Rapid Manufacturing of Highly Cytotoxic Clinical-Grade SARS-CoV-2-specific T Cell Products Covering SARS-CoV-2 and Its Variants for Adoptive T Cell Therapy.

Objectives: Evaluation of the feasibility of SARS-CoV-2-specific T cell manufacturing for adoptive T cell transfer in COVID-19 patients at risk to develop severe disease. Methods: Antiviral SARS-CoV-2-specific T cells were detected in blood of convalescent COVID-19 patients following stimulation with PepTivator SARS-CoV-2 Select using Interferon-gamma Enzyme-Linked Immunospot (IFN-γ ELISpot), SARS-CoV-2 T Cell Analysis Kit (Whole Blood) and Cytokine Secretion Assay (CSA) and were characterized with respect to memory phenotype, activation state and cytotoxic potential by multicolor flow cytometry, quantitative real-time PCR and multiplex analyses. Clinical-grade SARS-CoV-2-specific T cell products were generated by stimulation with MACS GMP PepTivator SARS-CoV-2 Select using CliniMACS Prodigy and CliniMACS Cytokine Capture System (IFN-gamma) (CCS). Functionality of enriched T cells was investigated in cytotoxicity assays and by multiplex analysis of secreted cytotoxic molecules upon target recognition. Results: Donor screening via IFN-γ ELISpot allows for pre-selection of potential donors for generation of SARS-CoV-2-specific T cells. Antiviral T cells reactive against PepTivator SARS-CoV-2 Select could be magnetically enriched from peripheral blood of convalescent COVID-19 patients by small-scale CSA resembling the clinical-grade CCS manufacturing process and showed an activated and cytotoxic T cell phenotype. Four clinical-grade SARS-CoV-2-specific T cell products were successfully generated with sufficient cell numbers and purities comparable to those observed in donor pretesting via CSA. The T cells in the generated products were shown to be capable to replicate, specifically recognize and kill target cells in vitro and secrete cytotoxic molecules upon target recognition. Cell viability, total CD3+ cell number, proliferative capacity and cytotoxic potential remained stable throughout storage of up to 72 h after end of leukapheresis. Conclusion: Clinical-grade SARS-CoV-2-specific T cells are functional, have proliferative capacity and target-specific cytotoxic potential. Their function and phenotype remain stable for several days after enrichment. The adoptive transfer of partially matched, viable human SARS-CoV-2-specific T lymphocytes collected from convalescent individuals may provide the opportunity to support the immune system of COVID-19 patients at risk for severe disease.

COVID-19 immune signatures reveal stable antiviral T cell function despite declining humoral responses.

Cellular and humoral immunity to SARS-CoV-2 is critical to control primary infection and correlates with severity of disease. The role of SARS-CoV-2-specific T cell immunity, its relationship to antibodies, and pre-existing immunity against endemic coronaviruses (huCoV), which has been hypothesized to be protective, were investigated in 82 healthy donors (HDs), 204 recovered (RCs), and 92 active COVID-19 patients (ACs). ACs had high amounts of anti-SARS-CoV-2 nucleocapsid and spike IgG but lymphopenia and overall reduced antiviral T cell responses due to the inflammatory milieu, expression of inhibitory molecules (PD-1, Tim-3) as well as effector caspase-3, -7, and -8 activity in T cells. SARS-CoV-2-specific T cell immunity conferred by polyfunctional, mainly interferon-γ-secreting CD4+ T cells remained stable throughout convalescence, whereas humoral responses declined. Immune responses toward huCoV in RCs with mild disease and strong cellular SARS-CoV-2 T cell reactivity imply a protective role of pre-existing immunity against huCoV.

Low serum neutralizing anti-SARS-CoV-2 S antibody levels in mildly affected COVID-19 convalescent patients revealed by two different detection methods.

Neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells via surface-expressed angiotensin-converting enzyme 2 (ACE2). We used a surrogate virus neutralization test (sVNT) and SARS-CoV-2 S protein-pseudotyped vesicular stomatitis virus (VSV) vector-based neutralization assay (pVNT) to assess the degree to which serum antibodies from coronavirus disease 2019 (COVID-19) convalescent patients interfere with the binding of SARS-CoV-2 S to ACE2. Both tests revealed neutralizing anti-SARS-CoV-2 S antibodies in the sera of ~90% of mildly and 100% of severely affected COVID-19 convalescent patients. Importantly, sVNT and pVNT results correlated strongly with each other and to the levels of anti-SARS-CoV-2 S1 IgG and IgA antibodies. Moreover, levels of neutralizing antibodies correlated with the duration and severity of clinical symptoms but not with patient age. Compared to pVNT, sVNT is less sophisticated and does not require any biosafety labs. Since this assay is also much faster and cheaper, sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.