Virus Activity Research Articles

VEGF-induced Nrdp1 deficiency in vascular endothelial cells promotes cancer metastasis by degrading vascular basement membrane.

Vascular endothelial cells (VECs) are key players in the formation of neovessels and tumor metastasis, the ultimate cause of the majority of cancer-related human death. However, the crosstalk between VECs and metastasis remain greatly elusive. Based on our finding that tumor-associated VECs present significant decrease of Nrdp1 protein which is closely correlated with higher metastatic probability, herein we show that the conditional medium from hypoxia-incubated cancer cells induces extensive Nrdp1 downregulation in human and mouse VECs by vascular endothelial growth factor (VEGF), which activates CHIP, followed by Nrdp1 degradation in ubiquitin-proteasome-dependent way. More importantly, lung metastases of cancer cells significantly increase in conditional VECs Nrdp1 knockout mice. Mechanically, Nrdp1 promotes degradation of Fam20C, a secretory kinase involved in phosphorylating numerous secreted proteins. Reciprocally, deficiency of Nrdp1 in VECs (ecNrdp1) results in increased secretion of Fam20C, which induces degradation of extracellular matrix and disrupts integrity of vascular basement membrane, thus driving tumor metastatic dissemination. In addition, specific overexpression of ecNrdp1 by Nrdp1-carrying adeno-associated virus or chemical Nrdp1 activator ABPN efficiently mitigates tumor metastasis in mice. Collectively, we explore a new mechanism for VEGF to enhance metastasis and role of Nrdp1 in maintaining the integrity of vascular endothelium, suggesting that ecNrdp1-mediated signaling pathways might become potential target for anti-metastatic therapies.

Collection and transportation of SARS-CoV-2 and influenza A virus diagnostic samples: Optimizing the usage of guanidine-based chaotropic salts for enhanced biosafety and viral genome preservation

Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.

Comparative Inactivation of the RNA of the Delta and Omicron Variants of SARS-CoV-2 in Wastewater of Five Municipalities in Southeast Germany

During the COVID-19 pandemic, wastewater-based epidemiology has been extensively used as a helpful tool for evaluation of the epidemic situation in catchments of wastewater treatment plants. In this context, knowledge about the rate of virus inactivation in wastewater is important for characterization of the influence of retention times in sewers on virus concentrations, of the storage periods before analysis in the laboratory, on virus elimination during treatment and for modeling approaches. In the present study, we genotyped SARS-CoV-2 in the respiratory tract specimens of infected patients and spiked the raw wastewater of five treatment plants with aliquots of these samples. The test preparations were incubated under practically relevant temperature conditions (14 °C) over 31 days. The linear decay rates resulted in comparable T90 values (30.5 d) for the Delta and the Omicron BA1 variants of SARS-CoV-2 without differences in using the E or S gene of the virus as the target for amplification. In contrast, the origin of wastewater influenced the inactivation rate of both variants significantly, with the mean T90 values varying between 24.3 and 53.1 days in the wastewater from the five plants. The data suggest that the inactivation rate of SARS-CoV-2 in wastewater is more strongly determined by the composition of the water than by the virus variant.

Descriptive Epidemiology of Pathogens Associated with Acute Respiratory Infection in a Community-Based Study of K-12 School Children (2015-2023).

School-based outbreaks often precede increased incidence of acute respiratory infections in the greater community. We conducted acute respiratory infection surveillance among children to elucidate commonly detected pathogens in school settings and their unique characteristics and epidemiological patterns. The ORegon CHild Absenteeism due to Respiratory Disease Study (ORCHARDS) is a longitudinal, laboratory-supported, school-based, acute respiratory illness (ARI) surveillance study designed to evaluate the utility of cause-specific student absenteeism monitoring for early detection of increased activity of influenza and other respiratory viruses in schools from kindergarten through 12th grade. Eligible participants with ARIs provided demographic, epidemiologic, and symptom data, along with a nasal swab or oropharyngeal specimen. Multipathogen testing using reverse-transcription polymerase chain reaction (RT-PCR) was performed on all specimens for 18 respiratory viruses and 2 atypical bacterial pathogens (Chlamydia pneumoniae and Mycoplasma pneumoniae). Between 5 January 2015 and 9 June 2023, 3498 children participated. Pathogens were detected in 2455 of 3498 (70%) specimens. Rhinovirus/enteroviruses (36%) and influenza viruses A/B (35%) were most commonly identified in positive specimens. Rhinovirus/enteroviruses and parainfluenza viruses occurred early in the academic year, followed by seasonal coronaviruses, RSV, influenza viruses A/B, and human metapneumovirus. Since its emergence in 2020, SARS-CoV-2 was detected year-round and had a higher median age than the other pathogens. A better understanding of the etiologies, presentations, and patterns of pediatric acute respiratory infections can help inform medical and public health system responses.

Dual antibacterial/antiviral performance of ZIFs for highly contaminated sanitary surfaces

Effective antimicrobial activity on highly contaminated surfaces, like in sanitary, requires the development of novel functional nanomaterials. In this work, we synthesized zeolitic imidazolate frameworks (ZIFs, 7 and 8) and validated their dual antibacterial/antiviral activity for real applications in sanitary. ZIF-7 and ZIF-8 presented the polygonal morphology associated with the organic ligand arrangement with a crystalline sodalite conformation, and significant surface area (SA) differences were found (38 vs. 1456 m2/g) and associated with particle agglomeration in ZIF-7. The in-vitro antibacterial performance indicated that the high SA of ZIF-8 enabled activity at early and late interaction stages promoted by the controlled Zn ion release in solution. Similarly, ZIF-8 induces the HSV1 virus inactivation during the host infection, demonstrating a high antiviral performance (ca. 96 %). This was also validated when depositing onto flush buttons and door handles in a sanitary.

An allosteric inhibitor of sirtuin 2 blocks hepatitis B virus covalently closed circular DNA establishment and its transcriptional activity

296 million people worldwide are predisposed to developing severe end-stage liver diseases due to chronic hepatitis B virus (HBV) infection. HBV forms covalently closed circular DNA (cccDNA) molecules that persist as episomal DNA in the nucleus of infected hepatocytes and drive viral replication. Occasionally, the HBV genome becomes integrated into host chromosomal DNA, a process that is believed to significantly contribute to circulating HBsAg levels and HCC development. Neither cccDNA accumulation nor expression from integrated HBV DNA are directly targeted by current antiviral treatments. In this study, we investigated the antiviral properties of a newly described allosteric modulator, FLS-359, that targets sirtuin 2 (SIRT2), an NAD+-dependent deacylase. Our results demonstrate that SIRT2 modulation by FLS-359 and by other tool compounds inhibits cccDNA synthesis following de novo infection of primary human hepatocytes and HepG2 (C3A)-NTCP cells, and FLS-359 substantially reduces cccDNA recycling in HepAD38 cells. While pre-existing cccDNA is not eradicated by short-term treatment with FLS-359, its transcriptional activity is substantially impaired, likely through inhibition of viral promoter activities. Consistent with the inhibition of viral transcription, HBsAg production by HepG2.2.15 cells, which contain integrated HBV genomes, is also suppressed by FLS-359. Our study provides further insights on SIRT2 regulation of HBV infection and supports the development of potent SIRT2 inhibitors as HBV antivirals.

Mimicking bacterial infection in male mice changes sperm small RNA profiles and multigenerationally alters offspring behavior and physiology

Paternal pre-conceptual exposures, including stress, diet, substance abuse, parasite infection, and viral immune activation via Poly I:C, have been reported to influence the brains and behavior of offspring through sperm epigenetic changes. However, the effects of paternal (F0) pre-conceptual exposure to bacterial-induced immune activation on the behavior and physiology of F1 and F2 generations remain unexplored. We examined this using C57BL/6J mice. Eight-week-old males (F0) received a single intraperitoneal injection of the bacterial mimetic lipopolysaccharide (LPS: 5 mg/kg) or 0.9 % saline (vehicle control) before mating with naïve females at four weeks post-injection. Comprehensive behavioral assessments were conducted to investigate anxiety, social behaviors, depressive-like behaviors and cognition in both the F1 and F2 generations within the age range of 8 to 14 weeks. Results demonstrated that only female offspring of LPS-exposed fathers exhibited reduced anxiety levels in the light/dark box, large open field, and novelty-suppressed feeding test. These F1 female offspring also exhibited heightened sociability in the 3-chambered social interaction test and a reduced preference for saccharin in the saccharin preference test. Additionally, the F1 male offspring of LPS-challenged males demonstrated an increased total distance traveled in the light/dark box and a longer distance covered in the light zone. They also exhibited diminished preference for social novelty in the 3-chambered social interaction test and an elevated novel arm preference index in the Y-maze. In the F2 generation, male descendants of LPS-treated fathers showed reduced latency to feed in the novelty-suppressed feeding test. Additionally, the F2 generation of LPS-challenged fathers, but not the F1 generation, displayed enhanced immune response in both sexes after an acute LPS immune challenge (5 mg/kg). Analysis of sperm small noncoding RNA profiles from LPS-treated F0 mice revealed significant changes at 4 weeks after administration of LPS. These changes included three microRNAs, eight PIWI-interacting RNAs, and two transfer RNAs, exhibiting significant upregulation (mmu-miR-146a-5p, mmu-piR-27082 and mmu-piR-29102) or downregulation (mmu-miR-5110, mmu-miR-467e-3p, mmu-piR-22583, mmu-piR-23548, mmu-piR-36341, mmu-piR-50293, mmu-piR-16583, mmu-piR-36507, Mus_musculus_tRNA-Ile-AAT-2-1 and Mus_musculus_tRNA-Tyr-GTA-1-1). Additionally, we detected 52 upregulated small noncoding RNAs (including 9 miRNAs, 41 piRNAs, and 2 tRNAs) and 7 downregulated small noncoding RNAs (3 miRNAs, 3 piRNAs, and 1 tRNA) in the sperm of F1 offspring from LPS-treated males. These findings provide compelling evidence for the involvement of epigenetic mechanisms in the modulation of brain function and immunity, and associated behavioral and immunological traits, across generations, in response to bacterial infection.

In vitro anti-HSV1 activity of aqueous extract of Areca catechu L.

Background and Objectives: HSV-1 is known as a very contagious virus and the main cause of cold sores or fever blisters. Herein, the aqueous extract of Areca catechu L. was evaluated for its anti-HSV-1 activity, compared to the standard control (acyclovir). Also, the effect of extract on the expression of UL46 and US6 genes that accumulate late in viral infection, was studied. Materials and Methods: The aqueous extract was obtained by the maceration of powdered plant in boiling water. Its anti- viral activity was evaluated on Vero cells infected with HSV-1 at different times: 2 h pre-infection, simultaneous infection, and 4 h post-infection, using MTT assay. The effect of extract on the expression of genes was investigated with quantitative real-time PCR. Results: The aqueous extract of A. catechu induced the inhibition of infection with the IC value of 110.52 ± 1.36 μg/ml. Also, it reduced the expression of UL46 when it was added 2 h pre-infection at 100 μg/ml. Moreover, reduction of expression of US6 was observed at the same concentration when the extract was used simultaneously with the occurrence of infection and 4 h post-infection. Conclusion: A. catechu can be considered an essential element of natural-based anti-HSV-1 agents.

Merging total synthesis and NMR technology for deciphering the realistic structure of natural 2,6-dideoxyglycosides.

The structural identification and efficient synthesis of bioactive 2,6-dideoxyglycosides are daunting challenges. Here, we report the total synthesis and structural revision of a series of 2,6-dideoxyglycosides from folk medicinal plants Ecdysanthera rosea and Chonemorpha megacalyx, which feature pregnane steroidal aglycones bearing an 18,20-lactone and glycans consisting of 2,6-dideoxy-3-O-methyl-β-pyranose residues, including ecdysosides A, B, and F and ecdysantheroside A. All the eight possible 2,6-dideoxy-3-O-methyl-β-pyranoside stereoisomers (of the proposed ecdysantheroside A) have been synthesized that testify the effective gold(I)-catalyzed glycosylation methods for the synthesis of various 2-deoxy-β-pyranosidic linkages and lays a foundation via nuclear magnetic resonance data mapping to identify these sugar units which occur promiscuously in the present and other natural glycosides. Moreover, some synthetic natural compounds and their isomers have shown promising anticancer, immunosuppressive, anti-inflammatory, and anti-Zika virus activities.

Clinical experience in the use of local antiviral therapy for glossitis caused by the herpes virus type IV

Herpetic infections are one of the most common anthroponotic diseases. The prevalence of herpes virus types, their combined manifestation in the body of one person, as well as the increase in the number of people with secondary immunodeficiency make the problem of diagnosis and treatment of herpes-associated diseases the most urgent [1,2].As is known, herpes virus type IV (Epstein-Barr, EBV) is one of the frequently encountered members of the herpesvirus family [3]. According to the researches of many authors, by the age of 45 they have infected up to 100% of the world's population [4,5]. Persisting in the tissues of the mucous membrane of the oral cavity (lymphoepithelial pharyngeal ring of Pirogov-Valdeier, stroma of salivary glands, epithelial cells), reactivation of the virus occurs in the case of a decrease in immunity. [8,9]. At the same time, the number of diseases caused by EBV increases annually and depends on the phase of virus activity [10,11].One of the relatively "young" EBV-associated diseases is the corresponding glossitis, the clinical and laboratory characteristics of which are poorly presented in the literature. For this reason, the therapy of oral manifestations of chronic EBV infection is considered from the position of treating infectious mononucleosis [6,7], which is ineffective. Ignorance of dentists about the reliable clinical and laboratory signs of EBV caused by glossitis leads to incorrect diagnosis and inadequate treatment, generalization of infection, and the appearance of opportunistic diseases.

SARS-CoV-2 inactivation by supercritical carbon dioxide coupled with hydrogen peroxide

The recent COVID-19 pandemic has underscored the need for innovative decontamination techniques capable of treating sensitive materials potentially contaminated. Combining supercritical carbon dioxide (scCO2) with sterilant agents has shown promise in this regard. This study aimed at testing scCO2 as a virus inactivation method for biomedical materials contaminated with the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). The virus was inoculated on a stainless-steel carrier and treated at 45 °C and 8 MPa. No inactivation was detected when only scCO2 was used, even after long treatment times (60 min). The addition of 50 ppm of H2O2 to the process allowed the inactivation of more than 5 Log PFU (Plaque Forming Unit) of the virus by only pressurising and depressurising the vessel, while a 20-min process is needed by only using H2O2. Overall, the study demonstrates a synergistic effect when H2O2 is added to the scCO2 process for the inactivation of SARS-CoV-2.

Engineered Luminescent Oncolytic Vaccinia Virus Activation of Photodynamic-Immune Combination Therapy for Colorectal Cancer.

Oncolytic virus therapy is currently regarded as a promising approach in cancer immunotherapy. It has greater therapeutic advantages for colorectal cancer that is prone to distant metastasis. However, the therapeutic efficacy and clinical application of viral agents alone for colorectal cancer remain suboptimal. In this study, an engineered oncolytic vaccinia virus (OVV-Luc) that expresses the firefly luciferase gene is developed and loaded Chlorin e6 (Ce6) onto the virus surface through covalent coupling, resulting in OVV-Luc@Ce6 (OV@C). The OV@C infiltrates tumor tissue and induces endogenous luminescence through substrate catalysis, resulting in the production of reactive oxygen species. This unique system eliminates the need for an external light source, making it suitable for photodynamic therapy (PDT) in deep tissues. Moreover, this synergistic effect between PDT and viral immunotherapy enhances dendritic cell maturation, macrophage polarization, and reversal of the immunosuppressive microenvironment. This synergistic effect has the potential to convert a "cold" into a "hot" tumor, it offers valuable insights for clinical translation and application.

Abstract LB422: Specific activation of the Epstein-Barr Virus BGLF4 locus via lowered DNA methylation in EBV-associated CNS lymphoproliferative disease

Abstract Introduction: Epstein-Barr virus positive (EBV+) central nervous system lymphoproliferative diseases (CNS-LPD) are aggressive clinical conditions with poor prognosis. We have previously reported use of a ganciclovir (GCV), zidovudine (AZT), rituximab and dexamethasone regimen (GARD) that induced complete and durable responses in patients with primary CNS post-transplant lymphoproliferative disease (PTLD). Responses were associated with detection of two GCV/AZT viral drug targets, the EBV protein kinases BGLF4 and BXLF1. These viral proteins are normally expressed in lytic phase EBV, and the molecular basis for expression in latently infected EBV+ CNS-LPD is unknown. Methods: RNA expression of EBV genes LMP1, BZLF1, BXLF1, and BGLF4 was measured in 12 CNS-LPD biopsies (n=8/12 DLBCL-type) via qRT-PCR. Mass spectrometry-based EpiTYPER assay was used to quantify DNA methylation in EBV gene promoters. Biopsies from 25 patients with systemic PTLD were used for comparison. The BGLF4 locus was investigated in detail using luciferase reporters and 5’RACE mapping of transcription start sites in 3 EBV-infected cell lines and 4 CNS-LPD biopsy samples. Overall EBV methylation was assessed in a screening assay covering 31 EBV loci. Mechanistic involvement of active demethylation via TET2 was studied in a HEK293 CRISPR-Cas9 gene knockout model under EBV (strain M81) infection. Results: Gene expression analysis of biopsy specimens showed expression of LMP1, BXLF1, and BGLF4, but not BZLF1. Expression of BXLF1 and BGLF4 was significantly higher in CNS-LPD when compared to systemic EBV+ PTLD (p=0.043 and p=0.00022, respectively). EpiTYPER data showed significantly decreased promoter methylation at BGLF4 (p=0.00013). Luciferase reporter analysis of the BGLF4 upstream sequence revealed 3 regions of promoter activity which were silenced by in vitro methylation of reporters. 5’RACE identified transcription start sites at all 3 BGLF4 promoters and detected their activity in EBV+ cell lines and CNS-LPD biopsies. By overlaying transcriptional activity, 5’RACE and DNA methylation data, we identified DNA methylation loss at single CpG dinucleotides correlated with BGLF4 expression (r2=0.73). These sites were specifically demethylated in CNS-LPD, while surrounding EBV methylation remained high. Knockout of TET2 followed by EBV infection revealed that active demethylation is required for demethylation at BGLF4 promoters. Conclusions: The molecular determinant for success of the GARD regimen in CNS-LPD has remained poorly understood, with immunohistochemical detection of BGLF4/BXLF1 as the only predictor of treatment response. Our results provide the molecular basis for expression of BGLF4 in CNS-LPD by showing decreased promotor DNA methylation. We identified specific BGLF4 methylation sites that hold promise as potential DNA biomarkers for EBV+ CNS-LPD. Citation Format: Christoph Weigel, Haley L. Klimaszewski, Selamawit Addissie, Sarah Schlotter, Fode Tounkara, James P. Dugan, Bradley M. Haverkos, Lynda Villagomez, Mark Lustberg, Pierluigi Porcu, Timothy Voorhees, Michael A. Caligiuri, Ginny Bumgardner, Christopher C. Oakes, Robert Baiocchi. Specific activation of the Epstein-Barr Virus BGLF4 locus via lowered DNA methylation in EBV-associated CNS lymphoproliferative disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB422.

Antiviral Rotenoids and Isoflavones Isolated from Millettia oblata ssp. teitensis.

Three new (1-3) and six known rotenoids (5-10), along with three known isoflavones (11-13), were isolated from the leaves of Millettia oblata ssp. teitensis. A new glycosylated isoflavone (4), four known isoflavones (14-18), and one known chalcone (19) were isolated from the root wood extract of the same plant. The structures were elucidated by NMR and mass spectrometric analyses. The absolute configuration of the chiral compounds was established by a comparison of experimental ECD and VCD data with those calculated for the possible stereoisomers. This is the first report on the use of VCD to assign the absolute configuration of rotenoids. The crude leaves and root wood extracts displayed anti-RSV (human respiratory syncytial virus) activity with IC50 values of 0.7 and 3.4 μg/mL, respectively. Compounds 6, 8, 10, 11, and 14 showed anti-RSV activity with IC50 values of 0.4-10 μM, while compound 3 exhibited anti-HRV-2 (human rhinovirus 2) activity with an IC50 of 4.2 μM. Most of the compounds showed low cytotoxicity for laryngeal carcinoma (HEp-2) cells; however compounds 3, 11, and 14 exhibited low cytotoxicity also in primary lung fibroblasts. This is the first report on rotenoids showing antiviral activity against RSV and HRV viruses.

The importance of monitoring viral activity and viral load of SARS-CoV-2

The COVID-19 pandemic has made it possible to study many aspects of the laboratory diagnosis of this infection, including the assessment of the viral load and viral activity of SARS-CoV-2. The review presents data on modern views on the virological characteristics of the SARS-CoV-2 virus, however, many aspects of this problem remain debatable. The issues of the relationship between the level of viral load and the clinical profile of patients are proposed for discussion, and the possibility of using the obtained data in practical healthcare is discussed. The control of viral load in patients with COVID-19 has not only a certain clinical significance, but is also undoubtedly important in the epidemiological aspect. Monitoring of a number of SARS-CoV-2 parameters. will allow to solve practical problems in the context of the ongoing pandemic.

Monitoring the sensitivity of influenza viruses to oseltamivir, in Romania, from 2016 to 2023

Abstract Introduction: influenza is an acute viral disease caused by infection of the upper respiratory tract - difficult to control, which affects a large part of the population. Neuraminidase inhibitors (NAIs), such as oseltamivir and zanamivir, are the antiviral drugs utilized in Romania for the treatment of respiratory infections caused by the influenza viruses. The objective of the study is to monitor and analyze influenza viruses circulating in Romania, in terms of their susceptibility to oseltamivir, with the aim of signalling the emergence of viral strains resistant to antivirals. Methods: The phenotypic method was performed to evaluate the inhibitory effect of oseltamivir on the neuraminidase enzyme activity of influenza viruses. Genotypic methods (SNP-based assays or sequencing of neuraminidase/whole genome) were used for detecting amino acid substitutions associated with influenza antiviral resistance. The study period is from 2016 to 2023. Results: from the total number of influenza viruses isolated during the period (833 isolated), 670 strains were characterized antigenically and genetically, and the phenotypic susceptibility to antivirals was performed for 245 isolated viral strains. According to WHO criteria categories antiviral susceptibility based on IC50 data, none of the test results indicated reduced or highly reduced inhibition by oseltamivir. Conclusion: This monitoring, carried out in the National Influenza Center Romania - Cantacuzino NMMIRD, is extremely important to maintain effective flu control and further development of other antiviral agents.

Pilot-scale production of a highly efficacious and stable monoglycosylated influenza split virus vaccine

The yearly epidemics and unpredictable outbreaks of influenza have raisedserious concernsglobally and led to prioritizing the development of an effective vaccine toprotectagainst newly emerging variants. Previously, we demonstrated that monoglycosylated influenza virus vaccines derived from A/California/7/2009 or an updated A/Brisbane/02/2018 (IVR-190) vaccine strain recommended by WHO are superior to fully glycosylated vaccines and could broadly protect against past and new coming H1N1 variants. However, whether such a monoglycosylated virus vaccine can be mass-produced to meet clinical demands and stable enough to provide consistent efficacy against H1N1 viruses remains unclear. Herein, we developed a platform for the pilot-scale production of the monoglycosylated split virus vaccine from the IVR-190 strain (IVR-190mg) with a robust and cost-effective manufacturing process. The critical parameters of inoculum dose, concentration of kifunensine, and optimized Endo H treatment process were comprehensively investigated. Several aims for preclinical studies of IVR-190mg were achieved, including [i] the execution of three engineering batch runs to validate lot-to-lot consistency, [ii] the establishment of IVR-190mg specifications to meet the acceptance criteria of a conventional influenza vaccine, [iii] an investigation of the stability profile of IVR-190mg, and completion of a safety evaluation by conducting an animal toxicology study. The toxicology study under GLP guidance found no systemic toxicity after rabbits were vaccinated with IVR-190mg. The serological data showed that IVR-190mg is highly immunogenic and effective in inducing a cross-strain protective level of antibody immune responses, including hemagglutination-inhibition titers, viral neutralization activity, and broad HA- and NA-inhibiting antibody titers against past and new H1N1 viruses. In conclusion, this study provides efficacy and safety profiles of IVR-190mg for further clinical study and shows that this vaccine without a glycan shield has great potential to be safe and protective against H1N1 variants.

GMP implementation of a hybrid continuous manufacturing process for a recombinant non-mAb protein-A case study.

Advances in manufacturing technology coupled with the increased potency of new biotherapeutic modalities have created an external environment where continuous manufacturing (CM) can address a growing need. Amgen has successfully implemented a hybrid CM process for a commercial lifecycle program. In this process, the bioreactor, harvest, capture column, and viral inactivation/depth filtration unit operations were integrated together in an automated, continuous module, while the remaining downstream unit operations took place in stand-alone batch mode. CM operations are particularly suited for so-called "high mix, low volume" manufacturing plants, where a variety of molecules are manufactured in relatively low volumes. The selected molecule fit this mold and was manufactured in a low-capital micro-footprint suite attached to an existing therapeutic production facility. Use of a hybrid process within an already operating facility required less capital and minimized complexity. To enable this hybrid CM process, an established fed-batch process was converted to a perfusion process with continuous harvest. Development efforts included both process changes and the generation of a novel cell line adapted to long-term perfusion. Chromatography resins were updated, and purification processes adapted to handle variable inputs due to the fluctuations in harvest titer from the lengthy production process. A novel automated single-use (SU) viral inactivation (VI) skid was introduced, which entailed the development of a robust pH verification and alarm system, along with procedures for product isolation to allow discard of specific cycles. The CM process demonstrated consistent performance, meaning it met predefined performance criteria (including product quality attributes, or PQAs) when operated within established process parameters and manufactured according to applicable procedures. Using a 75% reduction in scale, it resulted in a five-fold reduction in process media and buffer usage, a fifteen-fold increase in mass per thaw, and an overall process productivity increase of 45-fold (as measured by grams drug substance per liter per day.) The hybrid CM process also enabled increased material demand to be met with no change in cost of goods manufactured or plant capacity, due to the repurposing of existing facility space and the flexible duration of the hybrid CM harvest. Overall, the success of the hybrid CM platform represents an exciting opportunity to reduce costs and increase process efficiency in industry.

Prevalence, tropism, and activity of cutavirus in circulating blood lymphocytes, stool, and skin biopsy specimens of patients with cutaneous T-cell lymphoma and parapsoriasis en plaques.

A significant association has been established between a newly emerging human parvovirus, cutavirus (CuV), and cutaneous T-cell lymphoma/mycosis fungoides (CTCL/MF) and its precursor parapsoriasis en plaques (PP). CTCL is a heterogeneous group of skin malignancies of T cells, the cause of which remains unknown. This study aimed to determine the activity, spread, and cell tropism of the skin-persistent CuV. CuV DNA was detected in both skin biopsies (6/20, 30%) and peripheral blood mononuclear cells (PBMCs) (4/29, 13.8%) from 49 CTCL/MF or PP patients, while none from 33 patients with any other type of skin disease or healthy subjects harbored CuV DNA. CuV DNA persisted in the skin or PBMCs for up to 15 years, despite circulating CuV-specific IgG. Spliced CuV mRNA was expressed in skin, indicating viral activity. Also, both of two available stool samples contained encapsidated CuV genomes, suggesting that the patients excrete infectious virus into the environment. Finally, CuV was observed to target circulating and skin-resident CD4 + T cells and some skin keratinocytes and macrophages. This is especially intriguing as malignant T cells in CTCL develop from CD4 + T cells. Hence, CuV should be further investigated for the overall role it plays in the complex tumor microenvironment of CTCL/MF.

Effects of Strain Differences, Humidity Changes, and Saliva Contamination on the Inactivation of SARS-CoV-2 by Ion Irradiation.

One of the methods to inactivate viruses is to denature viral proteins using released ions. However, there have been no reports detailing the effects of changes in humidity or contamination with body fluids on the inactivation of viruses. This study investigated the effects of humidity changes and saliva contamination on the efficacy of SARS-CoV-2 inactivation with ions using multiple viral strains. Virus solutions with different infectious titers were dropped onto a circular nitrocellulose membrane and irradiated with ions from 10 cm above the membrane. After the irradiation of ions for 60, 90, and 120 min, changes in viral infectious titers were measured. The effect of ions on virus inactivation under different humidity conditions was also examined using virus solutions containing 90% mixtures of saliva collected from 10 people. A decrease in viral infectivity was observed over time for all strains, but ion irradiation further accelerated the decrease in viral infectivity. Ion irradiation can inactivate all viral strains, but at 80% humidity, the effect did not appear until 90 min after irradiation. The presence of saliva protected the virus from drying and maintained infectiousness for a longer period compared with no saliva. In particular, the Omicron strain retained its infectivity titer longer than the other strains. Ion irradiation demonstrated a consistent reduction in the number of infectious viruses when compared to the control across varying levels of humidity and irradiation periods. This underscores the notable effectiveness of irradiation, even when the reduction effect is as modest as 50%, thereby emphasizing its crucial role in mitigating the rapid dissemination of SARS-CoV-2.