Automated imaging and other developments in whole-organism anthelmintic screening.

Helminth infections still represent a huge public health problem throughout the developing world and in the absence of vaccines control is based on periodic mass drug administration. Poor efficacy of some anthelmintics and concerns about emergence of drug resistance has highlighted the need for new drug discovery. Most current anthelmintics were discovered through in vivo screening of selected compounds in animal models but recent approaches have shifted towards screening for activity against adult or larval stages in vitro. Larvae are normally available in greater numbers than adults, can often be produced in vitro and are small enough for microplate assays. However, the manual visualization of drug effects in vitro is subjective, laborious and slow. This can be overcome by application of automated readouts including high-content imaging. Incorporated into robotically controlled HTS platforms such methods allow the very large compound collections being made available by the pharmaceutical industry or academic organizations to be screened against helminths for the first time, invigorating the drug discovery pipeline. Here, we review the status of whole-organism screens based on in vitro activity against living worms and highlight the recent progress towards automated image-based readouts.

Radiation-attenuated schistosome vaccination--a brief historical perspective.

The high level of protection which can be induced by vaccination of a range of hosts, from rodents to primates, with live radiation-attenuated schistosome larvae offers great promise for development of a human schistosome vaccine. Studies of the irradiated vaccine models benefitted from significant funding during the 1970-90s and much was learned concerning the inducers, targets and mechanisms of immunity. Less progress was made in definition of the protective antigens involved. The application of new techniques for identifying membrane and secreted antigens has recently provided new vaccine candidates and a new impetus for schistosome vaccine development. This article is intended as an overview of some of the main lessons learned from the studies of the irradiated vaccines as a backdrop to renewed interest in schistosome vaccine development.

IFN-gamma is associated with risk of Schistosoma japonicum infection in China.

Before the start of the schistosomiasis transmission season, 129 villagers resident on a Schistosoma japonicum-endemic island in Poyang Lake, Jiangxi Province, 64 of whom were stool-positive for S. japonicum eggs by the Kato method and 65 negative, were treated with praziquantel. Forty-five days later the 93 subjects who presented for follow-up were all stool-negative. Blood samples were collected from all 93 individuals. S. japonicum soluble worm antigen (SWAP) and soluble egg antigen (SEA) stimulated IL-4, IL-5 and IFN-gamma production in whole-blood cultures were measured by ELISA. All the subjects were interviewed nine times during the subsequent transmission season to estimate the intensity of their contact with potentially infective snail habitats, and the subjects were all re-screened for S. japonicum by the Kato method at the end of the transmission season. Fourteen subjects were found to be infected at that time. There was some indication that the risk of infection might be associated with gender (with females being at higher risk) and with the intensity of water contact, and there was evidence that levels of SEA-induced IFN-gamma production were associated with reduced risk of infection.

Down-regulation of specific antigen-driven cytokine production in a population with endemic Schistosoma japonicum infection.

Schistosome antigen-driven cytokine responses and antischistosome antibody levels of residents of a Schistosoma japonicum endemic island in Poyang Lake, Jiangxi Province were studied before and 45 days after treatment with praziquantel. IL-4, IL-5, IL-10 and INF-gamma were all detected in the supernatants of whole-blood cultures after stimulation with schistosome soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP). The percentages of subjects producing detectable amounts of each cytokine assayed were higher in the group who were negative by stool examination at the start of the study than in those who were initially stool positive. After praziquantel treatment the percentages of subjects producing both type I and type II cytokines increased. This suggests that the production of both types of cytokine was down-regulated in the presence of live, egg-laying S. japonicum adult worms but that this was reversible by treatment. In contrast, the antibody studies showed higher levels of SWAP and SEA-specific antibodies (IgE, total IgG, IgG4, IgM) in subjects who were originally stool-positive than in those who were stool-negative. After treatment specific IgE responses were elevated, but total IgG and IgG4 anti-SEA and IgM anti-SWAP antibody levels all fell significantly.

Vaccination of mice with a cocktail DNA vaccine induces a Th1-type immune response and partial protection against Schistosoma japonicum infection.

Several defined vaccine candidate antigens of Schistosoma japonicum have shown promise in large animal vaccination experiments. However, vaccination of mice in the laboratory with either single recombinant antigens or DNA encoding forms of the individual antigens has so far failed to induce significant protection against S. japonicum cercarial challenge infection as judged by worm reduction, although specific antibodies were generated. This is in contrast to the results achieved using radiation-attenuated vaccines which are highly protective. Even in large animal vaccination experiments, the protection levels obtained with single defined antigens were far below those achieved using the attenuated vaccines. One possible interpretation is that the immune responses induced by single antigen vaccination may not be strong enough to combat the challenging infection. We, therefore, carried out mouse vaccination experiments using a cocktail DNA vaccine comprising four DNA plasmids encoding four different S. japonicum antigens, Sj62, Sj28, Sj23 and Sj14-3-3, respectively. We, also investigated whether co-injection of the mouse IL-12 encoding plasmid with the cocktail DNA vaccine was able to enhance the Th1 responses and hence the protective immunity. Three intramuscular injections of the cocktail DNA vaccine induced a significant Th1-type cellular response with high level of IFN-gamma production by splenocytes upon in vitro stimulation with recombinant antigens. Importantly, significant IgG antibody responses were also induced against crude worm antigens. In two out of three experiments, significant resistance (34-37 and 44-45%, respectively) was demonstrated while another experiment did not show any protection against S. japonicum cercarial challenge infection. Co-injection of the IL-12 encoding DNA did not further enhance these responses, nor the level of resistance, compared with the cocktail DNA alone.

Laboratory and field evaluation of Schistosoma japonicum DNA vaccines in sheep and water buffalo in China.

Vaccines are needed to control zoonotic Schistosoma japonicum infection and several vaccine candidates have now been identified. Two of these (Sj28GST and Sj23) have shown particular promise in sheep when injected with Freund's adjuvants. The objective of the present work was to find a vaccine formulation which may have potential for widespread use in the field. DNA vaccine formulations of these antigens were produced and tested first in sheep under laboratory conditions and then in both the laboratory and the field in water buffalo. In both host species partial protection as evidenced by a reduction in parasite counts in vaccinated compared with control animals was induced by both vaccines, and in water buffalo the vaccines were shown to be partially protective in the field as well as in the laboratory. These results suggest that the two DNA vaccines tested here may have potential for large-scale field use.

Comparison of the vaccine efficacy of gamma-irradiated Schistosoma japonicum cercariae with the defined antigen Sj62(IrV-5) in pigs.

Development of a vaccine against Schistosoma japonicum which can protect both man and the domestic animal zoonotic reservoirs of infection would be an invaluable tool in attempts to control this infection in those areas in which conventional control methods have failed to break transmission. The pig is a natural host of S. japonicum and because of its anatomical and immunological similarities to humans, it is a potentially valuable host for studies on S. japonicum in particular and schistosomes in general. Radiation-attenuated cercariae are highly effective in inducing immunity in experimental schistosomosis and there are promising reports of partial protection against schistosomes with recombinant-derived individual antigens. In the present study we have set out to establish a protocol for inducing protection with gamma-irradiated cercariae in pigs and to assess the protective capacity of recombinant and naked DNA formulations of Sj62, a 62kDa region of S. japonicum myosin. The corresponding S. mansoni version or Sj62, recombinant IrV-5, has previously been implicated in irradiated vaccine immunity in S. mansoni infections and has been shown to induce high levels of immunity in a variety of hosts. Groups of pigs were immunised three times at 2-week intervals with 2000 cercariae irradiated at 20krad, with Sj62 as a recombinant (rSj62) incorporated in Freund's adjuvant, a micellar preparation, or as a naked DNA construct. Vaccination with irradiated cercariae did not induce significant anti-Sj62 antibody but following intramuscular challenge with 2000 cercariae, the vaccinated pigs showed >95% resistance as assessed by reduced faecal egg output, worm tissue egg burdens and also reduced septal fibrosis. Immunisation with each of the Sj62 formulations induced significant anti-Sj62 antibody responses, the highest titre (>12,800) being with the Freund's preparation but none of the Sj62-immunised groups showed significant resistance to challenge. The data suggest that Sj62 shows little promise as a vaccine candidate for schistosomosis.

Cloning of Schistosoma japonicum 14-3-3 epsilon (Sj14-3-3 epsilon), a new member of the 14-3-3 family of proteins from schistosomes.

A new member of the 14-3-3 protein family from Schistosoma japonicum has been identified. Phylogenetic analysis showed that this member belongs to the epsilon subfamily of the 14-3-3 proteins, and it is therefore named Sj14-3-3 epsilon. Consistent with the findings for the previously reported S. japonicum 14-3-3 protein (Sj14-3-3), Southern analysis suggested the presence of more than one gene, and/or introns or allelic polymorphism in this epsilon isoform. By RT-PCR, Sj14-3-3 epsilon was shown to be stage-specifically transcribed, being abundant in adults, present in sporocysts but absent in cercariae. Furthermore, mRNA of the epsilon isoform seemed to be much less abundant in the sporocyst stage, compared with Sj14-3-3. This suggests varying requirements of the different 14-3-3 isoforms at different stages of the life cycle.

Analysis of human antibodies to erythrocyte binding antigen 175 of Plasmodium falciparum.

Invasion of human erythrocytes by Plasmodium falciparum merozoites is a multistep process. For many strains of the parasite, part of this process requires that the erythrocyte binding antigen 175 (EBA-175) of the merozoite binds to sialic acid residues of glycophorin A on the erythrocyte surface, a receptor-ligand interaction which represents a potential target for inhibition by antibodies. This study characterizes the reactivity of naturally acquired human antibodies with four recombinant proteins representing parts of EBA-175 (region II, regions III to V, and the dimorphic C and F segment region) in populations in which the organism is endemic. Serum immunoglobulin G (IgG) recognizing the recombinant proteins is predominantly of the IgG1 and IgG3 subclasses, and its prevalence increases with age. In a large population study in The Gambia, serum positivity for IgG or IgG1 and IgG3 subclass antibodies to each of the EBA-175 recombinant antigens was not significantly associated with subsequent protection from clinical malaria. However, there was a trend indicating that individuals with high levels of IgG to region II may have some protection.

Schistosoma mansoni phosphofructokinase: immunolocalization in the tegument and immunogenicity.

Schistosoma mansoni depends for its survival on glycolysis. Two glycolytic enzymes, glyceraldehyde-3P-dehydrogenase and triose-phosphate dehydrogenase, found in both the adult and schistosomular tegument, have been reported to confer partial protection against cercarial infection. This paper describes the immunogenic properties of phosphofructokinase (PFK), a rate-limiting enzyme of glycolysis, and its localization in the tegument and adjacent tissues. Recombinant schistosome PFK was used as antigen. A polyclonal antibody against purified PFK from Fasciola hepatica was affinity purified using recombinant PFK and used in combination with immunogold labelling to identify PFK by transmission electron microscopy in cryosections. In both adult worms and in schistosomula most immunogold label localized in the cytoplasmic syncytial region with less being found in the tegument. There was no significant PFK localization within or external to the outer membrane. Sera from mice immunized with recombinant S. mansoni PFK with Freund's adjuvant or alum plus rIL-12 demonstrated high titres of anti-PFK IgG, but no protection against cercarial infection. Sera from mice that were acutely or chronically infected or multiply exposed to irradiated cercariae did not recognize recombinant schistosome PFK in either Western blotting or ELISA. Similarly, sera from humans infected with S. mansoni did not recognize PFK. We conclude that in spite of the high immunogenicity of rPFK in mice, it is not a significant immunogen during the course of infection and does not confer protection from schistosomiasis. One main difference between PFK and the other 2 glycolytic enzymes seems to be the inaccessibility of PFK to the outside surface of the tegument.

Immunogenicity of plasmid DNA encoding the 62 kDa fragment of Schistosoma japonicum myosin.

The recombinant Schistosoma mansoni 62 kDa myosin fragment, rIrV-5, is highly protective in experimental animals, however, vaccination of mice and rats with the recombinant Schistosoma japonicum homologue, rSj62, did not induce significant resistance against S. japonicum infection. To explore alternative ways of presenting this antigen, we further constructed a plasmid (VRSj62) which encodes Sj62 using the VR1020 vector and tested it in vaccination experiments. Four immunisations with 10 microg VRSj62 DNA alone were sufficient to induce high and progressively increasing levels of IgG antibodies against rSj62 with increasing numbers of injections in CBA/Ca mice (IgG titre > or =1:25000), and three injections with 50 microg VRSj62 DNA alone induced significant IgG responses in C57Bl/6 mice (IgG titre, 1:1600). However, vaccination with plasmid DNA entrapped in cationic liposomes or together with pUC19 DNA as a source of CpG motifs, both of which have been reported to enhance immune responses, did not enhance specific antibody production. In spite of the stimulation of specific antibodies against rSj62 with the naked DNA construct no resistance to challenge was demonstrated.

Molecular cloning and characterization of a novel Schistosoma japonicum "irradiated vaccine-specific" antigen, Sj14-3-3.

A Schistosoma japonicum cDNA coding for a full length S. japonicum 14-3-3 protein was obtained by antibody screening of an adult worm cDNA library using sera taken from mice vaccinated with UV-attenuated cercariae, which are capable of transferring high levels of passive immunity to this parasite. The deduced amino acid sequence consists of 254 amino acids and is highly homologous with 14-3-3 family of proteins from a variety of species (55-69% identity). The recombinant S. japonicun 14-3-3 protein (rSj14-3-3) was expressed and purified in pGEX/E. coli, and in Western blotting was strongly recognised by sera from mice, rats and bovines vaccinated with irradiated S. japonicum cercariae. Analysis of mRNA showed that Sj14-3-3 is expressed in sporocysts and adult worms, but not in cercariae, however mouse antisera against rSj14-3-3 recognised a 29 kDa native antigen in antigen preparations made from eggs, cercariae, schistosomula and adult worms of S. japonicum indicating that this antigen is present in all life-cycle stages. The presence of the native antigen in detergent extracts of intact schistosomula suggests that it is also present in the schistosomular tegument which is the most vulnerable target for immune attack. However, antisera against rSj14-3-3 did not recognise a similar band in S. mansoni or S. haematobium antigens, indicating that, like the UV-attenuated vaccines, this protein induced species-specific immune responses. Southern blot analysis suggested that there may exist more than one gene copy and/or polymorphism for Sj14-3-3. Immunoelectron microscopy confirmed that the native antigen is present throughout the body of adult worms including the tegument, but is less abundant in the muscles. The potential of rSj14-3-3 as a vaccine is now under further investigation.

Characterization of a stage-specific Mr16000 schistosomular surface glycoprotein antigen of Schistosoma mansoni.

A 16 kDa Schistosoma mansoni schistosomular surface antigen (Sm16) was originally described as the target of a passively protective mAb (B3A). It appeared on the schistosomular surface after transformation of cercariae and was uniquely recognised by sera from animals exposed to attenuated cercariae. In this work sequential extractions of schistosomula with Triton X-114 and sodium dodecyl sulphate showed Sm16 to be an integral membrane structure which did not appear to be glycosylphosphatidylinositol-anchored as judged by experiments using phosphatidyl inositol-specific phospholipase C. The antigen was strongly reactive in Western blotting with rabbit irradiated vaccine sera. Sm16 was demonstrated in the hepatopancreas of S. mansoni-infected snails and was equally abundant in cercariae and mechanically- transformed schistosomula but was undetected in liver stage worms or eggs. Immunoelectron microscopy showed Sm16 to be localised, in cercariae, to what are believed to be subtegumental cell bodies packed with membraneous vesicles. Treatment with proteases and with sodium metaperiodate showed Sm16 to be a glycoprotein of which the epitope recognised by B3A was periodate sensitive. Two-dimensional electrophoresis gave a PI of 6. Neither the size or the recognition by B3A was affected by treatment with N-glycosidase F, endoglycosidase F or endo-alpha-N-acetylgalactosaminidase. Western blotting using a wide range of biotinylated lectins showed recognition only by peanut agglutinin and Ricinus communis agglutinin II (ricin). It is concluded that Sm16 has antigenic surface-exposed O-linked complex oligosaccharides which lack mannose/glucose, GlcNAc, L-fucose and sialic acid but contain terminal Gal beta (1-3) GalNAc and/or galactose.

Vaccination of mice with gamma-irradiated Schistosoma japonicum cercariae.

gamma-irradiated cercarial vaccines induce high levels of protection in mice against Schistosoma mansoni infection, however, the same has not been well established for S. japonicum. Here we describe vaccination studies in mice with gamma-irradiated S. japonicum cercariae testing the effectiveness of different irradiation doses, number of vaccinations, and mouse strains. In CBA/Ca mice, a single percutaneous exposure to 500 S. japonicum cercariae previously attenuated by 10, 20, 30, 40 or 50 krad gamma-irradiation induced significant, but comparable levels of protection (34-46%) against challenge infection. In a repeat experiment in C57Bl/6 mice, only groups vaccinated with 10 or 20 krad gamma-irradiated cercariae showed statistically significant, but lower levels of resistance (20-24%). Multiple vaccination of CBA/Ca mice with 500 20 krad gamma-irradiated cercariae did not improve the resistance level (40%). Analysis of IgG responses showed no clear correlation between antibody levels and levels of protection. Western blot analysis suggested that recognition of a 200-kDa antigen might be correlated with protection, that antigens of 42 and 50 kDa may be involved in the protection induced by single vaccination, but that different antigens might be protective in single vs multiple vaccinations. Sera from mice vaccinated with gamma-irradiated cercariae recognized many fewer antigens than more protective sera from mice vaccinated with UV-attenuated cercariae. These results suggest that the mouse may not be a suitable host for studies involving gamma-irradiated S. japonicum vaccines.

Rapid purification and characterization of L-dopachrome-methyl ester tautomerase (macrophage-migration-inhibitory factor) from Trichinella spiralis, Trichuris muris and Brugia pahangi.

Macrophage-migration-inhibition factor (MIF) is an essential stimulator of mammalian T-lymphocyte-dependent adaptive immunity, hence MIF orthologues might be expressed by infectious organisms as an immunosubversive stratagem. Since MIF actively catalyses the tautomerization of the methyl ester of l-dopachrome (using dopachrome tautomerase), the occurrence of MIF orthologues in several parasitic helminths was investigated by assaying and characterizing such activity. Evidence of MIF orthologues (dopachrome tautomerase) was found in the soluble fraction of the nematodes Trichinella spiralis (stage 4 larvae) and Trichuris muris (adults), and the filarial nematode Brugia pahangi (adults). The MIF orthologues of Tr. muris (TmMIF) and B. pahangi (BpMIF) were purified to homogeneity using phenyl-agarose chromatography, that of T. spiralis (TsMIF) required a further step: cation-exchange FPLC. Retention time on reverse-phase HPLC and Mr on SDS/PAGE of the nematode MIFs were similar to those of human MIF. N-terminal sequences (19 residues) of TsMIF and TmMIF showed 47 and 36% identity, respectively, with human MIF. The N-terminal sequence of BpMIF (14 residues) was identical to that of an MIF orthologue in the genome of B. malayi (Swiss-Prot, P91850) and showed 43% identity to either human or TsMIF. TsMIF had 10-fold higher dopachrome tautomerase activity than MIF from the other sources. The enzyme activities of TsMIF, BpMIF and TmMIF were less sensitive to inhibition by haematin (I50: >15 microM, >15 microM and 2.6 microM, respectively) than that of human MIF (I50 0.2 microM). Significant dopachrome tautomerase or phenyl-agarose-purifiable MIF-like protein was not detected in the soluble fraction of the nematodes Heligmosomoides polygyrus and Nippostrongylus brasiliensis, the cestode Hymenolepis diminuta, or the trematodes Schistosoma mansoni, S. japonicum and S. haematobium, or the free-living nematode, Caenorhabditis elegans, which does contain an MIF-related gene.

Production and testing of Schistosoma japonicum candidate vaccine antigens in the natural ovine host.

The objectives of this work were to clone and express Chinese strain Schistosoma japonicum antigens and evaluate their immunogenicity and protective efficacy in the natural ovine host in China. Recombinant antigens selected for testing were: isoforms of glutathione S-transferase Sj28GST and Sj26GST; the large hydrophilic domain of Sj23, the homologue of the protective S. mansoni membrane antigen Sm23; and a 3' fragment of S. japonicum paramyosin. In addition, Chinese strain S. japonicum native paramyosin and GST were purified and used for vaccination. Antigens were co-administered with Freund's adjuvants or BCG. We also examined the effects of co-administration of native unfractionated GSTs with keyhole limpet haemocyanin (KLH), which shares a cross-reactive protective epitope with schistosomes. These are the first side-by-side comparisons of candidate defined-antigen schistosomiasis vaccines in a natural host. Significant partial protection was obtained with each of the antigens tested. Less protection was obtained with a recombinant fragment of S. japonicum paramyosin compared with native paramyosin. Co-administration of native GST and KLH was no more effective than vaccination with either antigen alone. Although encouraging levels of protection against S. japonicum were demonstrated using each of these antigens, further work is needed to optimise vaccine delivery and vaccination schedules.

Schistosoma japonicum myosin: cloning, expression and vaccination studies with the homologue of the S. mansoni myosin fragment IrV-5.

The Schistosoma japonicum homologue of the 62 kDa fragment of S. mansoni myosin (SmIrV-5), which has proved highly protective against S. mansoni infection in mice and rats, has been cloned and expressed as the full length 62 kDa equivalent, Sj62, and a truncated 44 kDa version, Sj44. DNA sequencing showed the Sj62 sequence to be 88.4% identical at the nucleic acid level and 96% identical in deduced amino acid sequence to that of SmIrV-5. The recombinant proteins (rSj44 and rSj62) were strongly recognized in Western blotting by sera from mice multiply vaccinated with UV-irradiated S. japonicum cercariae and weakly recognized by S. japonicum chronic infection mouse sera. Unlike SmIrV-5, mouse antisera against the recombinant S. japonicum proteins did not give positive recognition in immunofluorescence assay with the surface of newly transformed schistosomula of the homologous species, S. japonicum, nor did they react with S. mansoni schistosomula. However, the anti-rSj62 sera clearly localized the native antigen to the subtegumental muscle layers in male adult worm sections by immunoelectron microscopy. Vaccination of several groups of mice and/or rats with rSj44 and rSj62 incorporated into different adjuvants induced high titres of specific IgG but in only one experimental group was there a significant reduction in worm burden (27%, P < 0.05). The possible reasons for the disparity between the vaccination results presented here and those demonstrated in experiments using rSm62 (IrV-5) are discussed.

Higher levels of passive compared with active immunity in rats immunized with larval antigens of Schistosoma mansoni.

We have previously shown that it is the lung stage schistosomulum which is both the inducer and the target of immunity in rats vaccinated with irradiated cercariae but that rats can also be partially protected by immunization with detergent extracts of mechanically-transformed schistosomula. In the present study we therefore compared the immunogenicity of these and intermediate schistosomular stages. In the first experiment low levels of protection (13-24%) were induced by extracts of mechanically-transformed schistosomula cultured for 3 hours or 2 days but not by extracts of lung schistosomula. In a repeat experiment insignificant levels of protection were induced by extracts of 3 h schistosomula and lung schistosomula were again non-protective. Nevertheless, high levels of anti-schistosomula antibodies were demonstrated and sera from actively immunized rats conferred significant passive protection in five out of six trials. The levels of protection conferred by passive immunization (26-51%) were in each case higher than the levels of protection demonstrated in the respective serum donor groups, showing that some form of immunological blockade suppression is operating to prevent expression of protective immunity in the actively immunized rats.

Resistance induced by drug abbreviated Schistosoma mansoni infections: treatment with the drug Ro11-3128 leads to enhanced antigen presentation.

Treatment of mice with the benzodiazepine derivative Ro11-3128 1-2 days post-infection with Schistosoma mansoni leads to arrest of virtually all schistosomula at the skin stage, and results in the development of protective immunity to challenge infection. A characteristic feature of Ro11-3128 treatment in vitro is the formation of exudates and membranous blebs at the schistosomular surface; other drugs tested, such as Ro15-5458 and oxamniquine which are also effective against the skin stages but relatively ineffective in inducing protection, do not induce this reaction. Here, we have examined whether such in vitro treatment causes enhanced presentation of schistosomular antigens by host antigen-presenting cells (APC) using an in vitro assay with activated peritoneal adherent cells as APC and T cells from S. mansoni antigen-sensitized mice. We have shown that viable mechanically transformed schistosomula (MS) can be processed and presented with similar kinetics to soluble antigen. However, in vitro drug treatment leads to enhanced presentation of MS. Experiments in which membranous blebs and antigen released by Ro11-3128-treated parasites during in vitro culture were separated from the remaining intact schistosomula, demonstrated significant stimulatory activity in the soluble and particulate-released antigen fractions. Filtration, antigen transfer experiments and SDS-PAGE analysis of the released material further suggested that most of the activity resided in the particulate fraction. Thus, quantitative and qualitative changes to antigen presentation by Ro11-3128 treatment early after infection may underlie the immunoprotective efficacy of Ro11-3128-abbreviated infections.

Diagnosis of Schistosoma haematobium infection: evaluation of ELISA using keyhole limpet haemocyanin or soluble egg antigen in comparison with detection of eggs or haematuria.

Keyhole limpet haemocyanin (KLH) was compared with Schistosoma haematobium soluble egg antigen (SEA) in the enzyme-linked immunosorbent assay (ELISA) for detection of S. haematobium infection, using 187 human sera collected from the S. haematobium endemic area of Pemba Island, Tanzania, and 30 normal sera from blood donors in Europe. There was a clear separation in terms of immunoglobulin (Ig) G and IgM titres between parasitologically positive patients and the blood donors, but titres of many parasitologically negative individuals in the endemic area were significantly high in comparison with the normal controls. Using as cut-off point the mean optical density +2 SD of sera from the blood donors to define ELISA positivity, and comparing the results with urine egg counts, the sensitivity of IgG-ELISA using KLH or SEA was high (91.11% and 95.56%, respectively) but the specificity was poor (43.30% and 31.90%, respectively. Similar results were obtained with IgM. When the 'gold standard' of haematuria and/or egg positivity as indicative of infection was used, the sensitivity of the ELISAs was similar but the specificity was increased to 59.25% and 44.44%, respectively. These results suggest that the patients with haematuria were probably infected with S. haematobium, which further supported the diagnostic value of haematuria detection for S. haematobium infection in endemic areas, and KLH was found to have a potential use in immunodiagnosis of S. haematobium infection in endemic areas. With both KLH and SEA antigens, the trend of reactivity in ELISA provided a correlate of the egg output (parasite burden) of infected patients.