ROS Induced by Aphrocallistes vastus Lectin Enhance Oncolytic Vaccinia Virus Replication and Induce Apoptosis in Hepatocellular Carcinoma Cells.

Oncolytic virotherapy is expected to provide a new treatment strategy for cancer. Aphrocallistes vastus lectin (AVL) is a Ca2+-dependent lectin receptor containing the conserved domain of C-type lectin and the hydrophobic N-terminal region, which can bind to the bird's nest glycoprotein and D-galactose. Our previous studies suggested that the oncolytic vaccinia virus (oncoVV) armed with the AVL gene exerted remarkable replication and antitumor effects in vitro and in vivo. In this study, we found that oncoVV-AVL may reprogram the metabolism of hepatocellular carcinoma cells to promote ROS, and elevated ROS subsequently promoted viral replication and induced apoptosis. This study will provide a new theoretical basis for the application of oncoVV-AVL in liver cancer.

Synthesis of LAVR-289, a new [(Z)-3-(acetoxymethyl)-4-(2,4-diaminopyrimidin-6-yl)oxy-but-2-enyl]phosphonic acid prodrug with pronounced antiviral activity against DNA viruses.

New acyclic pyrimidine nucleoside phosphonate prodrugs with a 4-(2,4-diaminopyrimidin-6-yl)oxy-but-2-enyl]phosphonic acid skeleton (O-DAPy nucleobase) were prepared through a convergent synthesis by olefin cross-metathesis as the key step. Several acyclic nucleoside 4-(2,4-diaminopyrimidin-6-yl)oxy-but-2-enyl]phosphonic acid prodrug exhibited in vitro antiviral activity in submicromolar or nanomolar range against varicella zoster virus (VZV), human cytomegalovirus (HCMV), human herpes virus type 1 (HSV-1) and type 2 (HSV-2), and vaccinia virus (VV), with good selective index (SI). Among them, the analogue 9c (LAVR-289) proved markedly inhibitory against VZV wild-type (TK+) (EC50 0.0035 µM, SI 740) and for thymidine kinase VZV deficient strains (EC50 0.018 µM, SI 145), with a low morphological toxicity in cell culture at 100 µM and acceptable cytostatic activity resulting in excellent selectivity. Compound 9c exhibited antiviral activity against HCMV (EC50 0.021 µM) and VV (EC50 0.050 µM), as well as against HSV-1 (TK-) (EC50 0.0085 µM). Finally, LAVR-289 (9c) deserves further (pre)clinical investigations as a potent candidate broad-spectrum anti-herpesvirus drug.

Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development.

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

An engineered oncolytic vaccinia virus encoding a single-chain variable fragment against TIGIT induces effective antitumor immunity and synergizes with PD-1 or LAG-3 blockade.

BACKGROUND: In addition to directly lysing tumors, oncolytic viruses also induce antitumor immunity by recruiting and activating immune cells in the local tumor microenvironment. However, the activation of the immune cells induced by oncolytic viruses is always accompanied by high-level expression of immune checkpoints in these cells, which may reduce the efficacy of the oncolytic viruses. The aim of this study is to arm the oncolytic vaccinia virus (VV) with immune checkpoint blockade to enhance its antitumor efficacy. METHODS: Through homologous recombination with the parental VV, an engineered VV-scFv-TIGIT was produced, which encodes a single-chain variable fragment (scFv) targeting T-cell immunoglobulin and ITIM domain (TIGIT). The antitumor efficacy of the VV-scFv-TIGIT was explored in several subcutaneous and ascites tumor models. The antitumor efficacy of VV-scFv-TIGIT combined with programmed cell death 1 (PD-1) or lymphocyte-activation gene 3 (LAG-3) blockade was also investigated. RESULTS: The VV-scFv-TIGIT effectively replicated in tumor cells and lysed them, and prompt the infected tumor cells to secret the functional scFv-TIGIT. Compared with control VV, intratumoral injection of VV-scFv-TIGIT in several mouse subcutaneous tumor models showed superior antitumor efficacy, accompanied by more T cell infiltration and a higher degree of CD8+ T cells activation. Intraperitoneal injection of VV-scFv-TIGIT in a mouse model of malignant ascites also significantly improved T cell infiltration and CD8+ T cell activation, resulting in more than 90% of the tumor-bearing mice being cured. Furthermore, the antitumor immune response induced by VV-scFv-TIGIT was dependent on CD8+ T cells which mediated a long-term immunological memory and a systemic antitumor immunity against the same tumor. Finally, the additional combination of PD-1 or LAG-3 blockade further enhanced the antitumor efficacy of VV-scFv-TIGIT, increasing the complete response rate of tumor-bearing mice. CONCLUSIONS: Oncolytic virotherapy using engineered VV-scFv-TIGIT was an effective strategy for cancer immunotherapy. Administration of VV-scFv-TIGIT caused a profound reshaping of the suppressive tumor microenvironment from 'cold' to 'hot' status. VV-scFv-TIGIT also synergized with PD-1 or LAG-3 blockade to achieve a complete response to tumors with poor response to VV or immune checkpoint blockade monotherapy.

The Efficacy of Disinfection on Modified Vaccinia Ankara and African Swine Fever Virus in Various Forest Soil Types.

African swine fever (ASF) has become a global threat to the pig industry and wild suids. Within Europe, including Germany, affected wild boar populations play a major role. Fencing and carcass removal in combination with the reduction in environmental contamination are key to control further spread. The handling of the ASF virus (ASFV) is restricted to high-containment conditions in Germany. According to the regulation of the German Veterinarian Society (DVG), modified vaccinia Ankara virus (MVAV) is the virus of choice to determine the efficacy of disinfection for enveloped viruses. The aim of this study was to use the MVAV as a guide to select the best possible disinfectant solution and concentration for the inactivation of ASFV in soil. Both viruses were tested simultaneously. In this study, two layers (top and mineral soil) of soil types from six different locations in Saxony, Germany, were collected. The tenacity of ASFV and MVAV were tested at various time points (0.5 to 72 h). The capabilities of different concentrations of peracetic acid and citric acid (approx. 0.1 to 2%) to inactivate the viruses in the selected soil types with spiked high protein load were examined under appropriate containment conditions. Around 2-3 Log10 (TCID50) levels of reduction in the infectivity of both ASFV and MVAV were observed in all soil types starting after two hours. For MVAV, a 4 Log10 loss was recorded after 72 h. A total of 0.1% of peracetic acid (5 L/m2) was sufficient to inactivate the viruses. A 4 log10 reduction in the infectivity of MVAV was noticed by applying 1% citric acid, while a 2 log10 decline was recorded with ASFV. In conclusion, comparing MVAV to ASFV for efficacy screening of disinfectant solutions has revealed many similarities. Peracetic acid reduced the infectivity of both viruses independently of the soil type and the existence of a high organic soiling.

Potential of an Eco-Sustainable Probiotic-Cleaning Formulation in Reducing Infectivity of Enveloped Viruses.

The COVID-19 pandemic has deeply influenced sanitization procedures, and high-level disinfection has been massively used to prevent SARS-CoV-2 spread, with potential negative impact on the environment and on the threat of antimicrobial resistance (AMR). Aiming to overcome these concerns, yet preserving the effectiveness of sanitization against enveloped viruses, we assessed the antiviral properties of the Probiotic Cleaning Hygiene System (PCHS), an eco-sustainable probiotic-based detergent previously proven to stably abate pathogen contamination and AMR. PCHS (diluted 1:10, 1:50 and 1:100) was tested in comparison to common disinfectants (70% ethanol and 0.5% sodium hypochlorite), in suspension and carrier tests, according with the European UNI EN 14476:2019 and UNI EN 16777:2019 standards. Human alpha- and beta-coronaviruses hCoV-229E and SARS-CoV-2, human herpesvirus type 1, human and animal influenza viruses, and vaccinia virus were included in the study. The results showed that PCHS was able to inactivate 99.99% of all tested viruses within 1-2 h of contact, both in suspension and on surface. Notably, while control disinfectants became inactive within 2 h after application, the PCHS antiviral action persisted up to 24 h post-application, suggesting that its use may effectively allow a continuous prevention of virus spread via contaminated environment, without worsening environmental pollution and AMR concern.

Protocol for analyzing and visualizing antiviral immune responses after acute infection of the murine oral mucosa.

The oral mucosa is an important site for virus infection and transmission, yet few animal models exist to examine the virology, pathology, and immunology of acute oral mucosal viral infection. Here, we provide a protocol for infecting and imaging the inner lip (labial mucosa) of mice with the poxvirus vaccinia virus (VACV). Inoculation of the labial mucosa with a bifurcated needle results in viral replication and priming of an adaptive antiviral response that can be imaged using intravital microscopy. For complete details on the use and execution of this protocol, please refer to Shannon et al. (2021).

Statins act as transient type I interferon inhibitors to enable the antitumor activity of modified vaccinia Ankara viral vectors.

BACKGROUND: Modified vaccinia virus Ankara (MVA) are genetically engineered non-replicating viral vectors. Intratumoral administration of MVA induces a cyclic GMP-AMP synthase-mediated type I interferon (IFN) response and the production of high levels of the transgenes engineered into the viral genome such as tumor antigens to construct cancer vaccines. Although type I IFNs are essential for establishing CD8-mediated antitumor responses, this cytokine family may also give rise to immunosuppressive mechanisms. METHODS: In vitro assays were performed to evaluate the activity of simvastatin and atorvastatin on type I IFN signaling and on antigen presentation. Surface levels of IFN α/ß receptor 1, endocytosis of bovine serum albumin-fluorescein 5 (6)-isothiocyanate, signal transducer and activator of transcription (STAT) phosphorylation, and real-time PCR of IFN-stimulated genes were assessed in the murine fibroblast cell line L929. In vivo experiments were performed to characterize the effect of simvastatin on the MVA-induced innate immune response and on the antitumor effect of MVA-based antitumor vaccines in B16 melanoma expressing ovalbumin (OVA) and Lewis lung carcinoma (LLC)-OVA tumor models. RNAseq analysis, depleting monoclonal antibodies, and flow cytometry were used to evaluate the MVA-mediated immune response. RESULTS: In this work, we identified commonly prescribed statins as potent IFNα pharmacological inhibitors due to their ability to reduce surface expression levels of IFN-α/ß receptor 1 and to reduce clathrin-mediated endocytosis. Simvastatin and atorvastatin efficiently abrogated for 8 hours the transcriptomic response to IFNα and enhanced the number of dendritic cells presenting an OVA-derived peptide bound to major histocompatibility complex (MHC) class I. In vivo, intraperitoneal or intramuscular administration of simvastatin reduced the inflammatory response mediated by peritumoral administration of MVA and enhanced the antitumor activity of MVA encoding tumor-associated antigens. The synergistic antitumor effects critically depend on CD8+ cells, whereas they were markedly improved by depletion of CD4+ lymphocytes, T regulatory cells, or NK cells. Either MVA-OVA alone or combined with simvastatin augmented B cells, CD4+ lymphocytes, CD8+ lymphocytes, and tumor-specific CD8+ in the tumor-draining lymph nodes. However, only the treatment combination increased the numbers of these lymphocyte populations in the tumor microenvironment and in the spleen. CONCLUSION: In conclusion, blockade of IFNα functions by simvastatin markedly enhances lymphocyte infiltration and the antitumor activity of MVA, prompting a feasible drug repurposing.

New methylene blue derivatives suggest novel anti-orthopoxviral strategies.

Decades after the eradication of smallpox and the discontinuation of routine smallpox vaccination, over half of the world's population is immunologically naïve to variola virus and other orthopoxviruses (OPXVs). Even in those previously vaccinated against smallpox, protective immunity wanes over time. As such, there is a concomitant increase in the incidence of human OPXV infections worldwide. To identify novel antiviral compounds with potent anti-OPXV potential, we characterized the inhibitory activity of PAV-866 and other methylene blue derivatives against the prototypic poxvirus, vaccinia virus (VACV). These compounds inactivated virions prior to infection and consequently inhibited viral binding, fusion and entry. The compounds exhibited strong virucidal activity at non-cytotoxic concentrations, and inhibited VACV infection when added before, during or after viral adsorption. The compounds were effective against other OPXVs including monkeypox virus, cowpox virus and the newly identified Akhmeta virus. Altogether, these findings reveal a novel mode of inhibition that has not previously been demonstrated for small molecule compounds against VACV. Additional studies are in progress to determine the in vivo efficacy of these compounds against OPXVs and further characterize the anti-viral effects of these derivatives.

Viral reduction of human blood by ultraviolet A-photosensitized vitamin K5.

Blood product transfusion can transmit viral pathogens. Pathogen reduction methods for blood products have been developed but, so far, are not available for whole blood. We evaluated if vitamin K5 (VK5) and ultraviolet A (UVA) irradiation could be used for virus inactivation in plasma and whole blood. Undiluted human plasma and whole blood diluted to 20% were spiked with high levels of vaccinia or Zika viruses. Infectious titers were measured by standard TCID50 assay before and after VK5/UVA treatments. Up to 3.6 log of vaccinia and 3.2 log of Zika were reduced in plasma by the combination of 500 µM VK5 and 3 J/cm2 UVA, and 3.1 log of vaccinia and 2.9 log of Zika were reduced in diluted human blood (20%) by the combination of 500 µM VK5 and 70 J/cm2 UVA. At end of whole blood treatment, hemolysis increased from 0.18% to 0.41% but remained below 1% hemolysis, which is acceptable to the Food and Drug Administration for red cell transfusion products. No significant alteration of biochemical parameters of red blood cells occurred with treatment. Our results provide proof of the concept that a viral pathogen reduction method based on VK5/UVA may be developed for whole blood.

Antiviral activity of Apigenin against buffalopox: Novel mechanistic insights and drug-resistance considerations.

We describe herein that Apigenin, which is a dietary flavonoid, exerts a strong in vitro and in ovo antiviral efficacy against buffalopox virus (BPXV). Apigenin treatment was shown to inhibit synthesis of viral DNA, mRNA and proteins, without affecting other steps of viral life cycle such as attachment, entry and budding. Although the major mode of antiviral action of Apigenin was shown to be mediated via targeting certain cellular factors, a modest inhibitory effect of Apigenin was also observed directly on viral polymerase. We also evaluated the selection of drug-resistant virus variants under long-term selection pressure of Apigenin. Wherein Apigenin-resistant mutants were not observed up to ~ P20 (passage 20), a significant resistance was observed to the antiviral action of Apigenin at ~ P30. However, a high degree resistance could not be observed even up to P60. To the best of our knowledge, this is the first report describing in vitro and in ovo antiviral efficacy of Apigenin against poxvirus infection. The study also provides mechanistic insights on the antiviral activity of Apigenin and selection of potential Apigenin-resistant mutants upon long-term culture.

Replication-inducible vaccinia virus vectors with enhanced safety in vivo.

Vaccinia virus (VACV) has been used extensively as the vaccine against smallpox and as a viral vector for the development of recombinant vaccines and cancer therapies. Replication-competent, non-attenuated VACVs induce strong, long-lived humoral and cell-mediated immune responses and can be effective oncolytic vectors. However, complications from uncontrolled VACV replication in vaccinees and their close contacts can be severe, particularly in individuals with predisposing conditions. In an effort to develop replication-competent VACV vectors with improved safety, we placed VACV late genes encoding core or virion morphogenesis proteins under the control of tet operon elements to regulate their expression with tetracycline antibiotics. These replication-inducible VACVs would only express the selected genes in the presence of tetracyclines. VACVs inducibly expressing the A3L or A6L genes replicated indistinguishably from wild-type VACV in the presence of tetracyclines, whereas there was no evidence of replication in the absence of antibiotics. These outcomes were reflected in mice, where the VACV inducibly expressing the A6L gene caused weight loss and mortality equivalent to wild-type VACV in the presence of tetracyclines. In the absence of tetracyclines, mice were protected from weight loss and mortality, and viral replication was not detected. These findings indicate that replication-inducible VACVs based on the conditional expression of the A3L or A6L genes can be used for the development of safer, next-generation live VACV vectors and vaccines. The design allows for administration of replication-inducible VACV in the absence of tetracyclines (as a replication-defective vector) or in the presence of tetracyclines (as a replication-competent vector) with enhanced safety.

Cell-to-cell spread of vaccinia virus is promoted by TGF-ß-independent Smad4 signalling.

The induction of Smad signalling by the extracellular ligand TGF-ß promotes tissue plasticity and cell migration in developmental and pathological contexts. Here, we show that vaccinia virus (VACV) stimulates the activity of Smad transcription factors and expression of TGF-ß/Smad-responsive genes at the transcript and protein levels. Accordingly, infected cells share characteristics to those undergoing TGF-ß/Smad-mediated epithelial-to-mesenchymal transition (EMT). Depletion of the Smad4 protein, a common mediator of TGF-ß signalling, results in an attenuation of viral cell-to-cell spread and reduced motility of infected cells. VACV induction of TGF-ß/Smad-responsive gene expression does not require the TGF-ß ligand or type I and type II TGF-ß receptors, suggesting a novel, non-canonical Smad signalling pathway. Additionally, the spread of ectromelia virus, a related orthopoxvirus that does not activate a TGF-ß/Smad response, is enhanced by the addition of exogenous TGF-ß. Together, our results indicate that VACV orchestrates a TGF-ß-like response via a unique activation mechanism to enhance cell migration and promote virus spread.

Study of Antiviral Activity of Metabolites of a New Serratia species K-57 Strain.

The results of studies of a newly isolated Serratia species K-57 strain are presented. The strain is characterized by antiviral activity towards human influenza A/Aichi/2/68/H3N2, vaccinia, mouse smallpox, and herpes simplex-2 viruses. The detected characteristics of the strain, including the data on activities on nucleolytic enzymes, recommend it for the development of therapeutic and preventive antiviral drugs.

Design and synthesis of novel Imidazo[2,1-b]thiazole derivatives as potent antiviral and antimycobacterial agents.

A series of novel acyl-hydrazone (4a-d) and spirothiazolidinone (5a-d, 6a-d) derivatives of imidazo[2,1-b]thiazole were synthesized and evaluated for their antiviral and antimycobacterial activity. The antituberculosis activity was evaluated by using the Microplate Alamar Blue Assay and the antiviral activity was evaluated against diverse viruses in mammalian cell cultures. According to the biological activity studies of the compounds, 5a-c displayed hope promising antitubercular activity, 6d was found as potent for Coxsackie B4 virus, 5d was found as effective against Feline corona and Feline herpes viruses. Consequently, the obtained results displayed that, 5a-d and 6d present a leading structure for future drug development due to its straightforward synthesis and relevant bioactivity.

Type I interferon-dependent CCL4 is induced by a cGAS/STING pathway that bypasses viral inhibition and protects infected tissue, independent of viral burden.

Type I interferons (T1-IFN) are critical in the innate immune response, acting upon infected and uninfected cells to initiate an antiviral state by expressing genes that inhibit multiple stages of the lifecycle of many viruses. T1-IFN triggers the production of Interferon-Stimulated Genes (ISGs), activating an antiviral program that reduces virus replication. The importance of the T1-IFN response is highlighted by the evolution of viral evasion strategies to inhibit the production or action of T1-IFN in virus-infected cells. T1-IFN is produced via activation of pathogen sensors within infected cells, a process that is targeted by virus-encoded immunomodulatory molecules. This is probably best exemplified by the prototypic poxvirus, Vaccinia virus (VACV), which uses at least 6 different mechanisms to completely block the production of T1-IFN within infected cells in vitro. Yet, mice lacking aspects of T1-IFN signaling are often more susceptible to infection with many viruses, including VACV, than wild-type mice. How can these opposing findings be rationalized? The cytosolic DNA sensor cGAS has been implicated in immunity to VACV, but has yet to be linked to the production of T1-IFN in response to VACV infection. Indeed, there are two VACV-encoded proteins that effectively prevent cGAS-mediated activation of T1-IFN. We find that the majority of VACV-infected cells in vivo do not produce T1-IFN, but that a small subset of VACV-infected cells in vivo utilize cGAS to sense VACV and produce T1-IFN to protect infected mice. The protective effect of T1-IFN is not mediated via ISG-mediated control of virus replication. Rather, T1-IFN drives increased expression of CCL4, which recruits inflammatory monocytes that constrain the VACV lesion in a virus replication-independent manner by limiting spread within the tissue. Our findings have broad implications in our understanding of pathogen detection and viral evasion in vivo, and highlight a novel immune strategy to protect infected tissue.

Tungsten carbide nanoparticles show a broad spectrum virucidal activity against enveloped and nonenveloped model viruses using a guideline-standardized in vitro test.

Five tungsten carbide nanoparticle preparations (denoted WC1-WC5) were investigated for broad spectrum virucidal activity against four recommended model viruses. These are modified vaccinia virus Ankara (MVA), human adenovirus type 5 (HAdV-5), poliovirus type 1 (PV-1) and murine norovirus (MNV). All virucidal tests were performed two to five times using the quantitative suspension test, which is a highly standardized test method to evaluate the virucidal efficacy of disinfectants in accordance with the European norm EN 14476+A1 and the German DVV/RKI guidelines. Quantitative detection of viruses was conducted by endpoint titration and quantitative real-time PCR. Results showed that three of the five tested compounds (WC1-WC3) were able to reduce the infectivity of all model viruses by at least four log10 of tissue culture infective dose 50% per ml after 15 min, whereas the other two compounds exhibited only limited efficacy (WC4) or showed cytotoxicity (WC5). Virucidal activity of nanoparticles increased with incubation time and a dose-effect curve showed dependence of virucidal activity with particle concentration. Whereas WC1-WC4 showed little cytotoxicity, WC5 which was doped with copper exhibited a significant cytotoxic effect. These findings propose tungsten carbide nanoparticles to be very promising in terms of new disinfection techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study investigates the virucidal activity of tungsten carbide nanoparticles using the quantitative suspension test in accordance with the European norm EN 14476+A1 and the German DVV/RKI guidelines. Due to highly standardized assay conditions, results of this test are considered very reliable for evaluation of the virucidal activity of disinfectants. Broad-spectrum activity and high efficacy of three different tungsten carbide nanoparticles preparations is concluded.

Screening and evaluation of potential inhibitors against vaccinia virus from 767 approved drugs.

The development of therapies for human smallpox is needed due to the increasing concern over the potential use of smallpox virus as a biological weapon. Here, we report a high-throughput screening for anti-smallpox virus drugs from a 767-small-molecule library, employing two vaccinia virus (VACV) strains containing firefly luciferase (VTT-Fluc and VG9-Fluc) as surrogate viruses. Using an eight-point dose response format assay, 26 compounds of different pharmacological classes were identified with in vitro anti-VACV activities. Mycophenolate mofetil (MMF) and tranilast (TRA) were detected to possess the highest anti-VACV potency (selectivity index values of >334 and >74, respectively); they could inhibit VTT-Fluc replication in nude mice at 5 days post-infection by 99% (10 mg/kg, P < .01) and 59% (45 mg/kg, P = .01), respectively, as indicated by bioluminescent intensity. In conclusion, MMF and TRA are promising anti-smallpox virus candidates for further optimization and repurposing for use in clinical practice.

Preemptive Tecovirimat Use in an Active Duty Service Member Who Presented With Acute Myeloid Leukemia After Smallpox Vaccination.

Smallpox vaccine is contraindicated in immunosuppression due to increased risk for adverse reactions (eg, progressive vaccinia). We describe the first-ever use of tecovirimat as a preemptive vaccinia virus treatment strategy during induction chemotherapy in an active duty service member who presented with acute leukemia and inadvertent autoinoculation after smallpox vaccination.

Mutation and structure guided discovery of an antiviral small molecule that mimics an essential C-Terminal tripeptide of the vaccinia D4 processivity factor.

The smallpox virus (variola) remains a bioterrorism threat since a majority of the human population has never been vaccinated. In the event of an outbreak, at least two drugs against different targets of variola are critical to circumvent potential viral mutants that acquire resistance. Vaccinia virus (VACV) is the model virus used in the laboratory for studying smallpox. The VACV processivity factor D4 is an ideal therapeutic target since it is both essential and specific for poxvirus replication. Recently, we identified a tripeptide (Gly-Phe-Ile) motif at the C-terminus of D4 that is conserved among poxviruses and is necessary for maintaining protein function. In the current work, a virtual screening for small molecule mimics of the tripeptide identified a thiophene lead that effectively inhibited VACV, cowpox virus, and rabbitpox virus in cell culture (EC50 = 8.4-19.7 µM) and blocked in vitro processive DNA synthesis (IC50 = 13.4 µM). Compound-binding to D4 was demonstrated through various biophysical methods and a dose-dependent retardation of the proteolysis of D4 proteins. This study highlights an inhibitor design strategy that exploits a susceptible region of the protein and identifies a novel scaffold for a broad-spectrum poxvirus inhibitor.