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1.
Free Radical Biology and Medicine ; 2022.
Article in English | ScienceDirect | ID: covidwho-2004074

ABSTRACT

Plasma medicine is a developing field that utilizes the effects of cold physical plasma on biological substrates for therapeutic purposes. Approved plasma technology is frequently used in clinics to treat chronic wounds and skin infections. One mode of action responsible for beneficial effects in patients is the potent antimicrobial activity of cold plasma systems, which is linked to their unique generation of a plethora of reactive oxygen and nitrogen species (ROS). During the SARS-CoV-2 pandemic, it became increasingly clear that societies need novel ways of passive and active protection from viral airway infections. Plasma technology may be suitable for superficial virus inactivation. Employing an optimized neon-driven micro plasma jet, treatment time-dependent ROS production and cytotoxic effects to different degrees were found in four different human cell lines with respect to their metabolic activity and viability. Using the murine hepatitis virus (MHV), a taxonomic relative of human coronaviruses, plasma exposure drastically reduced the number of infected murine fibroblasts by up to 3000-fold. Direct plasma contact (conductive) with the target maximized ROS production, cytotoxicity, and antiviral activity compared to non-conductive treatment with the remote gas phase only. Strikingly, antioxidant pretreatment reduced but not abrogated conductive plasma exposure effects, pointing to potential non-ROS-related mechanisms of antiviral activity. In summary, an optimized micro plasma jet showed antiviral activity and cytotoxicity in human cells, which was in part ROS-dependent. Further studies using more complex tissue models are needed to identify a safe dose-effect window of antiviral activity at modest toxicity.

2.
Am J Infect Control ; 50(8): 906-908, 2022 08.
Article in English | MEDLINE | ID: covidwho-2000223

ABSTRACT

Using the Murine Hepatitis Virus (MHV) A59 coronavirus as a SARS-CoV-2 animal surrogate, we validated that methylene blue (MB) in combination with sunlight exposure is a robust, fast, and low-cost decontamination method for PPE that should be added to the toolbox of practical pandemic preparedness.


Subject(s)
COVID-19 , Methylene Blue , Animals , COVID-19/prevention & control , Disinfection/methods , Mice , Personal Protective Equipment , SARS-CoV-2 , Sunlight
3.
Journal of General Internal Medicine ; 37:S453, 2022.
Article in English | EMBASE | ID: covidwho-1995835

ABSTRACT

CASE: 66yo woman with a past medical history of hypertension and monoclonal gammopathy of undetermined significance was sent from clinic in winter for 4 days of worsening fevers and sinus congestion unrelieved by over-the- counter medications. COVID and flu negative. Patient has had no sick contacts or recent travel and has pet cats but no recent scratches. Initial chest x-ray showed no acute processes, but patient was continuing to have fevers up to 103 with mild dyspnea and chills so a CT chest was completed which showed ground glass opacities in the right middle lobe. Blood and sputum cultures were obtained, and patient was started on ceftriaxone and azithromycin for community acquired pneumonia. Urine strep and legionella antigens were also acquired, both negative. Over the next two days, she continued to have high fevers and chills at nights with leukocytosis, thrombocytopenia, hyponatremia, and notable worsening of mild elevation of liver enzymes on admission. Cultures were negative and patient had no other indication of an infection aside from the cyclical fevers therefore empiric doxycycline was added for coverage of atypical infections. Over the next two days, she continued to have nightly fevers up to 103 so ID was consulted for fever of unknown origin. On repeat exposure assessment, patient revealed that she lived with multiple animals including cats, dogs, parakeets, chickens, geese and a pony. Patient was continued on doxycycline while additional lab tests were sent for atypical infections including Rickettsia typhi, Coxiella brunetti (Q fever), and Brucella spp given patient's history of exposure to multiple animals at home. Patient was discharged on doxycycline after being afebrile for 48hrs with declining white count and liver enzymes. Lab results confirmed the diagnosis with high titers for Rickettsia typhi IgG and IgM. IMPACT/DISCUSSION: This case illustrates an atypical presentation of murine typhus with pneumonia in winter. There are several key teaching points in this case: 1. Ricketssia typhi infections have largely nonspecific symptoms therefore it should should be included in differential diagnoses of febrile illnesses with thrombocytopenia and elevated liver enzymes 2. Although a complete history is acquired on admission, it is important to revisit and review information again when a clinical diagnosis has not been established 3. Defeverscence after starting doxycycline can take anywhere from 4 to 66hrs so fevers during this timeframe is not an indication of failure of therapy CONCLUSION: Murine typhus presents with non-specific symptoms so it should be included in the differential diagnosis of patients with fevers of unknown origin with potential exposure to flea-bearing animals. The optimal therapy is doxycycline 100mg twice a day for seven days. Patients should also be advised to treat their animals for fleas to prevent recurrent infections.

4.
Cancer Research ; 82(12), 2022.
Article in English | EMBASE | ID: covidwho-1986480

ABSTRACT

High redox potential reactive oxygen and nitrogen species (ROS/RNS), such as O2 free radicals, superoxide, and hypochlorous acid, generated by activities of the NADPH oxidase-2 (NOX2)/myeloperoxidase (MPO) axis and related enzymes, are key effector molecules of innate immunity in physiological and diseased inflammatory states. Other lower energy species (H2O2, NO) provide adjuvant signaling functions. NOX2- and MPO-derived high energy radicals are known to oxidize naphthol species, wherein the naphthol products bind to proximate proteins and activated myeloid cells. Herein, we present 4-[18F]fluoro-1-naphthol ([18F]4FN), a novel redox-tuned radiopharmaceutical that selectively detects by positron emission tomography (PET) high energy radicals produced by activated innate immunity. The products of human MPO plus H2O2 , but not H2O2 alone, rapidly and completely oxidized [18F]4FN. All-trans-retinoic acid-differentiated HL-60 'neutrophil-like' human cells activated with phorbol-12-myristate-13-acetate (PMA) retained [ 18F]4FN 5-fold over unstimulated cells. 4-ABAH, an MPO-specific inhibitor, or DPI, a broad oxidase inhibitor, blocked cellular retention by >95%. [18F]4FN PET/CT imaging readily discriminated foci of inflammation in vivo in three distinct murine models of acute inflammation: endotoxin-induced whole-body toxic shock, PMA-induced mild contact dermatitis of the ear, and lipopolysaccharide (LPS)-induced ankle arthritis. Mechanistically, in mice in vivo, 4-ABAH reduced inflammationinduced [18F]4FN retention, and Cybb-/- (Nox2-/-) gene-deletion strongly and significantly abrogated PMA-induced [18F]4FN retention. Thus, [18F]4FN shows promise as a robust redox-tuned reporter for imaging activation states of innate immunity by PET/CT, is ready for translation. [18F]4FN PET imaging may find application in a variety of inflammatory states associated with cancer therapy, immunotherapy-related adverse events, as well as other diseases, including arthritis, hepatitis, atherosclerosis, COVID-19, as well as up-staging and monitoring multi-organ inflammation.

5.
Embase; 2022.
Preprint in English | EMBASE | ID: ppcovidwho-342521

ABSTRACT

Interferon gamma may be a potential adjuvant immunotherapy for COVID-19 patients. In this work, we assessed gene expression profiles associated with the IFN-γ pathway in response to SARS-CoV-2 infection. Employing a case-control study from SARS-CoV-2 positive and negative patients, we identified IFN-γ-associated pathways to be enriched in positive patients. Bioinformatics analyses showed upregulation of MAP2K6, CBL, RUNX3, STAT1 and JAK2 in COVID-19 positive vs. negative patients. A positive correlation was observed between STAT1/JAK2, which varied alongside the patient’s viral load. Expression of MX1, MX2, ISG15 and OAS1 (4 well-known IFN-stimulated genes (ISGs)) displayed upregulation in COVID-19 positive vs. negative patients. Integrative analyses showcased higher levels of ISGs which were associated with increased viral load and STAT1/JAK2 expression. Confirmation of ISGs up-regulation was performed in vitro using the A549 lung cell line treated with Poly(I:C), a synthetic analog of viral double-stranded RNA;and in different pulmonary human cell lines and ferret tracheal biopsies infected with SARS-CoV-2. A preclinical murine model of coronavirus infection confirmed findings displaying increased ISGs in the liver and lungs from infected mice. Altogether, these results demonstrate the role of IFN-γ and ISGs in response to SARS-CoV-2 infection, highlighting alternative druggable targets that can boost the host response.

6.
Embase; 2022.
Preprint in English | EMBASE | ID: ppcovidwho-342113

ABSTRACT

To date, there is no effective oral antiviral against SARS-CoV-2 that is also anti-inflammatory. Herein, we show that the mitochondrial antioxidant mitoquinone/mitoquinol mesylate (Mito-MES), a dietary supplement, has potent antiviral activity against SARS-CoV-2 and its variants of concern in vitro and in vivo. Mito-MES had nanomolar in vitro antiviral potency against the Beta and Delta SARS-CoV-2 variants as well as the murine hepatitis virus (MHV-A59). Mito-MES given in SARS-CoV-2 infected K18-hACE2 mice through oral gavage reduced viral titer by nearly 4 log units relative to the vehicle group. We found in vitro that the antiviral effect of Mito-MES is attributable to its hydrophobic dTPP+ moiety and its combined effects scavenging reactive oxygen species (ROS), activating Nrf2 and increasing the host defense proteins TOM70 and MX1. Mito-MES was efficacious reducing increase in cleaved caspase-3 and inflammation induced by SARS-CoV2 infection both in lung epithelial cells and a transgenic mouse model of COVID-19. Mito-MES reduced production of IL-6 by SARS-CoV-2 infected epithelial cells through its antioxidant properties (Nrf2 agonist, coenzyme Q10 moiety) and the dTPP moiety. Given established safety of Mito-MES in humans, our results suggest that Mito-MES may represent a rapidly applicable therapeutic strategy that can be added in the therapeutic arsenal against COVID-19. Its potential long-term use by humans as diet supplement could help control the SARS-CoV-2 pandemic, especially in the setting of rapidly emerging SARS-CoV-2 variants that may compromise vaccine efficacy.

7.
Protein J ; 2022 Aug 06.
Article in English | MEDLINE | ID: covidwho-1982259

ABSTRACT

Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is the most frequently used enzyme in molecular biology for cDNA synthesis. To date, reverse transcription coupled with Polymerase Chain Reaction, known as RT-PCR, has been popular as an excellent approach for the detection of SARS-CoV-2 during the COVID-19 pandemic. In this study, we aimed to improve the enzymatic production and performance of MMLV-RT by optimizing both codon and culture conditions in E. coli expression system. By applying the optimized codon and culture conditions, the enzyme was successfully overexpressed and increased at high level based on the result of SDS-PAGE and Western blotting. The total amount of MMLV-RT has improved 85-fold from 0.002 g L-1 to 0.175 g L-1 of culture. One-step purification by nickel affinity chromatography has been performed to generate the purified enzyme for further analysis of qualitative and quantitative RT activity. Overall, our investigation provides useful strategies to enhance the recombinant enzyme of MMLV-RT in both production and performance. More importantly, the enzyme has shown promising activity to be used for RT-PCR assay.

8.
Chemical Engineering Journal ; : 138562, 2022.
Article in English | ScienceDirect | ID: covidwho-1977104

ABSTRACT

Metal-organic frameworks (MOFs) featuring composition and bandstructure diversity, are an emerging class of photoresponsive disinfectants. In this study, we demonstrated the superiority of core-shell arranged photoactive MOFs (prussian blue (PB) and zeolitic imidazolate framework (ZIF-8)) for pathogen inactivation in terms of biocidal efficiency and broad-spectrum sensitivity. Reactive oxygen species (ROS) production was significantly promoted after the integration of PB due to the photosensitization effect and initiation of in situ Fenton reaction. Favorably, another inactivation channel was also opened owing to the unique photothermal effect of PB. Attributed to the facilitated ROS intracellular penetration by heat, the composite outperforms not only individual component but anatase TiO2 in pathogen elimination. Specifically, the Staphylococcus aureus (S. aureus) inactivation efficiency of the composite (6.6 log) is 2, 1.8 and 5.1 times higher than that of PB (3.3 log), ZIF-8 (3.7 log) and TiO2 (1.3 log) over 45 min of simulated sunlight illumination. Significantly, the infectivity of Bacillus anthracis and murine coronavirus in droplets on composite-coated filter surface could be greatly reduced (approximately 3 log reduction in colony number/coronavirus titer) within few minutes of solar exposure, indicative of the great potential of MOF composites toward life-threatening microbial infection prevention.

9.
Int J Mol Sci ; 23(13)2022 Jun 29.
Article in English | MEDLINE | ID: covidwho-1934130

ABSTRACT

The effect of the antiviral peptide TAT-I24 on viral gene expression in cells infected with murine cytomegalovirus (MCMV) was investigated. The expression of immediate-early, early and late genes was highly induced upon infection with MCMV. In the presence of the peptide, the expression of all tested genes was sustainably reduced to a similar extent, independent of whether they were immediate-early, early or late genes. In contrast, the expression of host genes, such as NF-κB inhibitor alpha (Nfkbia), interferon-induced protein with tetratricopeptide repeats 1 (Ifit1), chemokine (C-X-C motif) ligand 10 (Cxcl10), chemokine (C-C motif) ligand 7 (Ccl7) and chemokine (C-C motif) ligand 5 (Ccl5), which are induced early upon virus infection, was only transiently suppressed in peptide-treated cells. The expression of other host genes which are affected by MCMV infection and play a role in endoplasmic reticulum stress or DNA-damage repair was not inhibited by the peptide. A combination of TAT-I24 with the nucleoside analogue cidofovir showed enhancement of the antiviral effect, demonstrating that viral replication can be more efficiently inhibited with a combination of drugs acting at different stages of the viral life-cycle.


Subject(s)
Muromegalovirus , Animals , Antiviral Agents/pharmacology , Gene Expression , Ligands , Mice , Muromegalovirus/genetics , Peptides/pharmacology , Virus Replication
10.
American Journal of Respiratory and Critical Care Medicine ; 205(1), 2022.
Article in English | EMBASE | ID: covidwho-1927733

ABSTRACT

Rationale: While oxygen therapy is standard for patients with pneumonia, a potential for increased oxidant damage exists. Understanding how oxygen therapy impacts inflammatory lung injury with SARS-CoV-2 infection (COVID-19) and related viruses will inform patient management. We investigated the effects of fractional inspired oxygen concentrations (FiO2s) of 30 or 60% in a mouse hepatitis virus-1 (MHV-1) model of acute lung injury we developed in A/J mice. Methods: MHV-1, a ß-coronavirus like SARS-CoV-2, can be studied at Biosafety Level-2. Intratracheal installation of MHV-1 in our model produces inflammatory lung injury, progressive arterial desaturation, and lethality over 14d, similar to COVID-19. Using this model, we compared outcomes in animals exposed in sealed chambers to atmospheric FiO2s of 21, 30 or 60% beginning 2h after of MHV-1 challenge and continuing for up to 14d. In each of three experiments, MHV-1 challenged animals were randomized to receive FiO2s of 21, 30 or 60% (10 animals per FiO2 group per experiment, 90 animals total). In another experiment, 30 animals challenged with noninfected viral culture medium were randomized to the same three FiO2s. Animals were observed for up to 14d. Results: Compared to FiO2 21%, chambers with FiO2 30 and 60% had similar humidities and temperatures but slightly lower carbon dioxide levels (CO2, p≤0.05) but all chamber CO2s were in the range of 400-2000 ppm. Compared to animals surviving with FiO2 21% in each of the three experiments [#survivors/#total animals (%)] [1/10 (10%);5/10 (50%);4/10 (40%)], and their survival times (Figure-1), survival was reduced in respective experiments with FiO2 30% [1/10 (10%);2/10 (20%);0/10 (0%)] and FiO2 60% [0/10 (0%);0/10 (0%);0/10 (0%)]. Patterns of survival were similar comparing the three experiments for each FiO2 and when combined, there was a significant dose-related difference in survival across the three FiO2's (p<0.0001) (Figure-1). Compared to FiO2 21%, survival decreased with FiO2 30% (p=0.06) and more so with FiO2 60% (p<0.0001) (log-rank test with Dunnett-Hsu adjustment). All animals challenged with noninfected viral culture medium and exposed similarly to FiO2s 21, 30 or 60% (n=10 per group) survived except one 30% animal that died at 12d despite appearing well. Conclusions: FiO2s of 30 and 60% that are considered therapeutic and relatively safe clinically, markedly worsened survival in mice with MHV-1 pneumonia, a ß-coronavirus like SARS-CoV-2. These findings emphasize the need to better understand how oxygen therapy impacts the pathogenesis of SARS-CoV-2 in patients.

11.
Metallomics ; 14(7)2022 07 20.
Article in English | MEDLINE | ID: covidwho-1901217

ABSTRACT

Severe acute respiratory syndrome (SARS) is a viral respiratory infection caused by human coronaviruses that include SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). Although their primary mode of transmission is through contaminated respiratory droplets from infected carriers, the deposition of expelled virus particles onto surfaces and fomites could contribute to viral transmission. Here, we use replication-deficient murine leukemia virus (MLV) pseudoviral particles expressing SARS-CoV-2, SARS-CoV, or MERS-CoV Spike (S) protein on their surface. These surrogates of native coronavirus counterparts serve as a model to analyze the S-mediated entry into target cells. Carboxymethyl cellulose (CMC) nanofibers that are combined with copper (Cu) exhibit strong antimicrobial properties. S-pseudovirions that are exposed to CMC-Cu nanoparticles (30 s) display a dramatic reduction in their ability to infect target Vero E6 cells, with ∼97% less infectivity as compared to untreated pseudovirions. In contrast, addition of the Cu chelator tetrathiomolybdate protects S-pseudovirions from CMC-Cu-mediated inactivation. When S-pseudovirions were treated with a hydrogen peroxide-based disinfectant (denoted SaberTM) used at 1:250 dilution, their infectivity was dramatically reduced by ∼98%. However, the combined use of SaberTM and CMC-Cu is the most effective approach to restrict infectivity of SARS-CoV-2-S, SARS-CoV-S, and MERS-CoV-S pseudovirions in Vero E6 cell assays. Together, these results show that cellulosic Cu nanoparticles enhance the effectiveness of diluted SaberTM sanitizer, setting up an improved strategy to lower the risk of surface- and fomite-mediated transmission of enveloped respiratory viruses.


Subject(s)
COVID-19 , Disinfectants , Middle East Respiratory Syndrome Coronavirus , Nanoparticles , Copper/pharmacology , Disinfectants/pharmacology , Humans , Hydrogen Peroxide/pharmacology , Mice , Middle East Respiratory Syndrome Coronavirus/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism
12.
J Appl Microbiol ; 2022 Jun 15.
Article in English | MEDLINE | ID: covidwho-1886681

ABSTRACT

AIMS: Fresh produce is often a vehicle for the transmission of foodborne pathogens such as human norovirus. Thus, it is recommended to wash the surface of produce before consumption, and one of the most common ways to wash produce is by rinsing under running tap water. This study determined the effectiveness of removal of human coronavirus-OC43 (HCoV-OC43), as a surrogate for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and murine norovirus-1 (MNV-1), as a surrogate for human norovirus, from contaminated lettuce, apples and cucumbers. METHODS AND RESULTS: The produce surfaces were artificially inoculated in conjunction with faecal material to represent natural contamination. Rinsing under tap water for 10 s at 40 ml/s removed 1.94 ± 0.44, 1.42 ± 0.00 and 1.42 ± 0.42 log of HCoV-OC43 from apple, cucumber and lettuce respectively. The same washing technique removed 1.77 ± 0.17, 1.42 ± 0.07 and 1.79 ± 0.14 log of MNV-1 from apple, cucumber and lettuce respectively. This washing technique was effective at reducing a significant amount of viral contamination, however, it was not enough to eliminate the entire contamination. There was no significant difference in the reduction of viral load between the two viruses, nor between the three surfaces tested in this study. CONCLUSIONS: Our data suggest that washing under tap water would be an efficient way of reducing the risk of foodborne viral transmission only if the level of contamination is less than 2 log PFU. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrates that running tap water was effective at reducing the amount of infectious HCoV-OC43 and MNV on produce surfaces, and washing produce continues to be an important task to perform prior to consumption to avoid infection by foodborne viruses, particularly for foods which are eaten raw.

13.
Journal of Urology ; 207(SUPPL 5):e415, 2022.
Article in English | EMBASE | ID: covidwho-1886501

ABSTRACT

INTRODUCTION AND OBJECTIVE: Neurodegenerative diseases, such as multiple sclerosis (MS), often lead to the development of neurogenic lower urinary tract symptoms (LUTS). We previously characterized neurogenic bladder dysfunction in a mouse model of MS induced by a coronavirus, mouse hepatitis virus (MHV). The objective of this study was to identify genes and pathways linking neuroinflammation in the central nervous system with urinary bladder dysfunction to enhance our understanding of the mechanisms underlying LUTS in demyelinating diseases. METHODS: Adult C57BL/6 male mice (N=12) received either an intracranial injection of MHV (6,000 PFU) or sterile saline (control). The lumbosacral (L6-S2) spinal cord (SC) segments and urinary bladders were collected during acute infection stage (week 1) and at the first peak of demyelination (week 4) after inoculation with the virus. Total RNA was isolated and analyzed using Nanostring nCounter Neuroinflammation panel. The expression levels of 770 genes associated with neuroinflammation were assessed and compared between the specimens. RESULTS: Transcriptome analysis of SC specimens confirmed a significantly increased expression of 132 genes in MHV mice (tens to hundreds fold change) involved in the regulation of astrocyte, microglia and oligodendrocyte functions, neuroinflammation and immune responses. Out of 132 genes up-regulated in the SC, only 2 genes (siglec1, 46-fold in the SC, 2.6-fold at 1 week and 1.8-fold at 4 weeks in the bladder;and zbp1, 568-fold in the SC, 2.8-fold at 1 week and 2.2-fold at 4 weeks in the bladder) were up-regulated in the urinary bladders of MHV-infected mice. Additionally, two genes were significantly up-regulated (ttr, 2.2-fold at 1week and 1.7-fold at 4 weeks;and ms4a4a, 2.3-fold at 1week and 1.6-fold at 4 weeks), and two were down-regulated (asb2, -1.8-fold at 1 week and -1.6-fold at 4 weeks, and myct1, -1.7-fold at 1week and -1.6-fold at 4 weeks) exclusively in the urinary bladders of MHV mice. CONCLUSIONS: Two genes, siglec1 (encodes type 1 transmembrane protein, expressed in microglia and macrophages, promotes neuroinflammation) and zbp1 (encodes a Z-DNA binding protein, plays role in the innate immune response) link the development of neuroinflammation in the central nervous system with neurogenic changes in the urinary bladders of MHV-infected mice. Further research is needed to establish a functional relationship between expression of these genes and neurogenic LUTS.

14.
Topics in Antiviral Medicine ; 30(1 SUPPL):88-89, 2022.
Article in English | EMBASE | ID: covidwho-1881034

ABSTRACT

Background: Rapid and large-scale deployment of COVID-19 mRNA vaccines highlights the potential utility of developing nucleic acid vaccines (such as RNA and DNA vaccines) against infectious diseases, including HIV-1. However, as compared to SARS-CoV-2, HIV-1 pose some unique challenges-induction of neutralizing antibodies (NAbs) against HIV-1 (frequently a correlate of protection) requires presentation of trimeric and highly conformational epitopes to the immune system, and whether nucleic acid vaccines can enable direct in vivo production of antigens that retain critical antigenic profile has not yet been elucidated. Additionally, it was previously reported that Tier 2 NAbs cannot be induced in mice due to a lack of antibody repertoire, and vaccine studies were suggested to be performed in larger mammals such as rabbits/NHPs, inadvertently slowing down and increasing the costs of preclinical HIV-1 vaccine studies. Methods: In our study, we used the Antigen Conformation Tracing In Vivo by ELISA (ACTIVE) assay developed in house to characterize antigenic profiles of vaccines produced in vivo (from transfected muscle tissues). We analyzed induced cellular responses, using stimulation with overlapping peptides followed by intracellular cytokine staining and IFN-g ELIspot assays. We analyzed induced humoral responses by using both binding ELISA assays and TZM-BL based neutralizing assays, and attempted to map induced NAb epitopes by engineering selectively mutated pseudovirus. We performed antigen-specific B-cell sorting, and used the 10x genomics pipeline to characterize antibody sequences of proliferating B-cell clones. Results: We confirmed that in vivo produced vaccines retained key trimeric conformational epitopes and glycan profiles. Compared to protein vaccination, DNA vaccination uniquely and strongly induced both TFH, CD4+, CD8+ T-cell responses, and Tier 2 NAbs mapped to a previously unreported Env C3/V5 epitope. 5 unique NAbs were isolated, and confirmed to bind to the epitope using a Cryo-EM structure of NAb-MD39 complex at 3.8Å resolution. Conclusion: Our study confirmed that with appropriate vaccine delivery technology, murine models can be appropriately used for HIV-1 vaccine studies aimed at generating NAb responses. In addition, beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.

15.
Topics in Antiviral Medicine ; 30(1 SUPPL):181, 2022.
Article in English | EMBASE | ID: covidwho-1880500

ABSTRACT

Background: PRTX007 is a prodrug of PRX034, a novel TLR7 agonist, for treatment of respiratory viral diseases including SARS-CoV-2. PRX034 activates plasmacytoid dendritic cells to preferentially synthesize poly sub-type interferons while minimizing NFκΒ-mediated proinflammatory factors. Pre-clinical studies have demonstrated decoupling of these two normally linked processes in human PBMCs in vitro and in cynomolgus monkeys in vivo. Antiviral activity is preserved, as demonstrated by inhibition of cytopathicity and viral replication in RNA viruses by conditioned media from PRX034-treated PBMCs. Efficacy and safety have been demonstrated in a murine model of RSV infection. An interim analysis of the first-in-human study in healthy volunteers is presented here. Methods: Phase I, single-center, prospective, randomized, double-blind study of 8 single-ascending dose (SAD) cohorts and 4 multiple-ascending dose (MAD) cohorts of PRTX007 administered orally to healthy adult subjects. Primary objective was safety and tolerability. Secondary objectives were to assess pharmacokinetics (PK) and pharmacodynamics (PD). This interim analysis focuses on the SAD portion of the study over 8 dose levels from 50-600mg. Safety through all SAD cohorts, PK up to the 500 mg cohort, PD up to the 400 mg cohort are presented. The MAD cohort safety data from 3 of 4 planned cohorts are also presented. Results: Treatment-related adverse events (AEs) include mild to moderate headache. Severity or frequency is not dose related, is of short duration and is not associated with systemic symptoms. One participant in the 400 mg cohort had asymptomatic mild sinus tachycardia, attributed to PRTX007. Two subjects in the second MAD cohort at 300 mg had mild increases in ALT that rapidly resolved after treatment. PK analysis demonstrated efficient conversion of PRTX007 to PRX034 with systemic exposure to drug and prodrug increasing proportionally with dose. Duration of systemic exposure to PRX034 at pharmacologically active levels is consistent with activation of innate immune response without counter-regulation (Panel A). Exposure-dependent PD response measured by induction of interferon stimulated genes in the blood is observed at drug AUC > 4,300 hr∗ng/ml (Panel B);duration of induction >24 hours (Panel C). Conclusion: Interim analysis of PRTX007 demonstrates a favorable safety profile with dose-dependent systemic exposure and demonstrated activation of innate immune response.

16.
Pathogens ; 11(5)2022 May 02.
Article in English | MEDLINE | ID: covidwho-1875721

ABSTRACT

Using an effective natural virucidal substance may be a feasible approach for preventing food-borne viral contamination. Here, the virucidal efficacy of theaflavins (TFs)-enriched tea leaf extract (TY-1) against feline calicivirus (FCV) and murine norovirus (MNV), surrogates of human norovirus (HuNoV), was evaluated. The virus solutions were mixed with various dosages of TY-1 and incubated at 25 °C for different contact times. TY-1 reduced the viral titer of both surrogate viruses in a time- and dosage-dependent manner. A statistically significant reduction in the viral titer of FCV by 5.0 mg/mL TY-1 and MNV by 25.0 mg/mL TY-1 was observed in 10 s and 1 min, respectively. Furthermore, TY-1 reduced the viral titer of FCV and MNV on the dry surface in 10 min. The multiple compounds in TY-1, including TFs and catechins, contributed to its overall virucidal activity. Furthermore, the effect of TY-1 on viral proteins and genome was analyzed using Western blotting, RT-PCR, and transmission electron microscopy. TY-1 was found to promote the profound disruption of virion structures, including the capsid proteins and genome. Our finding demonstrates the potential of using TY-1 as a nature-derived disinfectant in food processing facilities and healthcare settings to reduce viral load and HuNoV transmission.

17.
J Virol ; 96(11): e0036422, 2022 06 08.
Article in English | MEDLINE | ID: covidwho-1854234

ABSTRACT

Effective broad-spectrum antivirals are critical to prevent and control emerging human coronavirus (hCoV) infections. Despite considerable progress made toward identifying and evaluating several synthetic broad-spectrum antivirals against hCoV infections, a narrow therapeutic window has limited their success. Enhancing the endogenous interferon (IFN) and IFN-stimulated gene (ISG) response is another antiviral strategy that has been known for decades. However, the side effects of pegylated type-I IFNs (IFN-Is) and the proinflammatory response detected after delayed IFN-I therapy have discouraged their clinical use. In contrast to IFN-Is, IFN-λ, a dominant IFN at the epithelial surface, has been shown to be less proinflammatory. Consequently, we evaluated the prophylactic and therapeutic efficacy of IFN-λ in hCoV-infected airway epithelial cells and mice. Human primary airway epithelial cells treated with a single dose of IFN-I (IFN-α) and IFN-λ showed similar ISG expression, whereas cells treated with two doses of IFN-λ expressed elevated levels of ISG compared to that of IFN-α-treated cells. Similarly, mice treated with two doses of IFN-λ were better protected than mice that received a single dose, and a combination of prophylactic and delayed therapeutic regimens completely protected mice from a lethal Middle East respiratory syndrome CoV (MERS-CoV) infection. A two-dose IFN-λ regimen significantly reduced lung viral titers and inflammatory cytokine levels with marked improvement in lung inflammation. Collectively, we identified an effective regimen for IFN-λ use and demonstrated the protective efficacy of IFN-λ in MERS-CoV-infected mice. IMPORTANCE Effective antiviral agents are urgently required to prevent and treat individuals infected with SARS-CoV-2 and other emerging viral infections. The COVID-19 pandemic has catapulted our efforts to identify, develop, and evaluate several antiviral agents. However, a narrow therapeutic window has limited the protective efficacy of several broad-spectrum and CoV-specific antivirals. IFN-λ is an antiviral agent of interest due to its ability to induce a robust endogenous antiviral state and low levels of inflammation. Here, we evaluated the protective efficacy and effective treatment regimen of IFN-λ in mice infected with a lethal dose of MERS-CoV. We show that while prophylactic and early therapeutic IFN-λ administration is protective, delayed treatment is detrimental. Notably, a combination of prophylactic and delayed therapeutic administration of IFN-λ protected mice from severe MERS. Our results highlight the prophylactic and therapeutic use of IFN-λ against lethal hCoV and likely other viral lung infections.


Subject(s)
Antiviral Agents , Coronavirus Infections , Interferons , Middle East Respiratory Syndrome Coronavirus , Animals , Antiviral Agents/pharmacology , Coronavirus Infections/drug therapy , Coronavirus Infections/immunology , Humans , Interferons/pharmacology , Mice
18.
Respirology ; 27(SUPPL 1):73, 2022.
Article in English | EMBASE | ID: covidwho-1816629

ABSTRACT

Introduction: The induction of regulatory T cells (Tregs) is indicated as a potential therapeutic strategy in inflammatory lung diseases including, asthma, viral-induced pneumonia, viral-induced acute lung injury (ALI), severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and SARSCoV- 2-induced ALI. We previously identified that components of the bacteria Streptococcus pneumoniae (T + P) are able to increase Tregs to suppress experimental allergic airways disease, however, this mechanism of suppression and therapy has not been examined in ALI. Methods: We established a murine model of ALI using aerosolized LPS (100 μg/ml) in BALB/c mice. ALI was measured by the presence of neutrophils in the airways up to 96 hours post-exposure, and Tregs and dendritic cells were assessed by flow cytometry. To assess the therapeutic of T + P in ALI and the mechanisms involved, the combination was administered prior to LPS exposure in the absence or presence of anti-CD25. Results: Treatment with T + P significantly reduced total airway inflammation and suppressed the neutrophil chemokine C-X-C motif chemokine ligand 1 (Cxcl1) compared to Saline+LPS alone in experimental ALI. The numbers of Tregs were reduced in experimental ALI model and were restored by T + P treatment. Depletion of Tregs with anti- CD25 confirmed that the suppressive effects of T + P on ALI was through the induction of Tregs. Conclusion: Treatment with S. pneumoniae components T + P suppresses neutrophilic inflammation in ALI through immunoregulatory mechanisms that involve Tregs and may be a novel treatment for ALI including in COVID-19.

19.
Open Forum Infectious Diseases ; 8(SUPPL 1):S391-S392, 2021.
Article in English | EMBASE | ID: covidwho-1746419

ABSTRACT

Background. First-generation COVID-19 vaccines are matched to spike protein of the Wuhan-H1 (WT) strain. Convalescent and vaccinee samples show reduced neutralization of SARS-CoV-2 variants of concern (VOC). Next generation DNA vaccines could be matched to single variants or synthetically designed for broader coverage of multiple VOCs. Methods. The synthetic consensus (SynCon®) sequence for INO-4802 SARSCoV-2 spike with focused RBD changes and dual proline mutations was codon-optimized (Figure 1). Sequences for wild-type (pWT) and B.1.351 (pB.1.351) were similarly optimized. Immunogenicity was evaluated in BALB/c mice. Pre-clinical efficacy was assessed in the Syrian Hamster model. Figure 1. Design Strategy for INO-4802 Results. INO-4802 induced potent neutralizing antibody responses against WT, B.1.1.7, P.1, and B.1.351 VOC in a murine model. pWT vaccinated animals showed a 3-fold reduction in mean neutralizing ID50 for the B.1.351 pseudotyped virus. INO-4802 immunized animals had significantly higher (p = 0.0408) neutralizing capacity (mean ID50 816.16). ID50 of pB.1.351 serum was reduced 7-fold for B.1.1.7 and significantly lower (p = 0.0068) than INO-4802 (317.44). INO-4802 neutralized WT (548.28) comparable to pWT. INO-4802 also neutralized P.1 (1026.6) (Figure 2). pWT, pB.1.351 or INO-4802 induced similar T-cell responses against all variants. INO-4802 skewed towards a TH1-response. All hamsters vaccinated with INO-4802 or pB.1.351 were protected from weight loss after B.1.351 live virus challenge. 4/6 pWT immunized hamsters were completely protected. pWT immunized hamsters neutralized WT (1090) but not B.1.351 (39.16). INO-4802 neutralized both WT (672.2) and B.1.351 (1121) (Figure 3). We observed higher increase of binding titers following heterologous boost with INO-4802 (3.6 - 4.4 log2-fold change) than homologous boost with pWT (2.0 - 2.4 log2 fold change) (Figure 4). Conclusion. Vaccines matching single VOCs, like pB.1.351 and pWT, elicit responses against the matched antigen but have reduced cross-reactivity. Presenting a pan-SARS-CoV-2 approach, INO-4802 may offer substantial advantages in terms of cross-strain protection, reduced susceptibility to escape mutants and non-restricted geographical use.

20.
Tier..rztliche Praxis. Ausgabe K, Kleintiere/Heimtiere ; 49(3):229-235, 2021.
Article in German | CAB Abstracts | ID: covidwho-1744014

ABSTRACT

These proceedings contain 25 papers from the 64th Annual Meeting of the Pathology Section of the German Veterinary Medical Association. Topics include tumour infiltrating lymphocytes in mammary carcinomas in domestic rabbits;what decides good or bad? - global gene expression analysis of the adenoma of the hepatoid perianal glands and adenocarcinoma the canine apocrine anal sac glands;the canine cutaneous histiocytoma - boring or perspective in immuno-oncology?;impact of antibiotic pretreatment on ventilator-induced lung injury: contradiction between histology and transcriptome analysis?;characterization of murine satellite glial cells of the dorsal root ganglia - a unique cell population with potential regenerative capacities;impact of antibiotic pretreatment on ventilator-induced lung injury: contradiction between histology and transcriptome analysis?;primary diffuse leptomeningeals oligodendrogliomatosis in a cat;pathomorphological studies of fibroadnexal dysplasia in dogs;pyogranulomatous inflammation in multiple Organs of a dog with evidence of Corynebacterium tuberculostearicum;ovary tumors in cats - overview of the examination material from 2009-2020 and case report of a recurrent dysgerminoma;atherosclerosis in the dog;spinal neuroenteric cyst in one Saint Bernard;MENX - an endogenous model for pseudohypoxic pheochromocytomas;molecular Level Evolution II: similarities of CLCA2 in sauropsids and mammals;in vivo detection of double-stranded Ribonucleic acid (RNA) as an early detection marker unclear viral infections using the example of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2) in experimental infected hamsters;the role of different mast cell subtypes in the context of intestinal carcinogenesis - a species-comparative approach;an underestimated treasure in paraffin - establishment of a global transcriptome analysis canine tumors from FFPE material based on QuantSeq 3' technology;well researched? - an approximation of the role of CLCA1 in joints through usage molecular databases;integration of digitized historical and cytopathology into an open source DICOM database and viewer system;3R 3D: skin model for the study of viral infections;CARD9 signaling promotes hippocampal neurogenesis and cytokine balance in a mouse model of virus-induced encephalitis;neuropathological changes after intranasal infection with Rift Valley fever virus - a murine model for human encephalitis;a T-cell a day keeps Theiler away - the influence non-reactive T-cells on the course of a Theiler virus infection in mice with C57BL/6 background;digitization in pathology - new opportunities and their obstacles;and specific features of satellite glial cells of dog and pig.

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