ABSTRACT/SUMMARY: Single-cell genomic technology has rapidly transformed modern immunology. In relatively few short years, single-cell RNA sequencing has become near ubiquitous, primarily due to the availability of robust commercial platforms. The ability to combine multiple forms of information simultaneously (i.e. multi-modal readouts) has proven to be of significant utility, particularly for dissecting the diversity of the adaptive immune system. Single- cell methodology is now capable of acquiring information on multiple modalities simultaneously: surface receptor levels, paired immune receptor (BCR/TCR) clonotype identity, chromatin accessibility and tissue localization, and other parameters can now be measured alongside the transcriptome in relatively routine technology. The goal of Core B is to support the activities of Projects 1-3 by applying tailored advances in single-cell biology that are not commercially available. In Aim 1, we will to apply single-cell DNA bar-coding technology capable of assessing B cell specificity to SARS-CoV Spike protein epitopes from major variants. The application of this technology, capable of resolution of epitope specific responses would address several needs in SARS-CoV-2 research: (i) it accelerates the discovery of potential broadly neutralizing antibodies against SARS-CoV-2; (ii) it would afford comprehensive profiling of S protein variant specificity, (iii) it would provide a rich set of immunoglobulin sequence data to measure antibody evolution in distinct epitope specificities. In Aim 2, we will develop panels of DNA-linked antibodies to assay the protein abundance of master regulator transcription factors associated with the innate and adaptive immune system (NEAT-Seq). The ability to profile transcription factors provides the necessary granularity to dissect precise immune cell phenotypes and ontogeny, which will be critical in understanding the impact of senescence of SARS-CoV-2 specific cells in Project 2. Finally, in Aim 3, we will conduct spatial transcriptomics in support of Project 2, to profile the localization of macrophages in cadaver and NHP tissues from SARS-CoV-2 infected and vaccinated individuals. The activities of Core B leverage prior progress and parallel technology development by the MPIs. In the prior funding period, we developed novel methodology to obtain paired clonotype identity and transcriptome data in antigen-specific B cells, including development, validation and benchmarking of a novel bioinformatics algorithm capable of accurately reconstructing paired immunoglobulin gene sequences in vaccine-elicited B cells. Here, we extend our prior work to incorporate additional information: antigen specificity for SARS-CoV-2 epitopes. Additionally, in the prior period, we developed and NEAT-seq technology, a method for assaying the abundance of key transcription factors simultaneously with chromatin accessibility and the transcriptome. At the conclusion of the activities of Core B...