Keywords

Chloroplast transformation, Bacillus anthracis

Abstract

Anthrax, a fatal bacterial infection is caused by Bacillus anthracis, a gram-positive, spore forming, capsulated, rod shaped organism. Centers for Disease Control (CDC) lists anthrax as Category A biological agent due to its severity of impact on human health, high mortality rate, acuteness of the disease and potential for delivery as a biological weapon. The currently available human vaccine in the United States (AVA anthrax vaccine adsorbed) is prepared from Alum adsorbed formalin treated supernatant culture of toxigenic, non-encapsulated strain of Bacillus anthracis with the principle component being protective antigen (PA83). Evaluation of anthrax vaccine given to nearly 400,000 US military personnel by Vaccine Adverse Event Reporting System (VAERS) showed adverse effects such as flu-like symptoms, local pain, large degree of inflammation, edema, malaise, rash, arthralgia, and headache following vaccination. All the adverse reactions are attributed to the composition of vaccine components. These vaccine preparations contain trace contaminants of lethal and edema factors that contribute to the adverse side effects. Also, the current method of vaccine manufacture has limited production capacity.The production of PA83, in plants through chloroplast genetic engineering might eliminate the concerns of adverse side effects and the levels of expression would ensure the availability of vaccine for the human population in an environmentally friendly approach. The primary concern is whether the PA83 purified from transgenic chloroplasts is as immunogenic as the PA83 in the AVA. For this, PA83 has been expressed in transgenic chloroplasts of Nicotiana tabacum var. petit Havana, by inserting the pag (2205 bp) with the N-terminal 6X histidine tag, into the chloroplast genome by homologous recombination. Chloroplast integration of the pag was confirmed by PCR and Southern analysis. The PA83 protein was detected in transgenic chloroplasts by immunoblot analysis using anti-PA83 antibodies. Maximum expression levels of PA83 (14.17% TSP) were observed in mature leaves upon continuous illumination, due to the presence psbA 5'UTR, a light and developmentally regulated translation enhancer sequence. The PA83 has been purified by affinity chromatography using Ni resin columns. Chloroplast derived PA83 was functional in vitro and was able to lyse the mouse macrophages when combined with the lethal factor. The in vitro assays showed that the crude extracts contained up to 20ug/ml of functional PA83.The immunization studies of PA83 on Balb/c mice, revealed highly immunogenic IgG titers. Subcutaneous immunization with purified chloroplast derived PA83 with adjuvant yielded IgG titers up to 1:320,000, similar to that of the group immunized with PA83 derived from Bacillus anthracis. Immunization of groups with PA83 combined with alhydrogel adjuvant showed four - eight times higher immune response than the groups without adjuvant. The higher expression levels of PA83 in transgenic chloroplasts might ensure the availability of anthrax vaccine to the general public and the high immune response observed in the mouse model would enable the replacement of the current AVA with a cleaner and safer vaccine.

Notes

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Graduation Date

2004

Semester

Fall

Advisor

Daniell, Henry

Degree

Master of Science (M.S.)

College

Burnett College of Biomedical Sciences

Department

Molecular Biology and Microbiology

Degree Program

Molecular Biology and Microbiology

Format

application/pdf

Identifier

CFE0000298

URL

http://purl.fcla.edu/fcla/etd/CFE0000298

Language

English

Release Date

January 2006

Length of Campus-only Access

None

Access Status

Masters Thesis (Open Access)

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