Holly A. to the RBD and for their ability to block the interaction of the RBD and ACE2. Results Seven putative vaccine epitopes were identified. Memory B\cells (MBCs) specific for one of the epitopes were identified in the blood of convalescent patients. When used to vaccinate mice, six induced antibodies that bound recRBD and three induced antibodies that could partially block the interaction of the RBD and ACE2. However, when the sera were combined in pairs, we observed significantly enhanced inhibition of binding of RBD to ACE2. Two of the peptides were located in the main regions of the RBD known to contact ACE2. Of significant importance to vaccine development, two of the peptides were in regions that are invariant in the UK and South African strains. Conclusion COVID\19 convalescent patients have SARS\CoV\2\specific antibodies and MBCs, the specificities NADP of NADP which can be defined with short peptides. Epitope\specific antibodies synergistically block RBDCACE2 interaction. and characterisation of SARS\CoV2 peptide\specific memory B\cells (MBCs) in convalescent PBMCs To determine whether the three peptides were recognised by MBCs (CD3? CD20+ CD19+ IgD?CD27+) in the convalescent patients, we prepared tetramers to peptides\7, \13 and \17 and tested B\cells from available PBMCs collected from seven of the convalescent patients (see Methods). The number of MBCs correlated directly with the level of peptide\17\specific IgG in CP (Figure?4a), and we found that MBCs that recognised peptide\17 were significantly enriched in convalescent PBMCs in relation to healthy PBMCs (Figure?4b NADP and c). No difference between the healthy and the seven convalescent patients was noted for peptide\7 and peptide\13 tetramer+ MBCs (not shown); however, these seven convalescent patients had low levels of peptide\7 and \13\specific antibodies. Open in a separate window Figure 4 Peptide\tetramer staining for memory B cells. (a) Peptide 17 tetramer staining of memory B cells (assessed as CD19+CD20+CD27+IgD? cells) in healthy donors (healthy, and mouse studies. A random 20\mer peptide was designed using a random number generator (LEKLEAQRRARDKSELKVDA) and was used as a negative control. The recombinant SARS\CoV\2 Spike protein (RBD, His\Tag) encompassing amino acids Arg 319 \ Phe 541 (Accession # “type”:”entrez-protein”,”attrs”:”text”:”QHD43416.1″,”term_id”:”1791269090″,”term_text”:”QHD43416.1″QHD43416.1) was sourced from Genscript and used in ELISAs. The recombinant SARS\CoV\2 (2019\nCoV) Spike RBD\His protein used in the ACE2\binding inhibition assay was sourced from Sino Biological, Chesterbrook, PA, USA. Spike protein on virus\like particles (Cspike on VLPs), used Lox in SPR assay was produced using VSV system described below. Virus\like\particles Non\infectious lentivirus\like particles were produced in the presence or absence of SARS\CoV\2 spike protein to mimic the presentation of SARS\CoV\2 Spike proteins on the surface of an enveloped virus. SARS\CoV\2 spike protein mammalian cell expression vector was a kind gift from Prof Linfa Wang at Duke\NUS Medical School, Singapore. Production of VLPs and SARS\CoV\2 Spike pseudotyped VLPs were done by PEI mediated transfection of plasmid DNA into HEK 293 cells, and purification of these VLPs were done using virus purification procedures that we have previously described for HIV. 38 , 39 Peptide synthesis and conjugation Seven Cysteinyl peptides derived from the RBD peptide library were selected for conjugation to DT using 6\Maleimido\caproyl characterisation of memory B\cells (MBCs) was performed using antigen tetramers as previously described. 44 Biotinylated peptides synthesised at Genscript were tetramerised by serial addition of premium\grade streptavidin\PE and streptavidin\APC (Bio legend, San Diego, CA, USA) at a four\molar equivalent of biotinylated peptide to one molar equivalent of streptavidin during 2?h at room temperature. The prepared tetramer was diluted to a concentration of 2?m for use. Typically, 106 PBMCs were pre\incubated with Fc block (Bio legend) for 10?min and room temperature followed by tetramer staining on ice for 40?min. Cells were washed with PBS and then stained with NADP a mixture of Abs for 30?min on ice: LIVE/DEAD Fixable Near\IR (Molecular Probes, Waltham, Massachusetts, USA), anti\CD3CFITC for T cell dumping (clone UCHT1; BD Biosciences) anti\CD19CPerCP (clone HIB19; Bio legend); anti\CD20CAlexa Fluor 700 (clone 2H7; Bio Legend), anti\CD27CeFluor450 (clone O323; Molecular Probes).