselective glycosylation methods, appropriate timing for the introduction of the carboxylic acid groups on the ManpNAcA moieties, and the selection of appropriate conditions for the reduction of multiple azido moieties. The synthetic compounds were employed to uncover immunodominant moieties of ECA. Furthermore, a monoclonal antibody (mAb) was developed that binds to ECA and can selectively recognize a wide range of Enterobacteriaceae species.

A novel anti-PcrV antibody providing enhanced protection against Pseudomonas aeruginosa in multiple animal infection models.

Pseudomonas aeruginosa is a major cause of hospital-acquired infections, particularly in mechanically ventilated patients, and it is the leading cause of death in cystic fibrosis patients. A key virulence factor associated with disease severity is the P. aeruginosa type III secretion system (T3SS), which injects bacterial toxins directly into the cytoplasm of host cells. The PcrV protein, located at the tip of the T3SS injectisome complex, is required for T3SS function and is a well-validated target in animal models of immunoprophylactic strategies targeting P. aeruginosa. In an effort to identify a highly potent and protective monoclonal antibody (MAb) that inhibits the T3SS, we generated and characterized a panel of novel anti-PcrV MAbs. Interestingly, some MAbs exhibiting potent inhibition of T3SS in vitro failed to provide protection in a mouse model of P. aeruginosa infection, suggesting that effective in vivo inhibition of T3SS with anti-PcrV MAbs is epitope dependent. V2L2MD, while not the most potent MAb as assessed by in vitro cytotoxicity inhibition assays, provided strong prophylactic protection in several murine infection models and a postinfection therapeutic model. V2L2MD mediated significantly (P < 0.0001) better in vivo protection than that provided by a comparator antibody, MAb166, a well-characterized anti-PcrV MAb and the progenitor of a clinical candidate, KB001-A. The results described here support further development of a V2L2MD-containing immunotherapeutic and may suggest even greater potential than was previously recognized for the prevention and treatment of P. aeruginosa infections in high-risk populations.

Assessment of an anti-alpha-toxin monoclonal antibody for prevention and treatment of Staphylococcus aureus-induced pneumonia.

Alpha-toxin (AT) is a major virulence factor in the disease pathogenesis of Staphylococcus aureus. We previously identified a monoclonal antibody (MAb) against AT that reduced disease severity in a mouse dermonecrosis model. Here, we evaluate the activity of an affinity-optimized variant, LC10, in a mouse model of S. aureus pneumonia. Passive immunization with LC10 increased survival and reduced bacterial numbers in the lungs and kidneys of infected mice and showed protection against diverse S. aureus clinical isolates. The lungs of S. aureus-infected mice exhibited bacterial pneumonia, including widespread inflammation, whereas the lungs of mice that received LC10 exhibited minimal inflammation and retained healthy architecture. Consistent with reduced immune cell infiltration, LC10-treated animals had significantly lower (P < 0.05) proinflammatory cytokine and chemokine levels in the bronchoalveolar lavage fluid than did those of the control animals. This reduction in inflammation and damage to the LC10-treated animals resulted in reduced vascular protein leakage and CO2 levels in the blood. LC10 was also assessed for its therapeutic activity in combination with vancomycin or linezolid. Treatment with a combination of LC10 and vancomycin or linezolid resulted in a significant increase (P < 0.05) in survival relative to the monotherapies and was deemed additive to synergistic by isobologram analysis. Consistent with improved survival, the lungs of animals treated with antibiotic plus LC10 exhibited less inflammatory tissue damage than those that received monotherapy. These data provide insight into the mechanisms of protection provided by AT inhibition and support AT as a promising target for immunoprophylaxis or adjunctive therapy against S. aureus pneumonia.