Fusion expression of the recombinant protective antigen of Bacillus anthracis in E.coli

ABSTRACT Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 × 107 spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (≥100 μg/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracisspore-associated antigen(s) may contribute in a significant manner to protective immunity.

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Correlation between Lethal Toxin-Neutralizing Antibody Titers and Protection from Intranasal Challenge with Bacillus anthracis Ames Strain Spores in Mice after Transcutaneous Immunization with Recombinant Anthrax Protective Antigen

Infection and Immunity ◽

10.1128/iai.74.1.794-797.2006 ◽

2006 ◽

Vol 74 (1) ◽

pp. 794-797 ◽

Cited By ~ 43

Author(s):

Kristina K. Peachman ◽

Mangala Rao ◽

Carl R. Alving ◽

Robert Burge ◽

Stephen H. Leppla ◽

...

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Neutralizing Antibody ◽

Lethal Toxin ◽

Vaccine Strategy ◽

Transcutaneous Immunization ◽

Antibody Titers ◽

Anthrax Protective Antigen ◽

Ames Strain ◽

Recombinant Protective Antigen

ABSTRACT Transcutaneous immunization of mice with recombinant protective antigen (rPA) of Bacillus anthracis resulted in significantly higher lethal toxin-neutralizing antibody titers than did intramuscular injection of alum-adsorbed rPA. Immunized mice were partially protected against intranasal challenge with 235,000 (10 50% lethal doses) Ames strain B. anthracis spores. A highly significant correlation was observed between toxin-neutralizing antibody titer and survival after challenge. Future experiments with rabbits and nonhuman primates should confirm the significance of protection by this vaccine strategy.

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Two approaches for the stabilization of Bacillus anthracis recombinant protective antigen

Human Vaccines & Immunotherapeutics ◽

10.1080/21645515.2020.1772632 ◽

2020 ◽

pp. 1-6

Author(s):

Ekaterina M. Ryabchevskaya ◽

Ekaterina A. Evtushenko ◽

Dmitry L. Granovskiy ◽

Peter A. Ivanov ◽

Joseph G. Atabekov ◽

...

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Recombinant Protective Antigen

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Reference materials of catalytic activity as a means of ensuring the metrological traceability of measurement results. KATAL

Reference materials ◽

10.20915/2077-1177-2018-14-1-2-25-32 ◽

2019 ◽

Vol 14 (1-2) ◽

pp. 25-32

Author(s):

E. V. Kulyabina ◽

O. N. Melkova ◽

E. A. Guskova ◽

T. V. Grebennikova

Keyword(s):

Catalytic Activity ◽

Bacillus Anthracis ◽

Reference Materials ◽

Mass Concentration ◽

Ebola Virus ◽

Protective Antigen ◽

Metrological Traceability ◽

Measurement Results ◽

Traceability Of Measurement Results ◽

Recombinant Protective Antigen

Introduction. The article considers the problems of metrological support of catalytic activity measurements. The paper gives certain characteristics of the State Primary Special Measurement Standard for the unit of catalytic activity – KATAL and examines the role of reference materials (RMs) in ensuring the metrological traceability of measurement results.Materials and methods. A method for measuring the concentration of the recombinant protective antigen of Bacillus anthracis and the recombinant GP-protein of the Ebola virus, which means in using «sandwich» enzyme-linked immunosorbent assay, was certified by FGUP «VNIIMS» as a measurement procedure. The following RMs tested by FGUP «VNIIMS» can be used for the development and production of appropriate control samples when performing measurements for comparisons: the RM for the mass concentration of the recombinant protective antigen of Bacillus anthracis in phosphate-saline solution, the RM for the mass concentration of the recombinant GP-protein of the Ebola virus, and the RM for the mass concentration of recombinant Clostridium Difficile toxin.Results. The paper presents the main reactions of the method for measuring the catalytic activity of catalysts for heterogeneous processes, which are also used to carry out exhaust gas cleaning processes.Discussion and conclusions. Thus, conditions have been created for building hierarchies of calibrations of in-demand objects established on the basis of the List of Critical Technologies of the Russian Federation, state programs for the development of industrial sectors.

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Duration of protection of rabbits after vaccination with Bacillus anthracis recombinant protective antigen vaccine☆

Vaccine ◽

10.1016/j.vaccine.2005.12.028 ◽

2006 ◽

Vol 24 (14) ◽

pp. 2530-2536 ◽

Cited By ~ 43

Author(s):

S.F. Little ◽

B.E. Ivins ◽

W.M. Webster ◽

P.F. Fellows ◽

M.L.M. Pitt ◽

...

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Antigen Vaccine ◽

Duration Of Protection ◽

Recombinant Protective Antigen

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Comparative vaccine efficacy of different isoforms of recombinant protective antigen against Bacillus anthracis spore challenge in rabbits

Vaccine ◽

10.1016/j.vaccine.2006.02.013 ◽

2006 ◽

Vol 24 (17) ◽

pp. 3469-3476 ◽

Cited By ~ 19

Author(s):

W RIBOT ◽

B POWELL ◽

B IVINS ◽

S LITTLE ◽

W JOHNSON ◽

...

Keyword(s):

Bacillus Anthracis ◽

Vaccine Efficacy ◽

Protective Antigen ◽

Recombinant Protective Antigen

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Use of a Promoter Trap System in Bacillus anthracis and Bacillus subtilis for the Development of Recombinant Protective Antigen-Based Vaccines

Infection and Immunity ◽

10.1128/iai.71.2.801-813.2003 ◽

2003 ◽

Vol 71 (2) ◽

pp. 801-813 ◽

Cited By ~ 34

Author(s):

O. Gat ◽

I. Inbar ◽

R. Aloni-Grinstein ◽

E. Zahavy ◽

C. Kronman ◽

...

Keyword(s):

Bacillus Subtilis ◽

Bacillus Anthracis ◽

Protective Antigen ◽

Expression Plasmid ◽

Secretion Stress ◽

Trap System ◽

Promoter Trap ◽

Ability To Drive ◽

Practical Implications ◽

Recombinant Protective Antigen

ABSTRACT We have recently reported Bacillus anthracis attenuated live vaccine strains efficiently expressing recombinant protective antigen (rPA) and have shown a direct correlation between the level of rPA secreted by these cells and efficacy (S. Cohen, I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak, M. Fisher, C. Kronman, B. Velan, and A. Shafferman, Infect. Immun. 68:4549-4558, 2000). To isolate more potent Bacillus promoters for a further increase in the production of rPA, we developed a promoter trap system based on various gfp reporter genes adapted for use in both Bacillus subtilis and B. anthracis backgrounds. Accordingly, a B. anthracis library of 6,000 clones harboring plasmids with chromosomal B. anthracis DNA fragments inserted upstream from gfpuv was constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent B. anthracis promoter identified (Pntr; 271 bp) was 500 times more potent than the native pagA promoter and 70 times more potent than the α-amylase promoter (Pamy). This very potent promoter was tested along with the other promoters (which are three, six, and eight times more potent than Pamy) for the ability to drive expression of rPA in either B. subtilis or B. anthracis. The number of cell-associated pre-PA molecules in B. anthracis was found to correlate well with the strength of the promoter. However, there appeared to be an upper limit to the amount of mature PA secreted into the medium, which did not exceed that driven by Pamy. Furthermore, the rPA constructs fused to the very potent promoters proved to be deleterious to the bacterial hosts and consequently led to genetic instability of the PA expression plasmid. Immunization with attenuated B. anthracis expressing rPA under the control of promoters more potent than Pamy was less efficient in eliciting anti-PA antibodies than that attained with Pamy. The results are consistent with the notion that overexpression of PA leads to severe secretion stress and have practical implications for the design of second-generation rPA-based vaccines.

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