Validation and performance evaluation of a novel interferon-γ release assay for the detection of SARS-CoV-2 specific T-cell response

BackgroundThe reliable detection of the T-cell mediated response to COVID-19 or COVID-19 vaccination is important for individual patient care and for monitoring the immune response e.g. in COVID-19 vaccine trials in a standardized fashion. MethodsWe used blood samples from health care workers (HCW) with or without history of COVID-19 to define test accuracy of a novel interferon-release assay. Usefulness of qualitative and quantitative results after COVID-19 vaccination was examined in HCW receiving homologous or heterologous vaccination regimens. For a real-life performance evaluation, we analysed interferon-response to complete vaccination in 149 patients receiving immunosuppressive or immune modulating therapies. ResultsUsing a double-cut-off strategy integrating the result of background stimulation the assay had a specificity of 100%. Sensitivity of the IGRA was 83.5 and 100% in HCW after SARS-CoV-2 infection more or less than 6 months ago. Quantitative results showed significant differences between first and second vaccine dose, but no difference between homologous and heterologous vaccination regimen. The majority of immunocompromised patients showed no immune response or isolated T-cell or antibody response to complete vaccination. ConclusionsThe novel IGRA proved to be a highly specific and sensitive tool to detect the SARS-CoV-2 specific T-cell response to COVID-19 as well as COVID-19 vaccination. In perspective, it may serve as a standardized tool in COVID-19 vaccine trials and in clinical care of immunosuppressed patients.

Within-host evolution of SARS-CoV-2 in an immunosuppressed COVID-19 patient: a source of immune escape variants

The recent emergence of SARS-CoV-2 variants showing increased transmissibility and immune escape is a matter of global concern. Their origin remains unclear, but intra-host virus evolution during persistent infections could be a contributing factor. Here, we studied the long-term SARS-CoV-2 infection in an immunosuppressed organ transplant recipient. Frequent respiratory specimens were tested for variant viral genomes by RT-qPCR, next-generation sequencing (NGS), and virus isolation. Late in infection, several virus variants emerged which escaped neutralization by COVID-19 convalescent and vaccine-induced antisera and had acquired genome mutations similar to those found in variants of concern first identified in UK, South Africa, and Brazil. Importantly, infection of susceptible hACE2-transgenic mice with one of the patients escape variants elicited protective immunity against re-infection with either the parental virus, the escape variant or the South African variant of concern, demonstrating broad immune control. Upon lowering immunosuppressive treatment, the patient generated spike-specific neutralizing antibodies and resolved the infection. Our results indicate that immunocompromised patients are an alarming source of potentially harmful SARS-CoV-2 variants and open up new avenues for the updating of COVID-19 vaccines.

Ex vivo detection of SARS-CoV-2-specific CD8+ T cells: rapid induction, prolonged contraction, and formation of functional memory

CD8+ T cells are critical for the elimination and long-lasting protection of many viral infections, but their role in the current SARS-CoV-2 pandemic is unclear. Emerging data indicates that SARS-CoV-2-specific CD8+ T cells are detectable in the majority of individuals recovering from SARS-CoV-2 infection. However, optimal virus-specific epitopes, the role of pre-existing heterologous immunity as well as their kinetics and differentiation program during disease control have not been defined in detail. Here, we show that both pre-existing and newly induced SARS-CoV-2-specific CD8+ T-cell responses are potentially important determinants of immune protection in mild SARS-CoV-2 infection. In particular, our results can be summarized as follows: First, immunodominant SARS-CoV-2-specific CD8+ T-cell epitopes are targeted in the majority of individuals with convalescent SARS-CoV-2 infection. Second, MHC class I tetramer analyses revealed the emergence of phenotypically diverse and functionally competent pre-existing and newly induced SARS-CoV-2-specific memory CD8+ T cells that showed similar characteristics compared to influenza-specific CD8+ T cells. Third, SARS-CoV-2-specific CD8+ T-cell responses are more robustly detectable than antibodies against the SARS-CoV-2-spike protein. This was confirmed in a longitudinal analysis of acute-resolving infection that demonstrated rapid induction of the SARS-CoV-2-specific CD8+ T cells within a week followed by a prolonged contraction phase that outlasted the waning humoral immune response indicating that CD8+ T-cell responses might serve as a more precise correlate of antiviral immunity than antibody measurements after convalescence. Collectively, these data provide new insights into the fine specificity, heterogeneity, and dynamics of SARS-CoV-2-specific memory CD8+ T cells, potentially informing the rational development of a protective vaccine against SARS-CoV-2.

The landscape of SARS-CoV-2 RNA modifications

In 2019 the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the first documented cases of severe lung disease COVID-19. Since then, SARS-CoV-2 has been spreading around the globe resulting in a severe pandemic with over 500.000 fatalities and large economical and social disruptions in human societies. Gaining knowledge on how SARS-Cov-2 interacts with its host cells and causes COVID-19 is crucial for the intervention of novel therapeutic strategies. SARS-CoV-2, like other coronaviruses, is a positive-strand RNA virus. The viral RNA is modified by RNA-modifying enzymes provided by the host cell. Direct RNA sequencing (DRS) using nanopores enables unbiased sensing of canonical and modified RNA bases of the viral transcripts. In this work, we used DRS to precisely annotate the open reading frames and the landscape of SARS-CoV-2 RNA modifications. We provide the first DRS data of SARS-CoV-2 in infected human lung epithelial cells. From sequencing three isolates, we derive a robust identification of SARS-CoV-2 modification sites within a physiologically relevant host cell type. A comparison of our data with the DRS data from a previous SARS-CoV-2 isolate, both raised in monkey renal cells, reveals consistent RNA modifications across the viral genome. Conservation of the RNA modification pattern during progression of the current pandemic suggests that this pattern is likely essential for the life cycle of SARS-CoV-2 and represents a possible target for drug interventions.

Ad hoc laboratory-based surveillance of SARS-CoV-2 by real-time RT-PCR using minipools of RNA prepared from routine respiratory samples

We report a laboratory-based surveillance for SARS-CoV-2 using minipools of respiratory samples submitted for routine diagnostics. We tested a total of 70 minipools resembling 700 samples shortly before the upsurge of cases in Germany. We identified one SARS-CoV-2 positive patient. Our approach proved its concept, is easily adaptable and resource-saving.