Extracellular vesicles from Fasciola gigantica induce cellular response to stress of host cells.

Extracellular vesicles (EVs) from parasitic helminths play an important role in immunomodulation. However, EVs are little studied in the important parasite Fasciola gigantica. Here the ability of EVs from F. gigantica to induce cellular response to stress (reactive oxygen species generation, autophage and DNA damage response) in human intrahepatic biliary epithelial cells (HIBEC) was investigated. F. gigantica-derived EVs were isolated by ultracentrifugation, and identified with transmission electron microscopy, nanoparticle size analysis and parasite-derived EV markers. Internalization of EVs by HIBEC was determined by confocal immunofluorescence microscopy and flow cytometry. ROS levels in HIBEC were detected by molecular probing. EVs-induced autophagy and DNA-damaging effects were determined by evaluating expression levels of light chain 3B protein (LC3B), phosphor- H2A.X and phosphor-Chk1, respectively. Results revealed that EVs with sizes predominately ranging from 39 to 110 nm in diameter were abundant in adult F. gigantica and contained the parasite-derived marker proteins enolase and 14-3-3, and EVs were internalized by HIBEC. Further, uptake of EVs into HIBEC was associated with increased levels of reactive oxygen species, LC3Ⅱ, phosphor-H2A.X and phosphor-Chk1, suggesting EVs are likely to induce autophagy and DNA damage & repair processes. These results indicate F. gigantica EVs are associated with modulations of host cell responses and have a potential important role in the host-parasite interactions.

Broad specificity of immune helminth scFv library to identify monoclonal antibodies targeting Strongyloides.

Antibodies have different chemical properties capable of targeting a diverse nature of antigens. Traditionally, immune antibody libraries are perceived to be disease-specific with a skewed repertoire. The complexity during the generation of a combinatorial antibody library allows for a skewed but diverse repertoire to be generated. Strongyloides stercoralis is a parasite that causes strongyloidiasis, a potentially life-threatening disease with a complex diagnosis that impedes effective control and treatment of the disease. This study describes the isolation of monoclonal antibodies against S. stercoralis NIE recombinant protein using an immune antibody phage display library derived from lymphatic filaria-infected individuals. The isolated antibody clones showed both lambda and kappa light chains gene usage, with diverse amino acid distributions. Structural analysis showed that electropositivity and the interface area could determine the binding affinity of the clones with NIE. The successful identification of S. stercoralis antibodies from the filarial immune library highlights the breadth of antibody gene diversification in an immune antibody library that can be applied for closely related infections.

[Expression, purification and polyclonal antibody preparation of the Schistosoma japonicum SjGrpE protein].

OBJECTIVE: To investigate the biological properties of Schistosoma japonicum SjGrpE protein, and to express and purify the recombinant SjGrpE protein and test its immunogenicity. METHODS: The amino acid composition, molecular weight, hydrophilicity and hydrophobicity, transmembrane region, signal peptide, localization, phosphorylation site, ubiquitination site, glycosylation site, secondary and tertiary structures and B cell epitopes of the SjGrpE protein were predicted using bioinformatics analyses. The SjGrpE gene was amplified using PCR assay using S. japonicum cDNA as a template, double enzyme-digested and linked to the pET28a vector to yield the recombinant plasmid pET28a-SjGrpE. The recombinant plasmid pET28a-SjGrpE was transformed into Escherichia coli BL21, and then IPTG was employed to induce the expression of the target protein, which was purified by nickel ion affinity chromatography. After mice were immunized with the recombinant SjGrpE protein, mouse sera were collected, and the polyclonal antibody against the SjGrpE protein was characterized. RESULTS: SjGrpE protein, which was identified as a hydrophilic protein, was predicted to have a molecular weight of approximately 24.3 kDa without transmembrane regions or signal peptides, and locate in the mitochondrion. SjGrpE protein contained 18 phosphorylation sites and 2 ubiquitination sites, but had no glycosylation sites. In addition, SjGrpE protein contained 5 B-cell epitopes. The full length of SjGrpE gene was approximately 660 bp. The recombinant pET28a-SjGrpE plasmid was successfully generated, and the recombinant SjGrpE protein was obtained following the affinity chromatography, which stimulated mice to secrete high-titer antibodies. CONCLUSIONS: The recombinant SjGrpE protein has been successfully prepared and this recombinant protein has a high immunogenicity, which provides a basis for evaluating its value as a vaccine candidate for S. japonicum infections.

Production and characterization of monoclonal antibodies against highly immunogenic Opisthorchis viverrini proteins and development of coproantigen detection.

Opisthorchis viverrini and other foodborne trematode infections are major health concerns in the Greater Mekong Subregion. Currently, the gold-standard diagnostic method for opisthorchiasis is conventional stool examination for the presence of parasite eggs. This method lacks sensitivity and needs an experienced technician. We therefore produced monoclonal antibodies to highly immunogenic O. viverrini proteins aiming at detecting specific antigens in the feces. In this study, BALB/C mice were immunized using semi-purified somatic antigens and spleen cells were fused with a Sp2/0 myeloma cell line. Four hybridomas (1A2, 1E12, 2C7 and 8D6) were selected and cloned due to their strong reaction against O. viverrini somatic protein, resulting in three IgM clones and one IgG2 clone. Immunohistochemistry showed that 1A2, 1E12, 2C7 and 8D6 stained the parenchyma cells, gut, tegument and muscles, respectively. Western-blot analysis revealed that only antibody 1A2 could detect coproantigen (approx. 73 kDa protein) in feces of hamsters infected with O. viverrini. The 1A2 monoclonal antibody may be of value in the diagnosis of opisthorchiasis by coproantigen detection.

Correlation of salivary antibody to carbohydrate larval antigen (CarLA) with health and gastrointestinal nematode parasitism in sheep under Ontario grazing conditions.

Leveraging mucosal immunity is a promising method for controlling gastrointestinal nematode (GIN) parasitism in sheep. Salivary antibody to carbohydrate larval antigen (sCarLA), a heritable measure of immunity to third-stage GIN larvae (L3), has been successfully applied to genetic improvement programs in New Zealand. However, sCarLA levels wane in the absence of ongoing GIN exposure. New Zealand's temperate climate permits year-round exposure to L3, but cold winters in boreal regions such as Ontario, Canada interrupt exposure for five months or more. This study investigated associations between sCarLA levels, GIN parasitism, and indicators of overall health in sheep grazing under Ontario conditions. A commercial flock of 140 Rideau cross ewe lambs were followed from approximately 30 days of age in May 2016 until November 2017, including lambing and lactation in the spring of 2017. Every 6-8 weeks during the grazing season and at mid-gestation in March 2017, fecal egg counts were performed, blood collected to assess serum albumin, globulin, and hematocrit, and pasture samples obtained to confirm exposure to infective larvae. Measurements of sCarLA level were performed at the beginning, middle, and end of each grazing season, and at mid-gestation. Spearman's rank correlation coefficients were calculated to compare sCarLA levels over time, and general linear mixed models created to evaluate associations between sCarLA levels, GIN fecal egg count, hematocrit, serum albumin, and serum globulin. Levels of sCarLA followed a similar seasonal pattern to GIN fecal egg counts with a 6-8 week delay; much higher sCarLA levels were observed in the second grazing season. The proportion of the flock with detectable sCarLA (≥ 0.3 units/mL) was 68.3 % by the end of the first grazing season, declined over winter to 43.9 % at lambing, and approached 100 % after 3 months of grazing in the second grazing season. Correlations between sCarLA levels over time were consistently positive, of weak to moderate strength, and significant (p < 0.05). At all time points, sCarLA level was significantly (p < 0.001) and negatively associated with fecal egg counts. The flock displayed minimal variability in hematocrit, serum albumin, and serum globulin; none of which were significantly associated with sCarLA levels. These results suggest that sCarLA can be maintained over winter and is a useful measure of immunity to GINs in sheep under Ontario grazing conditions.

Donor-derived strongyloidiasis after organ transplantation in Norway.

Strongyloides stercoralis is an intestinal helminth which in humans can cause asymptomatic chronic infection maintained for decades through its auto-infective cycle. During solid organ transplantation, recipients may unintentionally receive an organ infected with strongyloides. This is a very rare complication but may have deadly outcome if not detected. We hereby report two transplant recipients whom developed Strongyloides hyperinfection syndrome after organ transplantation from the same deceased donor. Recipient 1 was kidney transplanted and presented at day 65 post engraftment with diarrhea and subsequent septicemia and gastric retention. Larvae were detected in gastric aspirate. Recipient 2 was simultaneously kidney and pancreas transplanted and presented at day 90 post engraftment also with gastric retention and septicemia. Larvae were demonstrated on duodenal biopsy and stool sample. The clinical course was complicated with severe duodenal bleedings, gastric retention, meningitis, and prolonged hospitalization. Retrospective testing of pre-transplant donor serum was positive for Strongyloides stercoralis antibodies. As a result of disease severity and gastric retention albenazole was administered via a jejunal tube and ivermectin subcutaneously in both recipients. S stercoralis was successfully eradicated and the transplants ended up with unaffected graft function. Following these two cases, we started systematic screening of all deceased donors for serum Strongyloides IgG in October 2016. After having screened 150 utilized donors one tested positive for Strongyloides, which initiated prophylactic ivermectin treatment to organ recipients. No symptoms or disease developed. Our center will continue to screen all donors as prophylactic treatment may avert this potentially lethal complication in cases of donor-derived Strongyloides infection.

Anti-α-Gal antibodies detected by novel neoglycoproteins as a diagnostic tool for Old World cutaneous leishmaniasis caused by Leishmania major.

Outbreaks of Old World cutaneous leishmaniasis (CL) have significantly increased due to the conflicts in the Middle East, with most of the cases occurring in resource-limited areas such as refugee settlements. The standard methods of diagnosis include microscopy and parasite culture, which have several limitations. To address the growing need for a CL diagnostic that can be field applicable, we have identified five candidate neoglycoproteins (NGPs): Galα (NGP3B), Galα(1,3)Galα (NGP17B), Galα(1,3)Galß (NGP9B), Galα(1,6)[Galα(1,2)]Galß (NGP11B), and Galα(1,3)Galß(1,4)Glcß (NGP1B) that are differentially recognized in sera from individuals with Leishmania major infection as compared with sera from heterologous controls. These candidates contain terminal, non-reducing α-galactopyranosyl (α-Gal) residues, which are known potent immunogens to humans. Logistic regression models found that NGP3B retained the best diagnostic potential (area under the curve from receiver-operating characteristic curve = 0.8). Our data add to the growing body of work demonstrating the exploitability of the human anti-α-Gal response in CL diagnosis.

Monoclonal Antibodies Production Against a 40KDa Band of Hydatid Cyst Fluid.

BACKGROUND AND OBJECTIVES: Hydatid cyst is the larval stage of the tapeworm Echinococcus granulosus. Hydatid cyst fluid, cyst membrane and Protoscolices, contain a complex mixture of antigens that can induce immune responses in the host. Anti-cancer properties of Protoscolices and hydatid cyst fluid has been shown. In order to identify antigens of hydatid cyst fluid that have anti-cancer effect, in this study production of monoclonal antibodies against one of the hydatid cyst fluid band (40KDa) has been investigated. There are many published patents about applications of monoclonal antibodies. METHODS: In this experimental study, 40KDa band of hydatid cyst fluid that has cross reaction with sera of patients with breast cancer was used as antigen. A group of mice were immunized with this antigen, and then their spleen cells were extracted and fused with SP2 cells. Monoclonal antibodies production was checked in wells with signs of cell growth using ELISA and western blotting. The reaction of the produced monoclonal antibodies with breast cancer cells was tested using flow cytometry method. Finally, effect of the monoclonal antibodies on growth of breast cancer cells was investigated in vitro. RESULTS: The results of this study showed that in the first plate antibody against 40KDa was detected in several wells. In the second plate monoclonal antibodies with high titer was detected in one well. The produced monoclonal antibodies reacted with the surface of breast cancer cells. However, they had no significant effect on growth of breast cancer cells in culture medium. CONCLUSION: Monoclonal antibodies against hydatid cyst fluid 40KDa band were produced. These antibodies reacted with the surface of breast cancer cells but had no significant effect on growth of these cells.

[Evaluation of ELISA kit for detection of serum specific IgG antibodies against Taenia solium in diagnosis of human cysticercosis].

OBJECTIVE: To evaluate the ELISA kit for detection of IgG antibodies against Taenia solium cysticercus in humans, so as to provide a reference for its application in clinical practice. METHODS: The sera collected from the patients with neurocysticercosis, echinococcosis, taeniasis and healthy people, respectively, were checked by ELISA for specific IgG antibodies against T. solium cysticercus, as described in the instruction of the kit. RESULTS: Of the 30 patients with neurocysticercosis at active stage, 28 showed seropositive reaction, indicating a sensitivity of 93.33%. There were no positive reactions in the 100 healthy people. Two of the 42 persons with taeniasis were seropositive with an infection rate of 4.76%. Forty-one of the 60 persons with echinococcosis showed positive reactions, suggesting that the cross reaction rate of the kit with echinococcosis was 68.33%. CONCLUSIONS: The assessed ELISA kit has a reasonably high sensitivity but a poor specificity, and the cross reaction rate with echinococcosis is very high. It is urgent to improve the sensitivity and specificity of the diagnostic kit for detection of T.solium cysticercosis in China.

Using the local immune response from the natural buffalo host to generate an antibody fragment library that binds the early larval stages of Schistosoma japonicum.

Antibodies isolated from the local draining inguinal lymph node of field exposed-water buffaloes following challenge with Schistosoma japonicum cercariae showed high reactivity towards S. japonicum antigen preparations and bound specifically to formaldehyde-fixed S. japonicum schistosomules. Using this specific local immune response we produced a series of single-chain antibody Fv domain libraries from the same lymph nodes. Removal of phage that cross reacted with epitopes on adult parasites yielded a single-chain antibody Fv domain-phage library that specifically bound to whole formaldehyde-fixed and live S. japonicum schistosomules. DNA sequencing indicated clear enrichment of the single-chain antibody Fv domain library for buffalo B-cell complementarity determining regions post-selection for schistosomule binding. This study also revealed that long heavy chain complementarity determining regions appear to be an important factor when selecting for antibody binding fragments against schistosomule proteins. The selected single-chain antibody Fv domain-phage were used to probe a schistosome-specific protein microarray, which resulted in the recognition of many proteins expressed across all schistosome life-cycle stages. Following absorption to adult worms, the single-chain antibody Fv domain-phage library showed significantly reduced binding to most proteins, whilst two proteins (NCBI GenBank accession numbers AY915878 and AY815196) showed increased binding. We have thus developed a unique set of host derived single-chain antibody Fv domains comprising buffalo B-cell variable regions that specifically bind to early S. japonicum life-stages.

Taenia solium metacestode preparation in rural areas of sub-Saharan Africa: a source for diagnosis and research on cysticercosis.

BACKGROUND: Taenia solium metacestodes/cysts obtained from pig carcasses constitute a primary source for diagnostic tools used for the detection of human cysticercosis. Data on T. solium cyst preparation in Africa is still scarce but required to establish independent reference laboratories. OBJECTIVES: The aim of the present study is a) to present the likely yield of T. solium cyst material by the use of two different preparation methods in the field and b) to investigate its suitability for immunodiagnosis of human cysticercosis. METHODS: In Zambia, Uganda and Tanzania 670 pigs were screened for T. solium infection. Cysts were prepared by 'shaking method' and 'washing method'. Generated crude antigens were applied in a standard western blot assay. RESULTS: 46 out of 670 pigs (6.9%) were found positive for T. solium (Zambia: 12/367, 3.3%; Uganda: 11/217, 5.1%; Tanzania 23/86, 26.7%). Mean values of 77.7 ml whole cysts, 61.8 ml scolices/membranes and 10.9 ml cyst fluid were obtained per pig. Suitability of collected material for the use as crude antigen and molecular diagnostic techniques was demonstrated. CONCLUSION: This study clearly shows that T. solium cyst preparation in African settings by simple field methods constitutes an effective way to obtain high quality material as source for diagnostic tools and research purposes.

A cross-reactive monoclonal antibody to nematode haemoglobin enhances protective immune responses to Nippostrongylus brasiliensis.

BACKGROUND: Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection. METHODOLOGY/PRINCIPAL FINDINGS: Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four -HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens. CONCLUSION: The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.

[Toxocariasis in children and adolescents with allergic and bronchopulmonary diseases, HIV infection, hepatitis B and C risk groups: results of serological screening].

Enzyme immunoassay was used to determine the presence of immunoglobulins class G to Toxocara canis antigens in the sera of children and adolescents (hereinafter referred to as children) with allergic and bronchopulmonary diseases from HIV infection and hepatitis B and C risk groups. A total of 422 dwellers of the Republic of Altai, including 144 subjects aged 1 to 17 years, were examined. Toxocara antibodies were found in 18.8 +/- 3.3% of the children and in 21.9 +/- 2.5% of the adults. The infection rate in children with bronchopulmonary and allergic diseases was 27.1 +/- 5.8 and 14.3 +/- 5.0%, respectively; that in the hepatitis B and C risk groups was 13.1 +/- 6.2%. The children (n = 6) from the HIV infection risk group were seronegative. The infection rate in the adults from the HIV infection and hepatitis risk group was 19.2 +/- 3.5 and 24.3 +/- 3.5%, respectively. Diagnostic antibody titers in the children and adults were determined in 9.0 +/- 2.3 and 8.3 +/- 1.6%, respectively. Immunological assays should be used to rule out toxocariasis in the examinees. If there are seropositive results, specific antiparasitic threatment should be performed.

[Surveillance of schistosome antibodies in population in mountainous schistosomiasis endemic regions in China].

OBJECTIVE: To understand the dynamics of schistosome antibodies in population in mountainous schistosomiasis low endemic regions, China, so as to provide the evaluation reference for immunodiagnosis. METHODS: The people who consecutively received the indirect heamagglutination assay (IHA) for schistosomiasis and Kato-Katz technique were enrolled in this study in Guixiang Village, Danling County, Sichuan Province from 2006 to 2011. The positive rates for schistosome antibodies, antibody titers and eggs per gram (EPG) were analyzed. RESULTS: A total of 353 people were investigated in this study from 2006 to 2011. The positive rates for schistosome antibodies decreased from 20.73% to 13.67%, and the geometric mean reciprocal titer (GMRT) decreased from 1.63 to 1.06. The antibodies tended to be negative in most antibody-positive people and the GMRT decreased gradually. The antibodies remained negative in most antibody-negative people. After chemotherapy in parasitology confirmed patients, the positive rate for schistosome antibodies decreased from 100% to 20%, and the GMRT decreased from 34.29 to 1.58. There was a highly positive correlation between GMRT and EPG (r = 0.94, P < 0.05). CONCLUSION: The positive rate for schistosome antibodies in population in mountainous schistosomiasis low endemic regions is relatively high (13.67%). The standardization of immunodiagnostic methods is necessary and the suitable diagnostic reagents should be developed in this region.

Monoclonal antibody-based dipstick assay: a reliable field applicable technique for diagnosis of Schistosoma mansoni infection using human serum and urine samples.

A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.

Production and characterization of a monoclonal antibody against recombinant saposin-like protein 2 of Fasciola gigantica.

A monoclonal antibody (MoAb) against recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with rFgSAP-2. This MoAb is an IgG1, κ light chain isotype. By immunoblotting and indirect ELISA, the MoAb reacted specifically with rFgSAP-2, the natural FgSAP-2 at 10kDa in whole body (WB) and excretory-secretory (ES) fractions of F. gigantica. It did not cross react with antigens in WB fractions from other parasites, including Opisthorchis viverrini, Schistosoma mansoni which are human parasites, Haemonchus placei, Setaria labiato-papillosa, Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gigantocotyle explanatum, Gastrothylax crumenifer, and Paramphistomum cervi which are ruminant parasites. By immunohistochemistry, the FgSAP-2 protein was localized only in the cytoplasm of caecal epithelial cells of 4-week-old juvenile and adult stages, but not in metacercariae, newly excysted juvenile (NEJ), 2- and 3-week-old juveniles. This finding indicated that FgSAP-2 is an abundantly expressed parasite protein that is released into the ES, hence SAP-2 and its MoAb may be used for immunodiagnosis of ruminant and human fasciolosis.

Strongyloidiasis in a high risk community of Dhaka, Bangladesh.

Residents of a slum community of Dhaka city, Bangladesh were tested by serological and faecal examination for evidence of Strongyloides stercoralis infection. In stool specimens from a total of 147 participants Strongyloides larvae were found in 34 (23.1%) by Harada-Mori culture, 15 (10.2%) by agar plate culture (APC) and one (0.7%) by microscopy. Strongyloides IgG, IgG1 and IgG4 antibodies were found in 90 (61.2%), 46 (31.3%) and 53 (36.1%) of participants, respectively. A positive correlation was observed between total IgG levels and the presence of isotypes IgG1 and IgG4 (p<0.001). Six sera (4.0%) reacted to the recombinant filaria antigen Bm 14, three of which were Strongyloides IgG positive. This indicates either there is cross reactivity or some participants are co-infected with lymphatic filariasis. No correlations were found between positive serology and Strongyloides infection in stool, socio- demographic factors or domestic hygienic practices. However, positive stool cultures showed significant associations with irregular nail trimming, walking bare-foot and irregular hand washing after defecation (p<0.05). Other enteric parasites were detected in stools of some participants but their presence showed no correlation with S. stercoralis infection or socio demographic factors. This study confirms that squatters in this slum community in Dhaka have a high prevalence of S. stercoralis infection identified both by serological and coprological methods.

Mimotope peptides selected from phage display combinatorial library by serum antibodies of pigs experimentally infected with Taenia solium as leads to developing diagnostic antigens for human neurocysticercosis.

Neurocysticercosis is caused by penetration of the tapeworm Taenia solium larvae into the central nervous system resulting in a diverse range of neurologic complications including epilepsy in endemic areas that globalization spreads worldwide. Sensitive and specific immunodiagnosis is needed for the early detection and elimination of the parasite, but the lack of standardized, readily obtainable antigens is a challenge. Here, we used the phage display for resolving the problem. The rationale of the strategy rests on the concept that the screening of combinatorial libraries with polyclonal serum to pathogens reveals families of peptides mimicking the pathogen most immunodominant epitopes indispensable for the successful diagnosis. The screening of a 7mer library with serum IgG of four pigs experimentally infected with parasite followed by computer aided segregation of the selected sequences resulted in the discovery of four clusters of homologous sequences of which one presented a family of ten mimotopes selected by three infected pig serum IgGs; the common motif sequence LSPF carried by the family was considered to be the core of an immunodominant epitope of the parasite critical for the binding with the antibody that selected the mimotopes. The immunoassay testing permitted to select a mimotope whose synthetic peptide free of the phage with the amino acid sequence Leu-Ser-Fen-Pro-Ser-Val-Val that distinguished well a panel of 21 cerebrospinal fluids of neurocysticercosis patients from the fluids of individuals with neurological complications of other etiology. This peptide is proposed as a lead for developing a novel molecularly defined diagnostic antigen(s) for the neurocysticercosis.