Short-Term Effectiveness of Oral Nirmatrelvir/Ritonavir Against the SARS-CoV-2 Omicron Variant and Culture-Positive Viral Shedding.

BACKGROUND: Information on the effectiveness of nirmatrelvir/ritonavir against the omicron is limited. The clinical response and viral kinetics to therapy in the real world need to be evaluated. METHODS: Mild to moderate coronavirus disease 2019 (COVID-19) patients with risk factors for severe illness were prospectively enrolled as a treatment group with nirmatrelvir/ritonavir therapy versus a control group with supportive care. Serial viral load and culture from the upper respiratory tract were evaluated for seven days, and clinical responses and adverse reactions were evaluated for 28 days. RESULTS: A total of 51 patients were analyzed including 40 in the treatment group and 11 in the control group. Faster symptom resolution during hospitalization (P = 0.048) was observed in the treatment group. Only minor adverse reactions were reported in 27.5% of patients. The viral load on Day 7 was lower in the treatment group (P = 0.002). The viral culture showed a positivity of 67.6% (25/37) vs. 100% (6/6) on Day 1, 0% (0/37) vs. 16.7 (1/6) on Day 5, and 0% (0/16) vs. 50.0% (2/4) on Day 7 in the treatment and control groups, respectively. CONCLUSIONS: Nirmatrelvir/ritonavir against the omicron was safe and resulted in negative viral culture conversion after Day 5 of treatment with better symptomatic resolution.

Can nirmatrelvir/ritonavir treatment shorten the duration of COVID-19 isolation?

Background: The impact of nirmatrelvir/ritonavir treatment on shedding of viable virus in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. Methods: A prospective cohort study evaluating mildly ill COVID-19 patients was conducted. Virologic responses were compared between nirmatrelvir/ritonavir-treatment and supportive care groups. Risk factors and relevant clinical factors for shedding of viable virus were investigated. Results: A total of 80 COVID-19 patients were enrolled and 222 sputum specimens were collected. Ten patients were dropped during follow-up, and 33 patients in the nirmatrelvir/ritonavir and 37 in the supportive care groups were compared. The median age was 67 years, and 67% were male. Clinical characteristics were similar between groups. Viral loads decreased significantly faster in the nirmatrelvir/ritonavir group compared with the supportive care group (P < 0.001), and the slope was significantly steeper (-2.99 ± 1.54 vs. -1.44 ± 1.52; P < 0.001). The duration of viable virus shedding was not statistically different between groups. In the multivariable analyses evaluating all collected specimens, male gender (OR 2.51, 95% CI 1.25-5.03, P = 0.010), symptom score (OR 1.41, 95% CI 1.07-1.87, P = 0.015), days from symptom onset (OR 0.72, 95% CI 0.59-0.88, P = 0.002), complete vaccination (OR 0.09, 95% CI 0.01-0.87, P = 0.038), and BA.2 subtype (OR 0.49, 95% CI 0.26-0.91, P = 0.025) were independently associated with viable viral shedding, while nirmatrelvir/ritonavir treatment was not. Conclusion: Nirmatrelvir/ritonavir treatment effectively reduced viral loads of SARS-CoV-2 Omicron variants but did not decrease the duration of viable virus shedding.

Discovery of New Potent anti-MERS CoV Fusion Inhibitors.

Middle East respiratory syndrome coronavirus (MERS-CoV), capable of zoonotic transmission, has been associated with emerging viral pneumonia in humans. In this study, a set of highly potent peptides were designed to prevent MERS-CoV fusion through competition with heptad repeat domain 2 (HR2) at its HR1 binding site. We designed eleven peptides with stronger estimated HR1 binding affinities than the wild-type peptide to prevent viral fusion with the cell membrane. Eight peptides showed strong inhibition of spike-mediated MERS-CoV cell-cell fusion with IC50 values in the nanomolar range (0.25-2.3 µM). Peptides #4-6 inhibited 95-98.3% of MERS-CoV plaque formation. Notably, peptide four showed strong inhibition of MERS-CoV plaques formation with EC50 = 0.302 µM. All peptides demonstrated safe profiles without cytotoxicity up to a concentration of 10 µM, and this cellular safety, combined with their anti-MERS-CoV antiviral activity, indicate all peptides can be regarded as potential promising antiviral agents.

MERS-CoV and SARS-CoV-2 replication can be inhibited by targeting the interaction between the viral spike protein and the nucleocapsid protein.

Background: The molecular interactions between viral proteins form the basis of virus production and can be used to develop strategies against virus infection. The interactions of the envelope proteins and the viral RNA-binding nucleocapsid (N) protein are essential for the assembly of coronaviruses including the Middle East respiratory syndrome coronavirus (MERS-CoV). Methods: Using co-immunoprecipitation, immunostaining, and proteomics analysis, we identified a protein interacting with the spike (S) protein in the cells infected with MERS-CoV or SARS-CoV-2. To confirm the interaction, synthetic peptides corresponding to the C-terminal domain of the S protein (Spike CD) were produced and their effect on the interaction was investigated in vitro. In vivo effect of the Spike CD peptides after cell penetration was further investigated using viral plaque formation assay. Phylogeographic analyses were conducted to deduce homology of Spike CDs and N proteins. Results: We identified a direct interaction between the S protein and the N protein of MERS-CoV that takes place during virus assembly in infected cells. Spike CD peptides of MERS-CoV inhibited the interaction between the S and N proteins in vitro. Furthermore, cell penetration by the synthetic Spike CD peptides inhibited viral plaque formation in MERS-CoV-infected cells. Phylogeographic analyses of Spike CDs and N proteins showed high homology among betacoronavirus lineage C strains. To determine if Spike CD peptides can inhibit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we used the same strategy and found that the SARS-CoV-2 Spike CD peptide inhibited virus replication in SARS-CoV-2-infected cells. Conclusions: We suggest that the interaction between the S protein and the N protein can be targeted to design new therapeutics against emerging coronaviruses, including SARS-CoV-2.

Differential Signaling and Virus Production in Calu-3 Cells and Vero Cells upon SARS-CoV-2 Infection.

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is responsible for the current coronavirus disease 2019 (COVID-19) pandemic. Signaling pathways that are essential for virus production have potential as therapeutic targets against COVID-19. In this study, we investigated cellular responses in two cell lines, Vero and Calu-3, upon SARS-CoV-2 infection and evaluated the effects of pathway-specific inhibitors on virus production. SARS-CoV-2 infection induced dephosphorylation of STAT1 and STAT3, high virus production, and apoptosis in Vero cells. However, in Calu-3 cells, SARS-CoV-2 infection induced long-lasting phosphorylation of STAT1 and STAT3, low virus production, and no prominent apoptosis. Inhibitors that target STAT3 phosphorylation and dimerization reduced SARS-CoV-2 production in Calu-3 cells, but not in Vero cells. These results suggest a necessity to evaluate cellular consequences upon SARS-CoV-2 infection using various model cell lines to find out more appropriate cells recapitulating relevant responses to SARS-CoV-2 infection in vitro.

Discovery of New Fusion Inhibitor Peptides against SARS-CoV-2 by Targeting the Spike S2 Subunit.

A novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), caused a worldwide pandemic. Our aim in this study is to produce new fusion inhibitors against SARS-CoV-2, which can be the basis for developing new antiviral drugs. The fusion core comprising the heptad repeat domains (HR1 and HR2) of SARS-CoV-2 spike (S) were used to design the peptides. A total of twelve peptides were generated, comprising a short or truncated 24-mer (peptide #1), a long 36-mer peptide (peptide #2), and ten peptide #2 analogs. In contrast to SARS-CoV, SARS-CoV-2 S-mediated cell-cell fusion cannot be inhibited with a minimal length, 24-mer peptide. Peptide #2 demonstrated potent inhibition of SARS-CoV-2 S-mediated cell-cell fusion at 1 µM concentration. Three peptide #2 analogs showed IC50 values in the low micromolar range (4.7-9.8 µM). Peptide #2 inhibited the SARS-CoV-2 pseudovirus assay at IC50=1.49 µM. Given their potent inhibition of viral activity and safety and lack of cytotoxicity, these peptides provide an attractive avenue for the development of new prophylactic and therapeutic agents against SARS-CoV-2.

A pilot study of the antiviral activity of anionic and cationic polyamidoamine dendrimers against the Middle East respiratory syndrome coronavirus.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus that causes infection with a potentially fatal outcome. Dendrimers are highly branched molecules that can be added to antiviral preparations to improve their delivery, as well as their intrinsic antiviral activity. Studies on identifying anti-MERS-CoV agents are few. Three types of polyanionic dendrimers comprising the terminal groups sodium carboxylate (generations 1.5, 2.5, 3.5, and 4.5), hydroxyl (generations 2, 3, 4, and 5), and succinamic acid (generations 2, 3, 4, and 5) and polycationic dendrimers containing primary amine (generations 2, 3, 4, and 5) were used to assess their antiviral activity with the MERS-CoV plaque inhibition assay. The hydroxyl polyanionic set showed a 17.36% to 29.75% decrease in MERS-CoV plaque formation. The most potent inhibition of MERS-CoV plaque formation was seen by G(1.5)-16COONa (40.5% inhibition), followed by G(5)-128SA (39.77% inhibition). In contrast, the cationic dendrimers were cytotoxic to Vero cells. Polyanionic dendrimers can be added to antiviral preparations to improve the delivery of antivirals, as well as the intrinsic antiviral activity.

Small Molecule Inhibitors of Middle East Respiratory Syndrome Coronavirus Fusion by Targeting Cavities on Heptad Repeat Trimers.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a newly emerging viral disease with fatal outcomes. However, no MERS-CoV-specific treatment is commercially available. Given the absence of previous structure-based drug discovery studies targeting MERS-CoV fusion proteins, this set of compounds is considered the first generation of MERS-CoV small molecule fusion inhibitors. After a virtual screening campaign of 1.56 million compounds followed by cell-cell fusion assay and MERS-CoV plaques inhibition assay, three new compounds were identified. Compound numbers 22, 73, and 74 showed IC50 values of 12.6, 21.8, and 11.12 µM, respectively, and were most effective at the onset of spike-receptor interactions. The compounds exhibited safe profiles against Human embryonic kidney cells 293 at a concentration of 20 µM with no observed toxicity in Vero cells at 10 µM. The experimental results are accompanied with predicted favorable pharmacokinetic descriptors and drug-likeness parameters. In conclusion, this study provides the first generation of MERS-CoV fusion inhibitors with potencies in the low micromolar range.