Project description:We studied the impact of a vaccine prime dose on CD8 T cell gene expression We first immunized mice with an Ad5-SARS CoV-2 spike vaccine and then evaluated gene expression on SARS CoV-2 specific CD8 T cells at week 4.
Project description:In this project, we characterized the antigen-specific CD8 T cells of people from five groups. We used HLA:A2 tetramers loaded with the GILGFVFTL (Influenza) or YLQPRTFLL (Sars-CoV-2) peptides to label cells. Cells were sorted based on CD3+CD8+TTM+. We distinguished between four groups: 1. SARS2-TTM+ cells 6 months after infection, 2. SARS2-TTM+ cells 1 motnhs after 2nd vaccination with the Pfizer vaccine, 3. SARS2-TTM+ cells of people that were both infected and vaccinated and 4. FLU-TTM+ cells. Cells were sorted and analyzed by bulk RNA sequencing.
Project description:RNA vaccines are efficient preventive measures to combat the SARS-CoV-2 pandemic. High levels of neutralizing SARS-CoV-2-antibodies are an important component of vaccine-induced immunity. Shortly after the initial two mRNA vaccine doses, the IgG response mainly consists of the pro-inflammatory subclasses IgG1 and IgG3. Here, we report that several months after the second vaccination, SARS-CoV-2-specific antibodies were increasingly composed of non-inflammatory IgG4, which were further boosted by a third mRNA vaccination and/or SARS-CoV-2 variant breakthrough infections. IgG4 antibodies among all spike-specific IgG antibodies rose on average from 0.04% shortly after the second vaccination to 19.27% late after the third vaccination. This induction of IgG4 antibodies was not observed after homologous or heterologous SARS-CoV-2 vaccination with adenoviral vectors. Single-cell sequencing and flow cytometry revealed substantial frequencies of IgG4-switched B cells within the spike-binding memory B-cell population (median 14.4%; interquartile range (ICR) 6.7-18.1%) compared to the overall memory B-cell repertoire (median 1.3%; ICR 0.9-2.2%) after three immunizations. Importantly, this class switch was associated with a reduced capacity of the spike-specific antibodies to mediate antibody-dependent cellular phagocytosis and complement deposition. Since Fc-mediated effector functions are critical for antiviral immunity, these findings may have consequences for the choice and timing of vaccination regimens using mRNA vaccines, including future booster immunizations against SARS-CoV-2.
Project description:RNA vaccines are efficient preventive measures to combat the SARS-CoV-2 pandemic. High levels of neutralizing SARS-CoV-2-antibodies are an important component of vaccine-induced immunity. Shortly after the initial two mRNA vaccine doses, the IgG response mainly consists of the pro-inflammatory subclasses IgG1 and IgG3. Here, we report that several months after the second vaccination, SARS-CoV-2-specific antibodies were increasingly composed of non-inflammatory IgG4, which were further boosted by a third mRNA vaccination and/or SARS-CoV-2 variant breakthrough infections. IgG4 antibodies among all spike-specific IgG antibodies rose on average from 0.04% shortly after the second vaccination to 19.27% late after the third vaccination. This induction of IgG4 antibodies was not observed after homologous or heterologous SARS-CoV-2 vaccination with adenoviral vectors. Single-cell sequencing and flow cytometry revealed substantial frequencies of IgG4-switched B cells within the spike-binding memory B-cell population (median 14.4%; interquartile range (ICR) 6.7-18.1%) compared to the overall memory B-cell repertoire (median 1.3%; ICR 0.9-2.2%) after three immunizations. Importantly, this class switch was associated with a reduced capacity of the spike-specific antibodies to mediate antibody-dependent cellular phagocytosis and complement deposition. Since Fc-mediated effector functions are critical for antiviral immunity, these findings may have consequences for the choice and timing of vaccination regimens using mRNA vaccines, including future booster immunizations against SARS-CoV-2.
Project description:Gene profiling and T cell clonotype analysis on SARS-CoV-2-M198-206-specific CD8+ T cells were performed by scRNA-seq and scTCR-seq.
Project description:We performed a study investigating donors previously infected with SARS-CoV-2 during the first wave of the COVID-19 pandemic to understand how vaccination may reshape T cell populations formed after infection. 10 donors were investigated using single-cell RNA-sequencing consisting of a) 3 mild non-hospitalised donors, b) 3 severe hospitalised donors, c) 1 mild recently vaccinated donor and d) 3 recently convalescent donors. For each of these groups we sampled at the following timepoints a) 6-9 months and 18 months after infection, b) 6-9 months and 18 months after infection, c) 13 months and 15 months after infection and d) 35 days after infection. All donors were unvaccinated at the first time point and vaccinated at the second time point. We stimulated PBMC with an overlapping peptide pool derived from the spike glycoprotein of the SARS-CoV-2 virus and sorted spike-specific CD4+ T cells (CD4+CD69+CD40L+) and spike-specific CD8+ T cells (CD8+CD69+4-1BB+) from every donor and time point using flow cytometry for 10X single-cell sequencing (5' protocol). Each time point from Dnr4868 was sequenced across two 10X reactions/libraries performed on the same day. Multiple samples were hashed and pooled together for sequencing using TotalSeq-C anti-human hashing antibodies from BioLegend. Individual library expression matrix files were generated using the CellRanger pipeline from 10X Genomics. The processed data file named immune.combined220929.rds is a Seurat Object containing all samples and generated using the Seurat package in R.
Project description:Clonal expansion is a hallmark of adaptive immunity, and appears to be driven by high antigen-specific receptor avidity as shown by research using murine in vivo models. In humans, however, the functionality of antigen-specific T cell clonotypes that are recruited into primary and recall immune responses remains surprisingly elusive. In this regard, the vaccination program during the SARS-CoV-2 pandemic represented a unique research opportunity by provision of highly standardized cohorts of healthy human individuals receiving immunizations against a previously unseen antigen. Here, we analyzed 30 HLA-typed individuals before, short-term and long-term after three mRNA vaccinations against SARS-CoV-2. We performed an in-depth characterization of the magnitude, phenotype, clonal composition and functionality of antigen-specific CD8 T cell responses by ELISPOT, flow cytometry and single-cell RNA sequencing, including CITE seq of 130 surface antigens and 16 T cell epitope specificities. 89 T cell receptors (TCRs) covering three complete epitope-specific repertoires were re-expressed by CRISPR/Cas9-mediated orthotopic TCR replacement and tested for their avidity. TCR repertoires underwent continuous tailoring, with high TCR avidity being linked to both clonal persistence and expansion. However, epitope-specific repertoires also maintained diversity by concomitant contraction and new emergence of functional T cell clones over time. These data on the phenotype and clonal selection within human antigen-specific T cell responses instruct our understanding of human T cell biology and may guide the development of enhanced or novel vaccines.
Project description:The COVID-19 pandemic caused by SARS-CoV-2 is a continuous challenge worldwide, and there is an urgent need to map the landscape of immunogenic and immunodominant epitopes recognized by CD8 T cells. Here, we analyze samples from 31 COVID-19 patients for CD8 T cell recognition of 500 peptide-HLA class I complexes, restricted by 10 common HLA alleles. We identify 18 CD8 T cell recognized SARS-CoV-2 epitopes, including an epitope with immunodominant features derived from ORF1ab and restricted by HLA-A*01:01. In-depth characterization of SARS-CoV-2-specific CD8 T cell responses of patients with acute critical and severe disease reveals high expression of NKG2A, lack of cytokine production and a gene expression profile inhibiting T cell re-activation and migration while sustaining survival. SARS-CoV-2-specific CD8 T cell responses are detectable up to 5 months post recovery from critical and severe disease, and these responses convert from dysfunctional effector to functional memory CD8 T cells during convalescence.
Project description:Despite extensive research on SARS-CoV-2 vaccination responses in healthy individuals, there is comparatively little known beyond antibody titers and T-cell responses in the vulnerable cohort of patients after allogeneic hematopoietic stem cell transplantation (ASCT). In this study, we assessed the serological response and performed longitudinal multimodal analyses including T cell functionality and single-cell RNA sequencing combined with TCR/BCR profiling in the context of BNT162b2 vaccination in ASCT patients. In addition, these data were compared to publicly available data sets of healthy vaccinees. Protective antibody titers were achieved in 40% of patients. We identified a distorted B and T cell distribution, a reduced TCR diversity, and increased levels of exhaustion marker expression as possible causes for the poorer vaccine response rates in ASCT patients. IGHV gene rearrangement after vaccination proved to be highly variable in ASCT patients. Changes in TCRα and TCRβ gene rearrangement after vaccination differed from patterns observed in healthy vaccinees as well as unvaccinated ASCT patients and associated transplant donors. Crucially, ASCT patients elicited comparable proportions of SARS-CoV-2 vaccine-induced (VI) CD8+ T cells, characterized by a distinct gene expression pattern that is associated with SARS-CoV-2 specificity in healthy individuals. Our study underlines the impaired immune system and thus the lower vaccine response rates in ASCT patients. However, since protective vaccine responses and VI-CD8+ T cells can be induced in part of ASCT patients, our data advocate early post-transplant vaccination due to the high risk of infection in this vulnerable group.
Project description:The experiment aims at characterizing the immune responses elicited by the BNT162b2 vaccine against SARS-CoV-2, initially administered in a two dose regimen (second dose after three weeks followinf the first dose) In particular the transcriptional landscape of circulating T and B lymphocytes has been profiled longitudinnaly by scRNA-seq coupleD with CITE-seq of 19 cell surface markers to better classify T cells subpopulations, LIBRA-seq to assess the Spike-specificity of BCRs and and V(D)J seq to also track T and B cell clones dynamics. Eeach sample was profiled before vaccination (T0), 21 days after the first dose (T1), 2 months after the first dose (1 month after the second dose) (T2). The immune responses were characterized using PBMC from 3 SARS-CoV-2 experienced donors (experiencing SARS-Cov-2 at least 4 months before the first vaccinatin) and 2 SARS-CoV-2 unexperienced donors.