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The demand for nucleic acid and protein derivatives from formalin-fixed paraffin-embedded (FFPE) tissue has greatly increased due to advances in extraction and purification methods, making these derivatives available for numerous genomic and proteomic platforms. Previously, DNA, RNA, microRNA (miRNA), or protein derived from FFPE tissue blocks were considered “unfit” for such platforms, as the process of tissue immobilization by FFPE resulted in cross-linked, fragmented, and chemically modified macromolecules. We conducted a systematic examination of nucleic acids and proteins co-extracted from 118 FFPE blocks sampled from the AIDS and Cancer Specimen Resource (ACSR) at The George Washington University after stratification by storage duration and the three most common tumor tissue types at the ACSR (adenocarcinoma, squamous cell carcinoma, and papillary carcinoma). DNA, RNA, miRNA, and protein could be co-extracted from 98% of the FFPE blocks sampled, with DNA and miRNA “fit” for diverse genomic purposes including sequencing. While RNA was the most labile of the FFPE derivatives, especially when assessed by RNA integrity number (RIN), it was still “fit” for genomic methods that use smaller sequence lengths, e.g., quantitative PCR. While more than half of the protein derivatives were fit for proteomic purposes, our analyses indicated a significant interaction effect on the absorbance values for proteins derived from FFPE, implying that storage duration may affect protein derivatives differently by tumor tissue type. The mean absorbance value for proteins derived from more recently stored FFPE was greater than protein derived from older FFPE, with the exception of adenocarcinoma tissue. Finally, the fitness of one type of derivative was weakly associated with the fitness of derivatives co-extracted from the same FFPE block. The current study used several novel quality assurance approaches and metrics to show that archival FFPE tissue blocks are a valuable resource for contemporary genomic and proteomic platforms. [ABSTRACT FROM AUTHOR]

Necator americanus Glutathione-S-Transferase-1 (Na-GST-1) plays a role in the digestion of host hemoglobin by adult N. americanus hookworms. Vaccination of laboratory animals with recombinant Na-GST-1 is associated with significant protection from challenge infection. Recombinant Na-GST-1 was expressed in Pichia pastoris and adsorbed to aluminum hydroxide adjuvant (Alhydrogel) according to current Good Manufacturing Practice. Two Phase 1 trials were conducted in 142 healthy adult volunteers in the United States and Brazil, first in hookworm-naïve individuals and then in residents of a N. americanus endemic area in Brazil. Volunteers received one of three doses of recombinant Na-GST-1 (10, 30, or 100 μg) adjuvanted with Alhydrogel, adjuvanted with Alhydrogel and co-administered with an aqueous formulation of Glucopyranosyl Lipid A (GLA-AF), or the hepatitis B vaccine. Vaccinations were administered via intramuscular injection on days 0, 56, and 112. Na-GST-1/Alhydrogel was well tolerated in both hookworm-naïve and hookworm-exposed adults, with the most common adverse events being mild to moderate injection site pain and tenderness, and mild headache and nausea; no vaccine-related severe or serious adverse events were observed. Antigen-specific IgG antibodies were induced in a dose-dependent fashion, with increasing levels observed after each vaccination in both trials. The addition of GLA-AF to Na-GST-1/Alhydrogel did not result in significant increases in specific IgG responses. In both the US and Brazil studies, the predominant IgG subclass induced against Na-GST-1 was IgG1, with lesser amounts of IgG3. Vaccination of both hookworm-naïve and hookworm-exposed adults with recombinant Na-GST-1 was safe, well tolerated, and resulted in significant antigen-specific IgG responses. Based on these results, this vaccine will be advanced into clinical trials in children and eventual efficacy studies. Trial registration: ClinicalTrials.gov ( for the Brazil trial and for the US trial) [ABSTRACT FROM AUTHOR]

A new generation of vaccines for the neglected tropical diseases (NTDs) have now advanced into clinical development, with the Na-GST-1/Alhydrogel Hookworm Vaccine already being tested in Phase 1 studies in healthy adults. The current manuscript focuses on the often overlooked critical aspects of NTD vaccine product development, more specifically, vaccine stability testing programs. A key measure of vaccine stability testing is "relative potency" or the immunogenicity of the vaccine during storage. As with most NTD vaccines, the Na-GST-1/Alhydrogel Hookworm Vaccine was not developed by attenuation or inactivation of the pathogen (Necator americanus), so conventional methods for measuring relative potency are not relevant for this investigational product. Herein, we describe a novel relative potency testing program and report for the first time on the clinical lot of this NTD vaccine during its first 60 months of storage at 2–8°C. We also describe the development of a complementary functional assay that measures the ability of IgG from animals or humans immunized with Na-GST-1/Alhydrogel to neutralize this important hookworm enzyme. While 90% inhibition of the catalytic activity of Na-GST-1 was achieved in animals immunized with Na-GST-1/Alhydrogel, lower levels of inhibition were observed in immunized humans. Moreover, anti-Na-GST-1 antibodies from volunteers in non-hookworm endemic areas were better able to inhibit catalytic activity than anti-Na-GST-1 antibodies from volunteers resident in hookworm endemic areas. The results described herein provide the critical tools for the product development of NTD vaccines. [ABSTRACT FROM AUTHOR]