Method: The target DNA was amplified from a patient sample, sequence confirmed, modified to achieve the desired design architecture as such that it harbors a suitable 5' UTR, ORF to express the S protein with G614 and double proline (2P) mutations (K986P and V987P) with a IgE-secretory signal sequence, a special 3' UTR constructed with modified alpha and beta globin in tandem, and finally a 130 residue-long poly-A tail.
Method: The SARS-CoV-2 N-terminal domain (NTD) (residues 14-305), receptor-binding domain (RBD) (residues 319-541), and ectodomain of the spike (S) protein (residues 14-1213 with R682G/R683G/R685G/K986P/V987P mutations) (GenBank: QHD43416.1), were cloned into a customized pFastBac vector.
Single-component, self-assembling, protein nanoparticles presenting the receptor binding domain and stabilized spike as SARS-CoV-2 vaccine candidates.
Introduction: We then probed the spike metastability by comparing two uncleaved spike antigens, S2P (K986P/V987P) and S2G (K986G/V987G).
Result: A similar 2P mutation (K986P/V987P) was introduced into the SARS-CoV-2 spi
Result: We first created uncleaved spike ectodomain (SECTO) constructs for SARS-CoV-1/2, both containing the 2P mutation (K968P/V969P and K986P/V987P, respectively), a 5-amino acid G4S linker, a trimerization motif (PDB: 1TD0), and a C-terminal His6 tag.
Introduction of Two Prolines and Removal of the Polybasic Cleavage Site Lead to Higher Efficacy of a Recombinant Spike-Based SARS-CoV-2 Vaccine in the Mouse Model.
Abstract: While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the K986P and V987P (PP) mutations completely protected from challenge in this mouse model.IMPORTANCE A vaccine for SARS-CoV-2 is urgently needed.
Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants.
Method: For S protein stability, six proline mutations (F817P, A892P, A899P, A942P, K986P, V987P) and a disulfide mutation between T883 and V705 (mutated to cysteines) were introduced.
BNT162b vaccines protect rhesus macaques from SARS-CoV-2.
Abstract: BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)).
D614G Mutation Alters SARS-CoV-2 Spike Conformation and Enhances Protease Cleavage at the S1/S2 Junction.
5Result: This is in contrast with cryo-EM results of the D614G mutation published in the context of a S-GSAS/PP S that show S populations with 2- or 3-RBDs in the ""up"" state, suggesting that the PP mutations (K986P, V987P) may have a role in increasing the propensity of the RBD ""up"" forms."
Figure: Each inset corresponds to the S regions understudy and is highlighted in red on the trimeric structure ( Figure: The K986P-V987P mutations between the HR1 and CH domains are indicated by a yellow star (red contour) on the S-GSAS/PP template.
SARS_CoV_2 mutation literature information.
PMID: 
2021
Human vaccines & immunotherapeutics
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