Repurposing of poly-gamma-glutamic acid, immunotherapeutic drug for cervical intraepithelial neoplasia 1, as covid-19 treatment

Covid-19 virus variants identified so far are due to viral genetic diversity, genetic evolution, and variable infectivity, suggesting that high infection rates and high mortality rates may be contributed by these mutations. And it has been reported that the targeting strategies for innate immunity should be less vulnerable to viral evolution, variant emergence and resistance. Therefore, the most effective solution to Covid-19 infection has been proposed to prevent and treat severe exacerbation of patients with moderate disease by enhancing human immune responses such as NK cell and T cell. In previous studies, we demonstrated for the first time that gamma-PGA induced significant antitumor activity and antiviral activity by modulating NK cell-mediated cytotoxicity. Especially intranasal administration of gamma-PGA was found to effectively induce protective innate and CTL immune responses against viruses and we found out that gamma-PGA can be an effective treatment for cervical intraepithelial neoplasia 1 through phase 2b clinical trial. In this study, the possibility of gamma-PGA as a Covid-19 immune modulating agent was confirmed by animal experiments infected with Covid-19 viruses. After oral administration of gamma-PGA 300mug/mouse once a day for 5 days in a K18-hACE2 TG mouse model infected with SARS-CoV-2 (NCCP 43326;original strain) and SARS-CoV-2 (NCCP 43390;Delta variant), virus titer and clinical symptom improvement were confirmed. In the RjHan:AURA Syrian hamster model infected with SARS-CoV-2 (NCCP 49930;Delta variant), 350 or 550 mug/head of gamma-PGA was administered orally for 10 days once a day. The virus for infection was administered at 5 x 104 TCID50, and the titer of virus and the improvement of pneumonia lesions were measured to confirm the effectiveness in terms of prevention or treatment. In the mouse model infected with original Covid-19 virus stain, the weight loss was significantly reduced and the survival rate was also improved by the administration of gamma-PGA. And gamma-PGA alleviated the pneumonic lesions and reduced the virus titer of lung tissue in mice infected with delta variant. In the deltavariant virus infected hamster model, gamma-PGA showed statistically significant improvement of weight loss and lung inflammation during administration after infection. This is a promising result for possibility of Covid-19 therapeutics along with the efficacy results of mouse model, suggesting gammaPGA can be therapeutic candidate to modulate an innate immune response for Covid-19.

Enabling mRNA medicine for brain tumors

mRNA is a new class of drugs that has the potential to revolutionize the treatment of brain tumors. Thanks to the COVID-19 mRNA vaccines and numerous therapy-based clinical trials, it is now clear that lipid nanoparticles (LNPs) are a clinically viable means to deliver RNA therapeutics. However, LNP-mediated mRNA delivery to brain tumors remains elusive. Over the past decade, numerous studies have shown that tumor cells communicate with each other via small extracellular vesicles, which are around 100 nm in diameter and consist of lipid bilayer membrane similar to synthetic lipidbased nanocarriers. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain tumors without undue toxicity. We synthesized LNPs using four components similar to the formulation used in the mRNA COVID19 vaccines (Moderna and Pfizer): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. For the in vitro screen, we tested ten classes of helper lipids based on their abundance in extracellular vesicle membranes, commercial availability, and large-scale production feasibility while keeping rest of the LNP components unchanged. The transfection kinetics of GFP mRNA encapsulated in LNPs and doped with 16 mol% of helper lipids was tested using GL261, U87 and SIM-A9 cell lines. Several LNP formations resulted in stable transfection (upto 5 days) of GFP mRNA in all the cell lines tested in vitro. The successful LNP candidates (enabling >80% transfection efficacy) were then tested in vivo to deliver luciferase mRNA to brain tumors via intrathecal administration in a syngeneic glioblastoma (GBM) mouse model, which confirmed luciferase expression in brain tumors in the cortex. LNPs were then tested to deliver Cre recombinase mRNA in syngeneic GBM mouse model genetically modified to express tdTomato under LoxP marker cassette that enabled identification of LNP targeted cells. mRNA was successfully delivered to tumor cells (70-80% transfected) and a range of different cells in the tumor microenvironment, including tumor-associated macrophages (80-90% transfected), neurons (31- 40% transfected), neural stem cells (39-62% transfected), oligodendrocytes (70-80% transfected) and astrocytes (44-76% transfected). Then, LNP formulations were assessed for delivering Cas9 mRNA and CD81 sgRNA (model protein) in murine syngeneic GBM model to enable gene editing in brain tumor cells. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~94% of brain tumor cells in vivo. In conclusion, we have developed a library of safe LNPs that can transfect GBM cells in vivo with high efficacy. This technology can potentially be used to develop novel mRNA therapies for GBM by delivering single or multiple mRNAs and holds great potential as a tool to study brain tumor biology.

SARS-CoV-2 spike S1 mediated lung tumor regression in a mouse model

Lung cancer is the leading cause of cancer related deaths worldwide, with a relatively low 5-year survival rate. Although there are some therapies against lung cancer, new effective treatment options are urgently required. Recently during the COVID-19 pandemic, we have seen that SARSCoV-2 binds to its receptor angiotensin-converting enzyme 2 (ACE2) via spike S1 to enter the cells. This study underlines the importance of SARS-CoV-2 spike S1 in inducing death in human lung cancer cells. Interestingly, we have seen that recombinant spike S1 treatment at very low doses led to death of human A549 lung cancer cells. On the other hand, boiled recombinant SARS-CoV-2 spike S1 remained unable to induce death, suggesting that the induction of cell death in A549 cells was due to native SARS-CoV-2 spike S1 protein. SARS-CoV-2 spike S1-induced A549 cell death was also inhibited by neutralizing antibodies against spike S1 and ACE2. Moreover, our newly designed wild type ACE2-interacting domain of SARS-CoV-2 (wtAIDS), but not mAIDS, peptide also attenuated SARS-CoV-2 spike S1-induced cell death, suggesting that SARS-CoV-2 spike S1- induced death in lung cancer cells depends on its interaction with ACE2 receptor. Similarly, recombinant spike S1 treatment also led to death of H1299 and H358 human lung cancer cells. Finally, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) intoxication led to the formation tumors in lungs of A/J mice and alternate day intranasal treatment with low dose of recombinant SARS-CoV-2 spike S1 from 22-weeks of NNK insult (late stage) led to induced apoptosis and tumor regression in the lungs. These studies indicate that recombinant SARS-CoV-2 Spike S1 protein may have implications in the treatment of lung cancer.

Determination of the Effects of Prenatal Remdesivir Exposure on Placenta Morphology, Placental IgG Transfer, and Fetal Growth in a Humanized Mouse Model

Remdesivir (RDV) is an antiviral medication used most recently for the treatment of COVID-19. Although no adverse effects were observed on perinatal parameters in reproductive and development toxicology studies at doses up to four-fold clinical area under the curve (AUC) exposures, some researchers have reported that therapeutic levels of RDV may impair early embryogenesis, as observed by in vitro studies. In addition, the influence of prenatal RDV exposure on maternal IgG transfer in the placenta is still unknown. Administration of RDV in pregnant humanized mouse model (Tg32), which expresses the human Fc gamma receptor and transporter (FCGRT) gene, was used to further evaluate potential effects on IgG transfer and concurrent perinatal endpoints. Animals were dosed daily from gestational days (GDs) 10- 14 with 25 mg/kg RDV (GS-5734) via intravenous injection (n=3-5 per group). Concurrent vehicle control animals were dosed intravenously with 12% sulfobutyl ether- beta-cyclodextrin in water (pH3.5;NaOH/HCl). All animals were administered 2 mg/kg human IgG via intravenous injection on GD 14. Placentae and fetuses were collected from dams on GD 14, 15, 16, and 18 and evaluated using histopathology and qPCR for inflammation markers. No abnormal morphologies (necrosis/apoptosis) of placentae were observed between the concurrent control and RDVdosed groups. Additionally, no differences in maternal body weights were observed. There were no statistically significant differences in placenta weights. There were no statistically significant changes in pregnancy parameters (implantation sites and dead fetuses/litter) and fetal weights between the RDV-dosed group and concurrent controls at GD 14, 15, 16, and 18. No changes were observed in transcript levels of inflammation markers in the RDV-dosed group when compared to the concurrent control group. There was a slightly lower ratio of fetal IgG level to maternal IgG levels in the RDV-dosed group;however, no statistically significant differences were observed between the RDV-dosed group and concurrent controls on GD 14, 15, 16, and 18. Our results suggest that a daily dose of 25 mg/kg RDV on GDs 10-14 in humanized mice did not cause adverse effects on placenta and fetal development. (Funded by the Perinatal Health Center of Excellence: E0300201.).

Pulmonary influenza infection promotes the awakening of dormant metastatic breast cancer cells

Breast cancer is the most common form of cancer and the second cancer-causing death in females. Although remission rates are high if detected early, survival rates drop substantially when breast cancer becomes metastatic. The most common sites of metastatic breast cancer are bone, liver and lung. Respiratory viral infections inflict illnesses on countless people. The latest pandemic caused by the respiratory virus, SARS-CoV-2, has infected more than 600 million worldwide, with documented COVID-related death upward of 1 million in the United States alone. Respiratory viral infections result in increased inflammation with immune cell influx and expansion to facilitate viral clearance. Prior studies have shown that inflammation, including through neutrophils, can contribute to dormant cancer cells reawakening and outgrowth. Moreover, inhibition of IL6 has been shown to decrease breast cancer lung metastasis in mouse models. However, how respiratory viral infections contribute to breast cancer lung metastasis remains to be unraveled. Using MMTV/PyMT and MMTV/NEU mouse models of breast cancer lung metastasis and influenza A virus as a model respiratory virus, we demonstrated that acute influenza infection and the accompanying inflammation and immune cell influx awakens and dramatically increased proliferation and expansion of dormant disseminated cancer cells (DCC) in the lungs. Acute influenza infection leads to immune influx and expansion, including neutrophils and macrophages, with increased proportion of MHCII+ macrophages in early time points, and a sustained decrease in CD206+ macrophages starting 6 days post-infection until 28 days after the initial infection. Additionally, we observed a sustained accumulation of CD4+ T cells around expanding tumor cells for as long as 28 days after the infection. Notably, neutrophil depletion or IL6 knockout reversed the flu-induced dormant cell expansion in the lung. Finally, awakened DCC exhibited downregulation of vimentin immunoreactivity, suggesting a role for phenotypic plasticity in DCC outgrowth following viral infection. In conclusion, we show that respiratory viral infections awaken and increase proliferation of dormant breast cancer cells in the lung, and that depletion of neutrophils or blocking IL6 reverses influenza-induced dormant cell awakening and proliferation.

Evaluation of the Antiviral Efficacy of Subcutaneous Nafamostat Formulated with Glycyrrhizic Acid against SARS-CoV-2 in a Murine Model.

The ongoing COVID-19 pandemic highlights the urgent need for effective antiviral agents and vaccines. Drug repositioning, which involves modifying existing drugs, offers a promising approach for expediting the development of novel therapeutics. In this study, we developed a new drug, MDB-MDB-601a-NM, by modifying the existing drug nafamostat (NM) with the incorporation of glycyrrhizic acid (GA). We assessed the pharmacokinetic profiles of MDB-601a-NM and nafamostat in Sprague-Dawley rats, revealing rapid clearance of nafamostat and sustained drug concentration of MDB-601a-NM after subcutaneous administration. Single-dose toxicity studies showed potential toxicity and persistent swelling at the injection site with high-dose administration of MDB-601a-NM. Furthermore, we evaluated the efficacy of MDB-601a-NM in protecting against SARS-CoV-2 infection using the K18 hACE-2 transgenic mouse model. Mice treated with 60 mg/kg and 100 mg/kg of MDB-601a-NM exhibited improved protectivity in terms of weight loss and survival rates compared to the nafamostat-treated group. Histopathological analysis revealed dose-dependent improvements in histopathological changes and enhanced inhibitory efficacy in MDB-601a-NM-treated groups. Notably, no viral replication was detected in the brain tissue when mice were treated with 60 mg/kg and 100 mg/kg of MDB-601a-NM. Our developed MDB-601a-NM, a modified Nafamostat with glycyrrhizic acid, shows improved protectivity against SARS-CoV-2 infection. Its sustained drug concentration after subcutaneous administration and dose-dependent improvements makes it a promising therapeutic option.

Coronavirus murine hepatitis virus strain 3 induces intestinal injury via caspase 3 dependent apoptosis

Background: Diarrhea was typical symptoms of the coronavirus disease 2019 (COVID-19). However, the underlying mechanism had not been fully understood. Aim(s): The study aimed to explore the mechanism of intestinal injury during COVID-19 in a coronavirus murine hepatitis virus strain 3 (MHV-3) induced acute mouse model. Method(s): MHV-3 induced acute infection Balb/cJ mice model was established. Intestine samples were collected at indicated time points as 0 h, 24 h, 48 h and 60 h post infection. The mRNA and protein expression of IL1b, TNFalpha, IL6, caspase 3 and cleaved caspase 3 were examined by real-time quantitative PCR (qPCR) and western blot respectively. The intestine injury and apoptosis were measured by HE staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Moreover, Z-DEVD-FMK (caspase 3 inhibitor) pre-treated MHV-3 infection mice model were established, in which the apoptosis of intestine was evaluated as well. Meanwhile, the murine intestinal cell MODE-K was infected by MHV-3 in vitro for evaluation of virus induced apoptosis. Result(s): Post MHV-3 infection, the histopathology of intestine tissue showed extraordinary injury with time dependence, as well as high level of TUNEL positivity. The mRNA levels of inflammatory cytokine IL1b, TNFalpha and IL6 were significantly increased. The protein expressions of caspase 3 and cleaved caspase 3 in the intestine was found significantly elevated from 24 to 48 h post MHV-3 infection. Z-DEVD-FMK pretreatment inhibited caspase 3 and cleaved caspase 3 expression and decreased TUNEL positivity. Meanwhile, alleviated gut injury and inhibited TNFalpha expression were observed. In vitro treated by MHV-3, intestinal cell line MODE-K showed nine-fold increase of apoptosis by comparison with saline treated ones. The expressions of apoptosis crucial protein caspase3 and cleaved caspase3 significantly elevated, as well as TNFalpha. Conclusion(s): Coronavirus murine hepatitis virus strain 3 induces intestinal injury via caspase 3 dependent apoptosis, which might shed light on the treatment of intestinal complications in COVID-19.

A Potential Therapeutic Target Underling Binge Alcohol-Triggered Spontaneous Arrhythmia Onset in the Aged Heart

Background: Aging and binge alcohol abuse are both known as independent risk factors for both atrial and ventricular arrhythmias. With the COVID-19 pandemic, increased social isolation has significantly increased alcohol consumption worldwide. Older adults are a high-risk drinking group and alcohol significantly enhances the risk of arrhythmia onset. Yet, how alcohol (a secondary stressor) drives spontaneous atrial and ventricular arrhythmia onset in the aged heart (a primary stressor) remains unclear. Objective(s): We recently reported the stress-response kinase c-jun N-terminal kinase 2 (JNK2) underlies alcohol-enhanced atrial arrhythmia vulnerability (pacing-induced) in healthy young hearts. Here, we reveal a critical role of JNK2 in alcohol-driven arrhythmia onset in the aged heart in vivo. Method(s): Ambulatory ECGs were recorded using wireless telemeters in binge alcohol-exposed aged (24 months) and young mice (2 months). Spontaneous premature atrial and ventricular contractions (PACs, PVCs), atrial and ventricular tachycardia (AT, VT) were quantified as previously described. The role of JNK2 in triggered arrhythmic activities was assessed using a well-evaluated JNK2-specific inhibitor and our unique cardiac-specific MKK7D and MKK7D-JNK2dn mouse models with tamoxifen inducible overexpression of constitutively active MKK7 (a JNK upstream activator) or co-expression of MKK7D and inactive dominant negative JNK2 (JNK2dn). Result(s): We found that binge alcohol exposure in aged mice (n=14) led to spontaneous PACs/PVCs (75% of the mice), and AT/VT episodes (50%) along with a 21% mortality rate. However, alcohol-exposed young (n=5) and non-alcohol-exposed aged mice (n=11) were absent of any spontaneous arrhythmic activities or premature death. Intriguingly, JNK2-specific inhibition in vivo abolished those alcohol-associated triggered activities and mortality in aged mice. The causative role of JNK2 in triggered arrhythmias and premature death was further supported by the high frequency of spontaneous PACs/PVCs and nonsustained AT/VT episodes along with a 50% mortality rate in MKK7D mice (n=10), which was strikingly alleviated in MKK7D-JNK2dn mice (n=5) with cardiac-specific JNK2 competitive inhibition. Conclusion(s): Our findings are the first to reveal that stress kinase JNK2 underlies binge alcohol-evoked atrial and ventricular arrhythmia initiation in aged mice. Modulating JNK2 could be a novel therapeutic strategy to treat and/or prevent binge drinking-evoked cardiac arrhythmias.Copyright © 2023

Nirmatrelvir and Molnupiravir Coadministration Modifies Preclinical Viral Exposure

Background: Implementation of vaccination programmes has had a transformational impact on control of the SARS-CoV-2 pandemic, but the need for effective antiviral drugs remains. Molnupiravir (MPV) targets viral RNA polymerase inhibiting replication via lethal mutagenesis and nirmatrelvir (NTV) is a protease inhibitor boosted with ritonavir when given clinically. This study aimed to assess the virological efficacy of NTV and MPV individually and in combination against the SARS-CoV-2 BA.1 Omicron variant in a K18-hACE2 mouse model. Method(s): K18-hACE2 mice were inoculated intranasally with 103 PFU of SARSCoV-2 BA.1 Omicron (B.1.1.529). After 24 hours, mice were orally dosed q12H, as outlined in Figure 1. At 2, 3, and 4-days post infection mice were sacrificed, and lung samples harvested. Animals were weighed and monitored daily throughout. Subsequently, viral replication in the lung was quantified using qRT-PCR to measure total (N-gene) and sub-genomic (E-gene) viral RNA. Data were normalized to 18S for quantitation. Viral exposures expressed as Areas Under viral load Curves (AUCs) were calculated by the trapezoidal method using mean values at each timepoint. Separate studies in Syrian golden hamsters using individual drugs were also conducted, and total serum IgG was measured by ELISA at 4-days post infection. Result(s): Mice gained weight in all groups post-treatment, with no significant difference between groups. A reduction in lung viral exposure was evident in all treatment groups compared to the vehicle control dosed mice (Figure 1). Coadministration of NTV with MPV displayed a trend towards lower lung viral exposure compared to the vehicle control with ~40-and ~45-fold reduction in AUC for N-and SgE-gene assays, respectively. Dosed individually, NTV and MPV reduced viral exposure 5.7-and 7.7-fold for the N-gene assay, respectively. Differences in total serum IgG concentrations were evident between vehicle and NTV-(34-fold reduction, P=0.018), and MPV-(4.2-fold reduction, P=0.053) treated hamsters. Conclusion(s): These data show virological efficacy of NTV and MPV against the SARS-CoV-2 BA.1 Omicron variant. The combination of NTV and MPV demonstrated a lower viral RNA exposure in the lung than either drug alone, albeit not statistically significant. Initial data indicate potential immune alterations in NTV and MPV dosed hamsters. Studies to clarify the utility of NTV/ MPV combinations and further characterize the impact of antiviral therapy on IgG are warranted.

Highly synergistic combinations of nanobodies that target SARS-CoV-2 reduce viral load in hACE2 transgenic mice

The ongoing emergence of SARS-CoV-2 variants threatens current vaccines and renders current therapeutic antibodies obsolete, demanding powerful new treatments that can resist viral escape. We therefore generated a large nanobody repertoire to saturate the distinct and highly conserved available epitope space of SARS-CoV-2 spike, including the S1 receptor binding domain, N-terminal domain, and the S2 subunit, to identify new nanobody binding sites that may reflect novel mechanisms of viral neutralization. Structural mapping and functional assays show that these highly stable monovalent nanobodies potently inhibit SARS-CoV-2 infection, display numerous neutralization mechanisms, are effective against past and present emerging variants of concern, and are resistant to mutational escape. Rational combinations of these nanobodies that bind dissimilar sites within and between spike subunits exhibit extraordinary synergy and suggest multiple tailored therapeutic and prophylactic strategies. All mouse involved experiments were performed in compliance with the Institutional Animal Care and Use Committee and mice were housed and maintained in a specific pathogen-free conditions at Seattle Children's Research Institute. Infected mice with SARSCoV- 2 were housed in a Biosafety Level 3 facility in an Animal Biohazard Containment Suite. Prophylactic intranasal application of a synergistic pair of unmodified nanobodies in 10-12 week-old female K18-hACE2 transgenic mice, a mouse model of SARS-CoV-2 infection, showed significant reduction in viral load after 3 days post-challenge with SARS-CoV-2, the first demonstration of synergy in vivo. In summary, our results show that our diverse repertoire of nanobodies can neutralize current variants of live SARS-CoV-2, pairs of nanobodies that bind distinct sites on spike show tremendous synergy in neutralizing efficacy in vitro, and the application of synergizing pair of nanobodies translates to an in vivo mouse model of SARSCoV- 2. Research funded by the Mathers Foundation, Robertson Foundation, NIH P41GM109824.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

IDWeek 2022

IDWeek is the joint annual meeting of the Infectious Diseases Society of America (IDSA), Society for Healthcare Epidemiology of America (SHEA), the HIV Medicine Association (HIVMA), the Pediatric Infectious Diseases Society (PIDS) and the Society of Infectious Diseases Pharmacists (SIDP). For the first time since the COVID-19 public health emergency began, IDWeek 2022 returned to in-person attendance. It was held in Washington, D.C., and the meeting comprised 5 days of live sessions and on-demand content that included posters and oral presentations.Copyright © 2023 Clarivate.

Quantification of functional antibody titers in a mouse model of SARS-CoV-2 infection

Towards the end of 2019 a novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) caused the ongoing global pandemic. The virus surface consists of spike proteins that mediate SARS-CoV-2 entry into cells through its receptor-binding domain (RBD) that attaches to the human receptor Angiotensin- Converting Enzyme 2 (ACE2). Upon infection with foreign material, like viruses and bacteria, the human immune system responds by producing a humoral response specific to the viral antigen. Cells from the innate immune system and antibodies generated in the humoral response work to destroy and block infectious antigens from causing damage to the human cells. The S protein of SARSCoV- 2 is the key protein that stimulates the immune system to generate neutralizing antibodies. To safely test and investigate SARS-CoV-2 in BSL-2 lab setting, we propagated a surrogate pseudo typed virus to evaluate the ability of antibodies to reduce viral cell entry and replication in SARS-CoV-2 infected mice model. Quantifying the functional ability of neutralizing antibodies would help us understand how they influence reinfection in recovered individuals. We hypothesize that antibodies generated in SARS-CoV-2 infected mice models will induce a protective immune response against the SARSCoV- 2 infection. To detect and quantify the protective immune response generated in mice, we performed two different serological assays and identified antibodies endpoint titers. Mice were infected with Delta and Beta at time points Day 3 and Day 4. We performed a SARS-CoV-2 Spike pseudo virus neutralization assay and measured luminescence to determine the percentage neutralization of functional antibodies induced in mice serum samples upon infection. Utilizing indirect ELISAs,' we measured absorbance for IgA antibodies in Bronchoalveolar lavage fluid (BALF) serum and total IgG antibodies in cardiac bleeds. Our results showed we did not obtain neutralizing activity of antibodies in mice serum samples taken at early time points, 24 hrs and 4 days, after infection with the Delta variant of SARS CoV2 virus using both the pseudo viruses Omicron andWA spike.We obtained 100% neutralizing activity in mice serum samples taken at day 21 and infected with Beta variant of SARS CoV2 virus using both the pseudo viruses Omicron and WA spike demonstrating that there is cross-neutralization against various variants of concern. Antibodies (IgA, IgM, IgG) generated in mice 3 weeks post infection with SARS CoV2 (Beta) virus are capable of neutralizing and inhibiting the entry of WA spike and Omicron pseudo viruses in human HEK293 T Ace2 cells. Moving forward utilizing samples with timepoints surpassing 3 weeks could possibly yield higher concentrations of IgA and IgM antibodies that can neutralize the SARS-CoV-2 pseudo virus. Thank you to Dr. Rhea Coler, the entire Coler lab, National Institutes of Health (NIH), and Seattle Children's Research Institute.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Novel Translational Animal Model Of Pediatric Viral Myocarditis

Myocarditis is a heart condition characterized by inflammation of cardiac myocytes. This inflammation is instigated by the activation of both the innate and acquired immune responses and is most often caused by viruses (flu, hand foot and mouth and COVID-19). Myocarditis may progress to dilated cardiomyopathy (DCM), a chronic heart condition in which fibrosis and remodeling weakens the ability of the heart to effectively pump. Myocarditis is a leading cause of sudden death in children and young adults. In this study, we investigated the progression and severity of myocarditis within the pediatric population as compared to an adult population. Prior to this study there were no mouse models for pediatric myocarditis but a large percent of patients with myocarditis are children. We utilized our knowledge from our adult mouse model of coxsackievirus B3 (CVB3) myocarditis to create a pediatric CVB3 myocarditis model in order to better understand the development of myocarditis in children. We hypothesized that myocarditis would have sex-specific differences in the manifestation and severity similar to the adult model but the mechanisms of disease would very between ages. We utilized 4-week-old male and female BALB/c mice to model pediatric myocarditis as compared to 8-week-old mice. Mice were be infected with heart-passaged CVB3 intraperitoneally (ip) on day (d) 0. Disease severity and progression was evaluated during acute myocarditis (d8-12 pi) and during DCM (d35pi). After anesthesia, body weight, heart weight and tibia length, blood, hearts, pancreas and spleens were harvested. Echocardiography was conducted on mice at the chronic DCM timepoint. We found that adult male and female mice develop myocarditis with male mice have more severe disease and progress to DCM as compared to female mice. The main immune cells and pathways involved in myocarditis severity were macrophages, complement and the inflammasome in male adult mice. This increase in disease was driven by testosterone and reduced by estrogen as seen utilizing gonadectomies. In the pediatric population we did not see as drastic of differences in sex hormone levels as the mice are pre-pubescent, this led to less dramatic sex differences in disease and altered immune mechanisms leading to disease in the pediatric population compared to the adults. We do see induction of myocarditis in both male and female pediatric mice compared to uninfected controls but severity in the pediatric population is less than adult population. Successful development of a pediatric translational mouse model of viral myocarditis will significantly impact the myocarditis field by allowing the ability to assess differences between pediatric and adult populations and develop targeted diagnostics and treatments.Copyright © 2022

Selection and validation of siRNAs preventing uptake and replication of SARS-CoV-2

The COVID-19 pandemic has dramatically underlined the desperate need for novel therapeutic options for treatment of respiratory viral infections to provide fast and efficacious drugs against new upcoming pathogens. RNA interference (RNAi)-based approaches depict a promising alternative to conventional medication, as they can be rapidly adjusted to the respective viral genome or its host cellular interaction partners. Here, we pursued both strategies. We designed and screened nine siRNAs targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90 % knockdown of the ACE2 mRNA and protein for at least six days, as assessed by a specially designed fluorescent reporter assay. siA1 application was found to protect Vero E6 and Huh-7 cells from infection with SARS-CoV-2 with an up to ~92 % reduction of the viral burden. In parallel, we exploited the respective sequence in generation miR30a-embedded lentivirally or AAV encoded shRNAs, which performed equally powerful, with shA1 being the most potent. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analyzed eight siRNAs directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). We identified siV1, which targets the nsp1-encoding sequence as particularly efficient. SiV1 inhibited SARS-CoV-2 replication in Vero E6 or Huh-7 cells by more than 99 % or 97 %, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention. Preliminary in vivo pilot experiments carried out in a K18-hACE-2 mice model showed first promising results. Thereby siRNAs complexed with nanoparticles (LP10Y) were applicated systemically by intravenous injection. Mice were intranasally infected with SARS-CoV-2, euthanized 48 hours later, and the viral burden was determined by RT-qPCR in lung homogenates. A positive trend in viral reduction was found in comparison to corresponding control group.

Evaluation Effectiveness of in Vitro Cultivation of Bezoar on Mouse Model Combining Disease with Syndrome of Coronavirus Pneumonia with Yidu Xifei Syndrome.

Objective:To determine the therapeutic effect of in vitro cultivation of bezoar on a mouse model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. Method: BALB/c mice were randomly divided into six groups according to their weight grade:normal group,HCoV-229E infection group,cold and damp group,a mouse model combining disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome,and high and low dose group of in vitro cultivation of bezoar. The combination model of human coronavirus pneumonia with Yidu Xifei syndrome mice was established by the method of cold dampness condition stimulation+coronavirus HCoV-229E infection. In vitro cultivation of bezoar (0.128,0.064 g.kg-1 )was administrated by gavage for 3 days from the day of infection. The observation indexes included:general state observation of mice,inhibition rate of lung index and lung index of mice. Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the viral load in the lung tissues of mice. Serum levels of motilin(MTL),gastrin(GAS),and cytokines interleukin(IL)-10,IL-6, tumor necrosis factor-alpha(TNF-alpha)and interferon-gamma(IFN-gamma)in lung tissue of mice were determined by enzyme-linked immunosorbent assay(ELISA). The percentages of CD4+ T lymphocytes,CD8+ T lymphocytes and B lymphocytes in the blood of mice were determined by flow cytometry. Result:The high and low dose group of in vitro cultivation of bezoar can significantly improve the general condition of model mice. Compared with blank group, model group mice lung index increased significantly(P<0.01), nucleic acids significantly increased expression of lung tissue in mice(P<0.01),significantly higher serum MTL content in mice,GAS content significantly decreased(P<0.05,P<0.01),lung tissue cells in the immune factor TNF-alpha,IL-10 and IL-6 were significantly increased(P<0.01),peripheral blood lymphocyte CD4+ T cells in mice,The percentages of CD8+ T cells and B cells were significantly decreased(P<0.01). Compared with model group, in vitro cultivation bezoar mice lung index of high and low dose group were significantly lower(P<0.01),the lung tissue of mice express nucleic acid decreased significantly(P<0.01),MTL content decreased significantly(P< 0.01),the lung tissue of mice in the IL-6,IL-10,the TNF-alpha,IFN-gamma levels were significantly lower(P<0.01), in vitro cultivation bezoar high dose group can significantly increase the CD4+ T cell percentage(P<0.05),in vitro cultivation bezoar can to a certain extent reduce model mice lung inflammatory exudation,pulmonary interstitial edema,as well as blood stasis symptoms. Conclusion:In vitro cultivation of bezoar has a significant therapeutic effect on a mice model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. It can be treated by reducing the lung index of the model mice,improving the pathological damage of the lung tissue,adjusting the immune effective and inhibiting the clearing of inflammatory factors,and to provide a laboratory basis for clinical medication.Copyright © 2021, China Academy of Chinese Medical Sciences Institute of Chinese Materia Medica. All rights reserved.

Effect of polycyclic aromatic hydrocarbons on skin pigmentation

Despite COVID-19 lockdowns, gradual restarting of the global economy has rapidly increased air quality index (AQI) values. With over 99 % of the world population living in areas exceeding air quality guidelines, air pollution is more so a threat to our health. Within particulate matter, a major air pollutant, lies polycyclic aromatic hydrocarbons (PAH). While recent studies explore the link between air pollution and pigmentation disorders, the molecular mechanisms responsible for this alteration remain largely unknown. To challenge our hypothesis that exposure to PAH leads to an increase in abnormal pigmentation, we have utilized in vitro and in vivo assays. In vitro, novel 2D and 3D co-culture assays were developed to analyze pigment production, transfer and total melanin content in human keratinocytes and melanocytes. Following that, bulk RNA-sequencing was also carried out on isolated melanocytes post co-culture to possibly elucidate the mechanism behind this phenomenon. In vivo, a mouse model bearing epidermal melanin was generated to investigate the effect of PAH exposure. Notably, our initial studies have indicated a significant increase in melanin production, transfer and total melanin content when exposed to PAH. From our transcriptome analysis, we have also pinpointed to several genes which have been differentially expressed, most significant being CYP1A1. This prompted us to look further into the AhR signaling pathway. Interestingly, we did not see an increase in classic melanogenesis genes, but instead genes which are usually associated with senescence-associated secretory phenotype (SASP). This hints to a possible alternative pathway leading to an eventual increase in melanin production. We believe that our findings highlight potential approaches for novel therapeutics the treatment of skin pigmentation disorders triggered by air pollution.Copyright © 2023

Evaluation Effectiveness of in Vitro Cultivation of Bezoar on Mouse Model Combining Disease with Syndrome of Coronavirus Pneumonia with Yidu Xifei Syndrome.

Objective:To determine the therapeutic effect of in vitro cultivation of bezoar on a mouse model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. Method: BALB/c mice were randomly divided into six groups according to their weight grade:normal group,HCoV-229E infection group,cold and damp group,a mouse model combining disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome,and high and low dose group of in vitro cultivation of bezoar. The combination model of human coronavirus pneumonia with Yidu Xifei syndrome mice was established by the method of cold dampness condition stimulation+coronavirus HCoV-229E infection. In vitro cultivation of bezoar (0.128,0.064 g.kg-1 )was administrated by gavage for 3 days from the day of infection. The observation indexes included:general state observation of mice,inhibition rate of lung index and lung index of mice. Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the viral load in the lung tissues of mice. Serum levels of motilin(MTL),gastrin(GAS),and cytokines interleukin(IL)-10,IL-6, tumor necrosis factor-alpha(TNF-alpha)and interferon-gamma(IFN-gamma)in lung tissue of mice were determined by enzyme-linked immunosorbent assay(ELISA). The percentages of CD4+ T lymphocytes,CD8+ T lymphocytes and B lymphocytes in the blood of mice were determined by flow cytometry. Result:The high and low dose group of in vitro cultivation of bezoar can significantly improve the general condition of model mice. Compared with blank group, model group mice lung index increased significantly(P<0.01), nucleic acids significantly increased expression of lung tissue in mice(P<0.01),significantly higher serum MTL content in mice,GAS content significantly decreased(P<0.05,P<0.01),lung tissue cells in the immune factor TNF-alpha,IL-10 and IL-6 were significantly increased(P<0.01),peripheral blood lymphocyte CD4+ T cells in mice,The percentages of CD8+ T cells and B cells were significantly decreased(P<0.01). Compared with model group, in vitro cultivation bezoar mice lung index of high and low dose group were significantly lower(P<0.01),the lung tissue of mice express nucleic acid decreased significantly(P<0.01),MTL content decreased significantly(P< 0.01),the lung tissue of mice in the IL-6,IL-10,the TNF-alpha,IFN-gamma levels were significantly lower(P<0.01), in vitro cultivation bezoar high dose group can significantly increase the CD4+ T cell percentage(P<0.05),in vitro cultivation bezoar can to a certain extent reduce model mice lung inflammatory exudation,pulmonary interstitial edema,as well as blood stasis symptoms. Conclusion:In vitro cultivation of bezoar has a significant therapeutic effect on a mice model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. It can be treated by reducing the lung index of the model mice,improving the pathological damage of the lung tissue,adjusting the immune effective and inhibiting the clearing of inflammatory factors,and to provide a laboratory basis for clinical medication.Copyright © 2021, China Academy of Chinese Medical Sciences Institute of Chinese Materia Medica. All rights reserved.

SARS-CoV-2 infection aggravates chronic comorbidities of cardiovascular diseases and diabetes in mice.

Background: Cardiovascular diseases (CVDs) and diabetes mellitus (DM) are top two chronic comorbidities that increase the severity and mortality of COVID-19. However, how SARS-CoV-2 alters the progression of chronic diseases remain unclear. Methods: We used adenovirus to deliver h-ACE2 to lung to enable SARS-CoV-2 infection in mice. SARS-CoV-2's impacts on pathogenesis of chronic diseases were studied through histopathological, virologic and molecular biology analysis. Results: Pre-existing CVDs resulted in viral invasion, ROS elevation and activation of apoptosis pathways contribute myocardial injury during SARS-CoV-2 infection. Viral infection increased fasting blood glucose and reduced insulin response in DM model. Bone mineral density decreased shortly after infection, which associated with impaired PI3K/AKT/mTOR signaling. Conclusion: We established mouse models mimicked the complex pathological symptoms of COVID-19 patients with chronic diseases. Pre-existing diseases could impair the inflammatory responses to SARS-CoV-2 infection, which further aggravated the pre-existing diseases. This work provided valuable information to better understand the interplay between the primary diseases and SARS-CoV-2 infection.

Rational development of multicomponent mRNA vaccine candidates against mpox.

The re-emerging mpox (formerly monkeypox) virus (MPXV), a member of Orthopoxvirus genus together with variola virus (VARV) and vaccinia virus (VACV), has led to public health emergency of international concern since July 2022. Inspired by the unprecedent success of coronavirus disease 2019 (COVID-19) mRNA vaccines, the development of a safe and effective mRNA vaccine against MPXV is of high priority. Based on our established lipid nanoparticle (LNP)-encapsulated mRNA vaccine platform, we rationally constructed and prepared a panel of multicomponent MPXV vaccine candidates encoding different combinations of viral antigens including M1R, E8L, A29L, A35R, and B6R. In vitro and in vivo characterization demonstrated that two immunizations of all mRNA vaccine candidates elicit a robust antibody response as well as antigen-specific Th1-biased cellular response in mice. Importantly, the penta- and tetra-component vaccine candidates AR-MPXV5 and AR-MPXV4a showed superior capability of inducing neutralizing antibodies as well as of protecting from VACV challenge in mice. Our study provides critical insights to understand the protection mechanism of MPXV infection and direct evidence supporting further clinical development of these multicomponent mRNA vaccine candidates.

COVID-19 spike polypeptide vaccine reduces the pathogenesis and viral infection in a mouse model of SARS-CoV-2.

The SARS-CoV-2 coronavirus, which causes a respiratory disease called COVID-19, has been declared a pandemic by the World Health Organization (WHO) and is still ongoing. Vaccination is the most important strategy to end the pandemic. Several vaccines have been approved, as evidenced by the ongoing global pandemic, but the pandemic is far from over and no fully effective vaccine is yet available. One of the most critical steps in vaccine development is the selection of appropriate antigens and their proper introduction into the immune system. Therefore, in this study, we developed and evaluated two proposed vaccines composed of single and multiple SARS-CoV-2 polypeptides derived from the spike protein, namely, vaccine A and vaccine B, respectively. The polypeptides were validated by the sera of COVID-19-vaccinated individuals and/or naturally infected COVID-19 patients to shortlist the starting pool of antigens followed by in vivo vaccination to hACE2 transgenic mice. The spike multiple polypeptide vaccine (vaccine B) was more potent to reduce the pathogenesis of organs, resulting in higher protection against the SARS-CoV-2 infection.