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An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen
Developing affordable and easily manufactured SARS-CoV-2 vaccines will be essential to achieve worldwide vaccine coverage and long-term control of the COVID-19 pandemic. Here the development is reported of a vaccine based on the SARS-CoV-2 receptor-binding domain (RBD), produced in the yeast Pichia...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Authors. Published by Elsevier B.V.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9359770/ https://www.ncbi.nlm.nih.gov/pubmed/35953030 http://dx.doi.org/10.1016/j.nbt.2022.08.002 |
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author | Limonta-Fernández, Miladys Chinea-Santiago, Glay Martín-Dunn, Alejandro Miguel Gonzalez-Roche, Diamile Bequet-Romero, Monica Marquez-Perera, Gabriel González-Moya, Isabel Canaan-Haden-Ayala, Camila Cabrales-Rico, Ania Espinosa-Rodríguez, Luis Ariel Ramos-Gómez, Yassel Andujar-Martínez, Ivan González-López, Luis Javier de la Iglesia, Mariela Perez Zamora-Sanchez, Jesus Cruz-Sui, Otto Lemos-Pérez, Gilda Cabrera-Herrera, Gleysin Valdes-Hernández, Jorge Martinez-Diaz, Eduardo Pimentel-Vazquez, Eulogio Ayala-Avila, Marta Guillén-Nieto, Gerardo |
author_facet | Limonta-Fernández, Miladys Chinea-Santiago, Glay Martín-Dunn, Alejandro Miguel Gonzalez-Roche, Diamile Bequet-Romero, Monica Marquez-Perera, Gabriel González-Moya, Isabel Canaan-Haden-Ayala, Camila Cabrales-Rico, Ania Espinosa-Rodríguez, Luis Ariel Ramos-Gómez, Yassel Andujar-Martínez, Ivan González-López, Luis Javier de la Iglesia, Mariela Perez Zamora-Sanchez, Jesus Cruz-Sui, Otto Lemos-Pérez, Gilda Cabrera-Herrera, Gleysin Valdes-Hernández, Jorge Martinez-Diaz, Eduardo Pimentel-Vazquez, Eulogio Ayala-Avila, Marta Guillén-Nieto, Gerardo |
author_sort | Limonta-Fernández, Miladys |
collection | PubMed |
description | Developing affordable and easily manufactured SARS-CoV-2 vaccines will be essential to achieve worldwide vaccine coverage and long-term control of the COVID-19 pandemic. Here the development is reported of a vaccine based on the SARS-CoV-2 receptor-binding domain (RBD), produced in the yeast Pichia pastoris. The RBD was modified by adding flexible N- and C-terminal amino acid extensions that modulate protein/protein interactions and facilitate protein purification. A fed-batch methanol fermentation with a yeast extract-based culture medium in a 50 L fermenter and an immobilized metal ion affinity chromatography-based downstream purification process yielded 30–40 mg/L of RBD. Correct folding of the purified protein was demonstrated by mass spectrometry, circular dichroism, and determinations of binding affinity to the angiotensin-converting enzyme 2 (ACE2) receptor. The RBD antigen also exhibited high reactivity with sera from convalescent individuals and Pfizer-BioNTech or Sputnik V vaccinees. Immunization of mice and non-human primates with 50 µg of the recombinant RBD adjuvanted with alum induced high levels of binding antibodies as assessed by ELISA with RBD produced in HEK293T cells, and which inhibited RBD binding to ACE2 and neutralized infection of VeroE6 cells by SARS-CoV-2. Additionally, the RBD protein stimulated IFNγ, IL-2, IL-6, IL-4 and TNFα secretion in splenocytes and lung CD3(+)-enriched cells of immunized mice. The data suggest that the RBD recombinant protein produced in yeast P. pastoris is suitable as a vaccine candidate against COVID-19. |
format | Online Article Text |
id | pubmed-9359770 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | The Authors. Published by Elsevier B.V. |
record_format | MEDLINE/PubMed |
spelling | pubmed-93597702022-08-09 An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen Limonta-Fernández, Miladys Chinea-Santiago, Glay Martín-Dunn, Alejandro Miguel Gonzalez-Roche, Diamile Bequet-Romero, Monica Marquez-Perera, Gabriel González-Moya, Isabel Canaan-Haden-Ayala, Camila Cabrales-Rico, Ania Espinosa-Rodríguez, Luis Ariel Ramos-Gómez, Yassel Andujar-Martínez, Ivan González-López, Luis Javier de la Iglesia, Mariela Perez Zamora-Sanchez, Jesus Cruz-Sui, Otto Lemos-Pérez, Gilda Cabrera-Herrera, Gleysin Valdes-Hernández, Jorge Martinez-Diaz, Eduardo Pimentel-Vazquez, Eulogio Ayala-Avila, Marta Guillén-Nieto, Gerardo N Biotechnol Article Developing affordable and easily manufactured SARS-CoV-2 vaccines will be essential to achieve worldwide vaccine coverage and long-term control of the COVID-19 pandemic. Here the development is reported of a vaccine based on the SARS-CoV-2 receptor-binding domain (RBD), produced in the yeast Pichia pastoris. The RBD was modified by adding flexible N- and C-terminal amino acid extensions that modulate protein/protein interactions and facilitate protein purification. A fed-batch methanol fermentation with a yeast extract-based culture medium in a 50 L fermenter and an immobilized metal ion affinity chromatography-based downstream purification process yielded 30–40 mg/L of RBD. Correct folding of the purified protein was demonstrated by mass spectrometry, circular dichroism, and determinations of binding affinity to the angiotensin-converting enzyme 2 (ACE2) receptor. The RBD antigen also exhibited high reactivity with sera from convalescent individuals and Pfizer-BioNTech or Sputnik V vaccinees. Immunization of mice and non-human primates with 50 µg of the recombinant RBD adjuvanted with alum induced high levels of binding antibodies as assessed by ELISA with RBD produced in HEK293T cells, and which inhibited RBD binding to ACE2 and neutralized infection of VeroE6 cells by SARS-CoV-2. Additionally, the RBD protein stimulated IFNγ, IL-2, IL-6, IL-4 and TNFα secretion in splenocytes and lung CD3(+)-enriched cells of immunized mice. The data suggest that the RBD recombinant protein produced in yeast P. pastoris is suitable as a vaccine candidate against COVID-19. The Authors. Published by Elsevier B.V. 2022-12-25 2022-08-08 /pmc/articles/PMC9359770/ /pubmed/35953030 http://dx.doi.org/10.1016/j.nbt.2022.08.002 Text en © 2022 The Authors Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. |
spellingShingle | Article Limonta-Fernández, Miladys Chinea-Santiago, Glay Martín-Dunn, Alejandro Miguel Gonzalez-Roche, Diamile Bequet-Romero, Monica Marquez-Perera, Gabriel González-Moya, Isabel Canaan-Haden-Ayala, Camila Cabrales-Rico, Ania Espinosa-Rodríguez, Luis Ariel Ramos-Gómez, Yassel Andujar-Martínez, Ivan González-López, Luis Javier de la Iglesia, Mariela Perez Zamora-Sanchez, Jesus Cruz-Sui, Otto Lemos-Pérez, Gilda Cabrera-Herrera, Gleysin Valdes-Hernández, Jorge Martinez-Diaz, Eduardo Pimentel-Vazquez, Eulogio Ayala-Avila, Marta Guillén-Nieto, Gerardo An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen |
title | An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen |
title_full | An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen |
title_fullStr | An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen |
title_full_unstemmed | An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen |
title_short | An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen |
title_sort | engineered sars-cov-2 receptor-binding domain produced in pichia pastoris as a candidate vaccine antigen |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9359770/ https://www.ncbi.nlm.nih.gov/pubmed/35953030 http://dx.doi.org/10.1016/j.nbt.2022.08.002 |
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