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The proteasome generates the majority of peptides presented on MHC class I molecules. The cleavage pattern of the proteasome has been shown to be changed via the proteasome activator (PA)28 alpha beta (PA28αß). In particular, several immunogenic peptides have been reported to be PA28αß-dependent. In contrast, we did not observe a major impact of PA28αß on the generation of different major histocompatibility complex (MHC) classI ligands. PA28αß-knockout mice infected with the lymphocytic choriomeningitis virus (LCMV) or vaccinia virus showed a normal cluster of differentiation (CD) 8 response and viral clearance. However, we observed that the adoptive transfer of wild-type cells into PA28αß-knockout mice led to graft rejection, but not vice versa. Depletion experiments showed that the observed rejection was mediated by CD8+ cytotoxic T cells. These data indicate that PA28αß might be involved in the development of the CD8+ T cell repertoire in the thymus. Taken together, our data suggest that PA28αß is a crucial factor determining T cell selection and, therefore, impacts graft acceptance.

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Immune check-point blockade (ICB) has revitalized cancer immunotherapy, showing unprecedented efficacy despite only a narrow number of indications and with limited long-term protection. Cancer vaccines are promising combination partners for ICB to widen the patient population profiting from these treatments. Therapeutic heterologous prime-boost vaccination with KISIMATM protein vaccine and VSV-GP-TAg oncolytic virus was shown to inflame the tumor microenvironment, promoting significant infiltration of antigen-specific CD8 T cells resulting in robust antitumoral efficacy in mouse tumor models, and clinical trials are currently ongoing. Here, we report the impact of NKG2A blockade on antitumoral CD8 T cell immune response elicited by KISIMA-VSV-GP-TAg vaccination in tumor mouse models. Combination therapy significantly reduced the amount of vaccine-induced exhausted CD8 T cells infiltrating the tumor, resulting in short-term improved tumor growth control and prolonged mouse survival, while it also influenced the establishment of systemic effector memory CD8 T cell response. Taken together, these data show a compartment-dependent effect of NKG2A blockade on cancer vaccine-induced T cell immunity, increasing intratumoral T cell efficacy and attenuating the development of peripheral effector memory CD8 T cell response.

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Generating balanced populations of CD8 effector and memory T cells is necessary for immediate and durable immunity to infections and cancer. Yet, a definitive understanding of CD8 differentiation remains unclear. We used CARLIN, a processive lineage recording mouse model with single-cell RNA-seq and TCR-seq to track endogenous antigen-specific CD8 T cells during acute viral infection. We identified a diverse repertoire of expanded T-cell clones represented by seven transcriptional states. TCR enrichment analysis revealed differential memory- or effector-fate biases within clonal populations. Shared Vb segments and amino acid motifs were found within biased categories despite high TCR diversity. Using single-cell CARLIN barcode-seq we tracked multi-generational clones and found that unlike unbiased or memory-biased clones, which stably retain their fate profiles, effector-biased clones could adopt memory- or effector-bias within subclones. Collectively, our study demonstrates that a heterogenous T-cell repertoire specific for a shared antigen is composed of clones with distinct TCR-intrinsic fate-biases.

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CD8 T cells are important antiviral effectors in the adaptive immune response to cytomegaloviruses (CMV). Naïve CD8 T cells can be primed by professional antigen-presenting cells (pAPCs) alternatively by "direct antigen presentation" or "antigen cross-presentation". In the case of direct antigen presentation, viral proteins are expressed in infected pAPCs and enter the classical MHC class-I (MHC-I) pathway of antigen processing and presentation of antigenic peptides. In the alternative pathway of antigen cross-presentation, viral antigenic material derived from infected cells of principally any cell type is taken up by uninfected pAPCs and eventually also fed into the MHC class-I pathway. A fundamental difference, which can be used to distinguish between these two mechanisms, is the fact that viral immune evasion proteins that interfere with the cell surface trafficking of peptide-loaded MHC-I (pMHC-I) complexes are absent in cross-presenting uninfected pAPCs. Murine cytomegalovirus (mCMV) models designed to disrupt either of the two presentation pathways revealed that both are possible in principle and can substitute each other. Overall, however, the majority of evidence has led to current opinion favoring cross-presentation as the canonical pathway. To study priming in the normal host genetically competent in both antigen presentation pathways, we took the novel approach of enhancing or inhibiting direct antigen presentation by using recombinant viruses lacking or overexpressing a key mCMV immune evasion protein. Against any prediction, the strongest CD8 T-cell response was elicited under the condition of intermediate direct antigen presentation, as it exists for wild-type virus, whereas the extremes of enhanced or inhibited direct antigen presentation resulted in an identical and weaker response. Our findings are explained by direct antigen presentation combined with a negative feedback regulation exerted by the newly primed antiviral effector CD8 T cells. This insight sheds a completely new light on the acquisition of viral immune evasion genes during virus-host co-evolution.

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Introduction: Significant evidence suggests a connection between transplant rejection and the presence of high levels of pre-existing memory T cells. Viral infection can elicit viral-specific memory T cells that cross-react with allo-MHC capable of driving allograft rejection in mice. Despite these advances, and despite their critical role in transplant rejection, a systematic study of allo-reactive memory T cells, their specificities, and the role of cross-reactivity with viral antigens has not been performed. Methods: Here, we established a model to identify, isolate, and characterize cross-reactive T cells using Nur77 reporter mice (C57BL/6 background), which transiently express GFP exclusively upon TCR engagement. We infected Nur77 mice with lymphocytic choriomeningitis virus (LCMV-Armstrong) to generate a robust memory compartment, where quiescent LCMV-specific memory CD8+ T cells could be readily tracked with MHC tetramer staining. Then, we transplanted LCMV immune mice with allogeneic hearts and monitored expression of GFP within MHC-tetramer defined viral-specific T cells as an indicator of their ability to cross-react with alloantigens. Results: Strikingly, prior LCMV infection significantly increased the kinetics and magnitude of rejection as well as CD8+ T cell recruitment into allogeneic, but not syngeneic, transplanted hearts, relative to non-infected controls. Interestingly, as early as day 1 after allogeneic heart transplant an average of ~8% of MHC-tetramer+ CD8+ T cells expressed GFP, in contrast to syngeneic heart transplants, where the frequency of viral-specific CD8+ T cells that were GFP+ was <1%. These data show that a significant percentage of viral-specific memory CD8+ T cells expressed T cell receptors that also recognized alloantigens in vivo. Notably, the frequency of cross-reactive CD8+ T cells differed depending upon the viral epitope. Further, TCR sequences derived from cross-reactive T cells harbored distinctive motifs that may provide insight into cross-reactivity and allo-specificity. Discussion: In sum, we have established a mouse model to track viral-specific, allo-specific, and cross-reactive T cells; revealing that prior infection elicits substantial numbers of viral-specific T cells that cross-react to alloantigen, respond very early after transplant, and may promote rapid rejection.

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For the development of new adenovirus (AdV)-based vectors, it is important to understand differences in immunogenicity. In a side-by-side in vitro analysis, we evaluated the effect of 40 AdV types covering human AdV (HAdV) species A through G on the expression of 11 activation markers and the secretion of 12 cytokines by AdV-transduced dendritic cells, and the effect on CD8+ T cell proliferation capacity. We found that the expression of activation markers and cytokines differed widely between the different HAdV types, and many types were able to significantly impair the proliferation capacity of CD8+ T cells. Univariate and multivariate regression analyses suggested an important role of type I interferons in mediating this suppression of CD8+ T cells, which we confirmed experimentally in a proliferation assay using a type I interferon receptor blocking antibody. Using Bayesian statistics, we calculated a prediction model that suggests HAdV types HAdV-C1, -D8, -B7, -F41, -D33, -C2, -A31, -B3 and -D65 as the most favorable candidates for vaccine vector development.

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Most licensed human vaccines are based on liquid dosage forms but have poor storage stability and require continuous and expensive cold-chain storage. In contrast, the use of solid vaccine dosage forms produced by for example spray drying, extends shelf life and eliminates the need for a cold chain. Zinc oxide (ZnO)-based nanoparticles display immunomodulatory properties, but their adjuvant effect as a dry powder formulation is unknown. Here, we show that reconstituted dry powder formulations of ZnO particles containing the model antigen ovalbumin (OVA) induce antigen-specific CD8+ T-cell and humoral responses. By systematically varying the ratio between ZnO and mannitol during spray drying, we manufactured dry powder formulations of OVA-containing ZnO particles that displayed: (i) a spherical or wrinkled surface morphology, (ii) an aerodynamic diameter and particle size distribution optimal for deep lung deposition, and (iii) aerosolization properties suitable for lung delivery. Reconstituted dry powder formulations of ZnO particles were well-tolerated by Calu-3 lung epithelial cells. Furthermore, almost equivalent OVA-specific serum antibody responses were stimulated by reconstituted ZnO particles, OVA adjuvanted with Alhydrogel®, and OVA adjuvanted with the cationic adjuvant formulation 01 (CAF®01). However, reconstituted dry powder ZnO particles and OVA adjuvanted with Alhydrogel® induced significantly lower OVA-specific CD8+CD44+ T-cell responses in the spleen than OVA adjuvanted with CAF®01. Similarly, reconstituted dry powder ZnO particles activated significantly lower percentages of follicular helper T cells and germinal center B cells in the draining lymph nodes than OVA adjuvanted with CAF®01. Overall, our results show that reconstituted dry powder formulations of ZnO nanoparticles can induce antigen-specific antibodies and can be used in vaccines to enhance antigen-specific humoral immune responses against subunit protein antigens.

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Little is known about the CD8+ T cell functionality in the coronavirus disease 2019 (COVID-19). Therefore, we examined twenty-five hospitalized COVID-19 patients with moderate (MD) or severe disease (SD) as well as seventeen SARS-CoV-2-unexposed persons regarding the cytolytic and cytokine-producing reactivity of their CD8+ T cells. Reactive CD8+ T cells were detectable in 90% of the unexposed persons, confirming high cross-reactive immune memory in the general population. Compared to unexposed persons and MD patients, SD patients had higher numbers of SARS-CoV-2 reactive CD8+ T cells with cytolytic function that can simultaneously produce inflammatory cytokines. In addition, SD patients showed higher CD8+ T cell reactivity against non-SARS-CoV-2-related viruses, which was mainly mediated by cytolytic response. Sequence alignments showed that cross-reactivities with the Spike protein could contribute to the expansion of such cells. Since insufficiently regulated cytolytic CD8+ T cells can damage peripheral and vascular tissue structures, high levels of both SARS-CoV-2-reactive and heterologously activated cytolytic CD8+ T cells could favor severe disease progression.

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Influenza, a respiratory disease mainly caused by influenza A and B, viruses of the Orthomyxoviridae, is still a burden on our society's health and economic system. Influenza A viruses (IAV) circulate in mammalian and avian populations, causing seasonal outbreaks with high numbers of cases. Due to the high variability in seasonal IAV triggered by antigenic drift, annual vaccination is necessary, highlighting the need for a more broadly protective vaccine against IAV. The safety tested Modified Vaccinia virus Ankara (MVA) is licensed as a third-generation vaccine against smallpox and serves as a potent vector system for the development of new candidate vaccines against different pathogens. Here, we generated and characterized recombinant MVA candidate vaccines that deliver the highly conserved internal nucleoprotein (NP) of IAV under the transcriptional control of five newly designed chimeric poxviral promoters to further increase the immunogenic properties of the recombinant viruses (MVA-NP). Infections of avian cell cultures with the recombinant MVA-NPs demonstrated efficient synthesis of the IAV-NP which was expressed under the control of the five new promoters. Prime-boost or single shot immunizations in C57BL/6 mice readily induced circulating serum antibodies' binding to recombinant IAV-NP and the robust activation of IAV-NP-specific CD8+ T cell responses. Moreover, the MVA-NP candidate vaccines protected C57BL/6 mice against lethal respiratory infection with mouse-adapted IAV (A/Puerto Rico/8/1934/H1N1). Thus, further studies are warranted to evaluate the immunogenicity and efficacy of these recombinant MVA-NP vaccines in other IAV challenge models in more detail.

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Despite the general agreement on the significance of T cells during SARS-CoV-2 infection, the clinical impact of specific and cross-reactive T-cell responses remains uncertain. Understanding this aspect could provide insights for adjusting vaccines and maintaining robust long-term protection against continuously emerging variants. To characterize CD8+ T-cell response to SARS-CoV-2 epitopes unique to the virus (SC2-unique) or shared with other coronaviruses (CoV-common), we trained a large number of T-cell receptor (TCR) - epitope recognition models for MHC-I-presented SARS-CoV-2 epitopes from publicly available data. These models were then applied to longitudinal CD8+ TCR repertoires from critical and non-critical COVID-19 patients. In spite of comparable initial CoV-common TCR repertoire depth and CD8+ T-cell depletion, the temporal dynamics of SC2-unique TCRs differed depending on the disease severity. Specifically, while non-critical patients demonstrated a large and diverse SC2-unique TCR repertoire by the second week of the disease, critical patients did not. Furthermore, only non-critical patients exhibited redundancy in the CD8+ T-cell response to both groups of epitopes, SC2-unique and CoV-common. These findings indicate a valuable contribution of the SC2-unique CD8+ TCR repertoires. Therefore, a combination of specific and cross-reactive CD8+ T-cell responses may offer a stronger clinical advantage. Besides tracking the specific and cross-reactive SARS-CoV-2 CD8+ T cells in any TCR repertoire, our analytical framework can be expanded to more epitopes and assist in the assessment and monitoring of CD8+ T-cell response to other infections.

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Antiretroviral therapy is capable of inhibiting HIV replication, but it fails to completely achieve a cure due to HIV persistence. The commonly used HIV cure approach is the "shock and kill" strategy, which employs latency-reversing agents to trigger viral reactivation and boost cellular immunity. Finding the appropriate drug combination for the "shock and kill" strategy would greatly facilitate clinical trials. The toll-like receptor (TLR) 7 agonist GS-9620 and nicotinamide (NAM) are reported as potential latency-reversing agents. Herein, we found the absence of viral reactivation when SHIVSF162P3-aviremic rhesus macaques were treated with GS-9620 monotherapy. However, our findings demonstrate that viral blips emerged in half of the macaques treated with the combination therapy of GS-9620 and NAM. Notably, an increase in the reactivation of the replication-competent latent virus was measured in monkeys treated with the combination therapy. These findings suggest that the GS-9620 and NAM combination could be used as a multipronged HIV latency stimulation approach, with potential for optimizing antiviral therapy design.

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Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense desmoplastic stroma that impedes drug delivery, reduces parenchymal blood flow, and suppresses the anti-tumor immune response. The extracellular matrix and abundance of stromal cells result in severe hypoxia within the tumor microenvironment (TME), and emerging publications evaluating PDAC tumorigenesis have shown the adenosine signaling pathway promotes an immunosuppressive TME and contributes to the overall low survival rate. Hypoxia increases many elements of the adenosine signaling pathway, resulting in higher adenosine levels in the TME, further contributing to immune suppression. Extracellular adenosine signals through 4 adenosine receptors (Adora1, Adora2a, Adora2b, Adora3). Of the 4 receptors, Adora2b has the lowest affinity for adenosine and thus, has important consequences when stimulated by adenosine binding in the hypoxic TME. We and others have shown that Adora2b is present in normal pancreas tissue, and in injured or diseased pancreatic tissue, Adora2b levels are significantly elevated. The Adora2b receptor is present on many immune cells, including macrophages, dendritic cells, natural killer cells, natural killer T cells, γδ T cells, B cells, T cells, CD4+ T cells, and CD8+ T cells. In these immune cell types, adenosine signaling through Adora2b can reduce the adaptive anti-tumor response, augmenting immune suppression, or may contribute to transformation and changes in fibrosis, perineural invasion, or the vasculature by binding the Adora2b receptor on neoplastic epithelial cells, cancer-associated fibroblasts, blood vessels, lymphatic vessels, and nerves. In this review, we discuss the mechanistic consequences of Adora2b activation on cell types in the tumor microenvironment. As the cell-autonomous role of adenosine signaling through Adora2b has not been comprehensively studied in pancreatic cancer cells, we will also discuss published data from other malignancies to infer emerging therapeutic considerations for targeting the Adora2b adenosine receptor to reduce the proliferative, invasive, and metastatic potential of PDAC cells.

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T cell recognition of SARS-CoV-2 antigens after vaccination and/or natural infection has played a central role in resolving SARS-CoV-2 infections and generating adaptive immune memory. However, the clinical impact of SARS-CoV-2-specific T cell responses is variable and the mechanisms underlying T cell interaction with target antigens are not fully understood. This is especially true given the virus' rapid evolution, which leads to new variants with immune escape capacity. In this study, we used the Omicron variant as a model organism and took a systems approach to evaluate the impact of mutations on CD8+ T cell immunogenicity. We computed an immunogenicity potential score for each SARS-CoV-2 peptide antigen from the ancestral strain and Omicron, capturing both antigen presentation and T cell recognition probabilities. By comparing ancestral vs. Omicron immunogenicity scores, we reveal a divergent and heterogeneous landscape of impact for CD8+ T cell recognition of mutated targets in Omicron variants. While T cell recognition of Omicron peptides is broadly preserved, we observed mutated peptides with deteriorated immunogenicity that may assist breakthrough infection in some individuals. We then combined our scoring scheme with an in silico mutagenesis, to characterise the position- and residue-specific theoretical mutational impact on immunogenicity. While we predict many escape trajectories from the theoretical landscape of substitutions, our study suggests that Omicron mutations in T cell epitopes did not develop under cell-mediated pressure. Our study provides a generalisable platform for fostering a deeper understanding of existing and novel variant impact on antigen-specific vaccine- and/or infection-induced T cell immunity.

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The present study sought to search for the immunodominance related to the N-terminal, Central and C-terminal regions of HTLV-1 Tax using novel, cutting-edge peptide microarray analysis. In addition, in silico predictions were performed to verify the presence of nine amino acid peptides present along Tax restricted to the human leukocyte antigen (HLA)-A2.02*01 haplotype, as well as to verify the ability to induce pro-inflammatory and regulatory cytokines, such as IFN-γ and IL-4, respectively. Our results indicated abundant dose-dependent reactivity for HLA-A*02:01 in all regions (N-terminal, Central and C-terminal), but with specific hotspots. Furthermore, the results of fold-change over the Tax11-19 reactivity obtained at lower concentrations of HLA-A*02:01 reveal that peptides from the three regions contain sequences that react 100 times more than Tax11-19. On the other hand, Tax11-19 has similar or superior HLA-A*02:01 reactivity at higher concentrations of this haplotype. The in silico analysis showed a higher frequency of IFN-γ-inducing peptides in the N-terminal portion, while the C-terminal portion showed a higher frequency of IL-4 inducers. Taken together, these results shed light on the search for new Tax immunodominant epitopes, in addition to the canonic Tax11-19, for the rational design of immunomodulatory strategies for HTLV-1 chronic diseases.

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Regulatory T cells overwhelm conventional T cells in the tumor microenvironment (TME) thanks to a FOXP3-driven metabolic program that allows them to engage different metabolic pathways. Using a melanoma model of adoptive T cell therapy (ACT), we show that FOXP3 overexpression in mature CD8 T cells improved their antitumor efficacy, favoring their tumor recruitment, proliferation, and cytotoxicity. FOXP3-overexpressing (Foxp3UP) CD8 T cells exhibited features of tissue-resident memory-like and effector T cells, but not suppressor activity. Transcriptomic analysis of tumor-infiltrating Foxp3UP CD8 T cells showed positive enrichment in a wide variety of metabolic pathways, such as glycolysis, fatty acid (FA) metabolism, and oxidative phosphorylation (OXPHOS). Intratumoral Foxp3UP CD8 T cells exhibited an enhanced capacity for glucose and FA uptake as well as accumulation of intracellular lipids. Interestingly, Foxp3UP CD8 T cells compensated for the loss of mitochondrial respiration-driven ATP production by activating aerobic glycolysis. Moreover, in limiting nutrient conditions these cells engaged FA oxidation to drive OXPHOS for their energy demands. Importantly, their ability to couple glycolysis and OXPHOS allowed them to sustain proliferation under glucose restriction. Our findings demonstrate a hitherto unknown role for FOXP3 in the adaptation of CD8 T cells to TME that may enhance their efficacy in ACT.

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We evaluated cellular immune responses induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in an immunized population based on HLA-E-restricted CD8+ T cell epitope identification. HLA-E-restricted SARS-CoV-2 CD8+ T cell nonamer peptides were predicted with software. An HLA-E-transfected K562 cell binding assay was used to screen for high-affinity peptides. IFN-γ enzyme-linked immunospot assays were used to identify HLA-E-restricted epitopes. An HLA-E/epitope tetramer was employed to detect the frequencies of epitope-specific CD8+ T cells. Four CD8+ T cell epitopes on the spike protein of SARS-CoV-2 restricted by both HLA-E*0101 and E*0103 were identified. HLA-E-restricted epitope-specific IFN-γ-secreting CD8+ T cell responses could be detected in individuals vaccinated with SARS-CoV-2 vaccines. Importantly, the frequencies of epitope-specific CD8+ T cells in Ad5-nCoV vaccinated individuals were higher than in individuals vaccinated with recombinant protein or inactivated vaccines. Moreover, the frequencies of epitope-specific CD8+ T cells could be maintained for at least 120 days after only one dose of Ad5-nCoV vaccine, while the frequencies of epitope-specific CD8+ T cells decreased in individuals after two doses of Ad5-nCoV vaccine. These findings may contribute to a more comprehensive evaluation of the protective effects of vaccines for SARS-CoV-2; meanwhile, they may provide information to characterize HLA-E-restricted CD8+ T cell immunity against SARS-CoV-2 infection.

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Introduction: The present work sought to identify MHC-I-restricted peptide signatures for arbovirus using in silico and in vitro peptide microarray tools. Methods: First, an in-silico analysis of immunogenic epitopes restricted to four of the most prevalent human MHC class-I was performed by identification of MHC affinity score. For that, more than 10,000 peptide sequences from 5 Arbovirus and 8 different viral serotypes, namely Zika (ZIKV), Dengue (DENV serotypes 1-4), Chikungunya (CHIKV), Mayaro (MAYV) and Oropouche (OROV) viruses, in addition to YFV were analyzed. Haplotype HLA-A*02.01 was the dominant human MHC for all arboviruses. Over one thousand HLA-A2 immunogenic peptides were employed to build a comprehensive identity matrix. Intending to assess HLAA*02:01 reactivity of peptides in vitro, a peptide microarray was designed and generated using a dimeric protein containing HLA-A*02:01. Results: The comprehensive identity matrix allowed the identification of only three overlapping peptides between two or more flavivirus sequences, suggesting poor overlapping of virus-specific immunogenic peptides amongst arborviruses. Global analysis of the fluorescence intensity for peptide-HLA-A*02:01 binding indicated a dose-dependent effect in the array. Considering all assessed arboviruses, the number of DENV-derived peptides with HLA-A*02:01 reactivity was the highest. Furthermore, a lower number of YFV-17DD overlapping peptides presented reactivity when compared to non-overlapping peptides. In addition, the assessment of HLA-A*02:01-reactive peptides across virus polyproteins highlighted non-structural proteins as "hot-spots". Data analysis supported these findings showing the presence of major hydrophobic sites in the final segment of non-structural protein 1 throughout 2a (Ns2a) and in nonstructural proteins 2b (Ns2b), 4a (Ns4a) and 4b (Ns4b). Discussion: To our knowledge, these results provide the most comprehensive and detailed snapshot of the immunodominant peptide signature for arbovirus with MHC-class I restriction, which may bring insight into the design of future virus-specific vaccines to arboviruses and for vaccination protocols in highly endemic areas.

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Background and aims: There is growing interest in T cell-based immune therapies for a functional cure of chronic HBV infection including check-point inhibition, T cell-targeted vaccines or TCR-grafted effector cells. All these approaches depend on recognition of HLA class I-presented viral peptides. The HBV core region 18-27 is an immunodominant target of CD8+ T cells and represents the prime target for T cell-based therapies. Here, a high-resolution analysis of the core18-27 specific CD8+ T cell and the selected escape pathways was performed. Methods: HLA class I typing and viral sequence analyses were performed for 464 patients with chronic HBV infection. HBV-specific CD8+ T-cell responses against the prototype and epitope variants were characterized by flow cytometry. Results: Consistent with promiscuous presentation of the core18-27 epitope, antigen-specific T cells were detected in patients carrying HLA-A*02:01, HLA-B*35:01, HLA-B*35:03 or HLA-B*51:01. Sequence analysis confirmed reproducible selection pressure on the core18-27 epitope in the context of these alleles. Interestingly, the selected immune escape pathways depend on the presenting HLA-class I-molecule. Although cross-reactive T cells were observed, some epitope variants achieved functional escape by impaired TCR-interaction or disturbed antigen processing. Of note, selection of epitope variants was exclusively observed in HBeAg negative HBV infection and here, detection of variants associated with significantly greater magnitude of the CD8 T cell response compared to absence of variants. Conclusion: The core18-27 epitope is highly variable and under heavy selection pressure in the context of different HLA class I-molecules. Some epitope variants showed evidence for impaired antigen processing and reduced presentation. Viruses carrying such escape substitutions will be less susceptible to CD8+ T cell responses and should be considered for T cell-based therapy strategies.

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BACKGROUND & AIMS: In immunosuppressed patients, persistent HEV infection is common and may lead to cirrhosis and liver failure. HEV clearance depends on an effective virus-specific CD8+ T-cell response; however, the knowledge gap around HEV-specific CD8+ T-cell epitopes has hindered analysis of the mechanisms of T-cell failure in persistent infection. METHODS: We comprehensively studied HEV-specific CD8+ T-cell responses in 46 patients with self-limiting (n = 34) or chronic HEV infection (n = 12), by epitope-specific expansion, functional testing, ex vivo peptide HLA class I tetramer multi-parametric staining, and viral sequence analysis. RESULTS: We identified 25 HEV-specific CD8+ T-cell epitopes restricted by 9 different HLA class I alleles. In self-limiting HEV infection, HEV-specific CD8+ T cells were vigorous, contracted after resolution of infection, and formed functional memory responses. In contrast, in chronic infection, the HEV-specific CD8+ T-cell response was diminished, declined over time, and displayed phenotypic features of exhaustion. However, improved proliferation of HEV-specific CD8+ T cells, increased interferon-γ production and evolution of a memory-like phenotype were observed upon reduction of immunosuppression and/or ribavirin treatment and were associated with viral clearance. In 1 patient, mutational viral escape in a targeted CD8+ T-cell epitope contributed to CD8+ T-cell failure. CONCLUSION: Chronic HEV infection is associated with HEV-specific CD8+ T-cell exhaustion, indicating that T-cell exhaustion driven by persisting antigen recognition also occurs in severely immunosuppressed hosts. Functional reinvigoration of virus-specific T cells is at least partially possible when antigen is cleared. In a minority of patients, viral escape also contributes to HEV-specific CD8+ T-cell failure and thus needs to be considered in personalized immunotherapeutic approaches. LAY SUMMARY: Hepatitis E virus (HEV) infection is usually cleared spontaneously (without treatment) in patients with fully functioning immune systems. In immunosuppressed patients, chronic HEV infection is common and can progress rapidly to cirrhosis and liver failure. Herein, we identified the presence of HEV-specific CD8+ T cells (a specific type of immune cell that can target HEV) in immunosuppressed patients, but we show that these cells do not function properly. This dysfunction appears to play a role in the development of chronic HEV infection in vulnerable patients.

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Background: Vaccine-induced immunity is essential for controlling the COVID-19 pandemic. Data on humoral and cellular immunogenicity and safety of different SARS-CoV-2 vaccines in patients with autoimmune rheumatic and musculoskeletal diseases (RMDs) are limited. Methods: A single center observational study evaluated the immunogenicity and safety of the two-dose regimen of the BBIBP-CorV inactivated, Gam-COVID-Vac and AZD1222 adenovirus-based, and BNT162b2 and mRNA-1273 mRNA-based vaccines in patients with RMDs (n = 89) compared with healthy controls (n = 74). Neutralizing anti-RBD (receptor binding domain) specific antibodies and SARS-CoV-2 specific T-cell response were measured one and four months after the second vaccine dose in parallel with vaccination efficacy and safety. Results: Disease-specific comparison showed that antibody response at four months was higher in spondylarthropathies compared to rheumatoid arthritis and autoimmune RMDs. Risk factors for reduced immunogenicity included longer disease duration, positive immunoserological profile and anti-CD20 therapy of patients. The rate of positive anti-RBD antibody response for healthy controls versus patients after 4 months post vaccination was 69% vs. 55% for the inactivated viral vaccine BBIBP-CorV, 97% vs. 53% for the pooled data of adenovirus vector-based vaccines Gam-COVID-Vac and AZD1222, or 100% vs. 81% for the pooled data of mRNA vaccines BNT162b2 and mRNA-1273, respectively. Patients who received the Gam-COVID-Vac or mRNA-1273 vaccines had a higher proportion of TNF-α producing CD4+ T-cells upon SARS-CoV-2 antigen stimulation compared to the inactivated viral vaccine. Conclusion: All five investigated vaccines were immunogenic in the majority of patients and healthy controls with variable antibody and T-cell response and an acceptable safety profile.

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