ANALYSIS OF TOXIN-NEUTRALIZING ACTIVITY OF MOUSE MONOCLONAL ANTIBODIES AGAINST PROTECTIVE ANTIGEN OF BACILLUS ANTHRACIS

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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A Monoclonal Antibody to Bacillus anthracis Protective Antigen Defines a Neutralizing Epitope in Domain 1

Infection and Immunity ◽

10.1128/iai.00150-06 ◽

2006 ◽

Vol 74 (7) ◽

pp. 4149-4156 ◽

Cited By ~ 38

Author(s):

Johanna Rivera ◽

Antonio Nakouzi ◽

Nareen Abboud ◽

Ekaterina Revskaya ◽

David Goldman ◽

...

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Proteolytic Digestion ◽

Potential Mechanism ◽

Host Protection ◽

Edema Toxin

ABSTRACT Antibody (Ab) responses to Bacillus anthracis toxins are protective, but relatively few protective monoclonal antibodies (MAbs) have been reported. Protective antigen (PA) is essential for the action of B. anthracis lethal toxin (LeTx) and edema toxin. In this study, we generated two MAbs to PA, MAbs 7.5G and 10F4. These MAbs did not compete for binding to PA, consistent with specificities for different epitopes. The MAbs were tested for their ability to protect a monolayer of cultured macrophages against toxin-mediated cytotoxicity. MAb 7.5G, the most-neutralizing MAb, bound to domain 1 of PA and reduced LeTx toxicity in BALB/c mice. Remarkably, MAb 7.5G provided protection without blocking the binding of PA or lethal factor or the formation of the PA heptamer complex. However, MAb 7.5G slowed the proteolytic digestion of PA by furin in vitro, suggesting a potential mechanism for Ab-mediated protection. These observations indicate that some Abs to domain 1 can contribute to host protection.

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Production and characterization of the monoclonal antibodies to Bacillus anthracis protective antigen

Russian Journal of Bioorganic Chemistry ◽

10.1134/s1068162011030162 ◽

2011 ◽

Vol 37 (3) ◽

pp. 316-321 ◽

Cited By ~ 3

Author(s):

N. V. Rudenko ◽

S. G. Abbasova ◽

E. V. Grishin

Keyword(s):

Monoclonal Antibodies ◽

Bacillus Anthracis ◽

Protective Antigen

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Characterization of Lethal Factor Binding and Cell Receptor Binding Domains of Protective Antigen of Bacillus anthracis using Monoclonal Antibodies

Microbiology ◽

10.1099/13500872-142-3-707 ◽

1996 ◽

Vol 142 (3) ◽

pp. 707-715 ◽

Cited By ~ 94

Author(s):

S. F. Little ◽

J. M. Novak ◽

J. R. Lowe ◽

S. H. Leppla ◽

Y. Singh ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Bacillus Anthracis ◽

Receptor Binding ◽

Protective Antigen ◽

Lethal Factor ◽

Cell Receptor ◽

Factor Binding ◽

Binding Domains

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Human Monoclonal Antibodies against Anthrax Lethal Factor and Protective Antigen Act Independently To Protect against Bacillus anthracis Infection and Enhance Endogenous Immunity to Anthrax

Infection and Immunity ◽

10.1128/iai.00261-07 ◽

2007 ◽

Vol 75 (11) ◽

pp. 5425-5433 ◽

Cited By ~ 81

Author(s):

Mark T. Albrecht ◽

Han Li ◽

E. Diane Williamson ◽

Chris S. LeButt ◽

Helen C. Flick-Smith ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Passive Immunization ◽

Cell Receptor ◽

Biological Action ◽

Anthrax Toxin ◽

Human Monoclonal Antibodies ◽

Receptor Binding Site

ABSTRACT The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-μg dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.

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