Evaluation of cross-reactive T cell responses between SARS-CoV-2 and common-cold human coronaviruses

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in high mortality and morbidity throughout the world.

Infections from SARS-CoV-2 have been reported to have various clinical presentations from asymptomatic to severe disease and death. However, most SARS-CoV-2 infections are found to result in mild symptoms.

Study: Broadly-recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles. Image Credit: CI Photos/ShutterstockStudy: Broadly-recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles. Image Credit: CI Photos/Shutterstock

Older individuals and individuals with comorbidities have been found to develop severe symptoms of disease with high mortality. Genetic polymorphism along with age-related variations in adaptive and innate immunity has been observed to play a critical role in the difference of clinical outcomes.


The local and systemic pathogenesis of SARS-CoV-2 can be attributed to reduced type I interferon, deficient anti-viral immunity in nasal epithelial cells, recruitment and activation of neutrophils, and atypical peripheral blood cytokine profile.

The role of T cells is not fully understood yet against this background although studies have shown that low CD4+ and CD8+ T cell counts are associated with severe disease. Peak severity has been observed to be inversely correlated with the frequency of SARS-CoV-2-specific IFN-γ-producing CD8+ T cells. Also, early CD4+ T cell responses were found to be associated with mild disease.

In addition to SARS-CoV-2 that has caused the current pandemic, six other coronaviruses are known to infect humans. These coronaviruses include the highly pathogenic SARS and MERS beta-coronaviruses, less pathogenic alpha coronaviruses 229E and NL63, and beta coronaviruses OC43 and HKU1. Reports after the discovery of the original SARS virus suggest that the T cells from unexposed individuals were capable of recognizing naturally processed and presented SARS antigens even before infection.

Studies have suggested that previous infections with common-cold coronaviruses (HCoVs) may have given rise to cross-reactive SARS-specific responses in unexposed individuals. However, T cells from individuals not previously infected with SARS were found to have an impaired response when compared with CD8+ T cells from previously SARS-infected individuals.

Studies on immune cross-reactivity among the human coronaviruses have gained importance due to the emergence of SARS-CoV-2. Both the SARS-CoV-2-infected individuals and the unexposed individuals were found to possess cross-reactive antibodies to both SARS-CoV-2 and HCoV spike protein. Moreover, individuals who had a recent infection with HCoV were found to have less severe COVID-19 infections.

Several studies suggested that T cell reactivity has been found in up to 50 percent of individuals who have not been exposed to SARS-CoV-2. A recent study reported that several people who had been exposed to SARS-CoV-2 did not test positive for the infection due to early T cell response. Although there is a high prevalence of HCoV infection and sequence homology with SARS-CoV-2, the definite relationship between the pre-infection T cell response and previous HCoV infection is not clear.

A new study published in the pre-print server bioRxiv* investigated SARS-CoV-2 spike protein responses targeted by cross-reactive T cells that were isolated from convalescent COVID-19 individuals along with previously uninfected donors that involved pre-pandemic donors sampled between 2015 to 2018 and seronegative asymptomatic individuals who were sampled during the pandemic.

About the study

The study involved generation of Peptide-pool or individual peptide expanded T cell lines from freshly isolated or frozen peripheral blood mononuclear cells (PBMCs) followed by IFN-γ ELISpot assay. Thereafter, Intracellular cytokine staining (ICS) was performed using the in vitro expanded T cells.

T cell clones were isolated from PBMCs followed by stimulation and blocking assay. A peptide binding assay was carried out to measure spike peptide binding. This was followed by tetramer staining, sorting of DP4-163/164 cells, T-cell receptor (TCR) sequencing, and clonotype analyses.

Study findings

The results indicated that a COVID-19 donor showed strong IFN-γ responses to peptide pools from SARS-CoV-2 spike (S), membrane (M), nucleocapsid (N) but not envelope (E) protein. However, responses to the spike (S) proteins of the four HCoVs were comparatively weaker. Moreover, responses to the SARS-CoV-2 S pool were observed to expand following stimulation with the HCoVs S pool. A pre-pandemic donor also reported IFN-γ T cell responses to S pools from each of the four HCoVs along with SARS-CoV-2 S peptides.

The results of the ex vivo studies were similar to the in vitro studies except for 42 percent of uninfected donors showed positive IFN-γ responses specific for SARS-CoV-2 S pool after in vitro expansion in comparison to 10 percent observed in the case of ex vivo. Ex vivo responses were also observed against HCoVs S pools in most COVID-19 and uninfected donors.

The results also indicated three pairs of overlapping peptides in the SARS-CoV-2 S protein sequence that could serve as epitopes for the cross-reactive T cell responses. These three overlapping peptides were 198/199, 190/191, and 163/164. Among 10 convalescent COVID-19 donors, 5 showed ex-vivo responses to 163/164, and one donor each recognized 190/191 and 198/199. Following in vitro expansion with HCoV S pools, another donor was found positive for peptide 163/164.

Furthermore, the three cross-reactive epitopes were identified from the S2’ domain of the S protein. The 163/164 sequence comprised the S2’ cleavage site and the fusion peptide (FP), 190/191 is located in the first heptapeptide repeat region, and 198/199 is located between heptapeptide repeats 1 and 2. These regions were found to be highly conserved among the SARS-CoV-2 variants including Omicron and Delta.

The T cells that responded to expansion were mostly reported to be CD4+. Therefore, it can be concluded that peptides 163/164, 190/191, and 198/90 contain epitopes presented predominantly by MHC-II proteins. Moreover, both the DP4- restricted clones and DQ-5 restricted clones were found to bind epitopes within the 163/164 sequence with a three-residue register shift between the core epitopes. DQ5-restricted clones were found to recognize 6 to 9 residues long minimal peptide sequences whereas DP4-restricted clones were found to recognize 9 residues long minimal peptide sequences.

Alignment of the DQ5 and DP4 core epitopes showed that the P2, P5, and P8 positions were 100 percent conserved in both SARS-CoV-2 and HCoV sequences. Other residues of DQ5 and DP4 are less conserved which accounts for the binding preference to several homologs. Also, the SARS-CoV-2 163/164 epitope was observed to be recognized broadly in unexposed, convalescent, as well as mRNA-vaccinated donors.

Tetramer staining validated the importance of DP4-163/164 tetramer in the detection of SARS-CoV-2 and HCoV-cross-reactive T cell populations. The results also identified a highly diverse repertoire of TCRs that recognized the 163/164 peptide. The TRAV and TRBV gene sharing among the donors was found to be quite limited.

The current study was capable of identifying a pan-coronavirus epitope that could be responsible for the cross-reactive T cell response. The epitope was found to be highly conserved among the human coronaviruses as well as the SARS-CoV-2 variants of concern. These conserved sequences can be useful in studies that are related to pre-existing HCoV immunity to COVID-19 severity or incidence. Moreover, they must also be considered for inclusion in pan-coronavirus vaccination.


The study had certain limitations. First, the non-IFN-γ-secreting populations were not considered in the determination of cross-reactive responses.

Second, in vitro culture conditions may have an impact on the cross-reactive T cell response regarding the involvement of mostly CD4+ T cells.

Third, the sample size of the study was small, and finally, the study did not determine which donors were previously exposed to which HCoV.

*Important notice

bioRxiv 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:
Suchandrima Bhowmik

Written by

Suchandrima Bhowmik

Suchandrima has a Bachelor of Science (B.Sc.) degree in Microbiology and a Master of Science (M.Sc.) degree in Microbiology from the University of Calcutta, India. The study of health and diseases was always very important to her. In addition to Microbiology, she also gained extensive knowledge in Biochemistry, Immunology, Medical Microbiology, Metabolism, and Biotechnology as part of her master's degree.


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