scholarly journals Using a Syrian (Golden) Hamster Biological Model for the Evaluation of Recombinant Anthrax Vaccines

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1388
Author(s):  
Tatiana Kravchenko ◽  
Galina Titareva ◽  
Irina Bakhteeva ◽  
Tatiana Kombarova ◽  
Alexander Borzilov ◽  
...  
Keyword(s):  
Mouse Model ◽  
Recombinant Proteins ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Biological Model ◽  
Specific Antibodies ◽  
Syrian Hamsters ◽  
Factor I ◽  
Anthrax Vaccines ◽  
Pa Domain

In this paper, we demonstrate that a Syrian hamster biological model can be applied to the study of recombinant anthrax vaccines. We show that double vaccination with recombinant proteins, such as protective antigen (PA) and fusion protein LF1PA4, consisting of lethal factor I domain (LF) and PA domain IV, leads to the production of high titers of specific antibodies and to protection from infection with the toxicogenic encapsulated attenuated strain B. anthracis 71/12. In terms of antibody production and protection, Syrian hamsters were much more comparable to guinea pigs than mice. We believe that Syrian hamsters are still underestimated as a biological model for anthrax research, and, in some cases, they can be used as a replacement or at least as a complement to the traditionally used mouse model.

scholarly journals Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

2013 ◽  
Vol 20 (7) ◽  
pp. 986-997 ◽  
Author(s):  
Hang Lu ◽  
Jason Catania ◽  
Katalin Baranji ◽  
Jie Feng ◽  
Mili Gu ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Neutralization Assay ◽  
Dominant Factor ◽  
Anthrax Lethal Toxin ◽  
Serum Samples ◽  
Native Form ◽  
Anthrax Lethal Factor ◽  
Methionine Residues ◽  
Anthrax Vaccines

ABSTRACTThe cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply.

scholarly journals Serology and anthrax in humans, livestock and Etosha National Park wildlife

1992 ◽  
Vol 108 (2) ◽  
pp. 299-313 ◽  
Author(s):  
P. C. B. Turnbull ◽  
M. Doganay ◽  
P. M. Lindeque ◽  
B. Aygen ◽  
J. McLaughlin
Keyword(s):  
National Park ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Epidemiological Studies ◽  
Anthrax Toxin ◽  
Serological Survey ◽  
Diagnostic Aid ◽  
Specific Antibodies ◽  
Enzyme Immunoassays

SUMMARYResults are presented from a number of epidemiological studies using enzyme immunoassays (EIA) based on the purified anthrax toxin antigens, protective antigen, lethal factor and oedema factor. Studies on sera from a group of 62 human anthrax patients in Turkey and from cattle in Britain following two unrelated outbreaks of anthrax show that EIA using protective antigen can be a useful diagnostic aid and will detect subclinical infections in appropriate circumstances. A serological survey on wildlife in the Etosha National Park, Namibia, where anthrax is endemic, showed that naturally acquired anthrax-specific antibodies are rare in herbivores but common in carnivores; in carnivores, titres appear to reflect the prevalence of anthrax in their ranges. Problems, as yet unresolved, were encountered in studies on sera from pigs following an outbreak of anthrax on a farm in Wales.Clinical details, including treatment, of the human and one of the bovine outbreaks are summarized and discussed in relation to the serological findings.

scholarly journals The chymotrypsin-sensitive site, FFD315, in anthrax toxin protective antigen is required for translocation of lethal factor.

1994 ◽  
Vol 269 (46) ◽  
pp. 29039-29046
Author(s):  
Y Singh ◽  
K R Klimpel ◽  
N Arora ◽  
M Sharma ◽  
S H Leppla
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Sensitive Site

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 Efficacy of a Vaccine Based on Protective Antigen and Killed Spores against Experimental Inhalational Anthrax

2008 ◽  
Vol 77 (3) ◽  
pp. 1197-1207 ◽  
Author(s):  
Yves P. Gauthier ◽  
Jean-Nicolas Tournier ◽  
Jean-Charles Paucod ◽  
Jean-Philippe Corre ◽  
Michèle Mock ◽  
...  
Keyword(s):  
Guinea Pigs ◽  
Protective Immunity ◽  
Protective Antigen ◽  
Virulent Strain ◽  
Neutralizing Antibody ◽  
Cutaneous Anthrax ◽  
Anthrax Vaccines ◽  
Mucosal Secretions ◽  
Highly Virulent ◽  
Lethal Doses

ABSTRACTProtective antigen (PA)-based anthrax vaccines acting on toxins are less effective than live attenuated vaccines, suggesting that additional antigens may contribute to protective immunity. Several reports indicate that capsule or spore-associated antigens may enhance the protection afforded by PA. Addition of formaldehyde-inactivated spores (FIS) to PA (PA-FIS) elicits total protection against cutaneous anthrax. Nevertheless, vaccines that are effective against cutaneous anthrax may not be so against inhalational anthrax. The aim of this work was to optimize immunization with PA-FIS and to assess vaccine efficacy against inhalational anthrax. We assessed the immune response to recombinant anthrax PA fromBacillus anthracis(rPA)-FIS administered by various immunization protocols and the protection provided to mice and guinea pigs infected through the respiratory route with spores of a virulent strain ofB. anthracis. Combined subcutaneous plus intranasal immunization of mice yielded a mucosal immunoglobulin G response to rPA that was more than 20 times higher than that in lung mucosal secretions after subcutaneous vaccination. The titers of toxin-neutralizing antibody and antispore antibody were also significantly higher: nine and eight times higher, respectively. The optimized immunization elicited total protection of mice intranasally infected with the virulentB. anthracisstrain 17JB. Guinea pigs were fully protected, both against an intranasal challenge with 100 50% lethal doses (LD50) and against an aerosol with 75 LD50of spores of the highly virulent strain 9602. Conversely, immunization with PA alone did not elicit protection. These results demonstrate that the association of PA and spores is very much more effective than PA alone against experimental inhalational 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 Injection of Staphylococcus aureus EDIN by the Bacillus anthracis Protective Antigen Machinery Induces Vascular Permeability

2009 ◽  
Vol 77 (9) ◽  
pp. 3596-3601 ◽  
Author(s):  
Monica Rolando ◽  
Patrick Munro ◽  
Caroline Stefani ◽  
Patrick Auberger ◽  
Gilles Flatau ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Vascular Permeability ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Vascular Leakage ◽  
Host Cells ◽  
Mitogen Activated Protein Kinase ◽  
Systemic Injection ◽  
Pulmonary Endothelium ◽  
Endothelium Permeability

ABSTRACT Systemic injection of Bacillus anthracis lethal toxin (LT) produces vascular leakage and animal death. Recent studies suggest that LT triggers direct endothelial cell cytotoxicity that is responsible for the vascular leakage. LT is composed of heptamers of protective antigen (PA), which drives the endocytosis and translocation into host cells of the lethal factor (LF), a mitogen-activated protein kinase kinase protease. Here we investigated the consequences of injection of an endothelium-permeabilizing factor using LT as a “molecular syringe.” To this end, we generated the chimeric factor LE, corresponding to the PA-binding domain of LF (LF1-254) fused to EDIN exoenzyme. EDIN ADP ribosylates RhoA, leading to actin cable disruption and formation of transcellular tunnels in endothelial cells. We report that systemic injection of LET (LE plus PA) triggers a PA-dependent increase in the pulmonary endothelium permeability. We also report that native LT induces a progressive loss of endothelium barrier function. We established that there is a direct correlation between the extent of endothelium permeability induced by LT and the cytotoxic activity of LT. This suggests new ways to design therapeutic drugs against anthrax directed toward vascular permeability.

scholarly journals Comparative Secretome Analyses of Three Bacillus anthracis Strains with Variant Plasmid Contents

2005 ◽  
Vol 73 (6) ◽  
pp. 3646-3658 ◽  
Author(s):  
Janine M. Lamonica ◽  
MaryAnn Wagner ◽  
Michel Eschenbrenner ◽  
Leanne E. Williams ◽  
Tabbi L. Miller ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Isocitrate Lyase ◽  
Lethal Factor ◽  
Protein A ◽  
Surface Protein ◽  
Computer Assisted ◽  
Two Dimensional Gel Electrophoresis ◽  
Peptide Mass ◽  
Proteomic Approach

ABSTRACT Bacillus anthracis, the causative agent of anthrax, secretes numerous proteins into the extracellular environment during infection. A comparative proteomic approach was employed to elucidate the differences among the extracellular proteomes (secretomes) of three isogenic strains of B. anthracis that differed solely in their plasmid contents. The strains utilized were the wild-type virulent B. anthracis RA3 (pXO1+ pXO2+) and its two nonpathogenic derivative strains: the toxigenic, nonencapsulated RA3R (pXO1+ pXO2−) and the totally cured, nontoxigenic, nonencapsulated RA3:00 (pXO1− pXO2−). Comparative proteomics using two-dimensional gel electrophoresis followed by computer-assisted gel image analysis was performed to reveal unique, up-regulated, or down-regulated secretome proteins among the strains. In total, 57 protein spots, representing 26 different proteins encoded on the chromosome or pXO1, were identified by peptide mass fingerprinting. S-layer-derived proteins, such as Sap and EA1, were most frequently observed. Many sporulation-associated enzymes were found to be overexpressed in strains containing pXO1+. This study also provides evidence that pXO2 is necessary for the maximal expression of the pXO1-encoded toxins lethal factor (LF), edema factor (EF), and protective antigen (PA). Several newly identified putative virulence factors were observed; these include enolase, a high-affinity zinc uptake transporter, the peroxide stress-related alkyl hydroperoxide reductase, isocitrate lyase, and the cell surface protein A.

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.

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