Jordan L. Plieskatt

Antibodies against a secreted protein from hookworm larvae reduce the intensity of hookworm infection in humans and vaccinated laboratory animals

The development of a vaccine would provide an important new tool for the control of human hookworm infection. On the basis of successful vaccination of laboratory animals with living irradiated, third‐stage hookworm larvae (L3), we examined the antibody responses of individuals from hookworm endemic areas of Brazil and China against the most abundant L3 secreted antigens, the ancylostoma secreted proteins, ASP‐1 and ASP‐2. Logistic regression was used to investigate the effects of antibody isotype responses to ASPs on the risk of an individual harboring heavy hookworm infection. A significant protective association was observed between increasing anti‐ASP‐2 IgE levels and the risk of heavy hookworm infection. To confirm that ASP‐2 is a protective antigen, laboratory dogs were immunized with recombinant ASP‐2 formulated with the GlaxoSmithKline Adjuvant, AS03. Sera obtained from the immunized dogs exhibited high geometric mean antibody titers, immunoprecipitated native ASP‐2 from L3 extracts and localized the site of ASP‐2 expression to the glandular esophagus and body channels exiting to the cuticle. The sera also exhibited an increased ability to inhibit migration of L3 through tissue in vitro relative to sera from AS03‐injected controls. Upon L3 challenge, the ASP‐2 vaccinated dogs exhibited significant reductions in fecal egg counts and intestinal hookworm burden. These findings provide strong support for the development of an effective recombinant vaccine against hookworm infection in humans.

Ancylostoma caninum MTP-1, an astacin-like metalloprotease secreted by infective hookworm larvae, is involved in tissue migration

ABSTRACT Infective larvae (L3) of nematodes secrete macromolecules that are critical to infection and establishment of the parasite in the host. The dog hookworm Ancylostoma caninum secretes an astacin-like metalloprotease, Ac-MTP-1, upon activation in vitro with host serum. Recombinant Ac-MTP-1 was expressed in the baculovirus/insect cell system as a secreted protein and was purified from culture medium by two separate methods, cation-exchange fast-performance liquid chromatography and gelatin-affinity chromatography. Recombinant MTP-1 was catalytically active and digested a range of native and denatured connective tissue substrates, including gelatin, collagen, laminin, and fibronectin. A dog was immunized with recombinant Ac-MTP-1 formulated with AS03 adjuvant, and the antiserum was used to immunolocalize the anatomic sites of expression within A. caninum L3 to secretory granules in the glandular esophagus and the channels that connect the esophagus to the L3 surface and to the cuticle. Antiserum inhibited the ability of recombinant MTP-1 to digest collagen by 85% and inhibited larval migration through tissue in vitro by 70 to 75%, in contrast to just 5 to 10% inhibition obtained with preimmunization serum. The metalloprotease inhibitors EDTA and 1,10-phenanthroline also reduced the penetration of L3 through skin in vitro by 43 to 61%. The data strongly suggest that Ac-MTP-1 is critical for the invasion process of hookworm larvae, and moreover, that antibodies against the enzyme can neutralize its function and inhibit migration.

An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection

Hookworms digest hemoglobin from erythrocytes via a proteolytic cascade that begins with the aspartic protease, APR‐1. Ac‐APR‐1 from the dog hookworm, Ancylostoma caninum, protects dogs against hookworm infection via antibodies that neutralize enzymatic activity and interrupt blood‐feeding. Toward developing a human hookworm vaccine, we expressed both wild‐type (Na‐APR‐1wt) and mutant (Na‐APR‐1mut—mutagenesis of the catalytic aspartic acids) forms of Na‐APR‐1 from the human hookworm, Necator americanus. Refolded Na‐APR‐1wt was catalytically active, and Na‐APR‐1mut was catalytically inactive but still bound substrates. Vaccination of canines with Na‐APR‐1mut and heterologous challenge with A. caninum resulted in significantly reduced parasite egg burdens (P=0.034) and weight loss (P=0.022). Vaccinated dogs also had less gut pathology, fewer adult worms, and reduced blood loss compared to controls but these did not reach statistical significance. Vaccination with Na‐APR‐1mut induced antibodies that bound the native enzyme in the parasite gut and neutralized enzymatic activity of Na‐APR‐1wt and APR‐1 orthologues from three other hookworm species that infect humans. IgG1 against Na‐APR‐1mut was the most prominently detected antibody in sera from people resident in high‐transmission areas for N. americanus, indicating that natural boosting may occur in exposed humans. Na‐APR‐1mut is now a lead antigen for the development of an antihematophagy vaccine for human hookworm disease.—Pearson, M. S., Bethony, J. M., Pickering, D. A., de Oliveira, L. M., Jariwala, A., Santiago, H., Miles, A. P., Zhan, B., Jiang, D., Ranjit, N., Mulvenna, J., Tribolet, L., Plieskatt, J., Smith, T., Bottazzi, M. E., Jones, K., Keegan, B., Hotez, P. J., Loukas, A. An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection. FASEB J. 23, 3007–3019 (2009). www.fasebj.org

Infection with the carcinogenic liver fluke Opisthorchis viverrini modifies intestinal and biliary microbiome

Opisthorchis viverrini is a fish‐borne trematode endemic in East Asia. Following ingestion, the flukes locate to the biliary tree, where chronic infection frequently leads to cholangiocarcinoma (CCA). The mechanisms by which O. viverrini infection culminates in CCA remain unknown. An unexplored aspect is its influence on the host microbiome. In the hamster, infection with this pathogen reliably leads to CCA. Genomic DNAs of microbiota from colorectal contents and bile of hamsters and from whole O. viverrini were examined in this model of fluke‐induced CCA. Microbial communities were characterized by high‐throughput sequencing of variable regions 7–9 of prokaryotic 16S ribosomal DNA Of ~1 million sequences, 536,009 with useable reads were assignable to 29,776 operational taxonomy units (OTUs) and, in turn, to 20 phyla and 273 genera of Bacteria or Archaea. Microbial community analyses revealed that fluke infection perturbed the gastrointestinal tract microbiome, increasing Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae, while decreasing Porphyromonadaceae, Erysipelotrichaceae, and Eubacteriaceae (P<0.05). More than 60 OTUs were detected in the biliary system, which confirmed bacteriobilia and a noteworthy community of microbes associated with the parasites. The fluke‐associated microorganisms included potential pathogens from the Enterobacteriaceae and Listeriaceae and others, including Cyanobacteria and Deinococci, usually found in external environments. Given that opisthorchiasis is distinguished from other helminth infections by a robust inflammatory phenotype with conspicuously elevated IL‐6, and that inflammation of the biliary system leads to periductal fibrosis, which is a precursor of CCA, the flukes and their microbiota may together drive this distinctive immune response.—Plieskatt, J. L., Raksawan, D., Mulvenna, J. P., Krause, L., Sripa, B., Bethony, J. M., Brindley, P. J. Infection with the carcinogenic liver fluke Opisthorchis viverrini modifies intestinal and biliary microbiome. FASEB J. 27, 4572–4584 (2013). www.fasebj.org

Expression, immunogenicity, histopathology, and potency of a mosquito-based malaria transmission-blocking recombinant vaccine

ABSTRACT Vaccines have been at the forefront of global research efforts to combat malaria, yet despite several vaccine candidates, this goal has yet to be realized. A potentially effective approach to disrupting the spread of malaria is the use of transmission-blocking vaccines (TBV), which prevent the development of malarial parasites within their mosquito vector, thereby abrogating the cascade of secondary infections in humans. Since malaria is transmitted to human hosts by the bite of an obligate insect vector, mosquito species in the genus Anopheles, targeting mosquito midgut antigens that serve as ligands for Plasmodium parasites represents a promising approach to breaking the transmission cycle. The midgut-specific anopheline alanyl aminopeptidase N (AnAPN1) is highly conserved across Anopheles vectors and is a putative ligand for Plasmodium ookinete invasion. We have developed a scalable, high-yield Escherichia coli expression and purification platform for the recombinant AnAPN1 TBV antigen and report on its marked vaccine potency and immunogenicity, its capacity for eliciting transmission-blocking antibodies, and its apparent lack of immunization-associated histopathologies in a small-animal model.

Reduction of Worm Fecundity and Canine Host Blood Loss Mediates Protection against Hookworm Infection Elicited by Vaccination with Recombinant Ac-16

ABSTRACT Hookworm infection is one of most important parasitic infection of humans, occurring in 740 million people. Here we report the protective vaccination of dogs with Ac-16, an immunodominant surface antigen from the hookworm Ancylostoma caninum. We show that immunization with Ac-16 formulated with AS03 elicited specific humoral and cellular immune responses and provided partial protection against hookworm infection and morbidity as evidenced by a significant reduction of hookworm egg counts (64% reduction; P = 0.0078) and worm-induced blood loss (P < 0.05). Moreover, specific anti-Ac-16 antibodies recognized the native protein on the surface of third-stage larvae and blocked their migration through tissue in vitro. Our data support the use of Ac-16 as a potential candidate for vaccination against hookworm infection.

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), non-tumour 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.

Methods and matrices: approaches to identifying miRNAs for Nasopharyngeal carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is a solid tumor of the head and neck. Multimodal therapy is highly effective when NPC is detected early. However, due to the location of the tumor and the absence of clinical signs, early detection is difficult, making a biomarker for the early detection of NPC a priority. The dysregulation of small non-coding RNAs (miRNAs) during carcinogenesis is the focus of much current biomarker research. Herein, we examine several miRNA discovery methods using two sample matrices to identify circulating miRNAs (c-miRNAs) associated with NPC.MethodsWe 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.ResultsBoth 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.ConclusionsWe 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.

Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): A production process developed for a lead candidate recombinant hookworm vaccine antigen

The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20 L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing.

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

BackgroundIntrahepatic 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.MethodsWe 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.ResultsDistinct 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.ConclusionsThe 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.