Ultra-fast, sensitive and low-cost real-time PCR system for nucleic acid detection.

Nucleic acid detection directly identifies the presence of pathogenic microorganisms and has various advantages, such as high sensitivity, commendable specificity and a short window period, and has been widely used in many fields, such as early tumor screening, prenatal diagnosis and infectious disease detection. Real-time PCR (polymerase chain reaction) is the most commonly used method for nucleic acid detection in clinical practice, but it always takes about 1-3 hours, severely limiting its application in particular scenarios such as emergency testing, large-scale testing and on-site testing. To solve the time-consuming problem, a real-time PCR system based on multiple temperature zones was proposed, which realized the speed of temperature change of biological reagents from 2-4 °C s-1 to 13.33 °C s-1. The system integrates the advantages of fixed microchamber-type and microchannel-type amplification systems, including a microfluidic chip capable of fast heat transfer and a real-time PCR device with a temperature control strategy based on the temperature difference. The detection of HCMV biological samples using the real-time PCR system in this research took only 15 min, which was 75% shorter compared to the commercial qPCR instrument such as BIO-RAD, and the detection sensitivity remained essentially the same. The system could complete nucleic acid detection within 9 min under extreme conditions, characterized by fast detection speed and high sensitivity, providing a promising solution for ultra-fast nucleic acid detection.

An Automated TW3-RUS Bone Age Assessment Method with Ordinal Regression-Based Determination of Skeletal Maturity.

The assessment of bone age is important for evaluating child development, optimizing the treatment for endocrine diseases, etc. And the well-known Tanner-Whitehouse (TW) clinical method improves the quantitative description of skeletal development based on setting up a series of distinguishable stages for each bone individually. However, the assessment is affected by rater variability, which makes the assessment result not reliable enough in clinical practice. The main goal of this work is to achieve a reliable and accurate skeletal maturity determination by proposing an automated bone age assessment method called PEARLS, which is based on the TW3-RUS system (analysis of the radius, ulna, phalanges, and metacarpal bones). The proposed method comprises the point estimation of anchor (PEA) module for accurately localizing specific bones, the ranking learning (RL) module for producing a continuous stage representation of each bone by encoding the ordinal relationship between stage labels into the learning process, and the scoring (S) module for outputting the bone age directly based on two standard transform curves. The development of each module in PEARLS is based on different datasets. Finally, corresponding results are presented to evaluate the system performance in localizing specific bones, determining the skeletal maturity stage, and assessing the bone age. The mean average precision of point estimation is 86.29%, the average stage determination precision is 97.33% overall bones, and the average bone age assessment accuracy is 96.8% within 1 year for the female and male cohorts.

Comprehensive Assessment of the Antigenic Impact of Human Papillomavirus Lineage Variation on Recognition by Neutralizing Monoclonal Antibodies Raised against Lineage A Major Capsid Proteins of Vaccine-Related Genotypes.

Human papillomavirus (HPV) is the causative agent of cervical and other epithelial cancers. Naturally occurring variants of HPV have been classified into lineages and sublineages based on their whole-genome sequences, but little is known about the impact of this diversity on the structure and function of viral gene products. The HPV capsid is an icosahedral lattice comprising 72 pentamers of the major capsid protein (L1) and the associated minor capsid protein (L2). We investigated the potential impact of this genome variation on the capsid antigenicity of lineage and sublineage variants of seven vaccine-relevant, oncogenic HPV genotypes by using a large panel of monoclonal antibodies (MAbs) raised against the L1 proteins of lineage A antigens. Each genotype had at least one variant that displayed a ≥4-fold reduced neutralizing antibody sensitivity against at least one MAb, demonstrating that naturally occurring variation can affect one or more functional antigenic determinants on the HPV capsid. For HPV16, HPV18, HPV31, and HPV45, the overall impact was of a low magnitude. For HPV33 (sublineages A2 and A3 and lineages B and C), HPV52 (lineage D), and HPV58 (lineage C), however, variant residues in the indicated lineages and sublineages reduced their sensitivity to neutralization by all MAbs by up to 1,000-fold, suggesting the presence of key antigenic determinants on the surface of these capsids. These determinants were resolved further by site-directed mutagenesis. These data improve our understanding of the impact of naturally occurring variation on the antigenicity of the HPV capsid of vaccine-relevant oncogenic HPV genotypes.IMPORTANCE Human papillomavirus (HPV) is the causative agent of cervical and some other epithelial cancers. HPV vaccines generate functional (neutralizing) antibodies that target the virus particles (or capsids) of the most common HPV cancer-causing genotypes. Each genotype comprises variant forms that have arisen over millennia and which include changes within the capsid proteins. In this study, we explored the potential for these naturally occurring variant capsids to impact recognition by neutralizing monoclonal antibodies. All genotypes included at least one variant form that exhibited reduced recognition by at least one antibody, with some genotypes affected more than others. These data highlight the impact of naturally occurring variation on the structure of the HPV capsid proteins of vaccine-relevant oncogenic HPV genotypes.

An immunoassay cassette with a handheld reader for HIV urine testing in point-of-care diagnostics.

Currently, most HIV tests are performed with blood samples, or alternatively saliva samples are used for HIV testing. Simple HIV tests need to be performed in hospitals or other medical agencies instead of more invasive HIV blood tests. To enable point-of-care (POC) HIV diagnostics, based on a recently developed lateral flow strip for HIV urine testing, a microfluidic immunoassay cassette with a handheld optical reader is developed. Based on lateral flow strip with gold colloid reporter, the integrated immunoassay cassette can perform sample introduction, metering, discharging, applying and detection which simplifies HIV testing. An indicator is incorporated into the cassette to guide sample introduction based on color change, and further, the excess test sample is stored inside the sealed cassette to avoid any contamination. The low-cost handheld optical reader can provide a test result within a few seconds, which is useful for simple, sensitive and affordable HIV onsite detection. Instead of using normal white LEDs, a customized back light module embedded with green LEDs is adopted to illuminate the lateral flow strip with an appropriate working current to achieve optimal performance. Compared to the standard lateral flow strips using a benchtop reader, with the disposable immunoassay cassette assisted by the handheld optical reader, more convenient, easier-to-operate, and more affordable HIV urine testing can be achieved in POC diagnostics.

An efficient isothermal PCR method for on-site detection of nucleic acid.

Convective PCR (CPCR) is an isothermal nucleic acid amplification technology; however, natural convection exhibits a chaotic and multiplex flow state, resulting in low amplification efficiency and specificity. We placed a polycarbonate strip (p-strip) inside reaction tubes to induce circumfluence by blocking the inner ring that originally allowed fluid to flow at suboptimal temperatures. Moreover, we constructed a dual-temperature instrument to provide appropriate denaturing and annealing zones for CPCR. Tubes containing p-strips exhibited significantly improved efficiency, sensitivity and specificity. For real-time detection, the variation coefficients of three replicates having the same concentrations were less than 2% in more than half of the cases, indicating improved CPCR amplification and potential as a commercial on-site nucleic acid diagnosis tool.

An automated microfluidic chemiluminescence immunoassay platform for quantitative detection of biomarkers.

A rapid, sensitive and quantitative biomarker detection platform is of great importance to the small clinic or point-of-care (POC) diagnosis. In this work, we realize that an automated diagnostic platform mainly includes two components: (1) an instrument that can complete all steps of the chemiluminescence immunoassay automatically and (2) an integrated microfluidic chip which is disposable and harmless. In the instrument, we adopt vacuum suction cups which are driven by linear motor to realize a simple, effective and convenient control. The method of acridine esterification chemiluminescence is adopted to achieve a quantitative detection, and a photomultiplier tube is used to detect photons from acridine ester producing in alkaline conditions. We use the laser cutting machine and hot press machine to accomplish the product of microfluidic chips. The automated microfluidics-based system is demonstrated by implementation of a chemiluminescence immunoassay for quantitative detection of ferritin. We observe alinear relationship between CL intensity and the concentration of ferritin from 5.1 to 1300 ng mL -1and the limit of detection (LoD) is 2.55 ng mL -1. At the same time, we also used the automated microfluidics-based system to test clinical serum samples. The whole process of chemiluminescence experiment can complete within 45 min. We realize that this lab-on-a-chip chemiluminescence immunoassay platform with features of automation and quantitation provides a promising strategy for POC diagnosis.

A low cost, membranes based serum separator modular.

To fulfill the requirement of sample preparation in a microfluidic analysis system designed for "sample in, answer out" testing which was urgently needed by resource limited clinical facilities, we proposed a critical low cost, membrane-based serum separator design in this article. With a specially designed microchip, this device can easily separate serum from the whole blood sample in 5 min. Different from techniques which have been reported earlier, this approach does not require either centrifugation or sample dilution which may cause hemolysis or decreased testing sensitivity. By applying 300 µl of the whole blood sample, 50-70 µl of serum can be recovered from each device, and the serum volume recovery rate compared with centrifuged control is around 73% which is sufficient for most of the microfluidic-based assays. The protein recovery rate ranged from 70% to 95% which was compared with centrifuged control. The evaluation results indicate that this sample preparation device can offer sufficient amount of purified serum sample for any kind of diagnostic assays such as immunoassay and serum nucleic acid assay.

Characterization of an Escherichia coli-derived human papillomavirus type 16 and 18 bivalent vaccine.

Human papillomavirus (HPV) types 16 and 18 account for approximately 70% of cervical cancer worldwide. Neutralizing HPV prophylactic vaccines offer significant benefit, as they block HPV infection and prevent subsequent disease. However, the three licensed HPV vaccines that cover these two genotypes were produced in eukaryotic cells, which is expensive, particularly for low-income countries where HPV is highest. Here, we report a new HPV16 and -18 bivalent candidate vaccine produced from Escherichia coli. We used two strategies of N-terminal truncation of HPV L1 proteins and soluble non-fusion expression to generate HPV16 and HPV18 L1-only virus-like particles (VLPs) in a scalable process. Through comprehensive characterization of the bivalent candidate vaccine, we confirm lot consistency in a pilot scale-up of 30L, 100L and 500L. Using cryo-EM 3D reconstruction, we found that HPV16 and -18VLPs present in a T=7 icosahedral arrangement, similar in shape and size to that of the native virions. This HPV16/18 bivalent vaccine shares comparable immunogenicity with the licensed vaccines. Overall, we show that the production of a HPV16/18 bivalent vaccine from an E. coli expression system is robust and scalable, with potentially good accessibility worldwide as a population-based immunization strategy.

Expanded strain coverage for a highly successful public health tool: Prophylactic 9-valent human papillomavirus vaccine.

Human papillomavirus is considered the causative factor for cervical cancer, which accounts for approximately 5% of the global cancer burden and more than 600,000 new cases annually that are attributable to HPV infection worldwide. The first-generation prophylactic HPV vaccines, Gardasil® and Cervarix®, were licensed approximately a decade ago. Both vaccines contain the most prevalent high-risk types, HPV16 and 18, which are associated with 70% of cervical cancer. To further increase the type coverage, 5 additional oncogenic HPV types (31, 33, 45, 52 and 58) were added to the existing Gardasil-4 to develop a 9-valent HPV vaccine (9vHPV), Gardasil 9®, increasing the potential level of protection from ∼70% to ∼90%. The efficacy of the vaccine lies primarily in its ability to elicit type-specific and neutralizing antibodies to fend off the viral infection. Therefore, type-specific and neutralizing murine monoclonal antibodies (mAbs) were used to quantitate the antigenicity of the individual vaccine antigens and to measure the antibody levels in the serum samples from vaccinees in a type- and epitope-specific manner in a competitive immunoassay. Assays for 9vHPV are extended from the proven platform used for 4vHPV by developing and adding new mAbs against the additional types. In Phase III clinical trials, comparable safety profile and immunogenicity against the original 4 types were demonstrated for the 9vHPV vaccine, and these were comparable to the 4vHPV vaccine. The efficacy of the 9vHPV vaccine was established in trials with young women. Immunobridging for younger boys and girls was performed, and the results showed higher immunogenicity in the younger age group. In a subsequent clinical trial, the 2-dose regimen of the 9vHPV vaccine used among girls and boys aged 9-14 y showed non-inferior immunogenicity to the regular 3-dose regimen for young women (aged 16-26 years). Overall, the clinical data and cost-effectiveness analysis for the 9vHPV vaccine support its widespread use to maximize the impact of this important, life-saving vaccine.

Bacterially expressed human papillomavirus type 6 and 11 bivalent vaccine: Characterization, antigenicity and immunogenicity.

Human papillomavirus (HPV)-6 and HPV11 are the major etiological causes of condylomata acuminate. HPV neutralization by vaccine-elicited neutralizing antibodies can block viral infection and prevent subsequent disease. Currently, two commercially available HPV vaccines cover these two genotypes, expressed by Saccharomyces cerevisiae. Here we describe another HPV6/11 bivalent vaccine candidate derived from Escherichia coli. The soluble expression of N-terminally truncated L1 proteins was optimized to generate HPV6- and HPV11 L1-only virus-like particles (VLPs) as a scalable process. In a pilot scale, we used various biochemical, biophysical and immunochemical approaches to comprehensively characterize the scale and lot consistency of the vaccine candidate at 30L and 100L. Cryo-EM structure analysis showed that these VLPs form a T=7 icosahedral lattice, imitating the L1 capsid of the authentic HPV virion. This HPV6/11 bivalent vaccine confers a neutralization titer and antibody production profile in monkey that is comparable with the quadrivalent vaccine, Gardasil. This study demonstrates the robustness and scalability of a potential HPV6/11 bivalent vaccine using a prokaryotic system for vaccine production.

A Low-Cost and Fast Real-Time PCR System Based on Capillary Convection.

A low-cost and fast real-time PCR system in a pseudo-isothermal manner with disposable capillary tubes based on thermal convection for point-of-care diagnostics is developed and tested. Once stable temperature gradient along the capillary tube has been established, a continuous circulatory flow or thermal convection inside the capillary tube will repeatedly transport PCR reagents through temperature zones associated with the DNA denaturing, annealing, and extension stages of the reaction. To establish stable temperature gradient along the capillary tube, a dual-temperature heating strategy with top and bottom heaters is adopted here. A thermal waveguide is adopted for precise maintenance of the temperature of the top heater. An optimized optical network is developed for monitoring up to eight amplification units for real-time fluorescence detection. The system performance was demonstrated with repeatable detection of influenza A (H1N1) virus nucleic acid targets with a limit of detection of 1.0 TCID50/mL within 30 min.

A fast and low-cost genotyping method for hepatitis B virus based on pattern recognition in point-of-care settings.

A fast and low-cost method for HBV genotyping especially for genotypes A, B, C and D was developed and tested. A classifier was used to detect and analyze a one-step immunoassay lateral flow strip functionalized with genotype-specific monoclonal antibodies (mAbs) on multiple capture lines in the form of pattern recognition for point-of-care (POC) diagnostics. The fluorescent signals from the capture lines and the background of the strip were collected via multiple optical channels in parallel. A digital HBV genotyping model, whose inputs are the fluorescent signals and outputs are a group of genotype-specific digital binary codes (0/1), was developed based on the HBV genotyping strategy. Meanwhile, a companion decoding table was established to cover all possible pairing cases between the states of a group of genotype-specific digital binary codes and the HBV genotyping results. A logical analyzing module was constructed to process the detected signals in parallel without program control, and its outputs were used to drive a set of LED indicators, which determine the HBV genotype. Comparing to the nucleic acid analysis to HBV viruses, much faster HBV genotyping with significantly lower cost can be obtained with the developed method.

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.

Virus-like particle-based human vaccines: quality assessment based on structural and functional properties.

Human vaccines against three viruses use recombinant virus-like particles (VLPs) as the antigen: hepatitis B virus, human papillomavirus, and hepatitis E virus. VLPs are excellent prophylactic vaccine antigens because they are self-assembling bionanoparticles (20 to 60 nm in diameter) that expose multiple epitopes on their surface and faithfully mimic the native virions. Here we summarize the long journey of these vaccines from bench to patients. The physical properties and structural features of each recombinant VLP vaccine are described. With the recent licensure of Hecolin against hepatitis E virus adding a third disease indication to prophylactic VLP-based vaccines, we review how the crucial quality attributes of VLP-based human vaccines against all three disease indications were assessed, controlled, and improved during bioprocessing through an array of structural and functional analyses.

An assessment of hepatitis E virus (HEV) in US blood donors and recipients: no detectable HEV RNA in 1939 donors tested and no evidence for HEV transmission to 362 prospectively followed recipients.

BACKGROUND: Hepatitis E virus (HEV) infection has become relevant to blood transfusion practice because isolated cases of blood transmission have been reported and because HEV has been found to cause chronic infection and severe liver disease in immunocompromised patients. STUDY DESIGN AND METHODS: We tested for immunoglobulin (Ig)G and IgM antibodies to the HEV and for HEV RNA in 1939 unselected volunteer US blood donors. Subsequently, we tested the same variables in pre- and serial posttransfusion samples from 362 prospectively followed blood recipients to assess transfusion risk. RESULTS: IgG anti-HEV seroprevalence in the total 1939 donations was 18.8%: 916 of these donations were made in 2006 at which time the seroprevalence was 21.8% and the remaining 1023 donations were in 2012 when the seroprevalence had decreased to 16.0% (p < 0.01). A significant (p < 0.001) stepwise increase in anti-HEV seroprevalence was seen with increasing age. Eight of 1939 donations (0.4%) tested anti-HEV IgM positive; no donation was HEV RNA positive. Two recipients had an apparent anti-HEV seroconversion, but temporal relationships and linked donor testing showed that these were not transfusion-transmitted HEV infections. CONCLUSION: No transfusion-transmitted HEV infections were observed in 362 prospectively followed blood recipients despite an anti-HEV seroprevalence among donations exceeding 16%.