Levels of 8-OxodG Predict Hepatobiliary Pathology in Opisthorchis viverrini Endemic Settings in Thailand.

Opisthorchis viverrini is distinct among helminth infections as it drives a chronic inflammatory response in the intrahepatic bile duct that progresses from advanced periductal fibrosis (APF) to cholangiocarcinoma (CCA). Extensive research shows that oxidative stress (OS) plays a critical role in the transition from chronic O. viverrini infection to CCA. OS also results in the excision of a modified DNA lesion (8-oxodG) into urine, the levels of which can be detected by immunoassay. Herein, we measured concentrations of urine 8-oxodG by immunoassay from the following four groups in the Khon Kaen Cancer Cohort study: (1) O. viverrini negative individuals, (2) O. viverrini positive individuals with no APF as determined by abdominal ultrasound, (3) O. viverrini positive individuals with APF as determined by abdominal ultrasound, and (4) O. viverrini induced cases of CCA. A logistic regression model was used to evaluate the utility of creatinine-adjusted urinary 8-oxodG among these groups, along with demographic, behavioral, and immunological risk factors. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive accuracy of urinary 8-oxodG for APF and CCA. Elevated concentrations of 8-oxodG in urine positively associated with APF and CCA in a strongly dose-dependent manner. Urinary 8-oxodG concentrations also accurately predicted whether an individual presented with APF or CCA compared to O. viverrini infected individuals without these pathologies. In conclusion, urinary 8-oxodG is a robust \u27candidate\u27 biomarker of the progression of APF and CCA from chronic opisthorchiasis, which is indicative of the critical role that OS plays in both of these advanced hepatobiliary pathologies. The findings also confirm our previous observations that severe liver pathology occurs early and asymptomatically in residents of O. viverrini endemic regions, where individuals are infected for years (often decades) with this food-borne pathogen. These findings also contribute to an expanding literature on 8-oxodG in an easily accessible bodily fluid (e.g., urine) as a biomarker in the multistage process of inflammation, fibrogenesis, and infection-induced cancer

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

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

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

Microproteinuria during Opisthorchis viverrini infection: A biomarker for advanced renal and hepatobiliary pathologies from chronic Opisthorchiasis

Approximately 680 million people are at risk of infection with Opisthorchis viverrini (OV) andClonorchis sinensis, with an estimated 10 million infected with OV in Southeast Asia alone. While opisthorchiasis is associated with hepatobiliary pathologies, such as advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA), animal models of OV infection show that immune-complex glomerulonephritis is an important renal pathology that develops simultaneously with hepatobiliary pathologies. A cardinal sign of immune-complex glomerulonephritis is the urinary excretion of immunoglobulin G (IgG) (microproteinuria). In community-based studies in OV endemic areas along the Chi River in northeastern Thailand, we observed that over half of the participants had urine IgG against a crude OV antigen extract (OV antigen). We also observed that elevated levels of urine IgG to OV antigen were not associated with the intensity of OV infection, but were likely the result of immune-complex glomerulonephritis as seen in animal models of OV infection. Moreover, we observed that urine IgG to OV antigen was excreted at concentrations 21 times higher in individuals with APF and 158 times higher in individuals with CCA than controls. We also observed that elevated urine IgG to OV antigen could identify APF+ and CCA+ individuals from non-cases. Finally, individuals with urine IgG to OV antigen had a greater risk of APF as determined by Odds Ratios (OR = 6.69; 95%CI: 2.87, 15.58) and a greater risk of CCA (OR = 71.13; 95%CI: 15.13, 334.0) than individuals with no detectable level of urine IgG to OV antigen. Herein, we show for the first time the extensive burden of renal pathology in OV endemic areas and that a urine biomarker could serve to estimate risk for both renal and hepatobiliary pathologies during OV infection, i.e., serve as a “syndromic biomarker” of the advanced pathologies from opisthorchiasis

Novel Neutralizing Antibody Assays for Recombinant Human Hookworm Na-GST-1 Vaccine

BACKGROUND Necator Americanus, a human hookworm causes approximately 85% of the global hookworm infections. Hookworm ingest hemoglobin containing erythrocytes. Hemoglobin is further digested to Heme and Globin by hookworm\u27s gut enzymes. Iron-containing Heme is a potent enzyme inhibitor and generates toxic reactive oxygen species which is toxic to hookworms. Hookworm\u27s gut enzyme Na-GST-1 (Necator Americanus Glutathione S-Transferase-1) has been hypothesized to detoxify Heme. Na-GST-1 adjuvanted with Alhydrogel® is a new vaccine which is currently under clinical development. Na-GST-1 has two active sites, the ligand binding or Heme detoxification site (H-site) and the catalytic active glutathione binding site (G-site). We have developed in-vitro assays to assess the neutralizing capacity of antibodies against the functional activity of these two active sites. The antibodies used in this assay were purified from serum of BALB/c mice vaccinated with Na-GST-1 vaccine. Here, we report the development and results of these two novel in-vitro assays. METHODS Heme detoxification function (H-site) of Na-GST-1 was evaluated by oxidizing and degrading hematin to release iron using a potent oxidizer, hydrogen peroxide. Free iron released from hematin was measured by iron detection reagent called ferrozine. BALB/c mice were vaccinated with Na-GST-1 vaccine and polyclonal IgG from the mice sera was purified using immuno-precipitation. The neutralizing capacity of IgG against the catalytic activity (G-site) of Na-GST-1 was assessed using the CDNB assay. Similarly, the neutralizing capacity of IgG against the H-site was assessed by the ferrozine iron releasing assay after incubating Na-GST-1 with Hematin and polyclonal IgG. RESULTS Na-GST-1 significantly inhibited free-iron release from hematin when incubated with hydrogen peroxide. No statistically significant reduction in iron release was found when other iron containing molecules like cytochrome C, ferredoxin or myoglobin were incubated with Na-GST-1 and H2O2. Four microgram of polyclonal IgG reversed 56.9% of the free-iron release from Hematin. Moreover, 4µg monoclonal Sj-GST IgG completely reversed the inhibition of the release of free-iron caused by Na-GST-1. The neutralizing antibody assay against G-site of Na-GST-1 generated a dose response (%Inhibition vs Negative-IgG) when 20µg (25.87%), 15µg (19.61%), 10µg (13.78%) and 5µg (6.32%) of the purified IgG was incubated with 500ng of Na-GST-1. CONCLUSIONS In-vitro assay showed that Na-GST-1 could prevent release of free-iron from hematin. Antibodies purified from Na-GST-1 vaccinated mice neutralized both the heme-detoxification activity (H-site) and catalytic activity (G-site) of Na-GST-1. These neutralizing antibody assay results of Na-GST-1 vaccine lay the foundation for a Phase 2 Clinical Trial in Brazil

Hematin Detoxification Assay for Necator americanus Glutathione S Transferase -1 (Na-GST-1): assessing the heme detoxification function of nu-class glutathione-s-transferases.

Human hookworm infects more than 700 million people worldwide and Necator americanus causes approximately 85% of those infections. The hookworm ingests erythrocytes containing hemoglobin and the hemoglobin is digested to Heme and Globin by the hookworm\u27s gut enzymes. The breakdown of Globin provides essential nutrition to the hookworm. However, Heme (ferroprotoporphyrin) which contains iron is a potent producer of reactive oxygen species (ROS) and is toxic to the hookworm. It has long been hypothesized that the nu-class hookworm Glutathione S-Transferase called the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) binds and detoxifies Heme, and therefore, plays a significant role in reducing the toxic reactive oxygen species (ROS) and iron burden of the hookworm. To prove this hypothesis, we have developed a unique Hematin Detoxification Assay, which measures the release of the free iron from the degraded Hematin (ferriprotoporphyrin) in presence of Na-GST-1. This assay utilizes hydrogen peroxide (H2O2), a potent oxidizer, to degrade Hematin to Biliverdin, ferric iron (Fe+3) and carbon monoxide. Ascorbic acid is then used to reduce the ferric iron (Fe+3) to ferrous iron (Fe+2). The levels of the ferrous iron (Fe+2) are then measured in the presence of ferrozine (free iron detection reagent) using a spectrophotometer (562 nm). Using this assay, a statistically significant (p = 0.0079) reduction in the iron released from 250 µM of Hematin was found in the presence of 5 µg of Na-GST-1 when compared to the iron released from 250 µM of Hematin alone. However, human placental GST did not (p = 0.578) prevent the release of free iron from 250 µM of Hematin. This in-vitro assay showed that Na-GST-1 specifically binds and protects Hematin from degradation. Based on neutralizing this detoxification function, an Alhydrogel® adjuvanted recombinant Na-GST-1 vaccine is currently under clinical development. We believe that this novel assay will also be very useful for assessing the detoxification activity of nu-class GSTs from other hematophagous helminths

A history of hookworm vaccine development

The human hookworms Necator americanus and Ancylostoma duodenale remain among the most common infections of humans in areas of rural poverty in the developing regions of the world, with an estimated 1 billion people infected with one or more of these parasites. Herein, we review the nearly 100 y of research, development, animal testing and fieldwork that haveled to our current progress in recombinant hookworm vaccines. We begin with the identification of hookworm at the start ofthe 20th century in Southern US, then discuss the progress in developed countries to eliminate human hookworm infection,and then the industrial development and field use in the 1970s a canine hookworm vaccine (Ancylostoma caninum), and finally our progress to date in the development and clinical testing of an array of recombinant antigens to prevent human hookworm disease from N. americanus infection. Special attention is given to the challenges faced in the development of a vaccine against a blood-feeding nematode, including the epidemiology of infection (high prevalence of infection), pathogenesis (chronic infection that increases with the age of the host), and a robust immune response that fails to confer the protection in thehost and a concomitant absence of correlates of protection by a successful vaccine could be developed and tested. Finally, we provide the optimal and acceptable profiles of a human hookworm vaccine, including the proposed indication, target population and route of administration, as developed by the Human Hookworm Vaccine Initiative, the only group currently working on vaccines targeting this parasite. © 2011 Landes Bioscience