Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks.
INTRODUCTION: Cholera toxin B subunit (CTB) is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to ki...
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ftdoajarticles:oai:doaj.org/article:a1d061edec6c445484c017d2acc5a6bc 2023-05-15T15:16:54+02:00 Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks. Krystal Teasley Hamorsky J Calvin Kouokam Lauren J Bennett Keegan J Baldauf Hiroyuki Kajiura Kazuhito Fujiyama Nobuyuki Matoba 2013-01-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0002046 https://doaj.org/article/a1d061edec6c445484c017d2acc5a6bc EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC3591335?pdf=render https://doaj.org/toc/1935-2735 1935-2735 doi:10.1371/journal.pntd.0002046 https://doaj.org/article/a1d061edec6c445484c017d2acc5a6bc PLoS Neglected Tropical Diseases, Vol 7, Iss 3, p e2046 (2013) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2013 ftdoajarticles https://doi.org/10.1371/journal.pntd.0002046 2022-12-31T03:28:38Z INTRODUCTION: Cholera toxin B subunit (CTB) is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to killed whole-cell bacteria provides superior short-term protection to whole-cell-only vaccines; however, challenges in CTB biomanufacturing (i.e., cost and scale) hamper its implementation to mass vaccination in developing countries. To provide a potential solution to this issue, we developed a rapid, robust, and scalable CTB production system in plants. METHODOLOGY/PRINCIPAL FINDINGS: In a preliminary study of expressing original CTB in transgenic Nicotiana benthamiana, the protein was N-glycosylated with plant-specific glycans. Thus, an aglycosylated CTB variant (pCTB) was created and overexpressed via a plant virus vector. Upon additional transgene engineering for retention in the endoplasmic reticulum and optimization of a secretory signal, the yield of pCTB was dramatically improved, reaching >1 g per kg of fresh leaf material. The protein was efficiently purified by simple two-step chromatography. The GM1-ganglioside binding capacity and conformational stability of pCTB were virtually identical to the bacteria-derived original B subunit, as demonstrated in competitive enzyme-linked immunosorbent assay, surface plasmon resonance, and fluorescence-based thermal shift assay. Mammalian cell surface-binding was corroborated by immunofluorescence and flow cytometry. pCTB exhibited strong oral immunogenicity in mice, inducing significant levels of CTB-specific intestinal antibodies that persisted over 6 months. Moreover, these antibodies effectively neutralized the cholera holotoxin in vitro. CONCLUSIONS/SIGNIFICANCE: Taken together, these results demonstrated that pCTB has robust producibility in Nicotiana plants and retains most, if not all, of major biological activities of the original protein. This rapid and ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLoS Neglected Tropical Diseases 7 3 e2046 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Krystal Teasley Hamorsky J Calvin Kouokam Lauren J Bennett Keegan J Baldauf Hiroyuki Kajiura Kazuhito Fujiyama Nobuyuki Matoba Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks. |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
INTRODUCTION: Cholera toxin B subunit (CTB) is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to killed whole-cell bacteria provides superior short-term protection to whole-cell-only vaccines; however, challenges in CTB biomanufacturing (i.e., cost and scale) hamper its implementation to mass vaccination in developing countries. To provide a potential solution to this issue, we developed a rapid, robust, and scalable CTB production system in plants. METHODOLOGY/PRINCIPAL FINDINGS: In a preliminary study of expressing original CTB in transgenic Nicotiana benthamiana, the protein was N-glycosylated with plant-specific glycans. Thus, an aglycosylated CTB variant (pCTB) was created and overexpressed via a plant virus vector. Upon additional transgene engineering for retention in the endoplasmic reticulum and optimization of a secretory signal, the yield of pCTB was dramatically improved, reaching >1 g per kg of fresh leaf material. The protein was efficiently purified by simple two-step chromatography. The GM1-ganglioside binding capacity and conformational stability of pCTB were virtually identical to the bacteria-derived original B subunit, as demonstrated in competitive enzyme-linked immunosorbent assay, surface plasmon resonance, and fluorescence-based thermal shift assay. Mammalian cell surface-binding was corroborated by immunofluorescence and flow cytometry. pCTB exhibited strong oral immunogenicity in mice, inducing significant levels of CTB-specific intestinal antibodies that persisted over 6 months. Moreover, these antibodies effectively neutralized the cholera holotoxin in vitro. CONCLUSIONS/SIGNIFICANCE: Taken together, these results demonstrated that pCTB has robust producibility in Nicotiana plants and retains most, if not all, of major biological activities of the original protein. This rapid and ... |
format |
Article in Journal/Newspaper |
author |
Krystal Teasley Hamorsky J Calvin Kouokam Lauren J Bennett Keegan J Baldauf Hiroyuki Kajiura Kazuhito Fujiyama Nobuyuki Matoba |
author_facet |
Krystal Teasley Hamorsky J Calvin Kouokam Lauren J Bennett Keegan J Baldauf Hiroyuki Kajiura Kazuhito Fujiyama Nobuyuki Matoba |
author_sort |
Krystal Teasley Hamorsky |
title |
Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks. |
title_short |
Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks. |
title_full |
Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks. |
title_fullStr |
Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks. |
title_full_unstemmed |
Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks. |
title_sort |
rapid and scalable plant-based production of a cholera toxin b subunit variant to aid in mass vaccination against cholera outbreaks. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2013 |
url |
https://doi.org/10.1371/journal.pntd.0002046 https://doaj.org/article/a1d061edec6c445484c017d2acc5a6bc |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 7, Iss 3, p e2046 (2013) |
op_relation |
http://europepmc.org/articles/PMC3591335?pdf=render https://doaj.org/toc/1935-2735 1935-2735 doi:10.1371/journal.pntd.0002046 https://doaj.org/article/a1d061edec6c445484c017d2acc5a6bc |
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https://doi.org/10.1371/journal.pntd.0002046 |
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PLoS Neglected Tropical Diseases |
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e2046 |
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