Efficacy and safety of single-dose ivermectin in mild-to-moderate COVID-19: the double-blind, randomized, placebo-controlled CORVETTE-01 trial.

Background: To investigate whether ivermectin inhibits SARS-CoV-2 proliferation in patients with mild-to-moderate COVID-19 using time to a negative COVID-19 reverse transcription-polymerase chain reaction (RT-PCR) test. Methods: CORVETTE-01 was a double-blind, randomized, placebo-controlled study (August 2020-October 2021) conducted in Japan. Overall, 248 patients diagnosed with COVID-19 using RT-PCR were assessed for eligibility. A single oral dose of ivermectin (200 µg/kg) or placebo was administered under fasting. The primary outcome was time to a negative COVID-19 RT-PCR test result for SARS-CoV-2 nucleic acid, assessed using stratified log-rank test and Cox regression models. Results: Overall, 112 and 109 patients were randomized to ivermectin and placebo, respectively; 106 patients from each group were included in the full analysis set (male [%], mean age: 68.9%, 47.9 years [ivermectin]; 62.3%, 47.5 years [placebo]). No significant difference was observed in the occurrence of negative RT-PCR tests between the groups (hazard ratio, 0.96; 95% confidence interval [CI] 0.70-1.32; p = 0.785). Median (95% CI) time to a negative RT-PCR test was 14.0 (13.0-16.0) and 14.0 (12.0-16.0) days for ivermectin and placebo, respectively; 82.1% and 84% of patients achieved negative RT-PCR tests, respectively. Conclusion: In patients with COVID-19, single-dose ivermectin was ineffective in decreasing the time to a negative RT-PCR test. Clinical Trial Registration: ClinicalTrials.gov, NCT04703205.

Studies on the Catechin Constituents of Bark of Cinnamomum sieboldii.

Screening for bioactivity related to anti-infective, anti-methicillin-resistant Staphylococcus aureus (MRSA) and anti-viral activity, led us to identify active compounds from a methanol extract of Litsea japonica (Thub.) Juss. and the hot water extract of bark of Cinnamomum sieboldii Meisn (also known as Karaki or Okinawa cinnamon). The two main components in these extracts were identified as the catechin trimers (+)-cinnamtannin B1 and pavetannin B5. Moreover, these extracts exhibited anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activity. The structures of these catechin trimers were previously determined by chemical and spectroscopic methods. Pavetanin B5 has never been reported to be isolated as a pure form and has been obtained as a mixture with another component. Although other groups have reported the putative structure of pavetannin B5, preparation of the methylated derivative of pavetannin B5 in this study allowed us to obtain the pure form for the first time as the undecamethyl derivative and confirm its exact structure. Commercially available (+)-cinnamtannin B1 and aesculitannin B (C2'-epimer of cinnamtannin B1) both of which contained pavetannin B5 as a minor component, and C. sieboldii bark extract (approx. 5/2 mixture of (+)-cinnamtannin B1/pavetannin B5) were assessed for anti-SARS-CoV-2 activity. Both C. sieboldii bark extract and commercially available aesculitannin B showed viral growth inhibitory activity.

T-Cell Response and Antibody Production Induced by the COVID-19 Booster Vaccine in Japanese Chronic Kidney Disease Patients Treated with Hemodialysis.

Humoral and cellular responses are critical in understanding immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. Here, we evaluated these responses in hemodialysis (HD) patients after the booster vaccination. SARS-CoV-2 immunoglobulin (IgG) levels, neutralizing antibody titers, and the T-SPOT®.COVID test (T-SPOT) were measured prior to, three weeks after, and three months after the booster administration. The HD group had significantly higher SARS-CoV-2 IgG levels and neutralizing antibody titers against the original strain at three weeks and three months after the booster vaccination compared to the control group, albeit the HD group had lower SARS-CoV-2 IgG levels and neutralizing antibody titers before the booster administration. Moreover, the HD group had significantly higher T-SPOT levels at all three time points compared to the control group. The HD group also had significantly higher local and systemic adverse reaction rates than the control group. By booster vaccination, HD patients could acquire more effective SARS-CoV-2-specific humoral and cellular immunity than the control group.

Correlation between anti-S IgG and neutralizing antibody titers against three live SARS-CoV-2 variants in BNT162b2 vaccine recipients.

We analyzed serially collected serum samples from healthy adults who underwent BNT162b2 vaccination to elucidate the association between spike (S)-IgG antibody titers determined by ELISA using the WHO international standard (NIBSC code 20/136) and neutralizing antibody titers against three live SARS-CoV-2 variants. This study included 53 health care workers who received two doses of the BNT162b2 vaccine. S-IgG and nucleocapsid (N)-IgG antibody titers were measured by ELISA. Neutralizing (NT) antibody responses against three variants (Wuhan D614 G: KUH003, Alpha, and Delta) were evaluated before and after the first and second vaccination. N-IgG were not detected in any serum samples. S-IgG antibody titers remarkably increased after two BNT162b2 vaccine doses in all participants. S-IgG antibody titers were strongly correlated with NT titers against three variants of live viruses: KUH003 (r = 0.86), Alpha (r = 0.72), and Delta (r = 0.84). Serum samples from participants after one dose of BNT162b2 neutralized Alpha efficiently (median titer, 113.0), but median NT titers against KUH003 and Delta variants were lower, 57.0 and 28.0, respectively (p < .01). Two doses of the BNT162b2 vaccine elicited a strong immune response in this study. The second dose was required for induction of a strong booster effect. Serum collected from BNT162b2 vaccine recipients contained significantly lower neutralizing activity against Delta than that of against KUH003 (p < .0001) and Alpha (p < .0001). If a new variant emerges, live virus-based NT titers should be examined in serum obtained from vaccine recipients to evaluate vaccine efficacy for protection against infection.

Investigation for the efficacy of COVID-19 vaccine in Japanese CKD patients treated with hemodialysis

Background Dialysis patients are predisposed to severe disease and have a high mortality rate in coronavirus disease 2019 (COVID-19) due to their comorbidities and immunocompromised conditions. Therefore, dialysis patients should be prioritized for vaccination. This study aimed to examine how long the effects of the vaccine are maintained and what factors affect antibody titers. Methods Hemodialysis patients (HD group) and age- and sex-matched non-dialysis individuals (Control group), receiving two doses of BNT162b2 vaccine, were recruited through the Japanese Society for Dialysis Therapy (JSDT) Web site in July 2021. Anti-SARS-CoV-2 immunoglobulin (IgG) (SARS-CoV-2 IgG titers) was measured before vaccination, 3 weeks after the first vaccination, 2 weeks after the second vaccination, and 3 months after the second vaccination, and was compared between Control group and HD group. Factors affecting SARS-CoV-2 IgG titers were also examined using multivariable regression analysis and stepwise regression analysis (least AIC). In addition, we compared adverse reactions in Control and HD groups and examined the relationship between adverse reactions and SARS-CoV-2 IgG titers. Results Our study enrolled 123 participants in the Control group (62.6% men, median age 67.0 years) and 206 patients in the HD group (64.1% men, median age 66.4 years). HD group had significantly lower SARS-CoV-2 IgG titers at 3 weeks after the first vaccination (p < 0.0001), 2 weeks after second vaccination (p = 0.0002), and 3 months after the second vaccination (p = 0.045) than Control group. However, the reduction rate of SARS-CoV-2 IgG titers between 2 weeks and 3 months after the second vaccination was significantly smaller in HD group than in Control (p = 0.048). Stepwise regression analysis revealed that dialysis time was identified as the significant independent factors for SARS-CoV-2 IgG titers at 2 weeks after the second vaccination in HD group (p = 0.002) and longer dialysis time resulted in higher maximum antibody titers. The incidences of fever and nausea after the second vaccination were significantly higher in the HD group (p = 0.039 and p = 0.020). Antibody titers in those with fever were significantly higher than those without fever in both groups (HD: p = 0.0383, Control: p = 0.0096). Conclusion HD patients had significantly lower antibody titers than age- and sex-matched non-dialysis individuals over 3 months after vaccination. Dialysis time was identified as a factor affecting SARS-CoV-2 IgG titers in HD group, with longer dialysis time resulting in higher maximum SARS-CoV-2 IgG titers.

Luciferase-based quantification of membrane fusion induced by SARS-CoV-2 S protein.

The ongoing COVID-19 pandemic is caused by SARS-CoV-2. Although several effective vaccines that target the Spike protein on the viral surface have been deployed, additional therapeutic agents are urgently needed. Here, we developed a system to measure the Spike protein function by quantifying cellular membrane fusion induced by the Spike protein. The system enables the evaluation of the effects of drugs and neutralizing antibodies against SARS-CoV-2 without using live viruses. Furthermore, the system characterizes membrane fusion activity of the Spike protein of each variant to reveal that Delta variant has more potent than Wuhan and Omicron. Our system could lead to develop high-throughput screening for drug candidates and neutralization antibodies that target virus entry and characterize Spike proteins from variants.

A highly photostable and bright green fluorescent protein.

The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.

Nasal delivery of single-domain antibody improves symptoms of SARS-CoV-2 infection in an animal model.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.

Direct derivation of human alveolospheres for SARS-CoV-2 infection modeling and drug screening.

Although the main cellular target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be alveolar cells, the absence of their tractable culture system precludes the development of a clinically relevant SARS-CoV-2 infection model. Here, we establish an efficient human alveolosphere culture method and sphere-based drug testing platform for SARS-CoV-2. Alveolospheres exhibit indolent growth in a Wnt- and R-spondin-dependent manner. Gene expression, immunofluorescence, and electron microscopy analyses reveal the presence of alveolar cells in alveolospheres. Alveolospheres express ACE2 and allow SARS-CoV-2 to propagate nearly 100,000-fold in 3 days of infection. Whereas lopinavir and nelfinavir, protease inhibitors used for the treatment of human immunodeficiency virus (HIV) infection, have a modest anti-viral effect on SARS-CoV-2, remdesivir, a nucleotide prodrug, shows an anti-viral effect at the concentration comparable with the circulating drug level. These results demonstrate the validity of the alveolosphere culture system for the development of therapeutic agents to combat SARS-CoV-2.