DNA methyltransferase inhibitor 5-azacytidine enhances neuroblastoma cell lysis by an oncolytic parainfluenza virus.

Studies with neuroblastoma have shown that the presence of aberrant DNA epigenetic modifications mediated by DNA methyltransferases correlates with poor prognosis, making these enzymes a target for therapeutics based on synthetic epigenetic modulators such as DNA methyltransferase inhibitors (DNMTi). Here, we have used a neuroblastoma cell line model to test the hypothesis that treatment with a DNMTi would enhance cell killing when used in combination with oncolytic Parainfluenza virus 5 (P/V virus), a cytoplasmic-replicating RNA virus. Pretreatment of SK-N-AS cells with the DNMTi 5-azacytidine substantially enhanced P/V virus-mediated cell death in a dose- and multiplicity of infection-dependent manner. Infection with the virus alone and the combination treatment with 5-azacytidine and P/V virus infection led to the activation of caspases-8, -9, and -3/7. Inhibition of caspases using a pan-caspase inhibitor minimally affected cell killing by P/V virus alone, but by contrast, largely reduced cell death mediated by 5-azacytidine treatment alone or in combination with P/V virus infection. 5-Azacytidine pretreatment dampened P/V virus gene expression and growth within the SK-N-AS cell population, which correlated with enhanced expression of important antiviral genes such as interferon-ß and OAS2 . Taken together, our data support the role of combination treatment using 5-azacytidine and an oncolytic P/V virus for neuroblastoma therapy.

Broad-Spectrum, Potent, and Durable Ceria Nanoparticles Inactivate RNA Virus Infectivity by Targeting Virion Surfaces and Disrupting Virus-Receptor Interactions.

There is intense interest in developing long-lasting, potent, and broad-spectrum antiviral disinfectants. Ceria nanoparticles (CNPs) can undergo surface redox reactions (Ce3+ ↔ Ce4+) to generate ROS without requiring an external driving force. Here, we tested the mechanism behind our prior finding of potent inactivation of enveloped and non-enveloped RNA viruses by silver-modified CNPs, AgCNP1 and AgCNP2. Treatment of human respiratory viruses, coronavirus OC43 and parainfluenza virus type 5 (PIV5) with AgCNP1 and 2, respectively, prevented virus interactions with host cell receptors and resulted in virion aggregation. Rhinovirus 14 (RV14) mutants were selected to be resistant to inactivation by AgCNP2. Sequence analysis of the resistant virus genomes predicted two amino acid changes in surface-located residues D91V and F177L within capsid protein VP1. Consistent with the regenerative properties of CNPs, surface-applied AgCNP1 and 2 inactivated a wide range of structurally diverse viruses, including enveloped (OC43, SARS-CoV-2, and PIV5) and non-enveloped RNA viruses (RV14 and feline calicivirus; FCV). Remarkably, a single application of AgCNP1 and 2 potently inactivated up to four sequential rounds of virus challenge. Our results show broad-spectrum and long-lasting anti-viral activity of AgCNP nanoparticles, due to targeting of viral surface proteins to disrupt interactions with cellular receptors.

Parainfluenza Virus Infection Sensitizes Cancer Cells to DNA-Damaging Agents: Implications for Oncolytic Virus Therapy.

A parainfluenza virus 5 (PIV5) with mutations in the P/V gene (P/V-CPI-) is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burdens in mouse model systems. Here we show that P/V-CPI- infection of HEp-2 human laryngeal cancer cells results in the majority of the cells dying, but unexpectedly, over time, there is an emergence of a population of cells that survive as P/V-CPI- persistently infected (PI) cells. P/V-CPI- PI cells had elevated levels of basal caspase activation, and viability was highly dependent on the activity of cellular inhibitor-of-apoptosis proteins (IAPs) such as Survivin and XIAP. In challenge experiments with external inducers of apoptosis, PI cells were more sensitive to cisplatin-induced DNA damage and cell death. This increased cisplatin sensitivity correlated with defects in DNA damage signaling pathways such as phosphorylation of Chk1 and translocation of damage-specific DNA binding protein 1 (DDB1) to the nucleus. Cisplatin-induced killing of PI cells was sensitive to the inhibition of wild-type (WT) p53-inducible protein 1 (WIP1), a phosphatase which acts to terminate DNA damage signaling pathways. A similar sensitivity to cisplatin was seen with cells during acute infection with P/V-CPI- as well as during acute infections with WT PIV5 and the related virus human parainfluenza virus type 2 (hPIV2). Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish PI as well as the potential for combining chemotherapy with oncolytic RNA virus vectors.IMPORTANCE There is intense interest in developing oncolytic viral vectors with increased potency against cancer cells, particularly those cancer cells that have gained resistance to chemotherapies. We have found that infection with cytoplasmically replicating parainfluenza virus can result in increases in the killing of cancer cells by agents that induce DNA damage, and this is linked to alterations to DNA damage signaling pathways that balance cell survival versus death. Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish persistent infection, the repurposing of drugs that target cellular IAPs as antivirals, and the combined use of DNA-damaging chemotherapy agents in conjunction with oncolytic RNA virus vectors.

A porcine xenograft-derived bone scaffold is a biocompatible bone graft substitute: An assessment of cytocompatibility and the alpha-Gal epitope.

BACKGROUND: Xenografts are an attractive alternative to traditional bone grafts because of the large supply from donors with predictable morphology and biology as well as minimal risk of human disease transmission. Clinical series involving xenograft bone transplantation, most commonly from bovine sources, have reported poor results with frequent graft rejection and failure to integrate with host tissue. Failures have been attributed to residual alpha-Gal epitope in the xenograft which humans produce natural antibody against. To the authors' knowledge, there is currently no xenograft-derived bone graft substitute that has been adopted by orthopedic surgeons for routine clinical use. METHODS: In the current study, a bone scaffold intended to serve as a bone graft substitute was derived from porcine cancellous bone using a tissue decellularization and chemical oxidation protocol. In vitro cytocompatibility, pathogen clearance, and alpha-Gal quantification tests were used to assess the safety of the bone scaffold intended for human use. RESULTS: In vitro studies showed the scaffold was free of processing chemicals and biocompatible with mouse and human cell lines. When bacterial and viral pathogens were purposefully added to porcine donor tissue, processing successfully removed these pathogens to comply with sterility assurance levels established by allograft tissue providers. Critically, 98.5% of the alpha-Gal epitope was removed from donor tissue after decellularization as shown by ELISA inhibition assay and immunohistochemical staining. CONCLUSIONS: The current investigation supports the biologic safety of bone scaffolds derived from porcine donors using a decellularization protocol that meets current sterility assurance standards. The majority of the highly immunogenic xenograft carbohydrate was removed from donor tissue, and these findings support further in vivo investigation of xenograft-derived bone tissue for orthopedic clinical application.

A Novel R848-Conjugated Inactivated Influenza Virus Vaccine Is Efficacious and Safe in a Neonate Nonhuman Primate Model.

Influenza virus infection of neonates poses a major health concern, often resulting in severe disease and hospitalization. At present, vaccines for this at-risk population are lacking. Thus, development of an effective vaccine is an urgent need. In this study, we have used an innovative nonhuman primate neonate challenge model to test the efficacy of a novel TLR 7/8 agonist R848-conjugated influenza virus vaccine. The use of the intact virus represents a step forward in conjugate vaccine design because it provides multiple antigenic targets allowing for elicitation of a broad immune response. Our results show that this vaccine induces high-level virus-specific Ab- and cell-mediated responses in neonates that result in increased virus clearance and reduced lung pathology postchallenge compared with the nonadjuvanted virus vaccine. Surprisingly, the addition of a second TLR agonist (flagellin) did not enhance vaccine protection, suggesting that combinations of TLR that provide increased efficacy must be determined empirically. These data support further exploration of this new conjugate influenza vaccine approach as a platform for use in the at-risk neonate population.

A novel factor I activity in Nipah virus inhibits human complement pathways through cleavage of C3b.

UNLABELLED: Complement is an innate immune system that most animal viruses must face during natural infections. Given that replication and dissemination of the highly pathogenic Nipah virus (NiV) include exposure to environments rich in complement factors, we tested the in vitro sensitivity of NiV to complement-mediated neutralization. Here we show that NiV was completely resistant to in vitro neutralization by normal human serum (NHS). Treatment of purified NiV with NHS activated complement pathways, but there was very little C3 deposition on virus particles. In in vitro reconstitution experiments, NiV particles provided time- and dose-dependent factor I-like protease activity capable of cleaving C3b into inactive C3b (iC3b). NiV-dependent inactivation of C3b only occurred with the cofactors factor H and soluble CR1 but not with CD46. Purified NiV particles did not support C4b cleavage. Electron microscopy of purified NiV particles showed immunogold labeling with anti-factor I antibodies. Our results suggest a novel mechanism by which NiV evades the human complement system through a unique factor I-like activity. IMPORTANCE: Viruses have evolved mechanisms to limit complement-mediated neutralization, some of which involve hijacking cellular proteins involved in control of inappropriate complement activation. Here we report a previously unknown mechanism whereby NiV provides a novel protease activity capable of in vitro cleavage and inactivation of C3b, a key component of the complement cascade. These data help to explain how an enveloped virus such as NiV can infect and disseminate through body fluids that are rich in complement activity. Disruption of the ability of NiV to recruit complement inhibitors could form the basis for the development of effective therapies and safer vaccines to combat these highly pathogenic emerging viruses.

Inclusion of Flagellin during Vaccination against Influenza Enhances Recall Responses in Nonhuman Primate Neonates.

UNLABELLED: Influenza virus can cause life-threatening infections in neonates and young infants. Although vaccination is a major countermeasure against influenza, current vaccines are not approved for use in infants less than 6 months of age, in part due to the weak immune response following vaccination. Thus, there is a strong need to develop new vaccines with improved efficacy for this vulnerable population. To address this issue, we established a neonatal African green monkey (AGM) nonhuman primate model that could be used to identify effective influenza vaccine approaches for use in young infants. We assessed the ability of flagellin, a Toll-like receptor 5 (TLR5) agonist, to serve as an effective adjuvant in this at-risk population. Four- to 6-day-old AGMs were primed and boosted with inactivated PR8 influenza virus (IPR8) adjuvanted with either wild-type flagellin or inactive flagellin with a mutation at position 229 (m229), the latter of which is incapable of signaling through TLR5. Increased IgG responses were observed following a boost, as well as at early times after challenge, in infants vaccinated with flagellin-adjuvanted IPR8. Inclusion of flagellin during vaccination also resulted in a significantly increased number of influenza virus-specific T cells following challenge compared to the number in infants vaccinated with the m229 adjuvant. Finally, following challenge infants vaccinated with IPR8 plus flagellin exhibited a reduced pathology in the lungs compared to that in infants that received IPR8 plus m229. This study provides the first evidence of flagellin-mediated enhancement of vaccine responses in nonhuman primate neonates. IMPORTANCE: Young infants are particularly susceptible to severe disease as a result of influenza virus infection. Compounding this is the lack of effective vaccines for use in this vulnerable population. Here we describe a vaccine approach that results in improved immune responses and protection in young infants. Incorporation of flagellin during vaccination resulted in increased antibody and T cell responses together with reduced disease following virus infection. These results suggest that flagellin may serve as an effective adjuvant for vaccines targeted to this vulnerable population.

Coinfection with Streptococcus pneumoniae negatively modulates the size and composition of the ongoing influenza-specific CD8⁺ T cell response.

Infection with influenza A virus can lead to increased susceptibility to subsequent bacterial infection, often with Streptococcus pneumoniae. Given the substantial modification of the lung environment that occurs following pathogen infection, there is significant potential for modulation of immune responses. In this study, we show that infection of mice with influenza virus, followed by the noninvasive EF3030 strain of Streptococcus pneumoniae, leads to a significant decrease in the virus-specific CD8(+) T cell response in the lung. Adoptive-transfer studies suggest that this reduction contributes to disease in coinfected animals. The reduced number of lung effector cells in coinfected animals was associated with increased death, as well as a reduction in cytokine production in surviving cells. Further, cells that retained the ability to produce IFN-γ exhibited a decreased potential for coproduction of TNF-α. Reduced cytokine production was directly correlated with a decrease in the level of mRNA. Negative regulation of cells in the mediastinal lymph node was minimal compared with that present in the lung, supporting a model of selective regulation in the tissue harboring high pathogen burden. These results show that entry of a coinfecting pathogen can have profound immunoregulatory effects on an ongoing immune response. Together, these findings reveal a novel dynamic interplay between concurrently infecting pathogens and the adaptive immune system.

An alphavirus-based adjuvant enhances serum and mucosal antibodies, T cells, and protective immunity to influenza virus in neonatal mice.

UNLABELLED: Neonatal immune responses to infection and vaccination are biased toward TH2 at the cost of proinflammatory TH1 responses needed to combat intracellular pathogens. However, upon appropriate stimulation, the neonatal immune system can induce adult-like TH1 responses. Here we report that a new class of vaccine adjuvant is especially well suited to enhance early life immunity. The GVI3000 adjuvant is a safe, nonpropagating, truncated derivative of Venezuelan equine encephalitis virus that targets dendritic cells (DCs) in the draining lymph node (DLN) and produces intracellular viral RNA without propagating to other cells. RNA synthesis strongly activates the innate immune response so that in adult animals, codelivery of soluble protein antigens induces robust humoral, cellular, and mucosal responses. The adjuvant properties of GVI3000 were tested in a neonatal BALB/c mouse model using inactivated influenza virus (iFlu). After a single immunization, mice immunized with iFlu with the GVI3000 adjuvant (GVI3000-adjuvanted iFlu) had significantly higher and sustained influenza virus-specific IgG antibodies, mainly IgG2a (TH1), compared to the mice immunized with antigen only. GVI3000 significantly increased antigen-specific CD4(+) and CD8(+) T cells, primed mucosal immune responses, and enhanced protection from lethal challenge. As seen in adult mice, the GVI3000 adjuvant increased the DC population in the DLNs, caused activation and maturation of DCs, and induced proinflammatory cytokines and chemokines in the DLNs soon after immunization, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6). In summary, the GVI3000 adjuvant induced an adult-like adjuvant effect with an influenza vaccine and has the potential to improve the immunogenicity and protective efficacy of new and existing neonatal vaccines. IMPORTANCE: The suboptimal immune responses in early life constitute a significant challenge for vaccine design. Here we report that a new class of adjuvant is safe and effective for early life immunization and demonstrate its ability to significantly improve the protective efficacy of an inactivated influenza virus vaccine in a neonatal mouse model. The GVI3000 adjuvant delivers a truncated, self-replicating viral RNA into dendritic cells in the draining lymph node. Intracellular RNA replication activates a strong innate immune response that significantly enhances adaptive antibody and cellular immune responses to codelivered antigens. A significant increase in protection results from a single immunization. Importantly, this adjuvant also primed a mucosal IgA response, which is likely to be critical for protection during many early life infections.

The Bordetella pertussis†Bps polysaccharide enhances lung colonization by conferring protection from complement-mediated killing.

Bordetella pertussis is a human-restricted Gram-negative bacterial pathogen that causes whooping cough or pertussis. Pertussis is the leading vaccine preventable disease that is resurging in the USA and other parts of the developed world. There is an incomplete understanding of the mechanisms by which B. pertussis evades killing and clearance by the complement system, a first line of host innate immune defence. The present study examined the role of the Bps polysaccharide to resist complement activity in vitro and in the mouse respiratory tract. The isogenic bps mutant strain containing a large non-polar in-frame deletion of the bpsA-D locus was more sensitive to serum and complement mediated killing than the WT strain. As determined by Western blotting, flow cytometry and electron microscopic studies, the heightened sensitivity of the mutant strain was due to enhanced deposition of complement proteins and the formation of membrane attack complex, the end-product of complement activation. Bps was sufficient to confer complement resistance as evidenced by a Bps-expressing Escherichia coli being protected by serum killing. Additionally, Western blotting and flow cytometry assays revealed that Bps inhibited the deposition of complement proteins independent of other B. pertussis factors. The bps mutant strain colonized the lungs of complement-deficient mice at higher levels than that observed in C57Bl/6 mice. These results reveal a previously unknown interaction between Bps and the complement system in controlling B. pertussis colonization of the respiratory tract. These findings also make Bps a potential target for the prevention and therapy of whooping cough.

Point mutations in the paramyxovirus F protein that enhance fusion activity shift the mechanism of complement-mediated virus neutralization.

Parainfluenza virus 5 (PIV5) activates and is neutralized by the alternative pathway (AP) in normal human serum (NHS) but not by heat-inactivated (HI) serum. We have tested the relationship between the fusion activity within the PIV5 F protein, the activation of complement pathways, and subsequent complement-mediated virus neutralization. Recombinant PIV5 viruses with enhanced fusion activity were generated by introducing point mutations in the F fusogenic peptide (G3A) or at a distal site near the F transmembrane domain (S443P). In contrast to wild-type (WT) PIV5, the mutant G3A and S443P viruses were neutralized by both NHS and HI serum. Unlike WT PIV5, hyperfusogenic G3A and S443P viruses were potent C4 activators, C4 was deposited on NHS-treated mutant virions, and the mutants were neutralized by factor B-depleted serum but not by C4-depleted serum. Antibodies purified from HI human serum were sufficient to neutralize both G3A and S443P viruses in vitro but were ineffective against WT PIV5. Electron microscopy data showed greater deposition of purified human antibodies on G3A and S443P virions than on WT PIV5 particles. These data indicate that single amino acid changes that enhance the fusion activity of the PIV5 F protein shift the mechanism of complement activation in the context of viral particles or on the surface of virus-infected cells, due to enhanced binding of antibodies. We present general models for the relationship between enhanced fusion activity in the paramyxovirus F protein and increased susceptibility to antibody-mediated neutralization.

Virion-associated complement regulator CD55 is more potent than CD46 in mediating resistance of mumps virus and vesicular stomatitis virus to neutralization.

Enveloped viruses can incorporate host cell membrane proteins during the budding process. Here we demonstrate that mumps virus (MuV) and vesicular stomatitis virus (VSV) assemble to include CD46 and CD55, two host cell regulators which inhibit propagation of complement pathways through distinct mechanisms. Using viruses which incorporated CD46 alone, CD55 alone, or both CD46 and CD55, we have tested the relative contribution of these regulators in resistance to complement-mediated neutralization. Virion-associated CD46 and CD55 were biologically active, with VSV showing higher levels of activity of both cofactors, which promoted factor I-mediated cleavage of C3b into iC3b as well as decay-accelerating factor (DAF) activity against the C3 convertase, than MuV. Time courses of in vitro neutralization with normal human serum (NHS) showed that both regulators could delay neutralization, but viruses containing CD46 alone were neutralized faster and more completely than viruses containing CD55 alone. A dominant inhibitory role for CD55 was most evident for VSV, where virus containing CD55 alone was not substantially different in neutralization kinetics from virus harboring both regulators. Electron microscopy showed that VSV neutralization proceeded through virion aggregation followed by lysis, with virion-associated CD55 providing a delay in both aggregation and lysis more substantial than that conferred by CD46. Our results demonstrate the functional significance of incorporation of host cell factors during virion envelope assembly. They also define pathways of virus complement-mediated neutralization and suggest the design of more effective viral vectors.

Incorporation of host complement regulatory proteins into Newcastle disease virus enhances complement evasion.

Newcastle disease virus (NDV), an avian paramyxovirus, is inherently tumor selective and is currently being considered as a clinical oncolytic virus and vaccine vector. In this study, we analyzed the effect of complement on the neutralization of NDV purified from embryonated chicken eggs, a common source for virus production. Fresh normal human serum (NHS) neutralized NDV by multiple pathways of complement activation, independent of neutralizing antibodies. Neutralization was associated with C3 deposition and the activation of C2, C3, C4, and C5 components. Interestingly, NDV grown in mammalian cell lines was resistant to complement neutralization by NHS. To confirm whether the incorporation of regulators of complement activity (RCA) into the viral envelope afforded complement resistance, we grew NDV in CHO cells stably transfected with CD46 or HeLa cells, which strongly express CD46 and CD55. NDV grown in RCA-expressing cells was resistant to complement by incorporating CD46 and CD55 on virions. Mammalian CD46 and CD55 molecules on virions exhibited homologous restriction, since chicken sera devoid of neutralizing antibodies to NDV were able to effectively neutralize these virions. The incorporation of chicken RCA into NDV produced in embryonated eggs similarly provided species specificity toward chicken sera.

Delivery of a novel membrane-anchored Fc chimera enhances NK cell-mediated killing of tumor cells and persistently virus-infected cells.

Antibody-dependent cellular cytotoxicity (ADCC) is one of the most powerful mechanisms for Natural Killer (NK) cells to kill cancer cells or virus-infected cells. A novel chimeric protein (NA-Fc) was created, which when expressed in cells, positions an IgG Fc domain on the plasma membrane, mimicking the orientation of IgG bound to the cell surface. This NA-Fc chimera was tested with PM21-NK cells, produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Real time viability assays revealed higher PM21-NK killing of both ovarian and lung cancer cells expressing NA-Fc, which correlated with increased release of TNF-α and IFN-γ cytokines from NK cells and was dependent on CD16-Fc interactions. Lentivirus delivery of NA-Fc to target cells increased the rate of PM21-NK cell killing of A549 and H1299 lung, SKOV3 ovarian and A375 melanoma cancer cells. This NA-Fc-directed killing was extended to virus infected cells, where delivery of NA-Fc to lung cells that were persistently infected with Parainfluenza virus resulted in increased killing by PM21-NK cells. In contrast to its effect on PM21-NK cells, the NA-Fc molecule did not enhance complement mediated lysis of lung cancer cells. Our study lays the foundation for application of the novel NA-Fc chimera that could be delivered specifically to tumors during oncolytic virotherapy to mark target cells for ADCC by co-treatment with adoptive NK cells. This strategy would potentially eliminate the need to search for unique cancer specific antigens for development of new antibody therapeutics.

Interactions of human complement with virus particles containing the Nipah virus glycoproteins.

Complement is an innate immune response system that most animal viruses encounter during natural infections. We have tested the role of human complement in the neutralization of virus particles harboring the Nipah virus (NiV) glycoproteins. A luciferase-expressing vesicular stomatitis virus (VSV) pseudotype that contained the NiV fusion (F) and attachment (G) glycoproteins (NiVpp) showed dose- and time-dependent activation of human complement through the alternative pathway. In contrast to our findings with other paramyxoviruses, normal human serum (NHS) alone did not neutralize NiVpp infectivity in vitro, and electron microscopy demonstrated no significant deposition of complement component C3 on particles. This lack of NiVpp neutralization by NHS was not due to a global inhibition of complement pathways, since complement was found to significantly enhance neutralization by antibodies specific for the NiV F and G glycoproteins. Complement components C4 and C1q were necessary but not sufficient by themselves for the enhancement of antibody neutralization. Human complement also enhanced NiVpp neutralization by a soluble version of the NiV receptor EphrinB2, and this depended on components in the classical pathway. The ability of complement to enhance neutralization fell into one of two profiles: (i) anti-F monoclonal antibodies showed enhancement only at high and not low antibody concentrations, and (ii) anti-G monoclonal antibodies and EphrinB2 showed enhancement at both high and very low levels of antibody (e.g., 3.1 ng) or EphrinB2 (e.g., 2.5 ng). Together, these data establish the importance of human complement in the neutralization of particles containing the NiV glycoproteins and will help guide the design of more effective therapeutics that harness the potency of complement pathways.

Differential In Vitro Growth and Cell Killing of Cancer versus Benign Prostate Cells by Oncolytic Parainfluenza Virus.

The development of effective oncolytic viruses will require understanding the differences in virus replication and killing between normal and cancer cells. Here, we have evaluated infections of metastatic cancer (22Rv1) and benign non-tumorigenic (BPH-1) prostate cell lines with a mutant parainfluenza virus 5 (P/V/F) encoding a defective V protein and a hyperfusogenic F protein. Under low multiplicity of infection (MOI), the P/V/F mutant efficiently spread in 22Rv1 cells but was restricted in BPH-1 cells due to type-I interferon (IFN-I) responses. In mixed co-cultures, the P/V/F mutant showed specificity towards and spread within the 22Rv1 cells versus BPH-1 cells. Under high MOI conditions, both BPH-1 and 22Rv1 cells showed efficient infection by the P/V/F mutant. However, compared to BPH-1 cells, the 22Rv1 cancer cells showed increased cytopathic effect, higher induction of caspase-8 and -9, and extensive syncytia formation. In 22Rv1 spheroid cultures, P/V/F infection was less efficient compared to monolayers, but the virus was able to spread through spheroids and induce death. These data indicate that IFN-I sensitivity is a major determinant of specificity of P/V/F spread through populations of cancer versus benign cells, and additionally, differences in activation of apoptotic pathways and syncytia formation can contribute to differential outcomes in cancer versus benign cells.

CD24 Expression Dampens the Basal Antiviral State in Human Neuroblastoma Cells and Enhances Permissivity to Zika Virus Infection.

Zika virus (ZIKV) exhibits distinct selectivity for infection of various cells and tissues, but how host cellular factors modulate varying permissivity remains largely unknown. Previous studies showed that the neuroblastoma cell line SK-N-AS (expressing low levels of cellular protein CD24) was highly restricted for ZIKV infection, and that this restriction was relieved by ectopic expression of CD24. We tested the hypothesis that CD24 expression allowed ZIKV replication by suppression of the antiviral response. SK-N-AS cells expressing an empty vector (termed CD24-low cells) showed elevated basal levels of phosphorylated STAT1, IRF-1, IKKE, and NFκB. In response to exogenously added type I interferon (IFN-I), CD24-low cells had higher-level induction of antiviral genes and activity against two IFN-I-sensitive viruses (VSV and PIV5-P/V) compared to SK-N-AS cells with ectopic CD24 expression (termed CD24-high cells). Media-transfer experiments showed that the inherent antiviral state of CD24-low cells was not dependent on a secreted factor such as IFN-I. Transcriptomics analysis revealed that CD24 expression decreased expression of genes involved in intracellular antiviral pathways, including IFN-I, NFκB, and Ras. Our findings that CD24 expression in neuroblastoma cells represses intracellular antiviral pathways support the proposal that CD24 may represent a novel biomarker in cancer cells for susceptibility to oncolytic viruses.

ALD based nanostructured zinc oxide coated antiviral silk fabric.

The COVID-19 pandemic has underscored the importance of research and development in maintaining public health. Facing unprecedented challenges, the scientific community developed antiviral drugs, virucides, and vaccines to combat the infection within the past two years. However, an ever-increasing list of highly infectious SARS-CoV-2 variants (gamma, delta, omicron, and now ba.2 stealth) has exacerbated the problem: again raising the issues of infection prevention strategies and the efficacy of personal protective equipment (PPE). Against this backdrop, we report an antimicrobial fabric for PPE applications. We have fabricated a nanofibrous silk-PEO material using electrospinning followed by zinc oxide thin film deposition by employing the atomic layer deposition technique. The composite fabric has shown 85% more antibacterial activity than the control fabric and was found to possess substantial superoxide dismutase-mimetic activity. The composite was further subjected to antiviral testing using two different respiratory tract viruses: coronavirus (OC43: enveloped) and rhinovirus (RV14: non-enveloped). We report a 95% reduction in infectious virus for both OC43 and RV14 from an initial load of ∼1 × 105 (sample size: 6 mm dia. disk), after 1 h of white light illumination. Furthermore, with 2 h of illumination, ∼99% reduction in viral infectivity was observed for RV14. High activity in a relatively small area of fabric (3.5 × 103 viral units per mm2) makes this antiviral fabric ideal for application in masks/PPE, with an enhanced ability to prevent antimicrobial infection overall.

Potent Inactivation of Human Respiratory Viruses Including SARS-CoV-2 by a Photoactivated Self-Cleaning Regenerative Antiviral Coating.

The COVID-19 pandemic marks an inflection point in the perception and treatment of human health. Substantial resources have been reallocated to address the direct medical effects of COVID-19 and to curtail the spread of the virus. Thereby, shortcomings of traditional disinfectants, especially their requirement for regular reapplication and the related complications (e.g., dedicated personnel and short-term activity), have become issues at the forefront of public health concerns. This issue became especially pressing when infection-mitigating supplies dwindled early in the progression of the pandemic. In consideration of the constant threat posed by emerging novel viruses, we report a platform technology for persistent surface disinfection to combat virus transmission through nanomaterial-mediated, localized UV radiation emission. In this work, two formulations of Y2SiO5-based visible-to-UV upconversion nanomaterials were developed using a facile sol-gel-based synthesis. Our formulations have shown substantial antiviral activities (4 × 104 to 0 TCID50 units in 30 min) toward an enveloped, circulating human coronavirus strain (OC43) under simple white light exposure as an analogue to natural light or common indoor lighting. Additionally, we have shown that our two formulations greatly reduce OC43 RNA recovery from surfaces. Antiviral activities were further demonstrated toward a panel of structurally diverse viruses including enveloped viruses, SARS-CoV-2, vaccinia virus, vesicular stomatitis virus, parainfluenza virus, and Zika virus, as well as nonenveloped viruses, rhinovirus, and calicivirus, as evidence of the technology's broad antiviral activity. Remarkably, one formulation completely inactivated 105 infectious units of SARS-CoV-2 in only 45 min. The detailed technology has implications for the design of more potent, long-lived disinfectants and modified/surface-treated personal protective equipment targeting a wide range of viruses.

Complement Inhibitors Vitronectin and Clusterin Are Recruited from Human Serum to the Surface of Coronavirus OC43-Infected Lung Cells through Antibody-Dependent Mechanisms.

Little is known about the role of complement (C') in infections with highly prevalent circulating human coronaviruses such as OC43, a group of viruses of major public health concern. Treatment of OC43-infected human lung cells with human serum resulted in C3 deposition on their surfaces and generation of C5a, indicating robust C' activation. Real-time cell viability assays showed that in vitro C'-mediated lysis of OC43 infected cells requires C3, C5 and C6 but not C7, and was substantially delayed as compared to rapid C'-mediated killing of parainfluenza virus type 5 (PIV5)-infected cells. In cells co-infected with OC43 and PIV5, C'-mediated lysis was delayed, similar to OC43 infected cells alone, suggesting that OC43 infection induced dominant inhibitory signals. When OC43-infected cells were treated with human serum, their cell surfaces contained both Vitronectin (VN) and Clusterin (CLU), two host cell C' inhibitors that can alter membrane attack complex (MAC) formation and C'-mediated killing. VN and CLU were not bound to OC43-infected cells after treatment with antibody-depleted serum. Reconstitution experiments with purified IgG and VN showed that human antibodies are both necessary and sufficient for VN recruitment to OC43-infected lung cells-novel findings with implications for CoV pathogenesis.