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1.
PLoS Pathog ; 14(3): e1006931, 2018 03.
Article in English | MEDLINE | ID: mdl-29566094

ABSTRACT

As part of on-going efforts to control hookworm infection, the "human hookworm vaccine initiative" has recognised blood feeding as a feasible therapeutic target for inducing immunity against hookworm infection. To this end, molecular approaches have been used to identify candidate targets, such as Necator americanus (Na) haemoglobinase aspartic protease-1 (APR-1), with immunogenicity profiled in canine and hamster models. We sought to accelerate the immune analysis of these identified therapeutic targets by developing an appropriate mouse model. Here we demonstrate that Nippostrongylus brasiliensis (Nb), a phylogenetically distant strongylid nematode of rodents, begins blood feeding early in its development and that immunisation with Na-APR-1 can block its growth and completion of its life cycle. Furthermore, we identify a new haem detoxification pathway in Nb required for blood feeding that can be blocked by drugs of the quinolone family, reducing both infection burden and the associated anaemia in rodents. Collectively, our findings show that haem metabolism has potential as a checkpoint for interrupting hookworm development in early stages of the hookworm life cycle and that the Nippostrongylus brasiliensis rodent model is relevant for identifying novel therapeutic targets against human hookworm.


Subject(s)
Antibodies, Helminth/pharmacology , Aspartic Acid Endopeptidases/antagonists & inhibitors , Erythrocytes/drug effects , Hookworm Infections/prevention & control , Necator americanus/enzymology , Nippostrongylus/growth & development , Strongylida Infections/prevention & control , Ancylostomatoidea/drug effects , Ancylostomatoidea/growth & development , Animals , Antigens, Helminth/immunology , Aspartic Acid Endopeptidases/immunology , Erythrocytes/parasitology , Female , Hookworm Infections/parasitology , Life Cycle Stages , Male , Mice , Mice, Inbred C57BL , Nippostrongylus/drug effects , Strongylida Infections/parasitology
2.
Article in English | MEDLINE | ID: mdl-23908024

ABSTRACT

Necator americanus is the major cause of human hookworm infection, which is a global cause of anemia in the developing world. Ongoing efforts to control hookworm infection include the identification of candidate vaccine antigens as well as potential therapeutic targets from the infective L3 larval stages and adult stages of the parasite. One promising family of proteins are the adult-stage-secreted cytosolic glutathione S-transferases (GSTs). Nematode GSTs facilitate the inactivation and degradation of a variety of electrophilic substrates (drugs) via the nucleophilic addition of reduced glutathione. Parasite GSTs also play significant roles in multi-drug resistance and the modulation of host immune defense mechanisms. Here, the structure of Na-GST-3, one of three GSTs secreted by adult-stage N. americanus, is reported. Unlike most GST structures, the Na-GST-3 crystal contains a monomer in the asymmetric unit. However, the monomer forms a prototypical GST dimer across the crystallographic twofold. A glutathione from the fermentation process is bound to the monomer. The overall binding cavity of Na-GST-3 is reminiscent of that of other N. americanus GSTs and is larger and capable of binding a wider array of ligands than GSTs from organisms that have other major detoxifying mechanisms. Furthermore, despite having low sequence identity to the host GST, Na-GST-3 has a greater tertiary-structure similarity to human sigma-class GST than was observed for the other N. americanus GSTs.


Subject(s)
Glutathione Transferase/chemistry , Helminth Proteins/chemistry , Necator americanus/enzymology , Amino Acid Sequence , Animals , Binding Sites/physiology , Crystallography, X-Ray , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Helminth Proteins/genetics , Helminth Proteins/metabolism , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Necator americanus/genetics
3.
Protein Expr Purif ; 83(2): 145-51, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22503665

ABSTRACT

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.


Subject(s)
Antigens, Helminth/isolation & purification , Glutathione Transferase/isolation & purification , Helminth Proteins/isolation & purification , Necator americanus/enzymology , Recombinant Proteins/isolation & purification , Animals , Antigens, Helminth/genetics , Antigens, Helminth/metabolism , Chromatography, Gel , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Helminth Proteins/genetics , Helminth Proteins/metabolism , Pichia/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
4.
J Infect Dis ; 201(10): 1561-9, 2010 May 15.
Article in English | MEDLINE | ID: mdl-20367477

ABSTRACT

The aspartic protease of Necator americanus, Na-APR-1, is a vaccine antigen that induces antibodies that neutralize hemoglobin proteolysis in the gut of the worm. To define the epitopes recognized by these antibodies, monoclonal antibodies (mAbs) were raised and assessed for neutralizing activity. Three immunoglobulin (Ig) G1 mAbs bound to the intestine of N. americanus and inhibited Na-APR-1 enzymatic activity. Overlapping fragments of Na-APR-1 were expressed, and one (APR-1/5B) was recognized by all 3 mAbs; the epitope was further characterized as AGPKAQVEAIQKY (A(291)Y). This same peptide with a Phe/Tyr(303) substitution was recognized by mAbs in APR-1 orthologues from Ancylostoma species hookworms. IgG from humans infected with hookworms did not recognize A(291)Y but, rather, recognized the S(107)L epitope. APR-1/5B was fused to other helminth vaccine antigens, including Schistosoma mansoni Sm-TSP-2 and N. americanus Na-GST-1; antibodies against both chimeras neutralized the enzymatic activity of Na-APR-1. These findings support the incorporation of Na-APR-1 into a multivalent vaccine against hookworm and/or schistosomiasis.


Subject(s)
Aspartic Acid Proteases/immunology , Hookworm Infections/prevention & control , Necator americanus/enzymology , Schistosomiasis/prevention & control , Vaccines/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Aspartic Acid Proteases/chemistry , Aspartic Acid Proteases/metabolism , Dose-Response Relationship, Immunologic , Epitopes , Immunoglobulin G/immunology , Mice , Molecular Sequence Data , Protein Conformation , Vaccines, Synthetic/immunology
5.
Infect Immun ; 78(4): 1552-63, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20145100

ABSTRACT

Hookworm glutathione S-transferases (GSTs) are critical for parasite blood feeding and survival and represent potential targets for vaccination. Three cDNAs, each encoding a full-length GST protein from the human hookworm Necator americanus (and designated Na-GST-1, Na-GST-2, and Na-GST-3, respectively) were isolated from cDNA based on their sequence similarity to Ac-GST-1, a GST from the dog hookworm Ancylostoma caninum. The open reading frames of the three N. americanus GSTs each contain 206 amino acids with 51% to 69% sequence identity between each other and Ac-GST-1. Sequence alignment with GSTs from other organisms shows that the three Na-GSTs belong to a nematode-specific nu-class GST family. All three Na-GSTs, when expressed in Pichia pastoris, exhibited low lipid peroxidase and glutathione-conjugating enzymatic activities but high heme-binding capacities, and they may be involved in the detoxification and/or transport of heme. In two separate vaccine trials, recombinant Na-GST-1 formulated with Alhydrogel elicited 32 and 39% reductions in adult hookworm burdens (P < 0.05) following N. americanus larval challenge relative to the results for a group immunized with Alhydrogel alone. In contrast, no protection was observed in vaccine trials with Na-GST-2 or Na-GST-3. On the basis of these and other preclinical data, Na-GST-1 is under possible consideration for further vaccine development.


Subject(s)
Antigens, Helminth/immunology , Antigens, Helminth/metabolism , Glutathione Transferase/immunology , Glutathione Transferase/metabolism , Heme/metabolism , Necator americanus/enzymology , Necator americanus/immunology , Necatoriasis/prevention & control , Adjuvants, Immunologic/administration & dosage , Aluminum Hydroxide/administration & dosage , Amino Acid Sequence , Animals , Antigens, Helminth/genetics , Cloning, Molecular , Cricetinae , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , DNA, Helminth/genetics , DNA, Helminth/isolation & purification , Gene Expression , Glutathione/metabolism , Glutathione Transferase/genetics , Humans , Lipid Peroxidation , Molecular Sequence Data , Necator americanus/genetics , Necatoriasis/immunology , Open Reading Frames , Pichia/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Vaccines, Subunit/immunology
6.
Trop Biomed ; 37(2): 536-541, 2020 Jun 01.
Article in English | MEDLINE | ID: mdl-33612820

ABSTRACT

Two female and one male adult hookworms were recovered from a female patient in Thailand. Based on gross and microscopic morphology, the three hookworms are members of Necator americanus. Phylogenetic reconstruction based on partial NADH dehydrogenase subunit 1 (nad1) mitochondrial gene sequences shows that these hookworms belong to the same genetic lineage as N. americanus adult worm from Zhejiang, China. The male and female hookworms were genetically distinct, belonging to two different nad1-haplotypes. This is the first report targeting the nad1 gene on the identification and genetic characterization of the human hookworms originated from infected patient. The nad1 gene marker is useful for species and higher taxa differentiation of hookworms.


Subject(s)
Genes, Mitochondrial , Genetic Variation , NADH Dehydrogenase/genetics , Necator americanus/enzymology , Aged , Animals , Base Sequence , DNA, Mitochondrial/genetics , Female , Haplotypes , Humans , Male , Necator americanus/genetics , Thailand
7.
Mol Biochem Parasitol ; 160(2): 90-9, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18501979

ABSTRACT

mRNAs encoding cathepsin B-like cysteine proteases (CatBs) are abundantly expressed in the genomes of blood-feeding nematodes. Recombinant CatBs have been partially efficacious in vaccine trials in animal models of hookworm infection, supporting further investigation of these enzymes as new control tools. We recently described a family of four distinct CatBs (Na-CP-2, -3, -4, -5) from the human hookworm, Necator americanus. Here we show that these N. americanus CatBs form a robust clade with other hookworm CatBs and are most similar to intestinal CatBs from Haemonchus contortus. All four mRNAs (Na-cp-2, -3, -4 and -5) are up-regulated during the transition from a free-living larva to a blood-feeding adult worm and are also expressed in gut tissue of adult N. americanus that was dissected using laser microdissection microscopy. Recombinant Na-CP-3 was expressed in soluble, secreted form in the yeast Pichia pastoris, while Na-CP-2, -4 and -5 were expressed in insoluble inclusion bodies in Escherichia coli. Recombinant Na-CP-3 was not catalytically active when secreted by yeast but underwent auto-activation to an active enzyme at low pH in the presence of dextran sulphate. Activated Na-CP-3 digested gelatin and cleaved the fluorogenic substrate Z-Phe-Arg-aminomethylcoumarin (AMC) but not Z-Arg-Arg-AMC. Recombinant Na-CP-3 did not digest intact hemoglobin but digested globin fragments generated by prior hydrolysis with N. americanus aspartic hemoglobinases. Antibodies raised in mice to all four recombinant proteins showed minimal cross-reactivity with each other, and each antiserum bound to the intestine of adult N. americanus, supporting the intestinal expression of their mRNAs. These data show that N. americanus expresses a family of intestinal CatBs, many of which are likely to be involved in nutrient acquisition and therefore are potential targets for chemotherapies and vaccines.


Subject(s)
Cathepsin B/biosynthesis , Necator americanus/enzymology , Up-Regulation , Amino Acid Sequence , Animals , Cathepsin B/metabolism , Cloning, Molecular , Coumarins/metabolism , Cricetinae , Dipeptides/metabolism , Escherichia coli/genetics , Gelatin/metabolism , Gene Expression , Gene Expression Profiling , Haemonchus/enzymology , Hemoglobins/metabolism , Mice , Molecular Sequence Data , Phylogeny , Pichia/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity
8.
J Leukoc Biol ; 73(1): 165-71, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12525574

ABSTRACT

The proteolytic activities frequently associated with sources of allergens and parasite secretions have been suggested as important immunomodulators. We have investigated whether the protease activity of the house dust mite allergen Der p1 and the secreted proteases of the hookworm Necator americanus are able to directly induce type 2 cytokine production by basophils. Der p1 and the secretions of N. americanus induced interleukin (IL)-4, IL-5, and IL-13 but not interferon-gamma mRNA in KU812 basophils. Enzyme-linked immunosorbent assay confirmed that IL-4 and IL-13 were secreted. A nonproteolytic antigen failed to induce cytokine expression, and preincubation of Der p1 or N. americanus secretions with protease inhibitors inhibited cytokine expression. Data were confirmed using basophils purified from human peripheral blood. We speculate that this innate mechanism may contribute to the development of a cytokine milieu that could promote immunoglobulin E synthesis, eosinophil recruitment, and the development of type 2 T cells.


Subject(s)
Basophils/immunology , Cytokines/biosynthesis , Endopeptidases/immunology , Helminths/enzymology , Pyroglyphidae/enzymology , Animals , Antigens, Dermatophagoides/immunology , Antigens, Dermatophagoides/pharmacology , Arthropod Proteins , Basophils/metabolism , Cysteine Endopeptidases , Cytokines/drug effects , Endopeptidases/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Helminths/immunology , Humans , Interferon-gamma , Interleukin-13/biosynthesis , Interleukin-13/metabolism , Interleukin-4/biosynthesis , Interleukin-4/metabolism , Interleukin-5/biosynthesis , Necator americanus/enzymology , Necator americanus/immunology , Pyroglyphidae/immunology , Th2 Cells/immunology
9.
ChemMedChem ; 10(10): 1647-54, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26269385

ABSTRACT

Necator americanus (hookworm) infects over half a billion people worldwide. Anthelminthic drugs are commonly used to treat the infection; however, vaccination is a more favorable strategy to combat this parasite. We designed new B-cell peptide epitopes based on the aspartic protease of N. americanus (Na-APR-1). The peptides were conjugated to self-adjuvanting lipid core peptide (LCP) systems via stepwise solid-phase peptide synthesis (SPPS) and copper catalyst azide-alkyne cycloaddition (CuAAC) reactions. The LCP vaccine candidates were able to self-assemble into nanoparticles, were administered to mice without the use of additional adjuvant, and generated antibodies that recognized the parent epitope. However, only one LCP derivative was able to produce a high titer of antibodies specific to Na-APR-1; circular dichroism analyses of this compound showed a ß-sheet conformation for the incorporated epitope. This study provides important insight in epitope and delivery system design for the development of a vaccine against hookworm infections.


Subject(s)
Aspartic Acid Proteases/immunology , Hookworm Infections/parasitology , Lipopeptides/immunology , Nanoparticles/chemistry , Necator americanus/immunology , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/immunology , Animals , Antibodies, Helminth/immunology , Aspartic Acid Proteases/chemistry , Female , Hookworm Infections/immunology , Lipopeptides/chemistry , Mice , Mice, Inbred BALB C , Molecular Conformation , Necator americanus/enzymology , Particle Size , Structure-Activity Relationship
10.
Hum Vaccin Immunother ; 11(6): 1474-88, 2015.
Article in English | MEDLINE | ID: mdl-25905574

ABSTRACT

Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.


Subject(s)
Antigens, Helminth/isolation & purification , Antigens, Helminth/metabolism , Aspartic Acid Endopeptidases/isolation & purification , Aspartic Acid Endopeptidases/metabolism , Necator americanus/enzymology , Vaccines, Synthetic/isolation & purification , Vaccines, Synthetic/metabolism , Animals , Antigens, Helminth/genetics , Aspartic Acid Endopeptidases/genetics , Biotechnology/methods , Gene Expression , Necator americanus/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Technology, Pharmaceutical/methods , Nicotiana/genetics , Nicotiana/metabolism , Vaccines, Synthetic/genetics
11.
Int J Parasitol ; 22(5): 563-72, 1992 Aug.
Article in English | MEDLINE | ID: mdl-1399239

ABSTRACT

Following the observation that live third-stage larvae (L3) could digest gelatin in vitro, gelatinolytic protease activity has been demonstrated at pH 8.5, in both exsheathing fluid (EF) and excretory/secretory (ES) products of infective L3 of Necator americanus. EF resolved as a single band of proteolytic activity, with a mol. wt of 116 kDa, while L3 ES products exhibited multiple bands of proteolysis, at 219, 200, 195, 166, 137, 92, 72 and 62 kDa; weak bands were detectable at 92 and 72 kDa. The EF protease was characterized as cysteine, whereas ES apparently possessed one serine (195 kDa) and seven (219, 200, 166, 137, 92, 72 and 62 kDa) cysteine protease bands and a combination of metallo- and cysteine proteases of approximately the same mol. wts (62, 137 and 219 kDa). Though EF was not able to cleave immunoglobulins, ES was shown to cleave IgG, IgA and IgM, but not IgD or IgE. The activity appeared to be directed toward the Fc portion of the molecule, and was inhibited by PMSF, which is indicative of serine protease activity. The significance of the presence of such apparently diverse proteases in larval products is discussed.


Subject(s)
Endopeptidases/metabolism , Necator americanus/enzymology , Animals , Electrophoresis, Polyacrylamide Gel , Immunoglobulins/metabolism , Larva/enzymology
12.
Am J Trop Med Hyg ; 60(5): 840-7, 1999 May.
Article in English | MEDLINE | ID: mdl-10344662

ABSTRACT

The infective larvae of Necator americanus were shown to secrete all mechanistic classes of proteolytic enzymes with two overall pH optima of 6.5 and 8.5 using fluorescein isothiocyanate-labeled casein as the substrate. Since infective larvae are obligate skin penetrators, the effect of each of these enzyme classes against macromolecules derived from human skin was examined. Larval secretions were shown to degrade collagen types I, III, IV, and V, fibronectin, laminin, and elastin. All the skin macromolecules tested were hydrolyzed by aspartyl proteinase activity, which was inhibitable by pepstatin A. Collagen and elastin was also hydrolyzed by metalloproteinase activity, while the serine proteinase activity hydrolyzed only elastin. As a consequence of these experiments, the effect of proteinase inhibitors on the penetration of live larvae through hamster skin was tested. Larval penetration was significantly inhibited only by pepstatin A, confirming the importance of the aspartyl proteinase activity during the skin penetration process.


Subject(s)
Aspartic Acid Endopeptidases/metabolism , Necator americanus/enzymology , Necator americanus/pathogenicity , Skin/metabolism , Skin/parasitology , Animals , Collagen/metabolism , Cricetinae , Elastin/metabolism , Fibronectins/metabolism , Helminth Proteins/metabolism , Humans , Hydrogen-Ion Concentration , Laminin/metabolism , Necator americanus/growth & development , Necatoriasis/parasitology
13.
Acta Trop ; 59(3): 259-63, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7572433

ABSTRACT

The difficulty in demonstrating protective immunity to human gastro-intestinal nematodes is thought to be a consequence of the expression of defences by the parasites directed against the toxic metabolites of leukocytes produced during inflammation (Brophy and Pritchard, 1992a). Parasite glutathione S-transferases (GSTs) may provide part of this defence by detoxifying the secondary products of lipid peroxidation produced via immune initiated free-radical attack on host or parasite membranes (Brophy and Pritchard, 1994; Taylor et al., 1988). Neutralisation of parasite immune defence components could tip the molecular balance in favour of the immune response during chronic infections. For example, GSTs have been extensively investigated from the digenean parasites Schistosoma and Fasciola hepatica and provide protection in animal-model systems (Mitchell, 1988; Wijffels et al., 1991). In contrast, although GSTs have been initially characterised in filarial nematodes (Salinas et al., 1994; Leibau et al., 1994; Jaffe and Lambert, 1986), there is limited information on GSTs from human gastro-intestinal nematode parasites. We were particularly interested in analysing the products of hookworms for evidence of the presence of excretory-secretory forms of this putative immune defence protein.


Subject(s)
Glutathione Transferase/physiology , Necator americanus/enzymology , Animals , Humans , Necatoriasis/immunology
14.
J Parasitol ; 78(5): 917-9, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1403440

ABSTRACT

Fresh living third-stage larvae of Necator americanus released a significant amount of label within 2 hr of their incubation on 125I-labeled gelatin-coated polystyrene plastic plates. This protease activity was primarily susceptible to o-phenanthroline, which identifies the activity as predominantly metalloprotease.


Subject(s)
Metalloendopeptidases/metabolism , Necator americanus/enzymology , Animals , Chromatography, Gel , Cricetinae , Gelatin/metabolism , Larva/enzymology , Metalloendopeptidases/antagonists & inhibitors
15.
J Parasitol ; 87(5): 1227-9, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11695411

ABSTRACT

Significant differences in the life histories of the human hookworms Ancylostoma duodenale and Necator americanus necessitate their differentiation for epidemiological studies and the design of control programs. Current methods of identification require time-consuming, labor-intensive techniques. A polymerase chain reaction (PCR)-based method that enables rapid species identification is described. The mitochondrial cytochrome oxidase I genes of both species were sequenced, and species-specific primer sets were designed. The primers were used in PCR to amplify 585-bp fragments of the cytochrome oxidase gene from individual hookworm eggs, larvae, and adults. The technique was also able to identify mixed infections containing equal amounts of eggs from each species. The technique is rapid, technically simple, and sensitive and will permit the accurate identification of human hookworms in epidemiological field studies.


Subject(s)
Ancylostoma/classification , Ancylostomiasis/parasitology , DNA, Helminth/genetics , Electron Transport Complex IV/genetics , Necator americanus/classification , Necatoriasis/parasitology , Ancylostoma/enzymology , Ancylostoma/genetics , Animals , Base Sequence , China , Cricetinae , DNA, Helminth/chemistry , DNA, Helminth/isolation & purification , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/genetics , DNA, Mitochondrial/isolation & purification , Electron Transport Complex IV/chemistry , Humans , Mesocricetus , Molecular Sequence Data , Necator americanus/enzymology , Necator americanus/genetics , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Sequence Homology, Nucleic Acid , Species Specificity
16.
Article in Zh | MEDLINE | ID: mdl-14628369

ABSTRACT

OBJECTIVE: To detect and compare the COI gene sequences of Necator americanus collected from the provinces of Sichuan, Hainan, Yunnan, Hubei and Jiangsu, and to analyze the genetic diversity of the geographic isolates. METHODS: COI genes of N. americanus were amplified from the genomic DNA by PCR and sequenced. RESULTS: The COI gene sequences of N. americanus from five provinces were 97%-99% identical over 595 bp, and base variation occurred in 19 nucleotide sites in which the transition was more frequent than the transversion. The difference between the sequences ranged from 1.34% to 2.18%. CONCLUSION: The COI gene sequences show high identity among the geographic isolates of N. americanus with some difference at specific nucleotide sites.


Subject(s)
Electron Transport Complex IV/genetics , Necator americanus/enzymology , Polymorphism, Genetic , Animals , Base Sequence , DNA, Helminth/genetics , Molecular Sequence Data , Necator americanus/genetics , Phylogeny , Polymerase Chain Reaction , Sequence Homology, Nucleic Acid
17.
Article in Zh | MEDLINE | ID: mdl-12563786

ABSTRACT

AIM: To identify the genetic diversity between Ancylostoma duodenale and Necator americanus. METHODS: Mitochondrial cytochrome C oxidase subunit 1 (CO1) gene was amplified from genomic DNA of human hookworms collected from infected patients in Hejiang County, Sichuan Province, and the purified PCR products were directly sequenced by using Licor auto-sequencer. RESULTS: The PCR products were about 700 bp. Alignment of CO1 gene fragment sequences showed 89.7% similarity between Ancylostoma duodenale and Necator americanus, but still certain nucleotide variations (10.3%) existed. CONCLUSION: CO1 gene sequence can be used as a marker to identify the two species of human hookworms.


Subject(s)
Ancylostoma/enzymology , Cytochrome-c Peroxidase/genetics , Necator americanus/enzymology , Amino Acid Sequence , Ancylostoma/classification , Ancylostoma/genetics , Animals , Base Sequence , Cytochrome-c Peroxidase/chemistry , DNA, Helminth/genetics , DNA, Helminth/isolation & purification , Humans , Molecular Sequence Data , Necator americanus/classification , Necator americanus/genetics , Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Species Specificity
18.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 9): 1162-6, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25195885

ABSTRACT

Glutathione S-transferase 1 from Necator americanus (Na-GST-1) is a vaccine candidate for hookworm infection that has a high affinity for heme and metal porphyrins. As part of attempts to clarify the mechanism of heme detoxification by hookworm GSTs, co-crystallization and soaking studies of Na-GST-1 with the heme-like molecules protoporphyrin IX disodium salt, hematin and zinc protoporphyrin were undertaken. While these studies did not yield the structure of the complex of Na-GST-1 with any of these molecules, co-crystallization experiments resulted in the first structures of the complex of Na-GST-1 with the substrate glutathione. The structures of the complex of Na-GST-1 with glutathione were solved from pathological crystalline aggregates comprising more than one crystal form. These first structures of the complex of Na-GST-1 with the substrate glutathione were solved by molecular replacement from data collected with a sealed-tube home source using the previously reported apo structure as the search model.


Subject(s)
Glutathione Transferase/chemistry , Glutathione/chemistry , Necator americanus/enzymology , Animals , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Molecular Structure
19.
Hum Vaccin Immunother ; 10(7): 1914-25, 2014.
Article in English | MEDLINE | ID: mdl-25424799

ABSTRACT

Infection by the human hookworm Necator americanus is a leading cause of anemia and disability in the developing countries of Africa, Asia, and the Americas. In order to prevent childhood hookworm disease in resource poor settings, a recombinant vaccine is under development by the Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, a Product Development Partnership (PDP). Previously, we reported on the expression and purification of a highly promising hookworm vaccine candidate, Na-GST-1, an N. americanus glutathione s-transferase expressed in Pichia pastoris (yeast), which led to production of 1.5 g of 95% pure recombinant protein at a 20L scale. (1) (,) (2) (,) (3) This yield and purity of Na-GST-1 was sufficient for early pilot manufacturing and initial phase 1 clinical testing. However, based on the number of doses which would be required to allow mass vaccination and a potential goal to deliver a vaccine as inexpensively as possible, a higher yield of expression of the recombinant antigen at the lowest possible cost is highly desirable. Here we report on modifications to the fermentation (upstream process) of the antigen expressed in P. pastoris, and to the purification (downstream process) of the recombinant protein that allowed for a 2-3-fold improvement in the final yield of Na-GST-1 purified protein. The major improvements included upstream process changes such as the addition of a sorbitol pulse and co-feed during methanol induction as well as an extension of the induction stage to approximately 96 hours; downstream process changes included modifying the UFDF to flat sheet with a 10 kDa Molecular Weight cut-off (MWCO), adjusting the capacity of an ion-exchange chromatography step utilizing a gradient elution as opposed to the original step elution, and altering the hydrophobic interaction chromatography conditions. The full process, as well as the purity and stability profiles of the target Na-GST-1, and its formulation on Alhydrogel(®), is described.


Subject(s)
Antigens, Helminth/isolation & purification , Glutathione Transferase/isolation & purification , Hookworm Infections/prevention & control , Necator americanus/enzymology , Technology, Pharmaceutical/methods , Vaccines, Synthetic/isolation & purification , Animals , Antigens, Helminth/genetics , Biotechnology/methods , Chemistry, Pharmaceutical , Chromatography, Liquid/methods , Culture Media/chemistry , Drug Stability , Glutathione Transferase/genetics , Hookworm Infections/immunology , Humans , Necator americanus/immunology , Pichia/genetics , Pichia/growth & development , Pichia/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Transcriptional Activation , Vaccines, Synthetic/genetics
20.
Expert Rev Vaccines ; 9(10): 1219-30, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20923271

ABSTRACT

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. Herein, we discuss the evolution of our thinking regarding the design of a potency assay along these newly defined lines and its application to the release of the experimental Necator americanus-glutathione-S- transferase-1 (Na-GST-1) vaccine to prevent human hookworm infection. We discuss the necessary steps to accomplish the design and implementation of such a new potency assay as a resource for the burgeoning NTD vaccine community. Our experience is that much of the existing information is proprietary and needs to be pulled together in a single source to aid in our overall understanding of potency testing.


Subject(s)
Ancylostomatoidea/immunology , Antigens, Helminth/immunology , Glutathione Transferase/immunology , Hookworm Infections/prevention & control , Necator americanus/enzymology , Vaccines, Synthetic/standards , Animals , Antigens, Helminth/genetics , Cricetinae , Disease Models, Animal , Dogs , Drug Design , Glutathione Transferase/genetics , Hookworm Infections/immunology , Humans , Necator americanus/immunology , Vaccines, Synthetic/administration & dosage
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