Ebola virus-induced eye sequelae: a murine model for evaluating glycoprotein-targeting therapeutics.

BACKGROUND: Ebola virus disease (EVD) survivors experience ocular sequelae including retinal lesions, cataracts, and vision loss. While monoclonal antibodies targeting the Ebola virus glycoprotein (EBOV-GP) have shown promise in improving prognosis, their effectiveness in mitigating ocular sequelae remains uncertain. METHODS: We developed and characterized a BSL-2-compatible immunocompetent mouse model to evaluate therapeutics targeting EBOV-GP by inoculating neonatal mice with vesicular stomatitis virus expressing EBOV-GP (VSV-EBOV). To examine the impact of anti-EBOV-GP antibody treatment on acute retinitis and ocular sequelae, VSV-EBOV-infected mice were treated with polyclonal antibodies or monoclonal antibody preparations with antibody-dependent cellular cytotoxicity (ADCC-mAb) or neutralizing activity (NEUT-mAb). FINDINGS: Treatment with all anti-EBOV-GP antibodies tested dramatically reduced viremia and improved survival. Further, all treatments reduced the incidence of cataracts. However, NEUT-mAb alone or in combination with ADCC-mAb reduced viral load in the eyes, downregulated the ocular immune and inflammatory responses, and minimized retinal damage more effectively. INTERPRETATION: Anti-EBOV-GP antibodies can improve survival among EVD patients, but improved therapeutics are needed to reduce life altering sequelae. This animal model offers a new platform to examine the acute and long-term effect of the virus in the eye and the relative impact of therapeutic candidates targeting EBOV-GP. Results indicate that even antibodies that improve systemic viral clearance and survival can differ in their capacity to reduce acute ocular inflammation, and long-term retinal pathology and corneal degeneration. FUNDING: This study was partly supported by Postgraduate Research Fellowship Awards from ORISE through an interagency agreement between the US DOE and the US FDA.

Para-Methoxybenzylidene Acetal-Protected D-Glucosamine Derivatives as pH-Responsive Gelators and Their Applications for Drug Delivery.

Carbohydrate-based low molecular weight gelators (LMWGs) are compounds with the capability to self-assemble into complex molecular networks within a solvent, leading to solvent immobilization. This process of gel formation depends on noncovalent interactions, including Van der Waals, hydrogen bonding, and π-π stacking. Due to their potential applications in environmental remediation, drug delivery, and tissue engineering, these molecules have emerged as an important area of research. In particular, various 4,6-O-benzylidene acetal-protected D-glucosamine derivatives have shown promising gelation abilities. In this study, a series of C-2-carbamate derivatives containing a para-methoxy benzylidene acetal functional group were synthesized and characterized. These compounds exhibited good gelation properties in several organic solvents and aqueous mixtures. Upon removal of the acetal functional group under acidic conditions, a number of deprotected free sugar derivatives were also synthesized. Analysis of these free sugar derivatives revealed two compounds were hydrogelators while their precursors did not form hydrogels. For those protected carbamates that are hydrogelators, removal of the 4,6-protection will result in a more water-soluble compound that produces a transition from gel to solution. Given the ability of these compounds to form gels from solution or solution from gels in situ in response to acidic environments, these compounds may have practical applications as stimuli-responsive gelators in an aqueous medium. In turn, one hydrogelator was studied for the encapsulation and release of naproxen and chloroquine. The hydrogel exhibited sustained drug release over a period of several days, with the release of chloroquine being faster at lower pH due to the acid lability of the gelator molecule. The synthesis, characterization, gelation properties, and studies on drug diffusion are discussed.

Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients.

Unintended immunogenicity can affect the safety and efficacy of therapeutic proteins and peptides, so accurate assessments of immunogenicity risk can aid in the selection, development, and regulation of biologics. Product- and process- related impurities can act as adjuvants that activate the local or systemic innate immune response increasing the likelihood of product immunogenicity. Thus, assessing whether products have innate immune response modulating impurities (IIRMI) is a key component of immunogenicity risk assessments. Identifying trace levels of individual IIRMI can be difficult and testing individually for all potential impurities is not feasible. Therefore, to mitigate the risk, cell-based assays that use human blood cells or monocyte-macrophage reporter cell lines are being developed to detect minute quantities of impurities capable of eliciting innate immune activation. As these are cell-based assays, there is concern that excipients could blunt the cell responses, masking the presence of immunogenic IIRMI. Here, we explore the impact of frequently used excipients (non-ionic detergents, sugars, amino acids, bulking agents) on the sensitivity of reporter cell lines (THP-1- and RAW-Blue cells) and fresh human blood cells to detect purified TLR agonists as model IIRMI. We show that while excipients do not modulate the innate immune response elicited by TLR agonists in vivo, they can impact on the sensitivity of cell-based IIRMI assays. Reduced sensitivity to detect LPS, FSL-1, and other model IIRMI was also evident when testing 3 different recombinant drug products, product A (a representative mAb), B (a representative growth factor), C (a representative peptide), and their corresponding formulations. These results indicate that product formulations need to be considered when developing and validating cell-based assays for assessing clinically relevant levels of IIRMI in therapeutic proteins. Optimization of reporter cells, culture conditions and drug product concentration appear to be critical to minimize the impact of excipients and attain sensitive and reproducible assays.

BSL2-compliant lethal mouse model of SARS-CoV-2 and variants of concern to evaluate therapeutics targeting the Spike protein.

Since first reported in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is rapidly acquiring mutations, particularly in the spike protein, that can modulate pathogenicity, transmission and antibody evasion leading to successive waves of COVID19 infections despite an unprecedented mass vaccination necessitating continuous adaptation of therapeutics. Small animal models can facilitate understanding host-pathogen interactions, target selection for therapeutic drugs, and vaccine development, but availability and cost of studies in BSL3 facilities hinder progress. To generate a BSL2-compatible in vivo system that specifically recapitulates spike protein mediated disease we used replication competent, GFP tagged, recombinant Vesicular Stomatitis Virus where the VSV glycoprotein was replaced by the SARS-CoV-2 spike protein (rVSV-SARS2-S). We show that infection requires hACE2 and challenge of neonatal but not adult, K18-hACE2 transgenic mice (hACE2tg) leads to productive infection of the lungs and brains. Although disease progression was faster in SARS-CoV-2 infected mice, infection with both viruses resulted in neuronal infection and encephalitis with increased expression of Interferon-stimulated Irf7, Bst2, Ifi294, as well as CxCL10, CCL5, CLC2, and LILRB4, and both models were uniformly lethal. Further, prophylactic treatment targeting the Spike protein (Receptor Binding Domain) with antibodies resulted in similar levels of protection from lethal infection against rVSV-SARS2-S and SARS-CoV-2 viruses. Strikingly, challenge of neonatal hACE2tg mice with SARS-CoV-2 Variants of Concern (SARS-CoV-2-α, -ß, ϒ, or Δ) or the corresponding rVSV-SARS2-S viruses (rVSV-SARS2-Spike-α, rVSV-SARS2-Spike-ß, rVSV-SARS2-Spike-ϒ or rVSV-SARS2-Spike-Δ) resulted in increased lethality, suggesting that the Spike protein plays a key role in determining the virulence of each variant. Thus, we propose that rVSV-SARS2-S virus can be used to understand the effect of changes to SARS-CoV-2 spike protein on infection and to evaluate existing or experimental therapeutics targeting spike protein of current or future VOC of SARS-CoV-2 under BSL-2 conditions.

Sleep Habits of Early School-Aged Children with Type 1 Diabetes and Their Parents: Family Characteristics and Diabetes Management.

STUDY OBJECTIVES: School-aged children with type 1 diabetes (T1D) and their parents are at risk for sleep disturbances, yet few studies have used objective measures to assess sleep characteristics in young children with T1D. METHODS: Forty children (ages 5-9) with T1D and their parents wore actigraph watches and completed sleep diaries for 7 nights. Parents also completed questionnaires about demographic information, diabetes distress, fear of hypoglycemia, and family routines. Children's clinical data (HbA1c and blood glucose data) were extracted from the medical record. RESULTS: Most of the children and their parents obtained insufficient sleep. Based on actigraphy data, children slept an average of 7.9 hours/night and parents slept 6.7 hours/night, below the recommendations of 9-11 and 7-9 hours of sleep, respectively. Shorter child sleep latency was significantly associated with better glycemic levels, and parents' sleep duration and efficiency were related to child's glycemic levels. Parental fear of hypoglycemia and lack of family routines were associated with poorer sleep quality in parents and children, and with parental diabetes distress. CONCLUSIONS: Sleep duration and quality is a modifiable target for potentially improving glycemic levels and parental distress in early school-aged children with T1D.

Characterization of the therapeutic effect of antibodies targeting the Ebola glycoprotein using a novel BSL2-compliant rVSVΔG-EBOV-GP infection model.

Ebola virus (EBOV) infections cause haemorrhagic fever, multi-organ failure and death, and survivors can experience neurological sequelae. Licensing of monoclonal antibodies targeting EBOV glycoprotein (EBOV-GP) improved its prognosis, however, this treatment is primarily effective during early stages of disease and its effectiveness in reducing neurological sequela remains unknown. Currently, the need for BSL4 containment hinders research and therapeutic development; development of an accessible BSL-2 in vivo mouse model would facilitate preclinical studies to screen and select therapeutics. Previously, we have shown that a subcutaneous inoculation with replicating EBOV-GP pseudotyped vesicular stomatitis virus (rVSVΔG-EBOV-GP or VSV-EBOV) in neonatal mice causes transient viremia and infection of the mid and posterior brain resulting in overt neurological symptoms and death. Here, we demonstrate that the model can be used to test therapeutics that target the EBOV-GP, by using an anti-EBOV-GP therapeutic (SAB-139) previously shown to block EBOV infection in mice and primates. We show that SAB-139 treatment decreases the severity of neurological symptoms and improves survival when administered before (1 day prior to infection) or up to 3 dpi, by which time animals have high virus titres in their brains. Improved survival was associated with reduced viral titres, microglia loss, cellular infiltration/activation, and inflammatory responses in the brain. Interestingly, SAB-139 treatment significantly reduced the severe VSV-EBOV-induced long-term neurological sequalae although convalescent mice showed modest evidence of abnormal fear responses. Together, these data suggest that the neonatal VSV-EBOV infection system can be used to facilitate assessment of therapeutics targeting EBOV-GP in the preclinical setting.

Discovery of VU6028418: A Highly Selective and Orally Bioavailable M4 Muscarinic Acetylcholine Receptor Antagonist.

Herein, we report the SAR leading to the discovery of VU6028418, a potent M4 mAChR antagonist with high subtype-selectivity and attractive DMPK properties in vitro and in vivo across multiple species. VU6028418 was subsequently evaluated as a preclinical candidate for the treatment of dystonia and other movement disorders. During the characterization of VU6028418, a novel use of deuterium incorporation as a means to modulate CYP inhibition was also discovered.

Toward understanding the impact of mHealth features for people with HIV: a latent class analysis of PositiveLinks usage.

PositiveLinks (PL) is a multi-feature smartphone-based platform to improve engagement-in-care and viral suppression (VS) among clinic patients living with HIV. Features include medication reminders, mood/stress check-ins, a community board, and secure provider messaging. Our goal was to examine how PL users interact with the app and determine whether usage patterns correlate with clinical outcomes. Patients (N = 83) at a university-based Ryan White clinic enrolled in PL from June 2016 to March 2017 and were followed for up to 12 months. A subset (N = 49) completed interviews after 3 weeks of enrollment to explore their experiences with and opinions of PL. We differentiated PL members based on 6-month usage of app features using latent class analysis. We explored characteristics associated with class membership, compared reported needs and preferences by class, and examined association between class and VS. The sample of 83 PL members fell into four classes. "Maximizers" used all app features frequently (27%); "Check-in Users" tended to interact only with daily queries (22%); "Moderate All-Feature Users" used all features occasionally (33%); and "As-Needed Communicators" interacted with the app minimally (19%). VS improved or remained high among all classes after 6 months. VS remained high at 12 months among Maximizers (baseline and 12-month VS: 100%, 94%), Check-in Users (82%, 100%), and Moderate All-Feature Users (73%, 94%) but not among As-Needed Communicators (69%, 60%). This mixed-methods study identified four classes based on PL usage patterns that were distinct in characteristics and clinical outcomes. Identifying and characterizing mHealth user classes offers opportunities to tailor interventions appropriately based on patient needs and preferences as well as to provide targeted alternative support to achieve clinical goals.

Long-term persistence of infectious Zika virus: Inflammation and behavioral sequela in mice.

The neurodevelopmental defects associated with ZIKV infections early in pregnancy are well documented, however the potential defects and long-term consequences associated with milder infections in late pregnancy and perinatal period are less well understood. To model these, we challenged 1 day old (P1) immunocompetent C57BL/6 mice with ZIKV. The animals developed a transient neurological syndrome including unsteady gait, kinetic tremors, severe ataxia and seizures 10-15 days post-infection (dpi) but symptoms subsided after a week, and most animals survived. Despite apparent recovery, MRI of convalescent mice show reduced cerebellar volume that correlates with altered coordination and motor function as well as hyperactivity and impulsivity. Persistent mRNA levels of pro-inflammatory genes including Cd80, Il-1α, and Ifn-γ together with Cd3, Cd8 and perforin (PrfA), suggested persistence of low-grade inflammation. Surprisingly, the brain parenchyma of convalescent mice harbor multiple small discrete foci with viral antigen, active apoptotic processes in neurons, and cellular infiltrates, surrounded by activated astrocytes and microglia as late as 1-year post-infection. Detection of negative-sense strand viral RNA and isolation of infectious virus derived from these convalescent mice by blinded passage in Vero cells confirmed long-term persistence of replicating ZIKV in CNS of convalescent mice. Although the infection appears to persist in defined reservoirs within CNS, the resulting inflammation could increase the risk of neurodegenerative disorders. This raises concern regarding possible long-term effects in asymptomatic children exposed to the virus and suggests that long-term neurological and behavioral monitoring as well as anti-viral treatment to clear virus from the CNS may be useful in patients exposed to ZIKV at an early age.

Supporting Health Among Young Men Who Have Sex With Men and Transgender Women With HIV: Lessons Learned From Implementing the weCare Intervention.

Young gay, bisexual, and other men who have sex with men and transgender women with HIV, particularly those who are racial or ethnic minorities, often have poor health outcomes. They also utilize a wide array of social media. Accordingly, we developed and implemented weCare, an mHealth (mobile health) intervention where cyberhealth educators utilize established social media platforms (e.g., Facebook, texting, and GPS-based mobile applications ["apps"]) designed for social and sexual networking) to improve HIV-related care engagement and health outcomes. As part of the process evaluation of weCare, we conducted 32 interviews with intervention participants (n = 18) and HIV clinic providers and staff (n = 14). This article highlights three key intervention characteristics that promoted care engagement, including that weCare is (1) targeted (e.g., using existing social media platforms, similarity between intervention participants and cyberhealth educator, and implementation within a supportive clinical environment), (2) tailored (e.g., bidirectional messaging and trusting relationship between participants and cyberhealth educators to direct interactions), and (3) personalized (e.g., addressing unique care needs through messaging content and flexibility in engagement with intervention). In addition, interviewees' recommendations for improving weCare focused on logistics, content, and the ways in which the intervention could be adapted to reach a larger audience. Quality improvement efforts to ensure that mHealth interventions are relevant for young gay, bisexual, and other men who have sex with men and transgender women are critical to ensure care engagement and support health outcomes.

A peer navigation intervention to prevent HIV among mixed immigrant status Latinx GBMSM and transgender women in the United States: outcomes, perspectives and implications for PrEP uptake.

The Latinx population in the United States is disproportionately affected by HIV. Our community-based participatory research partnership developed, implemented and evaluated a Spanish-language peer navigation intervention designed to increase HIV testing and condom use among social networks of immigrant Spanish-speaking Latinx gay, bisexual and other men who have sex with men (GBMSM) and transgender women (TW). We randomized 21 social networks of Latinx GBMSM and TW, ages 18-55 years, to the intervention, known as HOLA, or a waitlist control group. Social network participants (n = 166) completed structured assessments at baseline and 12-month follow-up (24 months after baseline). Follow-up retention was 95%. Individual in-depth interviews with a sample of participants documented their intervention-related experiences, needs, and priorities to inform future research. At follow-up, HOLA participants reported increased HIV testing (adjusted odds ratio = 8.3; 95% CI = 3.0-23.0; P < 0.0001). All study participants reported increased condom use; there was no significant difference between HOLA and waitlist control participants. In-depth interviews identified critical intervention elements and impacts and community needs and priorities. The HOLA intervention is effective for increasing HIV testing among Latinx GBMSM and TW, an initial step within the HIV prevention and care continua, and may be adaptable to promote pre-exposure prophylaxis uptake.

A versatile microwave source for cold atom experiments controlled by a field programmable gate array.

We present a microwave source that is controlled by a commercially available field-programmable gate array (FPGA). Using an FPGA allows for precise control of the time dependent microwave-dressing applied to a sample of trapped cold atoms. We test our microwave source by exciting Rabi oscillations in a Na spinor Bose-Einstein condensate. We include, as supplements, the complete source code, parts' lists, pin connection diagrams, and schematics to make it easy for any group to build and use this device.

Community Perceptions of Comprehensive Harm Reduction Programs and Stigma Towards People Who Inject Drugs in Rural Virginia.

The spread of infectious diseases, including Human Immunodeficiency virus and Hepatitis C virus, is a major risk of injection drug use. The Center for Disease Control's 2015 ranking of counties vulnerable to rapid dissemination of HIV/HCV infection among people who inject drugs (PWID) places Dickenson County, Virginia at 29th in the nation and 2nd in Virginia. Comprehensive Harm Reduction is an evidence-based intervention shown to reduce the negative impacts of drug use including the spread of infectious diseases, overdose and death among people who are unable or not ready to stop using drugs. The aim of this study was to assess community perceptions of comprehensive harm reduction programs and stigma towards PWID in a rural community. Data were collected through the use of an anonymous survey and interviews with community stakeholders. Surveys were distributed online through email and social media, as well as in person. Participants were included based on their zip code as an indicator of residence in Dickenson County. Statistical analysis of survey results was conducted using Sigma Plot. The survey was completed by 153 participants. The perception of PWID in rural Virginia is strikingly negative. Participants consider injection drug use a major problem in their community but have little to no knowledge of harm reduction programs and display high levels of stigma towards PWID. Additionally, higher levels of stigma towards PWID was associated with lower support for CHR programs. The results of this study show a definite and perceived need for harm reduction programs in rural Virginia. Challenges to implementing harm reduction programs include low levels of knowledge and high levels of stigma.

Barriers to HIV Testing Within a Sample of Spanish-speaking Latinx Gay, Bisexual, and Other Men Who Have Sex with Men: Implications for HIV Prevention and Care.

Gay, bisexual, and other men who have sex with men (GBMSM) have higher rates of HIV infection compared to the general population in the United States, and the infection rate is growing among Latinx GBMSM, compared to a decline in most other demographic subgroups. Uptake of pre-exposure prophylaxis (PrEP), a biomedical strategy designed to reduce HIV transmission, is very low among Latinx GBMSM. HIV testing is a critical first step in the HIV prevention and care continua. We analyzed data from a community-based sample of Latinx GBMSM in the southeastern United States to identify the most common HIV testing barriers and the factors associated with barriers. The five most commonly reported HIV testing barriers included not knowing where to get tested, not having health insurance, fear of being HIV positive, practicing safer sex and perceiving not needing to be tested, and not being recommended to get tested. Using multivariable logistic regression modeling, speaking only Spanish, being unemployed, and adhering to traditional notions of masculinity were associated with increased barriers to HIV testing. We recommend that interventions to increase HIV testing among Latinx GBMSM be in Spanish and use culturally congruent messaging, be accessible to those who are unemployed, and incorporate positive risk-reducing aspects of masculinity.

Phenotypic Prioritization of Diphyllin Derivatives That Block Filoviral Cell Entry by Vacuolar (H+ )-ATPase Inhibition.

Many viruses use endosomal pathways to gain entry into cells and propagate infection. Sensing of endosomal acidification is a trigger for the release of many virus cores into the cell cytosol. Previous efforts with inhibitors of vacuolar ATPase have been shown to block endosomal acidification and affect viral entry, albeit with limited potential for therapeutic selectivity. In this study, four novel series of derivatives of the vacuolar ATPase inhibitor diphyllin were synthesized to assess their potential for enhancing potency and anti-filoviral activity over cytotoxicity. Derivatives that suitably blocked cellular entry of Ebola pseudotyped virus were further evaluated as inhibitors of endosomal acidification and isolated human vacuolar ATPase activity. Several compounds with significant increases in potency over diphyllin in these assays also separated from cytotoxic doses in human cell models by >100-fold. Finally, three derivatives were shown to be inhibitors of replication-competent Ebola viral entry into primary macrophages with similar potencies and enhanced selectivity toward antiviral activity.

Cell based assay identifies TLR2 and TLR4 stimulating impurities in Interferon beta.

Immunogenicity can have devastating consequences on the safety and efficacy of therapeutic proteins. Therefore, evaluating and mitigating the risk of product immunogenicity is critical for the development these products. This study, showed that Betaseron and Extavia, which are reported to be more immunogenic among IFNß products in clinical usage, contain residual innate immune response modulating impurities (IIRMIs) capable of activating NF-κB and induced expression of inflammatory mediators. These IIRMIs were undetectable in Rebif or Avonex. The stimulatory effect was attributed solely to IIRMIs because it was evident in murine cells lacking the interferon receptor (IFNAR). The IIRMIs in Betaseron and Extavia triggered NF-κB activation in HEK-293 cells bearing TLR2 and TLR4 in MyD88 dependent manner. Importantly, the IIRMIs in Betaseron induced up-regulation of IL-6, IL-1ß, and ccl5 in the skin of IFNAR knock out mice following subcutaneous administration. This indicates that trace level IIRMIs in Betaseron could contribute to the higher immunogenicity rates seen in clinics. Together these data suggest that cell based assays can reveal subtle but clinically relevant differences in IIRMIs following manufacturing changes or between products with the same active ingredients but different manufacturing processes. Appreciating these differences may inform immunogenicity risk assessments.

Detection of innate immune response modulating impurities in therapeutic proteins.

Therapeutic proteins can contain multiple impurities, some of which are variants of the product, while others are derived from the cell substrate and the manufacturing process. Such impurities, even when present at trace levels, have the potential to activate innate immune cells in peripheral blood or embedded in tissues causing expression of cytokines and chemokines, increasing antigen uptake, facilitating processing and presentation by antigen presenting cells, and fostering product immunogenicity. Currently, while products are tested for host cell protein content, assays to control innate immune response modulating impurities (IIRMIs) in products are focused mainly on endotoxin and nucleic acids, however, depending on the cell substrate and the manufacturing process, numerous other IIRMI could be present. In these studies we assess two approaches that allow for the detection of a broader subset of IIRMIs. In the first, we use commercial cell lines transfected with Toll like receptors (TLR) to detect receptor-specific agonists. This method is sensitive to trace levels of IIRMI and provides information of the type of IIRMIs present but is limited by the availability of stably transfected cell lines and requires pre-existing knowledge of the IIRMIs likely to be present in the product. Alternatively, the use of a combination of macrophage cell lines of human and mouse origin allows for the detection of a broader spectrum of impurities, but does not identify the source of the activation. Importantly, for either system the lower limit of detection (LLOD) of impurities was similar to that of PBMC and it was not modified by the therapeutic protein tested, even in settings where the product had inherent immune modulatory properties. Together these data indicate that a cell-based assay approach could be used to screen products for the presence of IIRMIs and inform immunogenicity risk assessments, particularly in the context of comparability exercises.