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

2022; Elsevier BV; Volume: 72; Linguagem: Inglês

10.1016/j.nbt.2022.08.002

1876-4347

Miladys Limonta-Fernández, Glay Chinea-Santiago, Alejandro Miguel Martín-Dunn, Diamile Gonzalez-Roche, Mónica Béquet‐Romero, Gabriel Márquez, Isabel González-Moya, Camila Canaan-Haden-Ayala, Ania Cabrales, Luis Ariel Espinosa, Yassel Ramos-Gómez, Ivan Andujar-Martínez, Luis Javier González, Mariela Pérez de la Iglesia, Jesús Zamora-Sánchez, Otto Cruz-Sui, Gilda Lemos, Gleysin Cabrera, Jorge Valdés-Hernández, Eduardo Martínez-Díaz, Eulogio Pimentel-Vázquez, Marta Ayala-Ávila, Gerardo Guillén,

Bacillus and Francisella bacterial research

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.