Differential haplotype expression in class I MHC genes during SARS-CoV-2 infection of human lung cell lines (preprint)

Cell entry of SARS-CoV-2 causes genome-wide disruption of the transcriptional profiles of genes and biological pathways involved in the pathogenesis of COVID-19. Expression allelic imbalance is characterized by a deviation from the Mendelian expected 1:1 expression ratio and is an important source of allele-specific heterogeneity. Expression allelic imbalance can be measured by allele-specific expression analysis (ASE) across heterozygous informative expressed single nucleotide variants (eSNVs). ASE reflects many regulatory biological phenomena that can be assessed by combining genome and transcriptome information. ASE contributes to the interindividual variability associated with disease. We aim to estimate the transcriptome-wide impact of SARS-CoV-2 infection by analyzing eSNVs. We compared ASE profiles in the human lung cell lines Calu-3, A459, and H522 before and after infection with SARS-CoV-2 using RNA-Seq experiments. We identified 34 differential ASE (DASE) sites in 13 genes (HLA-A, HLA-B, HLA-C, BRD2, EHD2, GFM2, GSPT1, HAVCR1, MAT2A, NQO2, SUPT6H, TNFRSF11A, UMPS), all of which are enriched in protein binding functions and play a role in COVID-19. Most DASE sites were assigned to the MHC class I locus and were predominantly upregulated upon infection. DASE sites in the MHC class I locus also occur in iPSC-derived airway epithelium basal cells infected with SARS-CoV-2. Using an RNA-Seq haplotype reconstruction approach, we found DASE sites and adjacent eSNVs in phase (i.e., predicted on the same DNA strand), demonstrating differential haplotype expression upon infection. We found a bias towards the expression of the HLA alleles with a higher binding affinity to SARS-CoV-2 epitopes. Independent of gene expression compensation, SARS-CoV-2 infection of human lung cell lines induces transcriptional allelic switching at the MHC loci. This suggests a response mechanism to SARS-CoV-2 infection that swaps HLA alleles with poor epitope binding affinity, an expectation supported by publicly available proteome data.

Human endogenous retrovirus K in the respiratory tract is associated with COVID-19 physiopathology (preprint)

Critically ill COVID-19 patients under invasive mechanical ventilation (IMV) are at greatly increased risk of death compared to the general population. While some drivers of COVID-19 disease progression, such as inflammation and hypercoagulability, have been identified, they do not completely explain the mortality of critically ill COVID-19 patients, making a search for overlooked factors necessary. A recent study examined the virome of tracheal aspirates from 25 COVID-19 patients under IMV. These samples were compared to tracheal aspirates from non-COVID patients and nasopharyngeal swabs from individuals with mild COVID-19. Critically ill COVID-19 patients had elevated expression of human endogenous retrovirus K (HERV-K), and elevated HERV-K expression in tracheal aspirate and plasma was associated with early mortality in those same patients. Among deceased patients, HERV-K expression was associated with IL-17-related inflammation, monocyte activation, and increased consumption of clotting factors. A subsequent in vitro experiment found that exposure to SARS-CoV-2 increased HERV-K expression in human primary monocytes from healthy donors. This preliminary study only included 25 individuals but implicates HERV-K in the physiopathology of COVID-19 and suggests that HERV-K could be used as a biomarker of disease severity in COVID-19 patients. 

Immunogenicity of trimetric spike protein associated to Poly(I:C) plus Alum (preprint)

The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid (Poly(I:C)) adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after one or two boosts. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation (S Ptn with Alum + Poly(I:C)) in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.

Apixaban, an orally available anticoagulant, inhibits SARS-CoV-2 replication by targeting its major protease in a non-competitive way (preprint)

Anticoagulants are associated with clinical benefit against the 2019 coronavirus disease (COVID-19), preventing COVID-19 associated coagulopathy. Blood coagulation factor Xa (FXa) and SARS-CoV-2 major protease (Mpro) share over 80% homology at the three-dimensional protein level. Thus, it is worth interrogating whether there is crosstalk between inhibitors and substrates between these enzymes. Here, we found that the clinically-approved FXa inhibitor apixaban targets SARS-CoV-2 Mpro with a 21-fold higher potency than boceprevir (GC376). Apixaban displayed a non-competitive mechanism of inhibition towards Mpro, since it targets the enzyme/substrate complex and the allosteric site onto the viral protease. Enzymatic assays were further validated in infected Calu-3 cells, which reveal that apixaban decreases the production of infectious viral particles in a dose-dependent manner, with an inhibitory potency in the micromolar range. Our results are in line with the proposed early use of anticoagulants, including FXa inhibitors, to improve clinical outcome of COVID-19 patients. In this context, apixaban may display a dual mechanism of action by targeting FXa to prevent coagulopathy and, at some level, SARS-CoV-2 Mpro.

Combination of Antiviral Drugs to Inhibit SARS-CoV-2 Polymerase and Exonuclease as Potential COVID-19 Therapeutics (preprint)

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.

Unlike Chloroquine, mefloquine inhibits SARS-CoV-2 infection in physiologically relevant cells and does not induce viral variants. (preprint)

Repositioning of clinical approved drugs could represent the fastest way to identify therapeutic options during public health emergencies, the majority of drugs explored for repurposing as antivirals for 2019 coronavirus disease (COVID-19) have failed to demonstrate clinical benefit. Without specific antivirals, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to cause major global mortality. Antimalarial drugs, such as chloroquine (CQ)/hydroxychloroquine (HCQ) and mefloquine have emerged as potential anti-SARS-CoV-2 antivirals. CQ/HCQ entered the Solidarity and RECOVERY clinical trials against COVID-19 and showed lack of efficacy. Importantly, mefloquine is not a 4-aminoquinoline like CQ and HCQ and has been previously repurposed for other respiratory diseases. Unlike the 4-aminoquinolines that accumulate in the high pH of intracellular lysosomes of the lung, the high respiratory tract penetration of mefloquine is driven by its high lipophilicity. While CQ and HCQ exhibit activity in Vero E6 cells, their activity is obviated in TMPRSS2-expressing cells, such as Calu-3 cells, which more accurately recapitulate in vivo entry mechanisms for SARS-CoV-2. Accordingly, here we report the anti-SARS-CoV-2 activity of mefloquine in Calu-3 type II pneumocytes and primary human monocytes. Mefloquine inhibited SARS-CoV-2 replication in Calu-3 cells with low cytotoxicity and EC50 and EC90 values of 1.2 and 5.3 {micro}M, respectively. In addition, mefloquine reduced up to 68% the SARS-CoV-2 RNA levels in infected monocytes, reducing viral-induced inflammation. Mefloquine blocked early steps of the SARS-CoV-2 replicative cycle and was less prone than CQ to induce drug-associated viral mutations and synergized with RNA polymerase inhibitor. The pharmacological parameters of mefloquine are consistent with its plasma exposure in humans and its tissue-to-plasma predicted coefficient points that this drug may accumulate in the lungs. These data indicate that mefloquine could represent an orally available clinically approved drug option against COVID-19 and should not be neglected on the basis of the failure of CQ and HCQ.

A cannabinoid receptor agonist shows anti-inflammatory and survival properties in human SARS-CoV-2-infected iPSC-derived cardiomyocytes (preprint)

Coronavirus disease 2019 (COVID-19) is caused by acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can infect several organs and lead to loss of vital organ function, especially impacting respiratory capacity. Among the extrapulmonary manifestations of COVID-19 is myocardial injury, caused both directly and indirectly by SARS-CoV-2, and which is associated with a high risk of mortality. One of the hallmarks of severe COVID-19 is the "cytokine storm", at which point the immune system malfunctions, leading to possible organ failure and death. Cannabinoids are known to have anti-inflammatory properties by negatively modulating the release of pro-inflammatory cytokines. Herein, we investigated the effects of the cannabinoid agonist WIN 55,212-2 (WIN) on SARS-CoV-2-infected human iPSC-derived cardiomyocytes (hiPSC-CMs). Although WIN did not modulate angiotensin-converting enzyme II, nor reduced SARS-CoV-2 infection and replication in hiPSC-CMs at the conditions tested, it had anti-inflammatory and protective effects by reducing the levels of interleukins 6, 8,18 and tumor necrosis factor-alpha (TNF-) and lactate dehydrogenase (LDH) activity in these cells without causing hypertrophic cardiac damage. These findings suggest that cannabinoids should be further investigated as an alternative therapeutic tool for the treatment of COVID-19. HighlightsO_LIHuman iPSC-derived cardiomyocytes (hiPSC-CMs) express CB1 receptor. C_LIO_LIThe cannabinoid receptor agonist, WIN 55,212-2 (WIN), does not influence SARS-CoV-2 infection in hiPSC-CMs. C_LIO_LIWIN reduces inflammation and death in SARS-CoV-2-infected hiPSC-CMs. C_LI

Inhibition of SARS-CoV-2 infection in human cardiomyocytes by targeting the Sigma-1 receptor disrupts cytoskeleton architecture and contractility (preprint)

Heart dysfunction, represented by conditions such as myocarditis and arrhythmia, has been reported in COVID-19 patients. Therapeutic strategies focused on the cardiovascular system, however, remain scarce. The Sigma-1 receptor (S1R) has been recently proposed as a therapeutic target because its inhibition reduces SARS-CoV-2 replication. To investigate the role of S1R in SARS-CoV-2 infection in the heart, we used human cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CM) as an experimental model. Here we show that the S1R antagonist NE-100 decreases SARS-CoV-2 infection and viral replication in hiPSC-CMs. Also, NE-100 reduces cytokine release and cell death associated with infection. Because S1R is involved in cardiac physiology, we investigated the effects of NE-100 in cardiomyocyte morphology and function. We show that NE-100 compromises cytoskeleton integrity and reduces beating frequency, causing contractile impairment. These results show that targeting S1R to challenge SARS-CoV-2 infection may be a useful therapeutic strategy but its detrimental effects in vivo on cardiac function should not be ignored.

The neuropeptides VIP and PACAP inhibit SARS-CoV-2 replication in monocytes and lung epithelial cells and decrease the production of proinflammatory cytokines in infected cells. (preprint)

Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), may elicit uncontrolled and damaging inflammatory reactions, due to an excessive immune response and dysregulated production of cytokines and chemokines. Thus, it is critical to identify compounds able to inhibit virus replication and thwart the excessive inflammatory reaction and tissue lesions secondary to SARS-CoV-2 infection. Here, we show that the neuropeptides VIP and PACAP, molecules endowed with immunoregulatory properties, were able to inhibit SARS-CoV-2 RNA synthesis/replication in human monocytes and viral production in lung epithelial cells. VIP and PACAP protected these cells from virus-induced cytopathicity, reduced the production of proinflammmatory mediators, and prevented the SARS-CoV-2-induced NF-kB activation, which is critically involved in the production of inflammatory mediators. Both neuropeptides promoted CREB activation in infected monocytes, a transcription factor with antiapoptotic activity and also a negative regulator of NF-kB. As a possible host response to control patient inflammation, we identified that VIP levels were elevated in plasma from patients with severe forms of COVID-19, correlating with the inflammatory marker CRP and survival on those patients. Since a synthetic form of VIP is clinically approved in Europe and under two clinical trials for patients with COVID-19, our results provide the scientific evidence to further support clinical investigation of these neuropeptides against COVID-19.

The in vitro antiviral activity of the anti-hepatitis C virus (HCV) drugs daclatasvir and sofosbuvir against SARS-CoV-2 (preprint)

The infection by the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes major public health concern and economic burden. Although clinically approved drugs have been repurposed to treat individuals with 2019 Coronavirus disease (COVID-19), the lack of safety studies and limited efficiency as well jeopardize clinical benefits. Daclatasvir and sofosbuvir (SFV) are clinically approved direct-acting antivirals (DAA) against hepatitis C virus (HCV), with satisfactory safety profile. In the HCV replicative cycle, daclatasvir and SFV target the viral enzymes NS5A and NS5B, respectively. NS5A is endowed with pleotropic activities, which overlap with several proteins from SARS-CoV-2. HCV NS5B and SARS-CoV-2 nsp12 are RNA polymerases that share homology in the nucleotide uptake channel. These characteristics of the HCV and SARS-CoV-2 motivated us to further study the activity of daclatasvir and SFV against the new coronavirus. Daclatasvir consistently inhibited the production of infectious SARS-CoV-2 virus particles in Vero cells, in the hepatoma cell line HuH-7 and in type II pneumocytes (Calu-3), with potencies of 0.8, 0.6 and 1.1 M, respectively. Daclatasvir targeted early events during SARS-CoV-2 replication cycle and prevented the induction of IL-6 and TNF-, inflammatory mediators associated with the cytokine storm typical of SARS-CoV-2 infection. Sofosbuvir, although inactive in Vero cells, displayed EC50 values of 6.2 and 9.5 M in HuH-7 and Calu-3 cells, respectively. Our data point to additional antiviral candidates, in especial daclatasvir, among drugs overlooked for COVID-19, that could immediately enter clinical trials.

Atazanavir inhibits SARS-CoV-2 replication and pro-inflammatory cytokine production (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors, such as atazanavir (ATV). ATV is of high interest because of its bioavailability within the respiratory tract. Our results show that ATV could dock in the active site of SARS-CoV-2 Mpro, with greater strength than LPV. ATV blocked Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells, human pulmonary epithelial cell line and primary monocytes, impairing virus-induced enhancement of IL-6 and TNF- levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19.