Cross-specificity of protective human antibodies against Klebsiella pneumoniae LPS O-antigen.

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.

Multiple Modes of Action of a Monoclonal Antibody against Multidrug-Resistant Escherichia coli Sequence Type 131-H30.

The multidrug-resistant H30 subclone of extraintestinal pathogenic Escherichia coli sequence type 131 (ST131-H30) has spread worldwide. This clone expresses a conserved lipopolysaccharide (LPS) O antigen, O25b. Previously, we described monoclonal antibodies (MAbs) specific to the O25b antigen and characterized them as diagnostic and therapeutic tools. In this study, evidence is provided that besides the previously shown complement-mediated bactericidal effect, an O25b-specific humanized MAb, A1124, also enhances opsonophagocytic uptake by the murine macrophage cell line RAW 264.7. Both phagocyte-dependent killing and phagocyte-independent killing, triggered by A1124, were confirmed in human whole blood. Furthermore, A1124 was shown to neutralize endotoxin activity of purified LPS of clinical isolates. This activity was demonstrated in vitro using both RAW 264.7 cells and a human Toll-like receptor 4 (TLR4) reporter cell line, as well as in a murine model of endotoxemia using purified LPS for challenge. Significant protective efficacy of A1124 at low doses (<1 mg/kg of body weight) was shown in murine and rat models of bacteremia. The contribution of the bactericidal and anti-inflammatory effects was dissected in the mouse bacteremia model through depletion of complement with cobra venom factor (CVF). Protective efficacy was lost in complement-depleted mice, suggesting the essential role of complement-mediated activities for protection in this model. These data suggest that A1124 exhibits different mechanisms of action, namely, direct complement-mediated and opsonophagocytic killing as well as endotoxin neutralization in various challenge models. Which of these activities are the most relevant in a clinical setting will need to be addressed by future translational studies.

Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.

Klebsiella pneumoniae ST258 is a globally disseminated, extremely drug resistant, nosocomial clone with limited treatment options. We show that the vast majority of ST258 isolates express modified d-galactan-I lipopolysaccharide O-antigen, termed hereinafter as D-galactan-III. The genetic determinant required for galactan-III synthesis was identified as a distinct operon adjacent to the rfb (wb) locus encoding D-galactan-I synthesis. The three genes within the operon encode predicted glycosyltransferases. Testing an isogenic transformant pair revealed that expression of D-galactan-III, in comparison to D-galactan-I, conferred improved survival in the presence of human serum. Eighty-three percent of the more than 200 ST258 draft genome sequences currently available carries the corresponding operon and hence these isolates are predicted to express galactan-III antigens. A D-galactan-III specific monoclonal antibody (mAb) was shown to bind to extracted LPS from a panel of ST258 isolates. The same mAb confirmed accessibility of galactan-III in surface staining of ST258 irrespective of the distinct capsular antigens expressed by both clades described previously. Based on these data, the galactan-III antigen may represent an attractive target for active and passive immunization approaches against K. pneumoniae ST258.

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.

Identification of d-Galactan-III As Part of the Lipopolysaccharide of Klebsiella pneumoniae Serotype O1.

Klebsiella pneumoniae is a Gram-negative, ubiquitous bacterium capable of causing severe nosocomial infections in individuals with impaired immune system. Emerging multi-drug resistant strains of this species and particularly carbapenem-resistant strains pose an urgent threat to public health. The lipopolysaccharide (LPS) O-antigen is the main surface antigen. It contributes to the virulence of this species and determines the O-serotype of K. pneumoniae isolates. Among the nine main O-serotypes of K. pneumoniae, O1-and O2-type pathogens are causative agents of over 50% of all infections. Serotype O1, the most common O-serotype, expresses complex LPS consisting of d-galactan-I (a polymer built of → 3)-ß-d-Galf-(1 → 3)-α-d-Galp-(1 → repeating units) capped by d-galactan-II (built of [ → 3)-α-d-Galp-(1 → 3)-ß-d-Galp-(1 →] repeating units). Galactan-I is present as the sole polymer in O2 serotype. Recently, in case of serotype O2, conversion of galactan-I to galactan-III (→ 3)-ß-d-Galf-(1 → 3)-[α-d-Galp-(1 → 4)]-α-d-Galp-(1 →) was reported. Substitution of → 3)-α-d-Galp by a branching terminal α-d-Galp was dependent on the presence of the gmlABC operon and had a major impact on the antigenicity of the galactan polymer. Genetic analysis indicated that 40% of the O1 clinical isolates also carry the gmlABC locus; therefore we aimed to characterize the corresponding phenotype of LPS O-antigens. The presence of galactan-III among O1 strains was proven using galactan-III-specific monoclonal antibodies and confirmed by structural analyses performed using sugar and methylation analysis as well as classical and high-resolution magic angle spinning NMR spectroscopy. By using an isogenic mutant pair, we demonstrated that galactan-III expression was dependent on the presence of glycosyltransferases encoded by gmlABC, as was shown previously for the O2 serotype. Furthermore, the galactan-II structures in O1gml+ strains remained unaffected corroborating no functional interactions between the biosynthesis of galactan-III and galactan-II polymers.

Discovery of monoclonal antibodies cross-reactive to novel subserotypes of K. pneumoniae O3.

Klebsiella pneumoniae is responsible for nosocomial infections causing significant morbidity and mortality. Treatment of newly emerging multi-drug resistant strains is hampered due to severely limited antibiotic choices. Passive immunization targeting LPS O-antigens has been proposed as an alternative therapeutic option, given the limited variability of Klebsiella O-antigens. Here we report that the O3 serogroup, previously considered to have uniform O-antigen built of mannan, represents three different subtypes differing in the number of mannose residues within the O-antigen repeating units. Genetic analysis of the genes encoding mannose polymerization revealed differences that underline the observed structural alterations. The O3 variants represent antigenically different types based on the different reactivity pattern of murine monoclonal antibodies raised against a K. pneumoniae O3 strain. Typing of a collection of K. pneumoniae O3 clinical isolates showed that strains expressing the novel O3b antigen, the tri-mannose form, were more prevalent than those having the penta-mannose form, traditionally called O3, while the tetra-mannose variant, termed here O3a, seems to be rare. A monoclonal antibody cross-reacting with all three O3 sub-serogroups was also selected and shown to bind to the surface of various K. pneumoniae strains expressing different O3 subtypes and capsular antigens.

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.

EVI1 inhibits apoptosis induced by antileukemic drugs via upregulation of CDKN1A/p21/WAF in human myeloid cells.

Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in acute myeloid leukemia (AML). Despite of its clinical importance, little is known about the mechanism through which EVI1 confers resistance to antileukemic drugs. Here, we show that a human myeloid cell line constitutively overexpressing EVI1 after infection with a retroviral vector (U937_EVI1) was partially resistant to etoposide and daunorubicin as compared to empty vector infected control cells (U937_vec). Similarly, inducible expression of EVI1 in HL-60 cells decreased their sensitivity to daunorubicin. Gene expression microarray analyses of U937_EVI1 and U937_vec cells cultured in the absence or presence of etoposide showed that 77 and 419 genes were regulated by EVI1 and etoposide, respectively. Notably, mRNA levels of 26 of these genes were altered by both stimuli, indicating that EVI1 regulated genes were strongly enriched among etoposide regulated genes and vice versa. One of the genes that were induced by both EVI1 and etoposide was CDKN1A/p21/WAF, which in addition to its function as a cell cycle regulator plays an important role in conferring chemotherapy resistance in various tumor types. Indeed, overexpression of CDKN1A in U937 cells mimicked the phenotype of EVI1 overexpression, similarly conferring partial resistance to antileukemic drugs.

The role of alveolar epithelial cells in initiating and shaping pulmonary immune responses: communication between innate and adaptive immune systems.

Macrophages and dendritic cells have been recognized as key players in the defense against mycobacterial infection. However, more recently, other cells in the lungs such as alveolar epithelial cells (AEC) have been found to play important roles in the defense and pathogenesis of infection. In the present study we first compared AEC with pulmonary macrophages (PuM) isolated from mice in their ability to internalize and control Bacillus Calmette-Guérin (BCG) growth and their capacity as APCs. AEC were able to internalize and control bacterial growth as well as present antigen to primed T cells. Secondly, we compared both cell types in their capacity to secrete cytokines and chemokines upon stimulation with various molecules including mycobacterial products. Activated PuM and AEC displayed different patterns of secretion. Finally, we analyzed the profile of response of AEC to diverse stimuli. AEC responded to both microbial and internal stimuli exemplified by TLR ligands and IFNs, respectively. The response included synthesis by AEC of several factors, known to have various effects in other cells. Interestingly, TNF could stimulate the production of CCL2/MCP-1. Since MCP-1 plays a role in the recruitment of monocytes and macrophages to sites of infection and macrophages are the main producers of TNF, we speculate that both cell types can stimulate each other. Also, another cell-cell interaction was suggested when IFNs (produced mainly by lymphocytes) were able to induce expression of chemokines (IP-10 and RANTES) by AEC involved in the recruitment of circulating lymphocytes to areas of injury, inflammation, or viral infection. In the current paper we confirm previous data on the capacity of AEC regarding internalization of mycobacteria and their role as APC, and extend the knowledge of AEC as a multifunctional cell type by assessing the secretion of a broad array of factors in response to several different types of stimuli.