Study characterizes SARS-CoV-2 spike-specific cluster of differentiation 4+ T-cell response

In a recent study posted to the Research Square* preprint server, and currently under consideration at a Nature Portfolio Journal, researchers characterized over 100 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific CD4+ T-cell epitopes.

Study: Broad SARS-CoV-2 spike-specific CD4+ T-cell response preserves recognition of variants of concern. Image Credit: Naeblys/Shutterstock
Study: Broad SARS-CoV-2 spike-specific CD4+ T-cell response preserves recognition of variants of concern. Image Credit: Naeblys/Shutterstock

Additionally, they mapped 21 CD4+ T cell epitopes to three SARS-CoV-2 proteins - spike (S), nucleocapsid (N), and membrane (M).


CD4+ T-cells, a key component of adaptive immunity, generate a more durable response to viral infections, including SARS-CoV-2 infections. They also dominate memory T-cell responses to SARS-CoV-2 S in vaccinated and previously infected individuals.

However, assay-based studies have mapped only a few SARS-CoV-2 CD4+ T cell epitopes using high concentrations of peptides, and in silico human leukocyte antigen (HLA) binding algorithms have helped infer HLA restrictions in the epitopes. The following three aspects of SARS-CoV-2-specific CD4+ T-cells are unknown -

  1. the specificity of CD4+ T-cell responses at the epitope level,
  2. the extent to which these responses cross-react with other human coronaviruses (HCoVs), and
  3. CD4+ T-cell sensitivity to mutations in SARS-CoV-2 variants of concern (VOC).

About the study

In the current study, researchers collected blood samples from 20 healthcare workers (HCW) infected with SARS-CoV-2 wild-type (wt) D614G strain during the first wave of the coronavirus disease 2019 (COVID-19) pandemic in the United Kingdom (UK) in 2020. Likewise, they gathered samples from the control group of individuals who were confirmed to be seronegative for SARS-CoV-2.

The researchers isolated peripheral blood mononuclear cells (PBMCs) and plasma from donor blood samples and generated CD8-depleted PBMCs using anti-CD8 Dynabeads to >94%, as measured by flow cytometry. They performed ex vivo interferon-gamma (IFNg) Elispot assays on whole or CD8-depleted PBMCs to quantify antigen-specific responses, expressed as spot-forming cells (sfc) per 106 cells.

Furthermore, the team used an enzyme-linked immunosorbent assay (ELISA) to test IFNg release into the supernatant medium used for polyclonal T cell lines, and a multiplex serology assay to quantify the immunoglobulin G (IgG) antibody titers against SARS-CoV-2 S and N and HCoV family S proteins.

The researchers also assessed SARS-CoV-2 S-specific CD4+ T-cell clones against SARS, Middle Eastern Respiratory Syndrome coronavirus (MERS), human coronavirus (HKU1), and OC43 S1 or S2 pepmixes. Wherever possible, they tested multiple T-cell clones per epitope.

Lastly, the team assessed the recognition of SARS-CoV-2 VOCs at the epitope level, in particular for VOCs - Alpha (B.1.1.7), Gamma (P1), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529).

Study findings

CD4+ T cell responses, to a control pepmix of HLA class I-restricted epitopes, decreased significantly in CD8-depleted PBMCs, whereas, these responses increased to S (S1 and S2), M, and N proteins, thus, indicating a predominance of CD4+ T-cell memory.

The authors detected weak responses to three SARS-CoV-2 proteins in CD8-depleted PBMCs from 14 control group individuals, with no S or N-specific antibody responses, most likely representing cross-reactive T cells primed by prior exposure to HCoVs.

Upon testing against individual 20-mer peptides, the authors noted that the S-specific polyclonal CD4+ T-cell line displayed responses against several peptides, including the receptor-binding domain (RBD), S1, and S2 regions. Similarly, it broadly responded to SARS-CoV-2 M and N proteins, indicating that SARS-CoV-2 induced a broad CD4+ T-cell response.

The researchers isolated CD4+ T-cell clones specific for 21 epitopes, 17 S, 2 N, and 2 M epitopes, from six study participants with varying HLA-II types. T-cell clones specific to 17 epitopes within S recognized autologous lymphoblastoid cell lines (LCL) pre-exposed to low concentrations of purified S protein, demonstrating that the major histocompatibility complex (MHC) II processing pathway generated all the 21 epitopes efficiently.

The authors also observed that SARS-CoV-2 infection and vaccination equally induced broad CD4+ T-cell responses to shared epitopes, and the HLA-II genotype was a key determinant of these responses.

All T cell clones showed similar levels of recognition against S1 or S2 SARS-CoV-2 pepmix and T cell clone’s-related epitope peptide. However, they detected no cross-reactivity against any other HCoV within the more divergent S1 region. Overall, S epitope-specific CD4+ T-cells isolated following SARS-CoV-2 infection could recognize highly homologous epitopes within the S2 region of SARS- but did not cross-react with MERS- or distant HCoVs, such as HKU1 and OC43.

VOC mutations in certain epitopes did not affect epitope-specific CD4+ T-cell recognition, while others diminished or abolished recognition. The epitopes with most mutations were present within the RBD region, the most common target of mutations in the VOCs.

Overall, data highlights the ultra-sensitivity of SARS-CoV-2 S-specific CD4+ T-cells to small amino acid (aa) changes in epitope sequences and the potential for epitope evasion through minor SARS-CoV-2 mutations.


The study data from T-cell clones and ex vivo PBMC experiments demonstrated variable effects of intra-epitope mutations on CD4+ T-cell recognition. Although the overall effect of point mutations was complex, triple aa deletions and multiple aa substitutions within individual epitopes greatly impacted CD4+ T-cell recognition.

The study also highlighted the significance of epitope mapping for rationalizing VOC risk stratification. Additionally, it emphasized the need for careful characterization of immunodominant CD4+ T-cell epitopes individually.

All HCW had antibody-based immunity against endemic beta-HCoVs, consistent with prior infection with these viruses, which indicated that T-cell clones were primed by SARS-CoV-2 infection. Importantly, broad CD4+ T-cell epitope targeted potentially limited evasion by current SARS-CoV-2 VOCs. Therefore, continual genomic surveillance efforts are warranted to help identify novel mutations conferring CD4+ T-cell immunity evasion capabilities to VOCs.

*Important notice

Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Neha Mathur

Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.


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