A Chimeric Protein That Functions as both an Anthrax Dual-Target Antitoxin and a 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.

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

Infection and Immunity ◽

10.1128/iai.72.11.6313-6317.2004 ◽

2004 ◽

Vol 72 (11) ◽

pp. 6313-6317 ◽

Cited By ~ 71

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.

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

Infection and Immunity ◽

10.1128/iai.00719-06 ◽

2006 ◽

Vol 74 (11) ◽

pp. 6067-6074 ◽

Cited By ~ 61

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.

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Inhibition of Anthrax Protective Antigen Outside and Inside the Cell

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00983-06 ◽

2006 ◽

Vol 51 (1) ◽

pp. 245-251 ◽

Cited By ~ 17

Author(s):

Marina V. Backer ◽

Vimal Patel ◽

Brian T. Jehning ◽

Kevin P. Claffey ◽

Vladimir A. Karginov ◽

...

Keyword(s):

Protective Antigen ◽

Fluorescent Microscopy ◽

Lethal Factor ◽

Fluorescent Dye ◽

Cell Uptake ◽

Raw 264.7 Cells ◽

Receptor Mediated Endocytosis ◽

Edema Factor

ABSTRACT In the course of Bacillus anthracis infection, B. anthracis lethal factor (LF) and edema factor bind to a protective antigen (PA) associated with cellular receptors ANTXR1 (TEM8) or ANTXR2 (CMG2), followed by internalization of the complex via receptor-mediated endocytosis. A new group of potential antianthrax drugs, β-cyclodextrins, has recently been described. A member of this group, per-6-(3-aminopropylthio)-β-cyclodextrin (AmPrβCD), was shown to inhibit the toxicity of LF in vitro and in vivo. In order to determine which steps in lethal factor trafficking are inhibited by AmPrβCD, we developed two targeted fluorescent tracers based on LFn, a catalytically inactive fragment of LF: (i) LFn site specifically labeled with the fluorescent dye AlexaFluor-594 (LFn-Al), and (ii) LFn-decorated liposomes loaded with the fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (LFn-Lip). Both tracers retained high affinity to PA/ANTXR complexes and were readily internalized via receptor-mediated endocytosis. Using fluorescent microscopy, we found that AmPrβCD inhibits receptor-mediated cell uptake but not the binding of LFn-Al to PA/ANTXR complexes, suggesting that AmPrβCD works outside the cell. Moreover, AmPrβCD and LFn-Al synergistically protect RAW 264.7 cells from PA-mediated LF toxicity, confirming that AmPrβCD did not affect the binding of LFn-Al to receptor-associated PA. In contrast, AmPrβCD did not inhibit PA-mediated internalization of LFn-Lip, suggesting that multiplexing of LFn on the liposomal surface overcomes the inhibiting effects of AmPrβCD. Notably, internalized LFn-Al and LFn-Lip protected cells that overexpressed anthrax receptor TEM8 from PA-induced, LF-independent toxicity, suggesting an independent mechanism for PA inhibition inside the cell. These data suggest the potential for the use of β-cyclodextrins in combination with LFn-Lip loaded with antianthrax drugs against intracellular targets.

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Mouse Monoclonal Antibodies to Anthrax Edema Factor Protect against Infection

Infection and Immunity ◽

10.1128/iai.05314-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4609-4616 ◽

Cited By ~ 20

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.

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Anthrax Protective Antigen Cleavage and Clearance from the Blood of Mice and Rats

Infection and Immunity ◽

10.1128/iai.00719-07 ◽

2007 ◽

Vol 75 (11) ◽

pp. 5175-5184 ◽

Cited By ~ 56

Author(s):

Mahtab Moayeri ◽

Jason F. Wiggins ◽

Stephen H. Leppla

Keyword(s):

Receptor Binding ◽

Protective Antigen ◽

Lethal Factor ◽

Dominant Negative ◽

Furin Cleavage ◽

Defective Mutant ◽

Tissue Surfaces ◽

Anthrax Protective Antigen

ABSTRACT Bacillus anthracis protective antigen (PA) is an 83-kDa (PA83) protein that is cleaved to the 63-kDa protein (PA63) as an essential step in binding and internalizing lethal factor (LF). To assess in vivo receptor saturating PA concentrations, we injected mice with PA variants and measured the PA remaining in the blood at various times using PA83- and PA63-specific enzyme-linked immunosorbent assays. We found that both wild-type PA (WT-PA) and a receptor-binding-defective mutant (Ub-PA) were cleaved to PA63 independent of their ability to bind cells. This suggested a PA-acting protease activity in the blood. The protease cleaved PA at the furin cleavage sequence because furin site-modified PA mutants were not cleaved. Cleavage measured in vitro was leupeptin sensitive and dependent on calcium. Cell surface cleavage was important for toxin clearance, however, as Ub-PA and uncleavable PA mutants were cleared at slower rates than WT-PA. The cell binding-independent cleavage of PA was also verified by using Ub-PA (which is still cleaved) to rescue mice from toxin challenge by competitively binding circulating LF. This mutant was able to rescue mice even when given 12 h before toxin challenge. Its therapeutic ability was comparable to that of dominant-negative PA, which binds cells but does not allow LF translocation, and to the protection afforded through receptor clearance by WT-PA and uncleavable receptor binding-competent mutants. The PA cleavage and clearance observed in mice did not appear to have a role in the differential mouse susceptibility as it occurred similarly in lethal toxin (LT)-resistant DBA/2J and LT-sensitive BALB/cJ mice. Interestingly, PA63 was not found in LT-resistant or -sensitive rats and PA83 clearance was slower in rats than in mice. Finally, to determine the minimum amount of PA required in circulation for LT toxicity in mice, we administered time-separated injections of PA and LF and showed that lethality of LF for mice after PA was no longer measurable in circulation, suggesting active PA sequestration at tissue surfaces.

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Structure of a dominant negative mutant reveals a stalled intermediate state of anthrax protective antigen pore maturation

10.1101/2020.06.15.152231 ◽

2020 ◽

Author(s):

Harry Scott ◽

Wei Huang ◽

Srinivas Gonti ◽

Kaiming Zhang ◽

Nurjahan Mehzabeen ◽

...

Keyword(s):

Pore Formation ◽

Protective Antigen ◽

Lethal Factor ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Edema Factor ◽

Beta Barrel ◽

Anthrax Protective Antigen ◽

Maturation State ◽

Membrane Spanning

AbstractAnthrax is a severe bacterial infection caused by Bacillus anthracis, which produces a tripartite toxin that includes protective antigen (PA), lethal factor (LF) and edema factor (EF). A series of dominant-negative mutations have been previously identified that prevent the heptameric PA prepore from forming the pH-induced, membrane spanning beta-barrel pore that is required for translocation of EF and LF to the cytoplasm of the infected cell. Here we show that the dominant negative D425A mutation stalls the formation of the pore at a reversible intermediate maturation state, which exhibits many of the structural aspects of the pore but fails to form the phi(ϕ)-clamp and beta-barrel structure needed for full pore maturation. Overall, this structure reveals that ϕ-clamp and beta-barrel pore formation are later steps in the pathway to pore formation, thereby providing a regulatory mechanism to prevent premature translocation of EF and LF.

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

10.1101/2021.01.22.427304 ◽

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.

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Investigation of New Dominant-Negative Inhibitors of Anthrax Protective Antigen Mutants for Use in Therapy and Vaccination

Infection and Immunity ◽

10.1128/iai.00264-09 ◽

2009 ◽

Vol 77 (10) ◽

pp. 4679-4687 ◽

Cited By ~ 10

Author(s):

Sha Cao ◽

Aizhen Guo ◽

Ziduo Liu ◽

Yadi Tan ◽

Gaobing Wu ◽

...

Keyword(s):

Protective Antigen ◽

Lethal Factor ◽

Therapeutic Agents ◽

Dominant Negative ◽

Raw264.7 Cells ◽

Necrosis Factor Alpha ◽

Anthrax Protective Antigen ◽

The Individual ◽

Novel Therapeutic

ABSTRACT The lethal toxin (LeTx) of Bacillus anthracis plays a key role in the pathogenesis of anthrax. The protective antigen (PA) is a primary part of the anthrax toxin and forms LeTx by combination with lethal factor (LF). Phenylalanine-427 (F427) is crucial for PA function. This study was designed to discover potential novel therapeutic agents and vaccines for anthrax. This was done by screening PA mutants that were mutated at the F427 residue for a dominant-negative inhibitory (DNI) phenotype which was nontoxic but inhibited the toxicity of the wild-type LeTx. For this, PA residue F427 was first mutated to each of the other 19 naturally occurring amino acids. The cytotoxicity and DNI phenotypes of the mutated PA proteins were tested in the presence of 1 μg/ml LF in RAW264.7 cells and were shown to be dependent on the individual amino acid replacements. A total of 16 nontoxic mutants with various levels of DNI activity were identified in vitro. Among them, F427D and F427N mutants had the highest DNI activities in RAW264.7 cells. Both mutants inhibited LeTx intoxication in mice in a dose-dependent way. Furthermore, they induced a Th2-predominant immune response and protected mice against a challenge with five 50% lethal doses of LeTx. The protection was correlated mainly with a low level of interleukin-1β (IL-1β) and with high levels of PA-specific immunoglobulin G1, IL-6, and tumor necrosis factor alpha. Thus, PA DNI mutants, such as F427D and F427N mutants, may serve in the development of novel therapeutic agents and vaccines to fight B. anthracis infections.

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Discriminating Virulence Mechanisms among Bacillus anthracis Strains by Using a Murine Subcutaneous Infection Model

Infection and Immunity ◽

10.1128/iai.00647-08 ◽

2008 ◽

Vol 77 (1) ◽

pp. 429-435 ◽

Cited By ~ 27

Author(s):

Hitendra S. Chand ◽

Melissa Drysdale ◽

Julie Lovchik ◽

Theresa M. Koehler ◽

Mary F. Lipscomb ◽

...

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Lethal Dose ◽

Infection Model ◽

Virulence Plasmid ◽

Edema Factor ◽

Lethal Infection ◽

Toxin Protein ◽

Ames Strain

ABSTRACT Bacillus anthracis strains harboring virulence plasmid pXO1 that encodes the toxin protein protective antigen (PA), lethal factor, and edema factor and virulence plasmid pXO2 that encodes capsule biosynthetic enzymes exhibit different levels of virulence in certain animal models. In the murine model of pulmonary infection, B. anthracis virulence was capsule dependent but toxin independent. We examined the role of toxins in subcutaneous (s.c.) infections using two different genetically complete (pXO1+ pXO2+) strains of B. anthracis, strains Ames and UT500. Similar to findings for the pulmonary model, toxin was not required for infection by the Ames strain, because the 50% lethal dose (LD50) of a PA-deficient (PA−) Ames mutant was identical to that of the parent Ames strain. However, PA was required for efficient s.c. infection by the UT500 strain, because the s.c. LD50 of a UT500 PA− mutant was 10,000-fold higher than the LD50 of the parent UT500 strain. This difference between the Ames strain and the UT500 strain could not be attributed to differences in spore coat properties or the rate of germination, because s.c. inoculation with the capsulated bacillus forms also required toxin synthesis by the UT500 strain to cause lethal infection. The toxin-dependent phenotype of the UT500 strain was host phagocyte dependent, because eliminating Gr-1+ phagocytes restored virulence to the UT500 PA− mutant. These experiments demonstrate that the dominant virulence factors used to establish infection by B. anthracis depend on the route of inoculation and the bacterial strain.

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The Major Neutralizing Antibody Responses to Recombinant Anthrax Lethal and Edema Factors Are Directed to Non-Cross-Reactive Epitopes

Infection and Immunity ◽

10.1128/iai.00749-09 ◽

2009 ◽

Vol 77 (11) ◽

pp. 4714-4723 ◽

Cited By ~ 17

Author(s):

Melissa L. Nguyen ◽

Simon Terzyan ◽

Jimmy D. Ballard ◽

Judith A. James ◽

A. Darise Farris

Keyword(s):

B Cell ◽

Protective Antigen ◽

Solid Phase ◽

Lethal Factor ◽

Neutralizing Antibody ◽

B Cell Epitopes ◽

Binding Domains ◽

Edema Factor ◽

Humoral Responses

ABSTRACT Anthrax lethal and edema toxins (LeTx and EdTx, respectively) form by binding of lethal factor (LF) or edema factor (EF) to the pore-forming moiety protective antigen (PA). Immunity to LF and EF protects animals from anthrax spore challenge and neutralizes anthrax toxins. The goal of the present study is to identify linear B-cell epitopes of EF and to determine the relative contributions of cross-reactive antibodies of EF and LF to LeTx and EdTx neutralization. A/J mice were immunized with recombinant LF (rLF) or rEF. Pools of LF or EF immune sera were tested for reactivity to rLF or rEF by enzyme-linked immunosorbent assays, in vitro neutralization of LeTx and EdTx, and binding to solid-phase LF and EF decapeptides. Cross-reactive antibodies were isolated by column absorption of EF-binding antibodies from LF immune sera and by column absorption of LF-binding antibodies from EF immune sera. The resulting fractions were subjected to the same assays. Major cross-reactive epitopes were identified as EF amino acids (aa) 257 to 268 and LF aa 265 to 274. Whole LF and EF immune sera neutralized LeTx and EdTx, respectively. However, LF sera did not neutralize EdTx, nor did EF sera neutralize LeTx. Purified cross-reactive immunoglobulin G also failed to cross-neutralize. Cross-reactive B-cell epitopes in the PA-binding domains of whole rLF and rEF occur and have been identified; however, the major anthrax toxin-neutralizing humoral responses to these antigens are constituted by non-cross-reactive epitopes. This work increases understanding of the immunogenicity of EF and LF and offers perspective for the development of new strategies for vaccination against anthrax.

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