Plasmacytoid dendritic cells stimulated with Lactococcus lactis strain Plasma produce soluble factors to suppress SARS-CoV-2 replication.

Innate immune responses are important in the control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. We have previously found a lactic acid bacteria species, Lactococcus lactis strain Plasma (LC-Plasma), which possesses specific feature to activate plasmacytoid dendritic cells (pDCs) and thus may affect innate immune responses. Here, we investigated the impact of pDC activation by LC-Plasma on SARS-CoV-2 replication in vitro. Addition of the culture supernatant of pDCs stimulated with LC-Plasma resulted in suppression of SARS-CoV-2 replication in Vero and Calu-3 cells. We confirmed interferon-α (IFN-α) secretion in the supernatant of pDCs stimulated with LC-Plasma and induction of IFN-stimulated genes in cells treated with the pDC supernatant. Anti-IFN-α antibody impaired the suppression of SARS-CoV-2 replication by the supernatant of LC-Plasma-stimulated pDCs, suggesting that IFN-α plays an important role in the SARS-CoV-2 suppression. Our results indicate the potential of LC-Plasma to induce inhibitory responses against SARS-CoV-2 replication through pDC stimulation with IFN-α secretion.

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.

SARS-CoV-2-specific CD4+ T cell longevity correlates with Th17-like phenotype.

Determinants of memory T cell longevity following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain unknown. In addition, phenotypes associated with memory T cell longevity, antibody titers, and disease severity are incompletely understood. Here, we longitudinally analyzed SARS-CoV-2-specific T cell and antibody responses of a unique cohort with similar numbers of mild, moderate, and severe coronavirus disease 2019 cases. The half-lives of CD4+ and CD8+ T cells were longer than those of antibody titers and showed no clear correlation with disease severity. When CD4+ T cells were divided into Th1-, Th2-, Th17-, and Tfh-like subsets, the Th17-like subset showed a longer half-life than other subsets, indicating that Th17-like cells are most closely correlated with T cell longevity. In contrast, Th2- and Tfh-like T cells were more closely correlated with antibody titers than other subsets. These results suggest that distinct CD4+ T cell subsets are associated with longevity and antibody responses.

Significant role of host sialylated glycans in the infection and spread of severe acute respiratory syndrome coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been transmitted across all over the world, in contrast to the limited epidemic of genetically- and virologically-related SARS-CoV. However, the molecular basis explaining the difference in the virological characteristics among SARS-CoV-2 and SARS-CoV has been poorly defined. Here we identified that host sialoglycans play a significant role in the efficient spread of SARS-CoV-2 infection, while this was not the case with SARS-CoV. SARS-CoV-2 infection was significantly inhibited by α2-6-linked sialic acid-containing compounds, but not by α2-3 analog, in VeroE6/TMPRSS2 cells. The α2-6-linked compound bound to SARS-CoV-2 spike S1 subunit to competitively inhibit SARS-CoV-2 attachment to cells. Enzymatic removal of cell surface sialic acids impaired the interaction between SARS-CoV-2 spike and angiotensin-converting enzyme 2 (ACE2), and suppressed the efficient spread of SARS-CoV-2 infection over time, in contrast to its least effect on SARS-CoV spread. Our study provides a novel molecular basis of SARS-CoV-2 infection which illustrates the distinctive characteristics from SARS-CoV.

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.

Subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques.

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10-17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure.

On-admission SARS-CoV-2 RNAemia as a single potent predictive marker of critical condition development and mortality in COVID-19.

BACKGROUND: This study aimed to clarify how SARS-CoV-2 RNAemia is related to COVID-19 critical condition development and mortality in comparison with other predictive markers and scoring systems. METHODS: This is a retrospective cohort study conducted at Yokohama Municipal Citizen's Hospital and National Institute of Infectious Diseases. We recruited adult patients with COVID-19 admitted between March 2020 and January 2021. We compared RNAemia with clinical status on admission including scoring systems such as the 4C Mortality, CURB-65, and A-DROP, as well as the Ct value of the nasopharyngeal PCR, in predicting COVID-19 mortality and critical condition development. RESULTS: Of the 92 recruited patients (median age, 58; interquartile range, 45-71 years), 14 (14.9%) had RNAemia. These patients had an older age (median, 68 years vs. 55.5 years; p = 0.011), higher values of lactated dehydrogenase (median, 381 U/L vs. 256.5 U/L, p < 0.001), C-reactive protein (median, 10.9 mg/dL vs. 3.8 mg/dL; p < 0.001), D-dimer (median, 2.07 µg/mL vs. 1.28 µg/mL; p = 0.015), lower values of lymphocyte (median, 802/µL vs. 1007/µL, p = 0.025) and Ct of the nasopharyngeal PCR assay (median, 20.59 vs. 25.54; p = 0.021) than those without RNAemia. Univariate analysis showed RNAemia was associated with mortality (odds ratio [OR], 18.75; 95% confidence interval [CI], 3.92-89.76; area under the receiver operating characteristic curve [AUC], 0.7851; p = 0.002) and critical condition (OR, 72.00; 95% CI, 12.98-399.29; AUC, 0.8198; p < 0.001). Plus, multivariate analysis also revealed the association of RNAemia with critical condition (adjusted OR, 125.71; 95% CI, 11.47-1377.32; p < 0.001). CONCLUSION: On-admission SARS-CoV-2 RNAemia is a potent predictive marker of COVID-19 critical condition and mortality. The adjusted OR for critical condition was as high as 125.71.

Temporal maturation of neutralizing antibodies in COVID-19 convalescent individuals improves potency and breadth to circulating SARS-CoV-2 variants.

Antibody titers against SARS-CoV-2 slowly wane over time. Here, we examined how time affects antibody potency. To assess the impact of antibody maturation on durable neutralizing activity against original SARS-CoV-2 and emerging variants of concern (VOCs), we analyzed receptor binding domain (RBD)-specific IgG antibodies in convalescent plasma taken 1-10 months after SARS-CoV-2 infection. Longitudinal evaluation of total RBD IgG and neutralizing antibody revealed declining total antibody titers but improved neutralization potency per antibody to original SARS-CoV-2, indicative of antibody response maturation. Neutralization assays with authentic viruses revealed that early antibodies capable of neutralizing original SARS-CoV-2 had limited reactivity toward B.1.351 (501Y.V2) and P.1 (501Y.V3) variants. Antibodies from late convalescents exhibited increased neutralization potency to VOCs, suggesting persistence of cross-neutralizing antibodies in plasma. Thus, maturation of the antibody response to SARS-CoV-2 potentiates cross-neutralizing ability to circulating variants, suggesting that declining antibody titers may not be indicative of declining protection.

Association of HLA-DRB1*09:01 with severe COVID-19.

HLA-A, -C, -B, and -DRB1 genotypes were analyzed in 178 Japanese COVID-19 patients to investigate the association of HLA with severe COVID-19. Analysis of 32 common HLA alleles at four loci revealed a significant association between HLA-DRB1*09:01 and severe COVID-19 (odds ratio [OR], 3.62; 95% CI, 1.57-8.35; p = 0.00251 [permutation p value = 0.0418]) when age, sex, and other common HLA alleles at the DRB1 locus were adjusted. The DRB1*09:01 allele was more significantly associated with risk for severe COVID-19 compared to preexisting medical conditions such as hypertension, diabetes, and cardiovascular diseases. These results indicate a potential role for HLA in predisposition to severe COVID-19.

Potential anti-COVID-19 agents, cepharanthine and nelfinavir, and their usage for combination treatment.

Antiviral treatments targeting the coronavirus disease 2019 are urgently required. We screened a panel of already approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new agents having higher antiviral potentials than the drug candidates such as remdesivir and chroloquine in VeroE6/TMPRSS2 cells: the anti-inflammatory drug cepharanthine and human immunodeficiency virus protease inhibitor nelfinavir. Cepharanthine inhibited SARS-CoV-2 entry through the blocking of viral binding to target cells, while nelfinavir suppressed viral replication partly by protease inhibition. Consistent with their different modes of action, synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation was highlighted. Mathematical modeling in vitro antiviral activity coupled with the calculated total drug concentrations in the lung predicts that nelfinavir will shorten the period until viral clearance by 4.9 days and the combining cepharanthine/nelfinavir enhanced their predicted efficacy. These results warrant further evaluation of the potential anti-SARS-CoV-2 activity of cepharanthine and nelfinavir.

Myeloid cell dynamics correlating with clinical outcomes of severe COVID-19 in Japan.

An expanded myeloid cell compartment is a hallmark of severe coronavirus disease 2019 (COVID-19). However, data regarding myeloid cell expansion have been collected in Europe, where the mortality rate by COVID-19 is greater than those in other regions including Japan. Thus, characteristics of COVID-19-induced myeloid cell subsets remain largely unknown in the regions with low mortality rates. Here, we analyzed cellular dynamics of myeloid-derived suppressor cell (MDSC) subsets and examined whether any of them correlate with disease severity and prognosis, using blood samples from Japanese COVID-19 patients. We observed that polymorphonuclear (PMN)-MDSCs, but not other MDSC subsets, transiently expanded in severe cases but not in mild or moderate cases. Contrary to previous studies in Europe, this subset selectively expanded in survivors of severe cases and subsided before discharge, but such transient expansion was not observed in non-survivors in Japanese cohort. Analysis of plasma cytokine/chemokine levels revealed positive correlation of PMN-MDSC frequencies with IL-8 levels, indicating the involvement of IL-8 on recruitment of PMN-MDSCs to peripheral blood following the onset of severe COVID-19. Our data indicate that transient expansion of the PMN-MDSC subset results in improved clinical outcome. Thus, this myeloid cell subset may be a predictor of prognosis in cases of severe COVID-19 in Japan.