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

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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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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Functional Analysis of Bacillus anthracis Protective Antigen by Using Neutralizing Monoclonal Antibodies

Infection and Immunity ◽

10.1128/iai.72.11.6313-6317.2004 ◽

2004 ◽

Vol 72 (11) ◽

pp. 6313-6317 ◽

Cited By ~ 71

Author(s):

Fabien Brossier ◽

Martine Lévy ◽

Annie Landier ◽

Pierre Lafaye ◽

Michèle Mock

Keyword(s):

Functional Analysis ◽

Monoclonal Antibodies ◽

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Cell Receptor ◽

Lethal Challenge ◽

Edema Factor

ABSTRACT Protective antigen (PA) is central to the action of the lethal and edema toxins produced by Bacillus anthracis. It is the common cell-binding component, mediating the translocation of the enzymatic moieties (lethal factor [LF] and edema factor) into the cytoplasm of the host cell. Monoclonal antibodies (MAbs) against PA, able to neutralize the activities of the toxins in vitro and in vivo, were screened. Two such MAbs, named 7.5 and 48.3, were purified and further characterized. MAb 7.5 binds to domain 4 of PA and prevents the binding of PA to its cell receptor. MAb 48.3 binds to domain 2 and blocks the cleavage of PA into PA63, a step necessary for the subsequent interaction with the enzymatic moieties. The epitope recognized by this antibody is in a region involved in the oligomerization of PA63; thus, MAb 48.3 does not recognize the oligomer form. MAbs 7.5 and 48.3 neutralize the activities of anthrax toxins produced by B. anthracis in mice. Also, there is an additive effect between the two MAbs against PA and a MAb against LF, in protecting mice against a lethal challenge by the Sterne strain. This work contributes to the functional analysis of PA and offers immunotherapeutic perspectives for the treatment of anthrax disease.

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00456-16 ◽

2016 ◽

Vol 24 (1) ◽

Cited By ~ 26

Author(s):

Elizabeth Diago-Navarro ◽

Isabel Calatayud-Baselga ◽

Donglei Sun ◽

Camille Khairallah ◽

Inderjit Mann ◽

...

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Intravital Microscopy ◽

Protective Antigen ◽

Protective Efficacy ◽

Mesenteric Lymph Nodes ◽

Content Type ◽

Anthrax Protective Antigen

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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In Vitro and In Vivo Characterization of Anthrax Anti-Protective Antigen and Anti-Lethal Factor Monoclonal Antibodies after Passive Transfer in a Mouse Lethal Toxin Challenge Model To Define Correlates of Immunity

Infection and Immunity ◽

10.1128/iai.00529-07 ◽

2007 ◽

Vol 75 (11) ◽

pp. 5443-5452 ◽

Cited By ~ 49

Author(s):

Herman F. Staats ◽

S. Munir Alam ◽

Richard M. Scearce ◽

Shaun M. Kirwan ◽

Julia Xianzhi Zhang ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Protective Antigen ◽

Lethal Factor ◽

Neutralization Assay ◽

Passive Transfer ◽

Lethal Toxin ◽

Significant Protection

ABSTRACT Passive transfer of antibody may be useful for preexposure prophylaxis against biological agents used as weapons of terror, such as Bacillus anthracis. Studies were performed to evaluate the ability of anthrax antiprotective antigen (anti-PA) and antilethal factor (anti-LF) neutralizing monoclonal antibodies (mAbs) to protect against an anthrax lethal toxin (LeTx) challenge in a mouse model and to identify correlates of immunity to LeTx challenge. Despite having similar affinities for their respective antigens, anti-PA (3F11) and anti-LF (9A11), passive transfer of up to 1.5 mg of anti-PA 3F11 mAb did not provide significant protection when transferred to mice 24 h before LeTx challenge, while passive transfer of as low as 0.375 mg of anti-LF 9A11 did provide significant protection. Serum collected 24 h after passive transfer had LeTx-neutralizing activity when tested using a standard LeTx neutralization assay, but neutralization titers measured using this assay did not correlate with protection against LeTx challenge. However, measurement of LeTx-neutralizing serum responses with an LeTx neutralization assay in vitro employing the addition of LeTx to J774A.1 cells 15 min before the addition of the serum did result in neutralization titers that correlated with protection against LeTx challenge. Our results demonstrate that only the LeTx neutralization titers measured utilizing the addition of LeTx to J774A.1 cells 15 min before the addition of sample correlated with protection in vivo. Thus, this LeTx neutralization assay may be a more biologically relevant neutralization assay to predict the in vivo protective capacity of LeTx-neutralizing antibodies.

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In VitroandIn VivoStudies of Monoclonal Antibodies with Prominent Bactericidal Activity against Burkholderia pseudomallei and Burkholderia mallei

Clinical and Vaccine Immunology ◽

10.1128/cvi.00533-10 ◽

2011 ◽

Vol 18 (5) ◽

pp. 825-834 ◽

Cited By ~ 32

Author(s):

Shimin Zhang ◽

Shaw-Huey Feng ◽

Bingjie Li ◽

Hyung-Yong Kim ◽

Joe Rodriguez ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Bactericidal Activity ◽

Burkholderia Pseudomallei ◽

Protective Efficacy ◽

Lethal Dose ◽

Burkholderia Mallei ◽

Content Type ◽

Bactericidal Activities

ABSTRACTOur laboratory has developed more than a hundred mouse monoclonal antibodies (MAbs) againstBurkholderia pseudomalleiandBurkholderia mallei. These antibodies have been categorized into different groups based on their specificities and the biochemical natures of their target antigens. The current study first examined the bactericidal activities of a number of these MAbs by anin vitroopsonic assay. Then, thein vivoprotective efficacy of selected MAbs was evaluated using BALB/c mice challenged intranasally with a lethal dose of the bacteria. The opsonic assay using dimethyl sulfoxide-treated human HL-60 cells as phagocytes revealed that 19 out of 47 tested MAbs (40%) have prominent bactericidal activities againstB. pseudomalleiand/orB. mallei. Interestingly, all MAbs with strong opsonic activities are those with specificity against either the capsular polysaccharides (PS) or the lipopolysaccharides (LPS) of the bacteria. On the other hand, none of the MAbs reacting to bacterial proteins or glycoproteins showed prominent bactericidal activity. Further study revealed that the antigenic epitopes on either the capsular PS or LPS molecules were readily available for binding in intact bacteria, while the epitopes on proteins/glycoproteins were less accessible to the MAbs. Ourin vivostudy showed that four MAbs reactive to either the capsular PS or LPS were highly effective in protecting mice against lethal bacterial challenge. The result is compatible with that of ourin vitrostudy. The MAbs with the highest protective efficacy are those reactive to either the capsular PS or LPS of theBurkholderiabacteria.

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Novel Chimpanzee/Human Monoclonal Antibodies That Neutralize Anthrax Lethal Factor, and Evidence for Possible Synergy with Anti-Protective Antigen Antibody

Infection and Immunity ◽

10.1128/iai.00200-09 ◽

2009 ◽

Vol 77 (9) ◽

pp. 3902-3908 ◽

Cited By ~ 39

Author(s):

Zhaochun Chen ◽

Mahtab Moayeri ◽

Devorah Crown ◽

Suzanne Emerson ◽

Inna Gorshkova ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Dissociation Constant ◽

Protective Antigen ◽

Lethal Factor ◽

Binding Assays ◽

Anthrax Lethal Factor ◽

Antigen Antibody ◽

Domain I

ABSTRACT Three chimpanzee Fabs reactive with lethal factor (LF) of anthrax toxin were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. In a macrophage toxicity assay, two of the MAbs, LF10E and LF11H, neutralized lethal toxin (LT), a complex of LF and anthrax protective antigen (PA). LF10E has the highest reported affinity for a neutralizing MAb against LF (dissociation constant of 0.69 nM). This antibody also efficiently neutralized LT in vitro, with a 50% effective concentration (EC50) of 0.1 nM, and provided 100% protection of rats against toxin challenge with a 0.5 submolar ratio relative to LT. LF11H, on the other hand, had a slightly lower binding affinity to LF (dissociation constant of 7.4 nM) and poor neutralization of LT in vitro (EC50 of 400 nM) and offered complete protection in vivo only at an equimolar or higher ratio to toxin. Despite this, LF11H, but not LF10E, provided robust synergistic protection when combined with MAb W1, which neutralizes PA. Epitope mapping and binding assays indicated that both LF10E and LF11H recognize domain I of LF (amino acids 1 to 254). Although domain I is responsible for binding to PA, neither MAb prevented LF from binding to activated PA. Although two unique MAbs could protect against anthrax when used alone, even more efficient and broader protection should be gained by combining them with anti-PA MAbs.

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Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

Clinical and Vaccine Immunology ◽

10.1128/cvi.00023-06 ◽

2006 ◽

Vol 13 (6) ◽

pp. 671-677 ◽

Cited By ~ 87

Author(s):

Robert Mabry ◽

Kathleen Brasky ◽

Robert Geiger ◽

Ricardo Carrion ◽

Gene B. Hubbard ◽

...

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Systemic Circulation ◽

Rapid Identification ◽

Anthrax Toxin ◽

Soluble Form ◽

Before And After

ABSTRACT Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.

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Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model

Journal of Virology ◽

10.1128/jvi.00652-17 ◽

2017 ◽

Vol 91 (16) ◽

Cited By ~ 18

Author(s):

James Duehr ◽

Teddy John Wohlbold ◽

Lisa Oestereich ◽

Veronika Chromikova ◽

Fatima Amanat ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Vesicular Stomatitis Virus ◽

Viral Vector ◽

Virus Disease ◽

Protective Efficacy ◽

Influenza Viruses ◽

Content Type ◽

Zaire Ebolavirus ◽

Vesicular Stomatitis

ABSTRACT Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2 −/− mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro. Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics.

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Inhibition of Anthrax Protective Antigen Outside and Inside the Cell

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00983-06 ◽

2006 ◽

Vol 51 (1) ◽

pp. 245-251 ◽

Cited By ~ 17

Author(s):

Marina V. Backer ◽

Vimal Patel ◽

Brian T. Jehning ◽

Kevin P. Claffey ◽

Vladimir A. Karginov ◽

...

Keyword(s):

Protective Antigen ◽

Fluorescent Microscopy ◽

Lethal Factor ◽

Fluorescent Dye ◽

Cell Uptake ◽

Raw 264.7 Cells ◽

Receptor Mediated Endocytosis ◽

Edema Factor

ABSTRACT In the course of Bacillus anthracis infection, B. anthracis lethal factor (LF) and edema factor bind to a protective antigen (PA) associated with cellular receptors ANTXR1 (TEM8) or ANTXR2 (CMG2), followed by internalization of the complex via receptor-mediated endocytosis. A new group of potential antianthrax drugs, β-cyclodextrins, has recently been described. A member of this group, per-6-(3-aminopropylthio)-β-cyclodextrin (AmPrβCD), was shown to inhibit the toxicity of LF in vitro and in vivo. In order to determine which steps in lethal factor trafficking are inhibited by AmPrβCD, we developed two targeted fluorescent tracers based on LFn, a catalytically inactive fragment of LF: (i) LFn site specifically labeled with the fluorescent dye AlexaFluor-594 (LFn-Al), and (ii) LFn-decorated liposomes loaded with the fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (LFn-Lip). Both tracers retained high affinity to PA/ANTXR complexes and were readily internalized via receptor-mediated endocytosis. Using fluorescent microscopy, we found that AmPrβCD inhibits receptor-mediated cell uptake but not the binding of LFn-Al to PA/ANTXR complexes, suggesting that AmPrβCD works outside the cell. Moreover, AmPrβCD and LFn-Al synergistically protect RAW 264.7 cells from PA-mediated LF toxicity, confirming that AmPrβCD did not affect the binding of LFn-Al to receptor-associated PA. In contrast, AmPrβCD did not inhibit PA-mediated internalization of LFn-Lip, suggesting that multiplexing of LFn on the liposomal surface overcomes the inhibiting effects of AmPrβCD. Notably, internalized LFn-Al and LFn-Lip protected cells that overexpressed anthrax receptor TEM8 from PA-induced, LF-independent toxicity, suggesting an independent mechanism for PA inhibition inside the cell. These data suggest the potential for the use of β-cyclodextrins in combination with LFn-Lip loaded with antianthrax drugs against intracellular targets.

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Association of Bacillus anthracis Capsule with Lethal Toxin during Experimental Infection

Infection and Immunity ◽

10.1128/iai.00764-08 ◽

2008 ◽

Vol 77 (2) ◽

pp. 749-755 ◽

Cited By ~ 28

Author(s):

J. W. Ezzell ◽

T. G. Abshire ◽

R. Panchal ◽

D. Chabot ◽

S. Bavari ◽

...

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Lethal Toxin ◽

Size Exclusion ◽

Terminal Phase ◽

Enzyme Linked Immunosorbent Assay ◽

Animal Blood

ABSTRACT Bacillus anthracis lethal toxin (LT) was characterized in plasma from infected African Green monkeys, rabbits, and guinea pigs. In all cases, during the terminal phase of infection only the protease-activated 63-kDa form of protective antigen (PA63) and the residual 20-kDa fragment (PA20) were detected in the plasma. No uncut PA with a molecular mass of 83 kDa was detected in plasma from toxemic animals during the terminal stage of infection. PA63 was largely associated with lethal factor (LF), forming LT. Characterization of LT by Western blotting, capture enzyme-linked immunosorbent assay, and size exclusion chromatography revealed that the antiphagocytic poly-γ-d-glutamic acid (γ-DPGA) capsule released from B. anthracis bacilli was associated with LT in animal blood in variable amounts. While the nature of this in vivo association is not understood, we were able to determine that a portion of these LT/γ-DPGA complexes retained LF protease activity. Our findings suggest that the in vivo LT complexes differ from in vitro-produced LT and that including γ-DPGA when examining the effects of LT on specific immune cells in vitro may reveal novel and important roles for γ-DPGA in anthrax pathogenesis.

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