SARS-CoV-2 Variant Delta Potently Suppresses Innate Immune Response and Evades Interferon-Activated Antiviral Responses in Human Colon Epithelial Cells.

The Delta variant of SARS-CoV-2 has caused more severe infections than its previous variants. We studied the host innate immune response to Delta, Alpha, and two earlier variants to map the evolution of the recent ones. Our biochemical and transcriptomic studies in human colon epithelial cell line Caco2 reveal that Alpha and Delta have progressively evolved over the ancestral variants by silencing the innate immune response, thereby limiting cytokine and chemokine production. Though Alpha silenced the retinoic acid-inducible gene (RIG)-I-like receptor (RLR) pathway just like Delta did, it failed to persistently silence the innate immune response, unlike Delta. Both Alpha and Delta have evolved to resist interferon (IFN) treatment, while they are still susceptible to RLR activation, further highlighting the importance of RLR-mediated, IFN-independent mechanisms in restricting SARS-CoV-2. Our studies reveal that SARS-CoV-2 Delta has integrated multiple mechanisms to silence the host innate immune response and evade the IFN response. We speculate that Delta's silent replication and sustained suppression of the host innate immune response, thereby resulting in delayed or reduced intervention by the adaptive immune response, could have potentially contributed to the severe symptoms and poor recovery index associated with it. It is likely that this altered association with the host would play an important role in the coevolution of SARS-CoV-2 with humans. IMPORTANCE Viruses generally learn to coexist with the host during the process of evolution. It is expected that SARS-CoV-2 would also evolve to coexist in humans by trading off its virulence for longer persistence, causing milder disease. Clinically, the fatality associated with COVID-19 has been declining due to vaccination and preinfections, but the Delta variant caused the most severe disease and fatality across several parts of the world. Our study identified an evolving trend of SARS-CoV-2 variants where the variants that emerged during early parts of the pandemic caused a more robust innate immune response, while the later emerging variant Delta showed features of suppression of the response. The features that Delta has acquired could have strongly influenced the distinct pathophysiology associated with its infection. How these changed associations with the host influence the long-term evolution of the virus and the disease outcome should be closely studied to understand the process of viral evolution.

A Novel Single-Stranded RNA-Based Adjuvant Improves the Immunogenicity of the SARS-CoV-2 Recombinant Protein Vaccine.

The research and development (R&D) of novel adjuvants is an effective measure for improving the immunogenicity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant protein vaccine. Toward this end, we designed a novel single-stranded RNA-based adjuvant, L2, from the SARS-CoV-2 prototype genome. L2 could initiate retinoic acid-inducible gene-I signaling pathways to effectively activate the innate immunity. ZF2001, an aluminum hydroxide (Al) adjuvanted SARS-CoV-2 recombinant receptor binding domain (RBD) subunit vaccine with emergency use authorization in China, was used for comparison. L2, with adjuvant compatibility with RBD, elevated the antibody response to a level more than that achieved with Al, CpG 7909, or poly(I:C) as adjuvants in mice. L2 plus Al with composite adjuvant compatibility with RBD markedly improved the immunogenicity of ZF2001; in particular, neutralizing antibody titers increased by about 44-fold for Omicron, and the combination also induced higher levels of antibodies than CpG 7909/poly(I:C) plus Al in mice. Moreover, L2 and L2 plus Al effectively improved the Th1 immune response, rather than the Th2 immune response. Taken together, L2, used as an adjuvant, enhanced the immune response of the SARS-CoV-2 recombinant RBD protein vaccine in mice. These findings should provide a basis for the R&D of novel RNA-based adjuvants.

Inhibition of Glycolysis Impairs Retinoic Acid-Inducible Gene I-Mediated Antiviral Responses in Primary Human Dendritic Cells.

Dendritic cells (DCs) are important mediators of the induction and regulation of adaptive immune responses following microbial infection and inflammation. Sensing environmental danger signals including viruses, microbial products, or inflammatory stimuli by DCs leads to the rapid transition from a resting state to an activated mature state. DC maturation involves enhanced capturing and processing of antigens for presentation by major histocompatibility complex (MHC) class I and class II, upregulation of chemokines and their receptors, cytokines and costimulatory molecules, and migration to lymphoid tissues where they prime naive T cells. Orchestrating a cellular response to environmental threats requires a high bioenergetic cost that accompanies the metabolic reprogramming of DCs during activation. We previously demonstrated that DCs undergo a striking functional transition after stimulation of the retinoic acid-inducible gene I (RIG-I) pathway with a synthetic 5' triphosphate containing RNA (termed M8), consisting of the upregulation of interferon (IFN)-stimulated antiviral genes, increased DC phagocytosis, activation of a proinflammatory phenotype, and induction of markers associated with immunogenic cell death. In the present study, we set out to determine the metabolic changes associated with RIG-I stimulation by M8. The rate of glycolysis in primary human DCs was increased in response to RIG-I activation, and glycolytic reprogramming was an essential requirement for DC activation. Pharmacological inhibition of glycolysis in monocyte-derived dendritic cells (MoDCs) impaired type I IFN induction and signaling by disrupting the TBK1-IRF3-STAT1 axis, thereby countering the antiviral activity induced by M8. Functionally, the impaired IFN response resulted in enhanced viral replication of dengue, coronavirus 229E, and Coxsackie B5.

A Retinol Derivative Inhibits SARS-CoV-2 Infection by Interrupting Spike-Mediated Cellular Entry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the global pandemic and life-threatening coronavirus disease 2019 (COVID-19). Although vaccines and therapeutic antibodies are available, their efficacy is continuously undermined by rapidly emerging SARS-CoV-2 variants. Here, we found that all-trans retinoic acid (ATRA), a vitamin A (retinol) derivative, showed potent antiviral activity against all SARS-CoV-2 variants in both human cell lines and human organoids of the lower respiratory tract. Mechanistically, ATRA directly binds in a deep hydrophobic pocket of the receptor binding domain (RBD) located on the top of the SARS-CoV-2 spike protein (S) trimer. The bound ATRA mediates strong interactions between the "down" RBDs and locks most of the S trimers in an RBD "all-down" and ACE2-inaccessible inhibitory conformation. In summary, our results reveal the pharmacological biotargets and structural mechanism of ATRA and other retinoids in SARS-CoV-2 infection and suggest that ATRA and its derivatives could be potential hit compounds against a broad spectrum of coronaviruses. IMPORTANCE Retinoids, a group of compounds including vitamin A and its active metabolite all-trans retinoic acid (ATRA), regulate serial physiological activity in multiple organ systems, such as cell growth, differentiation, and apoptosis. The ATRA analogues reported to date include more than 4,000 natural and synthetic molecules that are structurally and/or functionally related to ATRA. Here, we found that ATRA showed potent antiviral activity against all SARS-CoV-2 variants by directly binding in a deep hydrophobic pocket of the receptor binding domain (RBD) located on top of the SARS-CoV-2 spike protein (S) trimer. The bound ATRA mediates strong interactions between the "down" RBDs and locks most of the S trimers in an RBD "all-down" and ACE2-inaccessible inhibitory conformation, suggesting the pharmacological feasibility of using ATRA or its derivatives as a remedy for and prevention of COVID-19 disease.

All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity.

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.

Genomics-guided identification of potential modulators of SARS-CoV-2 entry proteases, TMPRSS2 and Cathepsins B/L.

SARS-CoV-2 requires serine protease, transmembrane serine protease 2 (TMPRSS2), and cysteine proteases, cathepsins B, L (CTSB/L) for entry into host cells. These host proteases activate the spike protein and enable SARS-CoV-2 entry. We herein performed genomic-guided gene set enrichment analysis (GSEA) to identify upstream regulatory elements altering the expression of TMPRSS2 and CTSB/L. Further, medicinal compounds were identified based on their effects on gene expression signatures of the modulators of TMPRSS2 and CTSB/L genes. Using this strategy, estradiol and retinoic acid have been identified as putative SARS-CoV-2 alleviation agents. Next, we analyzed drug-gene and gene-gene interaction networks using 809 human targets of SARS-CoV-2 proteins. The network results indicate that estradiol interacts with 370 (45%) and retinoic acid interacts with 251 (31%) human proteins. Interestingly, a combination of estradiol and retinoic acid interacts with 461 (56%) of human proteins, indicating the therapeutic benefits of drug combination therapy. Finally, molecular docking analysis suggests that both the drugs bind to TMPRSS2 and CTSL with the nanomolar to low micromolar affinity. The results suggest that these drugs can simultaneously target both the entry pathways of SARS-CoV-2 and thus can be considered as a potential treatment option for COVID-19.

Immunomodulatory agents as potential therapeutic or preventive strategies for COVID-19.

Currently, the COVID-19 pandemic, caused by the novel SARS-CoV-2 coronavirus, represents the greatest global health threat. Most people infected by the virus present mild to moderate respiratory symptoms and recover with supportive treatments. However, certain susceptible hosts develop an acute respiratory distress syndrome (ARDS), associated with an inflammatory "cytokine storm", leading to lung damage. Despite the current availability of different COVID-19 vaccines, the new emerging SARS-CoV-2 genetic variants represent a major concern worldwide, due to their increased transmissibility and rapid spread. Indeed, it seems that some mutations or combinations of mutations might confer selective advantages to the virus, such as the ability to evade the host immune responses elicited by COVID-19 vaccines. Several therapeutic approaches have been investigated but, to date, a unique and fully effective therapeutic protocol has not yet been achieved. In addition, steroid-based therapies, aimed to reduce inflammation in patients with severe COVID-19 disease, may increase the risk of opportunistic infections, increasing the hospitalization time and mortality rate of these patients. Hence, there is an unmet need to develop more effective therapeutic options. Here, we discuss the potential use of natural immunomodulators such as Thymosin α1 (Tα1), all-trans retinoic acid (ATRA), and lactoferrin (LF), as adjunctive or preventive treatment of severe COVID-19 disease. These agents are considered to be multifunctional molecules because of their ability to enhance antiviral host immunity and restore the immune balance, depending on the host immune status. Furthermore, they are able to exert a broad-spectrum antimicrobial activity by means of direct interactions with cellular or molecular targets of pathogens or indirectly by increasing the host immune response. Thus, due to the aforementioned properties, these agents might have a great potential in a clinical setting, not only to counteract SARS-CoV-2 infection, but also to prevent opportunistic infections in critically ill COVID-19 patients.

Retinoic acid improves baseline barrier function and attenuates TNF-α-induced barrier leak in human bronchial epithelial cell culture model, 16HBE 14o.

Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-α-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in 14C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-α-induced 16HBE barrier leak, attenuating 60% of the TNF-α-induced leak to 14C-mannitol and 80% of the leak to 14C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-α resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward "band-shift" in occludin Western immunoblots. The presence of RA partially reversed TNF-α's effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-α-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a "cytokine storm" may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.

In silico identification of Tretinoin as a SARS-CoV-2 envelope (E) protein ion channel inhibitor.

Viroporins are oligomeric, pore forming, viral proteins that play critical roles in the life cycle of pathogenic viruses. Viroporins like HIV-1 Vpu, Alphavirus 6 K, Influenza M2, HCV p7, and Picornavirus 2B, form discrete aqueous passageways which mediate ion and small molecule transport in infected cells. The alterations in host membrane structures induced by viroporins is essential for key steps in the virus life cycle like entry, replication and egress. Any disruption in viroporin functionality severely compromises viral pathogenesis. The envelope (E) protein encoded by coronaviruses is a viroporin with ion channel activity and has been shown to be crucial for the assembly and pathophysiology of coronaviruses. We used a combination of virtual database screening, molecular docking, all-atom molecular dynamics simulation and MM-PBSA analysis to test four FDA approved drugs - Tretinoin, Mefenamic Acid, Ondansetron and Artemether - as potential inhibitors of ion channels formed by SARS-CoV-2 E protein. Interaction and binding energy analysis showed that electrostatic interactions and polar solvation energy were the major driving forces for binding of the drugs, with Tretinoin being the most promising inhibitor. Tretinoin bound within the lumen of the channel formed by E protein, which is lined by hydrophobic residues like Phe, Val and Ala, indicating its potential for blocking the channel and inhibiting the viroporin functionality of E. In control simulations, tretinoin demonstrated a lower binding energy with a known target as compared to SARS-CoV-2 E protein. This work thus highlights the possibility of exploring Tretinoin as a potential SARS-CoV-2 E protein ion channel blocker and virus assembly inhibitor, which could be an important therapeutic strategy in the treatment for coronaviruses.

HPLC-MS/MS Shows That the Cellular Uptake of All-Trans-Retinoic Acid under Hypoxia Is Downregulated by the Novel Active Agent 5-Methoxyleoligin.

All-trans-retinoic acid (atRA) is the essential derivative of vitamin A and is of interest due to its various biological key functions. As shown in the recent literature, atRA also plays a role in the failing heart during myocardial infarction, the leading cause of death globally. To date insufficient mechanistic information has been available on related hypoxia-induced cell damage and reperfusion injuries. However, it has been demonstrated that a reduction in cellular atRA uptake abrogates hypoxia-mediated cell and tissue damage, which may offer a new route for intervention. Consequently, in this study, the effect of the novel cardio-protective compound 5-methoxyleoligin (5ML) on cellular atRA uptake was tested in human umbilical-vein endothelial cells (HUVECs). For this purpose, a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed to assess intra-cellular levels of the active substance and corresponding levels of vitamin A and its derivatives, including potential cis/trans isomers. This work also focused on light-induced isomerization and the stability of biological sample material to ensure sample integrity and avoid biased conclusions. This study provides evidence of the inhibitory effect of 5ML on cellular atRA uptake, a promising step toward a novel therapy for myocardial infarction.

COVID-19: Endogenous Retinoic Acid Theory and Retinoic Acid Depletion Syndrome.

This study presents two new concepts and definitions to the medical literature. One of those is "endogenous retinoic acid theory" and the other "retinoic acid depletion syndrome". A new classification will be provided for the immune system: "retinoic acid-dependent component" and "retinoic acid non-dependent component". If this theory is verified, all the diseases where the retinoic acid metabolism is defective and retinoic acid levels are low will be identified and new approaches will be developed fortreating such diseases. When the need for retinoic acids increases, such as acute infection, high fever, severe catabolic process, or chronic antigenic stimulation, cytochrome oxidase enzymes are inhibited by drugs or internal mechanisms. Metabolism and excretion of retinoic acids stored in the liver are prevented. In this way, retinoic acid levels in the blood are raised to therapeutic levels. This is called "Endogenous Retinoic Acid Theory". Retinoic acids also manage their metabolism through feedback mechanisms. Despite compensatory mechanisms, causes such as high fever, serious catabolic process and excessively large viral genome (SARS-CoV-2), excessive use of RIG-I and Type I interferon synthesis pathway using retinoic acid causes emptying of retinoic acid stores. As a result, the RIG-I pathway becomes ineffective, Type I IFN synthesis stops, and the congenital immune system collapses. Then the immune mechanism passes to TLR3, TLR7, TLR8, TLR9, MDA5 and UPS pathways in the monocyte, macrophage, neutrophil and dendritic cells of the adaptive immune defense system that do not require retinoic acid. This leads to excessive TNFα and cytokine discharge from the pathway. With the depletion of retinoic acid stores as a result of this overuse, the immune defense mechanism switches from the congenital immune system to the adaptive immune system, where retinoic acids cannot be used. As a result of this depletion of retinoic acids, the shift of the immune system to the NFκB arm, which causes excessive cytokine release, is called "retinoic acid depletion syndrome". COVID-19 and previously defined sepsis, SIRS and ARDS are each retinoic acid depletion syndrome. We claim that retinoic acid metabolism is defective in most inflammatory diseases, particularly COVID-19 (cytokine storm) sepsis, SIRS and ARDS. Finding a solution to this mechanism will bring a new perspective and treatment approach to such diseases.

Activation of C-Type Lectin Receptor and (RIG)-I-Like Receptors Contributes to Proinflammatory Response in Middle East Respiratory Syndrome Coronavirus-Infected Macrophages.

BACKGROUND: Human infection with Middle East respiratory syndrome coronavirus (MERS-CoV) poses an ongoing threat to public health worldwide. The studies of MERS patients with severe disease and experimentally infected animals showed that robust viral replication and intensive proinflammatory response in lung tissues contribute to high pathogenicity of MERS-CoV. We sought to identify pattern recognition receptor (PRR) signaling pathway(s) that mediates the inflammatory cascade in human macrophages upon MERS-CoV infection. METHODS: The potential signaling pathways were manipulated individually by pharmacological inhibition, small interfering ribonucleic acid (siRNA) depletion, and antibody blocking. The MERS-CoV-induced proinflammatory response was evaluated by measuring the expression levels of key cytokines and/or chemokines. Reverse transcription-quantitative polymerase chain reaction assay, flow cytometry analysis, and Western blotting were applied to evaluate the activation of related PRRs and engagement of adaptors. RESULTS: MERS-CoV replication significantly upregulated C-type lectin receptor (CLR) macrophage-inducible Ca2+-dependent lectin receptor (Mincle). The role of Mincle for MERS-CoV-triggered cytokine/chemokine induction was established based on the results of antibody blockage, siRNA depletion of Mincle and its adaptor spleen tyrosine kinase (Syk), and Syk pharmacological inhibition. The cytokine and/or chemokine induction was significantly attenuated by siRNA depletion of retinoic acid-inducible-I-like receptors (RLR) or adaptor, indicating that RLR signaling also contributed to MERS-CoV-induced proinflammatory response. CONCLUSIONS: The CLR and RLR pathways are activated and contribute to the proinflammatory response in MERS-CoV-infected macrophages.