Comparison of Two Different Methods for 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

Despite the availability of a vaccine, 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 candidates in vaccine development. OMVs obtained from Bordetella pertussis contain several antigens, which are considered immunogenic, and could make them a potential candidate for vaccine production. The current study aimed to compare the current OMV extraction method (with ultracentrifuge) and a modified extraction method (without ultracentrifuge) and to evaluate the physicochemical properties as well as the expression of their main virulence factors. Vaccinal strain BP134 grown on Bordet Gengo agar were inoculated in Modified Stainer-Scholte medium for mass cultivation. OMVs were prepared using two different methods. They were then stained and examined with a transmission electron microscope. Protein contents were measured by the Bradford method, and then the protein profile was evaluated by SDS-PAGE. The presence of immunogenic antigens was detected by Western blotting. The size and shape of the OMVs obtained from the modified method without the use of ultracentrifuge were similar to the current method and had a size between 40 and 200 nm. The total protein yields of the OMV isolated using the current and modified methods 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 was observed in OMVs extracted from both methods. Comparison of the 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 in protein yield as the modified method does not require ultracentrifuge, this extraction method can be used as a suitable alternative for extracting the outer membrane vesicles from 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.

Keywords

Main Subjects

Article Title [French]

Comparaison de Deux Méthodes Différentes pour l'extraction des Vésicules de la Membrane Externe de Bordetella pertussis en Tant Qu'un Candidat Vaccin

Abstract [French]

Malgré la disponibilité d'un vaccin, la coqueluche reste un problème de santé mondial. Les vésicules de la membrane externe (VME) chez les bactéries gram-négatives peuvent stimuler le système immunitaire grâce à plusieurs protéines de la membrane externe et sont de très bons candidats pour le développement de vaccins. Les VMEs obtenues à partir de Bordetella pertussis contiennent plusieurs antigènes, considérés comme immunogènes, et pourraient en faire un candidat potentiel pour la production de vaccins. La présente étude visait à comparer la méthode d'extraction actuelle de VME (avec ultracentrifugeuse) et une méthode d'extraction modifiée (sans ultracentrifugeuse) et à évaluer les propriétés physico-chimiques ainsi que l'expression de leurs principaux facteurs de virulence. La souche vaccinale BP134 cultivée sur gélose Bordet Gengo a été inoculée dans du milieu Stainer-Scholte modifié pour une culture de masse. Les VMEs ont été préparées en utilisant deux méthodes différentes. Elles ont ensuite été colorées et examinées au microscope électronique à transmission. Les teneurs en protéines ont été mesurées par la méthode de Bradford, puis le profil protéique a été évalué par SDS-PAGE. La présence d'antigènes immunogènes a été détectée par Western blot. La taille et la forme des VMEs obtenues à partir de la méthode modifiée sans l'utilisation d'ultracentrifugeuse étaient similaires à la méthode actuelle et avaient une taille comprise entre 40 et 200 nm. Les rendements protéiques totaux du VME isolé en utilisant les méthodes actuelles et modifiées étaient respectivement de 800 et 600 µg/ml. L'évaluation du profil protéique des VMEs extraites a montré la présence de différentes protéines. Enfin, la présence de PTX, PRN et FHA a été observée dans les VMEs extraites des deux méthodes. La comparaison des deux méthodes d'extraction de VME a montré que les vésicules obtenues ont une forme et une taille appropriées et similaires ainsi que l'expression de trois facteurs pathogènes importants en tant qu'immunogènes. Malgré la réduction relativement faible du rendement en protéines car la méthode modifiée ne nécessite pas d'ultracentrifugation, cette méthode d'extraction peut être utilisée comme une alternative appropriée pour extraire les vésicules de la membrane externe de B. pertussis, en particulier dans les pays en développement. Il convient de noter que d'autres expériences, y compris la détermination de l'immunogénicité des VMEs obtenues en tant que candidats vaccins dans des modèles animaux, sont nécessaires.

Keywords [French]

  • B. pertussis
  • Vésicule de membrane externe
  • facteurs de virulence

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Archives of Razi Institute
Volume 76, Issue 3
September 2021
Pages 411-419

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