Fluvoxamine with bromhexine combination to prevent clinical deterioration in SARS-CoV-2

Introduction/Aim: We conducted an open label, randomized, controlled trial to assess whether fluvoxamine combined with bromhexine, given during mild to moderate SARS-CoV-2 illness, prevented clinical deterioration due to their proposed immune modulatory effects. Method(s): Participants had confirmed SARS-CoV-2 infection, experiencing mild to moderate symptoms and oxygen saturation of >=92%. Participants were randomly assigned to receive fluvoxamine (100 mg days 1 and 2, followed by 150 mg daily till day 14) with bromhexine (FLU/BRO) (16 mg daily till day 10) or favipiravir alone (FAV) (3600 mg day 1 followed by 1600 mg daily till day 5). Primary outcome was clinical deterioration within 30 days of randomization defined as shortness of breath or hospitalization for shortness of breath or pneumonia and oxygen saturation >=92%, on room air or need for supplemental oxygen to achieve oxygen saturation of >=92%. Result(s): 158 participants were randomized (average age 50 years (range 18-68 years);68 [43%] women), and 142 (89%) completed the trial. 0/78 participants experience clinical deterioration with FLU/BRO and 18/64 patients with FAV. TNF-alpha, IL-6 IL-8 and IL-1beta levels were significantly (p < 0.005) reduced with FLU/BRO compared to FAV at day 3, 5, 7 and 14. 0/78 participants had long COVID symptoms with FLU/BRO compared to 32/64 (50%) with FAV (p < 0.005). One serious (clumsiness or unsteadiness) and 10 other adverse events were reported with FLU/BRO compared to 5 serious and 12 other adverse events with FAV. Conclusion(s): Results suggest there was significantly less clinical deterioration in symptomatic COVID-19 participants treated with FLU/BRO.

Interferon 1beta-1a ring prophylaxis to reduce household transmission of SARS-CoV-2

Introduction/Aim: Accumulating evidence indicates that an early, robust type 1 interferon (IFN) response to SARS-CoV-2 is critical for COVID-19 outcomes. Our objective was to examine the prophylactic potential of IFN treatment to limit viral transmission Methods: A cluster-randomised clinical trial was undertaken to determine effects of IFNbeta-1a treatment on SARS-CoV-2 household transmission (clinicaltrials.gov: NCT04552379). Index cases were identified from confirmed SARS-CoV-2 cases in Santiago, Chile, with 341 index cases and 831 household contacts enrolled. Households received 125 mug subcutaneous pegylated-IFNbeta-1a on days 1, 6, & 11 (172 households, 607 participants), or standard care (169 households, 565 participants). Primary outcomes included: (1) duration of viral shedding in infected cases (IC-INF), (2) transmission to treatment-eligible household contacts (EHC-ITT) at day 11. Result(s): Of 1172 individuals randomised, 53 individuals withdrew from the study (IFNbeta-1a = 35, SOC = 18). Eighty-two households (IFNbeta-1a = 36, SOC = 46) where the index case was SARS-CoV-2 negative on days 1 & 6, or with no SARS-CoV-2 negative contacts at recruitment, were excluded from exploratory analyses. Treatment with IFNbeta-1a: had no effect on duration of viral shedding in the IC-INF population (primary outcome 1), had no effect on transmission of SARS-CoV-2 at day 11 in the EHC-ITT population (primary outcome 2) but an effect was observed in a sensitivity analysis at day 6 (EHC-ITT: OR = 0.493, 95% CI = 0.256-0.949), reduced duration of hospitalisation in the IC-INF population and incidence of hospitalisation in per-protocol index cases (secondary outcome 3). In exploratory frequentist analysis, a significant effect of IFNbeta-1a treatment on SARS-CoV-2 transmission by day 11 (OR = 0.55, 95% CI = 0.36-0.99), and a Bayesian analysis identified a significant reduction in the odds of transmission (OR = -0.85, 95% credible interval = -1.59--0.16). Conclusion(s): Ring prophylaxis with IFNbeta-1a had no effect on duration of viral shedding but reduces the probability of SARS-CoV-2 transmission to uninfected, post-exposure contacts within a household.

SARS-CoV-2 spike fails to induce inflammation in paediatric airway cells

Introduction/Aim: The spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enables it to recognise and bind host receptors. These dynamics have been modelled in various cell types and immortalised lines, but rarely in primary airway epithelial cells (AEC), and especially not in children. Therefore, this study on AEC recapitulated earlier work testing the hypothesis that exposure to the spike protein would induce airway immune responses in airway cells of young children. Method(s): Primary AEC monolayer cultures from healthy children (n = 5, <10 years old, males = 5) were exposed to the spike protein S1 subunit (0.01, 1, and 10 mug/mL) over 48 h. Induced inflammatory cytokines, interleukin (IL) 6 and IL8, and viral-associated chemokines, CCL5 and CXCL10 were measured via ELISA. Basal receptor gene expression (ACE2 and TMPRSS2) was measured in monolayer (n = 5) and terminally differentiated (air-liquid interface [ALI];n = 5) cell models as well as in ex-vivo cells obtained directly from nasal brushings (n = 71). Generalised linear modelling, accounting for individual variability, identified any statistical difference (p < 0.05). Result(s): Exposure to the spike protein resulted in no increase in IL6 and IL8 production, however a significant (p < 0.05) decrease was observed at the highest dose tested (10 mug/mL). CXCL10 was only significantly induced at the highest dose (10 mug/mL) whereas CCL5 was not induced. When compared to ex-vivo samples, baseline expression of ACE2 and TMPRSS2 was significantly lower in monolayer cultures (~57- and ~4- fold respectively, p < 0.05), whereas ALI cultures had similar expression levels. Conclusion(s): The use of recombinant spike protein and monolayer cultures appears to not accurately model SARS-CoV-2 spike protein-host interactions. The lack of inflammatory responses may be attributed to the lower receptor gene expression in monolayer cultures. Future studies should utilise live virus and ALI cultures as a more biologically relevant model to study virus-host interactions.

Profiling viral receptors in nasal and amniotic samples at birth

Introduction/Aim: Children with wheeze and asthma present with airway epithelial vulnerabilities, such as impaired responses to viral infection. It is postulated that the in utero environment may contribute to the development of such airway epithelial vulnerabilities, that may predispose children to wheeze and asthma outcomes. To explore developmental mechanisms, further research is required using epithelial samples at birth. Our study asked whether amniotic epithelial samples from placentas show similar viral receptor expression to nasal epithelial cells at birth. We aimed to investigate expression of respiratory viral receptors for human rhinovirus (HRV), respiratory syncytial virus (RSV) and COVID-19-causing coronavirus (SARS-CoV-2) in nasal and amniotic epithelial samples. Methods: Unmatched nasal (n = 20 births) and amniotic (n = 33 newborns) epithelial samples were collected from ORIGINS cohort participants recruited into the AERIAL study. Using purified RNA, receptor expression for HRV (ICAM-1, LDLR, CDHR3), RSV (NCL, TLR4) and SARSCoV- 2 (ACE2, TMPRSS2) was assessed by qPCR. In addition, receptor protein expression was quantified through western blot and localized using immunohistochemistry in amniotic samples only. Results: Nasal epithelial and amniotic samples expressed various receptors for HRV, RSV and SARS-CoV-2 at the gene level in nasal (median(IQR) arbitrary units (AU);ICAM-1: 11.44(63.18);LDLR: 4.00(7.32);CDHR3: 0.40 (1.14);NCL: 2.32(2.18);CX3CR1: 2.17(2.33);TLR4: 2.20 (6.20);TMPRSS2: 1.99(4.85);ACE2: 0.36(0.52) AU) and amnion (ICAM-1: 0.69(2.21);LDLR: 0.39(1.38);CDHR3: 1.0 x 10-4(3.0x10-4);NCL: 1.03(0.55);CX3CR1: 0.12(0.24);TLR4: 0.10(0.13);TMPRSS2: 3.0 x 10-4 (16.0x10-4);ACE2: 0.01(0.02) AU). Amniotic samples also expressed these receptors at the protein level (ICAM-1: 0.03(0.05);LDLR: 0.06(0.03);CDHR3: 0.28(0.15);NCL: 0.96(1.19);CX3CR1: 0.08(0.08);TMPRSS2: 0.09(0.06);ACE2: 0.34(0.92) AU) and expression within the amniotic epithelium was confirmed by immunohistochemistry. Conclusion: Newborn nasal and amniotic epithelial samples expressed receptors for respiratory viruses, HRV, RSV, SARS-CoV-2. These findings warrant further investigation of the clinical significance of receptor expression in relation to prenatal and postnatal exposures, as well as childhood asthma development.

Late Breaking Abstract - ACE2 expression in lower airway epithelial cells is increased with age and in males, but is less in asthma

Introduction: COVID-19 is responsible for a global pandemic that is complicated by acute lung injury, and death. The virus SARS-CoV-2 requires the ACE2 receptor and serine proteases to enter airway epithelial cells. We sought to determine what factors were associated with levels of ACE2 expression and how this related to people with asthma and chronic obstructive pulmonary disease (COPD). Methods: We obtained airway epithelial cells from 146 people, aged 2 to 89 from Perth and Newcastle. The Newcastle cohort were enriched with people with asthma and COPD. Gene expression for ACE2 and other genes potentially associated with SARS-CoV-2 cell entry were assessed by quantitative PCR and immunohistochemistry. Results: Increased gene expression for ACE2 from lower airway epithelial cells was associated with older age (p=0.02) and male sex (p=0.03), but not packet years smoked. When we compared gene expression between adults with asthma, COPD and healthy controls, mean ACE2 was lower in asthma p=0.01 (Figure). Gene expression for Furin a protease that facilitates viral endocytosis was also lower in asthma (p=0.02), while ADAM-17, a disintegrin that has been shown to cleave ACE2 from the surface was increased p=0.02. We confirmed that ACE2 protein expression was lower in endobronchial biopsies from people with asthma. Conclusions: Increased ACE2 expression is seen in older people and males. While people with asthma appear to have reduced expression. Altered ACE2 expression in the lower airway may be an important factor in virus tropism to the lower airway.