Triteeraprapag et al 1994.pdf

Molecular and Biochemical Parasitology, 2004

Onchocerca volvulus is a tissue-dwelling, vector-borne nematode parasite of humans and the causative agent of onchocerciasis, or 'River Blindness'. Resistance to infection is associated with immune responses to the infective, third-stage (L3) larvae. The antigens of greatest interest for their vaccine potential are surface and secreted molecules. We have previously identified a family of Secreted Larval Acidic Proteins (SLAPs) from the L3 larvae of O. volvulus by biosynthetic labelling. Here, we provide further characterisation of these molecules following cloning and expression of the corresponding cDNAs. Using protein sequencing, we show that SLAPs are members of the alt gene family, first described in the lymphatic filarial parasite, Brugia malayi. Ov-ALT-1 and Ov-ALT-2 correspond with 20 and 18 kDa SLAPs. Both proteins are highly acidic and related by sequence, differing chiefly in an 8-amino acid deletion from Ov-ALT-2. By immunochemistry, we confirm that Ov-ALTs are highly stage-specific, being expressed exclusively in late L2 and L3 larvae during growth in the vector. They are synthesised and stored in the glandular oesophagus. Secretion is triggered by the resumption of development in the definitive host and occurs via the pseudocoelom and cuticle. Serological responses in humans to recombinant Ov-ALT-1 indicate that the level of IgG production may be governed by the force of transmission but does not overtly reflect infection status. Immunisation of mice with recombinant Ov-ALT-1 resulted in a modest level of protection against challenge with O. volvulus L3 larvae (P = 0.036). We conclude that Ov-ALT genes, like those of other filariae, are of interest from the standpoint of parasite transmission and infectivity. They may also offer promise as components of a future sub-unit vaccine should the means to enhance protection be achieved.

Clinical Immunology and Immunopathology, 1997

which kills the adult worms, there is considerable in-The localization of T and B cell epitopes on a well terest in elucidating immune reactions against the parcharacterized 33-kDa protein of the filarial nematode asite in order to evaluate possibilities of vaccine devel-Onchocerca volvulus (Ov33) was studied using periphopment. A requirement of potential vaccine antigens is eral blood mononuclear cells (PBMC) and sera from a that they should induce appropriate immune responses total of 52 onchocerciasis patients with the generalized in a large proportion of the target population. This form of infection. A proportion of the PBMC samples could be achieved using a subunit vaccine that contains proliferated in response to recombinant Ov33-GST fua combination of well defined T and B cell epitopes of sion protein and to fusion free Ov33-61His. Proliferavarious Onchocerca proteins. To characterize such B tive responses of patient PBMC to seven truncated and T cell epitopes, we analyzed the immunodominant Ov33-61His polypeptides and to three synthetic pep-33-kDa protein of O. volvulus (Ov33), which was cloned tides revealed at least one major and two minor T cell and characterized in earlier studies (2, 3). Ov33 is epitopes in the protein. The dominant T cell stimulating widely recognized by sera of individuals infected with domain was localized between amino acids 113 and 143.

Electrophoresis, 1999

1. Polypeptides of liver plasma membrane fractions enriched in three surface domains of hepatocytes, blood-sinusoidal, lateral and bile canalicular, were analysed by isoelectric focusing (IEF) and non-equilibrium pH gel electrophoresis (NEPHGE) across a wide pH range, followed by SDS/PAGE. The overall Coomassie Blue-stained polypeptide patterns in the fractions were different. Lateral plasma membrane fractions contained a characteristically higher number of polypeptides focusing at the basic pH range, whereas few basic polypeptides were present in sinusoidal plasma membrane fractions. The glycoproteins in these plasma membrane fractions stained by a lectin overlay technique with radioiodinated concanavalin A, wheat-germ agglutinin and a slug lectin, were also different. 2. The polypeptides and glycoproteins of 'early' and 'late' endosome fractions were also compared by two-dimensional electrophoresis. Their composition was shown by Coomassie Blue staining, lectin overlay staining and in membranes metabolically labelled with [35S]methionine to be generally similar. The glycoproteins of sinusoidal plasma membranes and early and late endosomes were generally similar, but major differences in polypeptides of molecular mass 20-50 kDa, pl 7.5-8.5, in plasma membranes and endosomes were demonstrated, with a specific population of basic (pI 8-9) low-molecular-mass polypeptides being present at highest levels in 'late' endosomal fractions (shown by Coomassie Blue staining). 3. Analysis of the distribution of three specific membrane glycoproteins identified by using immunoblotting techniques showed that the asialoglycoprotein and the divalent-cation-sensitive mannose 6-phosphate receptors were present in sinusoidal plasma membrane and in early and late endocytic fractions; they were not detected in canalicular plasma membrane fractions. In contrast, 5'-nucleotidase was detected in all fractions examined. The role of the endocytic compartment in regulating trafficking pathways between the plasma membrane domains of the hepatocyte is discussed.

Molecular and Biochemical Parasitology, 1998

A full length D. immitis cDNA (nDiCal) encoding a protein with significant similarity to the calreticulin protein family was isolated from a 6-day fourth-stage larval cDNA expression library by immunoscreening, using serum from a rabbit immunized by repeated injection of small numbers of third-stage larvae. nDiCal is 1538 bp long and contains the 21 bp nematode splice leader sequence SL1 at the 5′ end. nDiCal encodes for a protein (pDiCal) with a predicted molecular mass of 46 kDa. pDiCal sequence analysis revealed similarities with calreticulin, a protein that typically resides in the endoplasmic reticulum. pDiCal possesses three consensus sequences of the calreticulin family of proteins: a neutral N-terminal region with a putative signal sequence; a proline- and tryptophan-rich P region; and a highly acidic C-terminal region. A 45Ca2+-overlay assay showed that recombinant pDiCal (rDiCal) is a Ca2+-binding protein. Antibodies to rDiCal identified a 56 kDa native antigen in all developmental stages including the excretory-secretory products derived from larvae and adult worms. Localization studies demonstrated the ubiquitous presence of pDiCal with intense expression in the hypodermis and syncitial muscle cells in both male and female adult worms. Labeling was also seen in the developing embryos within the uterus of the female worms. Sera from immune as well as chronically-infected microfilaremic dogs contained antibodies that bind rDiCal. In addition, immunoblot analysis showed that serum from a rabbit immunized with L3 cuticles reacted with rDiCal.

Molecular and Biochemical Parasitology, 1991

We report here the development of in situ hybridization and immunohistochemistry protocols which permit the histological identification of gene expression of a cloned antigen of Onchocerca volvulus, O15, in the parasite. Skin nodules containing female adult worms were fixed in a modified Carnoy's fixative and embedded in paraffin. Histological staining of tissue sections revealed uniformly excellent morphology and RNA preservation. To localize mRNA by in situ hybridization, tissue sections were incubated with biotin-labeled pO15, the plasmid containing the genomic sequence of the antigen, and hybridization signals were histochemically visualized using a streptavidin-enzyme conjugate and chromogenic substrates. The protein antigen was localized immunohistochemically by incubating the sections with specific antibodies prepared against a recombinant fusion protein containing the O15 sequence (OI3), and visualized via a secondary antibody-biotin-enzyme conjugate procedure. The results reported here showed distinct localization of the O15 mRNA and O13 antigen in specific cellular and tissue regions of the adult parasite, and in microfilariae located within the uteri and in the surrounding host tissue. The specificity and high sensitivity of these histological detection methods should be generally applicable for the characterization of gene expression in the filarial parasite, particularly the insect-borne, infective filarial larvae, which are severely limited in quantity.

Mol Biochem Parasitol, 2003

Excretory-secretory (ES) products of Ostertagia ostertagi, an abomasal nematode of cattle, are considered to be important for the development and survival of the parasite within the host. To gain insight in the composition of these ES products of both larval (L3, L4) and adult life stages of Ostertagia cDNA libraries of the parasite were immunoscreened with polyclonal rabbit serum raised against these ES products. This approach led to the identification of 41 proteins, amongst which are structural proteins such as actin, kinesin and vitellogenin, housekeeping proteins such as those involved in protein folding, different metabolic pathways or mitochondrial functioning and proteins associated with stress (heat shock protein) or antioxidantia (thioredoxin peroxidase). A large number of the isolated proteins were similar to hypothetical proteins of the model nematode Caenorhabditis elegans. Because somatic proteins can be non-specifically released during in vitro culturing as nematodes deteriorate, it was checked if the isolated proteins are genuinely secreted. The amino acid sequences of the translated cDNAs were investigated for signal peptides and monospecific antibodies against the isolated proteins were purified and used to develop Western blots of ES and somatic extracts. In this manner it could be proven that 15 cDNAs code for genuine secreted proteins. The identification of these ES antigens allows to select proteins with potential protective capacities, which are targets for vaccine development.

International Journal for Parasitology, 2002

Presently, little is known of the mechanism by which Trichinella penetrates and modulates reprogramming of muscle cells. In light of evidence demonstrating strong protective characteristics of antigens derived from this stage, understanding this process may shed light on potential targets for effective abatement of infection. To this end, a PCR-derived cDNA expression library was constructed using 0.5 mg of total RNA from Trichinella spiralis newborn larvae. The library consisted of .125,000 insert-containing clones. Approximately 40-50 £ 10 3 clones were screened immunologically using sera from pigs experimentally infected with 7,000 Trichinella L1. Multiple clones reacting positively with the swine infection serum and encoding portions of a glutamic acid-rich protein were identified. Northern and Southern blots indicated at least two distinct genes that encoded the glutamic acid-rich proteins and that these genes were transcribed specifically in the newborn larvae stage. cDNA sequence data predicted open reading frames of 1,497 and 1,716 bp generating proteins of 498 amino acids and 571 amino acids, respectively. Both sequences consisted of approximately 39% glutamic acid and 16% serine residues, and differed by the presence of a 219 bp fragment present in the 1,716 bp sequence that was absent from the 1,497 bp sequence. PCR data indicated that additional isoforms exist within this gene family that are different in length from those described above. In addition, it was found that more than one isoform can exist within a single worm and that this pattern can vary between individual worms within a population. Mouse antibodies to recombinant antigen localised the glutamic acid-rich proteins to the periphery of the developing stichocyte cells within the newborn larvae consistent with the hypothesis that the newborn larval antigens are secreted. Published by

Molecular and Biochemical Parasitology, 2003

PLoS Pathogens, 2013

Gastrointestinal nematode parasites infect over 1 billion humans, with little evidence for generation of sterilising immunity. These helminths are highly adapted to their mammalian host, following a developmental program through successive niches, while effectively down-modulating host immune responsiveness. Larvae of Heligmosomoides polygyrus, for example, encyst in the intestinal submucosa, before emerging as adult worms into the duodenal lumen. Adults release immunomodulatory excretory-secretory (ES) products, but mice immunised with adult H. polygyrus ES become fully immune to challenge infection. ES products of the intestinal wall 4th stage (L4) larvae are similarly important in host-parasite interactions, as they readily generate sterile immunity against infection, while released material from the egg stage is ineffective. Proteomic analyses of L4 ES identifies protective antigen targets as well as potential tissue-phase immunomodulatory molecules, using as comparators the adult ES proteome and a profile of H. polygyrus egg-released material. While 135 proteins are shared between L4 and adult ES, 72 are L4 ES-specific; L4-specific proteins correspond to those whose transcription is restricted to larval stages, while shared proteins are generally transcribed by all life cycle forms. Two protein families are more heavily represented in the L4 secretome, the Sushi domain, associated with complement regulation, and the ShK/SXC domain related to a toxin interfering with T cell signalling. Both adult and L4 ES contain extensive but distinct arrays of Venom allergen/Ancylostoma secreted protein-Like (VAL) members, with acetylcholinesterases (ACEs) and apyrase APY-3 particularly abundant in L4 ES. Serum antibodies from mice vaccinated with L4 and adult ES react strongly to the VAL-1 protein and to ACE-1, indicating that these two antigens represent major vaccine targets for this intestinal nematode. We have thus defined an extensive and novel repertoire of H. polygyrus proteins closely implicated in immune modulation and protective immunity.

Parasite Immunology, 2004

Vaccination of mice with a recombinant protein, Ov-ASP-1, the Onchocerca volvulus homologue of the activation associated secreted gene family stimulated very high titres of both IgG1 and IgG2a without adjuvant. r Ov-ASP-1 was also immunoreactive with IgG isotypes from both O. volvulus-infected (INF) and putatively immune (PI) humans, with higher IgG4 in the former group. The protein also stimulated IFN-γ secretion by PBMC from INF and PI and IL-5 only in INF. Using a mouse diffusion chamber model, vaccination with r Ov-ASP-1 resulted in partial but significant protection against challenge with infective third-stage larvae (L3) but only when formulated with Freund's complete adjuvant (FCA) or alum. Protection was Th1dependent (highly elevated IgG2a) with FCA and contingent on a strongly Th2-skewed (IgG1) response with alum. IgE responses to r Ov-ASP-1 with or without adjuvant were weak or absent. When immunization using r Ov-ASP-1 in adjuvant failed to induce adequate Th1 (FCA) or Th2 (alum) responses, protection efficacy was compromised. The recombinant protein appears to stimulate a mixed Th1/Th2 response but the outcome in terms of protective immunity is the result of a subtle interplay of its intrinsic and adjuvant-augmented properties. Ov-ASP-1 is potentially secreted based on its localization in the secretory granules of L3.

Journal of bioscience and …, 2009

Transactions of the Royal Society of Tropical Medicine and Hygiene, 1990

The surface infective larvae polypeptide antigens of third-stage (LX) and adult males of Onchocerca volvul~~ have been compared after iodogen-catalysed radioiodination, separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in b-mercaptoethanol followed by autoradiography. L3 surfaces contained polypeptides with apparent molecular weights of 14, 18.5, 26, 34, 51, 68 and 130 kDa, whereas the adults contained 14, 19, 26, 34, 37.5 and 68 kDa components. By immunoprecipitation with patient's sera, only the 14 and 18.5 kDa components were shown to be antigenic on the Ls, the other components being unreactive with patients' antibodies. Under similar conditions, 4 of the 6 adult male polypeptides reacted with patients' antisera, confirming their antigenic nature. Lentil lectin and immunosorbent chromatography of the surface components revealed the 18.5 kDa and 68 kDa antigens of L3 and adult males respectively to be glycoproteins. The apparently weak reactivity of Ls surface components with host antibody may be part of an escape mechanism that favours the establishment of 0. volvulus in human hosts.

Molecular and Biochemical Parasitology, 1995

The complete sequence of the cDNA encoding the nematode polyprotein allergen/antigen (NPA) of the bovine lungworm Dictyocaulus viviparus was obtained by immunoscreening of cDNA expression libraries and by 5' RACE (rapid amplification of cDNA ends). The encoded polypeptide is similar in sequence to the ABA-l allergen of Ascaris, the gp15/400 'ladder' protein of Brugia malayi, Brugia pahangi and Wuchereria bancrofti, and a 15kDa antigen of Dirofilaria immitis. As with these, the predicted amino-acid sequence comprises a head-to-tail array of similar polypeptides with regularly spaced consensus proteinase cleavage sites. The D. viviparus protein was designated DvA-1 (D. vivipurus antigen-l) and the gene dva-1. The deduced amino-acid sequence of DvA-1 showed features not observed before in other NPAs: (i) a hydrophobic leader peptide is present, (ii) none of the 12 units in the array are identical and the sequences diverge to a degree hitherto unseen in the NPAs of other nematode parasites, (iii) the predicted proteinase cleavage sites are also diverse in sequence and, in two instances, no consensus cleavage site was identifiable at the expected position, (iu) a short repeat unit is present, which is the only one containing a consensus N-glycosylation site and (v) a C-terminal extension peptide is encoded which shows no similarity to that from A. mum ABA-l. Comparison of independent cDNAs revealed slight variations in the sequence of the gene within the parasite population. Antisera to recombinant DvA-1 polypeptide identified 14-15kDa antigens in both parasite somatic and excretory-secretory material. DvA-1 is the only NPA for which the complete coding sequence is available and the new principles which it illustrates may lie unsuspected in the NPA-encoding genes of all nematode parasites.

Biochemical and biophysical research communications, 2007

Molecular and Biochemical Parasitology, 1995

Filarial parasites release macromolecules into their environment both in vitro and in vivo. These excretory-secretory products (E-S) have been studied with respect to function, vaccination potential, pathogenicity, and ability to serve as antigen targets for diagnostic tests. We have recently described monoclonal antibody OV-1 which binds to an intermediate filament in E-S and circulating antigens of Onchocerca uolvulus. OV-1 also binds to cross-reactive antigens of Brugia malayi. Therefore, OV-1 was used to immunoscreen a B. maluyi adult worm cDNA library in an attempt to clone a homologue (BMIF). BMIF is a 1664-bp full-length transcript which codes for 505 amino acids. BMIF has 95% sequence homology at the amino-acid level to OVlCF, an 0. uoluulus intermediate filament that was also selected with OV-1, and 75% homology to Ascuris intermediate filament A. Southern blot analysis suggests that BMIF is confined to a single location in the genomic DNA of B. malayi. Antibodies raised to BMIF identified native antigens in immunoblots of B. malayi adult worms, infective larvae and adult E-S. In addition, the antibody also bound to a 60-kDa antigen in immunoblots of poly(ethylene glycol)-precipitated immune complexes in sera from B. maluyi infected patients. Localization studies showed that the antigen encoded by BMIF is present in the hypodermis, developing embryos and muscle of adult B. malayi. These studies show that BMIF is an E-S product of B. malayi adult worms which is detectable in sera from patients with brugian filariasis.