Circumventing qPCR inhibition to amplify miRNAs in plasma

Background: Circulating microRNAs (c-miRNAs) have be identified in saliva, urine and blood, which has led to increasing interest in their development as biomarkers for diverse diseases including cancers. One of the key advantages of c-miRNAs over other biomarkers is the ability to be amplified and quantified by quantitative PCR (qPCR). However, at phlebotomy when whole blood is dispensed into heparinized tubes, residual levels of the anti-coagulant lithium heparin may remain in the plasma and hence with RNA isolated from the plasma. This can confound the detection of c-miRNAs by qPCR because it inhibits reverse transcriptase (RT). Here we present a procedure, modified from earlier techniques, to detect c-miRNAs in plasma that improves sensitivity and streamlines performance.Findings: Treatment of total RNA isolated from human blood plasma with Bacteroides heparinase I during reverse transcription at 37°C for one hour improved sensitivity and performance of the qPCR. This is in comparison to no treatment or treatment of the RNA prior to RT, which is the current suggested method and exposes plasma to Flavobacterium heparinum heparinase I for up to 2 hours before RT. This modest alteration improved qPCR performance and resulted in lowered threshold cycles (C) for detection of the target sequence, candidate c-miRNA biomarkers, and controls. It also reduced the expense and number of processing steps, shortening the duration of the assay and minimizing exposure of RNA to elevated temperatures.Conclusion: Incorporating Bacteroides heparinase I treatment into conventional RT protocols targeting c-miRNA in plasma can be expected to expedite the discovery of biomarkers

Comparative Immunogenicity of Na-GST-1 Human Hookworm Vaccine with Synthetic Glucopyranosyl Lipid Adjuvant (GLA) in BALB/c Mice

More than 740 million people worldwide are infected with Hookworm. Hookworm infection is most prevalent in the poorest of the poor populations of the world, and has serious health effects. Hookworm infection causes blood loss leading to iron deficiency anemia and protein energy malnutrition, which results in a compromised immune response. Consequently, the target human population suffers from an increased susceptibility to infectious diseases including hookworm infection. We have developed recombinant adult hookworm vaccines against hookworm infection to break this vicious cycle. Toll-like receptor (TLR) 4 agonist are known to boost immune response in healthy and immunocompromised individuals. We believe that co-injecting Synthetic Glucopyranosyl Lipid Adjuvant (GLA) a novel TLR-4 agonist with adult hookworm Na-GST-1 + Alhydrogel® vaccine will produce a robust and sustainable immune response in this target human population. Here, we discuss the rationale of using GLA, study designs and the results of the pre-clinical immunogenicity studies of the Human Hookworm Na-GST-1 + Alhydrogel® Vaccine in BALB/c mice with and without GLA. We conclude that, GLA enhanced the immunogenicity of co-administered adult hookworm Na-GST-1 + Alhydrogel® vaccine, producing a strong anti-Na-GST-1 IgG response. These preclinical results lay the foundation of co-administrating GLA with adult hookworm Na-GST-1 + Alhydrogel® vaccine in Phase 1 clinical trial in Brazil

BioClojure: A functional library for the manipulation of biological sequences

Motivation: BioClojure is an open-source library for the manipulation of biological sequence data written in the language Clojure. BioClojure aims to provide a functional framework for the processing of biological sequence data that provides simple mechanisms for concurrency and lazy evaluation of large data sets. Results: BioClojure provides parsers and accessors for a range of biological sequence formats, including UniProtXML, Genbank XML, fasta and fastq. In addition it provides wrappers for key analysis programs, including BLAST, SignalP, TMHMM and InterProScan, and parsers for analyzing their output. All interfaces leverage Clojure\u27s functional style and emphasize laziness and composability, so that BioClojure, and user-defined, functions can be chained into simple pipelines that are thread-safe and seamlessly integrate lazy evaluation. Availability: BioClojure is distributed under the Lesser GPL (LGPL) and the source code is freely available from GitHub (https://github.com/s312569/clj-biosequence). Contact: [email protected]

Potency Testing for NTD Vaccines: Determining Relative Potency for the Na-GST-1 Human Hookworm Vaccine

Over the next decade, a new generation of vaccines will target the neglected tropical diseases (NTDs) . The goal of most NTD vaccines will be to reduce the morbidity and decrease the chronic debilitating nature of these often-forgotten infections - outcomes that are hard to measure in the traditional potency-testing paradigm . The absence of measurable correlates of protection, a lack of permissive animal models for lethal infection, and a lack of clinical indications that do not include the induction of sterilizing immunity required us to reconsider the traditional bioassay methods for determining vaccine potency . Owing to these limitations, potency assay design for NTD vaccines will increasingly rely on a paradigm where potency testing is one among many tools to ensure that a manufacturing process yields a product of consistent quality . This potency test is a bioassay using BALB/c mice, which evaluates the immunogenicity of the vaccine at set time interval post manufacture . Herein, we discuss the results of 12 month potency testing of Necator americanus-glutathione-S- transferase-1 (Na-GST-1) vaccine . The Effective Dose 50 (ED50), with its 95% fiducial limits (FL) for each time point was determined along with the Relative Potency with its 95% FL for 3, 6, 9 and 12 months post manufacture . Potency testing has shown that storage at 4° C decreases the ED50 and increases the relative potency of Na-GST-1 vaccine . We proposed that the change in ED50 and relative potency coincide with higher affinity binding of the Na-GST-1 to the Alhydrogel® that occurred during storage at 4° C . These preclinical results lay the foundation for moving forward with Phase 1 clinical trial in Brazil

A microRNA profile associated with Opisthorchis viverrini-induced cholangiocarcinoma in tissue and plasma

Background: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive tumor of the bile duct, and a significant public health problem in East Asia, where it is associated with infection by the parasite Opisthorchis viverrini. ICC is often detected at an advanced stage and with a poor prognosis, making a biomarker for early detection a priority. Methods: We have comprehensively profiled miRNA expression levels in ICC tumor tissue using small RNA-Seq and validated these profiles using quantitative PCR on matched plasma samples. Results: Distinct miRNA profiles were associated with increasing histological differentiation of ICC tumor tissue. We also observed that histologically normal tissue adjacent to ICC tumor displayed miRNA expression profiles more similar to tumor than liver tissue from healthy donors. In plasma samples, an eight-miRNA signature associated with ICC, regardless of the degree of histological differentiation of its matched tissue, forming the basis of a circulating miRNA-based biomarker for ICC. Conclusions: The association of unique miRNA profiles with different ICC subtypes suggests the involvement of specific miRNAs during ICC tumor progression. In plasma, an eight-miRNA signature associated with ICC could form the foundation of an accessible (plasma-based) miRNA-based biomarker for the early detection of ICC

Vaccination with Human Hookworm Vaccine Necator americanus Aspartic Protease-1 M74 Generates Neutralizing Antibodies and a Potent Immune Response in BALB/c Mice

Backgound: Human Hookworm Infection, a neglected tropical disease infects more than 600 million people around the world. Hookworms ingest hemoglobin containing erythrocytes and Necator americanus Aspartic Protease-1 wild type (Na-APR-1wt) a hemoglobinase cleaves hemoglobin to form Heme and Globin. Globin is further digested by other gut enzymes and the nutritional end products are absorbed by the hookworm’s gut wall. Also, Heme which is toxic to hookworm is detoxified by the Necator americanus Glutathione Transferase-1 (Na-GST-1) a detoxification enzyme secreted by the gut of the hookworm. Necator americanus Aspartic Protease-1 M74 (Na-APR-1 M74) is the new vaccine for the Human Hookworm Infection which is currently under pre-clinical development. Na-APR-1 M74 vaccine is an Alhydrogel® adjuvanted vaccine containing the mutant form of the Na-APR-1wt. Neutralizing Na-APR-1wt by potent antibodies (IgG) in the vaccinees will block the initiation of the hemoglobin digestion cascade and starve the hookworms from essential nutrition, leading to their death. Here, we report the results of the neutralizing capacity of antibodies and potency (immunogenicity) of Na-APR-1 M74 vaccine in BALB/c mice. Methods: Serum for IgG was generated by vaccinating BALB/c mice twice subcutaneously with Na-APR-1 M74 an enzymatically inactive mutant form of Na-APR-1wt formulated with Alhydrogel®. Assessment of neutralizing capacity of IgG was performed using the standard Cathepsin-D protease assay using MOCAc substrate. Dose response (% Inhibition vs Dose) was assessed using linear regression analysis. Potency testing of the Na-APR-1M74 clinical drug product was performed by standard bioassay. Median Effective Dose 50 (ED50) with the 95% fiducial limits (95%FL) was estimated using Probit Analysis (SAS® 9.3). Also, Relative Potency (RP) was estimated by the methods described in European Pharmacopeia\u27s Chapter 5.3. Results: Five microgram of IgG neutralized 51.06% of the enzymatic activity of 250ng of Na-APR-1wt. An excellent dose response was also observed. ED50 of 14.15μg (95%FL = 10.47μg -- 18.93μg) and 11.46μg (95%FL = 4.86μg --27.42μg) was estimated for time 1 and 7 month post manufacture respectively. RP at 7 months was found to be 1.23 (95%FL = 0.792--1.917). Conclusion: These preclinical results of the Na-APR-1 M74 vaccine lay the foundation for a Phase 1 Clinical Trial in USA and Brazil. This Na-APR-1 M74 vaccine will be subsequently combined with Necator americanus Glutathione transferase-1 (Na-GST-1) vaccine to form a multivalent human hookworm vaccine

Methods and matrices: Approaches to identifying miRNAs for nasopharyngeal carcinoma

We tested two miRNA discovery workflows on two sample sources for miRNAs associated with NPC. In the first workflow, we assumed that NPC tumor tissue would be enriched for miRNAs, so we compared miRNA expression in FFPE from NPC cases and controls using microarray and RNA-Seq technologies. Candidate miRNAs from both technologies were verified by qPCR in FFPE and sera from an independent NPC sample set. In a second workflow, we directly interrogated NPC case and control sera by RNA-Seq for c-miRNAs associated with NPC, with candidate c-miRNAs verified by qPCR in the sera from the same independent NPC sample set. Results Both microarray and RNA-Seq narrowed the miRNA signature to 1-5% of the known mature human miRNAs. Moreover, these two methods produced similar results when applied to the same sample type (FFPE), with RNA-Seq additionally indicating “unknown” miRNAs associated with NPC. However, we found different miRNA profiles in NPC sera compared to FFPE using RNA-Seq, with the few overlapping miRNAs found to be significantly up-regulated in FFPE significantly down-regulated in sera (and vice versa). Despite the different miRNA profiles found in FFPE and sera, both profiles strongly associated with NPC, providing two potential sources for biomarker signatures for NPC. Conclusions We determined that the direct interrogation of sera by RNA-Seq was the most informative method for identifying a c-miRNA signature associated with NPC. We also showed that there are different miRNA expression profiles associated with NPC for tumor tissue and sera. These results reflect on the methods and meaning of miRNA biomarkers for NPC in tissue and peripheral blood

The miRNAome of Opisthorchis viverrini induced intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer, arising in the biliary ducts that extend into the liver. The highest incidence of ICC occurs in Southeast Asia, particularly in the Mekong River Basin countries of Thailand, Laos, Cambodia, and Vietnam, where it is strongly associated with chronic infection by the food-borne liver fluke Opisthorchis viverrini (OV), one of only three eukaryote pathogens considered Group one carcinogens. Intrahepatic cholangiocarcinoma is usually diagnosed at an advanced stage, with a poor prognosis and survival often less than 24 months. Hence, biomarkers that enable the early detection of ICC would be desirable and have a potentially important impact on the public health in the resource-poor regions where this cancer is most prevalent. As microRNAs (miRNAs) remain well preserved after formalin fixation, there is much interest in developing them as biomarkers that can be investigated using tumor biopsy samples preserved in formalin fixed paraffin embedded (FFPE) tumor blocks. Recently, we reported the first comprehensive profiling of tissue-based miRNA expression using FFPE from the three most common subtypes of OV-induced ICC tumors: moderately differentiated ICC, papillary ICC, and well-differentiated ICC. We observed that each subtype of OV-induced ICC exhibited a distinct miRNA profile, which suggested the involvement of specific sets of miRNAs in the progression of this cancer. In addition, non-tumor tissue adjacent to ICC tumor tissue on the same FFPE block shared a similar miRNA dysregulation profile with the tumor tissue than with normal (non-tumor) liver tissue (individuals without ICC or OV infection). Herein, we provide a detailed description of the microarray analysis procedures used to derive these findings

Distinct miRNA signatures associate with subtypes of cholangiocarcinoma from infection with the tumourigenic liver fluke Opisthorchis viverrini

Background & Aims: Intrahepatic cholangiocarcinoma (ICC) is a significant public health problem in East Asia, where it is strongly associated with chronic infection by the food-borne parasite Opisthorchis viverrini (OV). We report the first comprehensive miRNA expression profiling by microarray of the most common histologic grades and subtypes of ICC: well differentiated, moderately differentiated, and papillary ICC. Methods: MicroRNA expression profiles from FFPE were compared among the following: ICC tumour tissue (n = 16), nontumour tissue distally macrodissected from the same ICC tumour block (n = 15), and normal tissue (n = 13) from individuals undergoing gastric bypass surgery. A panel of deregulated miRNAs was validated by qPCR. Results: Each histologic grade and subtype of ICC displayed a distinct miRNA profile, with no cohort of miRNAs emerging as commonly deregulated. Moderately differentiated ICC showed the greatest miRNA deregulation in quantity and magnitude, followed by the papillary subtype, and then well differentiated ICC. Moreover, when ICC tumour tissues were compared to adjacent non-tumour tissue, similar miRNA dysregulation profiles were observed. Conclusions: We show that common histologic grades and subtypes of ICC have distinct miRNA profiles. As histological grade and subtypes are associated with ICC aggressiveness, these profiles could be used to enhance the early detection and improve the personalised treatment for ICC. These findings also suggest the involvement of specific miRNAs during ICC tumour progression and differentiation. We plan to use these insights to (a) detect these profiles in circulation and (b) conduct functional analyses to decipher the roles of miRNAs in ICC tumour differentiation