Single-cell analyses and host genetics highlight the role of innate immune cells in COVID-19 severity.

Mechanisms underpinning the dysfunctional immune response in severe acute respiratory syndrome coronavirus 2 infection are elusive. We analyzed single-cell transcriptomes and T and B cell receptors (BCR) of >895,000 peripheral blood mononuclear cells from 73 coronavirus disease 2019 (COVID-19) patients and 75 healthy controls of Japanese ancestry with host genetic data. COVID-19 patients showed a low fraction of nonclassical monocytes (ncMono). We report downregulated cell transitions from classical monocytes to ncMono in COVID-19 with reduced CXCL10 expression in ncMono in severe disease. Cell-cell communication analysis inferred decreased cellular interactions involving ncMono in severe COVID-19. Clonal expansions of BCR were evident in the plasmablasts of patients. Putative disease genes identified by COVID-19 genome-wide association study showed cell type-specific expressions in monocytes and dendritic cells. A COVID-19-associated risk variant at the IFNAR2 locus (rs13050728) had context-specific and monocyte-specific expression quantitative trait loci effects. Our study highlights biological and host genetic involvement of innate immune cells in COVID-19 severity.

Clinical Characteristics and Risk Prediction Score in Patients With Mild-to-Moderate Coronavirus Disease 2019 in Japan.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has rapidly spread worldwide, causing widespread mortality. Many patients with COVID-19 have been treated in homes, hotels, and medium-sized hospitals where doctors were responsible for assessing the need for critical care hospitalization. This study aimed to establish a severity prediction score for critical care triage. METHOD: We analyzed the data of 368 patients with mild-to-moderate COVID-19 who had been admitted to Fussa Hospital, Japan, from April 2020 to February 2022. We defined a high-oxygen group as requiring ≥4 l/min of oxygen. Multivariable logistic regression was used to construct a risk prediction score, and the best model was selected using a stepwise selection method. RESULTS: Multivariable analysis showed that older age (≥70 years), elevated creatine kinase (≥127 U/L), C-reactive protein (≥2.19 mg/dL), and ferritin (≥632.7 ng/mL) levels were independent risk factors associated with the high-oxygen group. Each risk factor was assigned a score ranging from 0 to 4, and we referred to the final overall score as the Fussa score. Patients were classified into two groups, namely, high-risk (total risk factors, ≥2) and low-risk (total risk score, <2) groups. The high-risk group had a significantly worse prognosis (low-risk group, undefined vs. high-risk group, undefined; P< 0.0001). CONCLUSIONS: The Fussa score might help to identify patients with COVID-19 who require critical care hospitalization.

A sex-biased imbalance between Tfr, Tph, and atypical B cells determines antibody responses in COVID-19 patients.

Sex-biased humoral immune responses to COVID-19 patients have been observed, but the cellular basis for this is not understood. Using single-cell proteomics by mass cytometry, we find disrupted regulation of humoral immunity in COVID-19 patients, with a sex-biased loss of circulating follicular regulatory T cells (cTfr) at a significantly greater rate in male patients. In addition, a male sex-associated cellular network of T-peripheral helper, plasma blasts, proliferating and extrafollicular/atypical CD11c+ memory B cells was strongly positively correlated with neutralizing antibody concentrations and negatively correlated with cTfr frequency. These results suggest that sex-specific differences to the balance of cTfr and a network of extrafollicular antibody production-associated cell types may be a key factor in the altered humoral immune responses between male and female COVID-19 patients.

Efficacy and risk of mRNA vaccination in patients with autoimmune inflammatory rheumatic diseases.

Coronavirus disease 2019 (COVID-19), which spread worldwide from Wuhan, China, in 2019, appeared for a time to be overcome by the remarkable efficacy of mRNA vaccines; however, new variants of severe acute respiratory syndrome coronavirus 2 have emerged and remain rampant. The involvement of the virus in the emergence of variant strains and the relationship between vaccine efficacy and immunosuppressive drugs have attracted significant attention, particularly with regard to patients with autoimmune inflammatory rheumatic disease (AIRD) who take immunosuppressive drugs. This review outlines the relationship between mRNA vaccines, one of the key strategies against COVID-19, and AIRD and discusses the immune response elicited by mRNA vaccines. Furthermore, the impact of immunosuppressive agents on the mRNA vaccine-induced immune response in patients with AIRD and side effects of the vaccine, such as exacerbation of the underlying disease, is outlined.

Persistence of SARS-CoV-2 neutralizing antibodies and anti-Omicron IgG induced by BNT162b2 mRNA vaccine in patients with autoimmune inflammatory rheumatic disease: an explanatory study in Japan.

Background: Autoimmune inflammatory rheumatic disease (AIRD) patients are at high risk of the coronavirus disease 2019 (COVID-19), but the medium-term effects of immunosuppressants on vaccine efficacy are unknown. We investigated the duration of humoral responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wild-type and Omicron variant in AIRD patients administered with two doses of the BNT162b2 (Pfizer-BioNTech) vaccine. Methods: Serum-neutralizing antibody (NAb) and anti-receptor-binding domain (RBD)/spike antibody levels were measured. Short- and medium-term effects of immunosuppressants were analyzed pre-vaccination (Term 1) and 14-42 days (Term 2) and 100-200 days (Term 3) after the second vaccination. Findings: From Feb 1, 2021, to Feb 28, 2022, 439 AIRD patients and 146 healthy controls were investigated. The seropositivity rate and log10-NAb titers were significantly lower in AIRD patients than in controls at Terms 2 and 3. In rheumatoid arthritis patients, tumor necrosis factor-α inhibitors (TNFis) at Term 3, and older age, glucocorticoids, and abatacept at Terms 2 and 3 were risk factors for reduced responses. Anti-Omicron RBD/spike IgG levels strongly correlated with NAb titers. Interpretation: Glucocorticoids, TNFis, and abatacept treatments negatively affect the longevity of humoral responses to SARS-CoV-2, including Omicron, after two vaccine doses. These findings may inform the timing of additional vaccination for AIRD patients. Funding: Cloud Funding of Peace Winds Japan; Center of Innovation Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Japan Society for the Promotion of Science KAKENHI; Japan Agency for Medical Research and Development; Kansai Economic Federation; Mitsubishi Zaidan; and Research Grant from Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology.

Clinical Characteristics and Risk Prediction Score in Patients With Mild-to-Moderate Coronavirus Disease 2019 in Japan

Background: Coronavirus disease 2019 (COVID-19) has rapidly spread worldwide, causing widespread mortality. Many patients with COVID-19 have been treated in homes, hotels, and medium-sized hospitals where doctors were responsible for assessing the need for critical care hospitalization. This study aimed to establish a severity prediction score for critical care triage. Method: We analyzed the data of 368 patients with mild-to-moderate COVID-19 who had been admitted to Fussa Hospital, Japan, from April 2020 to February 2022. We defined a high-oxygen group as requiring ≥4 l/min of oxygen. Multivariable logistic regression was used to construct a risk prediction score, and the best model was selected using a stepwise selection method. Results: Multivariable analysis showed that older age (≥70 years), elevated creatine kinase (≥127 U/L), C-reactive protein (≥2.19 mg/dL), and ferritin (≥632.7 ng/mL) levels were independent risk factors associated with the high-oxygen group. Each risk factor was assigned a score ranging from 0 to 4, and we referred to the final overall score as the Fussa score. Patients were classified into two groups, namely, high-risk (total risk factors, ≥2) and low-risk (total risk score, <2) groups. The high-risk group had a significantly worse prognosis (low-risk group, undefined vs. high-risk group, undefined;P< 0.0001). Conclusions: The Fussa score might help to identify patients with COVID-19 who require critical care hospitalization.

Next-generation proteomics of serum extracellular vesicles combined with single-cell RNA sequencing identifies MACROH2A1 associated with refractory COVID-19.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is widespread; however, accurate predictors of refractory cases have not yet been established. Circulating extracellular vesicles, involved in many pathological processes, are ideal resources for biomarker exploration. METHODS: To identify potential serum biomarkers and examine the proteins associated with the pathogenesis of refractory COVID-19, we conducted high-coverage proteomics on serum extracellular vesicles collected from 12 patients with COVID-19 at different disease severity levels and 4 healthy controls. Furthermore, single-cell RNA sequencing of peripheral blood mononuclear cells collected from 10 patients with COVID-19 and 5 healthy controls was performed. RESULTS: Among the 3046 extracellular vesicle proteins that were identified, expression of MACROH2A1 was significantly elevated in refractory cases compared to non-refractory cases; moreover, its expression was increased according to disease severity. In single-cell RNA sequencing of peripheral blood mononuclear cells, the expression of MACROH2A1 was localized to monocytes and elevated in critical cases. Consistently, single-nucleus RNA sequencing of lung tissues revealed that MACROH2A1 was highly expressed in monocytes and macrophages and was significantly elevated in fatal COVID-19. Moreover, molecular network analysis showed that pathways such as "estrogen signaling pathway," "p160 steroid receptor coactivator (SRC) signaling pathway," and "transcriptional regulation by STAT" were enriched in the transcriptome of monocytes in the peripheral blood mononuclear cells and lungs, and they were also commonly enriched in extracellular vesicle proteomics. CONCLUSIONS: Our findings highlight that MACROH2A1 in extracellular vesicles is a potential biomarker of refractory COVID-19 and may reflect the pathogenesis of COVID-19 in monocytes.

Consecutive BNT162b2 mRNA vaccination induces short-term epigenetic memory in innate immune cells.

Consecutive mRNA vaccinations against SARS-CoV-2 reinforced both innate and adaptive immune responses. However, it remains unclear whether the enhanced innate immune responses are mediated by epigenetic regulation and, if so, whether these effects persist. Using mass cytometry, RNA-seq, and ATAC-seq, we show that BNT162b2 mRNA vaccination upregulated antiviral and IFN-stimulated gene expression in monocytes with greater effects after the second vaccination than those after the first vaccination. Transcription factor-binding motif analysis also revealed enriched IFN regulatory factors and PU.1 motifs in accessible chromatin regions. Importantly, although consecutive BNT162b2 mRNA vaccinations boosted innate immune responses and caused epigenetic changes in isolated monocytes, we showed that these effects occur only transiently and disappear 4 weeks after the second vaccination. Furthermore, single-cell RNA sequencing analysis revealed that a similar gene signature was impaired in the monocytes of unvaccinated COVID-19 patients with acute respiratory distress syndrome. These results reinforce the importance of the innate immune response in the determination of COVID-19 severity but indicate that, unlike adaptive immunity, innate immunity is not unexpectedly sustained even after consecutive vaccination. This study, which focuses on innate immmune memory, may provide novel insights into the vaccine development against infectious diseases.

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force.

Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection.

DOCK2 is involved in the host genetics and biology of severe COVID-19.

Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1-5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.

Marked increase of interferon-ß after BNT162b2 mRNA vaccination: a case of polyarthritis with pleurisy.

Exacerbation of rheumatic disease after vaccination against SARS-CoV-2 is being reported. However, there are only a few cases of new-onset rheumatic diseases. We present two cases of new-onset persistent polyarthritis that developed in patients after receiving the mRNA vaccine against SARS-CoV-2. One patient had bilateral pleural effusions with markedly elevated serum interferon (IFN)-ß, while the other had no effusion, with serum IFN-ß comparable with that in healthy subjects. Other cytokines were unaltered in association with effusion. Both patients responded well to treatment with 20 mg prednisolone. Although more investigations are needed, the marked increase in serum IFN-ß levels observed in the case with pleural effusion may reflect an excessive response from the innate immune system to mRNA vaccines.

Identification of conserved SARS-CoV-2 spike epitopes that expand public cTfh clonotypes in mild COVID-19 patients.

Adaptive immunity is a fundamental component in controlling COVID-19. In this process, follicular helper T (Tfh) cells are a subset of CD4+ T cells that mediate the production of protective antibodies; however, the SARS-CoV-2 epitopes activating Tfh cells are not well characterized. Here, we identified and crystallized TCRs of public circulating Tfh (cTfh) clonotypes that are expanded in patients who have recovered from mild symptoms. These public clonotypes recognized the SARS-CoV-2 spike (S) epitopes conserved across emerging variants. The epitope of the most prevalent cTfh clonotype, S864-882, was presented by multiple HLAs and activated T cells in most healthy donors, suggesting that this S region is a universal T cell epitope useful for booster antigen. SARS-CoV-2-specific public cTfh clonotypes also cross-reacted with specific commensal bacteria. In this study, we identified conserved SARS-CoV-2 S epitopes that activate public cTfh clonotypes associated with mild symptoms.

Radioligand Assay-Based Detection of Antibodies against SARS-CoV-2 in Hospital Workers Treating Patients with Severe COVID-19 in Japan.

This study aimed to clarify whether infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is prevalent among the staff of a hospital providing treatment to patients with severe coronavirus disease 2019 (COVID-19) using radioligand assay (RLA). One thousand samples from the staff of a general hospital providing treatment to patients with severe COVID-19 were assayed for SARS-CoV-2 nucleocapsid protein (N) IgG using RLA. Nine patients with COVID-19 who had been treated in inpatient settings and had already recovered were used as control subjects, and 186 blood donor samples obtained more than 10 years ago were used as negative controls. Four of the 1000 samples showed apparently positive results, and approximately 10 or more samples showed slightly high counts. Interestingly, a few among the blood donor samples also showed slightly high values. To validate the results, antibody examinations using ELISA and neutralizing antibody tests were performed on 21 samples, and chemiluminescence immunoassay (CLIA) was performed on 201 samples, both resulting in a very high correlation. One blood donor sample showed slightly positive results in both RLA and CLIA, suggesting a cross-reaction. This study showed that five months after the pandemic began in Japan, the staff of a general hospital with a tertiary emergency medical facility had an extremely low seroprevalence of the antibodies against SARS-CoV-2. Further investigation will be needed to determine whether the slightly high results were due to cross-reactions or a low titer of anti-SARS-CoV-2 antibodies. The quantitative RLA was considered sensitive enough to detect low titers of antibodies.