The combination of pleconaril, rupintrivir, and remdesivir efficiently inhibits enterovirus infections in vitro, delaying the development of drug-resistant virus variants.

Enteroviruses are a significant global health concern, causing a spectrum of diseases from the common cold to more severe conditions like hand-foot-and-mouth disease, meningitis, myocarditis, pancreatitis, and poliomyelitis. Current treatment options for these infections are limited, underscoring the urgent need for effective therapeutic strategies. To find better treatment option we analyzed toxicity and efficacy of 12 known broad-spectrum anti-enterovirals both individually and in combinations against different enteroviruses in vitro. We identified several novel, synergistic two-drug and three-drug combinations that demonstrated significant inhibition of enterovirus infections in vitro. Specifically, the triple-drug combination of pleconaril, rupintrivir, and remdesivir exhibited remarkable efficacy against echovirus (EV) 1, EV6, EV11, and coxsackievirus (CV) B5, in human lung epithelial A549 cells. This combination surpassed the effectiveness of single-agent or dual-drug treatments, as evidenced by its ability to protect A549 cells from EV1-induced cytotoxicity across seven passages. Additionally, this triple-drug cocktail showed potent antiviral activity against EV-A71 in human intestinal organoids. Thus, our findings highlight the therapeutic potential of the pleconaril-rupintrivir-remdesivir combination as a broad-spectrum treatment option against a range of enterovirus infections. The study also paves the way towards development of strategic antiviral drug combinations with virus family coverage and high-resistance barriers.

A pan-SARS-CoV-2-specific soluble angiotensin-converting enzyme 2-albumin fusion engineered for enhanced plasma half-life and needle-free mucosal delivery.

Immunocompromised patients often fail to raise protective vaccine-induced immunity against the global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Although monoclonal antibodies have been authorized for clinical use, most have lost their ability to potently neutralize the evolving Omicron subvariants. Thus, there is an urgent need for treatment strategies that can provide protection against these and emerging SARS-CoV-2 variants to prevent the development of severe coronavirus disease 2019. Here, we report on the design and characterization of a long-acting viral entry-blocking angiotensin-converting enzyme 2 (ACE2) dimeric fusion molecule. Specifically, a soluble truncated human dimeric ACE2 variant, engineered for improved binding to the receptor-binding domain of SARS-CoV-2, was fused with human albumin tailored for favorable engagement of the neonatal fragment crystallizable receptor (FcRn), which resulted in enhanced plasma half-life and allowed for needle-free transmucosal delivery upon nasal administration in human FcRn-expressing transgenic mice. Importantly, the dimeric ACE2-fused albumin demonstrated potent neutralization of SARS-CoV-2 immune escape variants.

Boosted production of antibodies that neutralized different SARS-CoV-2 variants in a COVID-19 convalescent following messenger RNA vaccination - a case study.

Vaccinated convalescents do not develop severe COVID-19 after infection with new SARS-CoV-2 variants. We questioned how messenger RNA (mRNA) vaccination of convalescents provides protection from emerging virus variants. From the cohort of 71 convalescent plasma donors, we identified a patient who developed immune response to infection with SARS-CoV-2 variant of 20A clade and who subsequently received mRNA vaccine encoding spike (S) protein of strain of 19A clade. We showed that vaccination increased the production of immune cells and anti-S antibodies in the serum. Serum antibodies neutralized not only 19A and 20A, but also 20B, 20H, 21J, and 21K virus variants. One of the serum antibodies (100F8) completely neutralized 20A, 21J, and partially 21K strains. 100F8 was structurally similar to published Ab188 antibody, which recognized non-conserved epitope on the S protein. We proposed that 100F8 and other serum antibodies of the patient which recognized non- and conserved epitopes of the S protein, could have additive or synergistic effects to neutralize various virus variants. Thus, mRNA vaccination could be beneficial for convalescents because it boosts production of neutralizing antibodies with broad-spectrum activity.

Combating pan-coronavirus infection by indomethacin through simultaneously inhibiting viral replication and inflammatory response.

Severe infections with coronaviruses are often accompanied with hyperinflammation, requiring therapeutic strategies to simultaneously tackle the virus and inflammation. By screening a safe-in-human broad-spectrum antiviral agents library, we identified that indomethacin can inhibit pan-coronavirus infection in human cell and airway organoids models. Combining indomethacin with oral antiviral drugs authorized for treating COVID-19 results in synergistic anti-coronavirus activity. Coincidentally, screening a library of FDA-approved drugs identified indomethacin as the most potent potentiator of interferon response through increasing STAT1 phosphorylation. Combining indomethacin with interferon-alpha exerted synergistic antiviral effects against multiple coronaviruses. The anti-coronavirus activity of indomethacin is associated with activating interferon response. In a co-culture system of lung epithelial cells with macrophages, indomethacin inhibited both viral replication and inflammatory response. Collectively, indomethacin is a pan-coronavirus inhibitor that can simultaneously inhibit virus-triggered inflammatory response. The therapeutic potential of indomethacin can be further augmented by combining it with oral antiviral drugs or interferon-alpha.

Broadly neutralizing humanized SARS-CoV-2 antibody binds to a conserved epitope on Spike and provides antiviral protection through inhalation-based delivery in non-human primates.

The COVID-19 pandemic represents a global challenge that has impacted and is expected to continue to impact the lives and health of people across the world for the foreseeable future. The rollout of vaccines has provided highly anticipated relief, but effective therapeutics are required to further reduce the risk and severity of infections. Monoclonal antibodies have been shown to be effective as therapeutics for SARS-CoV-2, but as new variants of concern (VoC) continue to emerge, their utility and use have waned due to limited or no efficacy against these variants. Furthermore, cumbersome systemic administration limits easy and broad access to such drugs. As well, concentrations of systemically administered antibodies in the mucosal epithelium, a primary site of initial infection, are dependent on neonatal Fc receptor mediated transport and require high drug concentrations. To reduce the viral load more effectively in the lung, we developed an inhalable formulation of a SARS-CoV-2 neutralizing antibody binding to a conserved epitope on the Spike protein, ensuring pan-neutralizing properties. Administration of this antibody via a vibrating mesh nebulization device retained antibody integrity and resulted in effective distribution of the antibody in the upper and lower respiratory tract of non-human primates (NHP). In comparison with intravenous administration, significantly higher antibody concentrations can be obtained in the lung, resulting in highly effective reduction in viral load post SARS-CoV-2 challenge. This approach may reduce the barriers of access and uptake of antibody therapeutics in real-world clinical settings and provide a more effective blueprint for targeting existing and potentially emerging respiratory tract viruses.

Metformin Therapy Changes Gut Microbiota Alpha-Diversity in COVID-19 Patients with Type 2 Diabetes: The Role of SARS-CoV-2 Variants and Antibiotic Treatment.

The gut microbiota play a crucial role in maintaining host health and have a significant impact on human health and disease. In this study, we investigated the alpha diversity of gut microbiota in COVID-19 patients and analyzed the impact of COVID-19 variants, antibiotic treatment, type 2 diabetes (T2D), and metformin therapy on gut microbiota composition and diversity. We used a culture-based method to analyze the gut microbiota and calculated alpha-diversity using the Shannon H' and Simpson 1/D indices. We collected clinical data, such as the length of hospital stay (LoS), C-reactive protein (CRP) levels, and neutrophil-to-lymphocyte ratio. We found that patients with T2D had significantly lower alpha-diversity than those without T2D. Antibiotic use was associated with a reduction in alpha-diversity, while metformin therapy was associated with an increase. We did not find significant differences in alpha-diversity between the Delta and Omicron groups. The length of hospital stay, CRP levels, and NLR showed weak to moderate correlations with alpha diversity. Our findings suggest that maintaining a diverse gut microbiota may benefit COVID-19 patients with T2D. Interventions to preserve or restore gut microbiota diversity, such as avoiding unnecessary antibiotic use, promoting metformin therapy, and incorporating probiotics, may improve patient outcomes.

Therapeutic Effectiveness of Interferon-α2b against COVID-19 with Community-Acquired Pneumonia: The Ukrainian Experience.

Despite several targeted antiviral drugs against SARS-CoV-2 currently being available, the application of type I interferons (IFNs) still deserves attention as an alternative antiviral strategy. This study aimed to assess the therapeutic effectiveness of IFN-α in hospitalized patients with COVID-19-associated pneumonia. The prospective cohort study included 130 adult patients with coronavirus disease (COVID-19). A dose of 80,000 IU of IFN-α2b was administered daily intranasally for 10 days. Adding IFN-α2b to standard therapy reduces the length of the hospital stay by 3 days (p < 0.001). The level of CT-diagnosed lung injuries was reduced from 35% to 15% (p = 0.011) and CT injuries decreased from 50% to 15% (p = 0.017) by discharge. In the group of patients receiving IFN-α2b, the SpO2 index before and after treatment increased from 94 (92-96, Q1-Q3) to 96 (96-98, Q1-Q3) (p < 0.001), while the percentage of patients with normal saturation increased (from 33.9% to 74.6%, p < 0.05), but the level of SpO2 decreased in the low (from 52.5% to 16.9%) and very low (from 13.6% to 8.5%) categories. The addition of IFN-α2b to standard therapy has a positive effect on the course of severe COVID-19.

Multi-OMICs landscape of SARS-CoV-2-induced host responses in human lung epithelial cells.

COVID-19 pandemic continues to remain a global health concern owing to the emergence of newer variants. Several multi-Omics studies have produced extensive evidence on host-pathogen interactions and potential therapeutic targets. Nonetheless, an increased understanding of host signaling networks regulated by post-translational modifications and their ensuing effect on the cellular dynamics is critical to expanding the current knowledge on SARS-CoV-2 infections. Through an unbiased transcriptomics, proteomics, acetylomics, phosphoproteomics, and exometabolome analysis of a lung-derived human cell line, we show that SARS-CoV-2 Norway/Trondheim-S15 strain induces time-dependent alterations in the induction of type I IFN response, activation of DNA damage response, dysregulated Hippo signaling, among others. We identified interplay of phosphorylation and acetylation dynamics on host proteins and its effect on the altered release of metabolites, especially organic acids and ketone bodies. Together, our findings serve as a resource of potential targets that can aid in designing novel host-directed therapeutic strategies.

Seven classes of antiviral agents.

The viral epidemics and pandemics have stimulated the development of known and the discovery of novel antiviral agents. About a hundred mono- and combination antiviral drugs have been already approved, whereas thousands are in development. Here, we briefly reviewed 7 classes of antiviral agents: neutralizing antibodies, neutralizing recombinant soluble human receptors, antiviral CRISPR/Cas systems, interferons, antiviral peptides, antiviral nucleic acid polymers, and antiviral small molecules. Interferons and some small molecules alone or in combinations possess broad-spectrum antiviral activity, which could be beneficial for treatment of emerging and re-emerging viral infections.

Antiviral Immunoglobulins of Chicken Egg Yolk for Potential Prevention of SARS-CoV-2 Infection.

Background: Some viruses cause outbreaks, which require immediate attention. Neutralizing antibodies could be developed for viral outbreak management. However, the development of monoclonal antibodies is often long, laborious, and unprofitable. Here, we report the development of chicken polyclonal neutralizing antibodies against SARS-CoV-2 infection. Methods: Layers were immunized twice with 14-day intervals using the purified receptor-binding domain (RBD) of the S protein of SARS-CoV-2/Wuhan or SARS-CoV-2/Omicron. Eggs were harvested 14 days after the second immunization. Polyclonal IgY antibodies were extracted. Binding of anti-RBD IgYs was analyzed by immunoblot and indirect ELISA. Furthermore, the neutralization capacity of anti-RBD IgYs was measured in Vero-E6 cells infected with SARS-CoV-2-mCherry/Wuhan and SARS-CoV-2/Omicron using fluorescence and/or cell viability assays. In addition, the effect of IgYs on the expression of SARS-CoV-2 and host cytokine genes in the lungs of Syrian Golden hamsters was examined using qRT-PCR. Results: Anti-RBD IgYs efficiently bound viral RBDs in situ, neutralized the virus variants in vitro, and lowered viral RNA amplification, with minimal alteration of virus-mediated immune gene expression in vivo. Conclusions: Altogether, our results indicate that chicken polyclonal IgYs can be attractive targets for further pre-clinical and clinical development for the rapid management of outbreaks of emerging and re-emerging viruses.

Broad-Spectrum Antivirals and Antiviral Combinations: An Editorial Update.

Our Special Issue received a great deal of attention, and several important papers have recently been added to it [...].

Novel Synergistic Anti-Enteroviral Drug Combinations.

Background: Enterovirus infections affect people around the world, causing a range of illnesses, from mild fevers to severe, potentially fatal conditions. There are no approved treatments for enterovirus infections. Methods: We have tested our library of broad-spectrum antiviral agents (BSAs) against echovirus 1 (EV1) in human adenocarcinoma alveolar basal epithelial A549 cells. We also tested combinations of the most active compounds against EV1 in A549 and human immortalized retinal pigment epithelium RPE cells. Results: We confirmed anti-enteroviral activities of pleconaril, rupintrivir, cycloheximide, vemurafenib, remdesivir, emetine, and anisomycin and identified novel synergistic rupintrivir-vemurafenib, vemurafenib-pleconaril and rupintrivir-pleconaril combinations against EV1 infection. Conclusions: Because rupintrivir, vemurafenib, and pleconaril require lower concentrations to inhibit enterovirus replication in vitro when combined, their cocktails may have fewer side effects in vivo and, therefore, should be further explored in preclinical and clinical trials against EV1 and other enterovirus infections.

DrugVirus.info 2.0: an integrative data portal for broad-spectrum antivirals (BSA) and BSA-containing drug combinations (BCCs).

Viruses can cross species barriers and cause unpredictable outbreaks in man with substantial economic and public health burdens. Broad-spectrum antivirals, (BSAs, compounds inhibiting several human viruses), and BSA-containing drug combinations (BCCs) are deemed as immediate therapeutic options that fill the void between virus identification and vaccine development. Here, we present DrugVirus.info 2.0 (https://drugvirus.info), an integrative interactive portal for exploration and analysis of BSAs and BCCs, that greatly expands the database and functionality of DrugVirus.info 1.0 webserver. Through the data portal that now expands the spectrum of BSAs and provides information on BCCs, we developed two modules for (i) interactive analysis of users' own antiviral drug and combination screening data and their comparison with published datasets, and (ii) exploration of the structure-activity relationship between various BSAs. The updated portal provides an essential toolbox for antiviral drug development and repurposing applications aiming to identify existing and novel treatments of emerging and re-emerging viral threats.

Mono- and combinational drug therapies for global viral pandemic preparedness.

Broadly effective antiviral therapies must be developed to be ready for clinical trials, which should begin soon after the emergence of new life-threatening viruses. Here, we pave the way towards this goal by reviewing conserved druggable virus-host interactions, mechanisms of action, immunomodulatory properties of available broad-spectrum antivirals (BSAs), routes of BSA delivery, and interactions of BSAs with other antivirals. Based on the review, we concluded that the range of indications of BSAs can be expanded, and new pan- and cross-viral mono- and combinational therapies can be developed. We have also developed a new scoring algorithm that can help identify the most promising few of the thousands of potential BSAs and BSA-containing drug cocktails (BCCs) to prioritize their development during the critical period between the identification of a new virus and the development of virus-specific vaccines, drugs, and therapeutic antibodies.

Broad-Spectrum Antivirals and Antiviral Drug Combinations.

Viral diseases consistently pose a substantial economic and public health burden worldwide [...].

Rapid elimination of SARS-CoV-2 in a fully vaccinated patient. / Rask eliminasjon av SARS-CoV-2 hos fullvaksinert pasient.

High SARS-CoV-2 viral loads in respiratory secretions detected by PCR technique are usually an indicator of high transmission risk, but not always. In this article, we present the case of a fully-vaccinated patient with rapid clearance of the alpha variant of the virus.

Recapitulating hepatitis E virus-host interactions and facilitating antiviral drug discovery in human liver-derived organoids.

Hepatotropic viruses naturally have narrow host and tissue tropisms, challenging the development of robust experimental models. The advent of organoid technology provides a unique opportunity for moving the field forward. Here, we demonstrate that three-dimensional cultured organoids from fetal and adult human liver with cholangiocyte or hepatocyte phenotype support hepatitis E virus (HEV) replication. Inoculation with infectious HEV particles demonstrates that human liver­derived organoids support the full life cycle of HEV infection. By directing organoids toward polarized monolayers in a transwell system, we observed predominantly apical secretion of HEV particles. Genome-wide transcriptomic and tRNAome analyses revealed robust host responses triggered by viral replication. Drug screening in organoids identified brequinar and homoharringtonine as potent HEV inhibitors, which are also effective against the ribavirin resistance variant harboring G1634R mutation. Thus, successful recapitulation of HEV infection in liver-derived organoids shall facilitate the study of virus-host interactions and development of antiviral therapies.

Drug Combinations as a First Line of Defense against Coronaviruses and Other Emerging Viruses.

The world was unprepared for coronavirus disease 2019 (COVID-19) and remains ill-equipped for future pandemics. While unprecedented strides have been made developing vaccines and treatments for COVID-19, there remains a need for highly effective and widely available regimens for ambulatory use for novel coronaviruses and other viral pathogens. We posit that a priority is to develop pan-family drug cocktails to enhance potency, limit toxicity, and avoid drug resistance. We urge cocktail development for all viruses with pandemic potential both in the short term (<1 to 2 years) and longer term with pairs of drugs in advanced clinical testing or repurposed agents approved for other indications. While significant efforts were launched against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in vitro and in the clinic, many studies employed solo drugs and had disappointing results. Here, we review drug combination studies against SARS-CoV-2 and other viruses and introduce a model-driven approach to assess drug pairs with the highest likelihood of clinical efficacy. Where component agents lack sufficient potency, we advocate for synergistic combinations to achieve therapeutic levels. We also discuss issues that stymied therapeutic progress against COVID-19, including testing of agents with low likelihood of efficacy late in clinical disease and lack of focus on developing virologic surrogate endpoints. There is a need to expedite efficient clinical trials testing drug combinations that could be taken at home by recently infected individuals and exposed contacts as early as possible during the next pandemic, whether caused by a coronavirus or another viral pathogen. The approach herein represents a proactive plan for global viral pandemic preparedness.

Synergistic Interferon-Alpha-Based Combinations for Treatment of SARS-CoV-2 and Other Viral Infections.

BACKGROUND: There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. METHODS: Here, we tested the antiviral properties of interferons (IFNs), alone and with other drugs in vitro. RESULTS: While IFNs alone were insufficient to completely abolish replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), IFNα, in combination with remdesivir, EIDD-2801, camostat, cycloheximide, or convalescent serum, proved to be more effective. Transcriptome and metabolomic analyses revealed that the IFNα-remdesivir combination suppressed SARS-CoV-2-mediated changes in Calu-3 cells and lung organoids, although it altered the homeostasis of uninfected cells and organoids. We also demonstrated that IFNα combinations with sofosbuvir, telaprevir, NITD008, ribavirin, pimodivir, or lamivudine were effective against HCV, HEV, FLuAV, or HIV at lower concentrations, compared to monotherapies. CONCLUSIONS: Altogether, our results indicated that IFNα can be combined with drugs that affect viral RNA transcription, protein synthesis, and processing to make synergistic combinations that can be attractive targets for further pre-clinical and clinical development against emerging and re-emerging viral infections.

Nafamostat-Interferon-α Combination Suppresses SARS-CoV-2 Infection In Vitro and In Vivo by Cooperatively Targeting Host TMPRSS2.

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNα) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNα and that both Serpin E1 and nafamostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.