![]() Mild COVID-19 is characterized by ageusia, fever, sore throat, cough, and mild pneumonia. When symptomatic, COVID-19 can range from a mild, common flu-like sickness in ∼85% to a severe respiratory disease in ∼15% of affected patients ( 12, 13). Furthermore, even asymptomatic and presymptomatic SARS-CoV-2–infected individuals can produce high viral loads sufficient for human-to-human transmission ( 8– 10).ĭiagnosis of COVID-19 is routinely achieved by detection of SARS-CoV-2 RNA in nasopharyngeal swabs via quantitative RT-PCR (RT-qPCR) ( 11) however, even symptomatic SARS-CoV-2 infections frequently remain unrecognized. These characteristics include the capability to transmit already during the asymptomatic phase of infection and its variable incubation time of ∼3–14 d (S. Several attributes of SARS-CoV-2 have contributed to its rapid spread. First cases occurred in December 2019, and by October 1, 2020, more than 1 million deaths and 35 million cases of SARS-CoV-2 infection had been reported worldwide (Johns Hopkins University). The appearance of SARS-CoV-2 has led to a rapidly spreading pandemic. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of a respiratory disease termed COVID-19, is a betacoronavirus related to severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) ( 1– 6). We are presenting a highly specific diagnostic tool for the detection of SARS-CoV-2–reactive T cells. Our comparison showed that the stimulation conditions can profoundly impact the activation readout in unexposed individuals. When we stimulated the same samples overnight, we measured significant numbers of cytokine-producing cells even in unexposed individuals. Strikingly, we were not able to detect SARS-CoV-2–specific T cells in 18 unexposed healthy individuals. Although some Ags were detected by CD4 + and CD8 + T cells, VME1 was mainly recognized by CD4 + T cells. All patients had reactive T cells against at least 1 of 12 analyzed viral Ags, and all patients had Spike-specific T cells. We were able to detect SARS-CoV-2–specific T cells in 10 of 10 COVID-19 patients with mild symptoms. The assay readout was an intracellular cytokine staining and flow cytometric analysis detecting four functional markers simultaneously (CD154, TNF, IL-2, and IFN-γ). Both CD4 + and CD8 + T cells were detected after 6 d of in vitro expansion using overlapping peptide libraries representing the whole viral protein. For the present work, we have adapted a protocol designed for the detection of rare neoantigen-specific memory T cells in cancer patients for studying cellular immune responses against SARS-CoV-2. Although studies analyzing humoral immune responses against SARS-CoV-2 were available rather early during the pandemic, cellular immunity came into focus of investigations just recently. Gaining detailed insights into the role of host immune responses in viral clearance is critical for understanding COVID-19 pathogenesis and future treatment strategies.
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