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

2010 ◽  
Vol 78 (4) ◽  
pp. 1610-1617 ◽  
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.

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

2004 ◽  
Vol 72 (11) ◽  
pp. 6313-6317 ◽  
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.

scholarly journals Association of Bacillus anthracis Capsule with Lethal Toxin during Experimental Infection

2008 ◽  
Vol 77 (2) ◽  
pp. 749-755 ◽  
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.

scholarly journals Revisiting the Concept of Targeting Only Bacillus anthracis Toxins as a Treatment for Anthrax

2016 ◽  
Vol 60 (8) ◽  
pp. 4878-4885 ◽  
Author(s):  
Itai Glinert ◽  
Elad Bar-David ◽  
Assa Sittner ◽  
Shay Weiss ◽  
Josef Schlomovitz ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Protein Fraction ◽  
Neutralizing Antibodies ◽  
Pathogenic Bacteria ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Immunofluorescence Assay ◽  
Secreted Protein ◽  
Content Type

ABSTRACTProtective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. TheBacillus anthracistoxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditionsin vitro, may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets.

scholarly journals Retrocyclins Kill Bacilli and Germinating Spores of Bacillus anthracis and Inactivate Anthrax Lethal Toxin

2006 ◽  
Vol 281 (43) ◽  
pp. 32755-32764 ◽  
Author(s):  
Wei Wang ◽  
Chandrika Mulakala ◽  
Sabrina C. Ward ◽  
Grace Jung ◽  
Hai Luong ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Lethal Toxin ◽  
Raw 264.7 Cells ◽  
Protective Effects ◽  
Anthrax Lethal Toxin ◽  
Arginine Residues

θ-defensins are cyclic octadecapeptides encoded by the modified α-defensin genes of certain nonhuman primates. The recent demonstration that human α-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of θ-defensins on Bacillus anthracis (Sterne). We tested rhesus θ-defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of θ-defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of θ-defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that θ-defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins.

scholarly journals 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

2007 ◽  
Vol 75 (11) ◽  
pp. 5443-5452 ◽  
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.

scholarly journals Protective Antigen-Mediated Antibody Response against a Heterologous Protein Produced In Vivo by Bacillus anthracis

2000 ◽  
Vol 68 (10) ◽  
pp. 5731-5734 ◽  
Author(s):  
Fabien Brossier ◽  
Martine Weber-Levy ◽  
Michèle Mock ◽  
Jean-Claude Sirard
Keyword(s):  
Antibody Response ◽  
Bacillus Anthracis ◽  
Tetanus Toxin ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Specific Response ◽  
Lethal Challenge ◽  
Heterologous Antigen

ABSTRACT Bacillus anthracis secretes a lethal toxin composed of two proteins, the lethal factor (LF) and the protective antigen (PA), which interact within the host or in vitro at the surfaces of eukaryotic cells. Immunization with attenuated B. anthracisstrains induces an antibody response against PA and LF. The LF-specific response is potentiated by the binding of LF to PA. In this study, we investigated the capacity of PA to increase the antibody response against a foreign antigen. We constructed a chimeric gene encoding the PA-binding part of LF (LF254) fused to the C fragment of tetanus toxin (ToxC). The construct was introduced by allelic exchange into the locus encoding LF. Two recombinant B. anthracis strains secreting the hybrid protein LF254-ToxC were generated, one in a PA-producing background and the other in a PA-deficient background. Mice were immunized with spores of the strains, and the humoral response and protection against tetanus toxin were assessed. The B. anthracis strain producing both PA and LF254-ToxC induced significantly higher antibody titers and provided better protection against a lethal challenge with tetanus toxin than did its PA-deficient counterpart. Thus, PA is able to potentiate protective immunity against a heterologous antigen, demonstrating the potential of B. anthracis recombinant strains for use as live vaccine vehicles.

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

2008 ◽  
Vol 77 (1) ◽  
pp. 162-169 ◽  
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.

scholarly journals A Chimeric Protein That Functions as both an Anthrax Dual-Target Antitoxin and a Trivalent Vaccine

2010 ◽  
Vol 54 (11) ◽  
pp. 4750-4757 ◽  
Author(s):  
Gaobing Wu ◽  
Yuzhi Hong ◽  
Aizhen Guo ◽  
Chunfang Feng ◽  
Sha Cao ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Vaccine Candidate ◽  
Lethal Factor ◽  
Lethal Dose ◽  
Chimeric Protein ◽  
Dominant Negative ◽  
Lethal Challenge ◽  
Edema Factor ◽  
Trivalent Vaccine

ABSTRACT Effective measures for the prophylaxis and treatment of anthrax are still required for counteracting the threat posed by inhalation anthrax. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing the protective antigen (PA)-binding domain of lethal factor (LFn) to PA, retained the functions of the respective molecules. On the basis of this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF) to PA and the transportation of LF/EF. Therefore, we replaced PA in LFn-PA with a dominant-negative inhibitory PA (DPA), i.e., PAF427D. In in vitro models of anthrax intoxication, the LFn-DPA chimera showed 3-fold and 2-fold higher potencies than DPA in protecting sensitive cells against anthrax lethal toxin (LeTx) and edema toxin (EdTx), respectively. In animal models, LFn-DPA exhibited strong potency in rescuing mice from lethal challenge with LeTx. We also evaluated the immunogenicity and immunoprotective efficacy of LFn-DPA as an anthrax vaccine candidate. In comparison with recombinant PA, LFn-DPA induced significantly higher levels of the anti-PA immune response. Moreover, LFn-DPA elicited an anti-LF antibody response that could cross-react with EF. Mice immunized with LFn-DPA tolerated a LeTx challenge that was 5 times its 50% lethal dose. Thus, LFn-DPA represents a highly effective trivalent vaccine candidate for both preexposure and postexposure vaccination. Overall, we have developed a novel and dually functional reagent for the prophylaxis and treatment of anthrax.

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

2006 ◽  
Vol 13 (6) ◽  
pp. 671-677 ◽  
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.

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

2010 ◽  
Vol 17 (11) ◽  
pp. 1753-1762 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document