Project description:This study aims to under the characteristics and diversitification of Dengue speicific immunoglobulin repertoires after different immunization strategies in mouse. We propose two different immunization strategies in dengue vaccine development, one is repeated strategy, which DENV1 virus and administrated three times. The other one is epitope-decreased sequential strategy, which sequetially administrated DENV1 virus, DENV1 E protein and DENV1 E protein domain III subunit.
Project description:This study aims to understand the characteristics and diversification of Dengue specific immunoglobulin repertoires after different immunization strategies in mouse. We propose two different immunization strategies in dengue vaccine development. One is tetravalent strategy, which pre-mixed four serotype dengue DNA vaccine and administrated four times. The other one is sequential strategy, which sequentially administrated four time, each serotype vaccine for each dose as sequences of Den1, Den2, Den3 and Den4.
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: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:We developed an improved library prep protocol and standardized the data analysis pipeline for accurate repertoire profiling. In addition, two metrics were implemented to assess repertoire clone properties. We then studied systemically the effects of two adjuvants, CpG and Alum, on the Ig heavy chain repertoire using the ovalbumin (OVA) challenged mouse model. Ig repertoires of different tissues (spleen and bone marrow) and isotypes (IgG and IgM) were examined and compared in terms of sequence mutation frequency, IGHV gene usage, CDR3 length, rescaled Hill numbers for clonal diversity, and clone selection strength. As a result, Ig repertoires of different tissues or isotypes exhibited distinguishable profiles at the non-immunized steady state. Adjuvanted immunizations further resulted in statistically significant alterations in Ig repertoire compared with PBS or OVA alone immunized groups. Lastly, we applied unsupervised machine learning techniques – multiple factor analysis and clustering – to identify Ig repertoire signatures in different compartments and under varying immunizations.
Project description:We used 454 sequencing to assess the repertoire of B cell subsets from bone marrow, spleen, and small intestinal lamina propria from two mouse strains. We used a RAG2-GFP reporter mouse strain (129Sve background) to isolate CD19+ RAG2+ B lineage cells from bone marrow and small intestinal lamina propria and total splenic B cells. We used 5' RACE to amplify cDNA libraries using primers specific for the mu constant region of IgH and the Ig kappa constant region. We also used this technique to analyze total B cell libraries from Swiss Webster germ-free mice to compare to littermate controls that were cohoused with regular specific pathogen free (SPF) mice for 7 days. Examination of the Ig kappa repertoire and IgH repertoire in RAG2+ bone marrow B lineage cells compared to RAG2+ small intestinal lamina propria B lineage cells or total splenic B cells. There are 8 (Ig kappa) or 4 (IgH) independent experiments comparing repertoires in RAG2-GFP mice. Each experiment in RAG2-GFP+ mice consisted of a pool of 8-12 mice. There are 3 experiments comparing germ-free to colonized mouse total B cell repertoires, each consisting of one mouse per condition.
Project description:We performed single-cell transcriptome and antibody repertoire sequencing of bone marrow plasma cells following protein (OVA and TNFR2) immunizations or infection with high dose LCMV clone 13.
Project description:We performed single-cell transcriptome and antibody repertoire sequencing of bone marrow and splenic B cells from mice of different age following protein (TNFR2) immunizations