scholarly journals Killed but Metabolically Active Bacillus anthracis Vaccines Induce Broad and Protective Immunity against Anthrax

2009 ◽  
Vol 77 (4) ◽  
pp. 1649-1663 ◽  
Author(s):  
Justin Skoble ◽  
John W. Beaber ◽  
Yi Gao ◽  
Julie A. Lovchik ◽  
Laurie E. Sower ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Neutralizing Antibodies ◽  
Protective Immunity ◽  
Protective Antigen ◽  
Excision Repair ◽  
Uv Light ◽  
Lethal Factor ◽  
Point Mutations ◽  
Anthrax Vaccine ◽  
Edema Factor

ABSTRACTBacillus anthracisis the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizingB. anthracisthat is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting thespoIIEanduvrABgenes, renderingB. anthracisextremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into thelefandcyagenes, which allowed inactive but immunogenic toxins to be produced. Photochemically inactivated vaccine strains maintained a high degree of metabolic activity and secreted protective antigen (PA), lethal factor, and edema factor. KBMAB. anthracisvaccines were avirulent in mice and induced less injection site inflammation than recombinant PA adsorbed to aluminum hydroxide gel. KBMAB. anthracis-vaccinated animals produced antibodies against numerous anthrax antigens, including high levels of anti-PA and toxin-neutralizing antibodies. Vaccination with KBMAB. anthracisfully protected mice against challenge with lethal doses of toxinogenic unencapsulated Sterne 7702 spores and rabbits against challenge with lethal pneumonic doses of fully virulent Ames strain spores. Guinea pigs vaccinated with KBMAB. anthraciswere partially protected against lethal Ames spore challenge, which was comparable to vaccination with the licensed vaccine anthrax vaccine adsorbed. These data demonstrate that KBMA anthrax vaccines are well tolerated and elicit potent protective immune responses. The use of KBMA vaccines may be broadly applicable to bacterial pathogens, especially those for which the correlates of protective immunity are unknown.

scholarly journals Neutralizing Antibodies and Persistence of Immunity following Anthrax Vaccination

2006 ◽  
Vol 13 (2) ◽  
pp. 208-213 ◽  
Author(s):  
James F. Hanson ◽  
Sarah C. Taft ◽  
Alison A. Weiss
Keyword(s):  
Neutralizing Antibodies ◽  
Protective Antigen ◽  
Colorimetric Assay ◽  
Lethal Factor ◽  
Neutralization Activity ◽  
Anthrax Vaccine ◽  
Edema Factor ◽  
Low Levels ◽  
Antibody Levels ◽  
Toxic Components

ABSTRACT Anthrax toxin consists of protective antigen (PA) and two toxic components, lethal factor (LF) and edema factor (EF). PA binds to mammalian cellular receptors and delivers the toxic components to the cytoplasm. PA is the primary antigenic component of the current anthrax vaccine. Immunity is due to the generation of antibodies that prevent the PA-mediated internalization of LF and EF. In this study, we characterized sera obtained from vaccinated military personnel. Anthrax vaccine is administered in a series of six injections at 0, 2, and 4 weeks and 6, 12, and 18 months, followed by annual boosters. The vaccination histories of the subjects were highly varied; many subjects had not completed the entire series, and several had not received annual boosters. We developed a simple colorimetric assay using alamarBlue dye to assess the antibody-mediated neutralization of LF-mediated toxicity to the J774A.1 murine macrophage cell line. Recently vaccinated individuals had high antibody levels and neutralizing activity. One individual who had not been boosted for 5 years had low immunoglobulin G antibody levels but a detectable neutralization activity, suggesting that this individual produced low levels of very active antibodies.

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.

scholarly journals Toxin-Deficient Mutants of Bacillus anthracis Are Lethal in a Murine Model for Pulmonary Anthrax

2006 ◽  
Vol 74 (11) ◽  
pp. 6067-6074 ◽  
Author(s):  
Sara Heninger ◽  
Melissa Drysdale ◽  
Julie Lovchik ◽  
Julie Hutt ◽  
Mary F. Lipscomb ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Murine Model ◽  
Parent Strain ◽  
Protective Antigen ◽  
Histopathological Examination ◽  
Lethal Factor ◽  
Lethal Dose ◽  
Etiologic Agent ◽  
Time To Death ◽  
Edema Factor

ABSTRACT Bacillus anthracis, the etiologic agent of anthrax, produces at least three primary virulence factors: lethal toxin, edema toxin, and a capsule. The capsule is absolutely required for dissemination and lethality in a murine model of inhalation anthrax, yet the roles for the toxins during infection are ill-defined. We show in a murine model that when spores of specific toxin-null mutants are introduced into the lung, dissemination and lethality are comparable to those of the parent strain. Mutants lacking one or more of the structural genes for the toxin proteins, i.e., protective antigen, lethal factor, and edema factor, disseminated from the lung to the spleen at rates similar to that of the virulent parental strain. The 50% lethal dose (LD50) and mean time to death (MTD) of the mutants did not differ significantly from those of the parent. The LD50s or MTDs were also unaffected relative to those of the parent strain when mice were inoculated intravenously with vegetative cells. Nonetheless, histopathological examination of tissues revealed subtle but distinct differences in infections by the parent compared to some toxin mutants, suggesting that the host response is affected by toxin proteins synthesized during infection.

scholarly journals Very Early Blood Diffusion of the Active Lethal and Edema Factors of Bacillus anthracis After Intranasal Infection

2019 ◽  
Vol 221 (4) ◽  
pp. 660-667 ◽  
Author(s):  
Clémence Rougeaux ◽  
François Becher ◽  
Pierre L Goossens ◽  
Jean-Nicolas Tournier
Keyword(s):  
Bacillus Anthracis ◽  
Quantitative Methods ◽  
Protective Antigen ◽  
Early Stage ◽  
Lethal Factor ◽  
Wild Type ◽  
Intranasal Infection ◽  
Edema Factor ◽  
Systemic Dissemination ◽  
Few Data

Abstract Background Lethal and edema toxins are critical virulence factors of Bacillus anthracis. Few data are available on their presence in the early stage of intranasal infection. Methods To investigate the diffusion of edema factor (EF) and lethal factor (LF), we use sensitive quantitative methods to measure their enzymatic activities in mice intranasally challenged with a wild-type B anthracis strain or with an isogenic mutant deficient for the protective antigen. Results One hour after mouse challenge, although only 7% of mice presented bacteremia, LF and EF were detected in the blood of 100% and 42% of mice, respectively. Protective antigen facilitated the diffusion of LF and EF into the blood compartment. Toxins played a significant role in the systemic dissemination of B anthracis in the blood, spleen, and liver. A mouse model of intoxination further confirmed that LT and ET could diffuse rapidly in the circulation, independently of bacteria. Conclusions In this inhalational model, toxins have disseminated rapidly in the blood, playing a significant and novel role in the early systemic diffusion of bacteria, demonstrating that they may represent a very early target for the diagnosis and the treatment of anthrax.

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 Plant-Based Vaccine: Mice Immunized with Chloroplast-Derived Anthrax Protective Antigen Survive Anthrax Lethal Toxin Challenge

2005 ◽  
Vol 73 (12) ◽  
pp. 8266-8274 ◽  
Author(s):  
Vijay Koya ◽  
Mahtab Moayeri ◽  
Stephen H. Leppla ◽  
Henry Daniell
Keyword(s):  
Protective Antigen ◽  
Expression System ◽  
Lethal Factor ◽  
Vaccine Antigen ◽  
Total Soluble Protein ◽  
Continuous Illumination ◽  
Anthrax Lethal Toxin ◽  
Anthrax Vaccine ◽  
Edema Factor ◽  
Subcutaneous Immunization

ABSTRACT The currently available human vaccine for anthrax, derived from the culture supernatant of Bacillus anthracis, contains the protective antigen (PA) and traces of the lethal and edema factors, which may contribute to adverse side effects associated with this vaccine. Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required. In an effort to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants, PA was expressed in transgenic tobacco chloroplasts by inserting the pagA gene into the chloroplast genome. Chloroplast integration of the pagA gene was confirmed by PCR and Southern analysis. Mature leaves grown under continuous illumination contained PA as up to 14.2% of the total soluble protein. Cytotoxicity measurements in macrophage lysis assays showed that chloroplast-derived PA was equal in potency to PA produced in B. anthracis. Subcutaneous immunization of mice with partially purified chloroplast-derived or B. anthracis-derived PA with adjuvant yielded immunoglobulin G titers up to 1:320,000, and both groups of mice survived (100%) challenge with lethal doses of toxin. An average yield of about 150 mg of PA per plant should produce 360 million doses of a purified vaccine free of bacterial toxins edema factor and lethal factor from 1 acre of land. Such high expression levels without using fermenters and the immunoprotection offered by the chloroplast-derived PA should facilitate development of a cleaner and safer anthrax vaccine at a lower production cost. These results demonstrate the immunogenic and immunoprotective properties of plant-derived anthrax vaccine antigen.

scholarly journals Direct imaging of anthrax intoxication in animals reveals shared and individual functions of CMG-2 and TEM-8 in cellular toxin entry

2021 ◽  
Author(s):  
Carly Merritt ◽  
Elizabeth M. Chun ◽  
Rasem J. Fattah ◽  
Mahtab Moayeri ◽  
Dennis Paliga ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Fluorescent Protein ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Chimeric Protein ◽  
Anthrax Toxin ◽  
Specific Cell ◽  
Anthrax Lethal Toxin ◽  
Surface Receptors ◽  
Edema Factor

SUMMARYThe virulence of Bacillus anthracis is linked to the secretion of anthrax lethal toxin and anthrax edema toxin. These binary toxins consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds either of two specific cell surface receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding, endocytosis, and cytoplasmic translocation of LF and EF. The cellular distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals is not fully elucidated. Herein, we describe a novel assay to image anthrax toxin intoxication in live animals, and we use the assay to visualize TEM-8- and CMG-2-dependent intoxication. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were co-administered to transgenic mice that ubiquitously express a red fluorescent protein in the absence of Cre activity and a green fluorescent protein in the presence of Cre activity, anthrax toxin intoxication could be visualized at single-cell resolution by confocal microscopy. By using this assay, we show that CMG-2 is critical for intoxication in the liver and heart, whereas TEM-8 is required for full intoxication in the kidney and spleen. Other tissues examined were largely unaffected by single deficiences in either receptor, suggesting extensive overlap in TEM-8 and CMG-2 expression. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for identifying on- and off-targets for reengineered toxin variants in the clinical development of cancer treatments.BackgroundAssays for imaging of anthrax toxin intoxication in animals are not available.ResultsAnthrax toxin-Cre fusions combined with fluorescent Cre reporter mice enabled imaging of anthrax toxin intoxication in animals.ConclusionShared and distinct functions of toxin receptors in cellular entry were uncovered. Significance. A simple and versatile assay for anthrax toxin intoxication is described.

scholarly journals Autogenous Regulation of the Bacillus anthracis pag Operon

1999 ◽  
Vol 181 (15) ◽  
pp. 4485-4492 ◽  
Author(s):  
Alex R. Hoffmaster ◽  
Theresa M. Koehler
Keyword(s):  
Steady State ◽  
Bacillus Anthracis ◽  
Target Cell ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Repeat Sequence ◽  
Virulence Plasmid ◽  
Multicopy Plasmid ◽  
Transcription Terminator ◽  
Edema Factor

ABSTRACT Protective antigen (PA) is an important component of the edema and lethal toxins produced by Bacillus anthracis. PA is essential for binding the toxins to the target cell receptor and for facilitating translocation of the enzymatic toxin components, edema factor and lethal factor, across the target cell membrane. The structural gene for PA, pagA (previously known aspag), is located on the 182-kb virulence plasmid pXO1 at a locus distinct from the edema factor and lethal factor genes. Here we show that a 300-bp gene located downstream of pagA is cotranscribed with pagA and represses expression of the operon. We have designated this gene pagR (for protective antigen repressor). Two pagA mRNA transcripts were detected in cells producing PA: a short, 2.7-kb transcript corresponding to thepagA gene, and a longer, 4.2-kb transcript representing a bicistronic message derived from pagA and pagR. The 3′ end of the short transcript mapped adjacent to an inverted repeat sequence, suggesting that the sequence can act as a transcription terminator. Attenuation of termination at this site results in transcription of pagR. A pagR mutant exhibited increased steady-state levels of pagA mRNA, indicating that pagR negatively controls expression of the operon. Autogenous control of the operon may involve atxA, a trans-acting positive regulator of pagA. The steady-state level of atxA mRNA was also increased in thepagR mutant. The mutant phenotype was complemented by addition of pagR in trans on a multicopy plasmid.

scholarly journals Murine Innate Immune Response to Virulent Toxigenic and Nontoxigenic Bacillus anthracis Strains

2007 ◽  
Vol 75 (4) ◽  
pp. 1757-1764 ◽  
Author(s):  
Melissa Drysdale ◽  
Gwyneth Olson ◽  
Theresa M. Koehler ◽  
Mary F. Lipscomb ◽  
C. Rick Lyons
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Systemic Infection ◽  
Neutrophil Recruitment ◽  
Cell Recruitment ◽  
Edema Factor ◽  
Edema Toxin ◽  
Neutrophil Depletion ◽  
Intravenous Inoculation

ABSTRACT Effective treatment of anthrax is hampered by our limited understanding of the pathophysiology of Bacillus anthracis infection. We used a genetically complete (pXO1+ pXO2+) virulent B. anthracis strain and four isogenic toxin-null mutants to determine the effects of the anthrax edema toxin (ET; edema factor [EF] plus protective antigen [PA]) and lethal toxin (LT; lethal factor [LF] plus PA) on the host innate response during systemic infection. Using the spleen as an indicator for host response, we found that intravenous inoculation of LT-deficient mutants into C57BL/6 mice significantly increased production of several cytokines over that observed after infection with the parent strain or an EF-deficient mutant. Bacteria producing one or both of the toxins were capable of inducing significant apoptosis of cells present in spleens, whereas apoptosis was greatly reduced in mice infected with nontoxigenic mutants. Mice infected with toxin-producing strains also showed increased splenic neutrophil recruitment compared to mice infected with nontoxigenic strains and neutrophil depletion prior to infection with toxin-producing strains, leading to decreased levels of apoptosis. Together, these studies indicate that anthrax LT suppresses cytokine secretion during infection, but both EF and LF play roles in inducing neutrophil recruitment and enhancing apoptosis. Interestingly, in the absence of LF the effect of EF-induced cell recruitment is further enhanced, perhaps because LF so effectively suppresses the secretion of chemokines.

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.

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