Biomedical subjects

Alex Loukas

Publications and source records attributed to Alex Loukas.

50 records · Page 3Linked to original sources

PubMed2003

Helminth vaccines: from mining genomic information for vaccine targets to systems used for protein expression.

The control of helminth diseases of people and livestock continues to rely on the widespread use of anti-helminthic drugs. However, concerns with the appearance of drug resistant parasites and the presence of pesticide residues in food and the environment, has given further incentive to the goal of discovering molecular vaccines against these pathogens. The exponential rate at which gene and protein sequence information is accruing for many helminth parasites requires new methods for the assimilation and analysis of the data and for the identification of molecules capable of inducing immunological protection. Some promising vaccine candidates have been discovered, in particular cathepsin L proteases from Fasciola hepatica, aminopeptidases from Haemonchus contortus, and aspartic proteases from schistosomes and hookworms, all of which are secreted into the host tissues or into the parasite intestine where they play important roles in host-parasite interactions. Since secreted proteins, in general, are exposed to the immune system of the host they represent obvious candidates at which vaccines could be targeted. Therefore, in this article, we consider the potential values and uses of algorithms for characterising cDNAs amongst the collated helminth genomic information that encode secreted proteins, and methods for their selective isolation and cloning. We also review the variety of prokaryotic and eukaryotic cell expression systems that have been employed for the production and downstream purification of recombinant proteins in functionally active form, and provide an overview of the parameters that must be considered if these recombinant proteins are to be commercialised as vaccine therapeutics in humans and/or animals.

Animals↗

PubMed2003

Molecular characterisation of the Ancylostoma-secreted protein family from the adult stage of Ancylostoma caninum.

The Ancylostoma-secreted proteins are a family of nematode-specific cysteine-rich secreted proteins belonging to the pathogenesis-related protein superfamily. Previously we reported that third stage infective larvae of Ancylostoma caninum produce two different Ancylostoma-secreted proteins, a single and double-domain Ancylostoma-secreted protein, designated as Ancylostoma-secreted protein-1 and Ancylostoma-secreted protein-2, respectively. Here we report that adult A. caninum hookworms produce and release four additional Ancylostoma-secreted proteins (Ancylostoma-secreted protein-3-6). Using antiserum against adult excretory/secretory products, Ancylostoma-secreted protein cDNAs were isolated from cDNA expression libraries. Immunolocalisation experiments using specific antisera indicated that the single-domain Ac-Ancylostoma-secreted protein-3 is located in the adult pharyngeal and oesophageal glands. Ac-Ancylostoma-secreted protein-4, Ancylostoma-secreted protein-5 and Ancylostoma-secreted protein-6 are composed of two pathogenesis-related protein domains linked in tandem as a heterodimorphic repeat. Ac-Ancylostoma-secreted protein-4 is localised to the cuticular surface of the adult hookworm, whereas Ac-Ancylostoma-secreted protein-5 was found in the intestinal brush border membrane, and Ancylostoma-secreted protein-6 in the cephalic and excretory glands. All of the adult Ancylostoma-secreted proteins were identified in excretory/secretory products of adult hookworms by Western blotting and are presumably released by the parasite. None of the adult Ancylostoma-secreted proteins were detected by immunoblotting in L3 extracts, although mRNAs of Ac-Ancylostoma-secreted protein-3 and Ac-Ancylostoma-secreted protein-4 were present in the larval stage. The functions of the adult Ancylostoma-secreted proteins are unknown, although the secretion of multiple family members by the adult suggests an important role in the establishment or maintenance of the parasitic relationship.

Ancylostoma↗

PubMed2003

A gene family from Echinococcus granulosus differentially expressed in mature adult worms.

Differences in mRNA expression between immature adult worms (IAW) and mature adult worms (MAW) of Echinococcus granulosus were determined using polymerase chain reaction-based differential display (DDRT-PCR). Twenty-eight putative differential cDNA fragments were isolated, cloned and sequenced. mRNAs from IAW and MAW were probed with the labelled fragments. Six cDNA fragments (coded as egM12, egM13, egM22, egM26, egM30 and egM34) were putatively determined to be specific to MAW by Northern hybridisation. The stage-specificity of egM12, egM13 and egM34 was confirmed by RT-PCR. RNAs of IAW, MAW, protoscoleces and oncospheres, probed with egM13 and egM30, showed that the mRNAs were expressed exclusively in MAW, which implied involvement in the regulation of egg development. Using the labelled fragments to screen a cDNA library of MAW, 99 clones were identified and analysed. An alignment of selected clones showed that the MAW-specific mRNAs belonged to a family. Examination of the deduced amino acid sequence of three of the corresponding cDNAs (egM4, egM9 and egM123) indicated they were cysteine-rich and contained a 24 amino acid repeat sequence, repeated four to six times. The repeat regions were predominantly alpha helical in nature with interspersed turns, forming alternating zones of positive and negative charge. The functional significance of each of the cDNAs identified is unclear as none had significant sequence similarity to genes of known function. However, polypeptides encoded by egM4 and egM123 were recognised by antibodies in a serum pool from dogs experimentally infected with E. granulosus, suggesting they could prove of value in serodiagnosis of definitive hosts.

Amino Acid Sequence↗

PubMed2003

Mobile genetic elements colonizing the genomes of metazoan parasites.

A substantial fraction of the genome of most eukaryotes, including those of metazoan parasites, is predicted to comprise repetitive sequences. Mobile genetic elements (MGEs) will make up much of these repetitive sequences, particularly the interspersed sequences. This article reviews information on MGEs that have colonized the genomes of metazoan parasites (i.e. parasites of parasites). Helminth and mosquito genomes, in particular, are compared with those of better-understood model organisms. MGEs from the genomes of metazoan parasites can be expected to have practical uses in transgenesis and epidemiological studies.

Animals↗

PubMed2003

Digestive proteases of blood-feeding nematodes.

Blood-feeding parasites employ a battery of proteolytic enzymes to digest the contents of their bloodmeal. Host haemoglobin is a major substrate for these proteases and, therefore, a driving force in the evolution of parasite-derived proteolytic enzymes. This review will focus on the digestive proteases of the major blood-feeding nematodes - hookworms (Ancylostoma spp. and Necator americanus) and the ruminant parasite, Haemonchus contortus - but also compares and contrasts these proteases with recent findings from schistosomes and malaria parasites. Haematophagous nematodes express proteases of different mechanistic classes in their intestines, many of which have proven or putative roles in degradation of haemoglobin and other proteins involved in nutrition. Moreover, the fine specificity of the relationships between digestive proteases and their substrate proteins provides a new molecular paradigm for understanding host-parasite co-evolution. Numerous laboratories are actively investigating these molecules as antiparasite vaccine targets.

Ancylostomatoidea↗

PubMed2003

Isolation of cDNAs encoding secreted and transmembrane proteins from Schistosoma mansoni by a signal sequence trap method.

Surface and secreted proteins of schistosomes orchestrate the basic physiologic requirements of a parasitic existence. These proteins are often exposed to host tissues during penetration, migration, feeding, and immune evasion, and they are obvious targets for control strategies. Signal sequence trap (SST) represents a novel approach that selects for cDNAs encoding secreted and surface proteins with N-terminal signal peptides, so we constructed a randomly primed adult Schistosoma mansoni cDNA library fused to a signalless reporter gene encoding placental alkaline phosphatase. The library was used to transfect COS-7 cells, which were then assayed for the presence of reporter at the cell surface. Eighteen S. mansoni cDNA fragments were isolated and sequenced. Expression profiles of the novel clones were determined for different developmental stages; some transcripts were restricted to single-sex adult worms, while others were ubiquitously distributed. Most clones contained signal peptides or signal anchors as determined by the SignalP algorithm. Open reading frames (ORFs) were categorized as follows: (i) previously identified S. mansoni cDNAs encoding proteins of known function; (ii) cDNAs encoding proteins of known function in other organisms but novel for Schistosoma; (iii) S. mansoni expressed sequence tags (ESTs) of unknown function; and (iv) completely novel ORFs without homologues (including ESTs) from any phylum. Clones of particular interest included tetraspanins similar to human cell surface antigens, a protein kinase, and ORFs transcribed in the antisense orientation to previously characterized S. mansoni cDNAs. This is the first report describing the use of SST as a tool for identifying secreted proteins from any pathogenic organism.

Amino Acid Sequence↗

PubMed2003

Effect of vaccination with a recombinant fusion protein encoding an astacinlike metalloprotease (MTP-1) secreted by host-stimulated Ancylostoma caninum third-stage infective larvae.

Laboratory dogs were vaccinated intramuscularly with a recombinant fusion protein (expressed and isolated from Escherichia coli) formulated with the Glaxo SmithKline Adjuvant System 02 (AS02). The fusion protein encoded Ac-MTP-1, a developmentally regulated astacinlike metalloprotease secreted by host-stimulated Ancylostoma caninum third-stage larvae (L3). Control dogs were injected intramuscularly with an equivalent amount of AS02 adjuvant alone. The vaccinated and control dogs were then challenged by s.c. injection of 500 L3 of the canine hookworm A. caninum. The vaccinated dogs developed prechallenge immunoglobulin G2 (IgG2) antibody responses specific to anti-Ac-MTP-1-fusion protein with titers ranging between 1:40,000 and 1:364,000, whereas they developed antigen-specific immunoglobulin E antibody responses with titers ranging between 1:500 and 1:1,500. By immunoblotting, canine sera obtained from the vaccinated dogs recognized a protein of the estimated apparent molecular weight of Ac-MTP-1 in activated L3 secretory products. Spearman rank order correlations between the canine intestinal adult hookworm burden and quantitative egg counts at necropsy and anti-Ac-MTP-1 IgG2 antibody titers revealed a statistically significant inverse association (r = -0.89; P = 0.04), suggesting that this molecule offers promise as a recombinant vaccine.

Adjuvants, Immunologic↗

PubMed2003

Short report: Echinococcus granulosus from Xinjiang, PR China: cDNAS encoding the EG95 vaccine antigen are expressed in different life cycle stages and are conserved in the oncosphere.

The EG95-based vaccine protects sheep from infection with the dog tapeworm Echinococcus granulosus. The EG95 encoding gene is a member of a multigene family, several members of which are expressed in the oncosphere, believed to be the target of immunity induced by the vaccine. E. granulosus exhibits extensive intraspecific (strain) variation, and variability of the eg95 gene in different isolates of E. granulosus may directly impact the effectiveness of the EG95-based vaccine. We analyzed the eg95 gene from E. granulosus collected in Xinjiang, in northwest China, where hydatid disease is hyperendemic. The gene is expressed in oncospheres, protoscoleces, and immature and mature adult worms, and the eg95 gene family was shown to comprise two basic sequence types. Very limited sequence variation was evident in the EG95 protein from oncospheres. This high degree of sequence conservation predicts that the vaccine will continue to be effective in China and elsewhere.

Amino Acid Sequence↗

PubMed2002

Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity.

Hookworms routinely reach the gut of nonpermissive hosts but fail to successfully feed, develop, and reproduce. To investigate the effects of host-parasite coevolution on the ability of hookworms to feed in nonpermissive hosts, we cloned and expressed aspartic proteases from canine and human hookworms. We show here that a cathepsin D-like protease from the canine hookworm Ancylosotoma caninum (Ac-APR-1) and the orthologous protease from the human hookworm Necator americanus (Na-APR-1) are expressed in the gut and probably exert their proteolytic activity extracellularly. Both proteases were detected immunologically and enzymatically in somatic extracts of adult worms. The two proteases were expressed in baculovirus, and both cleaved human and dog hemoglobin (Hb) in vitro. Each protease digested Hb from its permissive host between twofold (whole molecule) and sixfold (synthetic peptides) more efficiently than Hb from the nonpermissive host, despite the two proteases' having identical residues lining their active site clefts. Furthermore, both proteases cleaved Hb at numerous distinct sites and showed different substrate preferences. The findings suggest that the paradigm of matching the molecular structure of the food source within a host to the molecular structure of the catabolic proteases of the parasite is an important contributing factor for host-parasite compatibility and host species range.

Ancylostomatoidea↗

PubMed2002

pido, a non-long terminal repeat retrotransposon of the chicken repeat 1 family from the genome of the Oriental blood fluke, Schistosoma japonicum.

A newly described non-long terminal repeat (non-LTR) retrotransposon element was isolated from the genome of the Oriental schistosome, Schistosoma japonicum. At least 1000 partial copies of the element, which was named pido, were dispersed throughout the genome of S. japonicum. As is usual with non-LTR retrotransposons, it is expected that many pido elements will be 5'-truncated. A consensus sequence of 3564 bp of the truncated pido element was assembled from several genomic fragments that contained pido-hybridizing sequences. The sequence encoded part of the first open reading frame (ORF), the entire second ORF and, at its 3'-terminus, a tandemly repetitive, A-rich (TA(6)TA(5)TA(8)) tail. The ORF1 of pido encoded a nucleic acid binding protein and ORF2 encoded a retroviral-like polyprotein that included apurinic/apyrimidinic endonuclease (EN) and reverse transcriptase (RT) domains, in that order. Based on its sequence and structure, and phylogenetic analyses of both the RT and EN domains, pido belongs to the chicken repeat 1 (CR1)-like lineage of elements known from the chicken, turtle, puffer fish, mosquitoes and other taxa. pido shared equal similarity with CR1 from chicken, an uncharacterized retrotransposon from Caenorhabditis elegans and SR1 (a non-LTR retrotransposon) from the related blood fluke Schistosoma mansoni; the level of similarity between pido and SR1 indicated that these two schistosome retrotransposons were related but not orthologous. The findings indicate that schistosomes have been colonized by at least two discrete CR1-like elements. Whereas pido did not appear to have a tight target site specificity, at least one copy of pido has inserted into the 3'-untranslated region of a protein-encoding gene (GenBank AW736757) of as yet unknown identity. mRNA encoding the RT of pido was detected by reverse transcription-polymerase chain reaction in the egg, miracidium and adult developmental stages of S. japonicum, indicating that the RT domain was transcribed and suggesting that pido was replicating actively and mobile within the S. japonicum genome.

Amino Acid Sequence↗

PubMed2002

Reverse transcriptase activity and untranslated region sharing of a new RTE-like, non-long terminal repeat retrotransposon from the human blood fluke, Schistosoma japonicum.

A new RTE-like, non-long terminal repeat retrotransposon, termed SjR2, from the human blood fluke, Schistosoma japonicum, is described. SjR2 is approximately 3.9 kb in length and is constituted of a single open reading frame encoding a polyprotein with apurinic/apyrimidinic endonuclease and reverse transcriptase domains. The open reading frame is bounded by 5'- and 3'-terminal untranslated regions and, at its 3'-terminus, SjR2 bears a short (TGAC)(3) repeat. Phylogenetic analyses based on conserved domains of reverse transcriptase or endonuclease revealed that SjR2 belonged to the RTE clade of non-long terminal repeat retrotransposons. Further, SjR2 was homologous, but probably not orthologous, to SR2 from the African blood fluke, Schistosoma mansoni; this RTE-like family of non-long terminal repeat retrotransposons appears to have arisen before the divergence of the extant schistosome species. Hybridisation analyses indicated that approximately 10,000 copies of SjR2 were dispersed throughout the S. japonicum chromosomes, accounting for up to 14% of the nuclear genome. Messenger RNAs encoding the reverse transcriptase and endonuclease domains of SjR2 were detected in several developmental stages of the schistosome, indicating that the retrotransposon was actively replicating within the genome of the parasite. Exploration of the coding and non-coding regions of SjR2 revealed two notable characteristics. First, the recombinant reverse transcriptase domain of SjR2 expressed in insect cells primed reverse transcription of SjR2 mRNA in vitro. By contrast, recombinant SjR2-endonuclease did not appear to cleave schistosome or plasmid DNA. Second, the 5'-untranslated region of SjR2 was >80% identical to the 3'-untranslated region of a schistosome heat shock protein-70 gene (hsp-70) in the antisense orientation, indicating that SjR2-like elements were probably inserted into the non-coding regions of ancestral S. japonicum HSP-70, probably after the species diverged from S. mansoni.

Amino Acid Sequence↗

PubMed2002

Na-ctl-2, a cDNA encoding a C-type lectin expressed exclusively in adult Necator americanus hookworms.

C-type lectins (C-TLs) are carbohydrate-binding proteins central to diverse physiological processes including immunity, venom-induced haemostasis and wound repair. Here we describe the cloning of Na-ctl-2, a cDNA encoding a secreted C-TL from the human hookworm Necator americanus. The transcript was detected in mRNA from adult worms but not infective larvae. The cDNA encoded an N-terminal secretory signal peptide followed by a long-form C-TL domain with sequence similarity to C-TL-like proteins from Caenorhabditis elegans and mammalian antigen presenting cell receptors, suggesting that hookworms might utilise this class of lectin to interrupt anti-parasite immune responses or interfere with host clotting mechanisms. This is the first report of a full-length cDNA encoding a lectin from hookworms. The unusually skewed representation of this protein family within different nematode genera and its subsequent impact on the evolution of nematode parasitism is discussed.

Amino Acid Sequence↗

PubMed2002

Effect of vaccinations with recombinant fusion proteins on Ancylostoma caninum habitat selection in the canine intestine.

Laboratory dogs were vaccinated subcutaneously with 3 different recombinant fusion proteins, each precipitated with alum or calcium phosphate. The vaccinated dogs were then challenged orally with 400 third-stage infective larvae (L3) of the canine hookworm, Ancylostoma caninum. The 3 A. caninum antigens selected were Ac-TMP, an adult-specific secreted tissue inhibitor of metalloproteases; Ac-AP, an adult-specific secreted factor Xa serine protease inhibitor anticoagulant; and Ac-ARR-1, a cathepsin D-like aspartic protease. Each of the 3 groups comprised 6 male beagles (8 +/- 1 wk of age). A fourth group comprised control dogs injected with alum. All of the dogs vaccinated with Ac-TMP or Ac-APR-1 exhibited a vigorous antigen-specific antibody response, whereas only a single dog vaccinated with Ac-AP developed an antibody response. Dogs with circulating antibody responses exhibited 4.5-18% reduction in the numbers of adult hookworms recovered from the small intestines at necropsy, relative to alum-injected dogs. In contrast, there was a concomitant increase in the number of adult hookworms recovered from the colon. The increase in colonic hookworms was as high as 500%, relative to alum-injected dogs. Female adult hookworms were more likely to migrate into the colon than were males. Anti-enzyme and anti-enzyme inhibitor antibodies correlated with an alteration in adult hookworm habitat selection in the canine gastroinntestinal tract.

Ancylostoma↗

PubMed2002

Proteolytic Enzymes as Therapeutic Targets - Keystone Symposium. Targeting ICE and ACE. 3-8 February 2002, Keystone, CA, USA.

The meeting covered a diverse range of topics, from therapeutic design of inhibitors of human proteases involved in disease, to the discovery of novel protease targets in infectious organisms. Various techniques were described, including the bioinformatics-based scanning of genomes for novel enzymes that might be linked to disease or virulence. In addition, crystal structures of well-known proteases were presented. The key focus however, was on designing inhibitors of proteolytic enzymes and their use in controlling disease processes mediated by proteases. Inhibitors described ranged from naturally occurring novel peptides such as Ascaris pepsin inhibitor, to trials with finely tuned peptidomimetics inhibiting human peptidases (interleukin converting enzyme; ICE and angiotensin converting enzyme; ACE) and rhinovirus 3C protease (antirhinovirus therapeutics; Pfizer Inc). The meeting also included presentations on the development of new techniques for detecting and analyzing protease activities such as luminescent protease assays, in vivo imaging of protease activity, surface plasmon resonance and novel peptide library screening methods.

Journal Article↗