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Two approaches for the stabilization of Bacillus anthracis recombinant protective antigen.

ABSTRACT: Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacteria Bacillus anthracis. There is a need for safe, highly effective, long-term storage vaccine formulations for mass vaccination. However, the development of new subunit vaccines based on recombinant protective antigen (rPA) faces the problem of vaccine antigen instability. Here, the potential of simultaneous application of two different approaches to stabilize rPA was demonstrated. Firstly, we employed spherical particles (SPs) obtained from the tobacco mosaic virus (TMV). Previously, we had reported that SPs can serve as an adjuvant and platform for antigen presentation. In the current work, SPs were shown to increase the stability of the full-size rPA without loss of its antigenic properties. The second direction was site-specific mutagenesis of asparagine residues to avoid deamidation that causes partial protein degradation. The modified recombinant protein comprising the PA immunogenic domains 3 and 4 (rPA3 + 4) was stable during storage at 4 and 25°C. rPA3 + 4 interacts with antibodies to rPA83 both individually and as a part of a complex with SPs. The results obtained can underpin the development of a recombinant vaccine with a full-size modified rPA (with similar amino acid substitutions that stabilize the protein) and SPs.

SUBMITTER: Ryabchevskaya EM

PROVIDER: S-EPMC7899644 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Anthrax is caused by Bacillus anthracis that produce two exotoxins, lethal toxin and edema toxin. The lethal toxin is composed of the lethal factor (LF) complexed with the cell binding protective antigen (PA83, 83 kDa). Likewise, the edema factor (EF) binds to the PA83 to form the edema toxin. Once PA83 is bound to the host cell surface, a furin-like protease cleaves the full-length, inactive protein into 63 kDa and 20 kDa antigens (PA63 and PA20). PA63 forms a heptamer and is internalized via receptor mediated endocytosis forming a protease-stable pore, which allows EF and LF to enter the cell and exert their toxic effects.Both proteolytically cleaved protective antigens (PA63 and PA20 fragments) are found in the blood of infected animals. The 63 kDa protective antigen PA63 fragment has been thoroughly studied while little is known about the PA20. METHODS: In this study we examined the role of PA20 using high throughput gene expression analysis of human peripheral blood mononuclear cells (PBMC) exposed to the PA20. We constructed a PA mutant in which a Factor Xa proteolytic recognition site was genetically engineered into the protective antigen PA83 to obtain PA20 using limited digestion of this recombinant PA83 with trypsin. RESULTS: Global gene expression response studies indicated modulation of various immune functions and showed gene patterns indicative of apoptosis via the Fas pathway in a subset of the lymphoid cells. This finding was extended to include observations of increased Caspase-3 enzymatic activity and the identification of increases in the population of apoptotic, but not necrotic cells, based on differential staining methods. We identified a list of approximately 40 inflammatory mediators and heat-shock proteins that were altered similarly upon exposure of PBMC to either rPA20 or B. anthracis spores/vegetative cells. CONCLUSION: This study shows that the PA20 has an effect on human peripheral blood leukocytes and can induce apoptosis in the absence of other PA components.

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Two approaches for the stabilization of Bacillus anthracis recombinant protective antigen.

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