Fasciola hepatica: an antigen fraction derived from newly excysted juveniles, containing an immunoreactive 32-kDa protein, induces strong protective immunity in rats

Crude antigens of adult Fasciola hepatica and of newly excysted juveniles (NEJ) and a low-molecular-weight fraction of antigen from NEJs were tested for inducing protective immunity in rats. Two routes of vaccination were applied. The results showed that intraperitoneal vaccination induced significantly better protection (P <0.05) than intramuscular vaccination. Intraperitoneal vaccination with antigens from NEJs induced more effective protection: after challenge infection, rats that were so vaccinated had 92.6% (+/-2.5% SEM) fewer parasites in their liver and 57.3% (+/-13.3% SEM) fewer parasites penetrating the gut wall than control rats. Rats that were vaccinated with a low-molecular-weight fraction of antigen from NEJs were also highly protected against a challenge. F. hepatica antigens that are immunoreactive were identified on immunoblots, using sera collected from highly protected rats that had been vaccinated with NEJ antigens and also sera from cattle and rats that were experimentally infected with F. hepatica. The low-molecular-weight fraction of antigen from NEJs contained an immunodominant 32-kDa protein that was recognized by serum antibodies of vaccinated rats and immune cattle. This 32-kDa protein was not detected in partially purified antigens from adult flukes. We conclude that antigens of NEJs of F. hepatica, when injected intraperitoneally in rats, are highly protective. In particular, the 32-kDa protein contained in these antigens may be highly valuable for the development of an effective vaccine against F. hepatica.     

Fasciola hepatica: molecular cloning, nucleotide sequence, and expression of a gene encoding a polypeptide homologous to a Schistosoma mansoni fatty acid-binding protein.

Immunization of mice with an antigenic polypeptide from Fasciola hepatica adult worms and having an apparent molecular mass of 12,000 Da (Fh12) has been shown to reduce the worm burden from challenge infection with Schistosoma mansoni by more than 50%. Moreover, mice infected with S. mansoni develop antibodies to Fh12 after 5-6 weeks of infection, indicating that this Fasciola-derived antigen is a cross-reactive, cross-protective protein. A lambda gt11 F. hepatica cDNA library was constructed from poly(A)+ RNA extracted from adult worms. A cDNA encoding a cross-reactive polypeptide (Fh15) was cloned by screening the F. hepatica lambda gt11 library with a monospecific, polyclonal rabbit antiserum against pure, native Fh12. The cDNA was sequenced and the predicted amino acid sequence revealed an open reading frame encoding a 132-amino-acid protein with a predicted molecular weight of 14,700 Da. This protein has significant homology to a 14-kDa S. mansoni fatty acid-binding protein. Comparison of the protective-inducing activity of recombinant Fh15 with that of purified Fh12 against schistosomes and Fasciola is warranted.     

Fasciola hepatica: identification of CD4+ T-helper epitopes from the 11.5 kDa saposin-like protein SAP-2 using synthetic peptides

Fasciola hepatica saposin-like protein (FhSAP-2) is a novel antigen expressed at an early stage of infection and has been shown to induce in rabbits a significant protection to infection with F. hepatica. There are no studies to identify the immunologically relevant regions of FhSAP-2. In this work the amino acid sequence of FhSAP-2 was analyzed to identify potential T-cell epitopes. A predictive algorithm identified four possible sites. Experimental determination of the T-cell epitopes was achieved using a panel of overlapping peptides spanning the entire sequence of FhSAP-2, which was evaluated for their ability to induce lymphoproliferative responses of spleen cells from 8 immunized BALB/c (H-2d) mice. Five different epitopes were identified. There was minimal agreement between theoretical and experimental approaches. It was found that peptides containing amino acid residues AVTFA and IDIDLCDICT as part of their structure induce high levels of IL-2 and IFNgammain vitro and was classified as Th1 epitopes. Peptides that contain the residues ADQTV, CIEFVQQEVD and YIIDHVDQHN induced significant amount of IL-4 and IL-2 were considered as containers of Th0 epitopes. Identification of prominent T-cell epitopes from FhSAP-2 offers the possibility of understanding how the CD4+ T-cell response is involved in protection against fasciolosis and how it is implicated in susceptibility to infection.     

Molecular dissection of the human B-cell response against Toxoplasma gondii infection by lambda display of cDNA libraries

The disorders generated by Toxoplasma gondii infection are closely associated with the competence of the host immune system and both humoral and cell mediated immunity are involved in response to parasite invasion. To identify antigens implicated in human B-cell responses, we screened a phage-display library of T. gondii cDNA fragments with sera of infected individuals. This approach identified a panel of recombinant phage clones carrying B-cell epitopes. All the peptide sequences selected by this procedure are regions of T. gondii gene products. These regions contain epitopes of the T. gondii antigens SAG1, GRA1, GRA7, GRA8 and MIC5, which are recognised by human immunoglobulins. Moreover, we report the isolation and characterisation of two additional immunodominant regions encoded by GRA3 and MIC3 genes, whose products have never been described as antigens of the human B-cell response against T. gondii infection. These results demonstrate potential of lambda-display technology for antigen discovery and for the study of the human antibody response against infectious agents.     

金黄色葡萄球菌肠毒素A蛋白的B细胞表位的预测

目的预测金黄色葡萄球菌肠毒素A蛋白（SEA）的B细胞表位。方法以金黄色葡萄球菌合肥乳源分离株M3基因组DNA为模板,PCR扩增SEA基因并进行序列测定与分析。应用DNAstar protean软件对SEA蛋白的二级结构、柔性、亲水性、表面可能性和抗原指数等多参数进行综合分析,预测其B细胞表位。结果M3分离株的SEA基因全长774bp,编码由257个氨基酸组成的相对分子量为29．67kDa的SEA蛋白,M3分离株SEA基因与标准株的核苷酸序列与氨基酸序列同源性分别为98．7％和98．4％。SEA蛋白的优势B细胞表位位于肽链的第64—68、100～107、138—141、156—160、166～173、213～217和237～244区段。结论预测出SEA蛋白的7个优势B细胞表位,为进而克隆表达表位蛋白,制备针对SEA表位的单克隆抗体奠定了基础。     

Sequence of membrane-associated thyroid hormone binding protein from bovine liver: its identity with protein disulphide isomerase

The complementary DNAs of the bovine liver membrane-associated 3,5,3'-triiodo-L-thyronine binding protein with 55 k-dalton (T3BP) were cloned and the nucleotide sequences were determined. Monospecific antibodies against T3BP were used to screen a bovine liver cDNA library in lambda gtll. We analyzed the sequences of two cloned T3BP cDNAs. The clones encoded a polypeptide of 510 amino acid residues, including a signal peptide of 20 amino acid. Northern blot analysis of bovine and human RNA showed that the mRNAs encoding T3BP are 2.7 kilobase in length. Amino acid sequence of N-terminal and other three peptides isolated from purified T3BP were found in the predicted amino acid sequence from the cDNA sequence. The predicted amino acid sequence is closely homologous (93%) with that of rat protein disulphide isomerase (EC 5.3.4.1), which catalyzes the isomerization of the protein disulphide bonds and has been shown to play an important role in post-translational regulation. The results suggest that T3BP and protein disulphide isomerase should be the same protein.     

Cloning and characterization of a lectin from the octocoral Sinularia lochmodes

In the present study, the entire amino acid sequence and cDNA structure encoding the d-galactose-binding lectin, SLL-2, isolated from the octocoral Sinularia lochmodes, were determined. SLL-2 regulates the morphology of symbiotic dinoflagellates Symbiodinium spp. through unknown mechanisms. Here, three cDNAs that encode SLL-2 were cloned and characterized. All the SLL-2 cDNAs encoded 142 amino acids with high similarity to each other. The mature subunit of SLL-2 was found to be composed of 94 amino acids and to contain one putative glycosylation site common to all three SLL-2. N-Glycopeptidase F treatment of SLL-2 resulted in a protein band shift from 16.5 to 9.5kDa in SDS-PAGE, confirming that SLL-2s are glycoproteins. Two-dimensional polyacrylamide gel electrophoresis analysis of the deglycosylated SLL-2 indicated a presence of three polypeptides as encoded in SLL-2 cDNAs. The deduced sequences of SLL-2 cDNAs had a similarity to the C-terminal region of discoidin I, the slime mold Dictyostelium discoideum lectin.     

Identification of fatty acid molecules in a Fasciola hepatica immunoprophylactic fatty acid-binding protein

Fasciola hepatica adult flukes have a native protein complex denoted nFh12 and consisting of fatty acid binding proteins that comprise at least 8 isoforms. It is a potent immunogen because in several animal hosts it induces an early antibody response to F. hepatica infection. It is also a potent cross-protective immunogen because it induces a protective immune response in mice to challenge infection with Schistosoma mansoni cercariae. The gene encoding this protein has been cloned and sequenced. It produces a polypeptide of 132 amino acids with a predicted molecular mass of 14.7 kDa and is denoted rFh15. It also has a significant homology to a 14-kDa S. mansoni fatty acid binding protein (Sm14). In the present study, nFh12 was delipidated with charcoal treatment and then studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Additionally, a lipid analysis of nFh12 was undertaken using gas chromatography-mass spectrometry to demonstrate that the nFh12 protein complex is, in fact, a complex of fatty acid binding proteins. Five long-chain saturated and unsaturated fatty acids were detected. The most abundant were palmitic acid (38%), stearic acid (24%), and oleic acid (13%). These fatty acid molecules do not have covalent bonds attached to the protein molecule. Because both nFh12 and Sm14 protect mice against challenge infection with F. hepatica and S. mansoni, it is possible that they have common protective epitopes in which fatty acids could be involved. Further studies are in progress to determine the chemical nature of these potential common epitopes.     

Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies

Antibody to the capsid (PORF2) protein of hepatitis E virus (HEV) is sufficient to confer immunity, but knowledge of B-cell epitopes in the intact capsid is limited. A panel of murine monoclonal antibodies (MAbs) was generated following immunization with recombinant ORF2.1 protein, representing the C-terminal 267 amino acids (aa) of the 660-aa capsid protein. Two MAbs reacted exclusively with the conformational ORF2.1 epitope (F. Li, J. Torresi, S. A. Locarnini, H. Zhuang, W. Zhu, X. Guo, and D. A. Anderson, J. Med. Virol. 52:289-300, 1997), while the remaining five demonstrated reactivity with epitopes in the regions aa 394 to 414, 414 to 434, and 434 to 457. The antigenic structures of both the ORF2.1 protein expressed in Escherichia coli and the virus-like particles (VLPs) expressed using the baculovirus system were examined by competitive enzyme-linked immunosorbent assays (ELISAs) using five of these MAbs and HEV patient sera. Despite the wide separation of epitopes within the primary sequence, all the MAbs demonstrated some degree of cross-inhibition with each other in ORF2. 1 and/or VLP ELISAs, suggesting a complex antigenic structure. MAbs specific for the conformational ORF2.1 epitope and a linear epitope within aa 434 to 457 blocked convalescent patient antibody reactivity against VLPs by approximately 60 and 35%, respectively, while MAbs against epitopes within aa 394 to 414 and 414 to 434 were unable to block patient serum reactivity. These results suggest that sequences spanning aa 394 to 457 of the capsid protein participate in the formation of strongly immunodominant epitopes on the surface of HEV particles which may be important in immunity to HEV infection.     

Molecular cloning of rat liver 3α-hydroxysteroid dehydrogenase and identification of structurally related proteins from rat lung and kidney

3α-Hydroxysteroid dehydrogenase and related enzymes play important roles in the metabolism of endogenous compounds including androgens, corticosteroid, prostaglandins and bile acids, as well as drugs and xenobiotics such as benzo(a)pyrene. Complementary DNA clones encoding 3α-hydroxysteroid dehydrogenase were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. A full-length cDNA clone of 1286 base pairs contained an open reading frame encoding a protein of 322 amino acids with an estimated M(w) of 37 kD. When expressed in E. coli, the encoded protein migrated to the same position on SDS-polycrylamide gels as the enzyme in rat liver cytosols. The protein expressed in bacteria was highly active in androsterone oxidation in the presence of NAD as cofactor and this activity was inhibited by indomethacin, a potent inhibitor of 3α-hydroxysteroid dehydrogenase. The predicted amino acid sequence of 3α-hydroxysteroid d dehydrogenase was related to sequences of several other aldo-keto reductases such as bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase and frog lens epsilon-crystallin, suggesting that these proteins belong to the same gene family. Recently, we have found that monoclonal antibodies against 3α-hydroxysteroid dehydrogenase also recognized multiple antigenically related proteins in rat lung, kidney and testis. Further screening of liver, lung and kidney cDNA libraries using these monoclonal antibodies as probes resulted in the isolation of additional five different cDNAs encoding proteins with high degree of structural homology to rat liver 3α-hydroxysteroid dehydrogenase.     

A组轮状病毒多个结构蛋白抗原表位的串联表达及其免疫原性的检测

从北京腹泻婴儿粪便提取的轮状病毒(rotavirus,RV)(T114株)的RNA中,克隆到轮状病毒结构蛋白基因vp4,vp6和vp7的全长cDNA,对它们编码的蛋白质序列和可能的抗原表位肽进行了预测,选择了RV主要抗原蛋白VP7、VP6和VP4的4个抗原表位肽,通过人工合成DNA的方式将这些抗原表位肽基因串联融合成一个阅读框RME(rotavirus multipleepitopes,RME)并构建原核表达载体.大肠杆菌表达的RME在ELISA反应中可被RV多克隆抗体识别,纯化的RME蛋白注射免疫小鼠可诱导特异性免疫应答,产生高滴度的同源氨基酸序列特异抗体和人RV抗体,其中针对RME的IgG抗体滴度达到l∶40 000,针对单个抗原表位EV7、EV6和EV4的IgG抗体滴度达l∶10 000～l∶20 000,针对RV Wa株的IgG抗体滴度较低为l∶2 500,但能特异地中和该病毒对MAC145细胞的侵染.上述结果为新型RV基因工程疫苗的研发提供了论据和基础.     

Babesia bovis expresses Bbo-6cys-E, a member of a novel gene family that is homologous to the 6-cys family of Plasmodium

A novel Babesia bovis gene family encoding proteins with similarities to the Plasmodium 6cys protein family was identified by TBLASTN searches of the B. bovis genome using the sequence of the P. falciparum PFS230 protein as query, and was termed Bbo-6cys gene family. The Bbo-cys6 gene family contains six genes termed Bbo-6cys-A, B, C, D, E and F encoding for proteins containing an arrangement of 6 cysteine residues. The Bbo-6cys genes A, B, C, D, and E are tandemly arranged as a cluster of Chromosome 2 in the B. bovis genome, whereas gene F is located in a distal region in the same chromosome. The Bbo-6cys-E gene, with higher homology to PFS230, was selected for further examination. Immunoblot analysis using recombinant Bbo-6cys-E protein and B. bovis-positive bovine serum demonstrated expression by the parasite and immunogenicity during B. bovis infection. Immunofluorescence analysis using anti-Bbo-6cys-E antibodies confirmed expression of Bbo-6cys-E in in vitro blood stages of B. bovis. In addition, polyclonal antisera against both recombinant Bbo-6cys-E and specific synthetic peptides containing predicted B-cell epitopes of Bbo-6cys-E, significantly inhibited erythrocyte invasion by B. bovis in in vitro neutralization assays, suggesting an important functional role for this protein. Identification of this new gene family in B. bovis and further investigation on its biological significance may aid our understanding of the bovine, tick and parasite relationships and the development of improved control methods against B. bovis infection in cattle.     

Plant viral genes in DNA idiotypic vaccines activate linked CD4+ T-cell mediated immunity against B-cell malignancies

DNA delivery of tumor antigens can activate specific immune attack on cancer cells. However, antigens may be weak, and immune capacity can be compromised. Fusion of genes encoding activating sequences to the tumor antigen sequence facilitates promotion and manipulation of effector pathways. Idiotypic determinants of B-cell tumors, encoded by the variable region genes, are clone-specific tumor antigens. When assembled as single-chain Fv (scFv) alone in a DNA vaccine, immunogenicity is low. Previously, we found that fusion of a sequence from tetanus toxin (fragment C; FrC) promoted anti-idiotypic protection against lymphoma and myeloma. We have now investigated an alternative fusion gene derived from a plant virus, potato virus X coat protein, a primary antigen in humans. When fused to scFv, the self-aggregating protein generates protection against lymphoma and myeloma. In contrast to scFv-FrC, protection against lymphoma is mediated by CD4+ T cells, as is protection against myeloma. Plant viral proteins offer new opportunities to activate immunity against linked T-cell epitopes to attack cancer.     

Computational characterization of epitopic region within the outer membrane protein candidate in Flavobacterium columnare for vaccine development

Abstract

Gram-negative bacteria is the main causative agents for columnaris disease outbreak to finfishes. The outer membrane proteins (OMPs) candidate of Flavobacterium columnare bacterial cell served a critical component for cellular invasion targeted to the eukaryotic cell and survival inside the macrophages. Therefore, OMPs considered as the supreme element for the development of promising vaccine against F. columnare. Implies advanced in silico approaches, the predicted 3-D model of targeted OMPs were characterized by the Swiss model server and validated through Procheck programs and Protein Structure Analysis (ProSA) web server. The protein sequences having B-cell binding sites were preferred from sequence alignment; afterwards the B cell epitopes prediction was prepared using the BCPred and amino acid pairs (AAP) prediction algorithms modules of BCPreds. Consequently, the selected antigenic amino acids sequences (B-cell epitopic regions) were analyzed for T-cell epitopes determination (MHC I and MHC II alleles binding sequence) performing the ProPred 1 and ProPred server respectively. The epitopes (9 mer: IKKYEPAPV, YGPNYKWKF and YRGLNVGTS) within the OMPs binds to both of the MHC classes (MHC I and MHC II) and covered highest number of MHC alleles are characterized. OMPs of F. columnare being conserved across serotypes and highly immunogenic for their exposed epitopes on the cell surface as a potent candidate focus to vaccine development for combating the disease problems in commercial aquaculture. The portrayed epitopes might be beneficial for practical designing of abundant peptide-based vaccine development against the columnaris through boosting up the advantageous immune responses.

Communicated by Ramaswamy H. Sarma     

Molecular cloning of grammistins, peptide toxins from the soapfish Pogonoperca punctata, by hemolytic screening of a cDNA library

A novel method, based on the hemolytic screening of a cDNA phage library, was developed to isolate cDNAs encoding grammistins (antibacterial peptide toxins) of the soapfish Pogonoperca punctata. As a result, cDNAs encoding six grammistins were isolated and elucidated for their nucleotide sequences. In common with the grammistins, the precursor protein is composed of a highly conserved signal peptide, a considerably conserved propeptide that is characterized to contain a pair of basic residues (Lys-Arg) at plural positions including the C-terminus and one copy of a mature peptide. This precursor organization is similar to those of dermaseptins, antibacterial peptides from the frog skin.     

Use of a pre-selected epitope of cathepsin-L1 in a highly specific peptide-based immunoassay for the diagnosis of Fasciola hepatica infections in cattle.

A peptide-based indirect ELISA to detect cattle antibodies against Fasciola hepatica was developed and evaluated for its sensitivity and specificity. An immunogenic antigen released in vitro by F. hepatica was purified. After purification the sequence of the first 20 N-terminal aa of this protein showed considerable homology with cathepsin L-like proteinase. Based on its homology with cathepsin-L1, we further focused on this protein for diagnostic purpose. Predicted B-cell epitopes of cathepsin-L1 were synthesised as single synthetic peptides and tested with respect to their diagnostic potential. An indirect ELISA based on one of these peptides was (i) evaluated further and (ii) compared to the potential of an indirect ELISA with excretion/secretion antigens from adult F. hepatica, or (iii) purified cathepsin-L1. Specificity and sensitivity of the three ELISAs were assessed using sera from calves experimentally infected with pure isolates of Dictyocaulus viviparus, Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus, Schistosoma mattheei, Ascaris suum, Taenia saginata or F. hepatica, respectively, and sera from parasite-naive calves. In addition, sera were analysed from calves naturally infected with F. hepatica. The sensitivities of all three ELISAs were also very high, 98.9% (i), 100% (ii) and 100% (iii). The specificity of the peptide ELISA was very high, 99.8%, whereas specificities of the ES antigens and cathepsin-L1 ELISAs were only 82.8% and 94.6%. In experimentally infected cattle, F. hepatica-specific antibodies were first detected between days 21 and 28 p.i. with all three ELISAs, and the antibody levels persisted in the peptide ELISA until day 183 p.i. All sera from naturally infected calves were positive in the peptide ELISA. These results demonstrate that the peptide-based F. hepatica ELISA is a useful method for detecting antibodies in the sera from cattle infected with F. hepatica. This type of immunodiagnostic will therefore contribute to more accurate diagnosis and to timely curative treatment of animals.     

Molecular cloning and characterization of cathepsin F gene from olive flounder Paralichthys Olivaceus

We isolated a homologue of cathepsin F from cDNA library of olive flounder liver. A 2,077 kb full-length cDNA encoding a predicted polypeptide of 474 amino acids was sequenced. The flounder cathepsin F exhibits a domain structure typical for papain-like cysteine proteases, a 17 amino acid N-terminal hydrophobic signal sequence followed by an extraordinarily long propeptide of 244 amino acids and the domain of the mature protease comprising 213 amino acids. The mature region contains all features characteristic of a papain-like cysteine protease, including the highly conserved cysteine, histidine and asparagine residues of the ‘catalytic triad’. The cathepsin F protein showed 49–99% amino acid sequence identity with other known cathepsin F sequences. An in vivo expression study showed that cathepsin F mRNA was expressed predominantly in brain, liver, eye and heart, and moderately in other tissues. The accumulation of cathepsin F mRNA in early stage of development increased with development. This expression pattern suggests that flounder cathepsin F has been implicated in the growth and reproduction regulation.     

Eimeria tenella: B-cell epitope mapping following primary and secondary infections

Immunisation against coccidiosis has become more reliable and effective with improved administration techniques for new vaccines. On the other hand, an ideal coccidial vaccine should contain both B- and T-cell immunogenic epitopes. Fine specificity of B-cell epitopes recognised by antibodies prepared following primary and secondary infections with Eimeria tenella were studied using "PepScan" techniques. Mapping of B-cell epitopes within an antigenic sequence from E. tenella showed that four distinct types of epitopes were recognised by the host immune system during the primary and secondary infections with the parasite. These observations demonstrated that new epitopes are also involved in induction of antibody responses following the secondary infection.