Comparison of Three Anthrax Toxin Neutralization Assays

ABSTRACT Different types of anthrax toxin neutralization assays have been utilized to measure the antibody levels elicited by anthrax vaccines in both nonclinical and clinical studies. In the present study, we sought to determine whether three commonly used toxin neutralization assays—J774A.1 cell-, RAW 264.7 cell-, and CHO cell-based assays—yield comparable estimates of neutralization activities for sera obtained after vaccination with anthrax vaccines composed of recombinant protective antigen (rPA). In order to compare the assays, sera were assayed alongside a common reference serum sample and the neutralization titers were expressed relative to the titer for the reference sample in each assay. Analysis of sera from rabbits immunized with multiple doses of the rPA vaccine showed that for later bleeds, the quantitative agreement between the assays was good; however, for early bleeds, some heterogeneity in relative neutralization estimates was observed. Analysis of serum samples from rabbits, nonhuman primates, and humans immunized with the rPA vaccine showed that the relative neutralization estimates obtained in the different assays agreed to various extents, depending on the species of origin of the sera examined. We identified differences in the magnitudes of the Fc receptor-mediated neutralization associated with the J774A.1 cell- and RAW 264.7 cell-based assays, which may account for some of the species dependence of the assays. The differences in the relative neutralization estimates among the assays were relatively small and were always less than 2.5-fold. However, because toxin neutralization assays will likely be used to establish the efficacies of new anthrax vaccines, our findings should be considered when assay outputs are interpreted.

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Analysis of the Fc Gamma Receptor-Dependent Component of Neutralization Measured by Anthrax Toxin Neutralization Assays

Clinical and Vaccine Immunology ◽

10.1128/cvi.00194-09 ◽

2009 ◽

Vol 16 (10) ◽

pp. 1405-1412 ◽

Cited By ~ 24

Author(s):

Anita Verma ◽

Miriam M. Ngundi ◽

Bruce D. Meade ◽

Roberto De Pascalis ◽

Karen L. Elkins ◽

...

Keyword(s):

Protective Antigen ◽

Neutralization Assay ◽

Anthrax Toxin ◽

Specific Cell ◽

Cell Type ◽

Fcγ Receptor ◽

Gamma Receptor ◽

Preclinical And Clinical Studies ◽

Antibody Levels ◽

Anthrax Vaccines

ABSTRACT Anthrax toxin neutralization assays are used to measure functional antibody levels elicited by anthrax vaccines in both preclinical and clinical studies. In this study, we investigated the magnitude and molecular nature of Fc gamma (Fcγ) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fcγ receptor-dependent neutralization was observed in the J774A.1 cell-based assay than in the RAW 264.7 cell-based assay, a finding that could be due to the larger numbers of Fcγ receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fcγ receptor blocking monoclonal antibodies, we found that at least three murine Fcγ receptor classes, IIB, III, and IV, can contribute to Fcγ receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fcγ receptor-dependent neutralization observed in the J774A.1 cell-based assay was dependent on the concentration of protective antigen utilized in the assay. Our results suggest that the characteristics of the antibodies analyzed in the assay (e.g., species of origin, isotype, and subclass), as well as the assay design (e.g., cell type and protective antigen concentration), could significantly influence the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by anthrax toxin neutralization assays. These findings should be considered when interpreting anthrax toxin neutralization assay output.

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Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

Clinical and Vaccine Immunology ◽

10.1128/cvi.00046-13 ◽

2013 ◽

Vol 20 (7) ◽

pp. 986-997 ◽

Cited By ~ 2

Author(s):

Hang Lu ◽

Jason Catania ◽

Katalin Baranji ◽

Jie Feng ◽

Mili Gu ◽

...

Keyword(s):

Protective Antigen ◽

Lethal Factor ◽

Neutralization Assay ◽

Dominant Factor ◽

Anthrax Lethal Toxin ◽

Serum Samples ◽

Native Form ◽

Anthrax Lethal Factor ◽

Methionine Residues ◽

Anthrax Vaccines

ABSTRACTThe cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply.

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Combining Anthrax Vaccine and Therapy: a Dominant-Negative Inhibitor of Anthrax Toxin Is Also a Potent and Safe Immunogen for Vaccines

Infection and Immunity ◽

10.1128/iai.73.6.3408-3414.2005 ◽

2005 ◽

Vol 73 (6) ◽

pp. 3408-3414 ◽

Cited By ~ 40

Author(s):

Benedikt A. Aulinger ◽

Michael H. Roehrl ◽

John J. Mekalanos ◽

R. John Collier ◽

Julia Y. Wang

Keyword(s):

Protective Antigen ◽

Present Report ◽

Point Mutations ◽

Dominant Negative ◽

Anthrax Toxin ◽

Central Component ◽

Endosomal Trafficking ◽

Dominant Negative Inhibitor ◽

Antigen Presenting ◽

Anthrax Vaccines

ABSTRACT Anthrax is caused by the unimpeded growth of Bacillus anthracis in the host and the secretion of toxins. The currently available vaccine is based on protective antigen (PA), a central component of anthrax toxin. Vaccination with PA raises no direct immune response against the bacilli and, being a natural toxin component, PA might be hazardous when used immediately following exposure to B. anthracis. Thus, we have sought to develop a vaccine or therapeutic agent that is safe and eliminates both secreted toxins and bacilli. To that end, we have previously developed a dually active vaccine by conjugating the capsular poly-γ-d-glutamate (PGA) with PA to elicit the production of antibodies specific for both bacilli and toxins. In the present report, we describe the improved potency of anthrax vaccines through the use of a dominant-negative inhibitory (DNI) mutant to replace PA in PA or PA-PGA vaccines. When tested in mice, DNI alone is more immunogenic than PA, and DNI-PGA conjugate elicits significantly higher levels of antibodies against PA and PGA than PA-PGA conjugate. To explain the enhanced immunogenicity of DNI, we propose that the two point mutations in DNI may have improved epitopes of PA allowing better antigen presentation to helper T cells. Alternatively, these mutations may enhance the immunological processing of PA by altering endosomal trafficking of the toxin in antigen-presenting cells. Because DNI has previously been demonstrated to inhibit anthrax toxin, postexposure use of DNI-based vaccines, including conjugate vaccines, may provide improved immunogenicity and therapeutic activity simultaneously.

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Prophylaxis and Therapy of Inhalational Anthrax by a Novel Monoclonal Antibody to Protective Antigen That Mimics Vaccine-Induced Immunity

Infection and Immunity ◽

10.1128/iai.00712-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5840-5847 ◽

Cited By ~ 66

Author(s):

Laura Vitale ◽

Diann Blanset ◽

Israel Lowy ◽

Thomas O'Neill ◽

Joel Goldstein ◽

...

Keyword(s):

Monoclonal Antibody ◽

Protective Antigen ◽

Fc Receptor ◽

Anthrax Toxin ◽

Functional Assay ◽

Human Mabs ◽

Anthrax Spores ◽

Anthrax Vaccines

ABSTRACT The neutralizing antibody response to the protective antigen (PA) component of anthrax toxin elicited by approved anthrax vaccines is an accepted correlate for vaccine-mediated protection against anthrax. We reasoned that a human anti-PA monoclonal antibody (MAb) selected on the basis of superior toxin neutralization activity might provide potent protection against anthrax. The fully human MAb (also referred to as MDX-1303 or Valortim) was chosen from a large panel of anti-PA human MAbs generated using transgenic mice immunized with recombinant PA solely on the basis of in vitro anthrax toxin neutralization. This MAb was effective in prophylactic and postsymptomatic treatment of rabbits exposed to aerosolized anthrax spores, and a single intramuscular injection of 1 mg/kg of body weight fully protected cynomolgus monkeys challenged with aerosolized anthrax spores. Importantly, MAb 1303 defines a novel neutralizing epitope that requires Fc receptor engagement for maximal activity. F(ab′)2 fragments of MAb 1303, which retain equivalent affinity for PA, are 10- to 100-fold less potent in neutralizing anthrax toxin in vitro. Addition of Fc receptor-blocking antibodies also greatly reduced the activity of MAb 1303. Moreover, we found that the neutralizing activity of mouse, rabbit, and human antisera elicited by PA vaccines was effectively abrogated by blocking Fc receptors. Selection of an anti-PA MAb by using a functional assay that is a surrogate for protection has resulted in the identification of a fully human MAb with potent activity in vivo and uncovered a previously unrecognized mechanism of antibody-mediated toxin neutralization that is important for currently used anthrax vaccines.

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Anthrax Spores Make an Essential Contribution to Vaccine Efficacy

Infection and Immunity ◽

10.1128/iai.70.2.661-664.2002 ◽

2002 ◽

Vol 70 (2) ◽

pp. 661-664 ◽

Cited By ~ 99

Author(s):

Fabien Brossier ◽

Martine Levy ◽

Michèle Mock

Keyword(s):

Guinea Pigs ◽

Protective Antigen ◽

Humoral Response ◽

Anthrax Toxin ◽

Experimental Animals ◽

Challenge Strain ◽

Anthrax Spores ◽

Acellular Vaccines ◽

Essential Contribution ◽

Anthrax Vaccines

ABSTRACT Anthrax is caused by Bacillus anthracis, a gram-positive spore-forming bacterium. Septicemia and toxemia rapidly lead to death in infected mammal hosts. Currently used acellular vaccines against anthrax consist of protective antigen (PA), one of the anthrax toxin components. However, in experimental animals such vaccines are less protective than live attenuated strains. Here we demonstrate that the addition of formaldehyde-inactivated spores (FIS) of B. anthracis to PA elicits total protection against challenge with virulent B. anthracis strains in mice and guinea pigs. The toxin-neutralizing activities of sera from mice immunized with PA alone or PA plus FIS were similar, suggesting that the protection conferred by PA plus FIS was not only a consequence of the humoral response to PA. A PA-deficient challenge strain was constructed, and its virulence was due solely to its multiplication. Immunization with FIS alone was sufficient to protect mice partially, and guinea pigs totally, against infection with this strain. This suggests that spore antigens contribute to protection. Guinea pigs and mice had very different susceptibilities to infection with the nontoxigenic strain, highlighting the importance of verifying the pertinence of animal models for evaluating anthrax vaccines.

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Neutralization of the anthrax toxin by antibody-mediated stapling of its membrane-penetrating loop

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798321007816 ◽

2021 ◽

Vol 77 (9) ◽

Author(s):

F. Hoelzgen ◽

R. Zalk ◽

R. Alcalay ◽

S. Cohen-Schwartz ◽

G. Garau ◽

...

Keyword(s):

Pore Formation ◽

Protective Antigen ◽

Neutralizing Antibody ◽

Anthrax Toxin ◽

Structural Basis ◽

Severe Illness ◽

Central Component ◽

Important Target ◽

Anthrax Infection ◽

Anthrax Vaccines

Anthrax infection is associated with severe illness and high mortality. Protective antigen (PA) is the central component of the anthrax toxin, which is one of two major virulence factors of Bacillus anthracis, the causative agent of anthrax disease. Upon endocytosis, PA opens a pore in the membranes of endosomes, through which the cytotoxic enzymes of the toxin are extruded. The PA pore is formed by a cooperative conformational change in which the membrane-penetrating loops of PA associate, forming a hydrophobic rim that pierces the membrane. Due to its crucial role in anthrax progression, PA is an important target for monoclonal antibody-based therapy. cAb29 is a highly effective neutralizing antibody against PA. Here, the cryo-EM structure of PA in complex with the Fab portion of cAb29 was determined. It was found that cAb29 neutralizes the toxin by clamping the membrane-penetrating loop of PA to the static surface-exposed loop of the D3 domain of the same subunit, thereby preventing pore formation. These results provide the structural basis for the antibody-based neutralization of PA and bring into focus the membrane-penetrating loop of PA as a target for the development of better anti-anthrax vaccines.

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Neutralization of the anthrax toxin by antibody-mediated stapling of its membrane penetrating loop

10.1101/2021.04.18.440036 ◽

2021 ◽

Author(s):

Fabian Hoelzgen ◽

Ran Zalk ◽

Ron Alcalay ◽

Sagit Cohen Schwartz ◽

Gianpiero Garau ◽

...

Keyword(s):

Pore Formation ◽

Protective Antigen ◽

Neutralizing Antibody ◽

Anthrax Toxin ◽

Structural Basis ◽

Severe Illness ◽

Central Component ◽

Important Target ◽

Anthrax Infection ◽

Anthrax Vaccines

Anthrax infection is associated with severe illness and high mortality. Protective antigen (PA) is the central component of the anthrax toxin, which is the main virulent factor of anthrax. Upon endocytosis, PA opens a pore in the membranes of endosomes, through which the toxin's cytotoxic enzymes are extruded. The PA pore is formed by a cooperative conformational change where PA's membrane-penetrating loops associate, forming a hydrophobic rim that pierces the membrane. Due to its crucial role in anthrax progression, PA is an important target of monoclonal antibodies-based therapy. cAb29 is a highly effective neutralizing antibody against PA. We determined the cryo-EM structure of PA in complex with the Fab portion of cAb29. We found that cAb29 neutralizes the toxin by clamping the membrane-penetrating loop of PA to a static region on PA's surface, thereby preventing pore formation. Therefore, our results provide the structural basis for the antibody-based neutralization of PA and bring to focus the membrane-penetrating loop of PA as a target for the development of better anti-anthrax vaccines.

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