Comparison of two different methods in the extraction of outer membrane vesicles from the Bordetella pertussis as a vaccine candidate

Document Type: Original Articles

Authors

1 Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.

2 Department of Human Bacterial Vaccines Production and Research, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

3 Department of Immunology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

4 Department of Bacteriology, Pasteur Institute of Iran, Tehran, Iran

Abstract

Background & Objective: Despite vaccination, pertussis is still a worldwide health problem. Outer membrane vesicles (OMVs) in gram-negative bacteria can stimulate the immune system due to several outer membrane proteins and are very good candidate in vaccine development, OMVs obtain from Bordetella pertussis contain several antigens, which are considered immunogenic that could make them a potential candidate for vaccine production. The aim of this study was to compare the current OMVs extraction method (with ultracentrifuge) and the modified extraction method (without ultracentrifuge) and to evaluate the physicochemical properties as well as the expression of their main virulence factors.
Materials & Methods: Vaccinal strain BP134 were grown on Bordet Gengo agar were inoculated in Modified Stainer-Scholte medium for mass cultivation. OMVs were prepared by using two different methods. The OMVs obtained were stained and examined with a transmission electron microscope. Protein contents were measured by the Bradford method and then the protein profile evaluated by SDS-PAGE. The presence of immunogenic antigens were detected by Western blotting. 
Results: The size and shape of the OMVs obtained from the modified method without the use of ultracentrifuge were similar to the current method and with a size between 40 to 200 nm. The total protein yield of the OMV isolated using the current and modified method were 800 and 600 µg/ml respectively. Evaluating the protein profile of extracted OMVs, showed the presence of different proteins. Finally, the presence of PTX, PRN, and FHA in OMVs extracted from both methods was observed.
Conclusions: Comparison results of two OMV extraction methods showed that the obtained vesicles have a suitable and similar shape and size as well as the expression of three important pathogenic factors as immunogens. Despite the relatively low reduction of protein yield since the modified method does not require ultracentrifuge, this extraction method can be used as a suitable alternative for extracting the outer membrane vesicles from the B. pertussis, especially in developing countries. It should be noted that further experiments, including immunogenicity determination of OMVs, obtained as vaccine candidates in animal models are required.

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