Alejandro Miguel Martín-Dunn

Centro de Ingeniería Genética y Biotecnología

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Luis Ariel Espinosa

Centro de Ingeniería Genética y Biotecnología

Glay Chinea-Santiago

Centro de Ingeniería Genética y Biotecnología

Gabriel Márquez

Centro de Ingeniería Genética y Biotecnología

Gilda Lemos

Centro de Ingeniería Genética y Biotecnología

Eulogio Pimentel-Vázquez

Centro Nacional de Investigaciones Científicas

Mónica Béquet‐Romero

University of California, Berkeley

Camila Canaan-Haden-Ayala

Centro de Ingeniería Genética y Biotecnología

Ania Cabrales

Centro de Ingeniería Genética y Biotecnología

Gleysin Cabrera

Centro de Ingeniería Genética y Biotecnología

Eduardo Martínez-Díaz

Ministerio de Ciencia, Tecnología y Medio Ambiente

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Centro de Ingeniería Genética y Biotecnología

Institut thématique Génétique, génomique et bioinformatique

Centro Nacional de Investigaciones Científicas

ORCID

National Center for Genetic Engineering and Biotechnology

International Vaccine Institute

University of Camagüey

Cuban Neuroscience Center

Wound Healing Society

Fundación Ciencias Exactas y Naturales

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An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen

New Biotechnology (2022)

Miladys Limonta-Fernández Glay Chinea-Santiago Alejandro Miguel Martín-Dunn Diamile Gonzalez-Roche Mónica Béquet‐Romero

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.

10.1016/j.nbt.2022.08.002

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