A promising new target to control fasciolosis: Fasciola hepatica leucine aminopeptidase 2.

Parasite M17 leucine aminopeptidases (LAPs) have been associated with critical roles in different key functions such as the nutrition, migration, and invasion of the natural host. Native or recombinant LAP used as a vaccine antigen has proved effective to elicit protection against Fasciola hepatica infection in sheep, pointing to potential vaccine candidates against fascioliasis in ruminant species. Previously, the FhLAP1, abundantly secreted in vitro by the mature adult parasite was used as a vaccine antigen obtaining promising protection results against F. hepatica challenge in small ruminants. Here, we report the biochemical characterization of a second recombinant LAP (FhLAP2) associated with the juvenile stage of F. hepatica. FhLAP2 showed aminopeptidase activity using different synthetic substrates, including leucine, arginine, and methionine and was increased in the presence of Mn+ 2 and Mg+ 2. The activity was inhibited by bestatin, 1,10-phenanthroline, and EDTA, specific inhibitors of aminopeptidase and/or metalloproteases. Finally, the recombinant FhLAP2 functional form was tested in combination with Freund's incomplete adjuvant in an immunization trial in mice followed by an experimental challenge with F. hepatica metacercariae. The immunization with FhLAP2/FIA resulted in a significant reduction of parasite recovery compared to control groups. The immunized group elicited total specific IgG and subclasses IgG1 and IgG2 antibody responses. This study highlights the potential of a new candidate vaccine formulation with potential applications in natural ruminant hosts, especially those targeting the juvenile stage.

Protective immune potential of multiple antigenic peptide (MAP) constructs comprising peptides that are shared by several cysteine peptidases against Schistosoma mansoni infection in mice.

In vaccine trials, Schistosoma mansoni cathepsin B1 (SmCB1), helminth cathepsins of the L family (e.g., SmCL3), and papain consistently induce highly significant reductions in challenge worm burden and egg viability, but generated no additive protective effects when used in combination. The protective capacity of the cysteine peptidases is associated with modest (SmCB1) and poor (cathepsins L) production of cytokines and antibodies, essentially of the type 2 axis, and is only marginally reduced upon use of proteolytically inactive enzymes. In this work, peptides shared by SmCB1, cathepsins of the L family, papain and other allergens were selected, synthesized as tetrabranched multiple antigen peptide constructs (MAP-1 and MAP-2), and used in two independent experiments to immunize outbred mice, in parallel with papain. The two peptides elicited significant (P < 0.05) reduction in challenge worm burden when compared to unimmunized mice, albeit lower than that achieved by papain. Protection was associated with modest serum type 2 cytokines and antibody levels in MAP-, and papain-immunized mice. Immunization with papain also elicited a reduction in parasite egg load, viability, and granuloma numbers in liver and intestine. MAP-1 and MAP-2 immunogens displayed some opposite effects- MAP-1 leading to higher egg numbers with poor vitality, whereas MAP-2 immunization yielded fewer eggs. Cysteine peptidase thus appear to carry peptides that elicit opposing outcomes, highlighting the difficulty of reaching fully fledged protection, unless a vaccine is based on carefully selected peptides and combined with an effective adjuvant.

Evaluation of the therapeutic effect of propolis on Fasciola gigantica and Clostridium novyi type B infections in sheep.

The use of natural products for disease control is a promising approach to solving the problem of drug resistance. The aim of the research reported here was to evaluate the fasciolicidal and anti-Clostridium novyi type B activities of propolis administered orally to sheep infected with Fasciola gigantica and C. novyi type B. Sheep infected with both pathogens were divided into two groups: an infected treated group and an infected non-treated group. The treatment was oral administration of 50 mg propolis extract/kg daily for 15 days. The body weight of the sheep, fecal egg counts of F. gigantica, serum levels of F. gigantica IgG, concentrations of cytokines (IL-2, IL-10, and IL-17), and bacterial counts of C. novyi were evaluated. Following treatment, the sheep had increased body weight and a significant decrease in the egg count, which was reduced by 54.54% at 15 days post treatment. The level of anti- Fasciola IgG increased, whereas levels of IL-2, IL-10, and IL-17 decreased in propolistreated sheep. Treatment of sheep with propolis produced a significant reduction in fecal count of C. novyi, from 8 × 109 to 3 × 103 colony units per gram at 15 days post treatment. This research highlights the therapeutic potential of Egyptian propolis extract as a treatment against F. gigantica and C. novyi type B infections, and investigated its mode of action through its effect on some cellular and humoral responses in sheep with both infections.

Saccharomyces cerevisiae (YT001) supplementation for the control of Haemonchus contortus and modulation of the immune response of sheep.

Studies aiming at the development and evaluation of alternative methods to minimise losses caused by the gastrointestinal nematode Haemonchus contortus are extremely important. Such research is essential, given the high morbidity rates among sheep and the significant mortality rates of lambs, allied to the low efficacy of commercial products for the control of this parasite. The purpose of this study was to evaluate the effect of the Saccharomyces cerevisiae (YT001 - YEASTECH) on the control of H. contortus and its modulation of the immune response in experimentally infected sheep. Eighteen sheep were divided into two groups. Group 1, the control group, comprised animals infected with H. contortus and supplemented with distilled water, while Group 2, the treated group, consisted of animals infected and supplemented with S. cerevisiae (400 million cfu/day of suspension for 49 days). The following parasitological parameters were evaluated: number of eggs per gram of faeces, number of infective larvae (L3) recovered per faecal culture, and parasitic load of the abomasum. The following immunological parameters were quantified: immunoglobulin (Ig)A in the mucous secretions and serum IgG; cytokines interleukin (IL)-4, IL-5 and IL-10; number of eosinophils in the abomasal mucosa and groups of cells positive for the markers: MHCII, CD4+CD25+, CD5+CD8+, WC4, CD5+CD4+, CD8+CD11b+ and CD5+WC1 by whole blood flow cytometry. The results revealed a significant decrease (P<0.05) in the number of larvae and significantly higher serum IgG levels (P<0.05) in the group supplemented with S. cerevisiae. The supplemented animals showed significantly larger numbers of eosinophils (P<0.05), as well as more cells positive for MHCII, CD4+CD25+, CD5+CD8+ than the control animals. This study confirmed the beneficial action of S. cerevisiae on the host immune response to H. contortus, as evidenced mainly by the smaller number of L3 recovered from the faeces of sheep supplemented with S. cerevisiae.

Process Development of Sj-p80: A Low-Cost Transmission-Blocking Veterinary Vaccine for Asiatic Schistosomiasis.

Asiatic schistosomiasis caused by Schistosoma japonicum is a neglected tropical disease resulting in significant morbidity to both humans and animals - particularly bovines - in endemic areas. Infection with this parasite leads to less healthy herds, causing problems in communities which rely on bovines for farming, milk and meat production. Additionally, excretion of parasite eggs in feces perpetuates the life cycle and can lead to human infection. We endeavored to develop a minimally purified, inexpensive, and effective vaccine based on the 80 kDa large subunit of the calcium activated neutral protease (calpain) from S. japonicum (Sj-p80). Here we describe the production of veterinary vaccine-grade Sj-p80 at four levels of purity and demonstrate in a pilot study that minimally purified antigen provides protection against infection in mice when paired with a low-cost veterinary adjuvant, Montanide™ ISA61 VG. Preliminary data demonstrate that the vaccine is immunogenic with robust antibody titers following immunization, and vaccination resulted in a reduction of parasite eggs being deposited in the liver (23.4-51.4%) and intestines (1.9-55.1%) depending on antigen purity as well as reducing the ability of these eggs to hatch into miracidia by up to 31.6%. We therefore present Sj-p80 as a candidate vaccine antigen for Asiatic schistosomiasis which is now primed for continued development and testing in bovines in endemic areas. A successful bovine vaccine could play a major role in reducing pathogen transmission to humans by interrupting the parasitic life cycle and improving quality of life for people living in endemic countries.

Immunogenicity, antibody responses and vaccine efficacy of recombinant annexin B30 against Schistosoma mansoni.

AIMS: Schistosomes infect approximately 250 million people worldwide. To date, there is no effective vaccine available for the prevention of schistosome infection in endemic regions. There remains a need to develop means to confer long-term protection of individuals against reinfection. In this study, an annexin, namely annexin B30, which is highly expressed in the tegument of Schistosoma mansoni was selected to evaluate its immunogenicity and protective efficacy in a mouse model. METHODS AND RESULTS: Bioinformatics analysis showed that there were three potential linear B-cell epitopes and four conformational B-cell epitopes predicted from annexin B30, respectively. Full-length annexin B30 was cloned and expressed in Escherichia coli BL21(DE3). In the presence of adjuvants, the soluble recombinant protein was evaluated for its protective efficacy in two independent vaccine trials. Immunization of CBA mice with recombinant annexin B30 formulated either in alum only or alum/CpG induced a mixed Th1/Th2 cytokine profile but no significant protection against schistosome infection was detected. CONCLUSION: Recombinant annexin B30 did not confer significant protection against the parasite. The molecule may not be suitable for vaccine development. However, it could be an ideal biomarker recommended for immunodiagnostics development.

Detection of Echinococcus granulosus sensu lato infection by using extracts derived from a protoscoleces G1 cell line.

Cystic echinococcosis (CE) can be diagnosed by means of several serological approaches, but their results vary among laboratories due to the molecular characteristics of the reference antigens used. Thus, this study aimed to address both the relevance of an EGPE cell line previously obtained from Echinococcus granulosus protoscoleces G1 and the complexity of the immune response by using two different in vitro growth stages as separate sources of parasite antigens. The serum reactivity was investigated by western blotting (WB) in 21 CE patients from an endemic area in a matched case-control design and also in seven experimentally infected sheep and five healthy control sheep. EGPE-antigen-human serum sensitivity by WB was higher than that of hydatid fluid (HF) WB, ELISA and DD5 (P < .05, Chi-square test). EGPE protein extract was immunogenic in mice and hyperimmune plasma reacted with HF proteins, and AgB2 expression was detected by molecular analysis. Proteins of 37 to 60 kDa were recognized by 95.24% of the CE patients' sera but, with poor specificity. Statistically significant differences were found between serum protein extract recognition at 7 and 20 days of cell growth. The EGPE cell line is a laboratory source of antigens for improvement of CE serological diagnosis.

Moderate protection is induced by a chimeric protein composed of leucine aminopeptidase and cathepsin L1 against Fasciola hepatica challenge in sheep.

Leucine aminopeptidase (FhLAP) and cathepsin L1 (FhCL1) of Fasciola hepatica play a critical role in parasite feeding, migration through host tissue, and immune evasion. These antigens have been tested for immune protection as single components with variable degrees of success. The chimeric-protein approach could improve protection levels against fasciolosis. Previously, we reported the design and construction of a chimeric protein composed of antigenic sequences of FhLAP and FhCL1 of F. hepatica. The goal of the present study was to express and evaluate the immune-protective capacity of this chimeric protein (rFhLAP-CL1) in sheep. Animals were randomly allocated into five groups with five animals in each group. Groups 1, 2 and 3 were immunized twice with 100 µg, 200 µg and 400 µg of rFhLAP-CL1 emulsified with Quil A adjuvant, whereas groups 4 and 5 were the adjuvant control and infection control groups, respectively. The animals were then challenged with 200 metacercariae two weeks after the rFhLAP-CL1 booster. The fluke burden was reduced by 25.5%, 30.7% (p < 0.05) and 46.5% (p < 0.01) in sheep immunized with 100 µg, 200 µg and 400 µg of chimeric protein, respectively, in comparison to the infection control group. There was a reduction of 22.7% (p < 0.05) and 24.4% (p < 0.01) in fecal egg count in groups 2 and 3, respectively, compared to the infection control group. Sheep immunized with chimeric protein produced F. hepatica excretion-secretion product-specific total IgG antibody, which were increased after challenge. Moreover, the levels of rFhLAP-CL1-specific IgG1 and IgG2 isotypes in immunized sheep increased rapidly two weeks after the first immunization and were significantly more elevated than those of the control groups, indicating a mixed Th1/Th2 response. This is a preliminary evaluation of the chimeric protein rFhLAP-CL1 as a possible immunogen against F. hepatica infection in sheep.

IgE antibodies from schistosomiasis patients to recognize epitopes in potato apyrase.

INTRODUCTION: High percentages of structural identity and cross-immunoreactivity have been reported between potato apyrase and Schistosoma mansoni ATP diphosphohydrolase (SmATPDases) isoforms, showing the existence of particular epitopes shared between these proteins. METHODS: Potato apyrase was employed using ELISA, western blot, and mouse immunization methods to verify IgE reactivity. RESULTS: Most of the schistosomiasis patient's (75%) serum was seropositive for potato apyrase and this protein was recognized using western blotting, suggesting that parasite and plant proteins share IgE-binding epitopes. C57BL/6 mice immunized with potato apyrase showed increased IgE antibody production. CONCLUSIONS: Potato apyrase and SmATPDases have IgE-binding epitopes.

Immunodiagnostic potential of Wuchereria bancrofti L1 antigen-based filarial immunoglobulin G4 detection assay.

Background: After mass drug administration to eliminate human lymphatic filariasis, there is a need for surveillance to detect the measurable endpoint of the program. Methods: An immunodominant seroreactive clone, WbL1, was identified through immunoscreening of a Wuchereria bancrofti L3 complementary DNA expression library. Recombinant WbL1 (rWbL1) was analysed with sera from W. bancrofti patients. Diagnostic evaluation was carried out by developing an enzyme-linked immunosorbent assay (ELISA) to detect the filarial-specific antibodies in various categories of filarial sera samples against recombinant WbL1 (rWbL1) protein. Results: Performance parameters of the test in terms of immunoglobulin G (IgG) and IgG4 detection displayed significant sensitivity and specificity values up to 77% and 100%, respectively. Our results showed filarial antibodies against rWbL1 to be highly reactive with microfilaremic and clinical filarial sera samples compared with the endemic and non-endemic control sera samples. Reasonably satisfactory performance of the test was also confirmed from the multicentric evaluation of an anti-WbL1 IgG4 detection ELISA. This test was found to be minimally reactive with other nematode parasites and protozoan infections. Conclusions: The anti-WbL1 IgG4 detection test can be considered as a field test for initial screening and epidemiological monitoring of filarial infections in filariasis-endemic areas.

Comparison of the Impact of Annual and Semiannual Mass Drug Administration on Lymphatic Filariasis Prevalence in Flores Island, Indonesia.

We compared the impact of annual and semiannual mass drug administration (MDA) on the prevalence of Brugia timori and Wuchereria bancrofti in Flores Island. Two villages (Paga, B. timori only; Lewomada, co-endemic) received annual MDA with diethylcarbamazine/albendazole and a larger village (Pruda, co-endemic) received semiannual MDA. Infection parameters (microfilariae [Mf], antibodies to recombinant filarial antigen BmR1 [Brugia Rapid (BR)], and a test for W. bancrofti antigenemia [immunochromatographic test (ICT)]) were assessed before and after treatment. The crude Mf prevalence in Pruda decreased after five semiannual treatments from 14.2% to 1.2%, whereas the Mf prevalence in the other two villages decreased after three annual treatments from 3.9% to 0% and from 5% to 0.3%, respectively. ICT positivity prevalence in Pruda and Lewomada decreased from 22.9% and 6.5% to 7% and 0.8%, respectively, whereas BR antibody prevalence in Pruda, Lewomada, and Paga decreased from 28.9%, 31.7%, and 12.5% to 3.6%, 4.1%, and 1.8%, respectively. Logistic regression analysis indicated that that Mf, BR, and ICT prevalence decreased significantly over time and that for the Mf and ICT outcomes the semiannual treatment had higher odds of positivity. Model-adjusted prevalence estimates revealed that apparent differences in treatment effectiveness were driven by differences in baseline prevalence and that adjusted prevalence declined more rapidly in the semiannual treatment group. We conclude that in this setting, annual MDA was sufficient to reduce Mf prevalence to less than 1% in areas with low to moderate baseline prevalence. Semiannual MDA was useful for rapidly reducing Mf prevalence in an area with higher baseline endemicity.

IgE antibodies from schistosomiasis patients to recognize epitopes in potato apyrase

Abstract INTRODUCTION: High percentages of structural identity and cross-immunoreactivity have been reported between potato apyrase and Schistosoma mansoni ATP diphosphohydrolase (SmATPDases) isoforms, showing the existence of particular epitopes shared between these proteins. METHODS: Potato apyrase was employed using ELISA, western blot, and mouse immunization methods to verify IgE reactivity. RESULTS: Most of the schistosomiasis patient's (75%) serum was seropositive for potato apyrase and this protein was recognized using western blotting, suggesting that parasite and plant proteins share IgE-binding epitopes. C57BL/6 mice immunized with potato apyrase showed increased IgE antibody production. CONCLUSIONS: Potato apyrase and SmATPDases have IgE-binding epitopes.

Trials with the Haemonchus vaccine, Barbervax®, in ewes and lambs in a tropical environment: Nutrient supplementation improves protection in periparturient ewes.

Haemonchus contortus is an economic problem in sheep farms worldwide, mainly in the tropics and subtropics. A vaccine against haemonchosis, called Barbervax®, was evaluated in ewes under two nutritional status, naturally infected with gastrointestinal nematodes. Ewes were divided into four groups: Supplemented Diet - Vaccine; Supplemented Diet - No vaccine; Basal Diet - Vaccine and Basal Diet - No vaccine. Their lambs were divided in Vaccinated and No vaccine. Ewes were immunised six times starting about 1 month of pregnancy with the first three doses at 3 week intervals and the last three shots at 4 week intervals. Supplemented ewes had higher body weight, body score and packed cell volume compared with those fed a basal diet. Both groups of vaccinated ewes showed a similar response in circulating anti-vaccine antibodies but the vaccine had no discernible effect on either body weight, body score and packed cell volume. There was a marked group difference in the number of ewes that received precautionary treatments with anthelmintic. All 14 Basal Diet - No vaccine ewes required treatment. In contrast only 7 ewes, in the Supplemented Diet - Vaccine group required anthelmintic treatment. In the Basal Diet - Vaccine and in the Supplemented Diet - No Vaccine groups, 12 and 13 ewes needed anthelmintic treatment, respectively. Vaccinated lambs showed much higher antibody titres resulting in 80% less Haemonchus spp. egg counts comparing with no vaccine lambs. Taken together these results clearly suggest that in pregnant and lactating ewes a combined protective effect between vaccination and improved nutrition resulted in fewer precautionary anthelmintic treatments. Thus, it was possible to achieve a more sustainable level of control of the haemonchosis, less dependent on anthelmintic drugs.

Ligand binding properties of two Brugia malayi fatty acid and retinol (FAR) binding proteins and their vaccine efficacies against challenge infection in gerbils.

Parasitic nematodes produce an unusual class of fatty acid and retinol (FAR)-binding proteins that may scavenge host fatty acids and retinoids. Two FARs from Brugia malayi (Bm-FAR-1 and Bm-FAR-2) were expressed as recombinant proteins, and their ligand binding, structural characteristics, and immunogenicities examined. Circular dichroism showed that rBm-FAR-1 and rBm-FAR-2 are similarly rich in α-helix structure. Unexpectedly, however, their lipid binding activities were found to be readily differentiated. Both FARs bound retinol and cis-parinaric acid similarly, but, while rBm-FAR-1 induced a dramatic increase in fluorescence emission and blue shift in peak emission by the fluorophore-tagged fatty acid (dansyl-undecanoic acid), rBm-FAR-2 did not. Recombinant forms of the related proteins from Onchocerca volvulus, rOv-FAR-1 and rOv-FAR-2, were found to be similarly distinguishable. This is the first FAR-2 protein from parasitic nematodes that is being characterized. The relative protein abundance of Bm-FAR-1 was higher than Bm-FAR-2 in the lysates of different developmental stages of B. malayi. Both FAR proteins were targets of strong IgG1, IgG3 and IgE antibody in infected individuals and individuals who were classified as endemic normal or putatively immune. In a B. malayi infection model in gerbils, immunization with rBm-FAR-1 and rBm-FAR-2 formulated in a water-in-oil-emulsion (®Montanide-720) or alum elicited high titers of antigen-specific IgG, but only gerbils immunized with rBm-FAR-1 formulated with the former produced a statistically significant reduction in adult worms (68%) following challenge with B. malayi infective larvae. These results suggest that FAR proteins may play important roles in the survival of filarial nematodes in the host, and represent potential candidates for vaccine development against lymphatic filariasis and related filarial infections.

Prevalence of Atopy following Mass Drug Administration with Albendazole: A Study in School Children on Flores Island, Indonesia.

BACKGROUND: In many rural areas of tropical countries such as Indonesia, the prevalence of soil-transmitted helminths (STH) infections remains high. At the same time, the burden of allergic disorders in such rural areas is reported to be low and inversely associated with helminth infections. To reduce the morbidity and transmission of helminth infections, the world health organization recommends preventive treatment of school children by providing mass drug administration (MDA) with albendazole. Here, we had an opportunity to evaluate the prevalence of skin reactivity to allergens before and after albendazole treatment to get an indication of the possible impact of MDA on allergic sensitization. METHODS: A study was conducted among 150 school children living in an area endemic for STH infections. Before and 1 year after anthelminthic treatment with albendazole, stool samples were examined for the presence of STH eggs, skin prick tests (SPT) for cockroach and house dust mites were performed, blood eosinophilia was assessed, and total immunoglobulin E (IgE) and C-reactive protein (CRP) were measured in plasma. RESULTS: Anthelminthic treatment significantly reduced the prevalence of STH from 19.6 before treatment to 6% after treatment (p < 0.001). Levels of total IgE (estimate: 0.30; 95% CI 0.22-0.42, p < 0.0001), CRP (estimate: 0.60; 95% CI 0.42-0.86, p = 0.006), and eosinophil counts (estimate: 0.70; 95% CI 0.61-0.80, p < 0.001) decreased significantly. The prevalence of SPT positivity increased from 18.7 to 32.7%. Multivariate analysis adjusted for confounding factors showed an increased risk of being SPT positive to any allergen (OR 3.04; 95% CI 1.338-6.919, p = 0.008). CONCLUSIONS: This study indicates that 1 year of MDA with albendazole was associated with a reduced prevalence of STH infections. This study shows that the prevalence of allergic sensitization increases after 1 year of albendazole treatment. Placebo-controlled and larger studies are needed to further substantiate a role of deworming treatment in an increased risk of allergic sensitization.

Antigenic cross-reactivity between Schistosoma mansoni and pollen allergens from the birch tree (Betula verrucosa) and Timothy grass (Phleum pratense): involvement of shared glycan epitopes and implications for the hygiene hypothesis.

Previous studies have shown that schistosome infection can protect against allergic symptoms, but the underlying mechanisms are still not fully understood. Here we have shown that rabbit IgG antibodies raised against Schistosoma mansoni soluble egg antigens (SmSEA) are cross-reactive with a wide array of molecules in Timothy grass pollen (TGP) and birch tree pollen (BTP). Five of the cross-reactive pollen molecules (two from TGP and three from BTP) were selected randomly and identified by tandem mass spectrometric (TMS) analysis to be, respectively, the TGP allergens Phl p 1 and Phl p 5b, and BTP glutathione S-transferase (GST), and the BTP allergens Bet v 1 and Bet v 6.0102. Rabbit anti-SmSEA IgG antibodies that cross-reacted with each of the five allergens were found to be reactive with three major S. mansoni egg antigens, IPSE/alpha-1, omega-1 and kappa-5. Pairwise alignment of the amino acid sequences of each of the five TMS-identified pollen allergens with each of the three egg antigens revealed a low level of amino acid sequence identity. Further experiments indicated that the schistosome antigen/allergen cross-reactivity was mostly due to similar glycans present in helminths and plants, but not in mammals: so called cross-reactive carbohydrate determinants (CCDs). Previously, CCDs have been implicated in the cross-reactivity between many plants and invertebrates. Furthermore, pollen-induced anti-CCD IgGs have been found in sera of patients undergoing allergen-specific immunotherapy (SIT) and implicated in the treatment of the allergy. Thus, our finding provides not only possible explanations for the allergy-protective effect of helminth/schistosome infections as explained by the hygiene hypothesis, but also a potential starting point for improved SIT.

Rational selection of immunodominant and preserved epitope Sm043300e from Schistosoma mansoni and design of a chimeric molecule for biotechnological purposes.

Human schistosomiasis is a neglected tropical disease of great importance in public health. A large number of people are infected with schistosomiasis, making vaccine development and effective diagnosis important control strategies. A rational epitope prediction workflow using Schistosoma mansoni hypothetical proteins was previously presented by our group, and an improvement to that approach is presented here. Briefly, immunodominant epitopes from parasite membrane proteins were predicted by reverse vaccinology strategy with additional in silico analysis. Furthermore, epitope recognition was evaluated using sera of individuals infected with S. mansoni. The epitope that stood out in both in silico and in vitro assays was used to compose a rational chimeric molecule to improve immune response activation. Out of 2185 transmembrane proteins, four epitopes with high binding affinities for human and mouse MHCII molecules were selected through computational screening. These epitopes were synthesized to evaluate their ability to induce TCD4+ lymphocyte proliferation in mice. Sm204830e and Sm043300e induced significant TCD4+ proliferation. Both epitopes were submitted to enzyme-linked immunosorbent assay to evaluate their recognition by IgG antibodies from the sera of infected individuals, and epitope Sm043300 was significantly recognized in most sera samples. Epitope Sm043300 also showed good affinity for human MHCII molecules in molecular docking, and its sequence is curiously highly conserved in four S. mansoni proteins, all of which are described as G-protein-coupled receptors. In addition, we have demonstrated the feasibility of incorporating this epitope, which showed low similarity to human sequences, into a chimeric molecule. The stability of the molecule was evaluated by molecular modeling aimed at future molecule production for use in diagnosis and vaccination trials.

Impact of heat treatment on antigen detection in sera of Angiostrongylus vasorum infected dogs.

BACKGROUND: In the last decade serological tests for detection of circulating Angiostrongylus vasorum antigen and specific antibodies have been developed and adopted for individual diagnosis and epidemiological studies in dogs. Although confirmed positive at necropsy, antigen detection was not possible in single experimentally, as well as naturally infected dogs, possibly due to immune complex formation. The aim of this study was to evaluate the effect of heat treatment on detection of A. vasorum antigen in sera of experimentally (n = 21, 119 follow-up sera) and naturally (n = 18) infected animals. In addition, sera of dogs showing clinical signs consistent with angiostrongylosis (n = 10), of randomly selected dogs (n = 58) and of dogs with other parasitic infections (n = 15) were evaluated. Sera were subjected to heat treatment at 100 °C after addition of 0.5 M EDTA (dilution 1:5) and tested with ELISAs for detection of circulating A. vasorum antigen before and after treatment. RESULTS: Between 5 and 11 weeks post-inoculation (wpi) the percentage of positive untreated samples (experimentally infected dogs) increased over time from 33.3 to 90%. Single samples were still negative between 12 and 15 wpi. Overall, between 5 and 15 wpi, 50.6% (45/89) of the available samples were seropositive. From 3 to 6 wpi EDTA/heat treatment caused a change in 8/34 (23.5%) of the samples, with most (n = 6, 17.6%) converting from positive to negative. In contrast, from 7 to 10 wpi, treatment induced a change in 19/52 (36.5%) samples, with all but one converting from negative to positive. Thirteen of 18 naturally infected dogs were antigen positive before and 15 after EDTA/heat treatment, respectively. Untreated samples of 3 dogs with suspected angiostrongylosis were antigen positive, of which only one remained positive after EDTA/heat treatment. One of 58 untreated random samples was antigen positive; this sample became negative after treatment, while another turned positive. One of 15 dogs infected with other parasites than A. vasorum was positive before but negative after treatment. CONCLUSION: Although heat treatment improves A. vasorum antigen detection between 7 and 10 wpi by immune complex disruption, we do not recommend systematic pretreating sera because of reduced antigen detection between 3 and 6 wpi and impairment of antibody detection, if performed contemporaneously.

Dietary cinnamaldehyde enhances acquisition of specific antibodies following helminth infection in pigs.

Dietary phytonutrients such as cinnamaldehyde (CA) may contribute to immune function during pathogen infections, and CA has been reported to have positive effects on gut health when used as feed additive for livestock. Here, we investigated whether CA could enhance antibody production and specific immune responses during infection with an enteric pathogen. We examined the effect of dietary CA on plasma antibody levels in parasite-naïve pigs, and subsequently acquisition of humoral immune responses during infection with the parasitic nematode Ascaris suum. Parasite-naïve pigs fed diets supplemented with CA had higher levels of total IgA and IgG in plasma, and A. suum-infected pigs fed CA had higher levels of parasite-specific IgM and IgA in plasma 14days post-infection. Moreover, dietary CA increased expression of genes encoding the B-cell marker CD19, sodium/glucose co-transporter1 (SCA5L1) and glucose transporter 2 (SLC2A2) in the jejunal mucosa of A.suum-infected pigs. Dietary CA induced only limited changes in the composition of the prokaryotic gut microbiota of A. suum-infected pigs, and in vitro experiments showed that CA did not directly induce proliferation or increase secretion of IgG and IgA from lymphocytes. Our results demonstrate that dietary CA can significantly enhance acquisition of specific immune responses in pigs. The underlying mechanism remains obscure, but apparently does not derive simply from direct contact between CA and host lymphocytes and appears to be independent of the gut microbiota.

Transcriptome profiling of gene expression during immunisation trial against Fasciola hepatica: identification of genes and pathways involved in conferring immunoprotection in a murine model.

BACKGROUND: Fasciolosis remains a significant food-borne trematode disease causing high morbidity around the world and affecting grazing animals and humans. A deeper understanding concerning the molecular mechanisms by which Fasciola hepatica infection occurs, as well as the molecular basis involved in acquiring protection is extremely important when designing and selecting new vaccine candidates. The present study provides a first report of microarray-based technology for describing changes in the splenic gene expression profile for mice immunised with a highly effective, protection-inducing, multi-epitope, subunit-based, chemically-synthesised vaccine candidate against F. hepatica. METHODS: The mice were immunised with synthetic peptides containing B- and T-cell epitopes, which are derived from F. hepatica cathepsin B and amoebapore proteins, as novel vaccine candidates against F. hepatica formulated in an adjuvant adaptation vaccination system; they were experimentally challenged with F. hepatica metacercariae. Spleen RNA from mice immunised with the highest protection-inducing synthetic peptides was isolated, amplified and labelled using Affymetrix standardised protocols. Data was then background corrected, normalised and the expression signal was calculated. The Ingenuity Pathway Analysis tool was then used for analysing differentially expressed gene identifiers for annotating bio-functions and constructing and visualising molecular interaction networks. RESULTS: Mice immunised with a combination of three peptides containing T-cell epitopes induced high protection against experimental challenge according to survival rates and hepatic damage scores. It also induced differential expression of 820 genes, 168 genes being up-regulated and 652 genes being down-regulated, p value <0.05, fold change ranging from -2.944 to 7.632. A functional study of these genes revealed changes in the pathways related to nitric oxide and reactive oxygen species production, Interleukin-12 signalling and production in macrophages and Interleukin-8 signalling with up-regulation of S100 calcium-binding protein A8, Matrix metallopeptidase 9 and CXC chemokine receptor 2 genes. CONCLUSION: The data obtained in the present study provided us with a more comprehensive overview concerning the possible molecular pathways implied in inducing protection against F. hepatica in a murine model, which could be useful for evaluating future vaccine candidates.