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

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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Sequential B-Cell Epitopes of Bacillus anthracis Lethal Factor Bind Lethal Toxin-Neutralizing Antibodies

Infection and Immunity ◽

10.1128/iai.00788-08 ◽

2008 ◽

Vol 77 (1) ◽

pp. 162-169 ◽

Cited By ~ 21

Author(s):

Melissa L. Nguyen ◽

Sherry R. Crowe ◽

Sridevi Kurella ◽

Simon Teryzan ◽

Brian Cao ◽

...

Keyword(s):

Monoclonal Antibodies ◽

B Cell ◽

Bacillus Anthracis ◽

Neutralizing Antibodies ◽

Lethal Factor ◽

Lethal Toxin ◽

B Cell Epitopes ◽

Protective Antibodies

ABSTRACT The bipartite anthrax lethal toxin (LeTx) consisting of protective antigen (PA) and lethal factor (LF) is a major virulence factor contributing to death from systemic Bacillus anthracis infection. The current vaccine elicits antibodies directed primarily to PA; however, in experimental settings serologic responses to LF can neutralize LeTx and contribute to protection against infection. The goals of the present study were to identify sequential B-cell epitopes of LF and to determine the capacity of these determinants to bind neutralizing antibodies. Sera of recombinant LF-immunized A/J mice exhibited high titers of immunoglobulin G anti-LF reactivity that neutralized LeTx in vitro 78 days after the final booster immunization and protected the mice from in vivo challenge with 3 50% lethal doses of LeTx. These sera bound multiple discontinuous epitopes, and there were major clusters of reactivity on native LF. Strikingly, all three neutralizing, LF-specific monoclonal antibodies tested bound specific peptide sequences that coincided with sequential epitopes identified in polyclonal antisera from recombinant LF-immunized mice. This study confirms that LF induces high-titer protective antibodies in vitro and in vivo. Moreover, the binding of short LF peptides by LF-specific neutralizing monoclonal antibodies suggests that generation of protective antibodies by peptide vaccination may be feasible for this antigen. This study paves the way for a more effective anthrax vaccine by identifying discontinuous peptide epitopes of LF.

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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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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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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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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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CD1d-Dependent B-Cell Help by NK-Like T Cells Leads to Enhanced and Sustained Production of Bacillus anthracis Lethal Toxin-Neutralizing Antibodies

Infection and Immunity ◽

10.1128/iai.00002-10 ◽

2010 ◽

Vol 78 (4) ◽

pp. 1610-1617 ◽

Cited By ~ 25

Author(s):

T. Scott Devera ◽

Lindsay M. Aye ◽

Gillian A. Lang ◽

Sunil K. Joshi ◽

Jimmy D. Ballard ◽

...

Keyword(s):

T Cells ◽

Bacillus Anthracis ◽

Neutralizing Antibodies ◽

Protective Antigen ◽

Lethal Factor ◽

Nkt Cells ◽

Lethal Toxin ◽

Type I ◽

Lethal Challenge

ABSTRACT The current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (α-galactosylceramide [α-GC]) at the time of immunization improves PA-specific Ab responses. We observed that α-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d−/− mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In Jα281−/− mice expressing CD1d but lacking type I α-GC-reactive NKT cells, α-GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.

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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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Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

10.1101/2021.04.05.438547 ◽

2021 ◽

Author(s):

Fatima Amanat ◽

Shirin Strohmeier ◽

Wen-Hsin Lee ◽

Sandhya Bangaru ◽

Andrew B Ward ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Mouse Model ◽

Neutralizing Antibodies ◽

Dependent Manner ◽

Receptor Binding Domain ◽

Wild Type ◽

Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

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Vaccination of Rhesus Macaques with the Anthrax Vaccine Adsorbed Vaccine Produces a Serum Antibody Response That Effectively Neutralizes Receptor-Bound Protective Antigen In Vitro

Clinical and Vaccine Immunology ◽

10.1128/cvi.00174-10 ◽

2010 ◽

Vol 17 (11) ◽

pp. 1753-1762 ◽

Cited By ~ 8

Author(s):

Kristin H. Clement ◽

Thomas L. Rudge ◽

Heather J. Mayfield ◽

Lena A. Carlton ◽

Arelis Hester ◽

...

Keyword(s):

Cell Surface ◽

Neutralizing Antibodies ◽

Protective Antigen ◽

Rhesus Macaques ◽

Lethal Factor ◽

Serum Antibody ◽

Target Cells ◽

Furin Cleavage ◽

Anthrax Vaccine

ABSTRACT Anthrax toxin (ATx) is composed of the binary exotoxins lethal toxin (LTx) and edema toxin (ETx). They have separate effector proteins (edema factor and lethal factor) but have the same binding protein, protective antigen (PA). PA is the primary immunogen in the current licensed vaccine anthrax vaccine adsorbed (AVA [BioThrax]). AVA confers protective immunity by stimulating production of ATx-neutralizing antibodies, which could block the intoxication process at several steps (binding of PA to the target cell surface, furin cleavage, toxin complex formation, and binding/translocation of ATx into the cell). To evaluate ATx neutralization by anti-AVA antibodies, we developed two low-temperature LTx neutralization activity (TNA) assays that distinguish antibody blocking before and after binding of PA to target cells (noncomplexed [NC] and receptor-bound [RB] TNA assays). These assays were used to investigate anti-PA antibody responses in AVA-vaccinated rhesus macaques (Macaca mulatta) that survived an aerosol challenge with Bacillus anthracis Ames spores. Results showed that macaque anti-AVA sera neutralized LTx in vitro, even when PA was prebound to cells. Neutralization titers in surviving versus nonsurviving animals and between prechallenge and postchallenge activities were highly correlated. These data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy. Furthermore, these data support the idea that the full-length PA83 monomer is an appropriate immunogen for inclusion in next-generation anthrax vaccines.

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