Scanning-fluorescence Reader Based on Embedded System

To measure the concentration of C-reactive protein (CRP) in serum, a portable, scanning-fluorescence reader based on time-resolved fluoroimmunoassays was developed. The scanning-fluorescence reader integrates with the AD7707 converter, which performs at a high accuracy. The photosensitive diode acts as the photoelectric conversion device, an optical module based on optical fibers, which is able to concentrate the excitation light from an LED into a line-shape beam, was designed to sendand receive the optical signal. The device subsequently addresses waveform data using a gradient, smoothing, and binarization method. When the device measures the CRP fluorescence test strip, the results exhibited a good linearity (0.99998) and the CVs (coefficient of variation) were below 5%, which indicate high accuracy. At the same time the system is low cost and small size

Functional assessment and structural basis of antibody binding to human papillomavirus capsid

Persistent high-risk human papillomavirus (HPV) infection is linked to cervical cancer. Two prophylactic virus-like particle (VLP)-based vaccines have been marketed globally for nearly a decade. Here, we review the HPV pseudovirion (PsV)-based assays for the functional assessment of the HPV neutralizing antibodies and the structural basis for these clinically relevant epitopes. The PsV-based neutralization assay was developed to evaluate the efficacy of neutralization antibodies in sera elicited by vaccination or natural infection or to assess the functional characteristics of monoclonal antibodies. Different antibody binding modes were observed when an antibody was complexed with virions, PsVs or VLPs. The neutralizing epitopes are localized on surface loops of the L1 capsid protein, at various locations on the capsomere. Different neutralization antibodies exert their neutralizing function via different mechanisms. Some antibodies neutralize the virions by inducing conformational changes in the viral capsid, which can result in concealing the binding site for a cellular receptor like 1A1D-2 against dengue virus, or inducing premature genome release like E18 against enterovirus 71. Higher-resolution details on the epitope composition of HPV neutralizing antibodies would shed light on the structural basis of the highly efficacious vaccines and aid the design of next generation vaccines. In-depth understanding of epitope composition would ensure the development of function-indicating assays for the comparability exercise to support process improvement or process scale up. Elucidation of the structural elements of the type-specific epitopes would enable rational design of cross-type neutralization via epitope re-engineering or epitope grafting in hybrid VLPs.The authors acknowledge the funding support from the Chinese government: National 863 Program of China (2014AA021302), National Natural Science Fund of China (81373061 and 81471934) and Fujian Provincial Program for Construction Plan of Science and Technology Innovation Platform (2014Y2101). This work was also supported by a Senior Research Fellowship from the Welcome Trust, grant number 101908/Z/13/Z, to Y.M.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/rmv.186

Functional assessment and structural basis of antibody binding to human papillomavirus capsid.

Persistent high-risk human papillomavirus (HPV) infection is linked to cervical cancer. Two prophylactic virus-like particle (VLP)-based vaccines have been marketed globally for nearly a decade. Here, we review the HPV pseudovirion (PsV)-based assays for the functional assessment of the HPV neutralizing antibodies and the structural basis for these clinically relevant epitopes. The PsV-based neutralization assay was developed to evaluate the efficacy of neutralization antibodies in sera elicited by vaccination or natural infection or to assess the functional characteristics of monoclonal antibodies. Different antibody binding modes were observed when an antibody was complexed with virions, PsVs or VLPs. The neutralizing epitopes are localized on surface loops of the L1 capsid protein, at various locations on the capsomere. Different neutralization antibodies exert their neutralizing function via different mechanisms. Some antibodies neutralize the virions by inducing conformational changes in the viral capsid, which can result in concealing the binding site for a cellular receptor like 1A1D-2 against dengue virus, or inducing premature genome release like E18 against enterovirus 71. Higher-resolution details on the epitope composition of HPV neutralizing antibodies would shed light on the structural basis of the highly efficacious vaccines and aid the design of next generation vaccines. In-depth understanding of epitope composition would ensure the development of function-indicating assays for the comparability exercise to support process improvement or process scale up. Elucidation of the structural elements of the type-specific epitopes would enable rational design of cross-type neutralization via epitope re-engineering or epitope grafting in hybrid VLPs.The authors acknowledge the funding support from the Chinese government: National 863 Program of China (2014AA021302), National Natural Science Fund of China (81373061 and 81471934) and Fujian Provincial Program for Construction Plan of Science and Technology Innovation Platform (2014Y2101). This work was also supported by a Senior Research Fellowship from the Welcome Trust, grant number 101908/Z/13/Z, to Y.M.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/rmv.186

Epidemiology of anal human papillomavirus infection and high-grade squamous intraepithelial lesions in 29 900 men according to HIV status, sexuality, and age: a collaborative pooled analysis of 64 studies

Age Factors; Anal human papillomavirus infection; SexualityFactores de edad; Infección anal por virus del papiloma humano; SexualidadFactors d'edat; Infecció anal pel virus del papil·loma humà; SexualitatBackground Robust age-specific estimates of anal human papillomavirus (HPV) and high-grade squamous intraepithelial lesions (HSIL) in men can inform anal cancer prevention efforts. We aimed to evaluate the age-specific prevalence of anal HPV, HSIL, and their combination, in men, stratified by HIV status and sexuality. Methods We did a systematic review for studies on anal HPV infection in men and a pooled analysis of individual-level data from eligible studies across four groups: HIV-positive men who have sex with men (MSM), HIV-negative MSM, HIV-positive men who have sex with women (MSW), and HIV-negative MSW. Studies were required to inform on type-specific HPV infection (at least HPV16), detected by use of a PCR-based test from anal swabs, HIV status, sexuality (MSM, including those who have sex with men only or also with women, or MSW), and age. Authors of eligible studies with a sample size of 200 participants or more were invited to share deidentified individual-level data on the above four variables. Authors of studies including 40 or more HIV-positive MSW or 40 or more men from Africa (irrespective of HIV status and sexuality) were also invited to share these data. Pooled estimates of anal high-risk HPV (HR-HPV, including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), and HSIL or worse (HSIL+), were compared by use of adjusted prevalence ratios (aPRs) from generalised linear models. Findings The systematic review identified 93 eligible studies, of which 64 contributed data on 29 900 men to the pooled analysis. Among HIV-negative MSW anal HPV16 prevalence was 1·8% (91 of 5190) and HR-HPV prevalence was 6·9% (345 of 5003); among HIV-positive MSW the prevalences were 8·7% (59 of 682) and 26·9% (179 of 666); among HIV-negative MSM they were 13·7% (1455 of 10 617) and 41·2% (3798 of 9215), and among HIV-positive MSM 28·5% (3819 of 13 411) and 74·3% (8765 of 11 803). In HIV-positive MSM, HPV16 prevalence was 5·6% (two of 36) among those age 15–18 years and 28·8% (141 of 490) among those age 23–24 years (ptrend=0·0091); prevalence was 31·7% (1057 of 3337) among those age 25–34 years and 22·8% (451 of 1979) among those age 55 and older (ptrend<0·0001). HPV16 prevalence in HIV-negative MSM was 6·7% (15 of 223) among those age 15–18 and 13·9% (166 of 1192) among those age 23–24 years (ptrend=0·0076); the prevalence plateaued thereafter (ptrend=0·72). Similar age-specific patterns were observed for HR-HPV. No significant differences for HPV16 or HR-HPV were found by age for either HIV-positive or HIV-negative MSW. HSIL+ detection ranged from 7·5% (12 of 160) to 54·5% (61 of 112) in HIV-positive MSM; after adjustment for heterogeneity, HIV was a significant predictor of HSIL+ (aPR 1·54, 95% CI 1·36–1·73), HPV16-positive HSIL+ (1·66, 1·36–2·03), and HSIL+ in HPV16-positive MSM (1·19, 1·04–1·37). Among HPV16-positive MSM, HSIL+ prevalence increased with age. Interpretation High anal HPV prevalence among young HIV-positive and HIV-negative MSM highlights the benefits of gender-neutral HPV vaccination before sexual activity over catch-up vaccination. HIV-positive MSM are a priority for anal cancer screening research and initiatives targeting HPV16-positive HSIL+.International Agency for Research on Cancer

Characterization and Analysis of Real-Time Capillary Convective PCR Toward Commercialization

Almost all the reported capillary convective polymerase chain reaction (CCPCR) systems to date are still limited to research use stemming from unresolved issues related to repeatability, reliability, convenience, and sensitivity. To move CCPCR technology forward toward commercialization, a couple of critical strategies and innovations are discussed here. First, single- and dual-end heating strategies are analyzed and compared between each other. Especially, different solutions for dual-end heating are proposed and discussed, and the heat transfer and fluid flow inside the capillary tube with an optimized dual-end heating strategy are analyzed and modeled. Second, real-time CCPCR is implemented with light-emitting diode and photodiode, and the real-time fluorescence detection method is compared with the post-amplification end-point detection method based on a dipstick assay. Thirdly, to reduce the system complexity, e.g., to simplify parameter tuning of the feedback control, an internal-model-control-based proportional-integral-derivative controller is adopted for accurate temperature control. Fourth, as a proof of concept, CCPCR with pre-loaded dry storage of reagent inside the capillary PCR tube is evaluated to better accommodate to point-of-care diagnosis. The critical performances of improved CCPCR, especially with sensitivity, repeatability, and reliability, have been thoroughly analyzed with different experiments using influenza A (H1N1) virus as the detection sample. Published by AIP Publishing

Efficient inhibition of HIV-1 replication by an artificial polycistronic miRNA construct

10.1186/1743-422X-9-118Virology Journal9

Real-time stability of a hepatitis E vaccine (Hecolin®) demonstrated with potency assays and multifaceted physicochemical methods

The first prophylactic vaccine against hepatitis E virus (HEV), Hecolin®, was licensed in China. Recombinant p239 virus-like particle (VLP) is its active component with dimeric protein as the basic building block harboring the immuno dominant and neutralizing epitopes. The real time and real condition stability of the prefilled syringes for the vaccine was demonstrated using both in vivo mouse potency and in vitro antigenicity assays. A total of 12 lots of Hecolin® were assessed with a set of assays after storage at 2-8 °C for 24 months. The particle characteristics of p239 VLP recovered from the aluminum-containing adjuvant was assessed with different methods including analytical ultracentrifugation, high performance size exclusion chromatography and transmission electron microscopy. The thermal and conformational stability of the adsorbed antigen was assessed using differential scanning calorimetry. The protein integrity of the recovered p239 antigen was demonstrated using SDS-PAGE with silvering staining, LC-MS and MALDI-TOF MS. Most importantly, the binding activity to the neutralizing antibody or vaccine antigenicity was measured using an epitope-specific and real-time SPR assay and a monoclonal antibody-based sandwich ELISA. Taken together, the overall good stability of the Hecolin® prefilled syringes was demonstrated with unaltered molecular and functional attributes after storage at 2-8 °C for 24 months

Case Report: Chronic hepatitis E virus Infection in an individual without evidence for immune deficiency

Chronic hepatitis E virus (HEV) infection occurs mainly in immunosuppressed populations. We describe an investigation of chronic HEV infection of genotype 3a in an individual without evidence for immune deficiency who presented hepatitis with significant HEV viremia and viral shedding. We monitored HEV RNA in plasma and stools, and assessed anti-HEV specific immune responses. The patient was without apparent immunodeficiency based on quantified results of white blood cell, lymphocyte, neutrophilic granulocyte, CD3+ T cell, CD4+ T cell, and CD8+ T cell counts and CD4/CD8 ratio, as well as total serum IgG, IgM, and IgA, which were in the normal range. Despite HEV specific cellular response and strong humoral immunity being observed, viral shedding persisted up to 109 IU/mL. After treatment with ribavirin combined with interferon, the indicators of liver function in the patient returned to normal, accompanied by complete suppression and clearance of HEV. These results indicate that HEV chronicity can also occur in individuals without evidence of immunodeficiency

Intravenous Injections of a Rationally Selected Oncolytic Herpes Virus as a Potent Virotherapy for Hepatocellular Carcinoma

As a clinical setting in which novel treatment options are urgently needed, hepatocellular carcinoma (HCC) exhibits intriguing opportunities for oncolytic virotherapy. Here we report the rational generation of a novel herpes simplex virus type 1 (HSV-1)-based oncolytic vector for targeting HCC, named Ld0-GFP, which was derived from oncolytic ICP0-null virus (d0-GFP), had a fusogenic phenotype, and was a novel killer against HCC as well as other types of cancer cells. Compared with d0-GFP, Ld0-GFP exhibited superior cancer cell-killing ability in vitro and in vivo . Ld0-GFP targets a broad spectrum of HCC cells and can result in significantly enhanced immunogenic tumor cell death. Intratumoral and intravenous injections of Ld0-GFP showed effective antitumor capabilities in multiple tumor models, leading to increased survival. We speculated that more active cell-killing capability of oncolytic virus and enhanced immunogenic cell death may lead to better tumor regression. Additionally, Ld0-GFP had an improved safety profile, showing reduced neurovirulence and systemic toxicity. Ld0-GFP virotherapy could offer a potentially less toxic, more effective option for both local and systemic treatment of HCC. This approach also provides novel insights toward ongoing efforts to develop an optimal oncolytic vector for cancer therapy

Transcriptional response of USP18 predicts treatment outcomes of interferon-alpha in HBeAg-positive chronic hepatitis B patientsefere.

Ubiquitin-specific protease 18 (USP18) is an important inhibitor of interferon (IFN) antiviral activity, and the aim of this study was to investigate the association between the USP18 mRNA level change in peripheral blood mononuclear cells (PBMCs) when stimulated with IFN in vitro before initiating treatment and the treatment outcomes in HBeAg-positive chronic hepatitis B (CHB) patients treated with IFN. A total of 44 patients who received standard IFN-based anti-HBV therapy and follow-up were enrolled in the study. The in vitro IFN-induced USP18 mRNA change (USP18IFN-N ) was measured via comparison of quantitative PCR-determined USP18 transcription levels of BPMCs cultured with and without IFN stimulation. Either for virological (VR) or serological response (SR), the baseline USP18IFN-N was significantly higher (P = 0.018 for VR, P = 0.008 for SR) among nonresponders (n = 23 for VR, n = 33 for SR) than that of responders (n = 21 for VR, n = 11 for SR). Multivariate analyses revealed baseline USP18IFN-N was a novel independent predictor for either VR (OR = 0.292, 95% CI = 0.102-0.835, P = 0.022) or SR (OR = 0.173, 95% CI = 0.035-0.849, P = 0.031) in our cohort. In addition, baseline USP18IFN-N in combination with HBV DNA loads or HBeAg levels showed improved accuracy of pretreatment prediction for VR or SR responders, respectively. Baseline USP18IFN-N levels are associated with both virological and serological response, and have the potential to become a clinical predictor for treatment outcomes in HBeAg-positive CHB patients before initiating IFN-α therapy