Immunodominant T cell responses to SARS-CoV-2 nucleocapsid protein in Omicron breakthrough infection post-inactivated vaccination

ABSTRACT

The widespread administration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has not eliminated breakthrough infections due to immune evasion by viral mutations. Whether individuals with breakthrough infections can elicit effective T cell responses against the circulating Omicron JN.1 variant, and the specific immunodominant characteristics of these responses, remain to be elucidated. To address this, we recruited 93 individuals who had experienced early Omicron subvariant breakthrough infections following a three-dose regimen of inactivated SARS-CoV-2 vaccines. Intracellular cytokine staining was employed to evaluate T cell responses to the JN.1 nucleocapsid (N) protein overlapping peptide pool in peripheral blood mononuclear cells. Immunodominant epitopes were identified using a three-dimensional matrix screening approach. Human leukocyte antigen (HLA) blocking assays and truncated peptides stimulation assays were conducted to define the minimal epitope and HLA restriction. We found that breakthrough infections elicited robust CD4⁺ and CD8⁺ T cell responses specific to the JN.1 N protein, characterized by Th1/Tc1-skewed phenotype and preserved polyfunctionality. The immunodominant epitope profile of the N protein has undergone a significant transformation following breakthrough infection. New epitopes targeting mutated sites have emerged, while previously dominant epitopes, such as N-4 and N-66, no longer maintained their immunodominance. Instead, highly conserved epitope N-24 has risen as the most immunodominant target for CD4⁺ T cells, restricted by HLA-DR, and with the minimal epitope identified as PKDHIGTRNPANNA. Our finding demonstrates that early Omicron strain breakthrough infections induce robust T cell responses targeting the circulating JN.1 subvariant and underscore the immunodominant features of these responses.

Introduction

Since the global use of coronavirus disease 2019 (COVID-19) vaccines approximately one year after the pandemic outbreak, the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been significantly curtailed, with notable reductions observed in both the incidence and severity of infections [1]. However, over the past five years, SARS-CoV-2 has undergone substantial evolutionary changes, acquiring the capacity to evade antibodies elicited by vaccination [2]. This immune evasion has contributed to the widespread occurrence of SARS-CoV-2 breakthrough infections [3]. As of 26 January 2025, there were 81,716 new confirmed COVID-19 cases and 3883 deaths reported worldwide over the past 28 days [4]. Furthermore, pre-infection vaccination against SARS-CoV-2 only partially mitigated the risks of long COVID (post-acute sequelae of COVID-19), which is a multisystemic debilitating illness characterized by a diverse array of issues, including heart disease, diabetes, chronic fatigue syndrome, and dysautonomia [5]. Symptoms of long COVID may endure for years and often become lifelong, with no validated treatments currently available [6]. Clearly, the COVID-19 pandemic remains a substantial burden on public health systems and the global economy.

The SARS-CoV-2 Omicron variant was first reported by the World Health Organization (WHO) on 24 November 2021 [7]. Since then, Omicron has further diversified into a range of sublineages, including BA.2.75, XBB, BA.5, BF.7, BA.2.86, JN.1, and others [8,9]. Given its extensive number of mutation sites, coupled with its high transmissibility and significant immune evasion potential, Omicron was rapidly reclassified from variants under monitoring (VUMs) to variants of concern (VOCs) in a remarkably short span of just 2 days [10]. To date, the Omicron sublineages JN.1 remain the predominant SARS-CoV-2 variants globally, underscoring their ongoing impact on the COVID-19 pandemic.

Despite early evidence showing that sequence mutations in SARS-CoV-2 variants can enable the escape of neutralizing antibodies, T cell responses appear to be less affected by this immune evasion [11,12]. Furthermore, memory T cells induced by vaccination can identify and react to Omicron subvariants [13–15], indicating that antigen-specific T cells may be critical in providing long-term protection against severe COVID-19 [16]. Our earlier studies demonstrated that the nucleocapsid (N) and spike (S) proteins of SARS-CoV-2 are immunodominant antigens compared to the envelope (E) and membrane (M) proteins among vaccination recipients [17]. The Omicron variant exhibits over 50 mutations in the S protein, while the N protein is comparatively conserved among its sublineages, with 90% amino acid homology [2,18,19]. Nonetheless, it remains to be elucidated whether memory T cells elicited by breakthrough infections with early Omicron subvariants can recognize the N protein of the currently circulating JN.1 strain.

The CD4⁺ and CD8⁺ T cell responses elicited by infection or vaccination predominantly target a restricted set of epitopes [20,21]. Although we have previously characterized the immunodominant epitopes of the N antigen in individuals vaccinated with inactivated vaccines, it remains to be determined whether the immunodominant epitopes profiles of the N protein have altered in individuals experiencing breakthrough infections, and whether any novel immunodominant epitopes have emerged.

In this study, we investigated T cell responses in 93 individuals who had received three doses of inactivated SARS-CoV-2 vaccines and subsequently experienced Omicron breakthrough infections. Our findings revealed that breakthrough infection with early Omicron subvariants elicited robust CD4⁺ and CD8⁺ T cell responses against the N protein of the JN.1 strain, characterized by a Th1/Tc1 phenotype and preserved polyfunctionality. The immunodominant epitope profile of the N protein has undergone a significant transformation following breakthrough infection, with highly conserved epitope N-24 emerging as the predominant target for CD4⁺ T cells, eliciting a dominant response in 35.1% of participants. Additionally, novel epitopes targeting mutated sites have emerged. Further analysis revealed that the N-24 epitope is highly conserved across SARS-CoV-2 variants and is restricted by human leukocyte antigen (HLA)-DR; its minimal core epitope sequence was mapped to PKDHIGTRNPANNA. These findings elucidate the T cell immune responses induced by Omicron breakthrough infections following inactivated vaccine immunization.

Materials and methods

Human donors

In this study, we enrolled adults aged 18–60 years who had completed a full three-dose regimen of the inactivated SARS-CoV-2 vaccine BBIBP-CorV, and subsequently experienced at least one breakthrough infection with the Omicron variant between 7 December 2022, and 11 November 2023, prior to the emergence of the JN.1 variant in China. Enrollment occurred at the Eighth Affiliated hospital of Sun Yat-sen University (n = 93; male = 44; female = 49; mean age = 43.00). The exclusion criteria are consistent with those previously described in our prior studies [17]. In brief, individuals who were pregnant or lactating, as well as juveniles, were not included in the study. Additionally, those with acute viral colds, autoimmune disorders, immunodeficiencies, cardiac insufficiency, hematological diseases, abnormal liver or kidney function, malignant tumors, terminal illnesses, or a history of drug or alcohol abuse were also excluded. This study was reviewed and approved by the Medical Ethical Committee of the Eighth Affiliated Hospital of Sun Yat-sen University (no. 2021–005–003). Written informed consent was obtained from each participant. This study was conducted according to the Declaration of Helsinki (1975).

Isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs)

Peripheral blood specimens (4-10 mL) were obtained using EDTA-treated blood collection tubes. The samples were diluted 1:1 with saline and carefully layered onto an equal volume of Ficoll-Paque (TBDscience, CHN, Cat#LTS1077) in 15 mL centrifuge tubes, ensuring the interface remained intact. The tubes were centrifuged at 800 g for 20 minutes with slow acceleration and deceleration. Following centrifugation, the plasma was removed, and the PBMCs was transferred to a new tube. PBMCs were washed twice with saline and centrifuged at 250 g for 10 mins. Finally, PBMCs were cryopreserved in liquid nitrogen for further detection.

Synthetic peptides

Overlapping peptide pools spanning the entire N protein were generated based on the coding sequence of the SARS-CoV-2 Wuhan-Hu-1 strain (YP_009724397.2) or JN.1 variant (XPD16435.1) as published by the National Center for Biotechnology Information (NCBI). Based on a stepwise overlapping strategy with a step size of 6-amino-acids and an overlap of 12-amino-acids, a total of 68 18mer peptides were synthesized for the N protein of Wuhan-Hu-1 and JN.1 strain, respectively. To identify smaller epitopes within the CD4⁺ T cell immunodominant epitope N-24, we generated two overlapping peptide sets: five 13mer offset by two residues (11-residue overlap) and five 14mer offset by a single residue (13-residue overlap). All peptides were synthesized by ChinaPeptides (CHN), with purity exceeded 90%. The lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO) (Sigma‒Aldrich, USA, Cat#D2650) to a storage concentration of 20 μM and stored at −80°C.

Three-dimensional peptide matrix construct

The overlapping peptides spanning the N protein of Wuhan-Hu-1 and JN.1 strains were assigned to a three-dimensional peptide matrix, as previously described [17]. Specifically, the 68 peptides encompassing the full-length N protein were systematically allocated into the matrix, forming 13 peptide pools. The matrix was divided into three quadrants: the first quadrants contained Pool 1 to 4, the second quadrants comprised Pool 5 to 8, and the third quadrant included Pool 9 to 13. Each peptide was distributed across three pools in different quadrants, with no more than 20 individual peptides in each pool. The three-dimensional matrix is illustrated in Figure S1.

Peptide stimulations

Peptide stimulation was performed as previously described [17]. Specifically, the cryopreserved PBMCs were thawed and resuspended in RPMI 1640 medium (Gibco, USA, Cat#C11875500BT) containing 10% FBS (Gibco, Cat#10099-141C) and 1% Pen/Strep (Gibco, Cat#15140122). Cell counts were determined using a hemocytometer (QiuJing, CHN, Cat#65657). Subsequently, a total of 1 × 10⁶ live PBMCs were plated into 96-well U-bottom plates and stimulated for 5 hours with premixed peptide pools N (68 peptides), the 13mer or 14mer sub-pools spanning N-24, or individual peptides, each at a concentration of 1.5 μM. Stimulation was performed in the presence of GolgiStop (BD, USA, Cat#554724).

Intracellular cytokine staining (ICS)

PBMCs were harvested post stimulations and stained with Zombie UV fixable viability kit to label live cells for 30 mins at room temperature. Cells were washed in PBS, followed by incubation with surface antibodies at 4°C for 30 mins, including anti-CD3-BV510, anti-CD8-FITC, and anti-CD4-AF700. After another round of wash, the cells were fixed using Fixation buffer (BioLegend, USA, Cat#420801) for 20 mins at 4°C, and then permeabilized using Intracellular Staining Perm Wash Buffer (BioLegend, USA, Cat#421002). Intracellular antibody cocktails were formulated in Perm buffer and cells were stained with these cocktails at 4°C for 30 mins, including anti-IL-2-BV650, anti-IL-10-BV421, anti-IL-4-PE, anti-TNF-α-PE-Dazzle594, and anti-IFN-γ-APC. Subsequently, the cells were washed and resuspended in PBS. PBMCs were acquired on an FACSCanto II LSRFortessa flow cytometer (Becton Dickinson, USA). Data analyses were done by using FlowJo X (FlowJo LLC, Ashland, OR). A well of cells stimulated in the absence of peptide was used as the negative control. All antibodies used in this study are listed in Table S1. Cytokines measurements were background subtracted prior to further analysis. The gating strategy for flow cytometry (FCM) analysis is delineated in Figure S2A.

Multi-sequence alignment

Sequences homology analyses were conducted using MEGA version 11, employing the ClustalW algorithm with default settings. The alignment results, exported in FASTA format from MEGA, were subsequently imported into ESPript 3.0 to generate sequence alignment diagrams and into Weblogo 3 to create sequence logos, as previously described [22]. Sequences of Omicron subvariants, including BA.2.75, XBB, BA.5, BF.7, BA.2.86, JN.1, KP.3, KP.3.1.1, XEC, LP.8.1, NB.1.8.1, and XFG were obtained from the COVID-19 database (RCoV19–2019 Novel Coronavirus Information Respository; available at: https://ngdc.cncb.ac.cn/ncov/). The N protein sequences of human β-coronaviruses SARS-CoV-2, 229E, OC43, SARS-CoV, HKU1, MERS-CoV, and NL63 can be found in the NCBI databases under the following accession numbers: YP_009724397.2, YP_073556.1, YP_009825–061.1, YP_009047211.1, YP_173242.1, YP_00955524–5.1, and YP_003771.1.

HLA blocking assays

Blocking assays using the HLA antibodies were assessed using the ICS, as previously described [23–26]. Specifically, PBMCs were co-culture with peptide in the presence of 2 μg/mL HLA antibodies, including anti-HLA-DP (clone B7/21: Abcam, USA, Cat#ab20897), anti-HLA-DQ (clone Tü169: BioLegend, USA, Cat#361502), and anti-HLA-DR (clone L243: Biolegend, USA, Cat#307602). Wells containing PBMCs alone or PBMCs with peptides served as controls.

Statistical analysis

Statistical analyses were conducted using GraphPad Prism 8 software. Data distribution was initially assessed via the Kolmogorov-Smirnov test. Subsequently, statistical significance was determined by nonparametric tests (matched-pairs Wilcoxon signed-rank test or Kruskal-Wallis one-way analysis of variance). To assess differences among the pie charts, permutations tests were conducted by SPICE 6 software. Values are presented as the median with interquartile range, with p ≤ 0.05 was considered statistically significant. Besides, P-value are depicted in the figures.

Result

Study and participants

Based on the established inclusion and exclusion criteria, a total of 93 volunteers were recruited for this study. Each participant had received three doses of inactivated SARS-CoV-2 vaccines derived from the original Wuhan-Hu-1 strain and had experienced at least once Omicron breakthrough infection between 7 December 2022 and 11 November 2023. During this period, all SARS-CoV-2 infections in China were caused by Omicron subvariants, with the exception of JN.1. Table 1 summarized the demographic and baseline characteristics of these volunteers. The average age of the participants was 43 years, and 49 individuals (52.69% of the cohort) were women.

Table 1.

Baseline table of the 93 participants.

Variables	Total (n = 93)
Age	43.00 (38.50, 51.00)
Sex, n%
Female	49 (52.69)
Male	44(47.31)
3-doses of previous SARS-CoV-2 vaccinations, n%
Yes	93 (100.00)
No	0 (0.00)
Breakthrough infection history, n%
Yes	93 (100.00)
No	0 (0.00)

Detection and comparison of T cell immunity to the N protein of Wuhan-Hu-1 and JN.1 subvariant

We synthesized a comprehensive set of 68 overlapping 18mer peptides spanning the entire length of the N protein for both the ancestral Wuhan-Hu-1 strain and the currently predominant Omicron subvariant JN.1. PBMCs were isolated and stimulated with the Wuhan-Hu-1 and JN.1 variants nucleocapsid peptide pool at a concentration of 1.5 μM, and N pool-specific CD4⁺ and CD8⁺ T cell responses were examined using ICS assays (Figure 1(A)). Additionally, a well of cells stimulated without peptide served as the negative control.

Figure 1.

Analysis of Wuhan-Hu-1 and JN.1 variant nucleocapsid-protein-specific T cell responses in individuals with omicron breakthrough infections post-three doses inactivated vaccination.

(A) Peripheral blood mononuclear cells (PBMCs) from individuals who experienced breakthrough infections with early Omicron subvariants following three doses of inactivated SARS-CoV-2 vaccination (n = 57), were stimulated with overlapping peptide pools for the full-length nucleocapsid of the original Wuhan-Hu-1 or JN.1 strains and analyzed by intracellular cytokine staining (ICS) assays. (B and E) Representative flow cytometry (FCM) plots showing IFN-γ-, TNF-α-, and IL-2-producing cells against the Wuhan-Hu-1 or JN.1 N pool among CD4⁺ (B) or CD8⁺ (E)T cells. (C and F) Comparison of the percentage of IFN-γ-, TNF-α-, and IL-2-producing CD4⁺ (C) or CD8⁺ (F)T cells upon the Wuhan-Hu-1 and JN.1 nucleocapsid peptide pool stimulation. (D and G) Pie graphs depict the proportion of CD4⁺ (C) or CD8⁺ (F) T cells exhibiting a specific number of functions among those with any functional activity. Permutation test = 10000 permutations. Data are shown as the median with interquartile range. P values were determined using a two-tailed Wilcoxon matched-pairs signed rank test. NS, indicates not significant.

The cytokines secretion profiles of T cells specific to the N protein pools derived from the ancestral Wuhan-Hu-1 strain and the JN.1 variant demonstrated that the frequency of IFN-γ-, TNF-α-, and IL-2-producing CD4⁺ T cells against the Omicron nucleocapsid was increased compared with that against the Wuhan-Hu-1 nucleocapsid (Figure 1(B,C)). Additionally, the production of IL-4 and IL-10 were also detected. Analysis of these five cytokines revealed that Omicron N-specific CD4⁺ T cells predominantly secreted IFN-γ, and TNF-α, with negligible levels of IL-4 and IL-10, indicating a Th1-skewed phenotype (Figure S2B). Similarly, for the CD8⁺ T cell responses, JN.1 N-specific T cells produced significantly higher levels of IFN-γ, TNF-α, and IL-2 compared to Wuhan-Hu-1 N-specific T cells (Figure 1(E,F)). Analysis further showed that JN.1-specific CD8⁺ T cells exhibited robust secretion of IFN-γ and TNF-α, with minimal secretion of IL-4 and IL-10, suggesting a Tc1-dominant profile (Figure S2C).

The polyfunctionality analysis focused on the cytokines IFN-γ, TNF-α, and IL-2 found that, for both CD4⁺ and CD8⁺ T cells, there were no statistically significant differences in the proportions of individuals secreting one, two or three cytokines in response to either Wuhan-Hu-1 or JN.1 specific stimulation (Figure 1(D,G)).

In summary, prior breakthrough infections with early Omicron subvariants have elicited robust memory CD4⁺ and CD8⁺ T cell responses against the N protein of the currently circulating JN.1 variant. These T cells are predominantly characterized by a Th1 and Tc1 phenotype with preserved polyfunctionality.

Identification of the immunodominant epitope profiled targeting the N protein of the prevalent JN.1 variant

In this study, we employed the three-dimensional peptide matrix previously described in our published work to distribute 68 peptides of the Omicron JN.1 variant’s N protein across 13 sub-peptide pools. These 13 sub-pools were used to stimulate PBMCs from 57 samples, and the proportions of IFN-γ-producing CD4⁺ and CD8⁺ T cells were assessed using ICS. By characterizing the most reactive peptide pools within the three-dimensional matrix, we identified the immunodominant peptide for each participant.

FCM plots from a presentative individual are presented in Figure 2(A). Compared with the negative control, a significant increase in the proportion of IFN-γ-producing CD4⁺ T cells was observed upon stimulation with the N pool of JN.1. Within the three dimensions, the sub-pools eliciting the strongest responses were identified as Pool 2 (1^st dimension, Pool 1–4), Pool 8 (2^nd, Pool 5–8), Pool 9 (3^rd, Pool 9–13). The only common peptide among Pool 2, Pool 8, and Pool 9 is N-24, support the conclusion that N-24 (N_139–156: LNTPKDHIGTRNPANNAA) is the dominant CD4⁺ T cell epitope for this individual. Moreover, stimulation with the N-24 single peptide elicited a significantly higher proportion of IFN-γ-producing CD4⁺ T cells compared to the negative control, thereby confirming N-24 as the immunodominant CD4⁺ T cell epitope for this individual.

Figure 2.

Screening and identification of CD4⁺ and CD8⁺ T cell epitopes for the nucleocapsid protein of the JN.1 variant using three-dimensional matrix.

(A) FCM plots showing CD4⁺ T cells IFN-γ responses from representative donors reactive with the N pool, each of the 13 sub-peptide pools, and a single N-24 peptide of the JN.1 nucleocapsid protein. (B) The dominant frequencies of screened CD4⁺ and CD8⁺ T cells epitopes are illustrated in the histogram (n = 57). Epitopes indicated by # symbol: mutated epitopes relative to Wuhan-Hu-1 strain.

By analyzing the frequency of dominant epitopes in T cell responses across 57 samples (Figure 2(B)), we compared the epitope profile to that previously characterized in vaccinated individuals and confirmed that considerable modifications had occurred. A multitude of epitopes harboring amino acid mutations were identified, including N-2, N-4, N-5, N-34, N-37, N-38, N-39, N-67, and N-68, several of which were newly identified in the current study. Notably, N-4 and N-66, previously identified as harboring dominant CD8⁺ and CD4⁺ T cell epitopes [17], respectively, no longer elicited predominant T cell responses among individuals with breakthrough infections. In contrast, N-24, which was identified as subdominant epitope, emerged as the predominant target for CD4⁺ T cells, with 20 out of 57 samples (35.09%) exhibiting a dominant response. Meanwhile, no epitope was identified as being significantly dominant in the CD8⁺ T cell response.

Evaluating the T cell response to vaccine-elicited immunodominant epitopes in individuals with omicron breakthrough infections

The immunodominant T cell epitopes among recipients of inactivated SARS-CoV-2 vaccines, as previously identified, display notable discrepancies when compared to those characterized in the current study among individuals with Omicron breakthrough infections, specifically targeting the JN.1 variant. To exclude the influence of SARS-CoV-2 mutations and to elucidate the current responsiveness of these previously dominant epitopes, we conducted single-peptide stimulation assays using 15 peptides that were previously identified as dominant epitopes with a dominant frequency greater than once for the Wuhan-Hu-1 strain. The peptides, including the previously predominant epitopes for CD4⁺ T cells (N-66) and CD8⁺ T cells (N-4), as well as the current immunodominant epitope for CD4⁺ T cells (N-24), were tested across 57 participants.

As shown in the representative FCM plots of Figure 3(A), compared to the negative control, stimulation with the N-pool of Wuhan-Hu-1 elicited a significantly elevated CD4⁺ T cells response. Among the 15 single-peptide stimulations, only N-24 induced a robust response of IFN-γ-producing CD4⁺ T cells. Upon evaluating the dominant frequency of these 15 previously identified T cell epitopes (Figure 3(B)), our results consistently highlight epitope N-24 as eliciting a significantly predominant CD4⁺ T cell response. Additionally, no epitope emerged as a significantly dominant target within the CD8⁺ T cell response profile.

Figure 3.

Validation of previously immunodominant epitopes induced by inactivated vaccines via single-peptide stimulation assays and amino acid sequence comparison.

(A) FCM plots showing IFN-γ responses of CD4⁺ T cells from representative donors, reactive with each of the 15 peptides previously identified as dominant epitopes with a frequency of dominant greater than once for the Wuhan-Hu-1 strain. (B) The histogram depicts the dominant frequencies of the screened CD4⁺ and CD8⁺ T cells epitopes (n = 57). Epitopes indicated by * symbol: previously immunodominant epitopes for CD4⁺ and CD8⁺ T cells induced by SARS-CoV-2 inactivated vaccine. (C) Comparison of the sequence of N-4 (i), N-24 (ii), and N-66 (iii) among Wuhan-Hu-1 and six Omicron subvariants, including BA.2.75, XBB, BA.5, BF.7, BA.2.86, and JN.1.

The amino acid sequence homology of epitopes N-24, N-4, and N-66 was evaluated across the ancestral Wuhan-Hu-1 strain and six Omicron subvariants, including BA.2.75, XBB, BA.5, BF.7, BA.2.86, JN.1. Notably, the former five subvariants were the predominant Omicron strains circulating in China prior to the emergence of JN.1 [27–29]. The analysis revealed that epitope N-4 exhibited a deletion mutation involving three amino acids (Figure 3(C-i)), while epitopes N-24 and N-66 were highly conserved ((Figure 3(C-ii,iii)). Further comparison of the amino acid sequences of epitopes N-4, N-24, and N-66 across SARS-CoV-2 and six other β-coronaviruses revealed that while epitope N-66 exhibited a greater number of deletion mutations, epitope N-24 displayed a higher degree of sequence conservation (Figure S3A-3SC). Specifically, sequence similarity was 11.11% for N-66 and 33.33% for N-24. Additionally, we compared the amino acid conservation of N-24 among JN.1 and the Variants Under Monitoring (VUMs), which are all sublineages of JN.1, including KP.3, KP.3.1.1, XEC, LP.8.1, NB.1.8.1, and XFG. The results showed that N-24 remains fully conserved across all these VUMs (Figure S3D).

To preliminarily assess the potential protective potential of the N-24 epitope, we profiled the cytokine signature of N-24-specific CD4⁺ T cells, quantifying IFN-γ, TNF-α, IL-2, IL-4, and IL-10. The results showed that these cells predominantly produced IFN-γ, TNF-α, and IL-2, indicating a Th1-biased phenotype (Figure S3E).

HLA restriction profiling and minimal epitope definition of N-24

To elucidate the HLA restriction of the N-24 epitope, we conducted HLA class II antibody-blocking experiments. As depicted in Figure 4(A), in three individuals with a dominant response to the N-24 epitope, the CD4⁺ T cell response to N-24 peptide stimulation was highly pronounced. Co-stimulation of PBMCs from these individuals with N-24 peptide and antibodies specific to HLA-DP, -DQ, and -DR revealed that the N-24 stimulation signal was significantly attenuated only in the presence of HLA-DR antibodies. These findings indicate that the HLA restriction of N-24 epitope is HLA-DR.

Figure 4.

Identification of the minimal epitope and HLA restriction for N-24.

(A) FCM plots from 3 representative donors illustrating IFN-γ responses against the N-24 epitope under co-culture conditions with anti-HLA-DP, -HLA-DQ, or -HLA-DR antibodies. (B) FCM plots depicting IFN-γ responses of CD4⁺ T cells from representative donors, stimulated with the N-24 epitope, pooled 14mer peptides, or five individual 14mer peptides. (C) A histogram depicts the immunodominant frequencies of the five N-24-derived 14mer peptides across the cohort (n = 26).

To delineate the epitope within the CD4⁺ T cell immunodominant epitope N-24, we first synthesized five overlapping 13mer peptides that spanned the entire length of N-24. PBMCs from samples were stimulated with either the pooled 13mer peptides or individual 13mer peptides. Cells without peptide stimulation served as negative controls, cells stimulated with N-24 was set as the positive control (Figure S4A). A total of 18 samples were tested, and the results from 7 samples that showed a significant increase in IFN-γ-producing CD4⁺ T cells in the positive control group were analyzed. The findings revealed that, in the presence of a robust CD4⁺ T cell response to N-24 peptide stimulation, neither the pooled 13mer peptides nor the five 13mer peptides elicited a significant response (Figure S4B). These results suggest that the minimal epitope length within N-24 is greater than 13 amino acids. Accordingly, we expanded the truncation series to a 14mer that covers the entire length of N-24. In addition to the original 57 samples, we incorporated an additional 36 eligible samples for the screening of the 14mer epitope. By testing 26 donors whose CD4⁺ T cell response was dominated by N-24, we found that 14mer-4 (PKDHIGTRNPANNA) exhibited the highest dominant response frequency (11/26), thereby identifying the minimal epitope of N-24 as PKDHIGTRNPANNA (Figure 4(B)).

Discussion

In this study, we characterized the N protein-specific T cell responses in individuals who experienced breakthrough infections with early Omicron subvariants following inactivated SARS-CoV-2 vaccination. We observed robust Th1/Tc1-skewed cellular immunity against the JN.1 strain and identified N-24 as a highly conserved and immunodominant CD4⁺ T cell epitope, restricted by HLA-DR. Our findings demonstrate that breakthrough infections with early Omicron variants elicit substantial T cell responses against the prevailing mutant strains JN.1, which may be crucial for preventing severe COVID-19. Moreover, these breakthrough infections significantly modulate the immune responses initially elicited by inactivated vaccines, as evidenced by marked alterations in the immunodominant epitope profile. The newly identified N-protein dominant epitopes N-24 could serve as potential candidates for vaccines targeting the evolving SARS-CoV-2 variants.

In our preceding study, we explored the structural protein (E, M, N, and S)-specific T cell responses among healthy adults who had received the inactivated SARS-CoV-2 vaccine derived from the Wuhan-Hu-1 strain. Our analyses revealed that the highly conserved N-protein-specific T cell responses were comparable to those elicited by the more mutation-prone S protein. Furthermore, we identified immunodominant epitopes based on the N protein antigen from Wuhan-Hu-1. However, vaccination did not fully prevent SARS-CoV-2 infection, with breakthrough infections occurring frequently.

The JN.1 variant has emerged as one of the most immune-evasive variants and remains the predominant circulating strain [30]. Additionally, all currently monitored Variants Under Monitoring (VUMs)-including KP.3, KP.3.1.1, XEC, LP.8.1, NB.1.8.1, and XFG-are sublineages of JN.1. The L455S mutation in the receptor-binding domain of the spike protein of JN.1 reduces the binding affinity between the host ACE2 receptor and the viral RBD, thereby enhancing its infectivity and immune evasion capacity [31]. Additionally, the F456L and Q493E mutations within the RBD further augment its ability to escape neutralizing antibodies [32]. However, due to the diverse T cell epitopes in viral proteins dictated by individual HLA allotypes, it is more challenging for novel variants to completely escape T cell recognition [33].

In light of these findings, we aimed to determine whether individuals with prior breakthrough infections maintain a robust T cell response specific to the N protein of the JN.1 variant. Regarding participant selection, pre-infection biospecimens from our earlier cohort (reference 17) had been exhausted, and the nationwide relaxation of containment measures in our country following December 2022 made the recruitment of infection-naïve controls virtually impossible. Consequently, recruitment of a pre-breakthrough population proved infeasible, which precluded a direct, longitudinal assessment of T-cell responses before and after breakthrough infection. We therefore quantified JN.1- and Wuhan-Hu-1-specific T cell responses in the same breakthrough-infected individuals. Although this cross-sectional design is not the most direct method of proof, it remains the most feasible approach under the prevailing conditions and provides valuable indirect evidence for the boosting efficacy of breakthrough infection. Employing PBMCs from individuals who had experienced early Omicron subvariant breakthrough infections, we found that the proportion of IFN-γ-, TNF-α-, and IL-2-producing CD4⁺ and CD8⁺ T cells specific to the N protein of JN.1 was significantly elevated compared to those responding to the ancestral Wuhan-Hu-1 strain. Besides, the JN.1 N protein-specific T cells are distinguished by a Th1/Tc1 phenotype and preserved polyfunctionality. These findings suggest that Omicron breakthrough infections following inactivated vaccine administration can elicit memory T cells capable of recognizing the circulating JN.1 variant, which may contribute to protection against severe disease progression.

Elucidating the immunodominance landscape can provide valuable insight for the development of vaccine to induce more comprehensive and protective T cell responses against SARS-CoV-2. Utilizing a three-dimensional matrix model, we previously characterized the T cell immunodominant epitope profiles targeting the N antigen among vaccinated individuals, identified N-66 as the dominant epitope for CD4⁺ T cells and N-4 as the dominant epitope for CD8⁺ T cells [17]. In the present study, employing the same three-dimensional matrix screening model, we identified the immunodominant epitope profiles targeting the N antigen among individuals with Omicron breakthrough infections, thereby allowing for a comparative investigation of the impact of these breakthrough infections on the immunodominant landscape in the population. Our results revealed substantial alterations in the epitope profile, with the emergence of several novel immunodominant epitopes, some of which have undergone mutations within the variants. Notably, N-4 and N-66, previously identified as immunodominant, no longer elicited dominant T cell responses. Instead, the previously identified subdominant epitope N-24 emerged as the predominant epitope for CD4⁺ T cells, while no epitope achieved a dominant response among CD8⁺ T cells. These findings underscore the dynamic nature of T cell responses in the context of evolving viral variants and highlight the need for adaptive vaccine strategies.

In order to elucidate the current immunodominance status of previously identified epitopes and to exclude potential confounding effects introduced by SARS-CoV-2 mutations within the three-dimensional matrix screening, single-peptide stimulations were performed using the previously characterized dominant epitopes. These experiments reconfirmed that N-24 is the currently predominant epitope, whereas epitopes N-4 and N-66 no longer exhibit immunodominant characteristics. Collectively, these findings underscore the substantial influence of breakthrough infections on the immunodominant epitope profile targeting the N antigen among individuals vaccinated with inactivated vaccines. Besides, amino acid sequence comparisons have confirmed that the N-24 epitope of the N protein is highly conserved across all current VUMs. This finding further underscores the potential application value of N-24. Furthermore, our investigation has demonstrated that the N-24 epitope effectively primes CD4⁺ T cells to secrete high levels of antiviral cytokine IFN-γ and adopt a Th1-biased phenotype, a clear indicator of protective potential. However, rigorous experiments that directly quantify its contribution to pathogen control and clinical benefit are essential before any translational application to vaccine design can be contemplated.

Tomas et al. reported that the deletion or inactivation of immunodominant epitopes, such as the removal of the RGPGRAFVTI epitope in HIV vaccines, can restore responsiveness to subdominant epitopes, suggesting the presence of competitive inhibition among epitopes [21]. A comparative sequence analysis of epitopes N-4, N-24 and N-66 between the ancestral Wuhan-Hu-1 strain and various Omicron subvariants (the variants that were widely circulating in China prior to the emergence of JN.1) revealed a consistent deletion of three amino acid in epitope N-4 across all Omicron subvariants. Further homology analysis of these epitopes across seven human β-coronaviruses revealed that N-24 is more conserved than N-66, with a similarity of 33.33% compared to 11.11%. This higher degree of conservation may contribute to the preferential recall of N-24 after repeated exposure to other coronaviruses. These findings suggest that mutations may have led to the loss of immunodominance for epitopes N-4 and N-66, while reactivating the responsiveness of the previously subdominant epitope N-24. Nevertheless, the phenomenon of immunodominance is also modulated by a complex interplay of additional factors, including the binding affinity of HLA molecules, the efficiency of antigen processing, and the repertoire of T cell receptors (TCRs) recognizing specific HLA-epitope combination [34]. The mechanisms underlying the observed changes in immunodominant epitopes remain to be fully elucidated and warrant further investigation.

Major histocompatibility complex (MHC) Class II molecules, including HLA-DR, HLA-DQ, and HLA-DP, bind antigenic peptides derived from the proteolysis of self and non-self-proteins in endosomes and lysosomes, and present them to antigen-specific CD4⁺ T cells [35,36]. Through antibody-blocking assays, we have confirmed that the N-24 epitope is presented by HLA-DR molecules to elicit an immune response. However, a notable limitation of our study is the absence of corroborative evidence from additional antigen presentation experiments or sequencing analyses.

Identifying shorter epitopes that retain the capacity to induce T cell responses-referred to as minimal optimal epitopes-is essential for precisely defining effective antigenic targets. This approach can reduce the necessity for synthesizing unnecessary peptides, thus facilitating more efficient vaccine design [37]. To identify the minimal optimal peptides within the 18mer N-24 peptide, we generated five overlapping 13mer peptide and five overlapping 14mer peptide spanning the entire N-24 sequence. Our finding revealed that in individuals with substantial N-24-specfic T cell responses, neither individual nor pooled 13mer peptides elicited a significant T cell response. This implies that the minimal epitope length within N-24 is probably greater than 13 amino acids. In contrast, the 14mer peptide pool elicited pronounced T-cell reactivity, with 14mer-4 (PKDHIGTRNPANNA) recognized most frequently (11 of 26). Collectively, these data establish PKDHIGTRNPANNA as the minimal epitope contained within N-24. Although HLA-DR grooves accept peptides of 9–30 amino acids, our results indicate that the functional epitope within N-24 is 14 residues long. We speculate that compared with the 13mer peptide, the 14mer peptide may achieve both stable anchoring and extended TCR engagement, thereby accounting for its superior immunogenicity. While the present study concentrates on T cell immunity, we recognize that a parallel investigation into neutralizing antibody landscapes and their cross-reactivity with JN.1 would provide valuable complementary data. Future studies should consider incorporating both humoral and cellular immune responses to offer a more comprehensive understanding of the immune mechanisms involved.

Conclusion

In summary, our study identifies a population of memory CD4⁺ and CD8⁺ T cells within individuals who experienced breakthrough infections with early Omicron variants, which can effectively respond to the circulating JN.1 strain. Moreover, these breakthrough infections have a profound impact on the immunodominant epitope profile targeting the N protein of SARS-CoV-2, with the highly conserved N-24 emerging as the predominant epitope for CD4⁺ T cell responses.

Funding Statement

This project was supported by the Postdoctoral Fellowship Program of CPSF under Grant Number [GZC20233232], and the Shenzhen Science and Technology Innovation Program [JCYJ20210324115204012].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The dataset generated in this study is publicly accessible in the Harvard Dataverse repository under the title “Immunodominant T cell responses to SARS-CoV-2 nucleocapsid protein in Omicron breakthrough infection post-inactivated vaccination” at https://doi.org/10.7910/DVN/GTXICD [38].

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2025.2566241