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 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 Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state

2020 ◽  
Author(s):  
Claudia Antoni ◽  
Dennis Quentin ◽  
Alexander E. Lang ◽  
Klaus Aktories ◽  
Christos Gatsogiannis ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Lethal Toxin ◽  
Anthrax Toxin ◽  
Anthrax Lethal Toxin ◽  
Terminal Domain ◽  
Edema Factor ◽  
Major Virulence Factor ◽  
Cell Substrate ◽  
Continuous Chain

AbstractAnthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the order of LF binding determines which factor is translocated first.

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 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 Structure of anthrax lethal toxin prepore complex suggests a pathway for efficient cell entry

2016 ◽  
Vol 148 (4) ◽  
pp. 313-324 ◽  
Author(s):  
Lucien Fabre ◽  
Eugenio Santelli ◽  
Driss Mountassif ◽  
Annemarie Donoghue ◽  
Aviroop Biswas ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Three Dimensional ◽  
Cell Entry ◽  
Anthrax Lethal Toxin ◽  
Edema Factor ◽  
Molecular Arrangement ◽  
Cryo Electron Microscopy ◽  
Translocation Efficiency ◽  
Terminal Domains

Anthrax toxin comprises three soluble proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA must be cleaved by host proteases before it oligomerizes and forms a prepore, to which LF and EF bind. After endocytosis of this tripartite complex, the prepore transforms into a narrow transmembrane pore that delivers unfolded LF and EF into the host cytosol. Here, we find that translocation of multiple 90-kD LF molecules is rapid and efficient. To probe the molecular basis of this translocation, we calculated a three-dimensional map of the fully loaded (PA63)7–(LF)3 prepore complex by cryo–electron microscopy (cryo-EM). The map shows three LFs bound in a similar way to one another, via their N-terminal domains, to the surface of the PA heptamer. The model also reveals contacts between the N- and C-terminal domains of adjacent LF molecules. We propose that this molecular arrangement plays an important role in the maintenance of translocation efficiency through the narrow PA pore.

scholarly journals Neutralizing Activity of Vaccine-Induced Antibodies to Two Bacillus anthracis Toxin Components, Lethal Factor and Edema Factor

2007 ◽  
Vol 15 (1) ◽  
pp. 71-75 ◽  
Author(s):  
Sarah C. Taft ◽  
Alison A. Weiss
Keyword(s):  
Animal Models ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Antibody Responses ◽  
Anthrax Toxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mouse Macrophage ◽  
Anthrax Vaccine ◽  
Neutralizing Activity ◽  
Edema Factor

ABSTRACT Anthrax vaccine adsorbed (AVA; BioThrax), the current FDA-licensed human anthrax vaccine, contains various amounts of the three anthrax toxin components, protective antigen (PA), lethal factor (LF), and edema factor (EF). While antibody to PA is sufficient to mediate protection against anthrax in animal models, it is not known if antibodies to LF or EF contribute to protection in humans. Toxin-neutralizing activity was evaluated in sera from AVA-vaccinated volunteers, all of whom had antibody responses to LF and EF, as well as PA. The contribution of antibodies to LF and EF was assessed using mouse macrophage J774A.1 cells by examining neutralization of LF-induced lysis using alamarBlue reduction and neutralization of EF-induced cyclic AMP increases by enzyme-linked immunosorbent assay. Antibody responses to LF and EF were low compared to those to PA, and the amount of LF or EF in the assay could exceed the amount of antibodies to LF or EF. Higher titers were seen for most individuals when the LF or EF concentration was limiting compared to when LF or EF was in excess, initially suggesting that antibody to LF or EF augmented protection. However, depletion of LF and EF antibodies in sera did not result in a significant decrease in toxin neutralization. Overall, this study suggests that AVA-induced LF and EF antibodies do not significantly contribute to anthrax toxin neutralization in humans and that antibodies to PA are sufficient to neutralize toxin activity.

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 Inactivation of Rho GTPases by Statins Attenuates Anthrax Lethal Toxin Activity

2008 ◽  
Vol 77 (1) ◽  
pp. 348-359 ◽  
Author(s):  
Aimee M. deCathelineau ◽  
Gary M. Bokoch
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Protective Antigen ◽  
Biological Effects ◽  
Lethal Factor ◽  
Lethal Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Gtpase Activity ◽  
Anthrax Lethal Toxin ◽  
Reductase Inhibitors

ABSTRACT Anthrax lethal factor (LF), secreted by Bacillus anthracis, interacts with protective antigen to form a bipartite toxin (lethal toxin [LT]) that exerts pleiotropic biological effects resulting in subversion of the innate immune response. Although the mitogen-activated protein kinase kinases (MKKs) are the major intracellular protein targets of LF, the pathology induced by LT is not well understood. The statin family of HMG-coenzyme A reductase inhibitors have potent anti-inflammatory effects independent of their cholesterol-lowering properties, which have been attributed to modulation of Rho family GTPase activity. The Rho GTPases regulate vesicular trafficking, cytoskeletal dynamics, and cell survival and proliferation. We hypothesized that disruption of Rho GTPase function by statins might alter LT action. We show here that statins delay LT-induced death and MKK cleavage in RAW macrophages and that statin-mediated effects on LT action are attributable to disruption of Rho GTPases. The Rho GTPase-inactivating toxin, toxin B, did not significantly affect LT binding or internalization, suggesting that the Rho GTPases regulate trafficking and/or localization of LT once internalized. The use of drugs capable of inhibiting Rho GTPase activity, such as statins, may provide a means to attenuate intoxication during B. anthracis infection.

scholarly journals Differential Contribution of Bacillus anthracis Toxins to Pathogenicity in Two Animal Models

2012 ◽  
Vol 80 (8) ◽  
pp. 2623-2631 ◽  
Author(s):  
Haim Levy ◽  
Shay Weiss ◽  
Zeev Altboum ◽  
Josef Schlomovitz ◽  
Itai Glinert ◽  
...  
Keyword(s):  
Animal Models ◽  
Guinea Pigs ◽  
Protective Antigen ◽  
Comprehensive Evaluation ◽  
Lethal Factor ◽  
Rabbit Model ◽  
Content Type ◽  
Edema Factor ◽  
Genes Encoding ◽  
Guinea Pig Model

ABSTRACTThe virulence ofBacillus anthracis, the causative agent of anthrax, stems from its antiphagocytic capsule, encoded by pXO2, and the tripartite toxins encoded by pXO1. The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play major roles in pathogenicity. We tested this assumption by a systematic study of mutants with combined deletions of thepag,lef, andcyagenes, encoding protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively. The resulting seven mutants (single, double, and triple) were evaluated following subcutaneous (s.c.) and intranasal (i.n.) inoculation in rabbits and guinea pigs. In the rabbit model, virulence is completely dependent on the presence of PA. Any mutant bearing apagdeletion behaved like a pXO1-cured mutant, exhibiting complete loss of virulence with attenuation indices of over 2,500,000 or 1,250 in the s.c. or i.n. route of infection, respectively. In marked contrast, in guinea pigs, deletion ofpagor even of all three toxin components resulted in relatively moderate attenuation, whereas the pXO1-cured bacteria showed complete attenuation. The results indicate that a pXO1-encoded factor(s), other than the toxins, has a major contribution to the virulence mechanism ofB. anthracisin the guinea pig model. These unexpected toxin-dependent and toxin-independent manifestations of pathogenicity in different animal models emphasize the importance and need for a comprehensive evaluation ofB. anthracisvirulence in general and in particular for the design of relevant next-generation anthrax vaccines.

scholarly journals Anthrax Toxin Characterization

2002 ◽  
Vol 45 (1) ◽  
pp. 3-5 ◽  
Author(s):  
Jiří Patočka ◽  
Miroslav Špliňo
Keyword(s):  
Blood Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
White Blood Cells ◽  
The Body ◽  
Anthrax Toxin ◽  
Signaling System ◽  
Animal Cells ◽  
The Third ◽  
Edema Factor

The anthrax toxin comprises three proteins. When they work together, they can kill humans, especially after spores of the bacteria have been inhaled. One anthrax protein, called protective antigen (PA), chaperones the two other toxins into human or animal cells and shields them from the body’s immune system. The second, lethal factor (LF), destroys the white blood cells that hosts send in defence. The third toxin molecule, edema factor (EF), hijacks the signaling system in the body. This disrupts the energy balance of cells and leads to them accumulating fluid and complete destroy of cells.

Sign in / Sign up

Export Citation Format

Share Document