Plant-based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge

Abstract

The currently available human vaccine for anthrax, derived from the culture supernatant of Bacillus anthracis, contains the protective antigen (PA) and traces of the lethal and edema factors, which may contribute to adverse side effects associated with this vaccine. Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required. In an effort to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants, PA was expressed in transgenic tobacco chloroplasts by inserting the pagA gene into the chloroplast genome. Chloroplast integration of the pagA gene was confirmed by PCR and Southern analysis. Mature leaves grown under continuous illumination contained PA as up to 14.2% of the total soluble protein. Cytotoxicity measurements in macrophage lysis assays showed that chloroplast-derived PA was equal in potency to PA produced in B. anthracis. Subcutaneous immunization of mice with partially purified chloroplast-derived or B. anthracis-derived PA with adjuvant yielded immunoglobulin G titers up to 1:320,000, and both groups of mice survived (100%) challenge with lethal doses of toxin. An average yield of about 150 mg of PA per plant should produce 360 million doses of a purified vaccine free of bacterial toxins edema factor and lethal factor from 1 acre of land. Such high expression levels without using fermenters and the immunoprotection offered by the chloroplast-derived PA should facilitate development of a cleaner and safer anthrax vaccine at a lower production cost. These results demonstrate the immunogenic and immunoprotective properties of plant-derived anthrax vaccine antigen.

FIG. 1.

Vector map and confirmation of transgene integration into chloroplast genome by PCR and Southern blotting. (a) Schematic representation of pLD-VK1 vector with protective antigen gene (pagA), aadA (selectable marker), 5′ UTR, and chloroplast flanking sequences for site-specific integration with the primers 3P/3M and 5P/2M annealing sites within the native chloroplast genome and the schematic diagram of expected products from digestion of plants transformed with pLD-VK1. (b) Schematic diagram of expected products from digestion of wild-type untransformed plant. (c) Confirmation of site-specific transgene cassette integration by PCR using primers (3P/3M) to yield a 1.65-kb product. Lane 1, 1-kb DNA ladder; lane 2, wild type; lanes 3 to 6, pLD-VK1 transgenic lines; lane 7, positive control (interferon clone). (d) Confirmation of gene integration by PCR using primers (5P/2M) to yield a 3.9-kb product. Lane 1, 1-kb-plus DNA ladder; lane 2, wild type; lanes 3 and 4, pLD-VK1 transgenic lines. (e) Southern blots with flanking sequence probe in pLD-VK1 transgenic lines showing the following: lane 1, wild type; lane 2, transgenic line 1; lane 3, transgenic line 2; lane 4, transgenic line 3; lane 5, transgenic line 4. (f) Southern blotting with PA gene-specific probe showing the presence of pagA in the transgenic plants. Lane 1, wild type; lanes 2, 3, and 4, pLD-VK1 transgenic lines.

FIG. 2.

Immunoblotting analysis and quantification of PA expressed in chloroplast of transgenic plants (pLD-VK1) in T₀ generation. (a) Immunoblotting demonstrating the expression of PA in transgenic plant crude extracts. Lane 1, wild type; lane 2, 100-ng standard; lane 4, transgenic line 5; lane 6, transgenic line 7; lane 8, transgenic line 8; lanes 3, 5, and 7, empty. (b) Expression levels in percent TSP of PA-expressing leaves (young, mature, and old) under normal and continuous illumination observed for 0 to 7 days.

FIG. 3.

Purification of PA by affinity chromatography from the crude extracts of plant leaves expressing PA. (a) Coomassie staining of the proteins in crude extract and purified protein: Lane 1, protein plus precision ladder; lane 2, wild-type leaf crude extract; lane 3, crude extract of transgenic plant expressing PA; lanes 4 and 5, purified chloroplast-derived PA; lane 6, flowthrough collected during purification. (b) Lane 1, ladder; lane 3, concentrated protein; lane 5, purified protein (before concentrating); lanes 2 and 4, overflow from lane 3.

FIG. 4.

Functional analysis of PA with macrophage cytotoxicity assay. The cytotoxicities of various PA preparations for mouse macrophage RAW264.7 cells were assayed in the presence of LF. Samples that were diluted serially were as follows: crude extract of plant leaves expressing PA with His tag, wild-type (WT) plant leaf crude extract, 20-μg/ml stock of purified chloroplast-derived PA, 20-μg/ml stock of purified PA derived from B. anthracis, and plant protein extraction buffer.

FIG. 5.

IgG antibody titers and toxin neutralization assay titers in serum samples obtained from mice after third and fourth doses. (a) Comparison of immune responses in serum samples of mice administered subcutaneously with chloroplast-derived PA (CpPA) with adjuvant (column 1), chloroplast-derived PA (CpPA) alone (column 2), Std-PA derived from B. anthracis with adjuvant (column 3), Std-PA alone (column 4), PA plant leaf crude extract with adjuvant (column 5), wild-type plant leaf crude extract with adjuvant (column 6), and unimmunized mice (column 7). (b) Toxin neutralization titers of sera collected from the mice on day 43 of post-initial immunization. Each symbol represents average EC₅₀ from three replicate assays of a single mouse serum. CHL, chloroplast; ADJ, adjuvant; B.A., B. anthracis; WT, wild type. (c) Toxin neutralization assays of serum samples collected from the mice on day 155 of post-initial immunization. Each symbol represents the average EC₅₀ from three replicate assays of a single mouse serum.

FIG. 5.

IgG antibody titers and toxin neutralization assay titers in serum samples obtained from mice after third and fourth doses. (a) Comparison of immune responses in serum samples of mice administered subcutaneously with chloroplast-derived PA (CpPA) with adjuvant (column 1), chloroplast-derived PA (CpPA) alone (column 2), Std-PA derived from B. anthracis with adjuvant (column 3), Std-PA alone (column 4), PA plant leaf crude extract with adjuvant (column 5), wild-type plant leaf crude extract with adjuvant (column 6), and unimmunized mice (column 7). (b) Toxin neutralization titers of sera collected from the mice on day 43 of post-initial immunization. Each symbol represents average EC₅₀ from three replicate assays of a single mouse serum. CHL, chloroplast; ADJ, adjuvant; B.A., B. anthracis; WT, wild type. (c) Toxin neutralization assays of serum samples collected from the mice on day 155 of post-initial immunization. Each symbol represents the average EC₅₀ from three replicate assays of a single mouse serum.

FIG. 6.

Toxin challenge of the mice with systemic anthrax lethal toxin. Shown is survival over time for different groups of mice after challenge with a 150-μg dose of lethal toxin. IP, intraperitoneal; CHLPST, chloroplast; ADJ, adjuvant; WT, wild type.