Available online 8 August 2022

A highly pure recombinant RBD protein (C-RBD-H6 PP) produced in P. pastoris.

Physico-chemical characterization confirmed correct protein folding.

The recombinant protein exhibited high reactivity with convalescent sera.

The sera from immunized animals inhibited RBD-ACE2 binding and neutralized virus.

C-RBD-H6 PP stimulated IFNγ, IL-2, IL-6, IL-4 and TNFα in mice.

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.

Receptor-binding domain

severe acute respiratory syndrome coronavirus 2

Chinese hamster ovary cells, HEK293T, human embryonic kidney cells

immobilized metal ion affinity chromatography

reversed-phase chromatography

viral neutralization titer

Electrospray ionization tandem mass spectrometry

SARS-CoV-2

P. pastoris

COVID-19

RBD

subunit vaccine

© 2022 The Authors. Published by Elsevier B.V.