Phage activity against Staphylococcus aureus is impaired in plasma and synovial fluid.

S. aureus is a pathogen that frequently causes severe morbidity and phage therapy is being discussed as an alternative to antibiotics for the treatment of S. aureus infections. In this in vitro and animal study, we demonstrated that the activity of anti-staphylococcal phages is severely impaired in 0.5% plasma or synovial fluid. Despite phage replication in these matrices, lysis of the bacteria was slower than phage propagation, and no reduction of the bacterial population was observed. The inhibition of the phages associated with a reduction in phage adsorption, quantified to 99% at 10% plasma. S. aureus is known to bind multiple coagulation factors, resulting in the formation of aggregates and blood clots that might protect the bacterium from the phages. Here, we show that purified fibrinogen at a sub-physiological concentration of 0.4 mg/ml is sufficient to impair phage activity. In contrast, dissolution of the clots by tissue plasminogen activator (tPA) partially restored phage activity. Consistent with these in vitro findings, phage treatment did not reduce bacterial burdens in a neutropenic mouse S. aureus thigh infection model. In summary, phage treatment of S. aureus infections inside the body may be fundamentally challenging, and more investigation is needed prior to proceeding to in-human trials.

Natural Bred ε2-Phages Have an Improved Host Range and Virulence against Uropathogenic Escherichia coli over Their Ancestor Phages.

Alternative treatments for Escherichia coli infections are urgently needed, and phage therapy is a promising option where antibiotics fail, especially for urinary tract infections (UTI). We used wastewater-isolated phages to test their lytic activity against a panel of 47 E. coli strains reflecting the diversity of strains found in UTI, including sequence type 131, 73 and 69. The plaquing host range (PHR) was between 13 and 63%. In contrast, the kinetic host range (KHR), describing the percentage of strains for which growth in suspension was suppressed for 24 h, was between 0% and 19%, substantially lower than the PHR. To improve the phage host range and their efficacy, we bred the phages by mixing and propagating cocktails on a subset of E. coli strains. The bred phages, which we termed evolution-squared ε2-phages, of a mixture of Myoviridae have KHRs up to 23% broader compared to their ancestors. Furthermore, using constant phage concentrations, Myoviridae ε2-phages suppressed the growth of higher bacterial inocula than their ancestors did. Thus, the ε2-phages were more virulent compared to their ancestors. Analysis of the genetic sequences of the ε2-phages with the broadest host range reveals that they are mosaic intercrossings of 2-3 ancestor phages. The recombination sites are distributed over the whole length of the genome. All ε2-phages are devoid of genes conferring lysogeny, antibiotic resistance, or virulence. Overall, this study shows that ε2-phages are remarkably more suitable than the wild-type phages for phage therapy.

ε2-Phages Are Naturally Bred and Have a Vastly Improved Host Range in Staphylococcus aureus over Wild Type Phages.

Due to the rapid spread of antibiotic resistance, and the difficulties of treating biofilm-associated infections, alternative treatments for S. aureus infections are urgently needed. We tested the lytic activity of several wild type phages against a panel of 110 S. aureus strains (MRSA/MSSA) composed to reflect the prevalence of S. aureus clonal complexes in human infections. The plaquing host ranges (PHR) of the wild type phages were in the range of 51% to 60%. We also measured what we called the kinetic host range (KHR), i.e., the percentage of strains for which growth in suspension was suppressed for 24 h. The KHR of the wild type phages ranged from 2% to 49%, substantially lower than the PHRs. To improve the KHR and other key pharmaceutical properties, we bred the phages by mixing and propagating cocktails on a subset of S. aureus strains. These bred phages, which we termed evolution-squared (ε2) phages, have broader KHRs up to 64% and increased virulence compared to the ancestors. The ε2-phages with the broadest KHR have genomes intercrossed from up to three different ancestors. We composed a cocktail of three ε2-phages with an overall KHR of 92% and PHR of 96% on 110 S. aureus strains and called it PM-399. PM-399 has a lower propensity to resistance formation than the standard of care antibiotics vancomycin, rifampicin, or their combination, and no resistance was observed in laboratory settings (detection limit: 1 cell in 1011). In summary, ε2-phages and, in particular PM-399, are promising candidates for an alternative treatment of S. aureus infections.

Preventing lung pathology and mortality in rabbit Staphylococcus aureus pneumonia models with cytotoxin-neutralizing monoclonal IgGs penetrating the epithelial lining fluid.

Staphylococcus aureus pneumonia is associated with high mortality irrespective of antibiotic susceptibility. Both MRSA and MSSA strains produce powerful cytotoxins: alpha-hemolysin(Hla) and up to five leukocidins - LukSF-PV, HlgAB, HlgCB, LukED and LukGH (LukAB) - to evade host innate defense mechanisms. Neutralizing cytotoxins has been shown to provide survival benefit in rabbit S. aureus pneumonia models. We studied the mechanisms of protection of ASN100, a combination of two human monoclonal antibodies (mAbs), ASN-1 and ASN-2, that together neutralize Hla and the five leukocidins, in rabbit MRSA and MSSA pneumonia models. Upon prophylactic passive immunization, ASN100 displayed dose-dependent increase in survival and was fully protective against all S. aureus strains tested at 5 or 20 mg/kg doses. Macroscopic and microscopic lung pathology, edema rate, and bacterial burden were evaluated 12 hours post infection and reduced by ASN100. Pharmacokinetic analysis of ASN100 in bronchoalveolar-lavage fluid from uninfected animals detected efficient penetration to lung epithelial lining fluid reaching peak levels between 24 and 48 hours post dosing that were comparable to the mAb concentration measured in serum. These data confirm that the ASN100 mAbs neutralize the powerful cytotoxins of S. aureus in the lung and prevent damage to the mucosal barrier and innate immune cells.

Disarming Staphylococcus aureus from destroying human cells by simultaneously neutralizing six cytotoxins with two human monoclonal antibodies.

Pathogenesis of Staphylococcus aureus is increasingly recognized to be driven by powerful toxins. Staphylococcus aureus employs up to six pore-forming toxins to subvert the human host defense and to promote bacterial invasion: alpha-hemolysin that disrupts epithelial and endothelial barriers and five leukocidins that lyse phagocytes involved in bacterial clearance. Previously, we described two human monoclonal antibodies (mAbs), ASN-1 that neutralizes alpha-hemolysin and four leukocidins (LukSF-PV, LukED, HlgAB, HlgCB), and ASN-2 that inactivates the 5th leukocidin, LukGH. In this study we tested the individual and combined effects of ASN-1 and ASN-2 in multiple in vitro models employing relevant human target cells. We found that diverse S. aureus isolates with different genetic backgrounds (based on MLST- and spa-typing) and antibiotic sensitivity (both MRSA and MSSA) displayed greatly different cytotoxin expression patterns influenced by the type of growth medium used. Both mAbs were required to fully prevent the lysis of human neutrophils exposed to the mixture of recombinant cytotoxins or native toxins present in the culture supernatants of S. aureus isolates. Flow cytometry confirmed the protective effects of ASN-1 + ASN-2 (known as ASN100) on granulocytes, monocytes, NK-cells and T-lymphocytes. ASN-1 alone preserved the integrity of a 3D-primary culture of human tracheal/bronchial mucociliary epithelial tissue infected with S. aureus. We conclude that simultaneous inhibition of alpha-hemolysin and five leukocidins by ASN100 blocks cytolytic activity of S. aureus towards human target cells in vitro.

Selective sensitization of human neutrophils to LukGH mediated cytotoxicity by Staphylococcus aureus and IL-8.

OBJECTIVES: Staphylococcus aureus produces up to five bi-component leukocidins - LukSF-PV, gamma-hemolysins AB and CB, LukGH (LukAB) and LukED - to evade innate immunity by lysing phagocytic cells. Species specificity of these leukocidins limits the relevance of animal models, therefore we assessed their individual contribution using human neutrophils. METHODS: Human polymorphonuclear leukocytes (PMNs) were activated with stimuli relevant during bacterial infections and sensitivity to recombinant leukocidins was measured in cell-viability assays. Leukocidin receptor expression was quantified by flow cytometry. RESULTS: We observed greatly variable sensitivities of different PMN preparations towards LukGH. Activation of PMNs by lipopolysaccharide (LPS) or S. aureus culture supernatant (CS) lacking all leukocidins resulted in higher surface expression of CD11b, the LukGH receptor, and greatly enhanced the sensitivity towards LukGH, eliminating the variability observed with unstimulated cells. In contrast, CS induced a decrease in sensitivity of PMNs to the other four leukocidins and reduced surface staining for their cognate receptors (CXCR1, CXCR2, C5aR, C5L2). Delta-toxin and peptidoglycan mimicked the effect of CS. Moreover, IL-8, an important cytokine in neutrophil activation, also selectively increased LukGH sensitivity. Deletion of lukGH, but not other leukocidin genes, prevented PMN killing upon infection with USA300 CA-MRSA. CONCLUSION: Inflammatory signals enhance the susceptibility of human PMNs to lysis by LukGH rendering this toxin dominant among the S. aureus leukocidins in vitro.

Endotoxin neutralization by an O-antigen specific monoclonal antibody: A potential novel therapeutic approach against Klebsiella pneumoniae ST258.

Klebsiella pneumoniae ST258 is a globally distributed multi-drug resistant pathogen responsible for severe invasive infections. In this study, the different virulence potential of K. pneumoniae ST258 isolates in endotoxin susceptible versus resistant animal models was shown. Furthermore, ST258 clinical isolates were found highly sensitive to the bactericidal effect of naive animal and human serum. These observations imply that LPS, released from the rapidly lysed bacteria, may contribute to the high mortality associated with ST258 bacteremia cases. A humanized version (mAb A1102) of a previously described murine mAb specific for the conserved LPS O-antigen, was tested for endotoxin neutralization. A1102 was able to neutralize TLR-4 activation by ST258-derived LPS in vitro with an efficacy exceeding that of polymyxin B by 3 orders of magnitude. Passive immunization with A1102 afforded a significant level of protection in a galactosamine-sensitized mouse model of endotoxemia, induced by ST258-derived LPS, or upon challenge with live bacteria. Efficacy was retained using an aglycosylated IgG, as well as upon complement depletion, suggesting that Fc-independent endotoxin neutralization may be the main protective mechanism in this model, in spite of the complement-dependent bactericidal and opsonic activities additionally observed for A1102 in vitro. Furthermore, rabbits that are naturally highly susceptible to endotoxin, were also significantly protected by low doses of A1102 when challenged with an ST258 strain. Given this unique mode of action and the high protective efficacy of this mAb, passive immunization, as prophylactic or adjunct therapeutic approach for the treatment of infections caused by ST258 isolates should be considered.

Improved Protection in a Rabbit Model of Community-Associated Methicillin-Resistant Staphylococcus aureus Necrotizing Pneumonia upon Neutralization of Leukocidins in Addition to Alpha-Hemolysin.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), especially the USA300 pulsotype, is a frequent cause of skin and soft tissue infections and severe pneumonia. Despite appropriate antibiotic treatment, complications are common and pneumonia is associated with high mortality. S. aureus strains express multiple cytotoxins, including alpha-hemolysin (Hla) and up to five bicomponent leukocidins that specifically target phagocytic cells for lysis. CA-MRSA USA300 strains carry the genes for all six cytotoxins. Species specificity of the leukocidins greatly contributes to the ambiguity regarding their role in S. aureus pathogenesis. We performed a comparative analysis of the leukocidin susceptibility of human, rabbit, and mouse polymorphonuclear leukocytes (PMNs) to assess the translational value of mouse and rabbit S. aureus models. We found that mouse PMNs were largely resistant to LukSF-PV, HlgAB, and HlgCB and susceptible only to LukED, whereas rabbit and human PMNs were highly sensitive to all these cytotoxins. In the rabbit pneumonia model with a USA300 CA-MRSA strain, passive immunization with a previously identified human monoclonal antibody (MAb), Hla-F#5, which cross-neutralizes Hla, LukSF-PV, HlgAB, HlgCB, and LukED, provided full protection, whereas an Hla-specific MAb was only partially protective. In the mouse USA300 CA-MRSA pneumonia model, both types of antibodies demonstrated full protection, suggesting that Hla, but not leukocidin(s), is the principal virulence determinant in mice. As the rabbit recapitulates the high susceptibility to leukocidins characteristic of humans, this species represents a valuable model for assessing novel, cytotoxin-targeting anti-S. aureus therapeutic approaches.

Bactericidal monoclonal antibodies specific to the lipopolysaccharide O antigen from multidrug-resistant Escherichia coli clone ST131-O25b:H4 elicit protection in mice.

The Escherichia coli sequence type 131 (ST131)-O25b:H4 clone has spread worldwide and become responsible for a significant proportion of multidrug-resistant extraintestinal infections. We generated humanized monoclonal antibodies (MAbs) that target the lipopolysaccharide O25b antigen conserved within this lineage. These MAbs bound to the surface of live bacterial cells irrespective of the capsular type expressed. In a serum bactericidal assay in vitro, MAbs induced >95% bacterial killing in the presence of human serum as the complement source. Protective efficacy at low antibody doses was observed in a murine model of bacteremia. The mode of action in vivo was investigated by using aglycosylated derivatives of the protective MAbs. The significant binding to live E. coli cells and the in vitro and in vivo efficacy were corroborated in assays using bacteria grown in human serum to mimic relevant clinical conditions. Given the dry pipeline of novel antibiotics against multidrug-resistant Gram-negative pathogens, passive immunization with bactericidal antibodies offers a therapeutic alternative to control infections caused by E. coli ST131-O25b:H4.

Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.

Monoclonal antibodies targeting different cell wall antigens of group B streptococcus mediate protection in both Fc-dependent and independent manner.

Group B streptococcus remains an important neonatal pathogen in spite of widely adopted intrapartum antibiotic administration; therefore immune prophylaxis for GBS infections is highly warranted. In passive immunization and lethal challenge studies with multiple GBS strains, we characterized the protective effect of rabbit polyclonal and murine monoclonal antibodies specific for four multi-functional cell wall anchored proteins, FbsA, BibA, PilA and PilB. Single specificity rabbit sera or mAbs induced high level, but strain dependent protection, while their combinations resulted in superior and broad efficacy against all GBS strains tested. Polyclonal and monoclonal antibodies specific for the pilus proteins exerted very potent opsonophagocytic killing activity in vitro and required the Fc domain for protection in vivo. In contrast, FbsA and BibA specific antibodies failed to show OPK activity, but their Fab fragments fully protected animals, suggesting that blocking the function of these proteins was the major mode of action. These data are supportive for developing immune prophylaxis with human mAbs for prematurely born neonates who receive low levels of antibodies by maternofetal transport and are characterized by not fully developed phagocytic and complement activity.

Immunological fingerprinting of group B streptococci: from circulating human antibodies to protective antigens.

Group B streptococcus is one of the most important pathogens in neonates, and causes invasive infections in non-pregnant adults with underlying diseases. Applying a genomic approach that relies on human antibodies we identified antigenic GBS proteins, among them most of the previously published protective antigens. In vitro analyses allowed the selection of conserved candidate antigens that were further evaluated in murine lethal sepsis models using several GBS strains. In active and passive immunization models, we identified four protective GBS antigens, FbsA and BibA, as well as two hypothetical proteins, all shown to contribute to virulence based on gene deletion mutants. These protective antigens have the potential to be components of novel vaccines or targets for passive immune prophylaxis against GBS disease.