Few studies have tracked human CD4+ T cell clones through repeated infections. We used longitudinal single-cell RNA and T cell receptor (TCR) tracking to study the functional stability and memory potential of CD4+ T cell clonotypes during repeated Plasmodium falciparum (Pf) infections in Ugandan children and adults. Nearly all clonotypes displayed a strong preference for one of seven CD4+ subsets. This phenomenon of "clonal fidelity" was influenced by clonal expansion, linking T cell polarization and proliferation in vivo. Using clone tracking, we characterized subset-specific activation trajectories and identified antigen-specific clones. Type 1 regulatory T (TR1) cells accounted for nearly 90% of Pf-specific CD4+ T cells in blood. Tracking these clones longitudinally for hundreds of days, we observed malaria-induced expansion of TR1 effectors, long-term persistence of TR1 memory cells, and high-fidelity recall responses after reinfection. This work establishes clonal fidelity as a natural phenomenon and demonstrates the stable, long-term memory potential of TR1 cells.

Infection with SARS-CoV-2 results in mild to severe COVID-19 disease courses. Several studies showed the association of impaired T-cell responses and certain HLA haplotypes with disease severity. However, it remained unclear if T-cell activation was compromised due to a general reduction of presented epitopes or other intrinsic factors within APCs or T cells. Furthermore, a potential reduction of presented epitopes would suggest if an upcoming SARS-CoV-2 variant could escape T-cell immunity. Hence, knowledge about the T-cell epitope landscape of SARS-CoV-2 would allow to better understand mechanisms leading to severe disease and to estimate the potential stability of the T-cell response in light of virus evolution, which might provide insights for future vaccine designs. Hence, in the present study, the T-cell epitope landscape of SARS-CoV-2 was determined via in vitro T-cell stimulation plus in silico prediction. HLAs associated with mild and severe disease courses showed almost the same potential in epitope presentation, suggesting intrinsic factors of APCs or T cells as contributors to the more severe disease courses. As T-cell epitopes did also not originate from regions of SARS-CoV-2 having shown high mutation rates in the past, a relatively stable T-cell response can be expected regarding new SARS-CoV-2 strains in the future. Analysis of the T-cell epitope landscape of SARS-CoV-2 suggests T-cell intrinsic factors as likely modulators of disease severity and that the capacity of MHC-peptide presentation remains stable among circulating SARS-CoV-2 viral strains.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has highlighted the critical importance of understanding protective long-lasting immune responses. This study investigates the epitope specificity, T cell receptor (TCR) usage, and phenotypic changes in SARS-CoV-2-specfic CD8+ and CD4+ T cells over time in convalescent individuals with COVID-19.

Peripheral blood mononuclear cells (PBMCs) were collected from 28 unvaccinated individuals with primary SARS-CoV-2 infection (6 identified as the D614G variant, clade 20C) and analyzed up to 12 months post-symptom onset. Antigen-specific CD8+ and CD4+ T cells were analyzed using flow cytometry and single-cell RNA sequencing (scRNAseq) using specific dextramer and antibody reagents. TCR clonotypes and activation markers were characterized to explore T cell dynamics.

SARS-CoV-2-specific CD8+ T cells exhibited waning frequencies long-term, transitioning from memory-like to a naïve-like state. scRNAseq revealed specificity against both spike and non-spike antigens with increased CD95 and CD127 expression over time, indicating that naïve-like T cells may represent stem cell memory T cells, which are multipotent and self-renewing, likely important for long-lived immunity. TCR clonal expansion was observed mainly in memory T cells, with overlapping TCR beta chain (TRB)-complementary determining region 3 (CDR3) sequences between participants, suggesting shared public TCR epitope-specific repertoires against SARS-CoV-2. Further, unique spike-specific CD4+ T cells with high CD95 and CD127 expression were identified, which may play a crucial role in long-term protection.

This study highlights epitope-specificity heterogeneity, with some immunodominant responses, and suggests a potential role for long-lived SARS-CoV-2-specific T cell immunity. Shared TCR repertoires offers insights into cross-reactive and protective T cell clones, providing valuable information for optimizing vaccine strategies against emerging SARS-CoV-2 variants. The findings underscore the critical role of cellular immunity in long-term protection against SARS-CoV-2 and emphasizes the importance of understanding T cell dynamics.

Clinical diagnosis typically incorporates physical examination, patient history, various laboratory tests, and imaging studies but makes limited use of the human immune system's own record of antigen exposures encoded by receptors on B cells and T cells. We analyzed immune receptor datasets from 593 individuals to develop MAchine Learning for Immunological Diagnosis, an interpretive framework to screen for multiple illnesses simultaneously or precisely test for one condition. This approach detects specific infections, autoimmune disorders, vaccine responses, and disease severity differences. Human-interpretable features of the model recapitulate known immune responses to severe acute respiratory syndrome coronavirus 2, influenza, and human immunodeficiency virus, highlight antigen-specific receptors, and reveal distinct characteristics of systemic lupus erythematosus and type-1 diabetes autoreactivity. This analysis framework has broad potential for scientific and clinical interpretation of immune responses.

Although emerging data have revealed the critical role of memory CD8+ T cells in preventing and controlling SARS-CoV-2 infection, virus-specific CD8+ T-cell responses against SARS-CoV-2 and its memory and innate-like subsets in unvaccinated COVID-19 patients with various disease manifestations in an HLA-restricted fashion remain to be understood. Here, we show the strong association of protective cellular immunity with mild COVID-19 and unique cell types against SARS-CoV-2 virus in an HLA-A2 restricted manner. ELISpot assays reveal that SARS-CoV-2-specific CD8+ T-cell responses in mild COVID-19 patients are significantly higher than in severe patients, whereas neutralizing antibody responses against SARS-CoV-2 virus significantly correlate with disease severity. Single-cell analyses of HLA-A2-restricted CD8+ T cells, which recognize highly conserved immunodominant SARS-CoV-2-specific epitopes, demonstrate divergent profiles in unvaccinated patients with mild versus severe disease. CD8+ T-cell types including cytotoxic KLRB1 + CD8αα cells with innate-like T-cell signatures, IFNG hi ID3 hi memory cells and IL7R + proliferative stem cell-like memory cells are preferentially observed in mild COVID-19, whereas distinct terminally-differentiated T-cell subsets are predominantly detected in severe COVID-19: highly activated FASL hi T-cell subsets and early-terminated or dysfunctional IL4R + GATA3 + stem cell-like memory T-cell subset. In conclusion, our findings suggest that unique and contrasting SARS-CoV-2-specific CD8+ T-cell profiles may dictate COVID-19 severity.

HLA-E expression plays a central role for modulation of NK cell function by interaction with inhibitory NKG2A and stimulatory NKG2C receptors on canonical and adaptive NK cells, respectively. Here, we demonstrate that infection of human primary lung tissue with SARS-CoV-2 leads to increased HLA-E expression and show that processing of the peptide YLQPRTFLL from the spike protein is primarily responsible for the strong, dose-dependent increase of HLA-E. Targeting the peptide site within the spike protein revealed that a single point mutation was sufficient to abrogate the increase in HLA-E expression. Spike-mediated induction of HLA-E differentially affected NK cell function: whereas degranulation, IFN-γ production, and target cell cytotoxicity were enhanced in NKG2C+ adaptive NK cells, effector functions were inhibited in NKG2A+ canonical NK cells. Analysis of a cohort of COVID-19 patients in the acute phase of infection revealed that adaptive NK cells were induced irrespective of the HCMV status, challenging the paradigm that adaptive NK cells are only generated during HCMV infection. During the first week of hospitalization, patients exhibited a selective increase of early NKG2C+CD57- adaptive NK cells whereas mature NKG2C+CD57+ cells remained unchanged. Further analysis of recovered patients suggested that the adaptive NK cell response is primarily driven by a wave of early adaptive NK cells during acute infection that wanes once the infection is cleared. Together, this study suggests that NK cell responses to SARS-CoV-2 infection are majorly influenced by the balance between canonical and adaptive NK cells via the HLA-E/NKG2A/C axis.

Next-generation T-cell-directed vaccines for COVID-19 focus on establishing lasting T-cell immunity against current and emerging SARS-CoV-2 variants. Precise identification of conserved T-cell epitopes is critical for designing effective vaccines. Here we introduce a comprehensive computational framework incorporating a machine learning algorithm-MHCvalidator-to enhance mass spectrometry-based immunopeptidomics sensitivity. MHCvalidator identifies unique T-cell epitopes presented by the B7 supertype, including an epitope from a + 1-frameshift in a truncated Spike antigen, supported by ribosome profiling. Analysis of 100,512 COVID-19 patient proteomes shows Spike antigen truncation in 0.85% of cases, revealing frameshifted viral antigens at the population level. Our EpiTrack pipeline tracks global mutations of MHCvalidator-identified CD8 + T-cell epitopes from the BNT162b4 vaccine. While most vaccine epitopes remain globally conserved, an immunodominant A*01-associated epitope mutates in Delta and Omicron variants. This work highlights SARS-CoV-2 antigenic features and emphasizes the importance of continuous adaptation in T-cell vaccine development.

CD8+ T-cell immunity, mediated through interactions between human leukocyte antigen (HLA) and the T-cell receptor (TCR), plays a pivotal role in conferring immune memory and protection against viral infections. The emergence of SARS-CoV-2 variants presents a significant challenge to the existing population immunity. While numerous SARS-CoV-2 mutations have been associated with immune evasion from CD8+ T cells, the molecular effects of most mutations on epitope-specific TCR recognition remain largely unexplored, particularly for epitope-specific repertoires characterized by common TCRs. In this study, we investigated an HLA-A*24-restricted NYN epitope (Spike448-456) that elicits broad and highly homologous CD8+ T cell responses in COVID-19 patients. Eleven naturally occurring mutations in the NYN epitope, all of which retained cell surface presentation by HLA, were tested against four transgenic Jurkat reporter cell lines. Our findings demonstrate that, with the exception of L452R and the combined mutation L452Q + Y453F, these mutations have minimal impact on the avidity of recognition by NYN peptide-specific TCRs. Additionally, we observed that a similar TCR responded differently to mutant epitopes and demonstrated cross-reactivity to the unrelated VYF epitope (ORF3a112-120). The results contradict the idea that immune responses with limited receptor diversity are insufficient to provide protection against emerging variants.

Identifying epitopes and their corresponding T-cell receptor (TCR) sequences is crucial in the face of rapidly mutating viruses. Peptide synthesis is often required to confirm the exact epitope sequences, which is time-consuming and expensive. In this study, we introduce a scalable workflow to identify the exact sequences of virus epitopes and reactive TCRs targeting the epitopes from memory T cells. Following the narrowing down of epitopes to specific regions via the tandem minigene (TMG) system, our workflow incorporates the utilization of peptide-major histocompatibility complex-displaying yeasts (pMHC-displaying yeasts) to rapidly screen immunogenic epitopes' precise sequences, obviating the necessity for the chemical synthesis of peptides. Focusing on SARS-CoV-2, we identify the precise sequences of reactive TCRs, targeting conserved epitopes across the Coronaviridae family, from the blood of COVID-19-recovered individuals over 8 months. Notably, we reveal that at least 75% (6/8) of the tested donors harbor T cells targeting a shared epitope, KTFPPTEPK, derived from the N protein. Furthermore, several identified TCRs exhibit cross-reactivity to mutant epitopes, suggesting a potential mechanism for sustained T-cell responses against emerging SARS-CoV-2 variants.

The difference in the immune response to severe acute respiratory syndrome coro-navirus 2 (SARS-CoV-2) in patients with mild versus severe disease remains poorly understood. Recent scientific advances have recognised the vital role of both B cells and T cells; however, many questions remain unanswered, particularly for T cell responses. T cells are essential for helping the generation of SARS-CoV-2 antibody responses but have also been recognised in their own right as a major factor influencing COVID-19 disease outcomes. The examination of T cell receptor (TCR) family differences over a 12-month period in patients with varying COVID-19 disease severity is crucial for understanding T cell responses to SARS-CoV-2.

We applied a machine learning approach to analyse TCR vb family responses in COVID-19 patients (n = 151) across multiple timepoints and disease severities alongside SARS-CoV-2 infection-naïve (healthy control) individ-uals (n = 62).

Blood samples from hospital in-patients with moderate, severe, or critical disease could be classified with an accuracy of 94%. Furthermore, we identified significant variances in TCR vb family specificities between disease and control subgroups.

Our findings suggest advantageous and disadvantageous TCR repertoire patterns in relation to disease severity. Following validation in larger cohorts, our methodology may be useful in detecting protective immunity and the assessment of long-term outcomes, particularly as we begin to unravel the immunological mechanisms leading to post-COVID complications.

Advances in mass spectrometry accelerates the characterization of HLA ligandome, necessitating the development of efficient methods for immunopeptidomics analysis and (neo)antigen prediction. We develop ImmuneApp, an interpretable deep learning framework trained on extensive HLA ligand datasets, which improves the prediction of HLA-I epitopes, prioritizes neoepitopes, and enhances immunopeptidomics deconvolution. ImmuneApp extracts informative embeddings and identifies key residues for pHLA binding. We also present a more accurate model-based deconvolution approach and systematically analyzed 216 multi-allelic immunopeptidomics samples, identifying 835,551 ligands restricted to over 100 HLA-I alleles. Our investigation reveals the effectiveness of the composite model, denoted as ImmuneApp-MA, which integrates mono- and multi-allelic data to enhance predictive performance. Leveraging ImmuneApp-MA as a pre-trained model, we built ImmuneApp-Neo, an immunogenicity predictor that outperforms existing methods for prioritizing immunogenic neoepitope. ImmuneApp demonstrates its utility across various immunopeptidomics datasets, which will promote the discovery of novel neoantigens and the development of new immunotherapies.

Cytotoxic CD8+ T lymphocytes (CTLs) have been implicated in the severity of COVID-19. The TCR-pMHC ternary complex, formed by the T cell receptor (TCR) and peptide-MHC (major histocompatibility complex), constitutes the molecular basis of CTL responses against SARS-CoV-2. While numerous studies have been conducted on T cell immunity, the molecular mechanisms underlying CTL-mediated immunity against SARS-CoV-2 infection have not been well elaborated. In this review, we described the association between HLA variants and different immune responses to SARS-CoV-2 infection, which may lead to varying COVID-19 outcomes. We also summarized the specific TCR repertoires triggered by certain SARS-CoV-2 CTL epitopes, which might explain the variations in disease outcomes among different patients. Importantly, we have highlighted the primary strategies used by SARS-CoV-2 variants to evade T-cell killing: disrupting peptide-MHC binding, TCR recognition, and antigen processing. This review provides valuable insights into the molecule mechanism of CTL responses during SARS-CoV-2 infection, aiding efforts to control the pandemic and prepare for future challenges.

The innate and adaptive immune systems collaborate to detect SARS-CoV-2 infection, minimize the viral spread, and kill infected cells, ultimately leading to the resolution of the infection. The adaptive immune system develops a memory of previous encounters with the virus, providing enhanced responses when rechallenged by the same pathogen. Such immunological memory is the basis of vaccine function. Here, we review the current knowledge on the immune response to SARS-CoV-2 infection and vaccination, focusing on the pivotal role of T cells in establishing protective immunity against the virus. After providing an overview of the immune response to SARS-CoV-2 infection, we describe the main features of SARS-CoV-2-specific CD4+ and CD8+ T cells, including cross-reactive T cells, generated in patients with different degrees of COVID-19 severity, and of Spike-specific CD4+ and CD8+ T cells induced by vaccines. Finally, we discuss T-cell responses to SARS-CoV-2 variants and hybrid immunity and conclude by highlighting possible strategies to improve the efficacy of COVID-19 vaccination.

COVID-19 remains a significant threat, particularly to vulnerable populations. The emergence of new variants necessitates the development of treatments and vaccines that induce both humoral and cellular immunity. This study aimed to identify potentially immunogenic SARS-CoV-2 peptides and to explore the intricate host-pathogen interactions involving peripheral immune responses, memory profiles, and various demographic, clinical, and lifestyle factors. Using in silico and experimental methods, we identified several CD8-restricted SARS-CoV-2 peptides that are either poorly studied or have previously unreported immunogenicity: fifteen from the Spike and three each from non-structural proteins Nsp1-2-3-16. A Spike peptide, LA-9, demonstrated a 57% response rate in ELISpot assays using PBMCs from 14 HLA-A*02:01 positive, vaccinated, and mild-COVID-19 recovered subjects, indicating its potential for diagnostics, research, and multi-epitope vaccine platforms. We also found that younger individuals, with fewer vaccine doses and longer intervals since infection, showed lower anti-Spike (ELISA) and anti-Wuhan neutralizing antibodies (pseudovirus assay), higher naïve T cells, and lower central memory, effector memory, and CD4hiCD8low T cells (flow cytometry) compared to older subjects. In our cohort, a higher prevalence of Vδ2-γδ and DN T cells, and fewer naïve CD8 T cells, seemed to correlate with strong cellular and lower anti-NP antibody responses and to associate with Omicron infection, absence of confusional state, and habitual sporting activity.

The architecture and dynamics of T cell populations are critical in orchestrating the immune response to SARS-CoV-2. In our study, we used T Cell Receptor sequencing (TCRseq) to investigate TCR repertoires in 173 post-infection COVID-19 patients.

The cohort included 98 mild and 75 severe cases with a median age of 53. We amplified and sequenced the TCR β chain Complementary Determining Region 3 (CDR3b) and performed bioinformatic analyses to assess repertoire diversity, clonality, and V/J allelic usage between age, sex and severity groups. CDR3b amino acid sequence inference was performed by clustering structural motifs and filtering validated reactive CDR3b to COVID-19.

Our results revealed a pronounced decrease in diversity and an increase in clonal expansion in the TCR repertoires of severe COVID-19 patients younger than 55 years old. These results reflect the observed trends in patients older than 55 years old (both mild and severe). In addition, we identified a significant reduction in the usage of key V alleles (TRBV14, TRBV19, TRBV15 and TRBV6-4) associated with disease severity. Notably, severe patients under 55 years old had allelic patterns that resemble those over 55 years old, accompanied by a skewed frequency of COVID-19-related motifs.

Present results suggest that severe patients younger than 55 may have a compromised TCR repertoire contributing to a worse disease outcome.

The COVID-19 pandemic caused by the rapid spread of the novel coronavirus SARS-CoV-2, has promoted an interest in studying the T-cell immune response. It was found that the polyclonal and cross-reactive T-cell response against seasonal coronaviruses and other SARS-CoV-2 strains reduced disease severity. We investigated the immunodominant T-cell epitope SPRWYFYYYL from the nucleocapsid protein of SARS-CoV-2. The immune response to this epitope is characterized by the formation of highly homologous (convergent) receptors that have been found in the T-cell receptor (TCR) repertoires of different individuals. This epitope belongs to a group of highly conserved peptides that are rarely mutated in novel SARS-CoV-2 strains and are homologous to the epitopes of seasonal coronaviruses. It has been suggested that the cross-reactive response to homologous peptides contributes to the reduction of COVID-19 severity. However, some investigators have questioned this hypothesis, suggesting that the low affinity of the cross-reactive receptors reduces the strength of the immune response. The aim of this study was to evaluate the effect of amino acid substitutions in the SPR epitope on its binding affinity to specific TCRs. For this, we performed antigen-dependent cellular expansions were performed using samples from four COVID-19-transfected donors and sequenced their TCR repertoires. The resulting SPR-specific repertoire of β-chains in TCRs had a greater sequence diversity than the repertoire of α-chains. However, the TCR repertoires of all four donors contained public receptors, three of which were cloned and used to generate the Jurkat E6-1 TPR cell line. Only one of these receptors was activated by the SPR peptide and recognized with the same affinity by its mutant homologue LPRWYFYYY from seasonal coronaviruses. This indicates that the presence of the mutation did not affect the strength of the immune response, which may explain why the cross-reactive response to the SPR epitope is so frequent and contributes positively to COVID-19 infection.

CD8+ T cell immunity, mediated by human leukocyte antigen (HLA) and T cell receptor (TCR), plays a critical role in conferring immune memory and protection against viral pathogens. The emergence of SARS-CoV-2 variants poses a serious challenge to the efficacy of current vaccines. Whereas numerous SARS-CoV-2 mutations associated with immune escape from CD8+ T cells have been documented, the molecular effects of most mutations on epitope-specific TCR recognition remain largely unexplored. Here, we studied an HLA-A24-restricted NYN epitope (Spike448-456) that elicits broad CD8+ T cell responses in COVID-19 patients characterized by a common TCR repertoire. Four natural mutations, N450K, L452Q, L452R, and Y453F, arose within the NYN epitope and have been transmitted in certain viral lineages. Our findings indicate that these mutations have minimal impact on the epitope's presentation by cell surface HLA, yet they diminish the affinities of their respective peptide-HLA complexes (pHLAs) for NYN peptide-specific TCRs, particularly L452R and Y453F. Furthermore, we determined the crystal structure of HLA-A24 loaded with the Y453F peptide (NYNYLFRLF), and subsequently a ternary structure of the public TCRNYN-I complexed to the original NYN-HLA-A24 (NYNYLYRLF). Our structural analysis unveiled that despite competent presentation by HLA, the mutant Y453F peptide failed to establish a stable TCR-pHLA ternary complex due to reduced peptide: TCR contacts. This study supports the idea that cellular immunity restriction is an important driving force behind viral evolution.

The accurate prediction of binding between T cell receptors (TCR) and their cognate epitopes is key to understanding the adaptive immune response and developing immunotherapies. Current methods face two significant limitations: the shortage of comprehensive high-quality data and the bias introduced by the selection of the negative training data commonly used in the supervised learning approaches. We propose a method, Transformer-based Unsupervised Language model for Interacting Peptides and T cell receptors (TULIP), that addresses both limitations by leveraging incomplete data and unsupervised learning and using the transformer architecture of language models. Our model is flexible and integrates all possible data sources, regardless of their quality or completeness. We demonstrate the existence of a bias introduced by the sampling procedure used in previous supervised approaches, emphasizing the need for an unsupervised approach. TULIP recognizes the specific TCRs binding an epitope, performing well on unseen epitopes. Our model outperforms state-of-the-art models and offers a promising direction for the development of more accurate TCR epitope recognition models.

The coronavirus disease 2019 (COVID-19) has merged as a global health threat since its outbreak in December 2019. Despite widespread recognition, there has been a paucity of studies focusing on the T cell receptor (TCR) bias in adaptive immunity induced by SARS-CoV-2. This research conducted a comparative analysis of the TCR immune repertoire to identify notable αβ TCR bias sequences associated with the SARS-CoV-2 virus antigen. The present study encompassed 73 symptomatic COVID-19 patients, categorized as moderate/mild or severe/critical, along with 9 healthy controls. Our findings revealed specific TCR chains prominently utilized by moderate and severe patients, identified as TRAV30-J34-TRBV3-1-J2-7 and TRAV12-3-J6-TRBV28-J1-1, respectively. Additionally, our research explored critical TCR preferences in the bronchoalveolar lavage fluid (BALF) of COVID-19 patients at various disease stages. Indeed, monitoring the dynamics of immune repertoire changes in COVID-19 patients could serve as a crucial biomarker for predicting disease progression and recovery. Furthermore, the study explored TCR bias in both peripheral blood mononuclear cells (PBMCs) and BALF. The most common αβ VJ pair observed in BALF was TRAV12-3-J18-TRBV7-6-J2-7. In addition, a comparative analysis with the VDJdb database indicated that the HLA-A*02:01 allele exhibited the widest distribution and highest frequency in COVID-19 patients across different periods. This comprehensive examination provided a global characterization of the TCR immune repertoire in COVID-19 patients, contributing significantly to our understanding of TCR bias induced by SARS-CoV-2.

Adenovirus vaccines, particularly the COVID-19 Ad5-nCoV adenovirus vaccine, have emerged as promising tools in the fight against infectious diseases. In this study, we investigated the structure of the T cell response to the Spike protein of the SARS-CoV-2 virus used in the COVID-19 Ad5-nCoV adenoviral vaccine in a phase 3 clinical trial (NCT04540419). In 69 participants, we collected peripheral blood samples at four time points after vaccination or placebo injection. Sequencing of T cell receptor repertoires from Spike-stimulated T cell cultures at day 14 from 17 vaccinated revealed a more diverse CD4+ T cell repertoire compared to CD8+. Nevertheless, CD8+ clonotypes accounted for more than half of the Spike-specific repertoire. Our longitudinal analysis showed a peak T cell response at day 14, followed by a decline until month 6. Remarkably, multiple T cell clonotypes persisted for at least 6 months after vaccination, as demonstrated by ex vivo stimulation. Examination of CDR3 regions revealed homologous sequences in both CD4+ and CD8+ clonotypes, with major CD8+ clonotypes sharing high similarity with annotated sequences specific for the NYNYLYRLF peptide, suggesting potential immunodominance. In conclusion, our study demonstrates the immunogenicity of the Ad5-nCoV adenoviral vaccine and highlights its ability to induce robust and durable T cell responses. These findings provide valuable insight into the efficacy of the vaccine against COVID-19 and provide critical information for ongoing efforts to control infectious diseases.

mRNA vaccines against the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) elicit strong T cell responses. However, a clonal-resolution analysis of T cell responses to mRNA vaccination has not been performed. Here, we temporally track the CD8+ T cell repertoire in individuals who received three shots of the BNT162b2 mRNA vaccine through longitudinal T cell receptor sequencing with peptide-human leukocyte antigen (HLA) tetramer analysis. We demonstrate a shift in T cell responses between the clonotypes with different kinetics: from early responders that expand rapidly after the first shot to main responders that greatly expand after the second shot. Although the main responders re-expand after the third shot, their clonal diversity is skewed, and newly elicited third responders partially replace them. Furthermore, this shift in clonal dominance occurs not only between, but also within, clonotypes specific for spike epitopes. Our study will be a valuable resource for understanding vaccine-induced T cell responses in general.

Global vaccination against COVID-19 has been widely successful; however, there is a need for complementary immunotherapies in severe forms of the disease and in immunocompromised patients. Cytotoxic CD8+ T cells have a crucial role in disease control, but their function can be dysregulated in severe forms of the disease. We report here a cell-based approach using a plasmacytoid dendritic cell line (PDC*line) to expand in vitro specific CD8+ responses against COVID-19 Ags. We tested the immunogenicity of eight HLA-A*02:01 restricted peptides derived from diverse SARS-Cov-2 proteins, selected by bioinformatics analyses in unexposed and convalescent donors. Higher ex vivo frequencies of specific T cells against these peptides were found in convalescent donors compared with unexposed donors, suggesting in situ T cell expansion upon viral infection. The peptide-loaded PDC*line induced robust CD8+ responses with total amplification rates that led up to a 198-fold increase in peptide-specific CD8+ T cell frequencies for a single donor. Of note, six of eight selected peptides provided significant amplifications, all of which were conserved between SARS-CoV variants and derived from the membrane, the spike protein, the nucleoprotein, and the ORF1ab. Amplified and cloned antiviral CD8+ T cells secreted IFN-γ upon peptide-specific activation. Furthermore, specific TCR sequences were identified for two highly immunogenic Ags. Hence, PDC*line represents an efficient platform to identify immunogenic viral targets for future immunotherapies.

Targeted synthetic vaccines have the potential to transform our response to viral outbreaks, yet the design of these vaccines requires a comprehensive knowledge of viral immunogens. Here, we report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides that are naturally processed and loaded onto human leukocyte antigen-II (HLA-II) complexes in infected cells. We identify over 500 unique viral peptides from canonical proteins as well as from overlapping internal open reading frames. Most HLA-II peptides colocalize with known CD4+ T cell epitopes in coronavirus disease 2019 patients, including 2 reported immunodominant regions in the SARS-CoV-2 membrane protein. Overall, our analyses show that HLA-I and HLA-II pathways target distinct viral proteins, with the structural proteins accounting for most of the HLA-II peptidome and nonstructural and noncanonical proteins accounting for the majority of the HLA-I peptidome. These findings highlight the need for a vaccine design that incorporates multiple viral elements harboring CD4+ and CD8+ T cell epitopes to maximize vaccine effectiveness.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hybrid immunity is more protective than vaccination or previous infection alone. To investigate the kinetics of spike-reactive T (TS) cells from SARS-CoV-2 infection through messenger RNA vaccination in persons with hybrid immunity, we identified the T cell receptor (TCR) sequences of thousands of index TS cells and tracked their frequency in bulk TCRβ repertoires sampled longitudinally from the peripheral blood of persons who had recovered from coronavirus disease 2019 (COVID-19). Vaccinations led to large expansions in memory TS cell clonotypes, most of which were CD8+ T cells, while also eliciting diverse TS cell clonotypes not observed before vaccination. TCR sequence similarity clustering identified public CD8+ and CD4+ TCR motifs associated with spike (S) specificity. Synthesis of longitudinal bulk ex vivo single-chain TCRβ repertoires and paired-chain TCRɑβ sequences from droplet sequencing of TS cells provides a roadmap for the rapid assessment of T cell responses to vaccines and emerging pathogens.

SARS-CoV-2, the etiological agent behind the coronavirus disease 2019 (COVID-19) pandemic, has continued to mutate and create new variants with increased resistance against the WHO-approved spike-based vaccines. With a significant portion of the worldwide population still unvaccinated and with waning immunity against newly emerging variants, there is a pressing need to develop novel vaccines that provide broader and longer-lasting protection. To generate broader protective immunity against COVID-19, we developed our second-generation vaccinia virus-based COVID-19 vaccine, TOH-VAC-2, encoded with modified versions of the spike (S) and nucleocapsid (N) proteins as well as a unique poly-epitope antigen that contains immunodominant T cell epitopes from seven different SARS-CoV-2 proteins. We show that the poly-epitope antigen restimulates T cells from the PBMCs of individuals formerly infected with SARS-CoV-2. In mice, TOH-VAC-2 vaccination produces high titers of S- and N-specific antibodies and generates robust T cell immunity against S, N, and poly-epitope antigens. The immunity generated from TOH-VAC-2 is also capable of protecting mice from heterologous challenge with recombinant VSV viruses that express the same SARS-CoV-2 antigens. Altogether, these findings demonstrate the effectiveness of our versatile vaccine platform as an alternative or complementary approach to current vaccines.

Antigen-specific CD8+ T cells mediate pathogen clearance. T cell phenotype is influenced by T cell receptor (TCR) sequences and environmental signals. Quantitative comparisons of these factors in human disease, while challenging to obtain, can provide foundational insights into basic T cell biology. Here, we investigate the phenotype kinetics of 679 CD8+ T cell clonotypes, each with specificity against one of three immunogenic viral antigens. Data were collected from a longitudinal study of 68 COVID-19 patients with antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), and influenza. Each antigen is associated with a different type of immune activation during COVID-19. We find TCR sequence to be by far the most important factor in shaping T cell phenotype and persistence for populations specific to any of these antigens. Our work demonstrates the important relationship between TCR sequence and T cell phenotype and persistence and helps explain why T cell phenotype often appears to be determined early in an infection.

The role of autoreactive T cells on the course of Coronavirus disease-19 (COVID-19) remains elusive. Type II pneumocytes represent the main target cells of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Autoimmune responses against antigens highly expressed in type II pneumocytes may influence the severity of COVID-19 disease.

The aim of this study was to investigate autoreactive T cell responses against self-antigens highly expressed in type II pneumocytes in the blood of COVID-19 patients with severe and non-severe disease.

We collected blood samples of COVID-19 patients with varying degrees of disease severity and of pre-pandemic controls. T cell stimulation assays with peptide pools of type II pneumocyte antigens were performed in two independent cohorts to analyze the autoimmune T cell responses in patients with non-severe and severe COVID-19 disease. Target cell lysis assays were performed with lung cancer cell lines to determine the extent of cell killing by type II PAA-specific T cells.

We identified autoreactive T cell responses against four recently described self-antigens highly expressed in type II pneumocytes, known as surfactant protein A, surfactant protein B, surfactant protein C and napsin A, in the blood of COVID-19 patients. These antigens were termed type II pneumocyte-associated antigens (type II PAAs). We found that patients with non-severe COVID-19 disease showed a significantly higher frequency of type II PAA-specific autoreactive T cells in the blood when compared to severely ill patients. The presence of high frequencies of type II PAA-specific T cells in the blood of non-severe COVID-19 patients was independent of their age. We also found that napsin A-specific T cells from convalescent COVID-19 patients could kill lung cancer cells, demonstrating the functional and cytotoxic role of these T cells.

Our data suggest that autoreactive type II PAA-specific T cells have a protective role in SARS-CoV-2 infections and the presence of high frequencies of these autoreactive T cells indicates effective viral control in COVID-19 patients. Type II-PAA-specific T cells may therefore promote the killing of infected type II pneumocytes and viral clearance.