A Novel Target and Approach for Identifying Antivirals against Molluscum Contagiosum Virus

The dermatological disease molluscum contagiosum (MC) presents as lesions restricted solely to the skin. The poxvirus molluscum contagiosum virus (MCV) is responsible for this skin disease that is easily transmitted through casual contact among all populations, with greater frequency in children and immunosuppressed individuals. In addition, sexual transmission of MCV in adolescents and adults is a health concern. Although the skin lesions ultimately resolve in immunocompetent individuals, they can persist for extended periods, be painful, and result in scarring. Treatment is problematic, and there is no drug that specifically targets MCV. The inability of MCV to propagate in cell culture has impeded drug development. To overcome these barriers, we integrated three new developments. First, we identified a new MCV drug target (mD4) that is essential for processive DNA synthesis in vitro. Second, we discovered a small chemical compound that binds to mD4 and prevents DNA synthesis in vitro. Third, and most significant, we engineered a hybrid vaccinia virus (mD4-VV) in which the natural vaccinia D4 (vD4) gene is replaced by the mD4 target gene. This hybrid virus is dependent on mD4 for viral growth in culture and is inhibited by the small compound. This target system provides, for the first time, a platform and approach for the discovery and evaluation of new therapeutics that can be used to treat MC.     

Identification of protein-protein interaction inhibitors targeting vaccinia virus processivity factor for development of antiviral agents

Poxvirus uracil DNA glycosylase D4 in association with A20 and the catalytic subunit of DNA polymerase forms the processive polymerase complex. The binding of D4 and A20 is essential for processive polymerase activity. Using an AlphaScreen assay, we identified compounds that inhibit protein-protein interactions between D4 and A20. Effective interaction inhibitors exhibited both antiviral activity and binding to D4. These results suggest that novel antiviral agents that target the protein-protein interactions between D4 and A20 can be developed for the treatment of infections with poxviruses, including smallpox.     

Friction and Mechanical Properties of Highly Filled Polybenzoxazine Composites: Nanosilica Particle Size and Surface Treatment

Frictional and mechanical properties of highly filled polybenzoxazine [poly(BA‐a)] composites which are influenced by nanosilica contents, particle sizes, and surface treatments are investigated. The coefficient of friction and wear resistance, storage moduli, and microhardness of the nanosilica‐filled poly(BA‐a) composites systematically increase with an increase of nanosilica content, while those values of the nanocomposites are improved with decreasing particle sizes at the equivalent nanosilica content. The modulus can be predicted by the Kerner model with the maximum packing fraction, while the microhardness of the nanocomposites is in agreement with the Halpin–Tsai model. The nanocomposites fabricated with untreated nanosilica particles exhibit higher frictional and mechanical properties when compared with the surface‐treated nanocomposites at the equivalent particle sizes. The interfacial interactions via covalent bond formation between the nanosilica and the poly(BA‐a) are determinative factors for the nanocomposite properties. Highly filled nanosilica‐poly(BA‐a) composites can be employed in various applications where wear‐resistance plays an important role.     

Sensitivity of the modified Angoff standard‐setting method to variations in item content

Background: The faculty development community has been challenged to more rigorously assess program impact and move beyond traditional outcomes of knowledge tests and self ratings. Purpose: The purpose was to (a) assess our ability to measure supervisors' feedback skills as demonstrated in a clinical setting and (b) compare the results with traditional outcome measures of faculty development interventions. Methods: A pre–post study design was used. Resident and expert ratings of supervisors' demonstrated feedback skills were compared with traditional outcomes, including a knowledge test and participant self-evaluation. Results: Pre–post knowledge increased significantly (pre = 61%, post = 85%; p < .001) as did participant's self-evaluation scores (pre = 4.13, post = 4.79; p < .001). Participants' self-evaluations were moderately to poorly correlated with resident (pre r = .20, post r = .08) and expert ratings (pre r = .43, post r = ?.52). Residents and experts would need to evaluate 110 and 200 participants, respectively, to reach significance. Conclusions: It is possible to measure feedback skills in a clinical setting. Although traditional outcome measures show a significant effect, demonstrating change in teaching behaviors used in practice will require larger scale studies than typically undertaken currently.     

Identification of inhibitors that block vaccinia virus infection by targeting the DNA synthesis processivity factor D4

Smallpox was globally eradicated 30 years ago by vaccination. The recent threat of bioterrorism demands the development of improved vaccines and novel therapeutics to effectively preclude a reemergence of smallpox. One new therapeutic target is the vaccinia poxvirus processivity complex, comprising D4 and A20 proteins that enable the viral E9 DNA polymerase to synthesize extended strands. Five compounds identified from an AlphaScreen assay designed to disrupt A20:D4 binding were shown to be effective in: (i) blocking vaccinia processive DNA synthesis in vitro, (ii) preventing cellular infection with minimal cytotoxicity, and (iii) binding to D4, as evidenced by ThermoFluor. The EC(50) values for inhibition of viral infectivity ranged from 9.6 to 23 μM with corresponding selectivity indices (cytotoxicity CC(50)/viral infectivity EC(50)) of 3.9 to 17.8. The five compounds are thus potential therapeutics capable of halting smallpox DNA synthesis and infectivity through disruptive action against a component of the vaccinia processivity complex.     

Establishment of an Aerosol Challenge Model of Tuberculosis in Rhesus Macaques and an Evaluation of Endpoints for Vaccine Testing

The establishment of an aerosol challenge model in nonhuman primates (NHPs) for the testing of vaccines against Mycobacterium tuberculosis would assist the global effort to optimize novel vaccination strategies. The endpoints used in preclinical challenge studies to identify measures of disease burden need to be accurate and sensitive enough to distinguish subtle differences and benefits afforded by different tuberculosis (TB) vaccine regimens when group sizes are inevitably small. This study sought to assess clinical and nonclinical endpoints as potentially sensitive measures of disease burden in a challenge study with rhesus macaques by using a new protocol of aerosol administration of M. tuberculosis. Immunological and clinical readouts were assessed for utility in vaccine evaluation studies. This is the first example of TB vaccine evaluation with rhesus macaques where long-term survival was one of the primary endpoints. However, we found that in NHP vaccine efficacy studies with maximum group sizes of six animals, survival did not provide a valuable endpoint. Two approaches used in human clinical trials for the evaluation of the gamma interferon (IFN-γ) response to vaccination (enzyme-linked immunospot [ELISpot] assay and enzyme-linked immunosorbent assay [ELISA]) were included in this study. The IFN-γ profiles induced following vaccination were found not to correlate with protection, nor did the level of purified protein derivative (PPD)-specific proliferation. The only readout to reliably distinguish vaccinated and unvaccinated NHPs was the determination of lung lesion burden using magnetic resonance (MR) imaging combined with stereology at the end of the study. Therefore, the currently proposed key markers were not shown to correlate with protection, and only imaging offered a potentially reliable correlate.Tuberculosis (TB) is a reemerging infectious disease and is responsible for nearly 2 million deaths and 9 million new cases each year (36). The global TB pandemic has been exacerbated by the emergence of drug-resistant strains of Mycobacterium tuberculosis, which render treatment less effective, and by the HIV epidemic, where coinfection with HIV greatly increases the risk of reactivation of latent TB and susceptibility to active TB disease.The most effective means of controlling this global epidemic would be by prophylactic immunization. Mycobacterium bovis bacille Calmette-Guérin (BCG), the only licensed TB vaccine, is administered to neonates in high-risk populations as part of the WHO Expanded Programme on Immunization. BCG consistently protects against TB meningitis and disseminated TB in childhood (27, 30), but its efficacy wanes with time, and it affords only variable protection against pulmonary disease (10). A new, more effective TB vaccine is a major global health priority and is an important part of the WHO STOP TB partnership strategy.A large international effort is under way to develop a more effective vaccine. The leading TB vaccine development strategy involves vaccination with BCG followed by a heterologous subunit vaccine boost designed to enhance protective immunity. One such subunit vaccine is the virus-vectored subunit candidate TB vaccine developed at Oxford University, MVA85A (19) (live, replication-deficient, modified vaccinia virus Ankara [MVA] [7], expressing the highly conserved, immunodominant mycobacterial antigen 85A [Ag85A]). The enhancement of BCG with systemically administered (intradermal) MVA85A has been evaluated with several preclinical animal models and is currently the subject of ongoing evaluations in several clinical trials. This BCG-MVA85A vaccination regimen induces a high magnitude of cellular immunity in mice and cattle (19, 32) and can protect against M. tuberculosis in guinea pigs (34), nonhuman primates (NHPs) (31), and cattle (33). It is safe and highly immunogenic in healthy adults, adolescents, children, infants, and HIV- and M. tuberculosis-infected adults (6, 20, 21) and has recently entered a large-scale efficacy trial with South African infants (http://clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT00953927","term_id":"NCT00953927"}}NCT00953927).The lack of a defined immunological correlate of protection for TB means that in order to assess efficacy, candidate TB vaccines must enter large clinical trials involving thousands of at-risk individuals in countries where the disease is endemic. Therefore, there is a need for a validated preclinical animal model that can be utilized to accurately predict the effectiveness of a candidate vaccine in humans and to aid in the identification of correlates of protection through challenge studies. Mouse models are generally used as a first screen of vaccine candidates and are very useful for studying detailed immunological responses (24). Guinea pigs are considered a more stringent model than mice to discriminate between vaccines in terms of protective efficacy, since they show a variety of pulmonary and extrapulmonary lesion types that are similar to those observed for humans (4, 17). Although small-animal models are useful, it is widely accepted that larger animals such as cattle and NHPs are potentially the most relevant model species to predict safety, immunogenicity, and protective efficacy of vaccines prior to their large-scale evaluation in humans (8, 18). NHPs are naturally susceptible to infection with M. tuberculosis via the respiratory route and develop a disease that clinically closely mimics human disease. As with the other preclinical species, BCG vaccination of NHPs provides a limited level of protection against M. tuberculosis that can be quantified through a variety of clinical and nonclinical parameters (2, 3, 9, 12, 14).The establishment of an aerosol challenge model in NHPs, in which the M. tuberculosis challenge is delivered by the same route as that which occurs during natural infection, would assist the global effort in optimizing novel vaccination strategies. The endpoints used in preclinical challenge studies to identify measures of disease burden need to be accurate and sensitive enough to distinguish subtle differences and benefits afforded by different TB vaccine regimens, including those that enhance the protective efficacy of BCG, where partial protection is already conferred and the power to detect smaller incremental improvements in small numbers of animals is limited. The aims of this study were to establish an aerosol challenge model of TB in rhesus macaques and to assess clinical and nonclinical endpoints as potentially sensitive measures of disease burden in an NHP challenge study following vaccination with BCG or BCG boosted by MVA85A. The currently most widely used challenge model with macaques uses high-dose, intratracheal administration of M. tuberculosis, and it has been unclear whether low-dose aerosol administration would provide a better model, because although it should replicate the route of natural infection (31), larger group sizes may be necessary to compensate for interindividual heterogeneity to a low-dose challenge (8).     

Long-term renal outcome in pediatric glomerulonephritis associated with crescent formation

Background

Information on long-term renal outcome of pediatric glomerulonephritis associated with crescent formation is limited. A single center retrospective study was conducted to assess long-term renal survival and to determine whether the 2010 classification for antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis can predict renal outcome in pediatric glomerulonephritis associated with crescent formation.

Methods

Biopsy and clinical data of children, aged ≤ 18 years with ≥ 10 glomeruli and ≥ 10% crescentic glomeruli during January 1998 to December 2015, were reviewed. Biopsies were classified according to the 2010 classification into focal, crescentic, mixed, and sclerotic classes. The clinical endpoint was end-stage renal disease (ESRD).

Results

Of 72 children, 14 patients (19.4%) had positive ANCA. The biopsy indication was rapidly progressive glomerulonephritis in 38 patients (52.8%) and 22 patients (30.6%) required dialysis at onset. Lupus nephritis was the most common diagnosis (43.1%), followed by IgA nephropathy/Henoch–Schoenlein purpura (HSP) (22.2%). ESRD occurred in 18 patients (25%) and the risk of ESRD differed among the histological classifications (p < 0.001). Dialysis at onset and sclerotic class was independent predictors of ESRD in an adjusted model. The risk of ESRD was four-fold higher in patients requiring dialysis at onset and 7.7-fold higher in sclerotic patients than in crescentic patients.

Conclusions

The probability of ESRD was substantial in pediatric glomerulonephritis associated with crescent formation. The 2010 classification is useful for establishing long-term renal prognosis. Future research is required to validate whether histological classification could be a determinant in therapeutic guideline modification, since long-term renal prognosis is different in each class.