Clarified moderate was diluted 1:1 with protein A IgG Binding Buffer (Thermo Scientific) and filtered through 0.22 m filter to remove any precipitate before incubation with Protein A Plus Agarose (Thermo Scientific). titers, antibody avidity, antibody longevity, and B-cell epitopes targeted by the immune system. In conclusion, a recombinant subunit protein immunogen based on the RBD is usually a highly encouraging vaccine candidate. Continued evaluation of RBD immunogenicity using different adjuvants and vaccine regimens could further improve vaccine efficacy. Keywords: SARS-CoV-2, COVID-19, vaccine, RBD, neutralizing antibody Introduction In late 2019, an outbreak of severe pneumonia (referred to as COVID-19) began in China. The causative pathogen was identified as a novel coronavirus (CoV) designated as SARS-CoV-2 (of family. Its single strand RNA genome ranges from 29,825 to 29,902 bases long. The virus is usually most closely related to a bat coronavirus RaTG13 strain (nucleotide sequence identity of ~96%) (4). SARS-CoV-2 is usually more distantly related to SARS-CoV (82% identity). However, both viruses utilize angiotensin transforming enzyme 2 (ACE2) as a receptor (4). Binding of ACE2 and cellular Photochlor entry is usually mediated by spike (S) glycoprotein, which is usually DHRS12 1,273 amino acids long. S proteins of the two viruses are 76% identical. Cryo-EM structures of SARS-CoV-2 S protein as well as cocrystal structures of the receptor binding domain name (RBD) of the protein bound to ACE2 have been solved (5C8). S protein functions as a homotrimer (Physique 1A) and it is highly glycosylated. Glycosylation of all 22 potential N-linked glycosylation sites have been confirmed for SARS-CoV-2 (9). The RBD folds independently into a globular structure away from the rest of the S protein (Physique 1B). It exists in two different conformations around the trimer: open and closed says (Physique 1A). While it is usually in the open state, it can bind ACE2. Cocrystal structures of the RBD and ACE2 clearly identified amino acid residues on both proteins critical for binding (Figures 1CCE). Within the RBD, binding of ACE2 is usually mediated mostly by amino acid residues within a short linear segment called the receptor binding motif (RBM) (Figures 1D,E). Open in a separate windows Physique 1 Structural Photochlor features of SARS-CoV-2 S protein and RBD immunogen. (A) A cryo-EM structure of a trimer (PDB: 6VSB). Top and side views are shown, and the open and closed conformational says of the RBD are indicated. Three monomers are shown in different colors with the RBD portions highlighted in darker shades. Glycans (N-acetylglucosamine) are shown in green. (B) A monomeric S Photochlor protein in an open state. (C) A cocrystal structure of the RBD with ACE2 (PDB: 6M0J). The RBM is usually shown in pink and the ACE2 binding residues are highlighted in reddish. (D) Details of amino acid residues involved in ACE2 binding. Side chains on ACE2 that bind the RBD are shown in cyan. (E) Schematic diagrams of S protein, RBD and RBM showing major functional domains and features. The RBM is usually shown in pink and ACE2-contact residues are shown in reddish. Glycosylation sites are shown as green hexagon. (F) SDS-PAGE of purified RBD and after treatment with endoglycosidase H and PNGase F. (G) ELISA showing binding of the RBD to neutralizing mAb CR3022. Many studies have shown that subunit protein antigens based on the RBD can elicit neutralizing antibodies (nAbs) against SARS-CoV (10C13). In this study, we examined immunogenicity of a SARS-CoV-2 recombinant RBD subunit protein vaccine candidate in mice using three different adjuvants. We focused on the humoral immune responses since nAbs are considered to be the major immune correlates of protection (14). In particular, we monitored around the potency, induction kinetics and durability of nAbs. The results indicate that potent, long-lasting nAbs can be induced using the RBD, that adjuvants can profoundly affect antibody responses, and that RBD-based subunit protein is usually a highly encouraging vaccine candidate against COVID-19. Results Immunogen Design, Production, and Characterization The RBD contains a single N-linked glycosylation site at N343. To generate an RBD immunogen that is as native as you possibly can, we constructed a plasmid encoding the RBD to be expressed in mammalian cells. The immunogen encompasses amino acids from T333 to K528, which are located three residues upstream or downstream of two cysteine residues, C336 and C525, respectively, that form a disulfide bridge and structurally define the RBD. The RBD gene is usually preceded by a signal peptide for secretion and followed by a hexahistidine tag (6H) for purification. The recombinant RBD protein was transiently expressed in HEK 293F cells and purified very easily from cell culture supernatant through.