Evidence for broad cross-reactivity of the SARS-CoV-2 NSP12-directed CD4+ T-cell response with pre-primed responses directed against common cold coronaviruses

Abstract

Introduction: The nonstructural protein 12 (NSP12) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has a high sequence identity with common cold coronaviruses (CCC).

Methods: Here, we comprehensively assessed the breadth and specificity of the NSP12-specific T-cell response after in vitro T-cell expansion with 185 overlapping 15-mer peptides covering the entire SARS-CoV-2 NSP12 at single-peptide resolution in a cohort of 27 coronavirus disease 2019 (COVID-19) patients. Samples of nine uninfected seronegative individuals, as well as five pre-pandemic controls, were also examined to assess potential cross-reactivity with CCCs.

Results: Surprisingly, there was a comparable breadth of individual NSP12 peptide-specific CD4⁺ T-cell responses between COVID-19 patients (mean: 12.82 responses; range: 0-25) and seronegative controls including pre-pandemic samples (mean: 12.71 responses; range: 0-21). However, the NSP12-specific T-cell responses detected in acute COVID-19 patients were on average of a higher magnitude. The most frequently detected CD4⁺ T-cell peptide specificities in COVID-19 patients were aa236-250 (37%) and aa246-260 (44%), whereas the peptide specificities aa686-700 (50%) and aa741-755 (36%), were the most frequently detected in seronegative controls. In CCC-specific peptide-expanded T-cell cultures of seronegative individuals, the corresponding SARS-CoV-2 NSP12 peptide specificities also elicited responses in vitro. However, the NSP12 peptide-specific CD4⁺ T-cell response repertoire only partially overlapped in patients analyzed longitudinally before and after a SARS-CoV-2 infection.

Discussion: The results of the current study indicate the presence of pre-primed, cross-reactive CCC-specific T-cell responses targeting conserved regions of SARS-CoV-2, but they also underline the complexity of the analysis and the limited understanding of the role of the SARS-CoV-2 specific T-cell response and cross-reactivity with the CCCs.

Keywords: CD4+ T-cells; Cross-reactivities; NSP12; RNA-dependant RNA polymerase; SARS-CoV-2; common cold coronaviruses; epitope analysis; sequence identities.

Figure 1

Experimental setup of the 15-mer single-peptide IFN-γ-ELISpot after 11–13 days of in vitro peptide-specific culture with different peptide pools, each spanning 46 to 47 peptides, and ICS after single-peptide re-stimulation for validation of positive peptide-specific T-cell responses.

Figure 2

(A–F) SARS-CoV-2 seronegative individuals demonstrated highly cross-reactive NSP12-specific CD4⁺ T-cell responses with low IFN-γ magnitude comparable to post-acute SARS-CoV-2 infection. (A) NSP12-specific T-cells were expanded in vitro with pools of overlapping NSP12 peptides in the presence of anti-CD28/anti-CD49d antibodies and IL-2. After 11–13 days, the cells were analyzed for IFN-γ production by ELISpot and validated by intracellular cytokine staining after restimulation with single peptides. (B) CD4⁺ T-cells predominated NSP12-specific IFN-γ responses in all groups. Representative flow cytometry plots of HH-N12-7 with acute SARS-CoV-2 infection. (C) Comparable numbers of recognized NSP12 peptides between individuals with acute or resolved SARS-CoV-2 infection and seronegative controls indicated relevant cross-reactivity of previously primed CD4⁺ T-cells. (D) The magnitude of IFN-γ production of NSP12-specific CD4⁺ T-cells in individuals with resolved SARS-CoV-2 infection and seronegative individuals compared to individuals with acute infection. (E) The breadth of cross-reactive NSP12-specific CD4⁺ T-cell responses in seronegative individuals is boosted by SARS-CoV-2 infection. (F) Increased breadth derives mostly from newly recognized specificities. Data are expressed as mean with a standard deviation. Mann–Whitney U test was performed to assess statistical significance. ns < 0.05; ^* p < 0.05.

Figure 3

(A, B) Regions of SARS-CoV-2 NSP12 frequently targeted by seronegative individuals show different degrees of sequence identity with common cold coronaviruses. Canonical protein amino acid sequences of SARS-CoV-2 and common cold coronaviruses were extracted from the UniProtKB database and aligned using the UniProt blast tool. (A) The SARS-CoV-2 NSP12 protein has a higher sequence identity with the corresponding proteins of the 229E, NL63, OC43, and HKU1 common cold coronaviruses than the structural spike glycoprotein and nucleocapsid protein. Serologic positivity for common cold coronaviruses was highly prevalent in the study cohort. (B) High response rates to SARS-CoV-2 NSP12 peptides in SARS-CoV-2-infected individuals and seronegative individuals are not restricted to regions with higher than overall sequence identity.

Figure 4

Experimental setup for CCC and SARS-CoV-2 T-cell cross-reactivity assessment.

Figure 5

(A–D) In vitro-expanded common cold coronavirus NSP12-specific CD4⁺ T-cells of seronegative controls cross-recognize corresponding SARS-CoV-2 NSP12 peptides. (A) PBMC of n = 3 seronegative controls were expanded in vitro with common cold coronavirus NSP12 peptides (according to their serologic positivity) as described before. After 11-13 days, the cells were analyzed for IFN-γ after individual restimulation with single peptides with the sequence of common cold coronaviruses and the corresponding SARS-CoV-2 peptides. (B–D) In all three individuals, considerable cross-recognition to corresponding SARS-CoV-2 peptides could be detected despite amino acid differences at certain positions (the asterisk shows sequences from 15-mer peptides used for stimulation that did not elicit a CD4⁺ T-cell response in the IFN-γ-ELISpot).