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.

Humoral immune responses to microbial polysaccharide surface antigens can prevent bacterial infection but are typically strain specific and fail to mediate broad protection against different serotypes. Here we describe a panel of affinity-matured monoclonal human antibodies from peripheral blood immunoglobulin M-positive (IgM) and IgA memory B cells and clonally related intestinal plasmablasts, directed against the lipopolysaccharide (LPS) O-antigen of Klebsiella pneumoniae, an opportunistic pathogen and major cause of antibiotic-resistant nosocomial infections. The antibodies showed distinct patterns of in vivo cross-specificity and protection against different clinically relevant K. pneumoniae serotypes. However, cross-specificity was not limited to K. pneumoniae, as K. pneumoniae-specific antibodies recognized diverse intestinal microbes and neutralized not only K. pneumoniae LPS but also non-K. pneumoniae LPS. Our data suggest that the recognition of minimal glycan epitopes abundantly expressed on microbial surfaces might serve as an efficient humoral immunological mechanism to control invading pathogens and the large diversity of the human microbiota with a limited set of cross-specific antibodies.

Background:LukGH is a member of the family of two-component bacterial toxins of Staphylococcus aureus that lyse human phagocytic cells. Results:The crystal structure of LukGH and mutagenesis revealed the molecular basis for heterodimer formation in solution. Conclusion:LukGH differs from other two-component leukocidins that interact only upon cell contact. Significance:These data might assist with development of therapeutics that counteract Staphylococcus aureus pathogenesis.

b 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.

MSSA strains with strong blood agar hemolysis and high α-hemolysin activity are markers for VAP, but not VAT, and might be considered in differential diagnosis and initiation of therapy.

LukGH (LukAB) is a potent leukocidin of Staphylococcus aureus that lyses human phagocytic cells and is thought to contribute to immune evasion. Unlike the other bi-component leukocidins of S. aureus, LukGH forms a heterodimer before binding to its receptor, CD11b expressed on professional phagocytic cells, and displays significant sequence variation. We employed a high diversity human IgG1 library presented on yeast cells to discover monoclonal antibodies (mAbs) neutralizing the cytolytic activity of LukGH. Recombinant LukG and LukH monomers or a LukGH dimer were used as capture antigens in the library selections. We found that mAbs identified with LukG or LukH as bait had no or very low toxin neutralization potency. In contrast, LukGH dimer-selected antibodies proved to be highly potent, and several mAbs were able to neutralize even the most divergent LukGH variants. Based on biolayer interferometry and mesoscale discovery, the high affinity antibody binding site on the LukGH complex was absent on the individual monomers, suggesting that it was generated upon formation of the LukG-LukH dimer. X-ray crystallography analysis of the complex between the LukGH dimer and the antigen-binding fragment of a very potent mAb (PDB code 5K59) indicated that the epitope is located in the predicted cell binding region (rim domain) of LukGH. The corresponding IgG inhibited the binding of LukGH dimer to target cells. Our data suggest that knowledge of the native conformation of target molecules is essential to generate high affinity and functional mAbs.

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.

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.