SARS-CoV-2-derived peptides define heterologous and COVID-19-induced T cell recognition.

T cell immunity is central for the control of viral infections. To characterize T cell immunity, but also for the development of vaccines, identification of exact viral T cell epitopes is fundamental. Here we identify and characterize multiple dominant and subdominant SARS-CoV-2 HLA class I and HLA-DR peptides as potential T cell epitopes in COVID-19 convalescent and unexposed individuals. SARS-CoV-2-specific peptides enabled detection of post-infectious T cell immunity, even in seronegative convalescent individuals. Cross-reactive SARS-CoV-2 peptides revealed pre-existing T cell responses in 81% of unexposed individuals and validated similarity with common cold coronaviruses, providing a functional basis for heterologous immunity in SARS-CoV-2 infection. Diversity of SARS-CoV-2 T cell responses was associated with mild symptoms of COVID-19, providing evidence that immunity requires recognition of multiple epitopes. Together, the proposed SARS-CoV-2 T cell epitopes enable identification of heterologous and post-infectious T cell immunity and facilitate development of diagnostic, preventive and therapeutic measures for COVID-19.

A COVID-19 peptide vaccine for the induction of SARS-CoV-2 T cell immunity.

T cell immunity is central for the control of viral infections. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T cell epitopes derived from various viral proteins1,2, combined with the Toll-like receptor 1/2 agonist XS15 emulsified in Montanide ISA51 VG, aiming to induce profound SARS-CoV-2 T cell immunity to combat COVID-19. Here we conducted a phase I open-label trial, recruiting 36 participants aged 18-80 years, who received a single subcutaneous CoVac-1 vaccination. The primary end point was safety analysed until day 56. Immunogenicity in terms of CoVac-1-induced T cell response was analysed as the main secondary end point until day 28 and in the follow-up until month 3. No serious adverse events and no grade 4 adverse events were observed. Expected local granuloma formation was observed in all study participants, whereas systemic reactogenicity was absent or mild. SARS-CoV-2-specific T cell responses targeting multiple vaccine peptides were induced in all study participants, mediated by multifunctional T helper 1 CD4+ and CD8+ T cells. CoVac-1-induced IFNγ T cell responses persisted in the follow-up analyses and surpassed those detected after SARS-CoV-2 infection as well as after vaccination with approved vaccines. Furthermore, vaccine-induced T cell responses were unaffected by current SARS-CoV-2 variants of concern. Together, CoVac-1 showed a favourable safety profile and induced broad, potent and variant of concern-independent T cell responses, supporting the presently ongoing evaluation in a phase II trial for patients with B cell or antibody deficiency.

Viral T-cell epitopes - Identification, characterization and clinical application.

T-cell immunity, mediated by CD4+ and CD8+ T cells, represents a cornerstone in the control of viral infections. Virus-derived T-cell epitopes are represented by human leukocyte antigen (HLA)-presented viral peptides on the surface of virus-infected cells. They are the prerequisite for the recognition of infected cells by T cells. Knowledge of viral T-cell epitopes provides on the one hand a diagnostic tool to decipher protective T-cell immune responses in the human population and on the other hand various prophylactic and therapeutic options including vaccination approaches and the transfer of virus-specific T cells. Such approaches have already been proven to be effective against various viral infections, particularly in immunocompromised patients lacking sufficient humoral, antibody-based immune response. This review provides an overview on the state of the art as well as current studies regarding the identification and characterization of viral T-cell epitopes and approaches of clinical application. In the first chapter in silico prediction tools and direct, mass spectrometry-based identification of viral T-cell epitopes is compared. The second chapter provides an overview of commonly used assays for further characterization of T-cell responses and phenotypes. The final chapter presents an overview of clinical application of viral T-cell epitopes with a focus on human immunodeficiency virus (HIV), human cytomegalovirus (HCMV) and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), being representatives of relevant viruses.

Antibody Binding and Angiotensin-Converting Enzyme 2 Binding Inhibition Is Significantly Reduced for Both the BA.1 and BA.2 Omicron Variants.

BACKGROUND: The rapid emergence of the Omicron variant and its large number of mutations led to its classification as a variant of concern (VOC) by the World Health Organization. Subsequently, Omicron evolved into distinct sublineages (eg, BA.1 and BA.2), which currently represent the majority of global infections. Initial studies of the neutralizing response toward BA.1 in convalescent and vaccinated individuals showed a substantial reduction. METHODS: We assessed antibody (immunoglobulin G [IgG]) binding, ACE2 (angiotensin-converting enzyme 2) binding inhibition, and IgG binding dynamics for the Omicron BA.1 and BA.2 variants compared to a panel of VOCs/variants of interest, in a large cohort (N = 352) of convalescent, vaccinated, and infected and subsequently vaccinated individuals. RESULTS: While Omicron was capable of efficiently binding to ACE2, antibodies elicited by infection or immunization showed reduced binding capacities and ACE2 binding inhibition compared to wild type. Whereas BA.1 exhibited less IgG binding compared to BA.2, BA.2 showed reduced inhibition of ACE2 binding. Among vaccinated samples, antibody binding to Omicron only improved after administration of a third dose. CONCLUSIONS: Omicron BA.1 and BA.2 can still efficiently bind to ACE2, while vaccine/infection-derived antibodies can bind to Omicron. The extent of the mutations within both variants prevents a strong inhibitory binding response. As a result, both Omicron variants are able to evade control by preexisting antibodies.

NeutrobodyPlex-monitoring SARS-CoV-2 neutralizing immune responses using nanobodies.

In light of the COVID-19 pandemic, there is an ongoing need for diagnostic tools to monitor the immune status of large patient cohorts and the effectiveness of vaccination campaigns. Here, we present 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD), of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following detailed epitope mapping and structural analysis, we select two inhibitory Nbs, one of which binds an epitope inside and one of which binds an epitope outside the RBD:ACE2 interface. Based on these, we generate a biparatopic nanobody (bipNb) with viral neutralization efficacy in the picomolar range. Using bipNb as a surrogate, we establish a competitive multiplex binding assay ("NeutrobodyPlex") for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in serum of convalescent or vaccinated patients. We demonstrate that NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, to monitor immune status or to guide vaccine design.

Upstream open reading frames regulate translation of cancer-associated transcripts and encode HLA-presented immunogenic tumor antigens.

BACKGROUND: Upstream open reading frames (uORFs) represent translational control elements within eukaryotic transcript leader sequences. Recent data showed that uORFs can encode for biologically active proteins and human leukocyte antigen (HLA)-presented peptides in malignant and benign cells suggesting their potential role in cancer cell development and survival. However, the role of uORFs in translational regulation of cancer-associated transcripts as well as in cancer immune surveillance is still incompletely understood. METHODS: We examined the translational regulatory effect of 29 uORFs in 13 cancer-associated genes by dual-luciferase assays. Cellular expression and localization of uORF-encoded peptides (uPeptides) were investigated by immunoblotting and immunofluorescence-based microscopy. Furthermore, we utilized mass spectrometry-based immunopeptidome analyses in an extensive dataset of primary malignant and benign tissue samples for the identification of naturally presented uORF-derived HLA-presented peptides screening for more than 2000 uORFs. RESULTS: We provide experimental evidence for similarly effective translational regulation of cancer-associated transcripts through uORFs initiated by either canonical AUG codons or by alternative translation initiation sites (aTISs). We further demonstrate frequent cellular expression and reveal occasional specific cellular localization of uORF-derived peptides, suggesting uPeptide-specific biological implications. Immunopeptidome analyses delineated a set of 125 naturally presented uORF-derived HLA-presented peptides. Comparative immunopeptidome profiling of malignant and benign tissue-derived immunopeptidomes identified several tumor-associated uORF-derived HLA ligands capable to induce multifunctional T cell responses. CONCLUSION: Our data provide direct evidence for the frequent expression of uPeptides in benign and malignant human tissues, suggesting a potentially widespread function of uPeptides in cancer biology. These findings may inspire novel approaches in direct molecular as well as immunotherapeutic targeting of cancer-associated uORFs and uPeptides.

The Peptide Vaccine of the Future.

The approach of peptide-based anticancer vaccination has proven the ability to induce cancer-specific immune responses in multiple studies for various cancer entities. However, clinical responses remain so far limited to single patients and broad clinical applicability was not achieved. Therefore, further efforts are required to improve peptide vaccination in order to integrate this low-side-effect therapy into the clinical routine of cancer therapy. To design clinically effective peptide vaccines in the future, different issues have to be addressed and optimized comprising antigen target selection as well as choice of optimal adjuvants and vaccination schedules. Furthermore, the combination of peptide-based vaccines with other immuno- and molecular targeted therapies as well as the development of predictive biomarkers could further improve efficacy. In this review, current approaches in the development of peptide-based vaccines and critical implications for optimal vaccine design are discussed.

SARS-CoV-2-reactive T-cell receptors isolated from convalescent COVID-19 patients confer potent T-cell effector function.

Both B cells and T cells are involved in an effective immune response to SARS-CoV-2, the disease-causing virus of COVID-19. While B cells-with the indispensable help of CD4+ T cells-are essential to generate neutralizing antibodies, T cells on their own have been recognized as another major player in effective anti-SARS-CoV-2 immunity. In this report, we provide insights into the characteristics of individual HLA-A*02:01- and HLA-A*24:02-restricted SARS-CoV-2-reactive TCRs, isolated from convalescent COVID-19 patients. We observed that SARS-CoV-2-reactive T-cell populations were clearly detectable in convalescent samples and that TCRs isolated from these T cell clones were highly functional upon ectopic re-expression. The SARS-CoV-2-reactive TCRs described in this report mediated potent TCR signaling in reporter assays with low nanomolar EC50 values. We further demonstrate that these SARS-CoV-2-reactive TCRs conferred powerful T-cell effector function to primary CD8+ T cells as evident by a robust anti-SARS-CoV-2 IFN-γ response and in vitro cytotoxicity. We also provide an example of a long-lasting anti-SARS-CoV-2 memory response by reisolation of one of the retrieved TCRs 5 months after initial sampling. Taken together, these findings contribute to a better understanding of anti-SARS-CoV-2 T-cell immunity and may contribute to paving the way toward immunotherapeutics approaches targeting SARS-CoV-2.

Guidance Document: Validation of a High-Performance Liquid Chromatography-Tandem Mass Spectrometry Immunopeptidomics Assay for the Identification of HLA Class I Ligands Suitable for Pharmaceutical Therapies.

For more than two decades naturally presented, human leukocyte antigen (HLA)-restricted peptides (immunopeptidome) have been eluted and sequenced using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since, identified disease-associated HLA ligands have been characterized and evaluated as potential active substances. Treatments based on HLA-presented peptides have shown promising results in clinical application as personalized T cell-based immunotherapy. Peptide vaccination cocktails are produced as investigational medicinal products under GMP conditions. To support clinical trials based on HLA-presented tumor-associated antigens, in this study the sensitive LC-MS/MS HLA class I antigen identification pipeline was fully validated for our technical equipment according to the current US Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines.The immunopeptidomes of JY cells with or without spiked-in, isotope labeled peptides, of peripheral blood mononuclear cells of healthy volunteers as well as a chronic lymphocytic leukemia and a bladder cancer sample were reliably identified using a data-dependent acquisition method. As the LC-MS/MS pipeline is used for identification purposes, the validation parameters include accuracy, precision, specificity, limit of detection and robustness.

The HLA ligandome landscape of chronic myeloid leukemia delineates novel T-cell epitopes for immunotherapy.

Antileukemia immunity plays an important role in disease control and maintenance of tyrosine kinase inhibitor (TKI)-free remission in chronic myeloid leukemia (CML). Thus, antigen-specific immunotherapy holds promise for strengthening immune control in CML but requires the identification of CML-associated targets. In this study, we used a mass spectrometry-based approach to identify naturally presented HLA class I- and class II-restricted peptides in primary CML samples. Comparative HLA ligandome profiling using a comprehensive dataset of different hematological benign specimens and samples from CML patients in deep molecular remission delineated a panel of novel frequently presented CML-exclusive peptides. These nonmutated target antigens are of particular relevance because our extensive data-mining approach suggests the absence of naturally presented BCR-ABL- and ABL-BCR-derived HLA-restricted peptides and the lack of frequent tumor-exclusive presentation of known cancer/testis and leukemia-associated antigens. Functional characterization revealed spontaneous T-cell responses against the newly identified CML-associated peptides in CML patient samples and their ability to induce multifunctional and cytotoxic antigen-specific T cells de novo in samples from healthy volunteers and CML patients. Thus, these antigens are prime candidates for T-cell-based immunotherapeutic approaches that may prolong TKI-free survival and even mediate cure of CML patients.

Cancer testis antigen Cyclin A1 harbors several HLA-A*02:01-restricted T cell epitopes, which are presented and recognized in vivo.

Cyclin A1 is a promising antigen for T cell therapy being selectively expressed in high-grade ovarian cancer (OC) and acute myeloid leukemia (AML) stem cells. For adoptive T cell therapy, a single epitope has to be selected, with high affinity to MHC class I and adequate processing and presentation by malignant cells to trigger full activation of specific T cells. In silico prediction with three algorithms indicated 13 peptides of Cyclin A1 9 to 11 amino acids of length to have high affinity to HLA-A*02:01. Ten of them proved to be affine in an HLA stabilization assay using TAP-deficient T2 cells. Their immunogenicity was assessed by repetitive stimulation of CD8+ T cells from two healthy donors with single-peptide-pulsed dendritic cells or monocytes. Intracellular cytokine staining quantified the enrichment of peptide-specific functional T cells. Seven peptides were immunogenic, three of them against both donors. Specific cell lines were cloned and used in killing assays to demonstrate recognition of endogenous Cyclin A1 in the HLA-A*02:01-positive AML cell line THP-1. Immunopeptidome analysis based on direct isolation of HLA-presented peptides by mass spectrometry of primary AML and OC samples identified four naturally presented epitopes of Cyclin A1. The immunopeptidome of HeLa cells transfected with Cyclin A1 and HLA-A*02:01 revealed six Cyclin A1-derived HLA ligands. Epitope p410-420 showed high affinity to HLA-A*02:01 and immunogenicity in both donors. It proved to be naturally presented on primary AML blast and provoked spontaneous functional response of T cells from treatment naïve OC and, therefore, warrants further development for clinical application.

MHCquant: Automated and Reproducible Data Analysis for Immunopeptidomics.

Personalized multipeptide vaccines are currently being discussed intensively for tumor immunotherapy. In order to identify epitopes-short, immunogenic peptides-suitable for eliciting a tumor-specific immune response, human leukocyte antigen-presented peptides are isolated by immunoaffinity purification from cancer tissue samples and analyzed by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Here, we present MHCquant, a fully automated, portable computational pipeline able to process LC-MS/MS data automatically and generate annotated, false discovery rate-controlled lists of (neo-)epitopes with associated relative quantification information. We could show that MHCquant achieves higher sensitivity than established methods. While obtaining the highest number of unique peptides, the rate of predicted MHC binders remains still comparable to other tools. Reprocessing of the data from a previously published study resulted in the identification of several neoepitopes not detected by previously applied methods. MHCquant integrates tailor-made pipeline components with existing open-source software into a coherent processing workflow. Container-based virtualization permits execution of this workflow without complex software installation, execution on cluster/cloud infrastructures, and full reproducibility of the results. Integration with the data analysis workbench KNIME enables easy mining of large-scale immunopeptidomics data sets. MHCquant is available as open-source software along with accompanying documentation on our website at https://www.openms.de/mhcquant/ .

Antigen Targets for the Development of Immunotherapies in Leukemia.

Immunotherapeutic approaches, including allogeneic stem cell transplantation and donor lymphocyte infusion, have significantly improved the prognosis of leukemia patients. Further efforts are now focusing on the development of immunotherapies that are able to target leukemic cells more specifically, comprising monoclonal antibodies, chimeric antigen receptor (CAR) T cells, and dendritic cell- or peptide-based vaccination strategies. One main prerequisite for such antigen-specific approaches is the selection of suitable target structures on leukemic cells. In general, the targets for anti-cancer immunotherapies can be divided into two groups: (1) T-cell epitopes relying on the presentation of peptides via human leukocyte antigen (HLA) molecules and (2) surface structures, which are HLA-independently expressed on cancer cells. This review discusses the most promising tumor antigens as well as the underlying discovery and selection strategies for the development of anti-leukemia immunotherapies.

The antigenic landscape of multiple myeloma: mass spectrometry (re)defines targets for T-cell-based immunotherapy.

Direct analysis of HLA-presented antigens by mass spectrometry provides a comprehensive view on the antigenic landscape of different tissues/malignancies and enables the identification of novel, pathophysiologically relevant T-cell epitopes. Here, we present a systematic and comparative study of the HLA class I and II presented, nonmutant antigenome of multiple myeloma (MM). Quantification of HLA surface expression revealed elevated HLA molecule counts on malignant plasma cells compared with normal B cells, excluding relevant HLA downregulation in MM. Analyzing the presentation of established myeloma-associated T-cell antigens on the HLA ligandome level, we found a substantial proportion of antigens to be only infrequently presented on primary myelomas or to display suboptimal degrees of myeloma specificity. However, unsupervised analysis of our extensive HLA ligand data set delineated a panel of 58 highly specific myeloma-associated antigens (including multiple myeloma SET domain containing protein) which are characterized by frequent and exclusive presentation on myeloma samples. Functional characterization of these target antigens revealed peptide-specific, preexisting CD8(+) T-cell responses exclusively in myeloma patients, which is indicative of pathophysiological relevance. Furthermore, in vitro priming experiments revealed that peptide-specific T-cell responses can be induced in response-naive myeloma patients. Together, our results serve to guide antigen selection for T-cell-based immunotherapy of MM.

iVAC-XS15-CLL01: personalized multi-peptide vaccination in combination with the TLR1/2 ligand XS15 in CLL patients undergoing BTK-inhibitor-based regimens.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia among adults in Western countries. Despite the introduction of targeted therapies, including first-line Bruton's tyrosine kinase inhibitor (BTKi) treatment, CLL remains largely incurable. Frequent disease relapses occur due to remaining treatment-resistant CLL cells, calling for novel therapies to eliminate minimal residual disease (MRD). Peptide-based vaccination targeting human leucocyte antigen (HLA)-presented CLL-associated antigens represents a promising, low-side-effect therapeutic option to optimize treatment responses and eliminate residual tumor cells by inducing an anti-leukemic immune response. The iVAC-XS15-CLL01 trial is an open-label, first-in-human (FIH) Phase I trial, evaluating the CLL-VAC-XS15 vaccine in CLL patients undergoing BTKi-based therapy. The vaccine was developed from HLA-presented CLL-associated antigen peptides, identified through comparative mass-spectrometry-based immunopeptidome analyses of CLL versus healthy samples in a previous study. To facilitate rapid and cost-effective deployment, vaccine peptides are selected for each patient from a pre-manufactured "peptide warehouse" based on the patient's individual HLA allotype and CLL immunopeptidome. The trial enrolls 20 CLL patients, who receive up to three doses of the vaccine, adjuvanted with the toll-like-receptor (TLR) 1/2 ligand XS15 and emulsified in Montanide ISA 51 VG. The primary objective of the iVAC-XS15-CLL01 trial is to assess the safety and immunogenicity of the CLL-VAC-XS15 vaccine. Secondary objectives are to evaluate the vaccine impact on MRD, progression-free survival, and overall survival, as well as comprehensive immunophenotyping to characterize vaccine-induced T-cell responses. This Phase I trial aims to advance CLL treatment by enhancing immune-mediated disease clearance and guiding the design of subsequent Phase II/III trials to implement a new therapeutic strategy for CLL patients.

AML-VAC-XS15-01: protocol of a first-in-human clinical trial to evaluate the safety, tolerability and preliminary efficacy of a multi-peptide vaccine based on leukemia stem cell antigens in acute myeloid leukemia patients.

Introduction: Acute myeloid leukemia (AML) has a dismal prognosis, mostly due to minimal residual disease-driven relapse, making an elimination of persisting therapy-resistant leukemia progenitor/stem cells (LPCs) the main goal for novel therapies. Peptide-based immunotherapy offers a low-side-effect approach aiming to induce T cell responses directed against human leukocyte antigen (HLA) presented tumor antigens on malignant cells by therapeutic vaccination. Mass spectrometry-based analysis of the naturally presented immunopeptidome of primary enriched LPC and AML samples enabled the selection of antigens exclusively expressed on LPC/AML cells, which showed de novo induction and spontaneous memory T cell responses in AML patients, and whose presentation and memory T cell recognition was associated with improved disease outcome. Methods: Based on these data the therapeutic vaccine AML-VAC-XS15 was designed, comprising two mutated HLA class I-restricted peptides from the common AML-specific mutation in NPM1 and seven HLA class II-restricted peptides (six non-mutated high-frequent AML/LPC-associated antigens and one mutated peptide from the AML-specific mutation R140Q in IDH2), adjuvanted with the toll like receptor 1/2 ligand XS15 and emulsified in Montanide ISA 51 VG. A phase I open label clinical trial investigating AML-VAC-XS15 was designed, recruiting AML patients in complete cytological remission (CR) or CR with incomplete blood count recovery. Patients are vaccinated twice with a six-week interval, with an optional booster vaccination four months after 2nd vaccination, and are then followed up for two years. The trial's primary objectives are the assessment of the vaccine's immunogenicity, safety and toxicity, secondary objectives include characterization of vaccine-induced T cell responses and assessment of preliminary clinical efficacy. Ethics and dissemination: The AML-VAC-XS15-01 study was approved by the Ethics Committee of the Bavarian State medical association and the Paul-Ehrlich Institut (P01392). Clinical trial results will be published in peer-reviewed journals.

FusionVAC22_01: a phase I clinical trial evaluating a DNAJB1-PRKACA fusion transcript-based peptide vaccine combined with immune checkpoint inhibition for fibrolamellar hepatocellular carcinoma and other tumor entities carrying the oncogenic driver fusion.

The DNAJB1-PRKACA fusion transcript was identified as the oncogenic driver of tumor pathogenesis in fibrolamellar hepatocellular carcinoma (FL-HCC), also known as fibrolamellar carcinoma (FLC), as well as in other tumor entities, thus representing a broad target for novel treatment in multiple cancer entities. FL-HCC is a rare primary liver tumor with a 5-year survival rate of only 45%, which typically affects young patients with no underlying primary liver disease. Surgical resection is the only curative treatment option if no metastases are present at diagnosis. There is no standard of care for systemic therapy. Peptide-based vaccines represent a low side-effect approach relying on specific immune recognition of tumor-associated human leucocyte antigen (HLA) presented peptides. The induction (priming) of tumor-specific T-cell responses against neoepitopes derived from gene fusion transcripts by peptide-vaccination combined with expansion of the immune response and optimization of immune function within the tumor microenvironment achieved by immune-checkpoint-inhibition (ICI) has the potential to improve response rates and durability of responses in malignant diseases. The phase I clinical trial FusionVAC22_01 will enroll patients with FL-HCC or other cancer entities carrying the DNAJB1-PRKACA fusion transcript that are locally advanced or metastatic. Two doses of the DNAJB1-PRKACA fusion-based neoepitope vaccine Fusion-VAC-XS15 will be applied subcutaneously (s.c.) with a 4-week interval in combination with the anti-programmed cell death-ligand 1 (PD-L1) antibody atezolizumab starting at day 15 after the first vaccination. Anti-PD-L1 will be applied every 4 weeks until end of the 54-week treatment phase or until disease progression or other reason for study termination. Thereafter, patients will enter a 6 months follow-up period. The clinical trial reported here was approved by the Ethics Committee II of the University of Heidelberg (Medical faculty of Mannheim) and the Paul-Ehrlich-Institute (P-00540). Clinical trial results will be published in peer-reviewed journals. Trial registration numbers: EU CT Number: 2022-502869-17-01 and ClinicalTrials.gov Registry (NCT05937295).

Identification of novel canonical and cryptic HCMV-specific T-cell epitopes for HLA-A∗03 and HLA-B∗15 via peptide-PRISM.

ABSTRACT: Human cytomegalovirus (HCMV) reactivation poses a substantial risk to patients receiving tranplants. Effective risk stratification and vaccine development is hampered by a lack of HCMV-derived immunogenic peptides in patients with common HLA-A∗03:01 and HLA-B∗15:01 haplotypes. This study aimed to discover novel HCMV immunogenic peptides for these haplotypes by combining ribosome sequencing (Ribo-seq) and mass spectrometry with state-of-the-art computational tools, Peptide-PRISM and Probabilistic Inference of Codon Activities by an EM Algorithm. Furthermore, using machine learning, an algorithm was developed to predict immunogenicity based on translational activity, binding affinity, and peptide localization within small open reading frames to identify the most promising peptides for in vitro validation. Immunogenicity of these peptides was subsequently tested by analyzing peptide-specific T-cell responses of HCMV-seropositive and -seronegative healthy donors as well as patients with transplants. This resulted in the direct identification of 3 canonical and 1 cryptic HLA-A∗03-restricted immunogenic peptides as well as 5 canonical and 1 cryptic HLA-B∗15-restricted immunogenic peptide, with a specific interferon gamma-positive (IFN-γ+)/CD8+ T-cell response of ≥0.02%. High T-cell responses were detected against 2 HLA-A∗03-restricted and 3 HLA-B∗15-restricted canonical peptides with frequencies of up to 8.77% IFN-γ+/CD8+ T cells in patients after allogeneic stem cell transplantation. Therefore, our comprehensive strategy establishes a framework for efficient identification of novel immunogenic peptides from both existing and novel Ribo-seq data sets.

Long-Term Follow-Up of COVID-19 Convalescents-Immune Response Associated with Reinfection Rate and Symptoms.

SARS-CoV-2 has spread worldwide, causing millions of deaths and leaving a significant proportion of people with long-term sequelae of COVID-19 ("post-COVID syndrome"). Whereas the precise mechanism of post-COVID syndrome is still unknown, the immune response after the first infection may play a role. Here, we performed a long-term follow-up analysis of 110 COVID-19 convalescents, analyzing the first SARS-CoV-2-directed immune response, vaccination status, long-term symptoms (approximately 2.5 years after first infection), and reinfections. A total of 96% of convalescents were vaccinated at least once against SARS-CoV-2 after their first infection. A reinfection rate of 47% was observed, and lower levels of anti-spike IgG antibodies after the first infection were shown to associate with reinfection. While T-cell responses could not be clearly associated with persistent postinfectious symptoms, convalescents with long-term symptoms showed elevated SARS-CoV-2-specific antibody levels at the first infection. Evaluating the immune response after the first infection might be a useful tool for identifying individuals with increased risk for re-infections and long-term symptoms.