Proximity‐dependent biotin identification (BioID) is a recently developed method that allows the identification of proteins in the close vicinity of a protein of interest in living cells. BioID relies on fusion of the protein of interest with a mutant form of the biotin ligase enzyme BirA (BirA*) that is capable of promiscuously biotinylating proximal proteins irrespective of whether these interact directly or indirectly with the fusion protein or are merely located in the same subcellular neighborhood. The covalent addition of biotin allows the labeled proteins to be purified from cell extracts on the basis of their affinity for streptavidin and identified by mass spectrometry. To date, BioID has been successfully applied to study a variety of proteins and processes in mammalian cells and unicellular eukaryotes and has been shown to be particularly suited to the study of insoluble or inaccessible cellular structures and for detecting weak or transient protein associations. Here, we provide an introduction to BioID, together with a detailed summary of where and how the method has been applied to date, and briefly discuss technical aspects involved in the planning and execution of a BioID study.

Objectives The role of innate lymphoid cells (ILCs) in coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), is unknown. Understanding the immune response in COVID‐19 could contribute to unravel the pathogenesis and identification of treatment targets. Here, we describe the phenotypic landscape of circulating ILCs in COVID‐19 patients and identified ILC phenotypes correlated to serum biomarkers, clinical markers and laboratory parameters relevant in COVID‐19. Methods Blood samples collected from moderately (n = 11) and severely ill (n = 12) COVID‐19 patients, as well as healthy control donors (n = 16), were analysed with 18‐parameter flow cytometry. Using supervised and unsupervised approaches, we examined the ILC activation status and homing profile. Clinical and laboratory parameters were obtained from all COVID‐19 patients, and serum biomarkers were analysed with multiplex immunoassays. Results Innate lymphoid cells were largely depleted from the circulation of COVID‐19 patients compared with healthy controls. Remaining circulating ILCs revealed decreased frequencies of ILC2 in severe COVID‐19, with a concomitant decrease of ILC precursors (ILCp) in all patients, compared with controls. ILC2 and ILCp showed an activated phenotype with increased CD69 expression, whereas expression levels of the chemokine receptors CXCR3 and CCR4 were significantly altered in ILC2 and ILCp, and ILC1, respectively. The activated ILC profile of COVID‐19 patients was associated with soluble inflammatory markers, while frequencies of ILC subsets were correlated with laboratory parameters that reflect the disease severity. Conclusion This study provides insights into the potential role of ILCs in immune responses against SARS‐CoV‐2, particularly linked to the severity of COVID‐19.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and has since become a global pandemic. Pathogen-specific Abs are typically a major predictor of protective immunity, yet human B cell and Ab responses during COVID-19 are not fully understood. In this study, we analyzed Ab-secreting cell and Ab responses in 20 hospitalized COVID-19 patients. The patients exhibited typical symptoms of COVID-19 and presented with reduced lymphocyte numbers and increased T cell and B cell activation. Importantly, we detected an expansion of SARS-CoV-2 nucleocapsid protein-specific Ab-secreting cells in all 20 COVID-19 patients using a multicolor FluoroSpot Assay. Out of the 20 patients, 16 had developed SARS-CoV-2-neutralizing Abs by the time of inclusion in the study. SARS-CoV-2-specific IgA, IgG, and IgM Ab levels positively correlated with SARS-CoV-2-neutralizing Ab titers, suggesting that SARS-CoV-2-specific Ab levels may reflect the titers of neutralizing Abs in COVID-19 patients during the acute phase of infection. Last, we showed that IL-6 and C-reactive protein serum concentrations were higher in patients who were hospitalized for longer, supporting the recent observations that IL-6 and C-reactive protein could be used as markers for COVID-19 severity. Altogether, this study constitutes a detailed description of clinical and immunological parameters in 20 COVID-19 patients, with a focus on B cell and Ab responses, and describes tools to study immune responses to SARS-CoV-2 infection and vaccination.

Objectives: The role of innate lymphoid cells (ILCs) in coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is unknown. Understanding the immune response in COVID-19 could contribute to unravel the pathogenesis and identification of treatment targets. To describe the phenotypic landscape of circulating ILCs in COVID-19 patients and to identify ILC phenotypes correlated to serum biomarkers, clinical markers, and laboratory parameters relevant in COVID-19. Methods: Blood samples collected from moderately (n=11) and severely ill (n=12) COVID-19 patients as well as healthy control donors (n=16), were analyzed with 18-parameter flow cytometry. Using supervised and unsupervised approaches, we examined the ILC activation status and homing profile. Clinical and laboratory parameters were obtained from all COVID-19 patients and serum biomarkers were analyzed with multiplex immunoassays. Results: ILCs were largely depleted from the circulation of COVID-19 patients compared with healthy controls. Remaining circulating ILCs from patients revealed increased frequencies of ILC2 in moderate COVID-19, with a concomitant decrease of ILC precursors (ILCp), as compared with controls. ILC2 and ILCp showed an activated phenotype with increased CD69 expression, whereas expression levels of the chemokine receptors CXCR3 and CCR4 were significantly altered in ILC2 and ILCp, and ILC1, respectively. The activated ILC profile of COVID-19 patients was associated with soluble inflammatory markers, while frequencies of ILC subsets were correlated with laboratory parameters that reflect the disease severity. Conclusion: This study provides insights into the potential role of ILCs in immune responses against SARS-CoV-2, particularly linked to the severity of COVID-19.

26Coronavirus disease 2019 , caused by severe acute respiratory syndrome 27 coronavirus 2 (SARS-CoV-2), emerged in late 2019 and has since become a global 28 pandemic. Pathogen-specific antibodies are typically a major predictor of protective 29immunity, yet B cell and antibody responses during COVID-19 are not fully understood. 30 Here, we analyzed antibody-secreting cell (ASC) and antibody responses in twenty 31 hospitalized COVID-19 patients. The patients exhibited typical symptoms of COVID-19, and 32 presented with reduced lymphocyte numbers and increased T cell and B cell activation. 33 Importantly, we detected an expansion of SARS-CoV-2 nucleocapsid protein-specific ASCs 34 in all twenty COVID-19 patients using a multicolor FluoroSpot assay. Out of the 20 patients, 35 16 had developed SARS-CoV-2-neutralizing antibodies by the time of inclusion in the study. 36 SARS-CoV-2-specific IgA, IgG and IgM antibody levels positively correlated with SARS-37CoV-2-neutralizing antibody titers, suggesting that SARS-CoV-2-specific antibody levels 38 may reflect the titers of neutralizing antibodies in COVID-19 patients during the acute phase 39 of infection. Lastly, we showed that interleukin 6 (IL-6) and C-reactive protein (CRP) 40 concentrations were higher in serum of patients who were hospitalized for longer, supporting 41 the recent observations that IL-6 and CRP could be used to predict COVID-19 severity. 42Altogether, this study constitutes a detailed description of clinical and immunological 43 parameters in twenty COVID-19 patients, with a focus on B cell and antibody responses, and 44 provides tools to study immune responses to SARS-CoV-2 infection and vaccination. 45 46

See the Acknowledgments section for all the members of The Karolinska COVID-19 Study Group.

Background Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic. The understanding of the transmission and the duration of viral shedding in SARS-CoV-2 infection is still limited. Objectives To assess the timeframe and potential risk of SARS-CoV-2 transmission from hospitalized COVID-19 patients in relation to antibody response. Method We performed a cross-sectional study of 36 COVID-19 patients hospitalized at Karolinska University Hospital. Patients with more than 8 days of symptom duration were sampled from airways, for PCR analysis of SARS-CoV-2 RNA and in vitro culture of replicating virus. Serum SARS-CoV-2-specific immunoglobulin G (IgG) and neutralizing antibodies titers were assessed by immunofluorescence assay (IFA) and microneutralization assay. Results SARS-CoV-2 RNA was detected in airway samples in 23 patients (symptom duration median 15 days, range 9–53 days), whereas 13 patients were SARS-CoV-2 RNA negative (symptom duration median 21 days, range 10–37 days). Replicating virus was detected in samples from 4 patients at 9–16 days. All but two patients had detectable levels of SARS-CoV-2-specific IgG in serum, and SARS-CoV-2 neutralizing antibodies were detected in 33 out of 36 patients. Total SARS-CoV-2-specific IgG titers and neutralizing antibody titers were positively correlated. High levels of both total IgG and neutralizing antibody titers were observed in patients sampled later after symptom onset and in patients where replicating virus could not be detected. Conclusions Our data suggest that the presence of SARS-Cov-2 specific antibodies in serum may indicate a lower risk of shedding infectious SARS-CoV-2 by hospitalized COVID-19 patients.

Tick-borne encephalitis virus (TBEV) is a flavivirus that belongs to the Flaviviridae family. TBEV is transmitted to humans primarily from infected ticks. The virus causes tick-borne encephalitis (TBE), an acute viral disease that affects the central nervous system (CNS). Infection can lead to acute neurological symptoms of significant severity due to meningitis or meningo(myelo)encephalitis. TBE can cause long-term suffering and has been recognized as an increasing public health problem. TBEV-affected areas currently include large parts of central and northern Europe as well as northern Asia. Infection with TBEV triggers a humoral as well as a cell-mediated immune response. In contrast to the well-characterized humoral antibody-mediated response, the cell-mediated immune responses elicited to natural TBEV-infection have been poorly characterized until recently. Here, we review recent progress in our understanding of the cell-mediated immune response to human TBEV-infection. A particular emphasis is devoted to studies of the response mediated by natural killer (NK) cells and CD8 T cells. The studies described include results revealing the temporal dynamics of the T cell- as well as NK cell-responses in relation to disease state and functional characterization of these cells. Additionally, we discuss specific immunopathological aspects of TBEV-infection in the CNS.