Real-time identification of epistatic interactions in SARS-CoV-2 from large genome collections.

BACKGROUND: The emergence of the SARS-CoV-2 virus has highlighted the importance of genomic epidemiology in understanding the evolution of pathogens and guiding public health interventions. The Omicron variant in particular has underscored the role of epistasis in the evolution of lineages with both higher infectivity and immune escape, and therefore the necessity to update surveillance pipelines to detect them early on. RESULTS: In this study, we apply a method based on mutual information between positions in a multiple sequence alignment, which is capable of scaling up to millions of samples. We show how it can reliably predict known experimentally validated epistatic interactions, even when using as little as 10,000 sequences, which opens the possibility of making it a near real-time prediction system. We test this possibility by modifying the method to account for the sample collection date and apply it retrospectively to multiple sequence alignments for each month between March 2020 and March 2023. We detected a cornerstone epistatic interaction in the Spike protein between codons 498 and 501 as soon as seven samples with a double mutation were present in the dataset, thus demonstrating the method's sensitivity. We test the ability of the method to make inferences about emerging interactions by testing candidates predicted after March 2023, which we validate experimentally. CONCLUSIONS: We show how known epistatic interaction in SARS-CoV-2 can be detected with high sensitivity, and how emerging ones can be quickly prioritized for experimental validation, an approach that could be implemented downstream of pandemic genome sequencing efforts.

A single-dose MCMV-based vaccine elicits long-lasting immune protection in mice against distinct SARS-CoV-2 variants.

Current vaccines against COVID-19 elicit immune responses that are overall strong but wane rapidly. As a consequence, the necessary booster shots have contributed to vaccine fatigue. Hence, vaccines that would provide lasting protection against COVID-19 are needed, but are still unavailable. Cytomegaloviruses (CMVs) elicit lasting and uniquely strong immune responses. Used as vaccine vectors, they may be attractive tools that obviate the need for boosters. Therefore, we tested the murine CMV (MCMV) as a vaccine vector against COVID-19 in relevant preclinical models of immunization and challenge. We have previously developed a recombinant MCMV vaccine vector expressing the spike protein of the ancestral SARS-CoV-2 (MCMVS). In this study, we show that the MCMVS elicits a robust and lasting protection in young and aged mice. Notably, spike-specific humoral and cellular immunity was not only maintained but also even increased over a period of at least 6 months. During that time, antibody avidity continuously increased and expanded in breadth, resulting in neutralization of genetically distant variants, like Omicron BA.1. A single dose of MCMVS conferred rapid virus clearance upon challenge. Moreover, MCMVS vaccination controlled two variants of concern (VOCs), the Beta (B.1.135) and the Omicron (BA.1) variants. Thus, CMV vectors provide unique advantages over other vaccine technologies, eliciting broadly reactive and long-lasting immune responses against COVID-19.

Dynamic monitoring of viral gene expression reveals rapid antiviral effects of CD8 T cells recognizing the HCMV-pp65 antigen.

Introduction: Human Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods. Methods: We developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi). Results: The timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes. Discussion: ARMATA does not only allow same day identification of antiviral T-cell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection.

Engineered HCMV-infected APCs enable the identification of new immunodominant HLA-restricted epitopes of anti-HCMV T-cell immunity.

Complications due to HCMV infection or reactivation remain a challenging clinical problem in immunocompromised patients, mainly due to insufficient or absent T-cell functionality. Knowledge of viral targets is crucial to improve monitoring of high-risk patients and optimise antiviral T-cell therapy. To expand the epitope spectrum, genetically-engineered dendritic cells (DCs) and fibroblasts were designed to secrete soluble (s)HLA-A*11:01 and infected with an HCMV mutant lacking immune evasion molecules (US2-6 + 11). More than 700 HLA-A*11:01-restricted epitopes, including more than 50 epitopes derived from a broad range of HCMV open-reading-frames (ORFs) were identified by mass spectrometry and screened for HLA-A*11:01-binding using established prediction tools. The immunogenicity of the 24 highest scoring new candidates was evaluated in vitro in healthy HLA-A*11:01+/HCMV+ donors. Thus, four subdominant epitopes and one immunodominant epitope, derived from the anti-apoptotic protein UL36 and ORFL101C (A11SAL), were identified. Their HLA-A*11:01 complex stability was verified in vitro. In depth analyses revealed highly proliferative and cytotoxic memory T-cell responses against A11SAL, with T-cell responses comparable to the immunodominant HLA-A*02:01-restricted HCMVpp65NLV epitope. A11SAL-specific T cells were also detectable in vivo in immunosuppressed transplant patients and shown to be effective in an in vitro HCMV-infection model, suggesting their crucial role in inhibiting viral replication and improvement of patient's outcome. The developed in vitro pipeline is the first to utilise genetically-engineered DCs to identify naturally presented immunodominant HCMV-derived epitopes. It therefore offers advantages over in silico predictions, is transferable to other HLA alleles, and will significantly expand the repertoire of viral targets to improve therapeutic options.

Serum cytokine dysregulation signatures associated with COVID-19 outcomes in high mortality intensive care unit cohorts across pandemic waves and variants.

The aim of this study was to characterize the systemic cytokine signature of critically ill COVID-19 patients in a high mortality setting aiming to identify biomarkers of severity, and to explore their associations with viral loads and clinical characteristics. We studied two COVID-19 critically ill patient cohorts from a referral centre located in Central Europe. The cohorts were recruited during the pre-alpha/alpha (November 2020 to April 2021) and delta (end of 2021) period respectively. We determined both the serum and bronchoalveolar SARS-CoV-2 viral load and identified the variant of concern (VoC) involved. Using a cytokine multiplex assay, we quantified systemic cytokine concentrations and analyzed their relationship with clinical findings, routine laboratory workup and pulmonary function data obtained during the ICU stay. Patients who did not survive had a significantly higher systemic and pulmonary viral load. Patients infected with the pre-alpha VoC showed a significantly lower viral load in comparison to those infected with the alpha- and delta-variants. Levels of systemic CTACK, M-CSF and IL-18 were significantly higher in non-survivors in comparison to survivors. CTACK correlated directly with APACHE II scores. We observed differences in lung compliance and the association between cytokine levels and pulmonary function, dependent on the VoC identified. An intra-cytokine analysis revealed a loss of correlation in the non-survival group in comparison to survivors in both cohorts. Critically ill COVID-19 patients exhibited a distinct systemic cytokine profile based on their survival outcomes. CTACK, M-CSF and IL-18 were identified as mortality-associated analytes independently of the VoC involved. The Intra-cytokine correlation analysis suggested the potential role of a dysregulated systemic network of inflammatory mediators in severe COVID-19 mortality.

HLA reduction of human T cells facilitates generation of immunologically multicompatible cellular products.

ABSTRACT: Adoptive cellular therapies have shown enormous potential but are complicated by personalization. Because of HLA mismatch, rejection of transferred T cells frequently occurs, compromising the T-cell graft's functionality. This obstacle has led to the development of HLA knock-out (KO) T cells as universal donor cells. Whether such editing directly affects T-cell functionality remains poorly understood. In addition, HLA KO T cells are susceptible to missing self-recognition through natural killer (NK) cells and lack of canonical HLA class I expression may represent a safety hazard. Engineering of noncanonical HLA molecules could counteract NK-cell recognition, but further complicates the generation of cell products. Here, we show that HLA KO does not alter T-cell functionality in vitro and in vivo. Although HLA KO abrogates allogeneic T-cell responses, it elicits NK-cell recognition. To circumvent this problem, we demonstrate that selective editing of individual HLA class I molecules in primary human T cells is possible. Such HLA reduction not only inhibits T-cell alloreactivity and NK-cell recognition simultaneously, but also preserves the T-cell graft's canonical HLA class I expression. In the presence of allogeneic T cells and NK cells, T cells with remaining expression of a single, matched HLA class I allele show improved functionality in vivo in comparison with conventional allogeneic T cells. Since reduction to only a few, most frequent HLA haplotypes would already be compatible with large shares of patient populations, this approach significantly extends the toolbox to generate broadly applicable cellular products.

Serine metabolism is crucial for cGAS-STING signaling and viral defense control in the gut.

Inflammatory bowel diseases are characterized by the chronic relapsing inflammation of the gastrointestinal tract. While the molecular causality between endoplasmic reticulum (ER) stress and intestinal inflammation is widely accepted, the metabolic consequences of chronic ER stress on the pathophysiology of IBD remain unclear. By using in vitro, in vivo models, and patient datasets, we identified a distinct polarization of the mitochondrial one-carbon metabolism and a fine-tuning of the amino acid uptake in intestinal epithelial cells tailored to support GSH and NADPH metabolism upon ER stress. This metabolic phenotype strongly correlates with IBD severity and therapy response. Mechanistically, we uncover that both chronic ER stress and serine limitation disrupt cGAS-STING signaling, impairing the epithelial response against viral and bacterial infection and fueling experimental enteritis. Consequently, the antioxidant treatment restores STING function and virus control. Collectively, our data highlight the importance of serine metabolism to allow proper cGAS-STING signaling and innate immune responses upon gut inflammation.

Human cytomegalovirus exploits STING signaling and counteracts IFN/ISG induction to facilitate infection of dendritic cells.

Human cytomegalovirus (HCMV) is a widespread pathogen that in immunocompromised hosts can cause life-threatening disease. Studying HCMV-exposed monocyte-derived dendritic cells by single-cell RNA sequencing, we observe that most cells are entered by the virus, whereas less than 30% of them initiate viral gene expression. Increased viral gene expression is associated with activation of the stimulator of interferon genes (STING) that usually induces anti-viral interferon responses, and with the induction of several pro- (RHOB, HSP1A1, DNAJB1) and anti-viral (RNF213, TNFSF10, IFI16) genes. Upon progression of infection, interferon-beta but not interferon-lambda transcription is inhibited. Similarly, interferon-stimulated gene expression is initially induced and then shut off, thus further promoting productive infection. Monocyte-derived dendritic cells are composed of 3 subsets, with one being especially susceptible to HCMV. In conclusion, HCMV permissiveness of monocyte-derived dendritic cells depends on complex interactions between virus sensing, regulation of the interferon response, and viral gene expression.

NFAT signaling is indispensable for persistent memory responses of MCMV-specific CD8+ T cells.

Cytomegalovirus (CMV) induces a unique T cell response, where antigen-specific populations do not contract, but rather inflate during viral latency. It has been proposed that subclinical episodes of virus reactivation feed the inflation of CMV-specific memory cells by intermittently engaging T cell receptors (TCRs), but evidence of TCR engagement has remained lacking. Nuclear factor of activated T cells (NFAT) is a family of transcription factors, where NFATc1 and NFATc2 signal downstream of TCR in mature T lymphocytes. We show selective impacts of NFATc1 and/or NFATc2 genetic ablations on the long-term inflation of MCMV-specific CD8+ T cell responses despite largely maintained responses to acute infection. NFATc1 ablation elicited robust phenotypes in isolation, but the strongest effects were observed when both NFAT genes were missing. CMV control was impaired only when both NFATs were deleted in CD8+ T cells used in adoptive immunotherapy of immunodeficient mice. Transcriptome analyses revealed that T cell intrinsic NFAT is not necessary for CD8+ T cell priming, but rather for their maturation towards effector-memory and in particular the effector cells, which dominate the pool of inflationary cells.

Decidual-tissue-resident memory T cells protect against nonprimary human cytomegalovirus infection at the maternal-fetal interface.

Congenital cytomegalovirus (cCMV) is the most common intrauterine infection, leading to infant neurodevelopmental disabilities. An improved knowledge of correlates of protection against cCMV is needed to guide prevention strategies. Here, we employ an ex vivo model of human CMV (HCMV) infection in decidual tissues of women with and without preconception immunity against CMV, recapitulating nonprimary vs. primary infection at the authentic maternofetal transmission site. We show that decidual tissues of women with preconception immunity against CMV exhibit intrinsic resistance to HCMV, mounting a rapid activation of tissue-resident memory CD8+ and CD4+ T cells upon HCMV reinfection. We further reveal the role of HCMV-specific decidual-tissue-resident CD8+ T cells in local protection against nonprimary HCMV infection. The findings could inform the development of a vaccine against cCMV and provide insights for further studies of the integrity of immune defense against HCMV and other pathogens at the human maternal-fetal interface.

SARS-CoV-2 BA.2.86 enters lung cells and evades neutralizing antibodies with high efficiency.

BA.2.86, a recently identified descendant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sublineage, contains ∼35 mutations in the spike (S) protein and spreads in multiple countries. Here, we investigated whether the virus exhibits altered biological traits, focusing on S protein-driven viral entry. Employing pseudotyped particles, we show that BA.2.86, unlike other Omicron sublineages, enters Calu-3 lung cells with high efficiency and in a serine- but not cysteine-protease-dependent manner. Robust lung cell infection was confirmed with authentic BA.2.86, but the virus exhibited low specific infectivity. Further, BA.2.86 was highly resistant against all therapeutic antibodies tested, efficiently evading neutralization by antibodies induced by non-adapted vaccines. In contrast, BA.2.86 and the currently circulating EG.5.1 sublineage were appreciably neutralized by antibodies induced by the XBB.1.5-adapted vaccine. Collectively, BA.2.86 has regained a trait characteristic of early SARS-CoV-2 lineages, robust lung cell entry, and evades neutralizing antibodies. However, BA.2.86 exhibits low specific infectivity, which might limit transmissibility.