SARS-CoV-2 E-PROTEIN VIROPORIN INHIBITOR BIT225 ACTIVE IN hACE2 TRANSGENIC MICE

Background: The CoV-2 envelope (E) protein plays an important role in virus assembly, budding, immunopathogenesis and disease severity. E protein has ion channel activity, is located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we report that BIT225, an investigational HIV clinical compound, inhibits E ion channel activity and prevents body weight loss and mortality and reduces inflammation in lethally infected K18-hACE2 transgenic mice. BIT225 efficacy was observed when dosing was initiated before or 24 h or 48 h after infection. Method(s): SARS-CoV-2 E protein ion channel activity and Xenopus TMEM16A were measured in Xenopus oocytes. K18-hACE2 transgenic mice were infected intranasally with 104 pfu SARS CoV 2 (US-WA1/2020) and dosed orally twice daily with BIT225 for up to 12 Days. Dosing was initiated 12 h pre-infection or 24 h or 48 h post-infection. Disease parameters measured were survival, body weight, viral RNA by qPCR and infectious virus titre (plaque assay) in lung tissue homogenates and serum. In addition, levels of pro-inflammatory cytokines (IL-6, IL-1alpha, IL-1beta, TNFalpha & TGFbeta, MCP-1) were measured in lung and serum samples. Result(s): BIT225 inhibited ion channel activity of E-protein, but not that of TMEM16A in Xenopus oocytes. BIT225 dosed at 300mg/kg BID for 12 days starting 12 h pre-infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (n=12), while all vehicle-dosed animals reached a mortality endpoint by day 9 across two studies (n=12). Figure 1 shows results from a time of addition study: When treatment with BIT225 started at 24 h post-infection, body weight loss and mortality was also prevented (100% survival, n=5). In the group of mice where treatment started at 48 h after infection, body weight loss and mortality were prevented in 4 of 5 mice. Treatment efficacy was associated with significant reduction in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. Conclusion(s): BIT225 is an inhibitor of SARS-CoV-2 E-protein viroporin activity. In the K18 model BIT225 protected mice from weight loss and death, inhibited virus replication and reduced inflammation. These effects were noted when treatment with BIT225 was initiated before or 24-48 hours after infection and validate viroporin E as a viable antiviral target and support the clinical study of BIT225 in treatment of SARS-CoV-2.

SARS-CoV-2 coinfection in immunocompromised host leads to the generation of recombinant strain.

OBJECTIVES: Recombination related to coinfection is a huge driving force in determining the virus genetic variability, particularly in conditions of partial immune control, leading to prolonged infection. Here, we characterized a distinctive mutational pattern, highly suggestive of Delta-Omicron double infection, in a lymphoma patient. METHODS: The specimen was characterized through a combined approach, analyzing the results of deep sequencing in primary sample, viral culture, and plaque assay. RESULTS: Bioinformatic analysis on the sequences deriving from the primary sample supports the hypothesis of a double viral population within the host. Plaque assay on viral culture led to the isolation of a recombinant strain deriving from Delta and Omicron lineages, named XS, which virtually replaced its parent lineages within a single viral propagation. CONCLUSION: It is impossible to establish whether the recombination event happened within the host or in vitro; however, it is important to monitor co-infections, especially in the exceptional intrahost environment of patients who are immunocompromised, as strong driving forces of viral evolution.

Enhancement of SARS-CoV-2 infection and growth by an ACE2-specific monoclonal antibody.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) pandemic and continues to pose a threat to global public health through genetic mutation. In this study, we have found that an angiotensin-converting enzyme 2-specific monoclonal antibody at low concentration was able to enhance SARS-CoV-2 infection and growth in cell culture. Strikingly, it promotes SARS-CoV-2 plaque formation, resulting in accurate titration of different SARS-CoV-2 variants, particularly the newly emerged Omicron variants, which otherwise cannot be determined by standard plaque assays. Quantification of infectious titers of the newly emerged variants will facilitate the development and evaluation of vaccines and antiviral drugs against SARS-CoV-2.

ACE2 IS INVOLVED IN SARS-COV-2 INDUCED ENDOTHELIAL CELL INFLAMMATION INDEPENDENT OF VIRAL REPLICATION

Objective: COVID-19 association with cardiovascular disease is thought to be due to endothelial cell inflammation. ACE2 interactions with SARS-CoV-2 spike protein S1 subunit is important to viral infection. Here we questioned whether SARS-CoV-2 induces vascular inflammation via ACE2 and whether this is related to viral infection. Design and Methods: Human microvascular endothelial cells (EC) were exposed to recombinant S1p (rS1p) 0.66 ug/mL for 10 min, 5 h and 24 h. Gene expression was assessed by RT-PCR and levels of IL6 and MCP1, as well as ACE2 activity, were assessed by ELISA. Expression of ICAM1 and PAI1 was assessed by immunoblotting. ACE2 activity was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Viral infection was assessed by exposing Vero E6 (kidney epithelial cells;pos ctl) and EC to 105 pfu of SARS-CoV-2 where virus titre was measured by plaque assay. Results: rS1p increased IL6 mRNA (14.2 ± 2.1 vs. C:0.61 ± 0.03 2-ddCT) and levels (1221.2 ± 18.3 vs. C:22.77 ± 3.2 pg/mL);MCP1 mRNA (5.55 ± 0.62 vs. C:0.65 ± 0.04 2-ddCT) and levels (1110 ± 13.33 vs. C:876.9 ± 33.4 pg/mL);ICAM1 (17.7 ± 3.1 vs. C:3.9 ± 0.4 AU) and PAI1 (5.6 ± 0.7 vs. C: 2.9 ± 0.2), p < 0.05. MLN4760, but not rS1p, decreased ACE2 activity (367.4 ± 18 vs. C: 1011 ± 268 RFU, p < 0.05) and blocked rS1p effects on ICAM1 and PAI1. ACE2 siRNA blocked rS1p-induced IL6 release, ICAM1, and PAI1 responses as well as rS1p-induced NFkB activation. EC were not susceptible to SARS-CoV-2 infection, while the virus replicated well in Vero E6. Conclusion: rS1p induces an inflammatory response through ACE2 in endothelial cells;an effect that was independent of viral infection.

An in vitro COVID-19 causing virus culture model: Assessment of SARS-CoV-2 infectivity in Vero E6 cell

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) a virus from the Coronaviridae family that causes the Coronavirus disease (COVID-19), has emerged and spread since December 2019. Since then, many in vitro and in vivo models for COVID-19 research has been developed. Objective(s): This study aimed to determine infectivity rate of various SARS-CoV-2 strains in the Vero E6 cell line. Material(s) and Method(s): Four SARS-CoV-2 strains (Wuhan, Alpha, Beta, and Delta) were isolated from clinical samples. Virus titre concentration of all strains were measured using Tissue Culture Infectious Dose (TCID50) assay and Plaque assay. At similar virus titre concentration, all strains were incubated in the Vero E6 cells at 37degreeC for 72 hours. At the end of incubation period, all virus cultures were terminated and analysed using TCID50 assay. Result(s) and Conclusion(s): It was found that the Wuhan strain has the highest infectivity rate (3601 PFU/mL/72hours) towards the Vero E6 cells, followed by Alpha (2946 PFU/mL/72hours), Beta (1780 PFU/mL/72hours) and Delta (571 PFU/mL/72hours). Vero E6 cell is commonly used for virus isolation and propagation, however this cell does not mimic the primary entry sites in the human respiratory track. The successful isolation and culture of SARS-CoV-2 in the Vero E6 cell is multifactorial, with high viral titre in source clinical samples and low passage number of cell culture as key factors. Vero E6 cell is susceptible towards all SARS-CoV-2 strains and can be used as in vitro COVID-19 culture model. Further studies can be conducted to determine the influence of different cell lines on the COVID-19 infectivity.

ZnO-chlorogenic acid nanostructured complex inhibits Covid-19 pathogenesis and increases hydroxychloroquine efficacy.

Objective: The study purpose was to compare the anti- novel coronavirus disease 2019 (COVID-19) property of chlorogenic acid (CGA) and Zinc oxide nanoparticles (ZnO-NP) with the new valid synthesized complex of ZnO /CGA-NPs. Methods: The facile mixing method was utilized to prepare ZnO/CGA-NPs. The in vitro effect of different ZnO/CGA-NPs concentrations on papain-like protease (PLpro) and spike protein- receptor-binding domain (RBD) was measured by ELISA technique. The compounds effects on SARS-CoV2 were determined on viral entry, replication, and assembly by using plaque reduction assay, qPCR, and ELISA techniques. Their individual effects or mixed with hydroxychloroquine (HCQ) on erythrocytes (RBCs) and leukocytes (WBCs) were evaluated by routine cell culture technique. Finally, turbidity and agar well diffusion assays were done to evaluate their antimicrobial properties against Escherichia. coli, klebsila pneumonia, Streptococcus pyogenes, Staphylococcus aureus, and Candida albicans. Results: The results confirmed that the uniformly dispersed ZnO-NPs were converted to aggregated form of ZnO/CGA-NPs upon the addition of CGA. The inhibitory concentration 50 (IC50) of ZnO /CGA-NPs against RBD, angiotensin-converting enzyme 2 (ACE2) and PLpro were 1647.7, 323.3 µg/mL and 38.7 µg/mL, respectively. Also, it inhibited E-gene, RdRp gene, E-protein, and spike protein with an IC50 of 0.11, 0.13, 0.48, and 0.37 µg/mL, respectively. It acted as an antimicrobial against all tested organisms with a minimum inhibitory concentration (MIC) of 26 µg/mL. Finally, ZnO/CGA-NPs Complex (0.1 IC50) prevented the cytotoxic effect of HCQ on RBCs and WBC by 92.3 and 90 %, respectively. Conclusion: ZnO/CGA-NPs Complex can be considered as a new anti- severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) compound.

Interferon-mediated Responses to Human Metapneumovirus

Background: Acute respiratory infection (ARI) is the leading infectious cause of pediatric death worldwide, comprising 15% of all deaths in children under 5 years old. Human metapneumovirus (HMPV) is a primary cause of ARI, and accounts for a major portion of ARI-related hospitalizations in infants and young children. Although nearly every person is infected with HMPV during early childhood, re-infections occur often, highlighting the difficulty in building long-term immunity. There are no approved vaccines or antiviral therapies. Early host responses to HMPV are poorly characterized, and further understanding could identify important antiviral pathways and potential therapeutic targets. Type I (IFN-α/β) and III interferons (IFN-λ) display antiviral activity against numerous respiratory viruses and are currently being investigated for therapeutic use in several respiratory infections including SARS-CoV-2. However, their roles in HMPV infection remain largely unknown. Our laboratory has previously shown that type I IFN is critical for HMPV pathogenesis, as loss of IFN-α/β signaling reduces lung inflammation and lessens HMPV disease severity in mice. Here, we describe distinct antiviral roles for type I and III IFNs during HMPV infection using an established mouse model. Methods: In vivo studies were conducted using mice lacking either the IFN-α/ β receptor (IFNAR-/-) or IFN-λ receptor (IFNLR-/-). Early immune responses to HMPV strains TN/94-49 and C2-202 were assessed by clinical disease scoring, plaque assay, Luminex immunoassay, and spectral cytometry of mouse lung samples. In vitro studies were performed using CMT 64-61 mouse bronchial epithelial cells. Responses to TN/94-49 and C2-202 were measured by qPCR, plaque assay, and Luminex immunoassay of cell lysates and supernatants. Results: IFNAR-/- mice exhibited lower clinical disease scores, reduced lung levels of inflammatory cytokines IL6, MIP-1α, and MCP-1, and decreased numbers of lung interstitial macrophages during HMPV infection, highlighting their critical role in HMPV immune-mediated pathogenesis. IFNLR-/- mice with intact IFNAR showed moderate clinical disease, higher lung levels of inflammatory cytokines IL-6, MCP-1, and IFN-γ, and increased lung interstitial macrophage recruitment. A reduction in HMPV disease was also recapitulated by IFNAR-neutralizing antibody treatment of IFNLR-/- mice. Interestingly, IFNLR-/- showed higher HMPV viral titers, while IFNAR-/- mice showed no differences or slightly lower viral titers, compared to wild-type mice. Moreover, IFN-λ pre-treatment of infected CMT 64-61 cells reduced HMPV viral titers and decreased supernatant levels of inflammatory cytokines IL-6, IL-1β, TNFα, and MCP-1. Conclusion: These findings suggest that type I IFN is necessary for HMPV pathogenesis, while type III IFN is critical for limiting HMPV replication in the lungs but does not contribute to HMPV inflammatory disease. This work uncovers key functional differences between type I and III IFNs during HMPV infection, an important feature of innate immune responses to HMPV that may be utilized to inform treatment.

Repurposing drugs to fight COVID-19: SARSCoV- 2 envelope targeting by clinically approved porphyrins and analogues

Despite mitigation measures and vaccination programs, there are still very few medicines to treat COVID-19. Porphyrins and analogues (P&A) usually present broad-spectrum antiviral activity. Some are clinically approved for photodynamic therapy in cancer. Therefore, repurposing clinically approved P&A might be an alternative to treat COVID-19. In this work, we evaluate the ability of the clinically approved temoporfin, verteporfin, talaporfin and redaporfin to inactivate SARS-CoV-2 infectious particles, characterizing their mechanism of action. Loss of infectivity of P&A treated SARS-CoV-2 was assessed by plaque assay. P&A photoactivation successfully inactivated SARS-CoV-2 with very low concentrations and light doses. However, only temoporfin and verteporfin were able to inactivate SARS-CoV-2 in the dark, verteporfin being the most effective. Next, P&A dark antiviral mechanism was characterized starting from P&A interaction with membrane models. P&A partition, membrane-insertion depth, lipid-membrane disruption and changes in membrane ordering were investigated using fluorescent spectroscopy. Among all tested P&A, verteporfin presented the highest partition coefficient, Kp. Curiously, temoporfin and redaporfin presented similar Kp values, although redaporfin did not present dark antiviral activity. Noteworthy, redaporfin was located closer to the surface of the lipid bilayer and both temoporfin and verteporfin were located closer to the centre. Finally, only temoporfin and verteporfin induced reduction of GP (laurdan-generalized polarization), with transition from an ordered phase to a liquidcrystalline phase. Our results suggest that dark antiviral activity is dependent on P&A interaction with viral envelope. Membrane affinity, penetration, and destabilization are critical for P&A dark antiviral activity. Furthermore, dark anti-SARS-CoV-2 activity opens the possibility for off-label P&A application in the systemic treatment of COVID-19.

Activation of immune receptor RIG-I protects human differentiated epithelial cells of the upper respiratory tract from SARS-CoV-2

SARS-CoV-2 virus has evolved non structural proteins (NSLs) to avoid recogniton by innate immune receptors such as RIG-I which induces an antiviral type I IFN response. A proper type I IFN response is essential for an effective defense against SARS-CoV-2. The aim of this project is the evaluation of the effect of a prophylactic activation of RIG-I by a specific synthetic RNA ligand (5-triphosphate RNA, 3pRNA) to an antiviral state in cells that protects against SARSCoV- 2. We analysed the effect of activation of RIG-I by 3pRNA on SARS-CoV-2 infection in primary upper airway cells. Primary nasal epithelial cells (PNEC) and primary bronchial cells (NHBEC) were cultivated in air-liquid interface and differentiated to cilia bearing epithelial cells. Viral replication was analyzed by plaque assay and qPCR. Furthermore, interferon-stimulated genes and ACE2/TMPRSS2 were determined on the transcriptional level and IP-10 protein was quantified in supernatants by ELISA. Only PNEC in a fully differentiated state could be infected with SARS-CoV-2, while undifferentiated cells were not susceptible to infection with this virus. Upon stimulation with 3pRNA at 6h prior to infection, PNEC produced more than 10.000 pg/ml IP-10. Infection with SARS-CoV-2 significantly decreased the average of 3pRNA-induced IP-10 production. 3pRNA pretreatment of PNEC significantly reduced SARS-CoV-2 replication at 48 h by up to 99,7 % as evaluated by plaque assay and up to 90 % as measured by qPCR. These results demonstrate for the first time that RIG-I activation protects primary fully differentiated epithelial cells against SARS-CoV-2 replication. Our results support the concept that RIG-I-mediated prophylaxis is a promising strategy to mitigate SARS-CoV-2 infection.

SARS-COV-2 IS DETECTED IN THE GASTROINTESTINAL TRACT OF ASYMPTOMATIC OUTPATIENTS USING ROUTINE ENDOSCOPY BUT POSES MINIMAL TRANSMISSION RISK

Objective: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected in multiple organ systems including the gastrointestinal (GI) tract using standard PCR techniques. However, whether the human gut supports active SARS-CoV-2 replication leading to shedding of infectious virions is still a matter of debate. Our study aimed to determine whether SARS-CoV-2 could be recovered from the GI tract of asymptomatic outpatients to assess the risk of SARS-CoV-2 exposure for healthcare workers performing routine endoscopies. Methods: Between April 2020 and February 2021, we enrolled 112 patients aged 19 – 70 years undergoing elective endoscopic procedures who had no known SARS-CoV-2 exposure or recent COVID-19 test result (n=100) or who had a history of previous SARS-CoV-2 infection but had recovered at the time of the procedure (n=12). None of the patients had gastrointestinal symptoms at the time of COVID-19 infection or respiratory complaints at the time of the endoscopy. Liquids and biopsies from the colon, ileum, duodenum, and stomach were collected during endoscopy following standard bowel preparation protocols. Samples were analyzed for SARS-CoV-2 by PCR, and PCR-positive samples were analyzed for the presence of infectious virus by VeroE6 plaque assays. We also used plaque assays to assess whether endoscopic colonic liquids could inactivate SARSCoV- 2. Results: Interestingly, one colonic biopsy out of the 255 tissue samples collected from patients with no known SARS-CoV-2 exposure tested positive for SARS-CoV-2 by PCR. Out of 12 patients who had recovered from COVID-19 between 2 and 21 weeks before the endoscopic procedure, three colonic fluid samples tested positive for SARS-CoV-2 (Fig. 1A). Positive PCR results were confirmed by an independent laboratory. Importantly, no replication-competent virus was detected in any of the tissue or liquid samples. In vitro treatment of SARS-CoV-2 with colonic liquid showed that SARS-CoV-2 was completely inactivated after 24 hours, but at 10 minutes and 1-hour viral inactivation varied considerably between samples (Fig. 1B). Discussion: In 25% (3 out of 12) of patients with previous COVID-19 history, virus was detected by PCR for up to 5 months following resolution of symptoms. Viral genomes were also detected in colonic biopsies from one subject with no known SARS-CoV-2 infection, consistent with a large proportion of asymptomatic infections in the US population. The persistent detection of SARS-CoV-2 genomes in endoscopy samples after resolution of COVID-19 points to the gut as a reservoir for SARS-CoV-2 and confirms previous reports of long-term SARS-CoV-2 shedding in fecal samples. However, the absence of infectious virions in the samples and the rapid inactivation of SARS-CoV-2 in colon liquids suggests that the risk to healthcare workers involved in endoscopy procedures is likely low. (Figure Presented)

Comparison among plaque assay, tissue culture infectious dose (TCID) 50 and real-time RT-PCR for SARS-CoV-2 variants quantification

Background and Objectives: SARS-CoV-2 variants of concern (VOC) and interest (VOI) pose a significant threat to public health because the rapid change in the SARS-CoV-2 genome can alter viral phenotypes such as virulence, transmissi-bility and the ability to evade the host response. Hence, SARS-CoV-2 quantification techniques are essential for timely diagnosis and follow-up. Besides, they are vital to understanding viral pathogenesis, antiviral evaluation, and vaccine de-velopment. Materials and Methods: Five isolates of SARS-CoV-2: D614G strain (B.1), three VOC (Alpha, Gamma and Delta), and one VOI (Mu) were used to compare three techniques for viral quantification, plaque assay, median tissue culture infectious dose (TCID) and real-time RT-PCR. 50 Results: Plaque assay showed viral titers between 0.15 ± 0.01×107 and 1.95 ± 0.09×107 PFU/mL while viral titer by TCID 50 assay was between 0.71 ± 0.01×106 to 4.94 ± 0.80×106 TCID /mL for the five SARS-CoV-2 isolates. The PFU/mL titer 50 obtained by plaque and the calculated from TCID assays differed by 0.61 log10, 0.59 log10, 0.59 log10 and 0.96 log10 50 for Alfa, Gamma, Delta, and Mu variants (p≤0.0007), respectively. No differences were observed for the D614G strain. Real-time PCR assay exhibited titers ranging from 0.39 ± 0.001×108 to 3.38 ± 0.04×108 RNA copies/µL for all variants. The relation between PFU/mL and RNA copies/mL was 1:29800 for D614G strain, 1:11700 for Alpha, 1:8930 for Gamma, 1:12500 for Delta, and 1:2950 for Mu. Conclusion: TCID assay was comparable to plaque assay for D614G but not for others SARS-CoV-2 variants. Our data 50 demonstrated a correlation among PFU/mL and E gene RNA copies/µL, units of measure commonly used to quantify the viral load in diagnostic and research fields. The results suggest that the proportion of infectious virions in vitro changes de-pending on the SARS-CoV-2 variant, being Mu, the variant reaching a higher viral titer with fewer viral copies.

Pulmonary Surfactant Phospholipids as Novel Anti-Virals Against SARSCoV- 2 Infection

Background: A novel coronavirus (SARS-CoV-2) has led to the worldwide spread of pandemic proportions and currently no effective therapy is available. The minor pulmonary surfactant lipids, palmitoyl-oleoyl-phosphatidylglycerol (POPG), and phosphatidylinositol (PI), are potent regulators of inflammatory processes, and are effective as anti-viral agents for multiple respiratory viruses including Respiratory syncytial virus (RSV), Influenza A virus (IAV) and Rhinoviruses (RVs). Objective: The primary objectives of this study are to determine whether POPG or PI are potent against SARS-CoV-2 in vitro, using human airway epithelial cells, and examine the potency of PI against SARS-CoV-2 in vivo, in a hamster model. Methods: We examined efficacies of POPG or PI against SARS-CoV-2 (USA WA/2020) in human bronchial epithelial cells, and nasal epithelial cells from healthy control subjects differentiated by ALI cultures. We quantified SARS-CoV-2 replication by quantitative plaque assays and qRT-PCR. We determined the potency of PI against SARS-CoV- 2 in golden Syrian hamster as in vivo model for SARS-CoV-2 infection. Results: We examined the efficacies of POPG and PI using primary human tracheal and nasal epithelial cells, differentiated in ALI culture. Cells were treated with POPG (10mg/ml) and PI (4mg/ml) added to apical media alone for 16hrs. Subsequently, cells were infected with SARS-CoV-2 at m.o.i = 0.02, for 48hrs, harvested for RNA extraction and qRT-PCR. SARS-CoV-2 replicated in tracheal cells with a 106-fold increase in mRNA. POPG and PI reduced viral mRNA expression by 70% and 85%, respectively (subject numbers n=3). In nasal epithelia, SARS-CoV-2 mRNA expression increased 105 -fold compared to sham infected cultures. Both POPG and PI attenuated the increase in viral mRNA expression by 70% - 82% (subject numbers n=6). We determined the PI effect in an in vivo study in hamsters. Hamsters were challenged with 103 pfu of SARS-CoV-2, either with, or without PI (2mg/hamster) administered intranasally. Hamsters were harvested at Day 3, and lungs were processed for histopathology. Pharyngeal swabs were used to examine viral burden by plaque assays. PI reduced plaque numbers compared to viral infection alone groups at day1 (Virus alone: 2.4±2.7(X104pfu/ml), Virus+PI: 0.9±2.1(X106pfu/ml), p<0.05). PI reduced lung histopathology score at day 3 (Virus alone: 28.0±15.6, Virus+PI: 6.7±7.0, p<0.05). Conclusions: POPG and PI significantly reduced SARS-CoV2 replication in human differentiated airway epithelial cells. PI inhibited SARS-CoV-2 infection in vivo in hamsters. These findings suggest that inhalation of POPG, or PI might be effective as novel anti-viral compounds for treating and preventing SARSCoV- 2 infection.

Dose-Dependent Respiratory Viral Inhibition by a Safe Inhaled Alkaline Treatment

Introduction: SARS-CoV-2 respiratory infection is pandemic and continues to cause significant mortality and morbidity worldwide. Respiratory viral infections in general are a leading cause of hospital admissions and mortality throughout the world as well. Most respiratory viral infections require an acidic intracellular and endosomal environment in order to enter host cells, replicate, and cause illness. We study the beneficial effects of airway alkalinization by an inhaled drug, Optate, that we currently have demonstrated is safe to inhale by healthy subjects and those with stable airways disease. We have recently shown that treatment with 4.5 mg/ml Optate safely inhibits SARS-CoV-2 infection in primary human airway epithelial cells (HAECs). We hypothesized that this inhibition would be dose dependent and that Optate would also inhibit other viral infections in a dosedependent manner. Methods: HAECs were infected with respiratory syncytial virus with green fluorescent protein (RSV-GFP) or SARS-CoV-2 virus. A dose-response curve of Optate was performed in each infection model and compared to a control group. Viral infection was quantified using fluorescence microscopy, plaque assays, and viral protein quantification. Optate pH was measured at each dose and a corresponding dose/pH curve was calculated to compare pH to dose-response. Results: SARS-CoV-2 infection was significantly inhibited by doses of Optate > 2.25 mg/ml, corresponding with an Optate pH > 9.2 (n = 4, p < 0.001). RSV infection was significantly inhibited by doses of Optate > 2 mg/ml, corresponding with an Optate pH > 9 (n = 3, p < 0.001). No significant difference was noted between control and Optate treated HAECs at lower concentrations of Optate. Conclusions: Optate inhibits SARS-CoV-2 and RSV viral infections in a dose-dependent manner that correlates with Optate pH. These findings suggest that Optate may be an inhaled therapeutic for patients with respiratory viral infections. (Table Presented).

Zebrafish embryos to study BSL-3 viruses and antiviral molecules

Emerging viruses are currently a burden with the recent SARS-CoV-2 pandemic resulting in more than 6 million death worldwide. Other viruses such as Arboviruses, transmitted by mosquitoes, can also emerge easily and represent a threat for humans and animals. These viruses are often RNA enveloped viruses and require to be studied in a BSL-3 (Bio-Safety- Laboratory of level 3 security), underlying the need to develop simple and rapid antiviral screenings. In recent years, the zebrafish has become a powerful tool in the biomedical sector to study viral infection and immunity. The optical transparency of the zebrafish embryo offers a major advantage for real time imaging of the infection process, and study hostpathogen interactions at subcellular levels in living systems using fluorescently labelled pathogens. While zebrafish embryos have been shown to be a successful vertebrate model to study a large panel of human disease, this model has been very little explored to study BSL-3 pathogen infection and propagation. This project was created to develop the zebrafish infection model for emerging BSL-3 viruses, and evolve towards high-content screening methods for antiviral molecules. Thanks to the setup of a microinjection system under a laminar flow hood in the BSL-3, we were able to inject in zebrafish embryos several emerging viruses, including the Chikungunya virus (CHIKV), Dengue virus (DENV) and SARS-CoV-2. Using fluorescently tagged viruses, infection was monitored in real time in vivo and confirmed with classical virology methods (RT-qPCR, plaque assays, TCID50). We then tested several nucleoside analogues described for their antiviral activity in vitro for these different viruses and we were able to validate the antiviral effect of some of these molecules in infected embryos. Altogether, our zebrafish infection model will provide us a better understanding of in vivo infection and propagation of these emergent BSL-3 viruses, and will be used as an intermediate model between in vitro antiviral screens and in vivo screens in mammals.

GALECTIN-9 PROTECTS HUMANIZED ACE2-IMMUNOCOMPETENT MICE from SARS-CoV-2 INFECTION

Background: Coronavirus disease 2019 (COVID19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) remains a global health emergency even with effective vaccines and limited FDA-approved therapies. To limit mortality and morbidity across the spectrum of disease, the need for therapeutics remains critical. Galectin9 (gal9) is a beta-galactoside binding protein that modulates cell-cell and cell-matrix interactions. In response to SARS-CoV2 infection, it has been shown that circulating gal9 levels are elevated in patient sera with moderate to severe disease. Additionally, it has been reported that gal9 unexpectedly may competitively bind the host ACE2 receptor, potentially impeding viral entry. Therefore, we hypothesized that early recombinant gal-9 treatment post infection may prevent binding of the virus to susceptible host cells resulting in decreased severity of SARS-CoV2-associated disease. Methods: To determine the therapeutic potential of gal9 for treating COVID19, we infected K18-hACE2 transgenic mice intranasally with 104 particle forming units (PFU) of SARS-CoV2. 6 hours post infection (hpi), mice were treated with a single dose of 30 ug of recombinant human gal9 (rhgal9) or PBS intraperitoneally and subsequently monitored 12 days for morbidity. Subgroups of mice were humanely euthanized at 2 and 5 days pi (dpi) for viral plaque assay, flow cytometry, and protein analysis from lung tissue and bronchial alveolar lavage (BAL). Results: We found that mice treated with rhgal9 during the acute phase of infection exhibit improved survival compared to PBS treated animals (25%, p<0.0001). We found that at 5 dpi, rhgal9 treated mice exhibited enhanced viral clearance in the BAL but not in the lung parenchyma. Additionally, we found increased CD8 T cell (p<0.001) and decreased neutrophil (p<0.05) frequencies in the lung at 5 dpi. Finally, we found that BAL fluid had elevated levels of Type 1 Interferon [IFNa (p<0.01) and IFNb (p<0.01)] at 2 dpi and increased MyD88 proinflammatory cytokines [IL1a (p<0.05), IL1b (p<0.01), TNFa (p<0.05), and MIP1a (p<0.05) at 5 dpi. Conclusion: Our study suggests that rhgal9 treatment may be potentially therapeutic for treating acute COVID19. Our data suggest that rhgal9 treatment in combination with other anti-inflammatory mediators may curtail damaging inflammation associated with SARS-CoV2 disease. Further studies are required to determine the optimal time, combination and duration of treatment pi to effectively target the gal9 pathways.

INVESTIGATING CONSERVED SEQUENCE in the SARS-CoV-2 GENOME AS NOVEL VACCINE IMMUNOGEN

Background: It is imperative to investigate novel, broadly conserved coronavirus immunogens as new SARS-CoV-2 variants of concern are continually emerging. The goal of this study was to generate a broadly protective long-term vaccine candidate against potential new variants of SARS-CoV-2 and novel, outbreak coronaviruses. The vaccine immunogen spanned portions of the highly conserved RNA replication machinery (nsp12 and nsp13) (CoV.Con). The vaccine was packaged into a rhesus adenoviral vector (RhAd52.CoV.Con) with the goal of generating robust long-lived CD8+ T-cell responses. Methods: The CoV.Con immunogen was generated by aligning coronavirus sequences to determine the most conserved region. ACE2 carrier and BALB/c mice were immunized intramuscularly with 109 RhAd52.CoV.Con and boosted four weeks later. Splenocytes were harvested four weeks after boost. Cellular immunity was determined through ELIspot and intracellular cytokine stain (ICS). BALB/c mice were primed and boosted with RhAd52.CoV.Con. Four weeks post boost mice were challenged intranasally with mouse adapted SARS-CoV-2. Protection was measured by weight loss and plaque assay. Results: Four weeks post RhAd52.CoV.Con boost immunization, ACE2 carrier and BALB/c mice developed cellular immunity as shown by ELIspot (Fig 1a) and ICS. ACE2 carrier mice cellular immunity showed bias toward nsp12 while BALB/c mice showed nsp13 preference. BALB/c mice were primed and boosted with RhAd52.CoV.Con. Four weeks after boost mice were challenged with mouse adapted SARS-CoV-2. RhAd52.CoV.Con was compared against and combined with a suboptimal dose of RhAd52.S.pp at 4 and 8 weeks post injection. Protection against weight loss (Fig 1b) and viral load (Fig 1c) was minimal although increased RhAd52.S.pp protection was observed from 4 to 8 weeks post immunization. Increased RhAd52.S.pp protection corresponded to increased spike antibody binding and neutralizing titers. Conclusion: Our work investigates a highly conserved coronavirus immunogen, CoV.Con, demonstrating immunogenicity in two mouse strains. While RhAd52. CoV.Con protection in the mouse model was minimal it demonstrates a schema for generating coronavirus immunogens that can protect against multiple different viruses. This work takes the first steps towards generating a long-lived broadly protective T-cell coronavirus vaccine.

Hit-to-Lead Short Peptides against Dengue Type 2 Envelope Protein: Computational and Experimental Investigations.

Data from the World Health Organisation show that the global incidence of dengue infection has risen drastically, with an estimated 400 million cases of dengue infection occurring annually. Despite this worrying trend, there is still no therapeutic treatment available. Herein, we investigated short peptide fragments with a varying total number of amino acid residues (peptide fragments) from previously reported dengue virus type 2 (DENV2) peptide-based inhibitors, DN58wt (GDSYIIIGVEPGQLKENWFKKGSSIGQMF), DN58opt (TWWCFYFCRRHHPFWFFYRHN), DS36wt (LITVNPIVTEKDSPVNIEAE), and DS36opt (RHWEQFYFRRRERKFWLFFW), aided by in silico approaches: peptide-protein molecular docking and 100 ns of molecular dynamics (MD) simulation via molecular mechanics using Poisson-Boltzmann surface area (MMPBSA) and molecular mechanics generalised Born surface area (MMGBSA) methods. A library of 11,699 peptide fragments was generated, subjected to in silico calculation, and the candidates with the excellent binding affinity and shown to be stable in the DI-DIII binding pocket of DENV2 envelope (E) protein were determined. Selected peptides were synthesised using conventional Fmoc solid-phase peptide chemistry, purified by RP-HPLC, and characterised using LCMS. In vitro studies followed, to test for the peptides' toxicity and efficacy in inhibiting the DENV2 growth cycle. Our studies identified the electrostatic interaction (from free energy calculation) to be the driving stabilising force for the E protein-peptide interactions. Five key E protein residues were also identified that had the most interactions with the peptides: (polar) LYS36, ASN37, and ARG350, and (nonpolar) LEU351 and VAL354; these residues might play crucial roles in the effective binding interactions. One of the peptide fragments, DN58opt_8-13 (PFWFFYRH), showed the best inhibitory activity, at about 63% DENV2 plague reduction, compared with no treatment. This correlates well with the in silico studies in which the peptide possessed the lowest binding energy (-9.0 kcal/mol) and was maintained steadily within the binding pocket of DENV2 E protein during the MD simulations. This study demonstrates the use of computational studies to expand research on lead optimisation of antiviral peptides, thus explaining the inhibitory potential of the designed peptides.

Mechanically Ventilated Patients Shed High-Titer Live Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) for Extended Periods From Both the Upper and Lower Respiratory Tract.

BACKGROUND: SARS-CoV-2 infection can lead to severe acute respiratory distress syndrome needing intensive care admission and may lead to death. As a virus that transmits by respiratory droplets and aerosols, determining the duration of viable virus shedding from the respiratory tract is critical for patient prognosis, and informs infection-control measures both within healthcare settings and the public domain. METHODS: We prospectively examined upper and lower airway respiratory secretions for both viral RNA and infectious virions in mechanically ventilated patients admitted to the intensive care unit (ICU) of the University Hospital of Wales. Samples were taken from the oral cavity (saliva), oropharynx (subglottic aspirate), or lower respiratory tract (nondirected bronchoalveolar lavage [NBAL] or bronchoalveolar lavage [BAL]) and analyzed by both quantitative PCR (qPCR) and plaque assay. RESULTS: 117 samples were obtained from 25 patients. qPCR showed extremely high rates of positivity across all sample types; however, live virus was far more common in saliva (68%) than in BAL/NBAL (32%). Average titers of live virus were higher in subglottic aspirates (4.5 × 107) than in saliva (2.2 × 106) or BAL/NBAL (8.5 × 106) and reached >108 PFU/mL in some samples. The longest duration of shedding was 98 days, while most patients (14/25) shed live virus for ≥20 days. CONCLUSIONS: ICU patients infected with SARS-CoV-2 can shed high titers of virus both in the upper and lower respiratory tract and tend to be prolonged shedders. This information is important for decision making around cohorting patients, de-escalation of personal protective equipment, and undertaking potential aerosol-generating procedures.

Propagation and Quantification of SARS-CoV-2.

In late 2019, the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. Since its emergence, SARS-CoV-2 has been responsible for a world-wide pandemic resulting in over 80 million infections and over 1.8 million deaths. The severity of the pandemic has prompted widespread research efforts to more fully understand SARS-CoV-2 and the disease it causes, COVID-19. Research into this novel virus will be facilitated by the availability of clearly described and effective protocols that enable the propagation and quantification of infectious virus. Here, we describe protocols for the propagation of SARS-CoV-2 in Vero E6 cells as well as two human cells lines, the intestinal epithelial Caco-2 cell line and the respiratory epithelial Calu-3 cell line. Additionally, we provide protocols for the quantification of SARS-CoV-2 by plaque assays and immunofocus forming assays in Vero E6 cells utilizing liquid overlays. These protocols provide a foundation for laboratories acquiring the ability to study SARS-CoV-2 to address this ongoing pandemic.

SARS-CoV-2 Viral Viability Culture and Sequencing from Immunocompromised Patients with Persistently Positive SARS-CoV-2 PCR Results

Background. Immunocompromised (IC) patients (pts) can have prolonged SARS-CoV-2 PCR positivity, even after resolution of COVID-19 symptoms. This study aimed to determine if viable virus could be detected in samples collected > 21 days after an initial positive (pos) SARS-CoV-2 PCR in IC pts. Methods. We obtained 20 remnant SARS-CoV-2 PCR pos nasopharyngeal swabs from IC pts (bone marrow or solid organ transplant, high dose steroids, immunosuppressive medications) with a pos repeat PCR within the previous 30 days. The repeat specimens were cultured on Vero-hACE2-TMPRSS2 cells and incubated for 96 hours to assess viral viability. Viable RNA and infectious virus in the cultured cells were measured by qPCR and infectious plaque assays. RNA sequencing was performed on a HiSeq platform (Illumina). Samples also underwent SARS-CoV-2 antigen (Ag) testing (BD Veritor). Clinical data were extracted from the electronic health record by chart review. Results. Pt characteristics are in Table 1. Viral cultures from the repeat specimen were negative (neg) for 18 pts and pos for 2 (Table 2). Pt 1 is a 60M treated with obinatuzumab 19 days prior to his first pos PCR test, with repeat specimen collected 21 days later (cycle threshold (Ct) not available). Pt 1 had a low viral titer (27 PFU/mL) & a D614G mutation on sequencing. Pt 2 is a 75M treated with rituximab 10 days prior to his first pos PCR test, with repeat specimen collected 23 days later (Ct 27.56/27.74). Pt 2 had a high viral titer (2e6 PFU/mL) and D614G, S98F, and S813I mutations. Demographics of Study Population (N=20) Characteristics of patients with a positive SARS-CoV-2 viral culture Conclusion. 90% of specimens collected > 21 days after an initial pos SARS-CoV-2 PCR did not have viable virus detected on their repeat specimen. The 2 pts with pos viral cultures had active hematologic malignancies treated with an anti-CD20 mAb at the time of COVID-19 diagnosis. One pt had a high concentration of active, viable virus. No known variants of concern were noted in this cohort, collected in Q2 2020, though prolonged replication is a risk for variant development. Further data are needed about risk factors for persistent viable viral shedding & methods to prevent transmission of viable virus from IC hosts.