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 American Journal of Tropical Medicine and Hygiene, Jan 2003

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.

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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

WENBAO ZHANG 0 1 JUN LI 0 1 HONG YOU 0 1 ZHUANGZHI ZHANG 0 1 GULINUL TURSON 0 1 ALEX LOUKAS 0 1 DONALD P. MCMANUS 0 1 0 Molecular Parasitology Laboratory, Australian Centre for International and Tropical Health and Nutrition, Queensland Institute of Medical Research and University of Queensland , Brisbane , Queensland , Australia ; Veterinary Research Institute, Xinjiang Academy of Animal Science , Urumqi, Xinjiang, China 1 Authors' addresses: Jun Li, Hong You, Alex Loukas, and Donald P. McManus. Molecular Parasitology Laboratory, Australian Centre for International and Tropical Health and Nutrition, The Queensland Institute of Medical Research and The University of Queensland , Brisbane , Queensland 4029. Wenbao Zhang , Zhuangzhi Zhang, and Gulinul Turson, Veterinary Research Institute, Xinjiang Academy of Animal Science , Urumqi, Xinjiang 830000, China 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. - solution to remove canine intestinal mucus, and then rinsed 10 times in PBS. Eggs were collected from mature adult worms. Oncospheres were hatched from eggs and activated13 and purified by density-gradient separation with 100% Percoll (Sigma, St Louis).14 All parasite materials were aliquoted into cryotubes immediately after a final PBS wash and stored in liquid nitrogen until used. Total RNAs from the different stages of E. granulosus were isolated using Trizol Reagent (Gibco, BRL Life Technologies Inc., Gaithersberg, MA) according to the manufacturers instructions. Approximately 1 mL of parasite material was used for extracting total RNA with 15 mL of Trizol reagent. The total RNAs (3 g were treated with RNase-free DNase (Promega), then reverse transcribed to cDNA using the SUPERSCRIPTTM Preamplification System (Gibco, BRL Life Technologies Inc., Gaithersberg, MD) for first-strand cDNA synthesis in a total reaction volume of 20 L. 1 L of the reaction was used as template DNA for polymerase chain reaction (PCR) analysis. Primers were designed according to theparent eg95 sequence (GenBank accession number X90928), upstream primer: 5 -AACAGAGACTCCGCTCCGTAAAC-3 (position 77-100 of the eg95 sequence); downstream primer: 5 - AAATGCAAGTTATGGGTCGTGCTA-3 (position 511534 of the eg95 sequence). An additional primer pair (5 -GTTGTGCTATGTGGCACTCGAC T-3 and 5 -CAATCCAGACAGAGTATTTGCGTTC-3 ) was also designed to amplify a fragment (379 bp) of actin I cDNA (GenBank accession number L07773) of E. granulosus15 to monitor the integrity of the RNA from each stage. PCR amplifications were carried out using a DNA Thermal cycler 2400 (Perkin Elmer Cetus, Emeryville, CA) in a 50- L reaction mixture (94C for 3 min, then 35 cycles of 94C for 1 min, 55C for 2 min, and 72C for 2 min, 30 sec, and finally 72C for 7 min) with Taq DNA polymerase (Promega). PCRamplified DNA fragments were purified by a MinElute Gel Extraction Kit (Qiagen, Hilden, Germany) after separation by agarose gel electrophoresis, ligated into the pGEM-T Easy plasmid (Promega) and sequenced using T7 and M13 reverse primers. Weamplified eg95 cDNAs by reverse-transcriptase (RT)PCR from each of thefour lifecyclestages of E. granulosus Echinococcus granulosus used Protoscoleces were aspirated from a number of sheep hydatid cysts and pooled. Worms were collected on day 35 postinfection from two dogs experimentally infected with a different pool of protoscoleces. The collected worms were pooled for analysis. Worms were collected on day 62 postinfection from 10 dogs experimentally infected with the same pool of protoscoleces. Theworms werepooled for analysis. Oncospheres were obtained from thematureadult worms collected on day 62 postinfection. The collected oncospheres were pooled for analysis. (Figure1). Thepredicted sizeof theRT-PCR product was 458 bp. The amplified sequence covers the linear immunogenic regions of the EG95 protein1617 that includethepeptidesequences TETPLRKHFNLTPV (peptide 6), SLKAVNPSDPLVYKRQTAKF (peptides 12 and 13), DIETPRAGKKESTVMTSGSA, (peptides 21 and 22), and SALTSAIAGFVFSC (peptide 24).16 The position of these sequences in the EG95 protein is highlighted in Figure 2. RT-PCR using 35 cycles of PCR indicated that eg95 is expressed in all four life-cycle stages examined (Figure 1A, II); more-abundant expression was noted in hatched/activated oncospheres after 26 cycles of PCR (Figure 1A, I). However, although an abundant 0.80.9-kbp RNA species was identified in hatched and activated oncospheres on a Northern blot by hybridization to the radiolabeled eg95 RT-PCR product from oncospheres, eg95 mRNA transcripts in thethreeother stages apparently were expressed at levels below the sensitivity limits of Northern analysis (Figure 1B). This is not unexpected as, although the precise function of EG95 is not known, it likely is involved in penetration of the epithelial border of the intestinal villi, with eg95 being expressed most abundantly in hatched and activated oncospheres that areinfcetiveto theintremediatehost. Theprimres used in the RT-PCR analysis, designed according to the parent eg95 sequence, span an intron of 215 bp in the eg95 gene. But sequencing (see below) of all clones arising from RT-PCR products of the predicted size (458 bp; arrowed, Figure 1A) indicated that they contained cDNA sequences only, without theintron. Additional products werevisiblein theRT-PCR reactions (Figure1A). Despitethefact that the original RNA preparations were treated with RNAse-free DNAse, these additional bands were probably generated from contaminating genomic DNA. Indeed, the band immediately above the eg95 cDNA band at 458 bp in thelane containing products obtained with protoscolex RNA was sequenced and was shown to have the eg95 sequence containing the intron. Further bands above this fragment evident in theimmatureadult worm and protoscolex stages may be non-specific bands dueto thecontaminating DNA or may bedueto primer-dimerization of the eg95 primers we designed. As the EG95 host-protective antigen is expressed by a family of genes, some of the genes may have different intron lengths and/or some may have repetitive DNA inserted into introns, which also may account for thelarger bands observed. RT-PCR products banding at 458 bp from each of the four life cycle stages were excised and ligated into T-ended plasmids; 1015 clones containing inserts were randomly selected for sequencing for each life cycle stage RT-PCR product. The different sequences were compared with other eg95 sequences available in the EMBL, GenBankTM, and DDBJ databases. Figure 2 shows an alignment of the deduced amino acid sequences. Although there are a number of amino acid substitutions in several of the individual sequences, the EG95 protein family comprises two basic sequence types. One is present in the protoscolex stage (the sequence designated as EG95-P7 in Figure 2), and the other occurs in oncospheres (sequences designated as EG95-O3, EG95-O4, and EG95-O6 in Figure2). It should beemphasized that theimmatureand mature adult worms and oncospheres used in the study resulted from the same collection of pooled protoscoleces originally used to experimentally infect dogs. Protoscolex RNA was extracted from a different pool of parasites collected on a separate occasion. Only one sequence (EGP5-P7) was evident in the clones originating from the protoscolex stage. It is noteworthy that this sequence was identical to a sequence present in mature adult worms (EG95-M7 in Figure 2), indicating that the two different E. granulosus sources were very similar genotypically. Of 13 clones sequenced from the oncosphere RT-PCR product, the most highly represented sequence was EG95-O4 (9/13) followed by EG95-O6 (3/13) and EG95-O3 (1/13). Over 20% amino acid differences are evident between the two types, but the most significant difference is that the former has an insertion of seven amino acids in the C-terminal (Figure 2). Both sequence types also occur in immature adult worms (sequences represented as EG95-I4 and EG95-I7 in Figure2) and matureadult worms (represented as EG95-M2, EG95-M5, and EG95-M7 in Figure 2). In addition to the two basic sequence types, a number of amino acid substitutions were evident between members of the EG95 protein family in different life cycle stages. It is well recognized that PCR analysis may cause errors in generating new duplicated fragments. We used the same source of Taq polymerase (Promega) to amplify a number of other cDNAs sized 1.51.8 kb for subcloning into expression vectors. Subsequent sequencing analysis of the clones did not indicate any nucleotidesubstitutions. (Data not shown.) Wearetherefore confident that the polymorphisms we recorded in the eg95 sequences did not result from PCR or some other technical artifact, particularly as thesamesubstitutions wereevident in sequences from different life cycle stages. One example is EG95-O6, present in oncospheres, and EG95-M5 from matureadult worms, wherethereis a histidine/argininesubstitution at the 20th position from the N-terminal end compared with the other EG95 sequences. Similarly, there is an aspartic acid/glutamic acid substitution at the24th residueposition from the N-terminal end in two oncosphere (EG95-O3/O6) and two mature worm (EG95-M2/M5) sequences compared with the other EG95 sequences (Figure 2). Neither of these substitutions occurs in linear immunogenic regions of the EG95 protein.16 With the exception of a single substitution (alanine/valine) in the end amino acid of peptide 21/22 in EG95-O3, the oncospheral EG95 sequences share absolute identity to the parent EG95 protein sequence1 in thefour immunogenic regions mapped previously.16 As weused samples of pooled parasites for each of the four life cycle stages, we do not know whether the variation we see in the eg95 cDNA sequences is due to genetic differences among the genes within an individual or among individuals in the sample population. However, it is noteworthy that, overall, in the oncospheral sequences examined, very limited sequence variation (03 amino acid substitutions) was evident in the EG95 protein. Although protection against hydatid disease induced with the EG95 vaccineis now thought to beassociated with conformational, rather than linear, epitopes,16,18 the high degree of sequence conservation, including the mapped immunodominant regions of the EG95 protein family, in oncospheres supports thewidescaleuseof theEG95-based vaccineand predicts that it will continue to be effective in China and elsewhere. Indeed, the vaccine already has proven highly effective (96100% protection) in challenge trials in China, Australia, New Zealand, and Argentina.19 Financial support: This work was supported by the Wellcome Trust (WZ and DPM), the Australian National Health and Medical Research Council (DPM), and by Australian International Postgraduate Research (IPRS) and University of Queensland International Postgraduate Research Scholarships (UQIPRS) to WZ.


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WENBAO ZHANG, JUN LI, HONG YOU, ZHUANGZHI ZHANG, GULINUL TURSON, ALEX LOUKAS, DONALD P. MCMANUS. 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 American Journal of Tropical Medicine and Hygiene, 2003, 40-43,