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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.

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 numbersEU CT Number: 2022-502869-17-01 and ClinicalTrials.gov Registry (NCT05937295).

Abstract T cell recognition of human leukocyte antigen (HLA)-presented tumor-associated peptides is central for cancer immune surveillance. Mass spectrometry (MS)-based immunopeptidomics represents the only unbiased method for the direct identification and characterization of naturally presented tumor-associated peptides, a key prerequisite for the development of T cell-based immunotherapies. This study reports on the implementation of ion mobility separation-based time-of-flight (TOFIMS) MS for next-generation immunopeptidomics, enabling high-speed and sensitive detection of HLA-presented peptides. Applying TOFIMS-based immunopeptidomics, a novel extensive benignTOFIMS dataset was generated from 94 primary benign samples of solid tissue and hematological origin, which enabled the expansion of benign reference immunopeptidome databases with > 150,000 HLA-presented peptides, the refinement of previously described tumor antigens, as well as the identification of frequently presented self antigens and not yet described tumor antigens comprising low abundant mutation-derived neoepitopes that might serve as targets for future cancer immunotherapy development.

Abstract T-cell immunity is central for control of COVID-19, particularly in patients incapable of mounting antibody responses. CoVac-1 is a peptide-based T-cell activator composed of SARS-CoV-2 epitopes with documented favorable safety profile and efficacy in terms of SARS-CoV-2-specific T-cell response. We here report a Phase I/II open-label trial (NCT04954469) in 54 patients with congenital or acquired B-cell deficiency receiving one subcutaneous CoVac-1 dose. Immunogenicity in terms of CoVac-1-induced T-cell responses and safety are the primary and secondary endpoints, respectively. No serious or grade 4 CoVac-1-related adverse events have been observed. Expected local granuloma formation has been observed in 94% of study subjects, whereas systemic reactogenicity has been mild or absent. SARS-CoV-2-specific T-cell responses have been induced in 86% of patients and are directed to multiple CoVac-1 peptides, not affected by any current Omicron variants and mediated by multifunctional T-helper 1 CD4+ T cells. CoVac-1-induced T-cell responses have exceeded those directed to the spike protein after mRNA-based vaccination of B-cell deficient patients and immunocompetent COVID-19 convalescents with and without seroconversion. Overall, our data show that CoVac-1 induces broad and potent T-cell responses in patients with B-cell/antibody deficiency with a favorable safety profile, which warrants advancement to pivotal Phase III safety and efficacy evaluation. ClinicalTrials.gov identifier NCT04954469.

Objective: (1) To assess the accuracy of a standard operating procedure (SOP) regarding the utilization of atrial fibrillation (AF) alarms in everyday clinical practice, and (2) to evaluate the performance of automated continuous surveillance for atrial fibrillation (AF) in hospitalized acute stroke patients. Design: Retrospective cohort study. Setting: Two stroke units from two tertiary care hospitals in Berlin, Germany. Participants: We identified 635 patients with ischemic stroke diagnosis for the time period between 01. January and 30. September 2021 of which 176 patients had recorded AF alarms during monitoring. Of those, 115 patients were randomly selected for evaluation. After excluding 6 patients with hemorrhagic stroke in their records, 109 patients (mean age: 79.1 years, median NIHSS at admission: 6, 57% female) remained for analysis. Intervention: Using a clinical data warehouse for comprehensive data storage we retrospectively downloaded and visualized ECG data segments of 65 s duration around the automated AF alarms. We restricted the maximum number of ECG segments to ten per patient. Each ECG segment plot was uploaded into a REDCap database and categorized as either AF, non-AF or artifact by manual review. Atrial flutter was subsumed as AF. These classifications were then matched with 1) medical history and known diseases before stroke, 2) discharge diagnosis, and 3) recommended treatment plan in the medical history using electronic health records. Main outcome measures: The primary outcome was the proportion of previously unknown AF diagnoses correctly identified by the monitoring system but missed by the clinical team during hospitalization. Secondary outcomes included the proportion of patients in whom a diagnosis of AF would likely have led to anticoagulant therapy. We also evaluated the accuracy of the automated detection system in terms of its positive predictive value (PPV). Results: We evaluated a total of 717 ECG alarm segments from 109 patients. In 4 patients (3.7, 95% confidence interval [CI] 1.18–9.68%) physicians had missed AF despite at least one true positive alarm. All four patients did not receive long-term secondary prevention in form of anticoagulant therapy. 427 out of 717 alarms were rated true positives, resulting in a positive predictive value of 0.6 (CI 0.56–0.63) in this cohort. Conclusion: By connecting a data warehouse, electronic health records and a REDCap survey tool, we introduce a path to assess the monitoring quality of AF in acute stroke patients. We find that implemented standards of procedure to detect AF during stroke unit care are effective but leave room for improvement. Such data warehouse-based concepts may help to adjust internal processes or identify targets of further investigations.

IntroductionMass spectrometry-based immunopeptidomics is the only unbiased method to identify naturally presented HLA ligands, which is an indispensable prerequisite for characterizing novel tumor antigens for immunotherapeutic approaches. In recent years, improvements based on devices and methodology have been made to optimize sensitivity and throughput in immunopeptidomics. However, developments in ligand isolation, mass spectrometric analysis, and subsequent data processing can have a marked impact on the quality and quantity of immunopeptidomics data.MethodsIn this work, we compared the immunopeptidome composition in terms of peptide yields, spectra quality, hydrophobicity, retention time, and immunogenicity of two established immunoprecipitation methods (column-based and 96-well-based) using cell lines as well as primary solid and hematological tumor samples.ResultsAlthough, we identified comparable overall peptide yields, large proportions of method-exclusive peptides were detected with significantly higher hydrophobicity for the column-based method with potential implications for the identification of immunogenic tumor antigens. We showed that column preparation does not lose hydrophilic peptides in the hydrophilic washing step. In contrast, an additional 50% acetonitrile elution could partially regain lost hydrophobic peptides during 96-well preparation, suggesting a reduction of the bias towards the column-based method but not completely equalizing it.DiscussionTogether, this work showed how different immunoprecipitation methods and their adaptions can impact the peptide repertoire of immunopeptidomic analysis and therefore the identification of potential tumor-associated antigens.

The DNAJB1-PRKACA fusion transcript is the oncogenic driver in fibrolamellar hepatocellular carcinoma, a lethal disease with limited therapeutic options. Here, the authors identify the DNAJB1-PRKACA protein as a source for immunogenic neoepitopes and a potential target of T cell-based immunotherapy.

Objectives: Besides SARS-CoV-2-directed humoral immune responses, T cell responses are indispensable for effective antiviral immunity. Recent data have shown a correlation between COVID-19 symptoms and humoral immune response, but so far, little is known about the association of SARS-CoV-2-directed T cell responses and disease severity. Herein, we evaluated the prevalence of different clinical COVID-19 symptoms in relation to SARS-CoV-2-directed humoral and cellular immune responses. Methods: The severity of eight different symptoms during acute infection were assessed using questionnaires from 193 convalescent individuals and were evaluated in relation to SARS-CoV-2 antibody levels and intensity of SARS-CoV-2-specific T cell responses 2–8 weeks after positive polymerase chain reaction. Results: Although increased IgG serum levels could be associated with severity of most symptoms, no difference in T cell response intensity between different symptom severities was observed for the majority of COVID-19 symptoms. However, when analyzing loss of smell or taste and cough, awareness of more severe symptoms was associated with reduced T cell response intensities. Conclusions: These data suggest that rapid virus clearance mediated by SARS-CoV-2-specific T cells prevents severe symptoms of COVID-19.

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.

Summary: HLA-presented antigenic peptides are central components of T cell-based immunity in infectious disease. Beside HLA molecules on cell surfaces, soluble HLA molecules (sHLA) are released in the blood suggested to impact cellular immune responses. We demonstrated that sHLA levels were significantly increased in COVID-19 patients and convalescent individuals compared to a control cohort and positively correlated with SARS-CoV-2-directed cellular immunity. Of note, patients with severe courses of COVID-19 showed reduced sHLA levels. Mass spectrometry-based characterization of sHLA-bound antigenic peptides, the so-called soluble immunopeptidome, revealed a COVID-19-associated increased diversity of HLA-presented peptides and identified a naturally presented SARS-CoV-2-derived peptide from the viral nucleoprotein in the plasma of COVID-19 patients. Of interest, sHLA serum levels directly correlated with the diversity of the soluble immunopeptidome. Together, these findings suggest an inflammation-driven release of sHLA in COVID-19, directly influencing the diversity of the soluble immunopeptidome with implications for SARS-CoV-2-directed T cell-based immunity and disease outcome.

With the routine use of effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, the number of life-threatening coronavirus disease 2019 (COVID-19) courses have largely been reduced. However, multiple COVID-19 convalescents, even after asymptomatic to moderate disease, suffer from post-COVID syndrome, with relevant limitations in daily life. The pathophysiologic mechanisms of post-COVID syndrome are still elusive, with dysregulation of the immune system suggested as a central mechanism. Here, we assessed COVID-19 post-infectious symptoms (5–6 months after PCR-confirmed acute infection) together with the humoral immune response against SARS-CoV-2 in non-hospitalized COVID-19 convalescents, early (5–6 weeks) and late (5–6 months) after their first positive SARS-CoV-2 PCR result. Convalescents reporting several post-infectious symptoms (>3) showed higher anti-spike and anti-nucleocapsid antibody levels 5–6 weeks after PCR-confirmed infection with the latter remained increased 5–6 months after positive PCR. Likewise, a higher post-infectious symptom score was associated with increased antibody levels. Of note, convalescents displaying neuro-psychiatric symptoms such as restlessness, palpitations, irritability, and headache, as well as general symptoms such as fatigue/reduced power had higher SARS-CoV-2-specific antibody levels compared with asymptomatic cases. The increased humoral immune response in convalescents with post-COVID syndrome might be useful for the detection of individuals with an increased risk for post-COVID syndrome.

Serology is an important way to monitor SARS-CoV-2 infection in the population and support vaccine development. Here the authors develop a multiplex immunoassay including spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses with high specificity and sensitivity.

Abstract Background Clear cell renal cell carcinoma (ccRCC) is the dominant subtype of renal cancer. With currently available therapies, cure of advanced and metastatic ccRCC is achieved only in rare cases. Here, we developed a workflow integrating different -omics technologies to identify ccRCC-specific HLA-presented peptides as potential drug targets for ccRCC immunotherapy. Methods We analyzed HLA-presented peptides by MS-based ligandomics of 55 ccRCC tumors (cohort 1), paired non-tumor renal tissues, and 158 benign tissues from other organs. Pathways enriched in ccRCC compared to its cell type of origin were identified by transcriptome and gene set enrichment analyses in 51 tumor tissues of the same cohort. To retrieve a list of candidate targets with involvement in ccRCC pathogenesis, ccRCC-specific pathway genes were intersected with the source genes of tumor-exclusive peptides. The candidates were validated in an independent cohort from The Cancer Genome Atlas (TCGA KIRC, n = 452). DNA methylation (TCGA KIRC, n = 273), somatic mutations (TCGA KIRC, n = 392), and gene ontology (GO) and correlations with tumor metabolites (cohort 1, n = 30) and immune-oncological markers (cohort 1, n = 37) were analyzed to characterize regulatory and functional involvements. CD8+ T cell priming assays were used to identify immunogenic peptides. The candidate gene EGLN3 was functionally investigated in cell culture. Results A total of 34,226 HLA class I- and 19,325 class II-presented peptides were identified in ccRCC tissue, of which 443 class I and 203 class II peptides were ccRCC-specific and presented in ≥ 3 tumors. One hundred eighty-five of the 499 corresponding source genes were involved in pathways activated by ccRCC tumors. After validation in the independent cohort from TCGA, 113 final candidate genes remained. Candidates were involved in extracellular matrix organization, hypoxic signaling, immune processes, and others. Nine of the 12 peptides assessed by immunogenicity analysis were able to activate naïve CD8+ T cells, including peptides derived from EGLN3. Functional analysis of EGLN3 revealed possible tumor-promoting functions. Conclusions Integration of HLA ligandomics, transcriptomics, genetic, and epigenetic data leads to the identification of novel functionally relevant therapeutic targets for ccRCC immunotherapy. Validation of the identified targets is recommended to expand the treatment landscape of ccRCC.

Chronic lymphocytic leukemia (CLL) is characterized by recurrent relapses and resistance to treatment, even with novel therapeutic approaches. Despite being considered as a disease with low mutational burden and thus poor immunogenic, CLL seems to retain the ability of eliciting specific T cell activation. Accordingly, we recently found non-mutated tumor-associated antigens to play a central role in CLL immunosurveillance. Here, we investigated the association of total and CLL-exclusive HLA class I and HLA class II peptide presentation in the mass spectrometry-defined immunopeptidome of leukemic cells with clinical features and disease outcome of 57 CLL patients. Patients whose CLL cells present a more diverse immunopeptidome experienced fewer relapses. During the follow-up phase of up to 10 years, patients with an HLA class I-restricted presentation of high numbers of total and CLL-exclusive peptides on their malignant cells showed a more favorable disease course with a prolonged progression-free survival (PFS). Overall, our results suggest the existence of an efficient T cell-based immunosurveillance mediated by CLL-associated tumor antigens, supporting ongoing efforts in developing T cell-based immunotherapeutic strategies for CLL.

Antigen-specific immunotherapies, in particular peptide vaccines, depend on the recognition of naturally presented antigens derived from mutated and unmutated gene products on human leukocyte antigens, and represent a promising low-side-effect concept for cancer treatment. So far, the broad application of peptide vaccines in cancer patients is hampered by challenges of time- and cost-intensive personalized vaccine design, and the lack of neoepitopes from tumor-specific mutations, especially in low-mutational burden malignancies. In this study, we developed an immunopeptidome-guided workflow for the design of tumor-associated off-the-shelf peptide warehouses for broadly applicable personalized therapeutics. Comparative mass spectrometry-based immunopeptidome analyses of primary chronic lymphocytic leukemia (CLL) samples, as representative example of low-mutational burden tumor entities, and a dataset of benign tissue samples enabled the identification of high-frequent non-mutated CLL-associated antigens. These antigens were further shown to be recognized by pre-existing and de novo induced T cells in CLL patients and healthy volunteers, and were evaluated as pre-manufactured warehouse for the construction of personalized multi-peptide vaccines in a first clinical trial for CLL (NCT04688385). This workflow for the design of peptide warehouses is easily transferable to other tumor entities and can provide the foundation for the development of broad personalized T cell-based immunotherapy approaches.