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.

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.

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.

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.

Ivermectin effectively inhibits hepatitis E virus replication, requiring the host nuclear transport protein importin α1.

We show that ivermectin, an FDA-approved anti-parasitic drug, effectively inhibits infection with hepatitis E virus (HEV) genotypes 1 and 3 in a range of cell culture models, including hepatic and extrahepatic cells. Long-term treatment showed no clear evidence of the development of drug resistance. Gene silencing of importin-α1, a cellular target of ivermectin and a key member of the host nuclear transport complex, inhibited viral replication and largely abolished the anti-HEV effect of ivermectin.

Safe-in-Man Broad Spectrum Antiviral Agents.

Emerging and re-emerging viral diseases occur with regularity within the human population. The conventional 'one drug, one virus' paradigm for antivirals does not adequately allow for proper preparedness in the face of unknown future epidemics. In addition, drug developers lack the financial incentives to work on antiviral drug discovery, with most pharmaceutical companies choosing to focus on more profitable disease areas. Safe-in-man broad spectrum antiviral agents (BSAAs) can help meet the need for antiviral development by already having passed phase I clinical trials, requiring less time and money to develop, and having the capacity to work against many viruses, allowing for a speedy response when unforeseen epidemics arise. In this chapter, we discuss the benefits of repurposing existing drugs as BSAAs, describe the major steps in safe-in-man BSAA drug development from discovery through clinical trials, and list several database resources that are useful tools for antiviral drug repositioning.

Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication.

Kaposi's sarcoma herpesvirus (KSHV) causes Kaposi's sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication.

Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages.

Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages to elucidate the intracellular signaling pathways and critical host factors activated upon influenza infection. We identified 1675 phosphoproteins, 4004 phosphopeptides and 4146 nonredundant phosphosites. The phosphorylation of 1113 proteins (66%) was regulated upon infection, highlighting the importance of such global phosphoproteomic profiling in primary cells. Notably, 285 of the identified phosphorylation sites have not been previously described in publicly available phosphorylation databases, despite many published large-scale phosphoproteome studies using human and mouse cell lines. Systematic bioinformatics analysis of the phosphoproteome data indicated that the phosphorylation of proteins involved in the ubiquitin/proteasome pathway (such as TRIM22 and TRIM25) and antiviral responses (such as MAVS) changed in infected macrophages. Proteins known to play roles in small GTPase-, mitogen-activated protein kinase-, and cyclin-dependent kinase- signaling were also regulated by phosphorylation upon infection. In particular, the influenza infection had a major influence on the phosphorylation profiles of a large number of cyclin-dependent kinase substrates. Functional studies using cyclin-dependent kinase inhibitors showed that the cyclin-dependent kinase activity is required for efficient viral replication and for activation of the host antiviral responses. In addition, we show that cyclin-dependent kinase inhibitors protect IAV-infected mice from death. In conclusion, we provide the first comprehensive phosphoproteome characterization of influenza A virus infection in primary human macrophages, and provide evidence that cyclin-dependent kinases represent potential therapeutic targets for more effective treatment of influenza infections.

Influenza virus NS1 protein binds cellular DNA to block transcription of antiviral genes.

Influenza NS1 protein is an important virulence factor that is capable of binding double-stranded (ds) RNA and inhibiting dsRNA-mediated host innate immune responses. Here we show that NS1 can also bind cellular dsDNA. This interaction prevents loading of transcriptional machinery to the DNA, thereby attenuating IAV-mediated expression of antiviral genes. Thus, we identified a previously undescribed strategy, by which RNA virus inhibits cellular transcription to escape antiviral response and secure its replication.

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.

Protein profiling of nasopharyngeal aspirates of hospitalized and outpatients revealed cytokines associated with severe influenza A(H1N1)pdm09 virus infections: A pilot study.

Influenza A viruses (IAV) mutate rapidly and cause seasonal epidemics and occasional pandemics, which result in substantial number of patient visits to the doctors and even hospitalizations. We aimed here to identify inflammatory proteins, which levels correlated to clinical severity of the disease. For this we analysed 102 cytokines and growth factors in human nasopharyngeal aspirate (NPA) samples of 27 hospitalized and 27 outpatients diagnosed with influenza A(H1N1)pdm09 virus infection. We found that the relative levels of monocyte differentiation antigen CD14, lipocalin-2 (LCN2), C-C-motif chemokine 20 (CCL20), CD147, urokinase plasminogen activator surface receptor (uPAR), pro-epidermal growth factor (EGF), trefoil factor 3 (TFF3), and macrophage migration inhibitory factor (MIF) were significantly lower (p<0.008), whereas levels of retinol-binding protein 4 (RBP4), C-X-C motif chemokine 5 (CXCL5), interleukin-8 (IL-8), complement factor D (CFD), adiponectin, and chitinase-3-like 1 (CHI3L1) were significantly higher (p<0.008) in NPA samples of hospitalized than non-hospitalized patients. While changes in CD14, LCN2, CCL20, uPAR, EGF, MIF, CXCL5, IL-8, adiponectin and CHI3L1 levels have already been correlated with severity of IAV infection in mice and humans, our study is the first to describe association of CD147, RBP4, TFF3, and CFD with hospitalization of IAV-infected patients. Thus, we identified local innate immune profiles, which were associated with the clinical severity of influenza infections.

Activation of Tryptophan and Phenylalanine Catabolism in the Remission Phase of Allergic Contact Dermatitis: A Pilot Study.

BACKGROUND: Allergic contact dermatitis (ACD) is an inflammatory skin disease caused by repeated skin exposure to contact allergens. The severity and duration of this disease are associated with many different factors. Some of these factors may represent markers for monitoring disease activity and the individual response to an intervention. METHODS: We used a targeted metabolomics approach to find such factors in the serum of individuals with ACD. Metabolomics profiles were examined and compared in the acute phase of the disease and also in the absence of disease activity. RESULTS: Our study identified a significant remission phase of ACD-associated systemic biochemical shifts in 2 metabolic pathways: tryptophan-kynurenine and phenylalanine-tyrosine. CONCLUSIONS: Although the responsible mechanisms are unclear, these results suggest that the remission phase of ACD is linked to tryptophan metabolism via kynurenine and phenylalanine-tyrosine pathways. However, further replication studies with a larger number of subjects and their subgroups are necessary to validate our results. These studies may provide a new perspective with which to understand the mechanism of and find potential biomarkers of ACD, as well as a new reference for personalized treatment.

The C terminus of NS1 protein of influenza A/WSN/1933(H1N1) virus modulates antiviral responses in infected human macrophages and mice.

Non-structural protein NS1 of influenza A viruses interacts with cellular factors through its N-terminal RNA-binding, middle effector and C-terminal non-structured domains. NS1 attenuates antiviral responses in infected cells and thereby secures efficient virus replication. Some influenza strains express C-terminally truncated NS1 proteins due to nonsense mutations in the NS1 gene. To understand the role of the NS1 C-terminal region in regulation of antiviral responses, we engineered influenza viruses expressing C-terminally truncated NS1 proteins using A/WSN/33(H1N1) reverse genetics and tested them in human macrophages and in mice. We showed that a WSN virus expressing NS1 with a 28 aa deletion from its C terminus is a more powerful inducer of antiviral responses than the virus expressing full-length NS1, or one with a 10 aa truncation of NS1 in vitro. Thus, our findings suggest that the C-terminal region of NS1 is essential for regulation of antiviral responses. Moreover, viruses expressing truncated NS1 proteins could be good vaccine candidates.

Serum Biomarkers of Allergic Contact Dermatitis: A Pilot Study.

BACKGROUND: Allergic contact dermatitis (ACD) is an inflammatory skin disease caused by repeated skin exposure to contact allergens. The goal of this pilot study was to identify inflammatory proteins which can serve as biomarkers for ACD. METHODS: We measured levels of 102 cytokines, chemokines, and growth factors in the sera of 16 ACD patients during acute and remission phases, and 16 healthy volunteers. RESULTS: Serum levels of adiponectin, chemokine (C-C motif) ligand 5 (CCL5), C-reactive protein (CRP), chitinase 3-like 1 (CHI3L1), complement factor D (CFD), endoglin, lipocalin-2, osteopontin, retinol-binding protein 4 (RBP4), and platelet factor 4 (PF4) were significantly higher, whereas levels of trefoil factor 3 (TFF3) were significantly lower, in ACD patients than in healthy controls. In ACD patients, serum levels of CCL5 were elevated, whereas levels of TFF3, soluble intercellular adhesion molecule-1 (sICAM-1), and platelet-derived growth factor (PDGF)-AB/BB were found to be lower during the remission phase of the disease. CONCLUSIONS: Serum levels of adiponectin, CCL5, CRP, CHI3L1, CFD, endoglin, lipocalin-2, osteopontin, RBP4, PF4, and TFF3 might be exploited as biomarkers for ACD, whereas levels of CCL5, TFF3, sICAM-1, and PDGF-AB/BB might be exploited for evaluation of disease progression and efficacy of ACD treatment.

Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution.

Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, 12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from 6, 8 and 13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in 8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops, whereas in 12 P4, a C-terminal residue, serine 282, forms a specific hydrogen bond to the N7 of purines ring to confer purine specificity for the 12 enzyme.

Akt inhibitor MK2206 prevents influenza pH1N1 virus infection in vitro.

The influenza pH1N1 virus caused a global flu pandemic in 2009 and continues manifestation as a seasonal virus. Better understanding of the virus-host cell interaction could result in development of better prevention and treatment options. Here we show that the Akt inhibitor MK2206 blocks influenza pH1N1 virus infection in vitro. In particular, at noncytotoxic concentrations, MK2206 alters Akt signaling and inhibits endocytic uptake of the virus. Interestingly, MK2206 is unable to inhibit H3N2, H7N9, and H5N1 viruses, indicating that pH1N1 evolved specific requirements for efficient infection. Thus, Akt signaling could be exploited further for development of better therapeutics against pH1N1 virus.

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.

Obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection.

Influenza A viruses (IAVs) infect humans and cause significant morbidity and mortality. Different treatment options have been developed; however, these were insufficient during recent IAV outbreaks. Here, we conducted a targeted chemical screen in human nonmalignant cells to validate known and search for novel host-directed antivirals. The screen validated saliphenylhalamide (SaliPhe) and identified two novel anti-IAV agents, obatoclax and gemcitabine. Further experiments demonstrated that Mcl-1 (target of obatoclax) provides a novel host target for IAV treatment. Moreover, we showed that obatoclax and SaliPhe inhibited IAV uptake and gemcitabine suppressed viral RNA transcription and replication. These compounds possess broad spectrum antiviral activity, although their antiviral efficacies were virus-, cell type-, and species-specific. Altogether, our results suggest that phase II obatoclax, investigational SaliPhe, and FDA/EMEA-approved gemcitabine represent potent antiviral agents.

Structural basis for group A trichothiodystrophy.

Patients with the rare neurodevelopmental repair syndrome known as group A trichothiodystrophy (TTD-A) carry mutations in the gene encoding the p8 subunit of the transcription and DNA repair factor TFIIH. Here we describe the crystal structure of a minimal complex between Tfb5, the yeast ortholog of p8, and the C-terminal domain of Tfb2, the yeast p52 subunit of TFIIH. The structure revealed that these two polypeptides adopt the same fold, forming a compact pseudosymmetric heterodimer via a beta-strand addition and coiled coils interactions between terminal alpha-helices. Furthermore, Tfb5 protects a hydrophobic surface in Tfb2 from solvent, providing a rationale for the influence of p8 in the stabilization of p52 and explaining why mutations that weaken p8-p52 interactions lead to a reduced intracellular TFIIH concentration and a defect in nucleotide-excision repair, a common feature of TTD cells.

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.