Alessandro Sette

COMBO ‐ RATE : An Experimentally Validated Bioinformatic Tool to Identify Promiscuous HLA Restrictions

Defining HLA restriction of T cell epitopes is essential for understanding immune responses in infectious disease, autoimmunity and vaccine design. Current bioinformatic programmes, including the IEDB RATE tool, enable inference of single‐HLA restrictions from immune response data of HLA‐typed donors. However, T cell epitopes are frequently presented by multiple HLA alleles, a phenomenon termed promiscuous restriction, limiting the utility of single‐allele approaches. To address this limitation, we developed COMBO‐RATE, an extension of RATE that systematically evaluates combinations of HLA alleles to identify multi‐allelic restriction patterns. Analysis of three independent datasets spanning distinct antigen systems and different epitope discovery strategies revealed that promiscuous restriction is a near‐universal feature of immunodominant epitopes. Focusing on the 43 immunodominant CD4 + T cell epitopes identified in a B. pertussis genome‐wide screen, COMBO‐RATE outperformed conventional RATE, identifying restrictions for 35 of 43 epitopes, compared to 24 by RATE alone, and uncovered 64 additional allele restrictions, including 29 unique alleles. Experimental validation using single‐HLA transfected cell lines and antigen presentation assays confirmed COMBO‐RATE‐inferred restrictions, demonstrating that a single epitope can be independently presented by distinct HLA alleles. Overall, COMBO‐RATE provides a robust and scalable framework for defining complete HLA restriction profiles from existing population response data, with important implications for the design of vaccines requiring broad HLA coverage across genetically diverse populations. This pipeline is available as both a Python package and a user‐friendly web application.

Phase 2 Evaluation of two GEO-CM04S1 Dose Levels Shows Comparable Safety and Broad Immunogenicity

Background GEO-CM04S1 is a synthetic modified vaccinia Ankara (sMVA)-based SARS-CoV-2 vaccine expressing Spike (S) and Nucleocapsid (N) antigens derived from the ancestral Wuhan-Hu-1 reference strain. This phase 2 study evaluated the safety and immunogenicity of two GEO-CM04S1 dose levels administered as a single booster in healthy adults previously vaccinated against COVID-19. Methods In this randomized, double-blind, parallel-group trial, participants received a single intramuscular booster of GEO-CM04S1 at either 1 × 10⁷ PFU (DL1) or 1 × 10⁸ PFU (DL2). Safety was assessed through adverse events and clinical evaluations. Humoral and cellular immune responses were measured for up to one year using SARS-CoV-2-specific IgG, neutralizing antibody assays against D614G, Omicron BA.1, and XBB.1.5, cytokine secretion, and activation-induced marker (AIM) assays. Results Sixty-three participants were enrolled (31 DL1; 32 DL2). GEO-CM04S1 was well tolerated at both dose levels, with no vaccine-related serious adverse events. Both doses elicited robust binding and neutralizing antibody responses, including cross-neutralization of Omicron variants, and durable Th1-biased cellular immunity. DL2 induced modestly higher anti-S and anti-RBD binding antibody responses at selected time points, whereas neutralizing antibody and T-cell responses were comparable between dose groups. Conclusions GEO-CM04S1 demonstrated a favourable safety profile and induced durable, broad humoral and cellular immune responses as a booster. Comparable functional immune responses between dose levels support the lower dose as a dose-sparing strategy for next-generation COVID-19 vaccination.

Allele-Skewed HLA-DR Immunopeptidomes of Bordetella pertussis

Human lungs maintain tissue-resident memory T cells against a broad spectrum of pathogens

Severe CAD is linked to increased CD8+ T-cell activation against five immunodominant APOB epitopes.

A booster dose of an inactivated SARS-CoV-2 vaccine targeting virus variants sustains protective humoral and cellular immunity

The Omicron lineage of SARS-CoV-2 showed a remarkable ability to escape vaccine-induced immunity. In a Phase 2 clinical trial, we investigated whether a third booster/fifth dose of one of three inactivated SARS-CoV-2 vaccine candidates, based on the Delta and Omicron BA.1 variants, enhanced humoral and cellular immune responses against SARS-CoV-2. Volunteers who received either four doses of CoronaVac® (homologous group, n = 228) or two doses of CoronaVac® followed by two mRNA vaccine doses (heterologous group, n = 298) were randomly assigned to receive an Omicron BA.1-based, trivalent (ancestral, Delta, Omicron BA.1) inactivated vaccine or an additional dose of CoronaVac® (heterologous group only). Local and systemic adverse events were recorded for 7 days after immunization with the fifth dose. Blood samples were collected at the time of immunization (day 0), 28 days post-immunization, and 180 days post-immunization. The immune response induced by vaccination was evaluated by quantifying total IgG against SARS-CoV-2 and neutralizing antibodies, IgG-producing B cells, relative antibody avidity, SARS-CoV-2-specific CD4 + and CD8 + T cells, and IFN-γ-producing cells. The most common local adverse event was pain at the site of inoculation (deltoid area), reported in 43.2% of the participants. No severe adverse events related to vaccination were recorded. Increased SARS-CoV-2-specific IgGs were observed exclusively in the homologous group at 28 days post-vaccination compared to the pre-immune status. Reduced neutralizing antibodies against the Omicron BA.1 variant were detected in both groups when compared to the ancestral (WT) virus, and no changes in IgG-producing B cells or relative antibody avidity were observed after vaccination. Furthermore, we observed sustained IFN-γ production and the frequency of SARS-CoV-2-specific CD4 + and CD8 + T cells before and after vaccination. Previous immunizations targeting the WT SARS-CoV-2 virus have induced a robust humoral and cellular response in the population, which is sustained by a fifth dose targeting either the Omicron BA.1, Delta, or WT virus. No. NCT05593042.

Symptomatic SARS-CoV-2 breakthrough infections broaden the repertoire of Spike-reactive CD4 T cells

Influenza A(H5N8) vaccine induces humoral and cell-mediated immunity against highly pathogenic avian influenza clade 2.3.4.4b A(H5N1) viruses in at-risk individuals

Impact of the bivalent COVID-19 booster on spike-specific t cell responses

The emergence of SARS-CoV-2 variants during the COVID-19 pandemic prompted updates to the original vaccines to improve immunogenicity against novel SARS-CoV-2 variants. Since 2022, vaccination guidelines introduced updated mRNA boosters, starting with a bivalent formulation that included the original and Omicron BA.4/BA.5 spike sequences, which emerged in late 2021. In this study, we characterized the spike-specific T cell response following the bivalent booster vaccination in humans. We performed an in-depth profiling of T cells, assessing activation markers, cytokine production and the impact of previous COVID-19 infection in the response to different spike variants. Overall, our results are consistent with the bivalent booster having a limited influence on spike-T cell responses, with similar magnitude and functionality observed toward the Omicron BA.4/BA.5 variant and the ancestral spike. Nonetheless, the booster proved to be beneficial for immunocompetent individuals with poor or declining T cell responses, increasing the frequency of specific T cells in blood.

Peptide-driven identification of TCRs reveals dynamics and phenotypes of CD4 T cells in tuberculosis

Assigning antigen specificity to T cell receptor (TCR) sequences is challenging due to the TCR repertoire's diversity and the complexity of TCR-antigen recognition. We developed the peptide-driven identification of TCRs (PDI-TCR) assay that combines in vitro expansion of cells with peptide pools, bulk TCR sequencing, and statistical analysis to identify antigen-specific TCRs from human blood. A key feature of PDI-TCR is the ability to distinguish true antigen-specific TCR clonotypes from TCRs associated with unspecific bystander activation by comparing responses to nonoverlapping peptide pools. We applied PDI-TCR to tuberculosis (TB) patients, sampling blood at diagnosis and throughout treatment, and Mycobacterium tuberculosis (Mtb)-sensitized healthy individuals (IGRA+). We identified hundreds of Mtb-specific TCRs, as well as unspecific TCRs, and characterized their phenotype in each cohort by single-cell RNA sequencing ex vivo. Mtb-specific T cells were highly diverse, with short-lived effector phenotypes only present in TB at diagnosis, while memory phenotypes were maintained through treatment. In contrast, unspecific expanded T cells were more clonally restricted, had a cytotoxic phenotype, and were maintained throughout treatment. While the PDI-TCR parameters used in this study are specific to Mtb, the underlying approach is broadly applicable to the study of antigen-specific T cells and can be adapted as needed for other antigen systems. Thus, PDI-TCR is a powerful tool for identifying antigen-specific TCRs and enables direct ex vivo identification and monitoring of antigen-specific T cells.

Mycobacterium tuberculosis peptide-specific T cells in pulmonary granulomas display broad effector functions

Mycobacterium tuberculosis (Mtb) peptide-specific T cell responses in granulomas are essential for host protection. Here we profiled macaque and human granuloma Mtb-specific T cells with single cell RNA sequencing after stimulation with peptide pools. Mtb peptide-specific T cells are primarily Th1 ∗ cells and express pro-inflammatory cytokines, mutiple chemokines, TNF superfamily molecules, granzyme B and perforin, SLAM family molecules, semaphorins, growth factors, proteases, and collagen. Peptide recognition by Mtb-specific T cells also drives responses in bystander and unconventional T cells including expression of FLT3LG , LTA , and XCL1 . In granulomas from a patient that underwent a pneumonectomy to remove tuberculosis-destroyed lung, we identify a population of peptide-specific T cells similar to macaque peptide-stimulated T cells. Thus, Mtb peptide-specific T cells may mediate protection against Mtb infection through the combined effects of many functions as well as the induction of bystander responses by neighboring T cells.

SetteCancer Immunology Immunotherapy

A meta-analysis of experimentally validated neo-epitopes: patterns, biases, and opportunities

Cancer cells harbor somatic mutations that generate novel amino acid sequences that are absent in the self-proteome. These mutation-derived cancer-specific peptides are defined as "neo-peptides". Neo-peptides eliciting immune responses, i.e. immunogenic neo-peptides, are defined as "neo-epitopes". Given their relevance to cancer immunotherapy, we conducted a meta-analysis to examine how experimental evidence informs our understanding of neo-epitopes. Our study is the largest reported to date. Using the cancer epitope database and analysis resource (CEDAR), we analyzed over 16,000 neo-peptides tested in more than 20,000 T cell assays across 180 studies. We found that validated neo-epitope frequencies varied across cancer types, with the highest rates in skin and lung and the lowest in colorectal cancer. Neo-epitopes were enriched in driver genes such as TP53 and KRAS. However, testing frequency correlated with mutation prevalence, revealing bias toward recurrent mutations. Despite the high sequence similarity among RAS family members, validated neo-epitope overlap was minimal, challenging pan-RAS strategies. Shared neo-epitopes across cancer types are rare, with only 16 validated in more than one cancer type. While most assays involved HLA class I, class II alleles presented a higher proportion of validated neo-epitopes. Specific alleles, including HLA-B*40:01 and HLA-DRB1*11:01, were enriched for neo-epitopes, whereas others, like HLA-A*02:01, were enriched for non-immunogenic neo-peptides. Finally, amino acid substitutions that altered hydrophobicity or charge were more common in neo-epitopes. Together, these findings define key features of neo-epitopes, expose methodological and biological biases in the literature, and highlight opportunities to improve the selection and prioritization of neo-epitopes for cancer immunotherapy.

Mpox-specific cellular and humoral immunity in mpox survivors living with HIV

The 2022 mpox virus (MPXV) outbreak caused life-threatening mpox disease in people living with HIV (PLWH). We compared immune responses to MPXV in PLWH who were either mpox-naive or were mpox survivors. We characterized the magnitude, polyfunctionality, and cytolytic potential of CD4 and CD8 T cells specific to MPXV and cross-reactive orthopoxviral antigens and compared them to HIV- and cytomegalovirus (CMV)-specific T cells. Mpox survivors developed MPXV-specific CD4 T cells capable of producing IFNγ, TNFα, and IL-2. Their polyfunctionality was superior to CMV-specific CD4 T cells. MPXV- and CMV-specific CD8 T cells had similar cytolytic potentials. Importantly, mpox survivors developed strong MPXV-specific antibodies with neutralizing activity. Prior vaccinia or JYNNEOS vaccination was associated with greater MPXV-specific antibody but not T cells. These data demonstrate that PLWH generate functional cellular and humoral immunity to MPXV post-mpox. Despite their immunocompromised state, mpox survivors with HIV are expected to have some protection against future MPXV exposures.

Minimal association between Th1-specific responses to COVID-19 vaccines and SARS-CoV-2 breakthrough infections in multiple sclerosis patients receiving disease-modifying therapies

Background: The COVID-19 pandemic highlighted challenges in managing patients with multiple sclerosis (PwMS), as disease-modifying therapies (DMTs) can interfere with immune responses to infections and vaccines. Objective: This study investigates the spike-specific T-cell response after the third dose of mRNA COVID-19 vaccines in PwMS undergoing DMTs, evaluating different cytokines, beyond IFN-γ, and exploring their potential association with SARS-CoV-2 breakthrough infections (BI). Methods: We prospectively enrolled 31 PwMS and 27 healthcare workers (HCWs). The spike-specific T-cell response was evaluated by measuring Th1 cytokines (IFN-γ, IL-2, TNF-α) and IP-10 using an easy-to-use whole-blood assay. Results: Most PwMS mounted a Wuhan spike-specific T-cell response by releasing Th1 cytokines (IFN-γ, IL-2, TNF-α) and IP-10, albeit with significantly reduced Th1 cytokine levels compared to HCWs. Fingolimod-treated patients showed the weakest response with significantly reduced IFN-γ and IL-2 levels compared to HCWs (both p<0.0001), as well as to ocrelizumab (p=0.0018 and p=0.0002, respectively) and cladribine/IFN-β-treated patients (p=0.041 and p<0.0001, respectively). Moreover, a cell-mediated response was observed against the Delta spike variant, and all cytokines correlated with each other. BI occurred in 38.7% of PwMS, with predominantly mild COVID-19 cases. Male sex (IRR: 4.05, p=0.017) and primary progressive MS (IRR: 3.65, p=0.052) were associated with a higher BI incidence rate. Spike-specific T-cell response did not associate with a higher protection against BI. Conclusions: This study provides an in-depth immunological characterization of the spike-specific T-cell response in PwMS under DMTs, evaluating immunological biomarkers whose relevance may extend beyond COVID-19 for studying immune responses to other infections and vaccinations.

Cancer Epitope Prediction Tools & Analysis Pipelines in CEDAR

Autoimmune response to C9orf72 protein in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a progressive loss of motor neurons. Neuroinflammation is apparent in affected tissues, including increased T cell infiltration and activation of microglia, particularly in the spinal cord1,2. Autoimmune responses are thought to have a key role in ALS pathology, and it is hypothesized that T cells contribute to the rapid loss of neurons during disease progression3,4. However, until now there has been no reported target for such an autoimmune response. Here we show that ALS is associated with recognition of the C9orf72 antigen, and we map the specific epitopes that are recognized. We show that these responses are mediated by CD4+ T cells that preferentially release IL-5 and IL-10, and that IL-10-mediated T cell responses are significantly greater in donors who have a longer predicted survival time. Our results reinforce the previous hypothesis that neuroinflammation has an important role in ALS disease progression, possibly because of a disrupted balance of inflammatory and counter-inflammatory T cell responses4. These findings highlight the potential of therapeutic strategies aimed at enhancing regulatory T cells5, and identify a key target for antigen-specific T cell responses that could enable precision therapeutics in ALS. An analysis of T cell responses in people with amyotrophic lateral sclerosis shows that the C9orf72 antigen is a key target of autoimmune responses in the disease, and identifies C9orf72 epitopes that are recognized.

Abstract B002: Meta-Analysis of Neoantigens: Insights from the Cancer Epitope Database and Analysis Resource (CEDAR)

Abstract PR-09: Meta-Analysis of Neoantigens: Insights from the Cancer Epitope Database and Analysis Resource (CEDAR)

Human CD4+ T cells recognize Mycobacterium tuberculosis–infected macrophages amid broader responses

Pediatric T cell and B cell responses to SARS-CoV-2 infection