RUXOLITINIB AS A TREATMENT STRATEGY FOR SARS-CoV-2 PNEUMONIA: CLINICAL EXPERIENCE IN A REAL-WORLD SETTING (preprint)

Introduction: Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) infection is characterised by a viral phase and a severe pro-inflammatory phase. The inhibition of the JAK/STAT pathway limits the pro-inflammatory state in moderate to severe COVID-19 cases. Methods: We analysed the data obtained for an observational cohort of patients with SARS-CoV-2 pneumonia treated with ruxolitinib in 22 hospitals of Mexico. The dose used was determined based on physician’s criteria. The benefit of ruxolitinib was evaluated using the 8-points ordinal scale developed by the NIH in the ACTT1 trial. Duration of hospital stay, changes in pro-inflammatory laboratory values, mortality, and toxicity were also measured. Results: A total of 287 patients administered ruxolitinib were reported at 22 sites in Mexico from March to June 2020; 80.8% received 5 mg BID and 19.16% received 10 mg BID ruxolitinib. At the beginning of treatment, 223 patients were on oxygen support, 59 on invasive ventilation. The percentage of patients on invasive ventilation was 53% in the 10 mg and 13% in the 5 mg cohort. There was a statistically significant improvement measured as a reduction by 2 points (initial 5.39 ± 0.93, final 3.67± 2.98, P value = 0.0001) on the 8-point ordinal scale. There were a total of 74 deaths. Serious adverse events were presented in 6.9% of the patients. Conclusion: Ruxolitinib appears to be safe in COVID-19 patients, with clinical benefits observed in terms of decrease in the 8-point ordinal scale and pro-inflammatory state. Further studies must be done to ensure efficacy against mortality.

Design and immunogenicity of a Pan-SARS-CoV-2 synthetic DNA vaccine (preprint)

First generation COVID-19 vaccines matched to the original Wuhan-Hu-1 (WT) strain are showing reduced efficacy against emerging SARS-CoV-2 variants of concern (VOC). In response, next generation vaccines either matched to a single variant or designed to provide broader coverage across the VOC group are being developed. The latter pan-SARS-CoV-2 approach may offer substantial advantages in terms of cross-strain protection, immune coverage, reduced susceptibility to escape mutants, and non-restricted geographical use. Here we have employed our SynCon(R) design technology to construct a DNA vaccine expressing a pan-Spike immunogen (INO-4802) to induce broad immunity across SARS-CoV-2 variants. Compared to WT and VOC-matched vaccines which showed limited cross-neutralizing activity, INO-4802 induced potent neutralizing antibodies and T cell responses against WT as well as B.1.1.7, P.1, and B.1.351 VOCs in a murine model. In addition, a hamster vaccination model showed enhanced humoral responses against VOCs in a heterologous pWT prime/INO-4802 boost setting. These results demonstrate the potential of the pan-SARS-CoV-2 vaccine, INO-4802 to induce cross-reactive immune responses against emerging VOCs as either a standalone vaccine, or as a potential boost for individuals previously immunized with WT-matched vaccines.

COVID-19 Disease Map, a computational knowledge repository of SARS-CoV-2 virus-host interaction mechanisms (preprint)

We hereby describe a large-scale community effort to build an open-access, interoperable, and computable repository of COVID-19 molecular mechanisms - the COVID-19 Disease Map. We discuss the tools, platforms, and guidelines necessary for the distributed development of its contents by a multi-faceted community of biocurators, domain experts, bioinformaticians, and computational biologists. We highlight the role of relevant databases and text mining approaches in enrichment and validation of the curated mechanisms. We describe the contents of the map and their relevance to the molecular pathophysiology of COVID-19 and the analytical and computational modelling approaches that can be applied to the contents of the COVID-19 Disease Map for mechanistic data interpretation and predictions. We conclude by demonstrating concrete applications of our work through several use cases.

Induced pulmonary comorbidities render CD-1 mice sensitive to SARS-CoV-2 (preprint)

Severe manifestations of COVID-19 are mostly restricted to persons with comorbidities, and they form a significantly high proportion of those which develop life-endangering lung injury. Nevertheless, COVID-19 animal models established to date are not based on preexistence of comorbidities. Here we report that mild pulmonary injury induced by administration of acute-lung-injury stimulants, renders outbred CD-1 mice to be sensitive to SARS-CoV-2. Following intranasal pretreatment of mice with low doses of ricin or bleomycin, SARS-CoV-2 infection caused a severe disease manifested by sustained body loss and mortality rates of >50%. Low-dose-ricin pretreated mice displayed markedly higher levels of viral RNA than mice not pretreated with ricin, not only in the nasal turbinate, trachea and lungs but also in the serum and heart. The deleterious effects of SARS-CoV-2 infection in ricin-pretreated mice were effectively alleviated by passive transfer of polyclonal and monoclonal antibodies generated against SARS-CoV-2 or SARS-CoV-2 RBD. Notably, viral cell entry in the sensitized mice model seems to involve viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. In summary, we present a novel animal model in mice that express native murine ACE2 yet are susceptible to genetically unaltered SARS-CoV-2, for the study of comorbidity-dependent COVID-19 pathology and treatment.

A genome-wide CRISPR/Cas9 knock-out screen identifies the DEAD box RNA helicase DDX42 as a broad antiviral inhibitor (preprint)

Genome-wide CRISPR/Cas9 knock-out genetic screens are powerful approaches to unravel new regulators of viral infections. With the aim of identifying new cellular inhibitors of HIV-1, we have developed a strategy in which we took advantage of the ability of type 1 interferon (IFN) to potently inhibit HIV-1 infection, in order to create a cellular environment hostile to viral replication. This approach led to the identification of the DEAD-box RNA helicase DDX42 as an intrinsic inhibitor of HIV-1. Depletion of endogenous DDX42 using siRNA or CRISPR/Cas9 knock-out increased HIV-1 infection, both in model cell lines and in physiological targets of HIV-1, primary CD4+ T cells and monocyte-derived macrophages (MDMs), and irrespectively of the IFN treatment. Similarly, the overexpression of a dominant-negative mutant of DDX42 positively impacted HIV-1 infection, whereas wild-type DDX42 overexpression potently inhibited HIV-1 infection. The positive impact of endogenous DDX42 depletion on HIV-1 infection was directly correlated to an increase in viral DNA accumulation. Interestingly, proximity ligation assays showed that DDX42, which can be mainly found in the nucleus but is also present in the cytoplasm, was in the close vicinity of HIV-1 Capsid during infection of primary monocyte-derived macrophages. Moreover, we show that DDX42 is also able to substantially decrease infection with other retroviruses and retrotransposition of long interspersed elements-1 (LINE-1). Finally, we reveal that DDX42 potently inhibits other pathogenic viruses, including Chikungunya virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Aglycone polyether ionophores as broad-spectrum agents inhibit multiple enveloped viruses including SARS-CoV-2 in vitro and successfully cure JEV infected mice (preprint)

Infections with zoonotic viruses, such as flaviviruses, influenza virus, and the SARS-CoV-2 pandemic coronavirus constitute an increasing global risk. Hence, an urgent need exists for the development of broad-spectrum antivirals to prevent such outbreaks. Here, we show that the maduramycin and CP-80,219 aglycone polyether ionophores exhibit effective broad-spectrum antiviral activity, against various viruses, including Japanese encephalitis virus (JEV), Dengue virus (DENV), Zika virus (ZIKV), and Chikungunya virus (CHIKV), while also exhibiting promising activity against PR8 influenza virus and SARS-CoV-2. Moreover, liposome-encapsulated maduramycin and CP-80,219 provide full protection for mice from infection with JEV in vivo. Mechanistic studies suggest that aglycone polyether ionophores primarily inhibit the viral replication step without blocking endosome acidification to promote the fusion between viral and cellular membranes. The successful application of liposomes containing aglycone polyether ionophores in JEV-infected mice offers hope to the development of broad-spectrum antiviral drugs like penicillin back to 1940s.

Extracellular vesicle-based vaccine platform displaying native viral envelope proteins elicits a robust anti-SARS-CoV-2 response in mice. (preprint)

Extracellular vesicles (EVs) emerge as essential mediators of intercellular communication. DNA vaccines encoding antigens presented on EVs efficiently induce T-cell responses and EV-based vaccines containing the Spike (S) proteins of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) are highly immunogenic in mice. Thus, EVs may serve as vaccine platforms against emerging diseases, going beyond traditional strategies, with the antigen displayed identically to the original protein embedded in the viral membrane and presented as such to the immune system. Compared to their viral and pseudotyped counterparts, EV-based vaccines overcome many safety issues including pre-existing immunity against these vectors. Here, we applied our technology in natural EV's engineering, to express the S proteins of SARS-CoV-2 embedded in the EVs, which mimic the virus with its fully native spikes. Immunizations with a two component CoVEVax vaccine, comprising DNA vector (DNAS-EV) primes, allowing in situ production of Spike harbouring EVs, and a boost using S-EVs produced in mammalian cells, trigger potent neutralizing and cellular responses in mice, in the absence of any adjuvants. CoVEVax would be the prototype of vaccines, where the sole exchange of the envelope proteins on EVs leads to the generation of new vaccine candidates against emerging viruses.

SARS-CoV-2 desensitizes host cells to interferon through inhibition of the JAK-STAT pathway (preprint)

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we performed global proteomic analysis of the virus-host interface in a newly established panel of phenotypically diverse, SARS-CoV-2-infectable human cell lines representing different body organs. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cell lines and primary-like cardiomyocytes, and found that several pathway components were targeted by SARS-CoV-2 leading to cellular desensitization to interferon. These findings indicate that the suppression of interferon signaling is a mechanism widely used by SARS-CoV-2 in diverse tissues to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19.

In vitro assessment of the virucidal activity of four mouthwashes containing Cetylpyridinium Chloride, ethanol, zinc and a mix of enzyme and proteins against a human coronavirus (preprint)

Background: saliva is established to contain high counts SARS-CoV-2 virus and contact with saliva droplets, contaminated surfaces or airborne particles are sources of viral transmission. The generation of infective aerosols during clinical procedures is of particular concern. Therefore, a fuller understanding of the potential of mouthwash to reduce viral counts and modulate the risk of transmission in medical professional and public context is an important research topic. Method: we determined the virucidal activity of four anti-bacterial mouthwashes against a surrogate for SARS-CoV-2, Human CoV-SARS 229E, using a standard ASTM suspension test, with dilution and contact times applicable to recommended mouthwash use. Results: the mouthwash formulated with 0.07% Cetylpyridinium Chloride exhibited virucidal effects providing a [≥]3.0 log reduction HCoV-229E viral count. Mouthwashes containing 15.7% ethanol, 0.2% zinc sulphate heptahydrate and a mix of enzymes and proteins did not demonstrate substantive virucidal activity in this test. Conclusion: mouthwash containing 0.07% Cetylpyridinium Chloride warrants further laboratory and clinical assessment to determine their potential benefit in reducing the risk of SARS-CoV-2.

Mechanism of SARS-CoV-2 polymerase inhibition by remdesivir (preprint)

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analogue and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3'-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3'-nucleotide of the RNA product is matched with the template base, and this may prevent proofreading by the viral 3'-exonuclease that recognizes mismatches. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

Ethacridine inhibits SARS-CoV-2 by inactivating viral particles in cellular models (preprint)

More than a million people have now died from COVID-19, because of infection with the SARS-CoV-2 coronavirus. Currently, the FDA has approved remdesivir, an inhibitor of SARS-CoV-2 replication, to treat COVID-19, though very recent data from WHO showed little if any COVID19 protective effect. Here we report that ethacridine, a safe and potent antiseptic use in humans, effectively inhibits SARS-CoV-2, at very low concentrations (EC50 ~ 0.08 M). Ethacridine was identified through a high-throughput screening of an FDA-approved drug library in living cells using a fluorescent assay. Interestingly, the main mode of action of ethacridine is to inactivate virus particles, preventing binding to the host cells. Thus, our work has identified a potent drug with a distinct mode of action against SARS-CoV-2.

Gamma-irradiated SARS-CoV-2 vaccine candidate, OZG-38.61.3, confers protection from SARS-CoV-2 challenge in human ACEII-transgenic mice (preprint)

The SARS-CoV-2 virus caused one of the severest pandemic around the world. The vaccine development for urgent use became more of an issue during the pandemic. An inactivated virus formulated vaccines such as Hepatitis A, inactivated polio, and influenza has been proven to be a reliable approach for immunization for long years. In this pandemic, we produced an inactivated SARS-CoV-2 vaccine candidate by modification of the oldest but the most experienced method that can be produced quickly and tested easily rather than the recombinant vaccines. Here, we optimized an inactivated virus vaccine which includes the gamma irradiation process for the inactivation as an alternative to classical chemical inactivation methods so that there is no extra purification required. Also, we applied the vaccine candidate (OZG-38.61.3) using the intradermal route in mice which decreased the requirement of a higher concentration of inactivated virus for proper immunization unlike most of the classical inactivated vaccine treatments. Thus, the novelty of our vaccine candidate (OZG-38.61.3) is a non-adjuvant added, gamma-irradiated, and intradermally applied inactive viral vaccine. We first determined the efficiency and safety dose (either 1013 or 1014 viral copy per dose) of the OZG-38.61.3 in Balb/c mice. Next, to test the immunogenicity and protective efficacy of the OZG-38.61.3, we immunized human ACE2-encoding transgenic mice and infected them with a dose of infective SARS-CoV-2 virus for the challenge test. We showed that the vaccinated mice showed lowered SARS-CoV-2 viral copy number in oropharyngeal specimens along with humoral and cellular immune responses against the SARS-CoV-2, including the neutralizing antibodies similar to those shown in Balb/c mice without substantial toxicity. This study encouraged us towards a new promising strategy for inactivated vaccine development (OZG-38.61.3) and the Phase 1 clinical trial for the COVID-19 pandemic.

SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third highly pathogenic coronavirus to spill over to humans in less than 20 years, after SARS-CoV-1 in 2002-2003 and Middle East respiratory syndrome (MERS)-CoV in 2012. SARS-CoV-2 is the etiologic agent of coronavirus disease 19 (COVID-19), which ranges from mild respiratory symptoms to severe lung injury and death in the most severe cases. The COVID-19 pandemic is currently a major health issue worldwide. Immune dysregulation characterized by altered innate cytokine responses is thought to contribute to the pathology of COVID-19 patients, which is a testimony of the fundamental role of the innate immune response against SARS-CoV-2. Here, we further characterized the host cell antiviral response against SARS-CoV-2 by using primary human airway epithelia and immortalized model cell lines. We mainly focused on the type I and III interferon (IFN) responses, which lead to the establishment of an antiviral state through the expression of IFN-stimulated genes (ISGs). Our results demonstrate that both primary airway epithelial cells and model cell lines elicit a robust immune response characterized by a strong induction of type I and III IFN through the detection of viral pathogen molecular patterns (PAMPs) by melanoma differentiation associated gene (MDA)-5. However, despite the high levels of type I and III IFNs produced in response to SARS-CoV-2 infection, the IFN response was unable to control viral replication, whereas IFN pre-treatment strongly inhibited viral replication and de novo production of infectious virions. Taken together, these results highlight the complex and ambiguous interplay between viral replication and the timing of IFN responses.