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.

Neutrophil Extracellular Traps Confine Pseudomonas aeruginosa Ocular Biofilms and Restrict Brain Invasion.

Bacterial biofilm infections are difficult to eradicate because of antibiotic insusceptibility and high recurrence rates. Biofilm formation by Pseudomonas aeruginosa, a leading cause of bacterial keratitis, is facilitated by the bacterial Psl exopolysaccharide and associated with heightened virulence. Using intravital microscopy, we observed that neutrophilic recruitment to corneal infections limits P. aeruginosa biofilms to the outer eye surface, preventing bacterial dissemination. Neutrophils moved to the base of forming biofilms, where they underwent neutrophil extracellular trap formation (NETosis) in response to high expression of the bacterial type-3 secretion system (T3SS). NETs formed a barrier "dead zone," confining bacteria to the external corneal environment and inhibiting bacterial dissemination into the brain. Once formed, ocular biofilms were resistant to antibiotics and neutrophil killing, advancing eye pathology. However, blocking both Psl and T3SS together with antibiotic treatment broke down the biofilm and reversed keratitis, suggesting future therapeutic strategies for this intractable infection.

Staphylococcus aureus drives expansion of low-density neutrophils in diabetic mice.

Diabetic individuals are at considerable risk for invasive infection by Staphylococcus aureus, however, the mechanisms underlying this enhanced susceptibility to infection are unclear. We observed increased mortality following i.v. S. aureus infection in diabetic mice compared with nondiabetic controls, correlating with increased numbers of low-density neutrophils (LDNs) and neutrophil extracellular traps (NETs). LDNs have been implicated in the inflammatory pathology of diseases such as lupus, given their release of large amounts of NETs. Our goal was to describe what drives LDN increases during S. aureus infection in the diabetic host and mechanisms that promote increased NET production by LDNs. LDN development is dependent on TGF-ß, which we found to be more activated in the diabetic host. Neutralization of TGF-ß, or the TGF-ß-activating integrin αvß8, reduced LDN numbers and improved survival during S. aureus infection. Targeting S. aureus directly with MEDI4893*, an α toxin-neutralizing monoclonal antibody, blocked TGF-ß activation, reduced LDNs and NETs, and significantly improved survival. A comparison of gene and protein expression in high-density neutrophils and LDNs identified increased GPCRs and elevated phosphatase and tensin homolog (PTEN) in the LDN subset. Inhibition of PTEN improved the survival of infected diabetic mice. Our data identify a population of neutrophils in infected diabetic mice that correlated with decreased survival and increased NET production and describe 3 therapeutic targets, a bacterial target and 2 host proteins, that prevented NET production and improved survival.

Mouse model of Gram-negative prosthetic joint infection reveals therapeutic targets.

Bacterial biofilm infections of implantable medical devices decrease the effectiveness of antibiotics, creating difficult-to-treat chronic infections. Prosthetic joint infections (PJI) are particularly problematic because they require prolonged antibiotic courses and reoperations to remove and replace the infected prostheses. Current models to study PJI focus on Gram-positive bacteria, but Gram-negative PJI (GN-PJI) are increasingly common and are often more difficult to treat, with worse clinical outcomes. Herein, we sought to develop a mouse model of GN-PJI to investigate the pathogenesis of these infections and identify potential therapeutic targets. An orthopedic-grade titanium implant was surgically placed in the femurs of mice, followed by infection of the knee joint with Pseudomonas aeruginosa or Escherichia coli. We found that in vitro biofilm-producing activity was associated with the development of an in vivo orthopedic implant infection characterized by bacterial infection of the bone/joint tissue, biofilm formation on the implants, reactive bone changes, and inflammatory immune cell infiltrates. In addition, a bispecific antibody targeting P. aeruginosa virulence factors (PcrV and Psl exopolysaccharide) reduced the bacterial burden in vivo. Taken together, our findings provide a preclinical model of GN-PJI and suggest the therapeutic potential of targeting biofilm-associated antigens.

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.

Publisher Correction: Anti-Psl Targeting of Pseudomonas aeruginosa Biofilms for Neutrophil-Mediated Disruption.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

S. aureus Evades Macrophage Killing through NLRP3-Dependent Effects on Mitochondrial Trafficking.

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-1ß/IL-18) but increased bactericidal function of macrophages. In vivo, neutralization of AT or NLRP3 improved bacterial clearance and survival, while blocking IL-1ß/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.

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.

Immune stealth-driven O2 serotype prevalence and potential for therapeutic antibodies against multidrug resistant Klebsiella pneumoniae.

Emerging multidrug-resistant bacteria are a challenge for modern medicine, but how these pathogens are so successful is not fully understood. Robust antibacterial vaccines have prevented and reduced resistance suggesting a pivotal role for immunity in deterring antibiotic resistance. Here, we show the increased prevalence of Klebsiella pneumoniae lipopolysaccharide O2 serotype strains in all major drug resistance groups correlating with a paucity of anti-O2 antibodies in human B cell repertoires. We identify human monoclonal antibodies to O-antigens that are highly protective in mouse models of infection, even against heavily encapsulated strains. These antibodies, including a rare anti-O2 specific antibody, synergistically protect against drug-resistant strains in adjunctive therapy with meropenem, a standard-of-care antibiotic, confirming the importance of immune assistance in antibiotic therapy. These findings support an antibody-based immunotherapeutic strategy even for highly resistant K. pneumoniae infections, and underscore the effect humoral immunity has on evolving drug resistance.

Anti-Psl Targeting of Pseudomonas aeruginosa Biofilms for Neutrophil-Mediated Disruption.

Bacterial biofilms are recalcitrant to antibiotic therapy and a major cause of persistent and recurrent infections. New antibody-based therapies may offer potential to target biofilm specific components for host-cell mediated bacterial clearance. For Pseudomonas aeruginosa, human monoclonal antibodies (mAbs) targeting the Psl biofilm exopolysaccharide exhibit protective activity against planktonic bacteria in acute infection models. However, anti-Psl mAb activity against P. aeruginosa biofilms is unknown. Here, we demonstrate that anti-Psl mAbs targeting three distinct Psl epitopes exhibit stratified binding in mature in vitro biofilms and bind Psl within the context of a chronic biofilm infection. These mAbs also exhibit differential abilities to inhibit early biofilm events and reduce biomass from mature biofilms in the presence of neutrophils. Importantly, a mAb mixture with neutrophils exhibited the greatest biomass reduction, which was further enhanced when combined with meropenem, a common anti-Pseudomonal carbapenem antibiotic. Moreover, neutrophil-mediated killing of biofilm bacteria correlated with the evident mAb epitope stratification within the biofilm. Overall, our results suggest that anti-Psl mAbs might be promising candidates for adjunctive use with antibiotics to inhibit/disrupt P. aeruginosa biofilms as a result of chronic infection.

An engineered bispecific DNA-encoded IgG antibody protects against Pseudomonas aeruginosa in a pneumonia challenge model.

The impact of broad-spectrum antibiotics on antimicrobial resistance and disruption of the beneficial microbiome compels the urgent investigation of bacteria-specific approaches such as antibody-based strategies. Among these, DNA-delivered monoclonal antibodies (DMAbs), produced by muscle cells in vivo, potentially allow the prevention or treatment of bacterial infections circumventing some of the hurdles of protein IgG delivery. Here, we optimize DNA-delivered monoclonal antibodies consisting of two potent human IgG clones, including a non-natural bispecific IgG1 candidate, targeting Pseudomonas aeruginosa. The DNA-delivered monoclonal antibodies exhibit indistinguishable potency compared to bioprocessed IgG and protect against lethal pneumonia in mice. The DNA-delivered monoclonal antibodies decrease bacterial colonization of organs and exhibit enhanced adjunctive activity in combination with antibiotics. These studies support DNA-delivered monoclonal antibodies delivery as a potential strategy to augment the host immune response to prevent serious bacterial infections, and represent a significant advancement toward broader practical delivery of monoclonal antibody immunotherapeutics for additional infectious pathogens.DNA-delivered monoclonal antibodies (DMAbs) can be produced by muscle cells in vivo, potentially allowing prevention or treatment of infectious diseases. Here, the authors show that two DMAbs targeting Pseudomonas aeruginosa proteins confer protection against lethal pneumonia in mice.

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.

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.

Anti-LPS antibodies protect against Klebsiella pneumoniae by empowering neutrophil-mediated clearance without neutralizing TLR4.

Initial promising results with immune sera guided early human mAb approaches against Gram-negative sepsis to an LPS neutralization mechanism, but these efforts failed in human clinical trials. Emergence of multidrug resistance has renewed interest in pathogen-specific mAbs. We utilized a pair of antibodies targeting Klebsiella pneumoniae LPS, one that both neutralizes LPS/TLR4 signaling and mediates opsonophagocytic killing (OPK) (54H7) and one that only promotes OPK (KPE33), to better understand the contribution of each mechanism to mAb protection in an acutely lethal pneumonia model. Passive immunization 24 hours prior to infection with KPE33 protected against lethal infection significantly better than 54H7, while delivery of either mAb 1 hour after infection resulted in similar levels of protection. These data suggest that early neutralization of LPS-induced signaling limits protection afforded by these mAbs. LPS neutralization prevented increases in the numbers of γδT cells, a major producer of the antimicrobial cytokine IL-17A, the contribution of which was confirmed using il17a-knockout mice. We conclude that targeting LPS for OPK without LPS signaling neutralization has potential to combat Gram-negative infection by engaging host immune defenses, rather than inhibiting beneficial innate immune pathways.

Enhancement of antibody functions through Fc multiplications.

Antibodies carry out a plethora of functions through their crystallizable fragment (Fc) regions, which can be naturally tuned by the adoption of several isotypes and post-translational modifications. Protein engineering enables further Fc function modulations through modifications of the interactions between the Fc and its functional partners, including FcγR, FcRn, complement complex, and additions of auxiliary functional units. Due to the many functions embedded within the confinement of an Fc, a suitable balance must be maintained for a therapeutic antibody to be effective and safe. The outcome of any Fc engineering depends on the interplay among all the effector molecules involved. In this report, we assessed the effects of Fc multiplication (or tandem Fc) on antibody functions. Using IgG1 as a test case, we found that, depending on the specifically designed linker, Fc multiplication led to differentially folded, stable molecules with unique pharmacokinetic profiles. Interestingly, the variants with 3 copies of Fc improved in vitro opsonophagocytic killing activity and displayed significantly improved protective efficacies in a Klebsiella pneumoniae mouse therapeutic model despite faster clearance compared with its IgG1 counterpart. There was no adverse effect observed or pro-inflammatory cytokine release when the Fc variants were administered to animals. We further elucidated that enhanced binding to various effector molecules by IgG-3Fc created a "sink" leading to the rapid clearance of the 3Fc variants, and identified the increased FcRn binding as one strategy to facilitate "sink" escape. These findings reveal new opportunities for novel Fc engineering to further expand our abilities to manipulate and improve antibody therapeutics.

Anti-MrkA Monoclonal Antibodies Reveal Distinct Structural and Antigenic Features of MrkA.

Antibody therapy against antibiotics resistant Klebsiella pneumoniae infections represents a promising strategy, the success of which depends critically on the ability to identify appropriate antibody targets. Using a target-agnostic strategy, we recently discovered MrkA as a potential antibody target and vaccine antigen. Interestingly, the anti-MrkA monoclonal antibodies isolated through phage display and hybridoma platforms all recognize an overlapping epitope, which opens up important questions including whether monoclonal antibodies targeting different MrkA epitopes can be generated and if they possess different protective profiles. In this study we generated four anti-MrkA antibodies targeting different epitopes through phage library panning against recombinant MrkA protein. These anti-MrkA antibodies elicited strong in vitro and in vivo protections against a multi-drug resistant Klebsiella pneumoniae strain. Furthermore, mutational and epitope analysis suggest that the two cysteine residues may play essential roles in maintaining a MrkA structure that is highly compacted and exposes limited antibody binding/neutralizing epitopes. These results suggest the need for further in-depth understandings of the structure of MrkA, the role of MrkA in the pathogenesis of Klebsiella pneumoniae and the protective mechanism adopted by anti-MrkA antibodies to fully explore the potential of MrkA as an efficient therapeutic target and vaccine antigen.

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties.

By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.

S. aureus blocks efferocytosis of neutrophils by macrophages through the activity of its virulence factor alpha toxin.

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.

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.