Antibody-Based Immunotherapy To Treat and Prevent Infection with Hypervirulent Klebsiella pneumoniae

ABSTRACT Hypervirulent Klebsiella pneumoniae (hvKp) strains are predicted to become a major threat in Asia if antibiotic resistance continues to spread. Anticapsular antibodies (Abs) were developed because disseminated infections caused by hvKp are associated with significant morbidity and mortality, even with antibiotic-sensitive strains. K1-serotype polysaccharide capsules (K1-CPS) are expressed by the majority of hvKp strains. In this study, K1-CPS-specific IgG Abs were generated by conjugation of K1-CPS to immunogenic anthrax protective antigen (PA) protein. Opsonophagocytic efficacy was measured in vitro and in vivo by intravital microscopy in murine livers. In vivo protection was tested in murine models, including a novel model for dissemination in hvKp-colonized mice. Protective efficacy of monoclonal antibodies (MAbs) 4C5 (IgG1) and 19A10 (IgG3) was demonstrated both in murine sepsis and pulmonary infection. In hvKp-colonized mice, MAb treatment significantly decreased dissemination of hvKp from the gut to mesenteric lymph nodes and organs. Intravital microscopy confirmed efficient opsonophagocytosis and clearance of bacteria from the liver. In vitro studies demonstrate that MAbs work predominantly by promoting FcR-mediated phagocytosis but also indicate that MAbs enhance the release of neutrophil extracellular traps (NETs). In anticipation of increasing antibiotic resistance, we propose further development of these and other Klebsiella-specific MAbs for therapeutic use.

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Combinations of Monoclonal Antibodies to Anthrax Toxin Manifest New Properties in Neutralization Assays

Infection and Immunity ◽

10.1128/iai.01328-12 ◽

2013 ◽

Vol 81 (6) ◽

pp. 1880-1888 ◽

Cited By ~ 13

Author(s):

Mary Ann Pohl ◽

Johanna Rivera ◽

Antonio Nakouzi ◽

Siu-Kei Chow ◽

Arturo Casadevall

Keyword(s):

Monoclonal Antibodies ◽

Protective Antigen ◽

Protective Efficacy ◽

Lethal Factor ◽

Current Treatment ◽

Emergent Properties ◽

Content Type ◽

Effective Form

ABSTRACTMonoclonal antibodies (MAbs) are potential therapeutic agents againstBacillus anthracistoxins, since there is no current treatment to counteract the detrimental effects of toxemia. In hopes of isolating new protective MAbs to the toxin component lethal factor (LF), we used a strain of mice (C57BL/6) that had not been used in previous studies, generating MAbs to LF. Six LF-binding MAbs were obtained, representing 3 IgG isotypes and one IgM. One MAb (20C1) provided protection from lethal toxin (LeTx) in anin vitromouse macrophage system but did not provide significant protectionin vivo. However, the combination of two MAbs to LF (17F1 and 20C1) provided synergistic increases in protection bothin vitroandin vivo. In addition, when these MAbs were mixed with MAbs to protective antigen (PA) previously generated in our laboratory, these MAb combinations produced synergistic toxin neutralizationin vitro. But when 17F1 was combined with another MAb to LF, 19C9, the combination resulted in enhanced lethal toxicity. While no single MAb to LF provided significant toxin neutralization, LF-immunized mice were completely protected from infection withB. anthracisstrain Sterne, which suggested that a polyclonal response is required for effective toxin neutralization. In total, these studies show that while a single MAb against LeTx may not be effective, combinations of multiple MAbs may provide the most effective form of passive immunotherapy, with the caveat that these may demonstrate emergent properties with regard to protective efficacy.

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Vaccination against Anthrax with Attenuated Recombinant Strains of Bacillus anthracis That Produce Protective Antigen

Infection and Immunity ◽

10.1128/iai.67.2.562-567.1999 ◽

1999 ◽

Vol 67 (2) ◽

pp. 562-567 ◽

Cited By ~ 53

Author(s):

John P. Barnard ◽

Arthur M. Friedlander

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Plasmid Stability ◽

Protective Efficacy ◽

Live Vaccines ◽

Antibody Titers ◽

Anthrax Protective Antigen ◽

Anthrax Vaccines

ABSTRACT The protective efficacy of several live, recombinant anthrax vaccines given in a single-dose regimen was assessed with Hartley guinea pigs. These live vaccines were created by transforming ΔANR and ΔSterne, two nonencapsulated, nontoxinogenic strains of Bacillus anthracis, with four different recombinant plasmids that express the anthrax protective antigen (PA) protein to various degrees. This enabled us to assess the effect of the chromosomal background of the strain, as well as the amount of PA produced, on protective efficacy. There were no significant strain-related effects on PA production in vitro, plasmid stability in vivo, survival of the immunizing strain in the host, or protective efficacy of the immunizing infection. The protective efficacy of the live, recombinant anthrax vaccine strains correlated with the anti-PA antibody titers they elicited in vivo and the level of PA they produced in vitro.

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Doripenem MICs andompK36Porin Genotypes of Sequence Type 258, KPC-Producing Klebsiella pneumoniae May Predict Responses to Carbapenem-Colistin Combination Therapy among Patients with Bacteremia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03894-14 ◽

2014 ◽

Vol 59 (3) ◽

pp. 1797-1801 ◽

Cited By ~ 23

Author(s):

Ryan K. Shields ◽

M. Hong Nguyen ◽

Brian A. Potoski ◽

Ellen G. Press ◽

Liang Chen ◽

...

Keyword(s):

Amino Acids ◽

Combination Therapy ◽

Klebsiella Pneumoniae ◽

Sequence Type ◽

Content Type

ABSTRACTTreatment failures of a carbapenem-colistin regimen among patients with bacteremia due to sequence type 258 (ST258), KPC-2-producingKlebsiella pneumoniaewere significantly more likely if both agents were inactivein vitro, as defined by a colistin MIC of >2 μg/ml and the presence of either a majorompK36porin mutation (guanine and alanine insertions at amino acids 134 and 135 [ins aa 134–135 GD], IS5promoter insertion [P= 0.007]) or a doripenem MIC of >8 μg/ml (P= 0.01). MajorompK36mutations among KPC-K. pneumoniaestrains are important determinants of carbapenem-colistin responsesin vitroandin vivo.

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In VitroandIn VivoActivities of OP0595, a New Diazabicyclooctane, against CTX-M-15-Positive Escherichia coli and KPC-Positive Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02704-15 ◽

2016 ◽

Vol 60 (5) ◽

pp. 3001-3006 ◽

Cited By ~ 11

Author(s):

Akihiro Morinaka ◽

Yuko Tsutsumi ◽

Keiko Yamada ◽

Yoshihiro Takayama ◽

Shiro Sakakibara ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Infection Model ◽

Gram Negative Bacteria ◽

Content Type ◽

Effective Combination ◽

Time Kill ◽

Lactamase Inhibitor

ABSTRACTGram-negative bacteria are evolving to produce β-lactamases of increasing diversity that challenge antimicrobial chemotherapy. OP0595 is a new diazabicyclooctane serine β-lactamase inhibitor which acts also as an antibiotic and as a β-lactamase-independent β-lactam “enhancer” againstEnterobacteriaceae. Here we determined the optimal concentration of OP0595 in combination with piperacillin, cefepime, and meropenem, in addition to the antibacterial activity of OP0595 alone and in combination with cefepime, inin vitrotime-kill studies and anin vivoinfection model against five strains of CTX-M-15-positiveEscherichia coliand five strains of KPC-positiveKlebsiella pneumoniae. An OP0595 concentration of 4 μg/ml was found to be sufficient for an effective combination with all three β-lactam agents. In bothin vitrotime-kill studies and anin vivomodel of infection, cefepime-OP0595 showed stronger efficacy than cefepime alone against all β-lactamase-positive strains tested, whereas OP0595 alone showed weaker or no efficacy. Taken together, these data indicate that combinational use of OP0595 and a β-lactam agent is important to exert the antimicrobial functions of OP0595.

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Mouse Monoclonal Antibodies to Anthrax Edema Factor Protect against Infection

Infection and Immunity ◽

10.1128/iai.05314-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4609-4616 ◽

Cited By ~ 20

Author(s):

Clinton E. Leysath ◽

Kuang-Hua Chen ◽

Mahtab Moayeri ◽

Devorah Crown ◽

Rasem Fattah ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Protective Antigen ◽

Lethal Factor ◽

Essential Element ◽

Content Type ◽

Time To Death ◽

Edema Factor ◽

Edema Toxin

ABSTRACTBacillus anthracisis the causative agent of anthrax, and the tripartite anthrax toxin is an essential element of its pathogenesis. Edema factor (EF), a potent adenylyl cyclase, is one of the toxin components. In this work, anti-EF monoclonal antibodies (MAb) were produced following immunization of mice, and four of the antibodies were fully characterized. MAb 3F2 has an affinity of 388 pM, was most effective for EF detection, and appears to be the first antibody reported to neutralize EF by binding to the catalytic CBdomain. MAb 7F10 shows potent neutralization of edema toxin activityin vitroandin vivo; it targets the N-terminal protective antigen binding domain. The four MAb react with three different domains of edema factor, and all were able to detect purified edema factor in Western blot analysis. None of the four MAb cross-reacted with the lethal factor toxin component. Three of the four MAb protected mice in both a systemic edema toxin challenge model and a subcutaneous spore-induced foreleg edema model. A combination of three of the MAb also significantly delayed the time to death in a third subcutaneous spore challenge model. This appears to be the first direct evidence that monoclonal antibody-mediated neutralization of EF alone is sufficient to delay anthrax disease progression.

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Serum Antibody Responses against Carbapenem-Resistant Klebsiella pneumoniae in Infected Patients

mSphere ◽

10.1128/msphere.01335-20 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Kasturi Banerjee ◽

Michael P. Motley ◽

Elizabeth Diago-Navarro ◽

Bettina C. Fries

Keyword(s):

Klebsiella Pneumoniae ◽

Capsular Polysaccharide ◽

Serum Antibody ◽

Antibody Responses ◽

Capsular Polysaccharides ◽

Content Type ◽

Carbapenem Resistant ◽

Reactive Properties

ABSTRACT Capsular polysaccharide (CPS) heterogeneity within carbapenem-resistant Klebsiella pneumoniae (CR-Kp) strain sequence type 258 (ST258) must be considered when developing CPS-based vaccines. Here, we sought to characterize CPS-specific antibody responses elicited by CR-Kp-infected patients. Plasma and bacterial isolates were collected from 33 hospital patients with positive CR-Kp cultures. Isolate capsules were typed by wzi sequencing. Reactivity and measures of efficacy of patient antibodies were studied against 3 prevalent CR-Kp CPS types (wzi29, wzi154, and wzi50). High IgG titers against wzi154 and wzi50 CPS were documented in 79% of infected patients. Patient-derived (PD) IgGs agglutinated CR-Kp and limited growth better than naive IgG and promoted phagocytosis of strains across the serotype isolated from their donors. Additionally, poly-IgG from wzi50 and wzi154 patients promoted phagocytosis of nonconcordant CR-Kp serotypes. Such effects were lost when poly-IgG was depleted of CPS-specific IgG. Additionally, mice infected with wzi50, wzi154, and wzi29 CR-Kp strains preopsonized with wzi50 patient-derived IgG exhibited lower lung CFU than controls. Depletion of wzi50 antibodies (Abs) reversed this effect in wzi50 and wzi154 infections, whereas wzi154 Ab depletion reduced poly-IgG efficacy against wzi29 CR-Kp. We are the first to report cross-reactive properties of CPS-specific Abs from CR-Kp patients through both in vitro and in vivo models. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is a rapidly emerging public health threat that can cause fatal infections in up to 50% of affected patients. Due to its resistance to nearly all antimicrobials, development of alternate therapies like antibodies and vaccines is urgently needed. Capsular polysaccharides constitute important targets, as they are crucial for Klebsiella pneumoniae pathogenesis. Capsular polysaccharides are very diverse and, therefore, studying the host’s capsule-type specific antibodies is crucial to develop effective anti-CPS immunotherapies. In this study, we are the first to characterize humoral responses in infected patients against carbapenem-resistant Klebsiella pneumoniae expressing different wzi capsule types. This study is the first to report the efficacy of cross-reactive properties of CPS-specific Abs in both in vitro and in vivo models.

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Strong Environment-Genotype Interactions Determine the Fitness Costs of Antibiotic Resistance In Vitro and in an Insect Model of Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01033-20 ◽

2020 ◽

Vol 64 (10) ◽

Author(s):

C. James Manktelow ◽

Elitsa Penkova ◽

Lucy Scott ◽

Andrew C. Matthews ◽

Ben Raymond

Keyword(s):

Antibiotic Resistance ◽

Resistance Mutations ◽

Fitness Costs ◽

Mechanisms Of Resistance ◽

Content Type ◽

Poor Predictor ◽

Fitness Consequences ◽

Rifampin Resistance

ABSTRACT The acquisition of antibiotic resistance commonly imposes fitness costs, a reduction in the fitness of bacteria in the absence of drugs. These costs have been quantified primarily using in vitro experiments and a small number of in vivo studies in mice, and it is commonly assumed that these diverse methods are consistent. Here, we used an insect model of infection to compare the fitness costs of antibiotic resistance in vivo to those in vitro. Experiments explored diverse mechanisms of resistance in a Gram-positive pathogen, Bacillus thuringiensis, and a Gram-negative intestinal symbiont, Enterobacter cloacae. Rifampin resistance in B. thuringiensis showed fitness costs that were typically elevated in vivo, although these were modulated by genotype-environment interactions. In contrast, resistance to cefotaxime via derepression of AmpC β-lactamase in E. cloacae resulted in no detectable costs in vivo or in vitro, while spontaneous resistance to nalidixic acid, and carriage of the IncP plasmid RP4, imposed costs that increased in vivo. Overall, fitness costs in vitro were a poor predictor of fitness costs in vivo because of strong genotype-environment interactions throughout this study. Insect infections provide a cheap and accessible means of assessing the fitness consequences of resistance mutations, data that are important for understanding the evolution and spread of resistance. This study emphasizes that the fitness costs imposed by particular mutations or different modes of resistance are extremely variable and that only a subset of these mutations is likely to be prevalent outside the laboratory.

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Anthrax Protective Antigen Cleavage and Clearance from the Blood of Mice and Rats

Infection and Immunity ◽

10.1128/iai.00719-07 ◽

2007 ◽

Vol 75 (11) ◽

pp. 5175-5184 ◽

Cited By ~ 56

Author(s):

Mahtab Moayeri ◽

Jason F. Wiggins ◽

Stephen H. Leppla

Keyword(s):

Receptor Binding ◽

Protective Antigen ◽

Lethal Factor ◽

Dominant Negative ◽

Furin Cleavage ◽

Defective Mutant ◽

Tissue Surfaces ◽

Anthrax Protective Antigen

ABSTRACT Bacillus anthracis protective antigen (PA) is an 83-kDa (PA83) protein that is cleaved to the 63-kDa protein (PA63) as an essential step in binding and internalizing lethal factor (LF). To assess in vivo receptor saturating PA concentrations, we injected mice with PA variants and measured the PA remaining in the blood at various times using PA83- and PA63-specific enzyme-linked immunosorbent assays. We found that both wild-type PA (WT-PA) and a receptor-binding-defective mutant (Ub-PA) were cleaved to PA63 independent of their ability to bind cells. This suggested a PA-acting protease activity in the blood. The protease cleaved PA at the furin cleavage sequence because furin site-modified PA mutants were not cleaved. Cleavage measured in vitro was leupeptin sensitive and dependent on calcium. Cell surface cleavage was important for toxin clearance, however, as Ub-PA and uncleavable PA mutants were cleared at slower rates than WT-PA. The cell binding-independent cleavage of PA was also verified by using Ub-PA (which is still cleaved) to rescue mice from toxin challenge by competitively binding circulating LF. This mutant was able to rescue mice even when given 12 h before toxin challenge. Its therapeutic ability was comparable to that of dominant-negative PA, which binds cells but does not allow LF translocation, and to the protection afforded through receptor clearance by WT-PA and uncleavable receptor binding-competent mutants. The PA cleavage and clearance observed in mice did not appear to have a role in the differential mouse susceptibility as it occurred similarly in lethal toxin (LT)-resistant DBA/2J and LT-sensitive BALB/cJ mice. Interestingly, PA63 was not found in LT-resistant or -sensitive rats and PA83 clearance was slower in rats than in mice. Finally, to determine the minimum amount of PA required in circulation for LT toxicity in mice, we administered time-separated injections of PA and LF and showed that lethality of LF for mice after PA was no longer measurable in circulation, suggesting active PA sequestration at tissue surfaces.

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Broad-Spectrum β-Lactam Antibiotics for Treating Experimental Peritonitis in Mice Due to Klebsiella pneumoniae Producing the Carbapenemase OXA-48

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06069-11 ◽

2012 ◽

Vol 56 (5) ◽

pp. 2759-2760 ◽

Cited By ~ 27

Author(s):

Olivier Mimoz ◽

Nicolas Grégoire ◽

Laurent Poirel ◽

Manuella Marliat ◽

William Couet ◽

...

Keyword(s):

Single Dose ◽

Klebsiella Pneumoniae ◽

Broad Spectrum ◽

Content Type ◽

Extended Spectrum ◽

Experimental Peritonitis ◽

Peritonitis Model

ABSTRACTA lethal peritonitis model was induced in mice with aKlebsiella pneumoniaeisolate producing the carbapenemase OXA-48. Administration of a single dose (up to 100 mg/kg) of the antibiotic piperacillin-tazobactam, imipenem-cilastatin, ertapenem, or cefotaxime had little or no impact on lethality. Ceftazidime had the highest efficacyin vivo, which mirrored itsin vitroactivity; this was not the case for carbapenems. Therefore, ceftazidime may be recommended for the treatment of infections due to OXA-48 producers if they do not coproduce an extended-spectrum β-lactamase or a plasmid-mediated AmpC cephalosporinase.

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Immunogenicity and Protective Efficacy against Enterotoxigenic Escherichia coli Colonization following Intradermal, Sublingual, or Oral Vaccination with EtpA Adhesin

Clinical and Vaccine Immunology ◽

10.1128/cvi.00248-16 ◽

2016 ◽

Vol 23 (7) ◽

pp. 628-637 ◽

Cited By ~ 16

Author(s):

Qingwei Luo ◽

Tim J. Vickers ◽

James M. Fleckenstein

Keyword(s):

Escherichia Coli ◽

Vaccine Development ◽

Protective Antigen ◽

Protective Efficacy ◽

Enterotoxigenic Escherichia Coli ◽

Oral Vaccination ◽

Content Type ◽

Degree Of Protection ◽

Alternative Routes

EnterotoxigenicEscherichia coli(ETEC) strains are a common cause of diarrhea. Extraordinary antigenic diversity has prompted a search for conserved antigens to complement canonical approaches to ETEC vaccine development. EtpA, an immunogenic extracellular ETEC adhesin relatively conserved in the ETEC pathovar, has previously been shown to be a protective antigen following intranasal immunization. These studies were undertaken to explore alternative routes of EtpA vaccination that would permit use of a double mutant (R192G L211A) heat-labile toxin (dmLT) adjuvant. Here, oral vaccination with EtpA adjuvanted with dmLT afforded significant protection against small intestinal colonization, and the degree of protection correlated with fecal IgG, IgA, or total fecal antibody responses to EtpA. Sublingual vaccination yielded compartmentalized mucosal immune responses with significant increases in anti-EtpA fecal IgG and IgA, and mice vaccinated via this route were also protected against colonization. In contrast, while intradermal (i.d.) vaccination achieved high levels of both serum and fecal antibodies against both EtpA and dmLT, mice vaccinated via the i.d. route were not protected against subsequent colonization and the avidity of serum IgG and IgA EtpA-specific antibodies was significantly lower after i.d. immunization compared to other routes. Finally, we demonstrate that antiserum from vaccinated mice significantly impairs binding of LT to cognate GM1 receptors and shows near complete neutralization of toxin delivery by ETECin vitro. Collectively, these data provide further evidence that EtpA could complement future vaccine strategies but also suggest that additional effort will be required to optimize its use as a protective immunogen.

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