---
title: "Conformational bias shapes SARS-CoV-2 Spike CD4+ T‑cell epitope dominance"
id: "frontiers-in-immunology-0-conformational-bias-in-sars-cov-2-spike-cd4-t-cell-epitope-dominance"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-0-conformational-bias-in-sars-cov-2-spike-cd4-t-cell-epitope-dominance"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1857103"
published_at: "2026-07-20T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Conformational bias shapes SARS-CoV-2 Spike CD4+ T‑cell epitope dominance
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/frontiers-in-immunology-0-conformational-bias-in-sars-cov-2-spike-cd4-t-cell-epitope-dominance
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1857103)
- **Published At:** 2026-07-20T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- The study examines how **spike** protein conformation influences CD4+ T‑cell epitope dominance via class II antigen processing and proteolysis. - Antigen processing likelihood (APL) models were compared across multiple pre‑ and post‑fusion **spike** conformations and to empiric epitope maps from the IEDB; APL predicted some epitopes but had modest overall accuracy for SARS‑CoV‑2 S1 and S2. - Limited proteolysis experiments on soluble recombinant spike ectodomains (stabilized by proline substitutions: spike‑2P and spike‑6P) identified cleavage‑sensitive sites that co-localize with conformationally unstable segments and domain boundaries. - Protease-sensitive regions included the NTD–RBD linker (N2R), subdomain 1 (SD1), the S1/S2 processing site, and regions flanking the upstream helix and fusion peptide in S2; some cleavage site usage differed between spike‑2P and spike‑6P. - The APL method was reasonably accurate for OC43 pre‑fusion S2 but not for SARS‑CoV‑2 pre‑fusion S2; accuracy improved modestly using a post‑fusion S2 conformation. - Mapping of CD4+ epitope dominance from IEDB showed that peptides from conformationally unstable, solvent‑exposed, protease‑sensitive regions more often prime responses after infection than after vaccination. - The ratio of CD4+ T‑cell responses to peptide pools derived from stable versus unstable spike regions distinguished infection versus vaccination in two non‑hospitalized cohorts. - Authors propose that differences in exposure circumstances (infection vs mRNA vaccination) alter antigen‑presenting cell types, activation states, endosomal processing, and spike conformational dynamics, thereby shaping spike fragmentation, peptide loading, and CD4+ epitope dominance. - The work links structural proteolytic sensitivity to epitope dominance and suggests that epitope dominance patterns may reflect exposure history, immune imprinting, and potential protective quality of CD4+ responses.
## Clinical Analysis & Structured Key Points
About us All journals All articles Submit your research Search Login Frontiers in Immunology Sections Articles Research Topics Editorial board About journal Published in Frontiers in Immunology Viral Immunology 7 impact factor 11.3 citescore Editor & Reviewers Edited by Kuan Rong Chan Reviewed by Miguel Alvaro-Benito Mengze Gan Outline Abstract Introduction Results Discussion Methods Data availability statement Ethics statement Author contributions Funding Acknowledgments Conflict of interest Generative AI statement Publisher’s note Supplementary material References Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Figure 4 View in article Figure 5 View in article Figure 6 View in article Figure 7 View in article ORIGINAL RESEARCH article Front. Immunol., 20 July 2026 Sec. Viral Immunology Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1857103 Conformational bias in SARS-CoV-2 Spike CD4+ T-cell epitope dominance Samuel J. Landry 1* N K N. Kalaya Steede 1 Yali Tiomkin 1 H S Haley Smith 1 Ramgopal R. Mettu 2 Loren Gragert 3 Judith H. Aberle 4 Kevin J. Zwezdaryk 5 C Z Crystal Zheng 6 Jay K. Kolls 7 Bronwyn M. Gunn 8 A E Amelie E. Murrell 5 I V Ivy V. Trinh 5 John S. Schieffelin 7 James E. Robinson 7† +7 more Elizabeth B. Norton 5† 1. Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA, United States 2. Department of Computer Science, Tulane University, New Orleans, Louisiana, United States, Tulane University School of Medicine, New Orleans, LA, United States See more Article metrics View details 149 Views Abstract Introduction: Epitope-specific T cells provide significant long-lived protection afforded by adaptive immunity to SARS-CoV-2 spike. CD4+ T-cell epitope peptides that are generated by non-ATP-dependent antigen-processing proteases bind with modest specificity to MHC class II molecules in the endo-lysosome. Studies document the influence of antigen-presenting cell type, manner of endocytosis, and antigen conformation on the strength of CD4+ T-cell response. Nevertheless, few studies report changes in epitope dominance due to circumstances of antigen exposure, which could shape proteolytic antigen processing in the class-II pathway because conformational domains limit proteolysis or MHCII binding. Methods: Processing of SARS-CoV-2 spike was modeled using limited proteolysis of soluble spike trimer, and the effect of spike conformation on CD4+ T-cell epitope dominance was analyzed using IL-2 Elispots responding to two nine-peptide pools from conformationally stable and unstable regions of spike. Results: Protease-sensitive sites coincided with domain boundaries and other conformationally unstable regions, confirming that structure limits proteolysis. The ratio of CD4+ T-cell response to stable and unstable peptide pools in two non-hospitalized human subjects cohorts distinguished whether exposure was by infection or vaccination. Discussion: Circumstances of exposure to spike, e.g., spike mRNA vaccination or SARS-CoV-2 infection, could influence populations of antigen presenting cells and their levels of activation, resulting in different patterns of spike fragmentation, peptide loading, and T-cell response. Circumstances of exposure also affect spike conformational changes that contribute to distinct dominance patterns. Thus, epitope dominance patterns potentially indicate exposure history, immune imprinting, and potentially the protectiveness of the CD4+ T-cell response. Introduction Seeking to mitigate the toll of COVID-19 disease caused by SARS-CoV-2, researchers continue to examine immune responses to this novel pathogen. As a major arm of the adaptive immune response, CD4+ T cells have attracted substantial attention for their potential in both positive and negative roles (1). CD4+ T cells generally promote antibody class-switch recombination and antibody affinity maturation in germinal centers, which are prominent in upper airway during COVID-19 infection (2). Apart from supporting antibody, CD4+ T cells promote CD8+ T-cell development, attract phagocytic cells, activate innate immune cell defenses, and deliver regulatory signals that mitigate immunopathology, all of which are thought to be relevant to recovery from COVID-19. Most of these roles have the support of direct evidence in animal models, as well as correlative studies in humans (1, 3, 4). Measuring the expansion of SARS-CoV-2-specific CD4+ T cells is challenged by heterogeneous cellular phenotypes and disparate tissue homing that depend on history and genetics of the host (1, 5). Weak or inhibited triggering of type-I interferon may be an important determinant of disease severity, but immune cell networks that link signaling pathways to tissue damage remain poorly defined (6, 7). Vaccination with the viral spike protein has provided a rapid and strikingly effective protection against severe disease, even though neutralizing antibody faded quickly, potentially due to the lack of long-lived plasma cells in the bone marrow (8). More durable protection may continue to be provided by CD4+ and CD8+ T cells (9–17). Animal models have provided crucial information, but the diversity of human immune systems and the variable history of exposure to viruses, vaccines, and other cross-reactive entities potentially hinders the development of T-cell-based vaccination and therapy (18–20). CD4+ T-cell epitope dominance refers to the strong or frequent response to a minor fraction of possible epitopes after priming with intact antigen. Peptides generally of 12–20 residues are displayed in class II MHC proteins (MHCII) for recognition by alpha/beta receptors on T cells that also express the CD4 molecule, which binds to a distinct domain of the MHCII (21). Antigen-specific T-cell response is quantified by the frequency of specific T cells, where specificity is established by restimulation of the cells with synthetic peptides that mimic the naturally processed peptides. In a screen for multiple CD4+ T-cell epitopes (hereafter, “CD4+ epitopes”), readout is typically by formation of Elispots by cells that express inflammatory cytokines (e.g., IFNγ or IL-2), or by flow cytometry of cells that express inflammatory cytokines or activation-induced markers (e.g., CD137 and OX40), or by quantitative TCR cloning or sequencing (22). A naturally processed CD4+ epitope typically occurs as a family of overlapping peptides that share a determinant core sequence of 8–9 residues (23). Much of the core is enclosed in the MHCII and is held there with H-bonds to the peptide backbone, and 3–4 peptide sidechains make favorable contacts with shallow pockets in the MHCII groove (24). The T-cell receptor (TCR) binds to the MHCII-peptide complex, typically making contact with 3–4 outward-facing sidechains of the peptide (25). Dominant CD4+ epitopes frequently occur in clusters of overlapping and neighboring sequences that are presented by different allotypes of MHCII or presented by the same MHCII using different binding registers (26–28). For a number of antigens, including HIV envelope glycoprotein and influenza hemagglutinin, this clustering of dominant CD4+ epitopes has been attributed to preferential pathways of proteolytic antigen processing (29–33). These observations led to a mechanistic model and bioinformatic approach to CD4+ epitope prediction based on antigen processing likelihood (APL), wherein the conformationally unstable antigen segments are preferentially cleaved, and then adjacent sequences are bound by MHCII proteins (34, 35). The complex structure and conformational behavior of the SARS-CoV-2 spike glycoprotein has the potential to modulate its class-II-pathway proteolytic processing and CD4+ epitope dominance. The trimeric SARS-CoV-2 spike glycoprotein is characterized by multiple structural domains connected by domain linkers that articulate conformational states. As a type 1 fusion protein, low pH triggers exposure of a fusion peptide that mediates transfer of the viral genome into the cell cytoplasm (36). For SARS-CoV-2 spike, two proteolytic steps are required. Cleavage of spike (S) into S1 and S2 chains primes the S2 spring-loaded mechanism, and then cleavage at the S2’ site exposes the adjacent fusion peptide (37). Cleavage at the S2’ site is blocked until the receptor-binding domains rotate up to engage the ACE2 receptor on target cells, which acts as a conformational signal mediated through S1 subdomains 1 and 2 (38). These conformational features could influence spike CD4+ epitope dominance. CD4+ epitope dominance in the influenza A hemagglutinin HA1 domain has been attributed to peptide abundance shaped by antigen processing, and the conformational change in HA2 was shown to affect the ability of the protein to restimulate specific T-cells (33). The rationale for the present study was that COVID-19 severity could be related to CD4+ epitope immunodominance. Severe disease could result from viral pathogenesis in the absence of protective CD4+ T-cell responses or from immunopathology caused by excess or misdirected CD4+ T cell responses. In both scenarios, the immediate cause of differential priming or recall of CD4+ epitope-specific T cells could be variations in the circumstances of antigen exposure, such as antigen dose (39, 40), platform (41), adjuvant (42, 43), age (44), and preexisting cross-reactive immunity (45, 46). The ways in which these circumstances could cause differential CD4+ epitope presentation include variations in antigen conformation, antigen-presenting cell type, and degree of endosomal acidification (47–49). In addition, human leukocyte antigen (HLA) genetics and expression inevitably have interactions with these variations. While a number of studies have reported HLA-allele associations with protection or susceptibility to COVID-19, few have been confirmed in the largest GWAS efforts. Most notably DQB1*06 was associated with vaccine-induced antibody against SARS-CoV-2 spike and reduced breakthrough infection (50). A recent large GWAS study resolved DQB1*06:04, DQA1*01:02, DQA1*01:01, DRB3*01:01, and DPB1*10:01 and found additive effects on antibody and protection (51). These results highlight the CD4+ T-cell helper function to B cells and raise the questions whether HLA polymorphism exerts its effects through CD4+ epitope peptide selectivity and whether some CD4+ epitopes are more protective than others. Nevertheless, studies linking CD4+ epitopes to immunological outcomes often find the CD4+ epitopes to be promiscuously presented by multiple HLA allotypes, a feature that seems to be common to many CD4+ epitopes in SARS-CoV-2 spike (52–55). The role of natural processing of dominant CD4+ epitopes comes to the fore when unravelling mechanisms of immune imprinting and, especially, of “hybrid immune damping” that arises from certain pairings of spike proteins from different viral strains during infection and vaccination (14). Following early reports of immunodominant CD4+ epitopes, we noted that some of the SARS-CoV-2 spike CD4+ epitopes were located in segments characterized by conformational flexibility, which is unusual for dominant CD4+ epitopes. To probe CD4+ epitope dominance, we designed spike peptide pools that in an IL-2 Elispot assay would discriminate circumstances of priming on the basis of APL score. In the second year of the pandemic, many people became vaccinated, some in their first exposure to SARS-CoV-2 spike and others having previously been infected by the virus. Differences in response to selected CD4+ epitopes following infection versus vaccination have previously been noted (18, 46). Here we highlight broad shifts in CD4+ epitope immunodominance in the course of multiple spike exposures and provide evidence that these shifts are based on antigen processing. We also find a similar shift in immunodominance between groups of moderate and mild disease in an unvaccinated, non-hospitalized cohort of infected individuals. Results Antigen processing likelihood In order to examine a potential relationship of SARS-CoV-2 spike structure to CD4+ T-cell immunodominance, APL-based CD4+ epitope-prediction profiles for multiple conformations of SARS-CoV-2 spike were compared to CD4+ epitope dominance as reported in the 2023 IEDB database (56). These immunodominant peptides obtained a response from 49-80% of previously infected subjects. The S1 and S2 chains were considered separately because the intact spike polypeptide is cleaved at the S1-S2 junction after shedding or during infection, or cleaved near the S1-S2 junction at an early stage of antigen processing following vaccination (Ref (57) and see below). Multiple S1 and S2 conformations were evaluated for APL, including the following: S1 in the all-RBD-down (PDB:6VXX) and 3-RBD-up (PDB:7DCC) conformations, and S2 in the pre-fusion (PDB:6VXX) or post-fusion (model based on PDB:6B3O) conformations. For S1, APL using any one structure predicted a subset of CD4+ epitopes but did not achieve a significant level of accuracy overall. Notably, scoring of the same overlapping-peptide set by MHCII binding (7-allele method) or by assignment according to observation in immunopeptidomics also did not yield significant accuracy (58, 59) (data not shown). The result with APL is unexpected in view of good accuracy for APL with HIV Env and Influenza HA (60). APL was reasonably accurate with the pre-fusion spike S2 of common cold coronavirus OC43 (and more accurate when combined with MHCII binding), but it was not accurate using the pre-fusion structure of S2 from SARS-CoV-2 (Supplementary Figure 1). APL was modestly accurate using the post-fusion conformation of S2 (Supplementary Figure 2). Limited proteolysis The modest performance of APL raised the possibility that proteolytic processing of spike was inadequately modeled using the available structures. Limited proteolysis was analyzed for soluble recombinant SARS-CoV-2 spike ectodomains containing proline residues that inhibit conversion to the fusogenic conformation and enhance protein production in cell culture (61, 62). Proteolytic enzymes were selected on the basis of association with class-II-pathway antigen processing (cathepsin S (63)) or of having weak sequence specificity (proteinase K). Large fragments, presumably generated from protease-sensitive sites in the intact spike or in early proteolytic fragments, were extracted from SDS-PAGE gels and subjected to exhaustive tryptic digestion and identification by mass spectrometry. Cleavage sites were mapped with precision limited by the frequency and size-range of non-glycosylated tryptic peptides contained within fragments, which generally place the sites 0–16 residues beyond the terminal tryptic peptide (Supplementary Tables 1, S2). Several PK- and CS-cleavage sites in spike-2P were mapped to the nearest residue by searching peptide masses with the “no enzyme” setting. The soluble recombinant spike polypeptide expressed as a trimer in mammalian cells is composed of 1274 amino acid residues (including tags) but migrates in SDS-PAGE as approximately 200 kDa due to extensive modification with glycans (64). In addition to proline substitutions (2P or 6P) that stabilize the pre-fusion conformation in S2, residues at the S1-S2 junction have been substituted in order to reduce cleavage by trypsin-like protease activities (61, 62). In spike-6P, the D614G substitution enhances the RBD-up conformation, most likely increasing its sensitivity to cleavage at the S1/S2 site (65). Both cathepsin S (CS) and proteinase K (PK) generated proteolytic fragments of spike-2P or spike-6P that were similar to S1 and S2 fragments (Figures 1, 2; Supplementary Figures S3, S4, Supplementary Table 2). Additional CS- and PK-cleavage sites within S1 and S2 were also evident (Supplementary Table 1). In S1 of spike-2P, large fragments were generated by cleavage at several sites in the NTD-to-RBD (N2R) linker and in subdomain 1 (SD1). Both of these protease-sensitive regions occur within conformationally unstable segments, according to the APL-based analysis of the structure (Figures 1A, C). In S1 of spike-6P, fragments indicated cleavage at similar sites in the N2R and SD1, but also at an unstable region of the RBD (Supplementary Figures 3C, D). In S2 of spike-2P, several large CS and PK fragments resulted from cleavage on the N-terminal flank of the upstream helix and at sites C-terminal to the beta hairpin (66) (Figures 2A, C). In S2 of spike-6P, major fragments extended from CS and PK cleavage sites on the C-terminal flanks of the upstream helix and fusion peptide, respectively, to PK sites in the central helix and subdomain 3 (Supplementary Figures 4 C, D). Thus, limited proteolysis detects several cleavage-sensitive regions that are common to spike-2P and spike-6P, including the regions of the S1/S2 processing site, N2R, and SD1 and other cleavage sites in spike-6P that were not observed or were shifted in position, compared to in spike-2P. All of the protease-sensitive sites were located in conformationally unstable segments of spike, and utilization of some sites was modulated by proline substitutions that affect spike conformation. Having confirmed that the APL analysis predicts regions of spike proteolytic sensitivity, we addressed the possibility that proteolytic antigen processing varies by individual subject and circumstances of exposure. Figure 1 Within the SARS-CoV-2 spike S1 fragment, conformationally unstable, solvent-exposed, protease-sensitive regions more frequently prime CD4+ epitopes during infection than during vaccination. (A) APL (black curve) and Aggregate Stability (green curve) for S1 by peptide. The broken horizontal line indicates the average APL for all spike peptides (0.29). Cleavage sites for cathepsin S (CS), proteinase K (PK), and cathepsin L (CL) are indicated. (B) Ribbon diagram of the intact spike prefusion trimer (PDB: 6VXX), indicating the dominant epitope-containing peptides and flanking protease cleavage sites. (C) Diagram of major proteolytic fragments generated by limited proteolysis of spike-2P with CS or PK. (D) Profiles of CD4+ epitope dominance generated with mapping data from SARS-CoV-2 infection or vaccination reported in the Immune Epitope Database (IEDB) and normalized by the all-epitope average frequency for the exposure type. Peptides with above- and below-average APL are identified as belonging to the stable and unstable categories (red and blue symbols, respectively). (E) Coomassie-stained bands generated by limited proteolysis of recombinant spike-2P with cathepsin S (CS: 1.8, 0.9, or 0.45 µg) or proteinase K (PK: 1.5, 0.06, or 0.03 µg), separated by SDS-PAGE, and identified by tryptic digestion and mass spectrometry. Banding patterns are typical of at least three experiments. The 9U peptides in S1 occur in unstable regions close to protease-sensitive sites (ex., peptides 64-65 at the C-terminus of the RBD); whereas the 9S peptides occur within the NTD and RBD domains (ex., peptides 50-51). IEDB data indicate a dominant response to peptides 64-65 after infection, and a dominant response to peptides 50-51 after vaccination. CL sites were reported by Zhao et al. (Ref. 91). Figure 2 Within the spike S2 fragment, conformationally unstable, solvent-exposed, protease-sensitive regions more frequently prime CD4+ epitopes during infection than during vaccination. (A) APL and Aggregate Stability for S2 annotated as in Figure 1A. (B) Ribbon diagram of the S2 portion of the intact prefusion trimer (PDB: 6VXX). (C) Diagram of major proteolytic fragments generated by limited proteolysis of spike-2P with cathepsin S (CS) or proteinase K (PK). Structural domains are as follows (Ref. 66): upstream helix (UH), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), beta-hairpin
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