Anthrax Vaccine Precipitated Induces Edema Toxin-Neutralizing, Edema Factor-Specific Antibodies in Human Recipients

ABSTRACT Edema toxin (ET), composed of edema factor (EF) and protective antigen (PA), is a virulence factor of Bacillus anthracis that alters host immune cell function and contributes to anthrax disease. Anthrax vaccine precipitated (AVP) contains low but detectable levels of EF and can elicit EF-specific antibodies in human recipients of AVP. Active and passive vaccination of mice with EF can contribute to protection from challenge with Bacillus anthracis spores or ET. This study compared humoral responses to ET in recipients of AVP (n = 33) versus anthrax vaccine adsorbed (AVA; n = 66), matched for number of vaccinations and time postvaccination, and further determined whether EF antibodies elicited by AVP contribute to ET neutralization. AVP induced higher incidence (77.8%) and titer (229.8 ± 58.6) of EF antibodies than AVA (4.2% and 7.8 ± 8.3, respectively), reflecting the reported low but detectable presence of EF in AVP. In contrast, PA IgG levels and ET neutralization measured using a luciferase-based cyclic AMP reporter assay were robust and did not differ between the two vaccine groups. Multiple regression analysis failed to detect an independent contribution of EF antibodies to ET neutralization in AVP recipients; however, EF antibodies purified from AVP sera neutralized ET. Serum samples from at least half of EF IgG-positive AVP recipients bound to nine decapeptides located in EF domains II and III. Although PA antibodies are primarily responsible for ET neutralization in recipients of AVP, increased amounts of an EF component should be investigated for the capacity to enhance next-generation, PA-based vaccines.

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Anthrax Edema Toxin Requires Influx of Calcium for Inducing Cyclic AMP Toxicity in Target Cells

Infection and Immunity ◽

10.1128/iai.70.9.4997-5007.2002 ◽

2002 ◽

Vol 70 (9) ◽

pp. 4997-5007 ◽

Cited By ~ 33

Author(s):

Praveen Kumar ◽

Nidhi Ahuja ◽

Rakesh Bhatnagar

Keyword(s):

Cyclic Amp ◽

Cell Function ◽

Immune Cell ◽

Protective Antigen ◽

Human Lymphocytes ◽

Host Cells ◽

Human Neutrophils ◽

Target Cells ◽

Edema Factor ◽

Edema Toxin

ABSTRACT The anthrax edema toxin comprises two proteins: protective antigen and edema factor. Anthrax protective antigen binds to the receptors on the surface of target cells and facilitates the entry of edema factor into these target cells. Edema factor (EF) is an adenylate cyclase that catalyzes the synthesis of cyclic AMP (cAMP) in the cytosol of the host cells. In this study, we examined the requirement of extracellular calcium for anthrax edema toxin-induced toxicity in host cells. The cAMP response generated by edema toxin was analyzed in a variety of cells, including CHO, macrophage-like RAW264.7, human neutrophils, and human lymphocytes. Our investigations reveal that after EF reaches the cell cytosol, a rapid influx of calcium is triggered in the host cell that has a pivotal role in determining the cAMP response of the affected cells. Although the cAMP response generated by edema toxin in different cell types varied in intensity and in the time of initiation, the influx of calcium invariably preceded cAMP accumulation. Agents that blocked the uptake of calcium also inhibited edema toxin-induced accumulation of cAMP in the host cells. This is the first report that demonstrates that edema toxin induces accumulation of cAMP in lymphocytes. By accumulating cAMP, a potent inhibitor of immune cell function, edema toxin may actually be poisoning the immune system and thus facilitating the survival of the bacteria in the host.

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Expression of either Lethal Toxin or Edema Toxin by Bacillus anthracis Is Sufficient for Virulence in a Rabbit Model of Inhalational Anthrax

Infection and Immunity ◽

10.1128/iai.06340-11 ◽

2012 ◽

Vol 80 (7) ◽

pp. 2414-2425 ◽

Cited By ~ 21

Author(s):

Julie A. Lovchik ◽

Melissa Drysdale ◽

Theresa M. Koehler ◽

Julie A. Hutt ◽

C. Rick Lyons

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Rabbit Model ◽

Lethal Toxin ◽

Content Type ◽

Edema Factor ◽

Systemic Dissemination ◽

Edema Toxin ◽

Ames Strain

ABSTRACTThe development of therapeutics against biothreats requires that we understand the pathogenesis of the disease in relevant animal models. The rabbit model of inhalational anthrax is an important tool in the assessment of potential therapeutics againstBacillus anthracis. We investigated the roles ofB. anthraciscapsule and toxins in the pathogenesis of inhalational anthrax in rabbits by comparing infection with the Ames strain versus isogenic mutants with deletions of the genes for the capsule operon (capBCADE), lethal factor (lef), edema factor (cya), or protective antigen (pagA). The absence of capsule or protective antigen (PA) resulted in complete avirulence, while the presence of either edema toxin or lethal toxin plus capsule resulted in lethality. The absence of toxin did not influence the ability ofB. anthracisto traffic to draining lymph nodes, but systemic dissemination required the presence of at least one of the toxins. Histopathology studies demonstrated minimal differences among lethal wild-type and single toxin mutant strains. When rabbits were coinfected with the Ames strain and the PA− mutant strain, the toxin produced by the Ames strain was not able to promote dissemination of the PA− mutant, suggesting that toxigenic action occurs in close proximity to secreting bacteria. Taken together, these findings suggest that a major role for toxins in the pathogenesis of anthrax is to enable the organism to overcome innate host effector mechanisms locally and that much of the damage during the later stages of infection is due to the interactions of the host with the massive bacterial burden.

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Contributions of Histamine, Prostanoids, and Neurokinins to Edema Elicited by Edema Toxin from Bacillus anthracis

Infection and Immunity ◽

10.1128/iai.01632-06 ◽

2007 ◽

Vol 75 (4) ◽

pp. 1895-1903 ◽

Cited By ~ 24

Author(s):

Jeffrey Tessier ◽

Candace Green ◽

Diana Padgett ◽

Wei Zhao ◽

Lawrence Schwartz ◽

...

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Cell Types ◽

Vascular Leakage ◽

Target Cells ◽

Edema Factor ◽

Edema Toxin ◽

Neurokinin 1

ABSTRACT Bacillus anthracis edema toxin (ET), composed of protective antigen and an adenylate cyclase edema factor (EF), elicits edema in host tissues, but the target cells and events leading from EF-mediated cyclic-AMP production to edema are unknown. We evaluated the direct effect of ET on several cell types in vitro and tested the possibility that mediators of vascular leakage, such as histamine, contribute to edema in rabbits given intradermal ET. ET increased the transendothelial electrical resistance of endothelial monolayers, a response that is mechanistically inconsistent with the in vivo vascular leakage induced by ET. Screening of several drugs by intradermal treatment prior to toxin injection demonstrated reduced ET-induced vascular leakage with a cyclo-oxygenase inhibitor (indomethacin), agents that interfere with histamine (pyrilamine or cromolyn), or a neurokinin antagonist (spantide). Systemic administration of indomethacin or celecoxib (cyclo-oxygenase inhibitors), pyrilamine, aprepitant (a neurokinin 1 receptor antagonist), or indomethacin with pyrilamine significantly reduced vascular leakage associated with ET. Although the effects of pyrilamine, cromolyn, or aprepitant on ET-induced vascular leakage suggest a possible role for mast cells (MC) and sensory neurons in ET-induced edema, ET did not elicit degranulation of human skin MC or substance P release from NT2N cells in vitro. Our results indicate that ET, acting indirectly or directly on a target yet to be identified, stimulates the production/release of multiple inflammatory mediators, specifically neurokinins, prostanoids, and histamine. These mediators, individually and through complex interactions, increase vascular permeability, and interventions directed at these mediators may benefit hosts infected with B. anthracis.

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Bacillus anthracis Edema Toxin Impairs Neutrophil Actin-Based Motility

Infection and Immunity ◽

10.1128/iai.00839-08 ◽

2009 ◽

Vol 77 (6) ◽

pp. 2455-2464 ◽

Cited By ~ 28

Author(s):

Sarah E. Szarowicz ◽

Russell L. During ◽

Wei Li ◽

Conrad P. Quinn ◽

Wei-Jen Tang ◽

...

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Fold Increase ◽

Polymorphonuclear Neutrophil ◽

Actin Assembly ◽

Anthrax Lethal Toxin ◽

Edema Factor ◽

Low Concentrations ◽

Inhalation Anthrax ◽

Edema Toxin

ABSTRACT Inhalation anthrax results in high-grade bacteremia and is accompanied by a delay in the rise of the peripheral polymorphonuclear neutrophil (PMN) count and a paucity of PMNs in the infected pleural fluid and mediastinum. Edema toxin (ET) is one of the major Bacillus anthracis virulence factors and consists of the adenylate cyclase edema factor (EF) and protective antigen (PA). Relatively low concentrations of ET (100 to 500 ng/ml of PA and EF) significantly impair human PMN chemokinesis, chemotaxis, and ability to polarize. These changes are accompanied by a reduction in chemoattractant-stimulated PMN actin assembly. ET also causes a significant decrease in Listeria monocytogenes intracellular actin-based motility within HeLa cells. These defects in actin assembly are accompanied by a >50-fold increase in intracellular cyclic AMP and a >4-fold increase in the phosphorylation of protein kinase A. We have previously shown that anthrax lethal toxin (LT) also impairs neutrophil actin-based motility (R. L. During, W. Li, B. Hao, J. M. Koenig, D. S. Stephens, C. P. Quinn, and F. S. Southwick, J. Infect. Dis. 192:837-845, 2005), and we now find that LT combined with ET causes an additive inhibition of PMN chemokinesis, polarization, chemotaxis, and FMLP (N-formyl-met-leu-phe)-induced actin assembly. We conclude that ET alone or combined with LT impairs PMN actin assembly, resulting in paralysis of PMN chemotaxis.

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

Infection and Immunity ◽

10.1128/iai.00530-08 ◽

2009 ◽

Vol 77 (4) ◽

pp. 1649-1663 ◽

Cited By ~ 24

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.

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

Infection and Immunity ◽

10.1128/iai.01712-06 ◽

2007 ◽

Vol 75 (4) ◽

pp. 1757-1764 ◽

Cited By ~ 12

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.

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Current understanding of Bacillus anthracis toxin molecules organization and approaches for blocking their cytotoxic action

Russian Journal of Infection and Immunity ◽

10.15789/2220-7619-2019-5-6-639-647 ◽

2020 ◽

Vol 9 (5-6) ◽

pp. 639-647

Author(s):

V. V. Firstova ◽

I. G. Shemyakin ◽

I. A. Dyatlov

Keyword(s):

Bacillus Anthracis ◽

Immune Cell ◽

Protective Antigen ◽

Functional Organization ◽

Lethal Factor ◽

Anthrax Toxin ◽

Eukaryotic Cells ◽

Receptor Interaction ◽

Cytotoxic Action ◽

Edema Factor

Here, we review the data on mechanisms inhibiting cytotoxic effect of anthrax toxin on the immune system cells. Various disease forms, immunopathogenesis and contemporary methods for anthrax treatment are discussed. In addition, an anthrax toxin was outlined, whereas structural and functional organization of the protective antigen, lethal and edema factors was detailed. A mechanism for association of a protective antigen and lethal factor, protective antigen and edema factor leading to formation of a lethal toxin and edema toxin, respectively, was described. Participation of protective antigen domains in the process of interaction with surface receptors of imunocompetent cells as well as features of binding a protective antigen with lethal factor and edema factor are discussed. A mechanism of endosomal toxin complex internalization and subsequent transfer of effector molecules to the cytosol are described. Effects of the lethal factor and the edema factor on components of eukaryotic cells as well as cytotoxicity mechanisms are analyzed. The approaches to block anthrax toxin action at various stages of toxicoemia have been analyzed based on previously uncovered sequential signs of cytotoxic activity for Bacillus anthracis toxins. Currently available chimeric and humanized monoclonal antibodies are capable of neutralizing B. anthracis toxins at diverse assembly stages, particularly considering the drugs inhibiting: inter-receptor interaction between protective antigen with eukaryotic cells; furin-like enzymes activating prepore assembly; protective antigen oligomerization; binding of the lethal factor or edema factor to the protective antigen; translocation of the lethal factor or the edema factor into cell cytosol; transport of protective antigen with lethal factor or edema factor from endosomes; enzymatic activity of lethal factor or edematous factor. The anti-toxin agents approved for anthrax prevention and treatment in Russia and worldwide are discussed. The limitations of anti-toxin agents and perspectives for their improvement are also described including inhibition of lethal factor activity, interference with integration of toxin components, blockade of interactions between toxic complexes and immune cell receptors.

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Anti-Protective Antigen IgG Enzyme-Linked Immunosorbent Assay for Diagnosis of Cutaneous Anthrax in India

Clinical and Vaccine Immunology ◽

10.1128/cvi.00154-12 ◽

2012 ◽

Vol 19 (8) ◽

pp. 1238-1242 ◽

Cited By ~ 15

Author(s):

N. Ghosh ◽

A. K. Goel

Keyword(s):

Bacillus Anthracis ◽

Immunosorbent Assay ◽

Predictive Value ◽

Protective Antigen ◽

Public Health Problem ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Animal Products ◽

Content Type ◽

Cutaneous Anthrax

ABSTRACTAnthrax caused byBacillus anthracisis a public health problem in several developing countries whose main source of income is farming. Anthrax is a disease of herbivorous animals, and humans can be infected by handling infected animals or contaminated animal products. Specific diagnostic tests are unavailable in India for the detection and confirmation of cutaneous anthrax in humans. Here, we describe the development of an enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies againstBacillus anthracisprotective antigen in the Indian population. A total of 405 serum samples collected from different groups were tested by the developed ELISA. The assay provided a specificity of 99.41% (95% confidence interval [CI], 97.89 to 99.93) and a sensitivity of 100% (CI, 94.4 to 100) using a cutoff value of 0.29 ELISA unit (EU). The positive predictive value (PPV) and negative predictive value (NPV) of the assay were 97% and 100%, respectively. The efficiency and J index for the reliability of the assay were 99.5% and 0.994, respectively. The assay can be a very useful tool for surveillance as well as for diagnosis of cutaneous anthrax cases in India.

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