Involvement of cell surface glycosaminoglycans in chebulagic acid's and punicalagin's antiviral activities against Coxsackievirus A16 infection.

BACKGROUND: Coxsackievirus A16 (CVA16) is responsible for several recent outbreaks of Hand, Foot, and Mouth Disease in the Asia-Pacific region, and there are currently no vaccines or specific treatments available. We have previously identified two tannins, chebulagic acid (CHLA) and punicalagin (PUG), as efficient entry inhibitors against multiple viruses known to engage cell surface glycosaminoglycans (GAGs). Interestingly, these two phytochemicals could also block enterovirus infection by directly inactivating CVA16 virions, which were recently reported to utilize GAGs to mediate its entry. PURPOSE: The aim of this study is to evaluate the involvement of GAGs in the anti-CVA16 activities of CHLA and PUG. METHODS: To explore a potential mechanistic link, the role of GAGs in promoting CVA16 entry was first confirmed by treating human rhabdomyosarcoma (RD) cells with soluble heparin or GAG lyases including heparinase and chondroitinase. We then performed a combination treatment of CHLA or PUG with the GAG interaction inhibitors to assess whether CHLA's and PUG's anti-CVA16 activities were related to GAG competition. Molecular docking and surface plasmon resonance (SPR) were conducted to analyze the interactions between CHLA, PUG, and CVA16 capsid. Lastly, CRISPR/Cas9 knockout (KO) of the Exostosin glycosyltransferase 1 (EXT1) gene, which encodes a transmembrane glycosyltransferase involved in heparan sulfate biosynthesis, was used to validate the importance of GAGs in CHLA's and PUG's antiviral effects. RESULTS: Intriguingly, combining GAG inhibition via heparin/GAG lyases treatments with CHLA and PUG revealed that their inhibitory activities against CVA16 infection were overlapping. Further molecular docking analysis indicated that the predicted binding sites of CHLA and PUG on the CVA16 capsid are in proximity to the putative residues recognized for GAG interaction, thus pointing to potential interference with the CVA16-GAG association. SPR analysis also confirmed the direct binding of CHLA and PUG to CVA16 capsid. Finally, RD cells with EXT1 KO decreased CHLA's and PUG's antiviral effect on CVA16 infection. CONCLUSION: Altogether, our results suggest that CHLA and PUG bind to CVA16 capsid and prevent the virus' interaction with heparan sulfate and chondroitin sulfate for its entry. This study provides mechanistic insight into the antiviral activity of CHLA and PUG against CVA16, which may be helpful for the development of antiviral strategies against the enterovirus.

Combination of Oncolytic Measles Virus and Ursolic Acid Synergistically Induces Oncolysis of Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) remains a difficult-to-treat cancer due to late diagnosis and limited curative treatment options. Developing more effective therapeutic strategies is essential for the management of HCC. Oncolytic virotherapy is a novel treatment modality for cancers, and its combination with small molecules merits further exploration. In this study, we combined oncolytic measles virus (MV) with the natural triterpenoid compound ursolic acid (UA) and evaluated their combination effect against HCC cells, including those harboring hepatitis B virus (HBV) or hepatitis C virus (HCV) replication. We found that the combination of MV and UA synergistically induced more cell death in Huh-7 HCC cells through enhanced apoptosis. In addition, increased oxidative stress and loss of mitochondrial potential were observed in the treated cells, indicating dysregulation of the mitochondria-dependent pathway. Similar synergistic cytotoxic effects were also found in HCC cells harboring HBV or HCV genomes. These findings underscore the potential of oncolytic MV and UA combination for further development as a treatment strategy for HCC.

Production and characterization of single-chain variable fragment antibodies targeting the breast cancer tumor marker nectin-4.

Background: Nectin-4 is a novel biomarker overexpressed in various types of cancer, including breast cancer, in which it has been associated with poor prognosis. Current literature suggests that nectin-4 has a role in cancer progression and may have prognostic and therapeutic implications. The present study aims to produce nectin-4-specific single-chain variable fragment (scFv) antibodies and evaluate their applications in breast cancer cell lines and clinical specimens. Methods: We generated recombinant nectin-4 ectodomain fragments as immunogens to immunize chickens and the chickens' immunoglobulin genes were amplified for construction of anti-nectin-4 scFv libraries using phage display. The binding capacities of the selected clones were evaluated with the recombinant nectin-4 fragments, breast cancer cell lines, and paraffin-embedded tissue sections using various laboratory approaches. The binding affinity and in silico docking profile were also characterized. Results: We have selected two clones (S21 and L4) from the libraries with superior binding capacity. S21 yielded higher signals when used as the primry antibody for western blot analysis and flow cytometry, whereas clone L4 generated cleaner and stronger signals in immunofluorescence and immunohistochemistry staining. In addition, both scFvs could diminish attachment-free cell aggregation of nectin-4-positive breast cancer cells. As results from ELISA indicated that L4 bound more efficiently to fixed nectin-4 ectodomain, molecular docking analysis was further performed and demonstrated that L4 possesses multiple polar contacts with nectin-4 and diversity in interacting residues. Conclusion: Overall, the nectin-4-specific scFvs could recognize nectin-4 expressed by breast cancer cells and have the merit of being further explored for potential diagnostic and therapeutic applications.

Validation of Viral Inactivation Protocols for Therapeutic Blood Products against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-COV-2).

Therapeutic blood products including convalescent plasma/serum and immunoglobulins concentrated from convalescent plasma, such as intravenous immunoglobulins or hyperimmune globulins, and monoclonal antibodies are passive immunotherapy options for novel coronavirus disease 2019 (COVID-19). They have been shown to improve the clinical status and biological and radiological parameters in some groups of COVID-19 patients. However, blood products are still potential sources of virus transmission in recipients. The use of pathogen reduction technology (PRT) should increase the safety of the products. The purpose of this study was to determine the impact of solvent/detergents (S/D) procedures on SARS-COV-2 infectivity elimination in the plasma of donors but also on COVID-19 convalescent serum (CCS) capacity to neutralize SARS-COV-2 infectivity. In this investigation, S/D treatment for all experiments was performed at a shortened process time (30 min). We first evaluated the impact of S/D treatments (1% TnBP/1% TritonX-45 and 1% TnBP/1% TritonX-100) on the inactivation of SARS-COV-2 pseudoparticles (SARS-COV-2pp)-spiked human plasma followed by S/D agent removal using a Sep-Pak Plus C18 cartridge. Both treatments were able to completely inactivate SARS-COV-2pp infectivity to an undetectable level. Moreover, the neutralizing activity of CCS against SARS-COV-2pp was preserved after S/D treatments. Our data suggested that viral inactivation methods using such S/D treatments could be useful in the implementation of viral inactivation/elimination processes of therapeutic blood products against SARS-COV-2.

Therapeutic Strategies against Ebola Virus Infection.

Since the 2014-2016 epidemic, Ebola virus (EBOV) has spread to several countries and has become a major threat to global health. EBOV is a risk group 4 pathogen, which imposes significant obstacles for the development of countermeasures against the virus. Efforts have been made to develop anti-EBOV immunization and therapeutics, with three vaccines and two antibody-based therapeutics approved in recent years. Nonetheless, the high fatality of Ebola virus disease highlights the need to continuously develop antiviral strategies for the future management of EBOV outbreaks in conjunction with vaccination programs. This review aims to highlight potential EBOV therapeutics and their target(s) of inhibition, serving as a summary of the literature to inform readers of the novel candidates available in the continued search for EBOV antivirals.

Pandemic strategies with computational and structural biology against COVID-19: A retrospective.

The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is the etiologic agent of the coronavirus disease 2019 (COVID-19) pandemic, has dominated all aspects of life since of 2020. Research studies on the virus and exploration of therapeutic and preventive strategies has been moving at rapid rates to control the pandemic. In the field of bioinformatics or computational and structural biology, recent research strategies have used multiple disciplines to compile large datasets to uncover statistical correlations and significance, visualize and model proteins, perform molecular dynamics simulations, and employ the help of artificial intelligence and machine learning to harness computational processing power to further the research on COVID-19, including drug screening, drug design, vaccine development, prognosis prediction, and outbreak prediction. These recent developments should help us better understand the viral disease and develop the much-needed therapies and strategies for the management of COVID-19.

Immunogenic cell death: The cornerstone of oncolytic viro-immunotherapy.

According to the World Health Organization, cancer is one of the leading global health concerns, causing nearly 10 million deaths in 2020. While classical chemotherapeutics produce strong cytotoxicity on cancer cells, they carry limitations of drug resistance and off-target effects and sometimes fail to elicit adequate antitumor protection against tumor relapse. Additionally, most cancer cells have developed various ways to escape immune surveillance. Nevertheless, novel anticancer strategies such as oncolytic viro-immunotherapy can trigger immunogenic cell death (ICD), which can quickly grasp the attention of the host defense machinery, resulting in an ensuing antitumor immune response. Specifically, oncolytic viruses (OVs) can infect and destroy targeted cancer cells and stimulate the immune system by exposing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to promote inflammatory reactions, and concomitantly prime and induce antitumor immunity by the release of neoantigens from the damaged cancer cells. Thus, OVs can serve as a novel system to sensitize tumor cells for promising immunotherapies. This review discusses the concept of ICD in cancer, centralizing ICD-associated danger signals and their consequence in antitumor responses and ICD induced by OVs. We also shed light on the potential strategies to enhance the immunogenicity of OVs, including the use of genetically modified OVs and their combination with ICD-enhancing agents, which are helpful as forthcoming anticancer regimens.

The Methanolic Extract of Perilla frutescens Robustly Restricts Ebola Virus Glycoprotein-Mediated Entry.

Ebola virus (EBOV), one of the most infectious human viruses and a leading cause of viral hemorrhagic fever, imposes a potential public health threat with several recent outbreaks. Despite the difficulties associated with working with this pathogen in biosafety level-4 containment, a protective vaccine and antiviral therapeutic were recently approved. However, the high mortality rate of EBOV infection underscores the necessity to continuously identify novel antiviral strategies to help expand the scope of prophylaxis/therapeutic management against future outbreaks. This includes identifying antiviral agents that target EBOV entry, which could improve the management of EBOV infection. Herein, using EBOV glycoprotein (GP)-pseudotyped particles, we screened a panel of natural medicinal extracts, and identified the methanolic extract of Perilla frutescens (PFME) as a robust inhibitor of EBOV entry. We show that PFME dose-dependently impeded EBOV GP-mediated infection at non-cytotoxic concentrations, and exerted the most significant antiviral activity when both the extract and the pseudoparticles are concurrently present on the host cells. Specifically, we demonstrate that PFME could block viral attachment and neutralize the cell-free viral particles. Our results, therefore, identified PFME as a potent inhibitor of EBOV entry, which merits further evaluation for development as a therapeutic strategy against EBOV infection.

Small molecules baicalein and cinnamaldehyde are potentiators of measles virus-induced breast cancer oncolysis.

BACKGROUND: Although the breast cancer mortality has slowed down from 2008 to 2017, breast cancer incidence rate continues to rise and thus, new and/or improved treatments are highly needed. Among them, oncolytic virotherapy which has the ability of facilitating the antitumor adaptive immunity, appears as a promising anticancer therapy. Oncolytic measles virus (MV) is particularly suitable for targeting breast cancer due to the upregulation of MV's receptor nectin-4. Nonetheless, with limited clinical success currently, ways of boosting MV-induced breast cancer oncolysis are therefore necessary. Oncolytic virotherapy alone and combined with chemotherapeutic drugs are two strategic areas with intensive development for the search of anticancer drugs. Considering that baicalein (BAI) and cinnamaldehyde (CIN) have demonstrated antitumor properties against multiple cancers including breast cancer, they could be good partners for MV-based oncolytic virotherapy. PURPOSE: To assess the in vitro effect of BAI and CIN with MV and assess their combination effects. METHODS: We examined the combinatorial cytotoxic effect of oncolytic MV and BAI or CIN on MCF-7 breast cancer cells. Potential anti-MV activities of the phytochemicals were first investigated in vitro to determine the optimal combination model. Synergism of MV and BAI or CIN was then evaluated in vitro by calculating the combination indices. Finally, cell cycle analysis and apoptosis assays were performed to confirm the mechanism of synergism. RESULTS: Overall, the viral sensitization combination modality using oncolytic MV to first infect MCF-7 breast cancer cells followed by drug treatment with BAI or CIN was found to produce significantly enhanced tumor killing. Further mechanistic studies showed that the combinations 'MV-BAI' and 'MV-CIN' display synergistic anti-breast cancer effect, mediated by elevated apoptosis. CONCLUSION: We demonstrated, for the first time, effective combination of oncolytic MV with BAI or CIN that could be further explored and potentially developed into novel therapeutic strategies targeting nectin-4-marked breast cancer cells.

Ursolic Acid and Its Nanoparticles Are Potentiators of Oncolytic Measles Virotherapy against Breast Cancer Cells.

Oncolytic viruses (OVs) and phytochemical ursolic acid (UA) are two efficacious therapeutic candidates in development against breast cancer, the deadliest women's cancer worldwide. However, as single agents, OVs and UA have limited clinical efficacies. As a common strategy of enhancing monotherapeutic anticancer efficacy, we explored the combinatorial chemovirotherapeutic approach of combining oncolytic measles virus (MV), which targets the breast tumor marker Nectin-4, and the anticancer UA against breast adenocarcinoma. Our findings revealed that in vitro co-treatment with UA synergistically potentiated the killing of human breast cancer cells by oncolytic MV, without UA interfering the various steps of the viral infection. Mechanistic studies revealed that the synergistic outcome from the combined treatment was mediated through UA's potentiation of apoptotic killing by MV. To circumvent UA's poor solubility and bioavailability and strengthen its clinical applicability, we further developed UA nanoparticles (UA-NP) by nanoemulsification. Compared to the non-formulated UA, UA-NP exhibited improved drug dissolution property and similarly synergized with oncolytic MV in inducing apoptotic breast cancer cell death. This oncolytic potentiation was partly attributed to the enhanced autophagic flux induced by the UA-NP and MV combined treatment. Finally, the synergistic effect from the UA-NP and MV combination was also observed in BT-474 and MDA-MB-468 breast cancer cells. Our study thus highlights the potential value of oncolytic MV and UA-based chemovirotherapy for further development as a treatment strategy against breast cancer, and the feasibility of employing nanoformulation to enhance UA's applicability.

Identification of the phytobioactive Polygonum cuspidatum as an antiviral source for restricting dengue virus entry.

Dengue virus (DENV) is a mosquito-borne pathogen that is becoming a serious global threat, owing to its rising incidence in inter-tropical regions that yield over 50 million annual infections. There are currently no approved antiviral agents for the management of dengue, and recent shortcomings in its immunization called for immediate action to develop effective drugs with prophylactic ability to better manage its infection. In an attempt to discover novel antiviral sources, we identified the medicinal herb Polygonum cuspidatum (PC) as a bioactive botanical material against DENV infectivity. Specifically, the methanolic extract from PC rhizomes (PCME) potently inhibited DENV infection without causing significant cytotoxicity. Further examination on the viral life cycle demonstrated that PCME particularly targeted the initial stages of DENV infection, while pre- and post-infection treatments had no effect. More importantly, the PCME could efficiently inactivate DENV free virus particles and block the viral attachment and entry/fusion events without apparently influencing viral replication, egress, and cell-to-cell spread. The antiviral effect of PCME was also recapitulated in infection analysis using DENV pseudoparticles displaying viral structural proteins that mediate DENV particle entry. Besides, PCME treatment also inhibited direct DENV entry into several cell types relevant to its infection and reduced viral infectivity of other members of the Flaviviridae family, including the hepatitis C virus (HCV) and Zika virus (ZIKV). Due to its potency against DENV entry, we suggest that the phytobioactive extract from PC is an excellent starting point as an antiviral source material for further development of therapeutic strategies in the prophylactic management of DENV infection.

Contribution of Human Retroviruses to Disease Development-A Focus on the HIV- and HERV-Cancer Relationships and Treatment Strategies.

Animal retroviruses are known for their transforming potential, and this is also true for the ones hosted by humans, which have gathered expanding attention as one of the potent causative agents in various disease, including specific cancer types. For instance, Human T Lymphotropic virus (HTLV) is a well-studied class of oncoviruses causing T cell leukemia, while human immunodeficiency virus (HIV) leads to acquired immunodeficiency syndrome (AIDS), which is linked to a series of defining cancers including Kaposi sarcoma, certain types of non-Hodgkin lymphoma, and cervical cancer. Of note, in addition to these "modern" exogenous retroviruses, our genome harbors a staggering number of human endogenous retroviruses (HERVs). HERVs are the genetic remnants of ancient retroviral germline infection of human ancestors and are typically silenced in normal tissues due to inactivating mutations and sequence loss. While some HERV elements have been appropriated and contribute to human physiological functions, others can be reactivated through epigenetic dysregulations to express retroviral elements and promote carcinogenesis. Conversely, HERV replication intermediates or protein products can also serve as intrinsic pathogen-associated molecular patterns that cause the immune system to interpret it as an exogenous infection, thereby stimulating immune responses against tumors. As such, HERVs have also been targeted as a potential internal strategy to sensitize tumor cells for promising immunotherapies. In this review, we discuss the dynamic role of human retroviruses in cancer development, focusing on HIV and HERVs contribution. We also describe potential treatment strategies, including immunotherapeutic targeting of HERVs, inhibiting DNA methylation to expose HERV signatures, and the use of antiretroviral drugs against HIV and HERVs, which can be employed as prospective anti-cancer modalities.

Targeting Autophagy Augments BBR-Mediated Cell Death in Human Hepatoma Cells Harboring Hepatitis C Virus RNA.

Hepatocellular carcinoma (HCC), including hepatitis C virus (HCV)-induced HCC, is a deadly disease highly refractory to chemotherapy, thus requiring the continuous identification of novel treatment strategies. Berberine (BBR) has been previously reported to inhibit hepatoma cell growth, but the main type of cell death elicited by BBR, and whether the alkaloid can inhibit hepatoma cells carrying HCV genomes, is unclear. Herein, we show that BBR treatment induced a biphasic cell death irrespective of the presence of HCV subgenomic replicon RNA, first triggering apoptosis that then progressed to necrosis between 24 and 48 h post-treatment. Furthermore, BBR treatment potentiated the HCV replicon-induced reactive oxygen species (ROS) production, inhibition of which with an antioxidant attenuated the cell death that was elicited by BBR in these cells. Moreover, BBR dampened the autophagic response in HCV RNA-positive or negative hepatoma cells, and pharmacological inhibition of autophagy conversely augmented the BBR-induced cell death. Finally, BBR inhibited the growth of Huh-7 cells that were persistently infected with the full-length genome HCV particles, and concomitant pharmacological inhibition of autophagy potentiated the killing of these cells by BBR. Our findings suggest that combining BBR with the inhibition of autophagy could be an attractive treatment strategy against HCC, irrespective of the presence of the HCV genome.

Update on Antiviral Strategies Against COVID-19: Unmet Needs and Prospects.

By December 2020, the COVID-19 pandemic had caused more than 74 million confirmed cases and 1.6 million related deaths around the world. However, only a few drugs have been approved in certain areas and for use in conditional patients, and the vaccine candidates were only recently approved or authorized for emergency use without being fully implemented worldwide, suggesting that we are yet to reach effective control of the current outbreak as its uninhibited transmission continues precariously. Over the past few months, several therapeutic candidates have been proven ineffective in large clinical trials, while some other agents exhibited promising preliminary results. Meanwhile, the investigation of SARS-CoV-2-specific antivirals is underway. Despite still being preclinical, these agents could be beneficial for the long-term control of COVID-19 and deserve more research focus. In this article, we update the current status of therapeutic candidates that have been examined for COVID-19 management, including the virus-targeting inhibitors and host-targeting agents, with their antiviral efficacy in vitro, in vivo, and in clinical studies. Finally, we highlight the current challenges and future prospect of developing potent therapeutic agents against COVID-19.

Virus-Like Particle Systems for Vaccine Development against Viruses in the Flaviviridae Family.

Viruses in the Flaviviridae family are important human and animal pathogens that impose serious threats to global public health. This family of viruses includes emerging and re-emerging viruses, most of which are transmitted by infected mosquito or tick bites. Currently, there is no protective vaccine or effective antiviral treatment against the majority of these viruses, and due to their growing spread, several strategies have been employed to manufacture prophylactic vaccines against these infectious agents including virus-like particle (VLP) subunit vaccines. VLPs are genomeless viral particles that resemble authentic viruses and contain critical repetitive conformational structures on their surface that can trigger the induction of both humoral and cellular responses, making them safe and ideal vaccine candidates against these viruses. In this review, we focus on the potential of the VLP platform in the current vaccine development against the medically important viruses in the Flaviviridae family.

Use of Viral Entry Assays and Molecular Docking Analysis for the Identification of Antiviral Candidates against Coxsackievirus A16.

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for the identification of candidate viral entry inhibitors. Here, we present the experimental approaches for the identification of small molecules capable of blocking infection by the non-enveloped coxsackievirus A16 (CVA16) through targeting the virus particles or specific steps in early viral entry. Assays include the time-of-drug-addition analysis, flow cytometry-based viral binding assay, and viral inactivation assay. We also present a molecular docking protocol utilizing virus capsid proteins to predict potential residues targeted by the antiviral compounds. These assays should help in the identification of candidate antiviral agents that act on viral entry. Future directions can explore these possible inhibitors for further drug development.

Chemovirotherapeutic Treatment Using Camptothecin Enhances Oncolytic Measles Virus-Mediated Killing of Breast Cancer Cells.

Oncolytic virotherapy represents an emerging development in anticancer therapy. Although it has been tested against a variety of cancers, including breast cancer, the efficacy of oncolytic viral vectors delivered as a monotherapy is limited. Enhancing viral oncolytic therapies through combination treatment with anticancer agents is a feasible strategy. In this study, we considered a chemovirotherapeutic approach for treating breast adenocarcinoma using oncolytic measles virus (MV) and the chemotherapeutic agent camptothecin (CPT). Our results demonstrated that co-treatment of MV with CPT yielded enhanced cytotoxicity against breast cancer cells. Low dosage CPT combined with MV was also found to elicit the same therapeutic effect as high doses of CPT. At the lower dosage used, CPT did not inhibit the early stages of MV entry, nor reduce viral replication. Further studies revealed that co-treatment induced significantly enhanced apoptosis of the breast cancer cells compared to either MV or CPT alone. Overall, our findings demonstrate the potential value of MV plus CPT as a novel chemovirotherapeutic treatment against breast cancer and as a strategy to enhance MV oncolytic activity.

Antiviral Activities of Silymarin and Derivatives.

Silymarin flavonolignans are well-known agents that typically possess antioxidative, anti-inflammatory, and hepatoprotective functions. Recent studies have also documented the antiviral activities of silymarin and its derivatives against several viruses, including the flaviviruses (hepatitis C virus and dengue virus), togaviruses (Chikungunya virus and Mayaro virus), influenza virus, human immunodeficiency virus, and hepatitis B virus. This review will describe some of the latest preclinical and clinical studies detailing the antiviral profiles of silymarin and its derivatives, and discuss their relevance for antiviral drug development.