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Principal Investigator: Rafi Ahmed
Organization: EMORY UNIVERSITY
Fiscal Year: 2024
Award: $280,751
Funding agency: National Institute of Allergy and Infectious Diseases
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, we will have applied novel, tailored technology to advance our understanding of variant-specific humoral
immunity, and the “in tissue” regional specialization of the innate response after SARS-CoV-2 vaccination.
Collectively, these activities will enhance the insight gained from the unique samples available to this consortium.
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