scholarly journals Enhanced Production of Protective Antigen, a Potent Diagnostic Protein of Bacillus anthracis, the Causative Agent of Anthrax

2019 ◽  
Vol 4 (4) ◽  
pp. 250-255
Author(s):  
Manoj Kumar ◽  
Nidhi Puranik ◽  
Nagesh Tripathi ◽  
Vijai Pal ◽  
Ajay Goel
Keyword(s):  
Western Blot ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enhanced Production ◽  
Diagnostic Potential ◽  
Anthrax Infection ◽  
Final Yield ◽  
Cutaneous Anthrax

Protective antigen (PA) produced by Bacillus anthracis is a highly immunogenic protein. Therefore, it has significant importance in serodiagnosis as well as a vaccine candidate for anthrax. In the present study, codons for PA gene were optimised and synthesised for its expression in Escherichia coli. Various expression conditions were optimised for scaled up production of rPA. The final yield of affinity chromatography purified protein was 40.8 mg/l during batch fermentation. For further purification, affinity purified protein was diafiltered and subjected to anion exchange chromatography. SDS-PAGE and Western blot was used to characterise the purified rPA protein. The diagnostic potential of purified rPA was evaluated in Western blot using standards reference serum AVR 801 and cutaneous anthrax clinical sera. The results of the present study established the optimum production of rPA in E. coli after codon optimisation for its subsequent use in diagnosis of anthrax infection.

scholarly journals A Recombinant Carboxy-Terminal Domain of the Protective Antigen of Bacillus anthracis Protects Mice against Anthrax Infection

2002 ◽  
Vol 70 (3) ◽  
pp. 1653-1656 ◽  
Author(s):  
Helen C. Flick-Smith ◽  
Nicola J. Walker ◽  
Paula Gibson ◽  
Helen Bullifent ◽  
Sarah Hayward ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Fusion Proteins ◽  
Protective Antigen ◽  
Protective Efficacy ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Anthrax Infection ◽  
Carboxy Terminal ◽  
Domain 4

ABSTRACT The immunogenicity and protective efficacy of overlapping regions of the protective antigen (PA) polypeptide, cloned and expressed as glutathione S-transferase fusion proteins, have been assessed. Results show that protection can be attributed to individual domains and imply that it is domain 4 which contains the dominant protective epitopes of PA.

scholarly journals Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection

PLoS ONE ◽  
2015 ◽  
Vol 10 (7) ◽  
pp. e0130952 ◽  
Author(s):  
Matthew D. Reed ◽  
Julie A. Wilder ◽  
William M. Mega ◽  
Julie A. Hutt ◽  
Philip J. Kuehl ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Pseudomonas Fluorescens ◽  
Protective Antigen ◽  
Mutant Form ◽  
Anthrax Infection

The Humoral Immune Response to Various Domains of Protective Antigen of Bacillus anthracis in Cutaneous Anthrax Cases in India

2016 ◽  
Vol 66 (6) ◽  
pp. 645 ◽  
Author(s):  
Anshul Varshney ◽  
Nidhi Puranik ◽  
M. Kumar ◽  
A.K. Goel
Keyword(s):  
Immune Response ◽  
Bacillus Anthracis ◽  
Humoral Immune Response ◽  
Vaccine Development ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Serum Samples ◽  
Public Health Importance ◽  
Humoral Immune ◽  
Cutaneous Anthrax

Anthrax, caused by Bacillus anthracis is known to occur globally since antiquity. Besides being an important biothreat agent, it is an important public health importance pathogen also in countries like India. B. anthracis secretes three distinct toxins, namely protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the central moiety of the anthrax toxin complex and therefore has been a molecule of choice for vaccine development. PA has four different domains with different functions. In this study, the major domains of PA were cloned and expressed in bacterial system. The purified recombinant proteins were used to determine the humoral immune response by ELISA using 43 human cutaneous anthrax serum samples. The maximum immunoreactivity was observed with the whole PA protein followed by domain 2, 4 and 1. The study corroborated that in addition to full PA, individual domain 2 and 4 can also be good target for vaccine development as well as for serodiagnostic assays for cutaneous anthrax

scholarly journals The Poly-γ-d-Glutamic Acid Capsule of Bacillus anthracis Enhances Lethal Toxin Activity

2011 ◽  
Vol 79 (9) ◽  
pp. 3846-3854 ◽  
Author(s):  
Jeyoun Jang ◽  
Minhui Cho ◽  
Jeong-Hoon Chun ◽  
Min-Hee Cho ◽  
Jungchan Park ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Immune Surveillance ◽  
Lethal Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Anthrax Infection ◽  
Processed Form

ABSTRACTThe poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors ofBacillus anthracis, which causes a highly lethal infectious disease. The PGA capsule disguisesB. anthracisfrom immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentration-dependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.

scholarly journals Cutaneous infection caused by Bacillus anthracis in Larissa, Thessaly, Central Greece, July 2012

2012 ◽  
Vol 17 (32) ◽  
Author(s):  
A Stefos ◽  
N K Gatselis ◽  
A Goudelas ◽  
M Mpakarosi ◽  
J Papaparaskevas ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Control Measures ◽  
Cutaneous Infection ◽  
Central Greece ◽  
Anthrax Infection ◽  
Cutaneous Anthrax

In July 2012, a confirmed case of cutaneous anthrax infection in a stockbreeder in the prefecture of Larissa, Thessaly, Central Greece was reported. The investigation revealed five related deaths in animals (two dogs and three sheep). Control measures have been taken immediately in order to prevent further spread in humans and animals.

scholarly journals Synthetic, structural and biological studies of the ubiquitin system: the total chemical synthesis of ubiquitin

1994 ◽  
Vol 299 (1) ◽  
pp. 151-158 ◽  
Author(s):  
R Ramage ◽  
J Green ◽  
T W Muir ◽  
O M Ogunjobi ◽  
S Love ◽  
...  
Keyword(s):  
Solid Phase ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Protecting Group ◽  
Ubiquitin System ◽  
Biological Studies ◽  
Final Yield ◽  
Fmoc Protecting Group ◽  
Optimized Conditions

The small protein ubiquitin (76 amino acids) has been synthesized under optimized conditions by Merrifield solid-phase methodology using the N alpha-Fmoc protecting group. The crude polypeptide mixture was purified to homogeneity by gel filtration, dialysis and a combination of cation- and anion-exchange chromatography to yield ubiquitin. Amino acid analysis, enzymic digestion and sequencing by automated Edman degradation were used to authenticate the primary structure. Isoelectric focusing and m.s. were used to demonstrate that the final product was greater than 98% pure with a final yield of 93 mg (4.3%) from a single synthesis on a 0.25 nmol scale.

scholarly journals A High-Affinity Monoclonal Antibody to Anthrax Protective Antigen Passively Protects Rabbits before and after Aerosolized Bacillus anthracis Spore Challenge

2005 ◽  
Vol 73 (2) ◽  
pp. 795-802 ◽  
Author(s):  
Nehal Mohamed ◽  
Michelle Clagett ◽  
Juan Li ◽  
Steven Jones ◽  
Steven Pincus ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Dose ◽  
Cell Binding ◽  
Anthrax Spore ◽  
Before And After ◽  
Anthrax Infection ◽  
Anthrax Protective Antigen ◽  
Binding Component

ABSTRACT We have developed a therapeutic for the treatment of anthrax using an affinity-enhanced monoclonal antibody (ETI-204) to protective antigen (PA), which is the central cell-binding component of the anthrax exotoxins. ETI-204 administered preexposure by a single intravenous injection of a dose of between 2.5 and 10 mg per animal significantly protected rabbits from a lethal aerosolized anthrax spore challenge (∼60 to 450 times the 50% lethal dose of Bacillus anthracis Ames). Against a similar challenge, ETI-204 administered intramuscularly at a 20-mg dose per animal completely protected rabbits from death (100% survival). In the postexposure setting, intravenous administration of ETI-204 provided protection 24 h (8 of 10) and 36 h (5 of 10) after spore challenge. Administration at 48 h postchallenge, when 3 of 10 animals had already succumbed to anthrax infection, resulted in the survival of 3 of 7 animals (43%) for the duration of the study (28 days). Importantly, surviving ETI-204-treated animals were free of bacteremia by day 10 and remained so until the end of the studies. Only 11 of 51 ETI-204-treated rabbits had positive lung cultures at the end of the studies. Also, rabbits that were protected from inhalational anthrax by administration of ETI-204 developed significant titers of PA-specific antibodies. Presently, the sole therapeutic regimen available to treat infection by inhalation of B. anthracis spores is a 60-day course of antibiotics that is effective only if administered prior to or shortly after exposure. Based upon results reported here, ETI-204 is an effective therapy for prevention and treatment of inhalational anthrax.

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

2012 ◽  
Vol 19 (8) ◽  
pp. 1238-1242 ◽  
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.

scholarly journals Mucosal or Parenteral Administration of Microsphere-Associated Bacillus anthracis Protective Antigen Protects against Anthrax Infection in Mice

2002 ◽  
Vol 70 (4) ◽  
pp. 2022-2028 ◽  
Author(s):  
Helen C. Flick-Smith ◽  
Jim E. Eyles ◽  
Richard Hebdon ◽  
Emma L. Waters ◽  
Richard J. Beedham ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Protective Efficacy ◽  
Dose Schedule ◽  
Mucosal Immune Response ◽  
Parenteral Administration ◽  
Routes Of Administration ◽  
Anthrax Infection ◽  
Anthrax Vaccines ◽  
Lethal Doses

ABSTRACT Existing licensed anthrax vaccines are administered parenterally and require multiple doses to induce protective immunity. This requires trained personnel and is not the optimum route for stimulating a mucosal immune response. Microencapsulation of vaccine antigens offers a number of advantages over traditional vaccine formulations, including stability without refrigeration and the potential for utilizing less invasive routes of administration. Recombinant protective antigen (rPA), the dominant antigen for protection against anthrax infection, was encapsulated in poly-l-lactide 100-kDa microspheres. Alternatively, rPA was loosely attached to the surfaces of microspheres by lyophilization. All of the microspheric formulations were administered to A/J mice with a two-dose schedule by either the intramuscular route, the intranasal route, or a combination of these two routes, and immunogenicity and protective efficacy were assessed. An intramuscular priming immunization followed by either an intramuscular or intranasal boost gave optimum anti-rPA immunoglobulin G titers. Despite differences in rPA-specific antibody titers, all immunized mice survived an injected challenge consisting of 103 median lethal doses of Bacillus anthracis STI spores. Immunization with microencapsulated and microsphere-associated formulations of rPA also protected against aerosol challenge with 30 median lethal doses of STI spores. These results show that rPA can be encapsulated and surface bound to polymeric microspheres without impairing its immunogenicity and also that mucosal or parenteral administration of microspheric formulations of rPA efficiently protects mice against both injected and aerosol challenges with B. anthracis spores. Microspheric formulations of rPA could represent the next generation of anthrax vaccines, which could require fewer doses because they are more potent, are less reactogenic than currently available human anthrax vaccines, and could be self-administered without injection.

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