Rudi Grams

Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens.

Liver flukes produce cathepsin B and cathepsin L in their excretory-secretory material. These proteases are proposed to be key virulence factors for parasite infection, and are therefore targets for vaccination. Cathepsin B is predominately released in the juvenile stage of the life cycle, while different cathepsin Ls are released throughout the cycle. Three proteases (cathepsin L5, cathepsin L1g and cathepsin B) were expressed in yeast from cDNA clones isolated from adult, metacercariae and newly excysted juvenile flukes respectively. Each was used singly or in combination to vaccinate rats that were subsequently challenged with Fasciola hepatica metercercariae. Each protein induced an immune response, and all groups vaccinated with recombinant protein yielded significantly fewer and smaller flukes than the control group. Maximal protection of 83% was seen in the group vaccinated with cathepsin B and cathepsin L5 in combination.

Molecular cloning and characterization of cathepsin L encoding genes from Fasciola gigantica

In this study cDNAs encoding cathepsin L-like proteins of Fasciola gigantica were cloned by the reverse transcription polymerase chain reaction method (RT-PCR) from total RNA of adult specimens. DNA sequence analyses revealed that six different cathepsin L cDNA fragments were isolated, which have DNA sequence identities of 87-99% towards the homologous genes from F. hepatica. Gene expression was studied at the RNA level by Northern and RNA in situ hybridizations. Northern analysis showed the cathepsin L genes to be strongly expressed in adult parasites as a group of 1050 nt sized RNAs. RNA in situ hybridization localized cathepsin L RNA to the cecal epithelial cells. Southern hybridization was used to determine the number of cathepsin L genes and indicated the presence of a family of closely related cathepsin L genes in the genome of F. gigantica.

Fasciola gigantica and Schistosoma mansoni: Vaccine potential of recombinant glutathione S-transferase (rFgGST26) against infections in mice

Recombinant Fasciola gigantica glutathione S-transferase (rFgGST26) was expressed in Escherichia coli. This protein had 86% and 56% sequence identity with 26 kDa GST from Fasciola hepatica and Schistosoma mansoni, respectively. Polyclonal antibody raised in ICR mice against rFgGST26 recognized immunoblotted 26 kDa native GSTs from F. gigantica and S. mansoni. rFgGST26 was used as a vaccine in combination with Freunds adjuvant to evaluate the induction of immune responses and protection against F. gigantica and S. mansoni infection in mice. Mice were immunized via subcutaneous (s.c.), intramuscular (i.m.) or intradermal (i.d.) routes. Strong protection (77-84%) against F. gigantica was observed in all routes. Immunization via s.c. route induced immune response with IgG1 isotype predominating, while i.m. and i.d. routes resulted in mixed IgG1/IgG2a immune responses. Passive intraperitoneal transfer of IgG1 predominating antisera from s.c. rFgGST26-immunized donors to naive recipient mice resulted in 47% protection against F. gigantica infection. This suggests that the mechanism of resistance depends on the presence of specific antibody against rFgGST26. Immunization with rFgGST26 via i.m. and i.d. routes resulted in significant cross protection (55%) against S. mansoni infection in the i.d. route with mixed IgG1/IgG2a response with IgG1 isotype predominating. This indicated that rFgGST26 is a good vaccine candidate against F. gigantica in mice and could also provide cross protection against S. mansoni.

Type I cystatin (stefin) is a major component of Fasciola gigantica excretion/secretion product.

In the present study we describe type 1 cystatin, a cysteine protease inhibitor, as a major released antigen of the tropical liver fluke Fasciola gigantica (FgStefin-1). Immunohistochemical analysis showed that FgStefin-1 is abundant in (a) tissue of tegumental type, including oral and ventral sucker, pharynx, genital atrium, metraterm, cirrus and (b) the intestinal epithelium. Faint staining was observed in the epithelia of ovary and proximal uterus. Immunoblots showed the presence of FgStefin-1 in the parasites excretion/secretion (ES) product and immunodepletion demonstrated that FgStefin-1 herein is partially complexed with cathepsin L. Furthermore, quantitation of FgStefin-1 in comparison to cathepsin L in ES product and crude worm extract of adults supports a major external function of FgStefin-1 with an estimated 50% being released in at least equimolar amounts to cathepsin L. Sera of an experimentally infected rabbit reacted with recombinant FgStefin-1 starting 8 weeks postinfection. Activity analyses of recombinant FgStefin-1 showed nanomolar inhibition constants for mammalian cathepsin B, L, and S cysteine proteases and released cysteine proteases of the parasite. The protein is active over a wide pH range and is heat stable. Our results suggest protective functions of FgStefin-1, regulating intracellular cysteine protease activity, and possibly protection against extracellular proteolytic damage to the parasites intestinal and tegumental surface proteins. Considering inhibition kinetics and previously demonstrated immunomodulatory properties of cystatin in parasitic nematodes a comparable function of FgStefin-1 is suggested and is at present under investigation.

Production and characterization of a monoclonal antibody against 28.5 kDa tegument antigen of Fasciola gigantica.

A monoclonal antibody (MoAb) against the 28.5 kDa tegumental antigen of Fasciola gigantica was produced by the hybridoma technique using spleen cells from BALB/c mice immunized with the tegumental extract from adult F. gigantica. This MoAb was found to be of the isotype IgG(1), kappa-light chain, and shown by immunoblotting to specifically react with the 28.5 kDa antigen present in the tegument, excretion-secretion material of the adult, whole-body extracts of newly excysted juveniles, 5-week-old juvenile and adult parasites. It did not cross-react with antigens from other trematode parasites, including Schistosoma mansoni, Eurytrema pancreaticum and Paramphistomum spp. Immunolocalization of this antigen by indirect immunofluorescence indicated that it was present as a major component of the adult tegument, particularly in its outer rim, tegumental cells, and their processes. Furthermore, the epithelium linings of the oral sucker, buccal tube, pharynx, caecal bifurcation, both male and female genital canals, which were the continuation of the tegumental-type epithelium, were also positively stained with this MoAb. A similar pattern of immunolocalization, but with weaker staining intensity, was observed in newly excysted, 5- and 7-week-old juveniles. Thus this antigen is expressed in all developmental stages of the parasite, and it could be a strong candidate for immunodiagnosis and vaccine development.

Adult and juvenile Fasciola cathepsin L proteases: Different enzymes for different roles

Cathepsin proteases are promising vaccine or drug targets for prophylaxis or therapy against Fasciola parasites which express cathepsin L and B proteases during their development. These proteases are believed to be involved in important functions for the parasite, including excystment, migration, feeding and host immune evasion. Several cathepsin L transcripts, including FhCatL5, have been isolated from adult Fasciola, while certain cathepsin L proteases, including FgCatL1G, have only been identified in the juvenile forms of the parasite. In this study, Fasciola hepatica cathepsin FhCatL5 and F. gigantica FgCatL1G were expressed in yeast and their biochemical properties characterised and compared. The pH profiles of activity and stability of the two recombinant cathepsins was shown to differ, differences that are likely to be functionally important and reflect the environments into which the cathepsins are expressed in vivo. Biochemical analysis indicates that FgCatL1G can cleave substrates with proline residues at P(2), a characteristic previously described for the adult cathepsin FhCatL2. FgCatL1G and FhCatL5 show differences in their host substrate digestion patterns, with different substrates cleaved at varying efficiencies. Functional analysis of a recombinant FhCatL5 L69W variant indicates that the residue at position 69 is important for the S(2) subsite architecture and can influence substrate specificity.

Efficient inhibition of cathepsin B by a secreted type 1 cystatin of Fasciola gigantica

Cysteine proteases are important antigens in the liver fluke genus Fasciola, essential for infection, protection and nutrition. While their biochemistry, biological roles and application as vaccines have been thoroughly studied there is a lack of data concerning their regulation. In the present study we have continued our investigation of cysteine protease inhibitors in Fasciola gigantica and demonstrate, in comparison with FgStefin-1 and human cystatin C, that a second type 1 cystatin of the parasite, FgStefin-2, has been evolutionary adapted to block cathepsin B. The protein, which unusually for a type 1 cystatin carries a signal peptide, is expressed from the metacercarial to adult stage and located in the epithelial cells of the intestinal tract in all stages and in the prostate gland cells in adults. Both cell types may contribute to the released FgStefin-2 observed in the ES product of the parasite. Distinct isoforms of cathepsin B are essential for host tissue penetration during the early infection process and FgStefin-2 may act as key regulator, required to protect the minute juvenile from autoproteolysis. Expression in the prostate gland in the adult stage suggests an additional regulative role of cysteine protease activity in the reproductive system.

Analysis of a calcium-binding EF-hand protein family in Fasciola gigantica.

Transcriptome data supports the notion of a Platyhelminthes-specific protein family that is characterized by combination of two N-terminal EF-hands and a C-terminal dynein light chain-like domain. Family members in schistosomes induce an IgE response that has been connected with resistance to reinfection in schistosomiasis and is considered as a marker of protection. In the present study, we have compared three homologs of the liver fluke Fasciola gigantica for their immunological properties in mouse. Antisera raised against the recombinant proteins detected the native proteins in tegumental type tissues and epithelial linings of excretory system and intestinal tract. The recombinant EF-hand domains induced strong IgG and IgE responses in immunised mice while only weak to moderate responses were observed against the complete recombinant proteins and their DLC-like domains. Parasite crude worm and tegumental extract antisera reacted predominantly with one isoform and its EF-hand domain. Sera of F. gigantica infected mice did not react with the recombinant proteins. The RNA products of the three genes were detected from the metacercarial up to the adult stage. These observations indicate that the investigated EF-hand proteins are not at the frontier of humoral host/parasite interaction in acute fascioliasis gigantica in mouse but are acting as intracellular proteins in tissues that interface with the parasites environment or tubular tracts.

Comparative analysis of two fatty acid binding proteins from Fasciola gigantica.

Fatty acid binding proteins are considered to be promising vaccine candidates against trematodiasis. In order to provide additional information about their function in Fasciola gigantica we performed a comparative analysis of FgFABP1 and FgFABP3, two isoforms with quite different isoelectric points of 4.9 and 9.9 and 67% sequence identity. Both are expressed in the juvenile and adult parasite but differ in their tissue-specific distribution. In addition, the sequence of FABP3 is identical in F. hepatica and F. gigantica indicating the proteins functional importance in this genus. Immune sera produced against soluble recombinant FgFABPs reacted with 14 kDa antigens in crude worm, soluble egg, cirrus sac extracts, and excretion/secretion product. Both FgFABPs were located in the parenchyma of the parasite but in addition, FgFABP1 was abundant in testes and spermatozoa while FgFABP3 was abundant in vitelline cells, eggs, and caecal epithelium. Mass spectrometry identified FgFABP1 and FgFABP3 in the ES product whereas only FgFABP3 was identified in egg extract. Serum samples of an experimentally infected rabbit reacted from week 6 post-infection with FgFABP3 and from week 12 with FgFABP1 while sera of infected sheep were not reactive. The results suggest differences in the biological functions of these 2 isoforms and differences in the host/parasite interaction that should be considered for their potential as vaccines against fascioliasis.

The mub gene encodes a protein containing three KH domains and is expressed in the mushroom bodies of Drosophila melanogaster

The ring gland function of Drosophila melanogaster is controlled by the CNS. To identify genes that are active in brain cells and are involved in the ring gland control, we analysed enhancer trap lines with respect to CNS- and/or ring gland-specific lacZ expression in third-instar larvae. From one of the enhancer trap lines, which shows specific lacZ expression in the CNS and prothoracic part of the ring gland, the mub gene was cloned. The gene is strongly expressed in the mushroom bodies throughout development. Nucleotide sequence analysis of cDNA clones revealed a high degree of similarity to vertebrate RNA binding KH domain proteins, suggesting a function of the MUB protein in binding and stabilizing of specific mRNAs in the mushroom bodies. Null mutants of the mub gene do not exhibit a visible mutant phenotype. We speculate, therefore, that the mub gene is involved in learning and memory processes.