Project description:Purpose: Construction of a SARSCoV2 vaccine TCR specific machine learning model using single-cell TCR technology sequencing Methods：PBMCs were isolated from peripheral venous blood of HLA-A2+ healthy donors. PBMCs were incubated with antibody cocktail and then RapidSpheres, then the magnet was applied and unbound CD8+ T cells were recovered from the supernatant. According to the above method of CD8+ T activation, CD8+ T cells specific for ancestral epitopes were obtained by stimulating with the corresponding mutant ancestral peptides. Activation-specific CD8+ T cells were labeled with tetramers-PE and CD8-APC and then sorted out by flow cytometer FACS Canto (BD). The following protocol describes surface protein staining with hashtag antibodies for protein detection outside of the single cell V(D)J signature barcoding technique for differentiating CD8+ T cells with different epitope specificities after mixing up samples. The following is the hashtag information corresponding to the ancestral epitopes of the mutant strains. B.1.1.7 corresponds to the ancestral epitope ORF1a 1707-16 , ORF1a 2225-34 , ORF1a 2230-38. B.1.617.2 corresponds to the ancestral epitope M 82-90 . B.1.617.3 corresponds to the ancestral epitope ORF1a 2240-49, ORF1a 3683-92 , and ancestral epitope S 2-11 of B.1.526.2 without labeled hashtag protein. Cell number and viability were checked after surface protein hashtag staining (cell viability > 80%). Then droplet-encapsulation single-cell sequencing experiments were performed, and 10,000 living single cells were loaded onto each of the Chromium Controller (10x Genomics). After droplet-encapsulation, single-cell cDNA synthesis, amplification and sequencing libraries were generated using Chromium Single Cell 5' Feature Barcode Library Kit (10x Genomics),Chromium The result showed the inactivated vaccine is less protective in older adults, who take longer to develop effective antibodies, and the TCR diversity of each epitope specific repertoire decreased in the elderly. In addition, we found inactivated vaccines could stimulate the proliferation of related B cells in the body, thereby reducing the diversity of BCR in the body. Compared with the young, the elderly is less likely to produce antibody related BCR clones and the same is true for TCR diversity. Conclusions: The result showed the inactivated vaccine is less protective in older adults, who take longer to develop effective antibodies, and the TCR diversity of each epitope specific repertoire decreased in the elderly. In addition, we found inactivated vaccines could stimulate the proliferation of related B cells in the body, thereby reducing the diversity of BCR in the body. Compared with the young, the elderly is less likely to produce antibody related BCR clones and the same is true for TCR diversity.

Project description:Purpose:Comprehensively compared the adaptive immune response of SARS-CoV-2 inactivated vaccines in young and elderly. Methods:CD8+ T, CD4+ T and B cells were purified from PBMCs with EasySep Human positive/negative selection . PBMCs were incubated with antibody cocktail and then RapidSpheres, then the magnet was applied and unbound CD8+ T, CD4+ T and B cells were recovered from the supernatant. Briefly, the cells were stained with the corresponding CD8+ T, CD4+ T and B-cell antibodies for 30 minutes at 4°C in the dark, and the purity of the cells was detected separately by flow cytometry, and all could reach more than 95%. Total RNA was isolated from CD8+ T, CD4+ T and B cells of 3 young and older people using TRIzol Reagent (Invitrogen) (7-days post second vaccination dose) . RNA purity was checked by the NanoPhotomerer spectrophotometer (IMPLEN), and integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies). Then cDNA libraries were constructed using 0.1 µg RNA per sample with the NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia). After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 250 bp paired-end reads were generated. The result showed the inactivated vaccine is less protective in older adults, who take longer to develop effective antibodies, and the TCR diversity of each epitope specific repertoire decreased in the elderly. In addition, we found inactivated vaccines could stimulate the proliferation of related B cells in the body, thereby reducing the diversity of BCR in the body. Compared with the young, the elderly is less likely to produce antibody related BCR clones and the same is true for TCR diversity. Conclusions: The result showed the inactivated vaccine is less protective in older adults, who take longer to develop effective antibodies, and the TCR diversity of each epitope specific repertoire decreased in the elderly. In addition, we found inactivated vaccines could stimulate the proliferation of related B cells in the body, thereby reducing the diversity of BCR in the body. Compared with the young, the elderly is less likely to produce antibody related BCR clones and the same is true for TCR diversity.

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Time of day influences immune response to an inactivated vaccine against SARS-CoV-2.

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