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Proc Natl Acad Sci U S A ; 118(38)2021 09 21.
Article in English | MEDLINE | ID: covidwho-1397979


Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

COVID-19 Vaccines/immunology , COVID-19/prevention & control , Protein Engineering/methods , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Animals , Antibodies, Viral/immunology , Antigens, Viral , Binding Sites , COVID-19/virology , COVID-19 Vaccines/economics , Humans , Immunogenicity, Vaccine , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Binding , Protein Conformation , Saccharomycetales/metabolism , Vaccines, Subunit
Sci Rep ; 10(1): 21779, 2020 12 11.
Article in English | MEDLINE | ID: covidwho-970872


The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.

SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Humans , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , SARS-CoV-2/genetics , Saccharomycetales/chemistry , Saccharomycetales/genetics , Saccharomycetales/metabolism , Spike Glycoprotein, Coronavirus/biosynthesis , Spike Glycoprotein, Coronavirus/genetics