B cell receptor repertoire analysis from autopsy samples of COVID-19 patients

Neutralizing antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being developed world over. We investigated the possibility of producing artificial antibodies from the formalin fixation and paraffin-embedding (FFPE) lung lobes of a patient who died by coronavirus disease 2019 (COVID-19). The B-cell receptors repertoire in the lung tissue where SARS-CoV-2 was detected were considered to have highly sensitive virus-neutralizing activity, and artificial antibodies were produced by combining the most frequently detected heavy and light chains. Some neutralizing effects against the SARS-CoV-2 were observed, and mixing two different artificial antibodies had a higher tendency to suppress the virus. The neutralizing effects were similar to the immunoglobulin G obtained from healthy donors who had received a COVID-19 mRNA vaccine. Therefore, the use of FFPE lung tissue, which preserves the condition of direct virus sensitization, to generate artificial antibodies may be useful against future unknown infectious diseases.

B cell receptor repertoire analysis from autopsy samples of COVID-19 patients.

Neutralizing antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being developed world over. We investigated the possibility of producing artificial antibodies from the formalin fixation and paraffin-embedding (FFPE) lung lobes of a patient who died by coronavirus disease 2019 (COVID-19). The B-cell receptors repertoire in the lung tissue where SARS-CoV-2 was detected were considered to have highly sensitive virus-neutralizing activity, and artificial antibodies were produced by combining the most frequently detected heavy and light chains. Some neutralizing effects against the SARS-CoV-2 were observed, and mixing two different artificial antibodies had a higher tendency to suppress the virus. The neutralizing effects were similar to the immunoglobulin G obtained from healthy donors who had received a COVID-19 mRNA vaccine. Therefore, the use of FFPE lung tissue, which preserves the condition of direct virus sensitization, to generate artificial antibodies may be useful against future unknown infectious diseases.

Multimodal single-cell analyses of peripheral blood mononuclear cells of COVID-19 patients in Japan.

SARS-CoV-2 continues to spread worldwide. Patients with COVID-19 show distinct clinical symptoms. Although many studies have reported various causes for the diversity of symptoms, the underlying mechanisms are not fully understood. Peripheral blood mononuclear cells from COVID-19 patients were collected longitudinally, and single-cell transcriptome and T cell receptor repertoire analysis was performed. Comparison of molecular features and patients' clinical information revealed that the proportions of cells present, and gene expression profiles differed significantly between mild and severe cases; although even among severe cases, substantial differences were observed among the patients. In one severely-infected elderly patient, an effective antibody response seemed to have failed, which may have caused prolonged viral clearance. Naïve T cell depletion, low T cell receptor repertoire diversity, and aberrant hyperactivation of most immune cell subsets were observed during the acute phase in this patient. Through this study, we provided a better understanding of the diversity of immune landscapes and responses. The information obtained from this study can help medical professionals develop personalized optimal clinical treatment strategies for COVID-19.

Single-cell analytical technologies: uncovering the mechanisms behind variations in immune responses.

The immune landscape varies among individuals. It determines the immune response and results in surprisingly diverse symptoms, even in response to similar external stimuli. However, the detailed mechanisms underlying such diverse immune responses have remained mostly elusive. The utilization of recently developed single-cell multimodal analysis platforms has started to answer this question. Emerging studies have elucidated several molecular networks that may explain diversity with respect to age or other factors. An elaborate interplay between inherent physical conditions and environmental conditions has been demonstrated. Furthermore, the importance of modifications by the epigenome resulting in transcriptome variation among individuals is gradually being revealed. Accordingly, epigenomes and transcriptomes are direct indicators of the medical history and dynamic interactions with environmental factors. Coronavirus disease 2019 (COVID-19) has recently become one of the most remarkable examples of the necessity of in-depth analyses of diverse responses with respect to various factors to improve treatment in severe cases and to prevent viral transmission from asymptomatic carriers. In fact, determining why some patients develop serious symptoms is still a pressing issue. Here, we review the current "state of the art" in single-cell analytical technologies and their broad applications to healthy individuals and representative diseases, including COVID-19.

Intensive single-cell analysis reveals immune-cell diversity among healthy individuals.

Immune responses are different between individuals and personal health histories and unique environmental conditions should collectively determine the present state of immune cells. However, the molecular systems underlying such heterogeneity remain elusive. Here, we conducted a systematic time-lapse single-cell analysis, using 171 single-cell libraries and 30 mass cytometry datasets intensively for seven healthy individuals. We found substantial diversity in immune-cell profiles between different individuals. These patterns showed daily fluctuations even within the same individual. Similar diversities were also observed for the T-cell and B-cell receptor repertoires. Detailed immune-cell profiles at healthy statuses should give essential background information to understand their immune responses, when the individual is exposed to various environmental conditions. To demonstrate this idea, we conducted the similar analysis for the same individuals on the vaccination of influenza and SARS-CoV-2. In fact, we detected distinct responses to vaccines between individuals, although key responses are common. Single-cell immune-cell profile data should make fundamental data resource to understand variable immune responses, which are unique to each individual.

Correlation Analysis between Gut Microbiota Alterations and the Cytokine Response in Patients with Coronavirus Disease during Hospitalization.

The role of the intestinal microbiota in coronavirus disease 2019 (COVID-19) is being elucidated. Here, we analyzed the temporal changes in microbiota composition and the correlation between inflammation biomarkers/cytokines and microbiota in hospitalized COVID-19 patients. We obtained stool specimens, blood samples, and patient records from 22 hospitalized COVID-19 patients and performed 16S rRNA metagenomic analysis of stool samples over the course of disease onset compared to 40 healthy individual stool samples. We analyzed the correlation between the changes in the gut microbiota and plasma proinflammatory cytokine levels. Immediately after admission, differences in the gut microbiota were observed between COVID-19 patients and healthy subjects, mainly including enrichment of the classes Bacilli and Coriobacteriia and decrease in abundance of the class Clostridia. The bacterial profile continued to change throughout the hospitalization, with a decrease in short-chain fatty acid-producing bacteria including Faecalibacterium and an increase in the facultatively anaerobic bacteria Escherichia-Shigella. A consistent increase in Eggerthella belonging to the class Coriobacteriia was observed. The abundance of the class Clostridia was inversely correlated with interferon-γ level and that of the phylum Actinobacteria, which was enriched in COVID-19, and was positively correlated with gp130/sIL-6Rb levels. Dysbiosis was continued even after 21 days from onset. The intestines tended to be an aerobic environment in hospitalized COVID-19 patients. Because the composition of the gut microbiota correlates with the levels of proinflammatory cytokines, this finding emphasizes the need to understand how pathology is related to the temporal changes in the specific gut microbiota observed in COVID-19 patients. IMPORTANCE There is growing evidence that the commensal microbiota of the gastrointestinal and respiratory tracts regulates local and systemic inflammation (gut-lung axis). COVID-19 is primarily a respiratory disease, but the involvement of microbiota changes in the pathogenesis of this disease remains unclear. The composition of the gut microbiota of patients with COVID-19 changed over time during hospitalization, and the intestines tended to be an aerobic environment in hospitalized COVID-19 patients. These changes in gut microbiota may induce increased intestinal permeability, called leaky gut, allowing bacteria and toxins to enter the circulatory system and further aggravate the systemic inflammatory response. Since gut microbiota composition correlates with levels of proinflammatory cytokines, this finding highlights the need to understand how pathology relates to the gut environment, including the temporal changes in specific gut microbiota observed in COVID-19 patients.