Montelukast and COVID-19-related complications in people with SARS-CoV-2 infection: A population-based observational study

Objective: We explore the association between montelukast use and the risk of developing COVID-19 complications in people with confirmed or suspected COVID- 19. Material and/or methods: Study based on electronic health records from the SIDIAP database, which includes 5,835,000 people in Catalonia (80% of total population). In a cohort of people with confirmed or suspected COVID-19 (diagnosis registry, positive PCR and/or a serologic test or non-confirmed diagnosis or test along with a record of hospitalization, pneumonia and/or death related to COVID-19), we identified people on montelukast treatment (an active prescription or a prescription ending 90 days before the index date) (confirmed or suspected Covid register) from March to June 2020 (exposed group). The non-exposed to montelukast cohort was built by pairing cases in a 1:4 ratio based on gender and age at the time of infection. We obtained socioeconomic risk factors and active comorbidities in the two previous years, and concomitant drugs. The COVID-19-related severe events analysed were hospitalization, pneumonia, death or any of the previous outcomes. For each outcome, we fitted a conditional logistic regression model to estimate the odds ratio with its 95% confidence interval (OR;CI95%) associated with montelukast use. The study was approved by the Clinical Research Ethics Committee IDIAPJGol. Results: During the study period, there were 183 (6.67%) hospitalizations in the exposed group and 554 (6.09%) in the non-exposed group, 40 (1.46%) and 176 (1.93%) pneumonias, 85 (3.1%) and 429 (4.72%) deaths and 258 (9.40%) and 996 (10.95%) of any of the outcomes, respectively. The corresponding multivariable ORs were 0.92 (CI95% 0.70-1.21) for hospitalization, 0.67 (0.44-1.00) for pneumonia, 0.79 (0.60-1.031) for death and 0.79 (0.63-0.99) for any of the outcomes. Conclusions: The results suggest that montelukast use could decrease the risk of complications in people with COVID-19.

Cetylpyridinium Chloride Mouthwash to Reduce Shedding of Infectious SARS-CoV-2: A Double-Blind Randomized Clinical Trial.

The airborne transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via respiratory fluids and droplets suggests that mouthwashes containing substances with virucidal activity can help reduce viral spread. We conducted a multicenter, double-blind, placebo-controlled, randomized trial to assess the virucidal activity of cetylpyridinium chloride (CPC) mouthwashes. Outpatients who tested positive for SARS-CoV-2 infection with or without symptoms were randomized to perform washes and gargles for 1 min with 15 mL of either colored distilled water or 0.07% CPC (Vitis CPC Protect) mouthwash. The study outcomes were the SARS-CoV-2 log10 viral RNA load and the nucleocapsid protein levels, both in saliva at 1 and 3 h after the intervention. In total, 118 patients were enrolled and randomized (mean [SD], age 46 [14] y). Thirteen of 118 participants (11%) did not complete follow-up or had insufficient sample volume for testing and were excluded from the analysis. The assessment of the viral load showed no significant differences between groups at any of the investigated points. However, the levels of SARS-CoV-2 nucleocapsid protein of lysed viruses were significantly higher in the CPC group compared with the control group at 1 h (adjusted difference 269.3 pg/mL; 95% confidence interval [CI], 97.1-441.5) and at 3 h postintervention (561.1 pg/mL; 95% CI, 380.0-742.2). In nonhospitalized patients with asymptomatic or mild symptomatic SARS-CoV-2 infection, a 0.07% CPC mouthwash, compared to placebo, was associated with a significant increase of nucleocapsid protein levels in saliva, indicating enhanced disruption of viral particles.

CETYLPYRIDINIUM CHLORIDE MOUTHWASHES to REDUCE the SHEDDING of VIABLE SARS-CoV-2

Background: SARS-CoV-2 is spread via airborne transmission. Mouthwashes containing virucidal compounds can help reduce viral spread. Here we show that cetylpyridinium chloride (CPC), a quaternary ammonium present in many oral mouthwashes, reduces SARS-CoV-2 infectivity by disrupting viral membranes both in vitro and in vivo. Methods: We tested the capacity of CPC-containing mouthwashes to inhibit SARS-CoV-2 entry into target cells by using a luciferase-based assay with a reporter lentivirus pseudotyped with the SARS-CoV-2 spike protein. The replication-competent SARS-CoV-2 B.1.1.7 and D614G variants were also assayed. Viral envelope disruption by CPC's virucidal effect was measured by dynamic light-scattering analyses (DSL). We confirmed these results by modifying an ELISA that detects the SARS-CoV-2 nucleocapsid (NC), which was used in the absence of its own lysis buffer. The effect of CPC in the saliva of individuals with CoVID-19 was assessed in a double-blind, placebo-controlled, randomized clinical trial. SARS-CoV-2 positive patients were randomized to gargle either water or 0.07% CPC mouthwash. The study outcomes were the SARS-CoV-2 log10 viral RNA load by RT-PCR and the NC protein levels by ELISA, both in saliva at 1h and 3h post-intervention. Results: CPC-containing mouthwashes inhibited SARS-CoV-2 viral fusion in vitro in a dose-dependent manner and decreased more than a 1000 times the viral TCID50 in target cells, regardless of the variant tested. The ELISA and the DSL analyses pointed to the effective disruption of the integrity of viral membranes after treatment with CPC. The clinical study performed with 105 patients showed no significant differences in viral RNA load at 1h and 3h post-treatment in saliva between placebo and CPC-treated groups. However, the levels of SARS-CoV-2 NC protein of lysed viruses were significantly higher in the CPC group at 1h and 3h post-intervention. Conclusion: CPC decreased more than a 1000 times the infectivity of SARS-CoV-2 in vitro and was effective against different SARS-CoV-2 variants. In CoVID-19 patients, the use of a 0.07% CPC mouthwash correlated with a statistically significant increase of NC protein levels in saliva, indicating enhanced disruption of viral particles. CPC-containing mouth rinses can represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals regardless of the variants they are infected with.

Early core signature of soluble factors in mild to moderate SARSCOV-2 infection

Background: Understanding the kinetics of early immune responses to SARSCoV-2 infection is critical to identify potentials biomarkers of disease outcome. A myriad of soluble mediators including, pro-inflammatory, immune-suppressors and growth factors, play a relevant role in the disease progression. However, to date, limited data is available about the role of soluble factors and most studies focus only in severe cases with limited follow-up. Here, we studied with high resolution the kinetics of soluble mediators in mild to moderate cases of SARSCoV-2 infection 1-90 days from symptom onset (DfSO). Methods: We selected individuals from the ProHEpiC-19 cohort study that included mainly healthcare workers with a PCR+ and mild or moderate disease within 1-14 DfSO. IgG and IgM levels were determined by ELISA. We selected plasma samples (n=30) in the range of 1-90 DfSO, and performed a Luminex multiplex assay including 45 soluble human factors. Results: We identified a core signature including 19 highly correlated soluble factors at 1-14 DfSO, based on clustering analysis. The core signature contained three sub-clusters: #1 (RANTES, IL13, TGFa, PDGF-AB, PDGF-AA, EGF, MIP1b, CD40L and GROb), #2 (G-CSF, PDL1-B7, Fractalkine, IL8, IFNg, Granzyme B and IL10) and #3 (IL7, IL6, and VEGF). We found major changes in #2 and #3 cluster composition between 1-14 and 30-45 DfSO, due to the loss of PDL1-B7, Fractalkine, IL8, IL7, IL6, and VEGF association. Moreover, by 60-75 DfSO, the soluble factor association in #2 and #3 disappeared from the core signature. In addition, we observed a negative correlation between IgG and IgM levels with IL4 production at 1-14 DfSO (IgG: ρ =-0.82, p=0.012;IgM ρ=-0.83, p=0.011). Similarly, a negative correlation was observed between Igs and Mip3a at 30-45 DfSO (IgG: ρ=-0.78, p=0.023;IgM: ρ =-0.81, p=0.022). Conclusion: We delineated a core signature of soluble factors in mild to moderate SARS-CoV-2 infection, including growth factors, chemokines and pro-inflammatory cytokines. The longitudinal follow-up of this signature revealed significant changes during the 1-90 DfSO. This information can provide new insights for the definition of biomarkers for patient stratification in mild or moderate SARS-Cov-2 infection. Further data is needed to understand the association between IL4 and Mip3a with low Igs levels.

High-resolution mapping of T-cell immunity to the entire sarscov-2 proteome

Background: Since the discovery of SARS-CoV-2, researchers have put major efforts towards the understanding of virus-specific cellular immunity. However, the identification of epitope-and protein specific T-cell responses is limited to bioinformatic approaches, use of total viral proteins or peptide mega pools. To overcome these current limitations, we performed a high-resolution mapping using IFN-y ELISpot and peptide sets covering the entire CoV-2 proteome. Methods: We synthetized a 15-mer peptide library of 2790 peptides (11 amino acid overlap) covering a CoV-2cons proteome sequence based on 1700 sequences. We designed a mega matrix of consecutive and non-consecutive peptide pools with 20 to 35 peptides per pool. We assessed T-cell responses in cryopreserved PBMCs from IgG+ SARS-CoV-2 infected individuals (N=13), who recovered from mild/moderate infection, 90-190 Days from off-set symptoms. Also, we expanded PBMCs in the presence of anti-CD3 and IL-2 during 3 weeks and performed a comparative ELISpot using total and expanded PBMCs. Results: Frequencies of T-cell responses from positive peptide pools revealed 40% of responses targeting S2, 20% against S1, 10% against M, and 6% against nsp3 and NP, respectively. The strongest responses were targeting S2 and S1 (median values of 540 and 240 IFN-y SFC/106, respectively), followed by nsp3, NP and M. We observed a median of 13 deconvoluted reactive peptides across the entire proteome per tested individual. The breadth of responses ranged from 1-8 targeted proteins with a median of 2. In addition, we mapped responses in subproteins 3C-LP, nsp6, nsp10 (Orf1ab), and alternative reading frames. We also identified responses to peptide sequences conserved across pan-coronavirus strains Orf1b (n=2), S (n=1) and M (n=1). Following expansion, we observed a loss of CD4+ T-cells in cultured cells and altered peptide-recognition profiles characterized by a loss of S2 and an increase of nsp3 responses. Conclusion: We characterize protein hierarchy in terms of breadth and magnitude by high-resolution mapping of T-cell responses against the entire CoV-2 proteome. The most frequently targeted and immunogenic regions were S2 and S1. We identify responses to small proteins, alternative reading frames and conserved regions across coronaviruses. This data brings new insight into the complexity of CoV-2 T-cell responses and crucial information for vaccine design.