scholarly journals Analysis of Defined Combinations of Monoclonal Antibodies in Anthrax Toxin Neutralization Assays and Their Synergistic Action

2012 ◽  
Vol 19 (5) ◽  
pp. 731-739 ◽  
Author(s):  
Miriam M. Ngundi ◽  
Bruce D. Meade ◽  
Stephen F. Little ◽  
Conrad P. Quinn ◽  
Cindi R. Corbett ◽  
...  
Keyword(s):  
Cell Culture ◽  
Monoclonal Antibodies ◽  
Protective Antigen ◽  
Neutralizing Antibody ◽  
Synergistic Action ◽  
Anthrax Toxin ◽  
Content Type ◽  
Antagonistic Effects ◽  
Antibody Interactions ◽  
New Vaccines

ABSTRACTAntibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused byBacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used. Synergistic toxin-neutralizing antibody interactions were examined in more detail. We found that one mechanism that can lead to antibody synergy is the bridging of PA monomers by one antibody, with resultant bivalent binding of the second antibody. These results may aid in optimal design of new vaccines and antibody therapies against anthrax.

scholarly journals Progress towards the Development of a NEAT Vaccine for Anthrax II: Immunogen Specificity and Alum Effectiveness in an Inhalational Model

2020 ◽  
Vol 88 (8) ◽  
Author(s):  
Joseph Jelinski ◽  
Austen Terwilliger ◽  
Sabrina Green ◽  
Anthony Maresso
Keyword(s):  
Neutralizing Antibodies ◽  
Protective Antigen ◽  
Anthrax Toxin ◽  
Cell Binding ◽  
Lethal Challenge ◽  
Anthrax Vaccine ◽  
Content Type ◽  
Heme Transport ◽  
Bacillus Spores ◽  
Culture Supernatants

ABSTRACT Bacillus anthracis is the causative agent of anthrax disease, presents with high mortality, and has been at the center of bioweapon efforts. The only currently U.S. FDA-approved vaccine to prevent anthrax in humans is anthrax vaccine adsorbed (AVA), which is protective in several animal models and induces neutralizing antibodies against protective antigen (PA), the cell-binding component of anthrax toxin. However, AVA requires a five-course regimen to induce immunity, along with an annual booster, and is composed of undefined culture supernatants from a PA-secreting strain. In addition, it appears to be ineffective against strains that lack anthrax toxin. Here, we investigated a vaccine formulation consisting of recombinant proteins from a surface-localized heme transport system containing near-iron transporter (NEAT) domains and its efficacy as a vaccine for anthrax disease. The cocktail of five NEAT domains was protective against a lethal challenge of inhaled bacillus spores at 3 and 28 weeks after vaccination. The reduction of the formulation to three NEATs (IsdX1, IsdX2, and Bslk) was as effective as a five-NEAT domain cocktail. The adjuvant alum, approved for use in humans, was as protective as Freund’s Adjuvant, and protective vaccination correlated with increased anti-NEAT antibody reactivity and reduced bacterial levels in organs. Finally, the passive transfer of anti-NEAT antisera reduced mortality and disease severity, suggesting the protective component is comprised of antibodies. Collectively, these results provide evidence that a vaccine based upon recombinant NEAT proteins should be considered in the development of a next-generation anthrax vaccine.

scholarly journals Mouse Monoclonal Antibodies to Anthrax Edema Factor Protect against Infection

2011 ◽  
Vol 79 (11) ◽  
pp. 4609-4616 ◽  
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.

scholarly journals Structural and Immunological Analysis of Anthrax Recombinant Protective Antigen Adsorbed to Aluminum Hydroxide Adjuvant

2012 ◽  
Vol 19 (9) ◽  
pp. 1465-1473 ◽  
Author(s):  
Leslie Wagner ◽  
Anita Verma ◽  
Bruce D. Meade ◽  
Karine Reiter ◽  
David L. Narum ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Neutralizing Antibodies ◽  
Tertiary Structure ◽  
Protective Antigen ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Neutralizing Antibody ◽  
Fluorescence Emission Spectra ◽  
Recombinant Protective Antigen

ABSTRACTNew anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies.

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

2013 ◽  
Vol 81 (6) ◽  
pp. 1880-1888 ◽  
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.

scholarly journals Anthrax toxin-neutralizing antibody reconfigures the protective antigen heptamer into a supercomplex

2010 ◽  
Vol 107 (32) ◽  
pp. 14070-14074 ◽  
Author(s):  
M. Radjainia ◽  
J.-K. Hyun ◽  
C. E. Leysath ◽  
S. H. Leppla ◽  
A. K. Mitra
Keyword(s):  
Protective Antigen ◽  
Neutralizing Antibody ◽  
Anthrax Toxin

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

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.

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

2007 ◽  
Vol 75 (11) ◽  
pp. 5425-5433 ◽  
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.

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

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.

scholarly journals Efficient Neutralization of Anthrax Toxin by Chimpanzee Monoclonal Antibodies against Protective Antigen

2006 ◽  
Vol 193 (5) ◽  
pp. 625-633 ◽  
Author(s):  
Zhaochun Chen ◽  
Mahtab Moayeri ◽  
Yi‐Hua Zhou ◽  
Stephen Leppla ◽  
Suzanne Emerson ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Protective Antigen ◽  
Anthrax Toxin

scholarly journals Symmetry Requirements for Effective Blocking of Pore-Forming Toxins: Comparative Study with α-, β-, and γ-Cyclodextrin Derivatives

2011 ◽  
Vol 55 (7) ◽  
pp. 3594-3597 ◽  
Author(s):  
Konstantina Yannakopoulou ◽  
Laszlo Jicsinszky ◽  
Crysie Aggelidou ◽  
Nikolaos Mourtzis ◽  
Tanisha M. Robinson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Comparative Study ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Toxin ◽  
Anthrax Toxin ◽  
Cyclodextrin Derivatives ◽  
Content Type

ABSTRACTWe compared the abilities of structurally related cationic cyclodextrins to inhibitBacillus anthracislethal toxin andStaphylococcus aureusα-hemolysin. We found that both β- and γ-cyclodextrin derivatives effectively inhibited anthrax toxin action by blocking the transmembrane oligomeric pores formed by the protective antigen (PA) subunit of the toxin, whereas α-cyclodextrins were ineffective. In contrast, α-hemolysin was selectively blocked only by β-cyclodextrin derivatives, demonstrating that both symmetry and size of the inhibitor and the pore are important.

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