Dataset Information

Identification of linear epitopes in SjSP-13 of Schistosoma japonicum using a GST-peptide fusion protein microplate array.

ABSTRACT:

Background

The identification and characterization of epitopes facilitate the discovery and development of new therapeutics, vaccines and diagnostics for infectious diseases. In this study, we developed a glutathione S-transferase (GST)-peptide fusion protein microplate array for the identification of linear B-cell epitopes and applied this novel method to the identification of linear B-cell epitopes of SjSP-13, an immunodiagnostic biomarker of schistosomiasis japonica.

Methods

SjSP-13 was divided into 17 overlapped peptides (p1-17), and the coding sequence of each peptide was obtained by annealing two complementary oligonucleotides. SjSP-13 peptides were expressed by fusion with an N-terminal GST tag and a C-terminal 6xHis tag. The GST-peptide-His fusion protein was specifically bound to the Immobilizer Glutathione MicroWell 96-well plates without purification. SjSP-13 peptides and core epitopes that could be recognized by sera from schistosomiasis patients were identified by ELISA and confirmed by Western blot analysis. The receiver operating characteristic (ROC) analysis was performed to determine the diagnostic validity of the identified peptide.

Results

Full-length GST-peptide-His fusion proteins were successfully expressed and specifically bound to the Immobilizer Glutathione MicroWell 96-well plates. Two adjacent peptides (p7 and p8) were found to be highly immunogenic in humans. The core epitope of p7 and p8 is an 11-aa peptide (₈₀KCLDVTDNLPE₉₀) and an 8-aa peptide (₉₀EKIIQFAE₉₇), respectively. The area under the ROC curve (AUC) value of the peptide which contains the two identified epitopes is 0.947?±?0.019. The diagnostic sensitivity and specificity of the peptide is 76.7% (95% CI: 68.8-84.5%) and 100%, respectively.

Conclusions

₉₀EKIIQFAE₉₇ and ₈₀KCLDVTDNLPE₉₀ are the two linear epitopes of SjSP-13 recognized by patient sera, and could be potential serological markers for schistosomiasis japonica.

SUBMITTER: Ma L

PROVIDER: S-EPMC6822365 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification of linear epitopes in SjSP-13 of Schistosoma japonicum using a GST-peptide fusion protein microplate array.

Ma Li L Zhao Wenrong W Hou Xunya X Liu Mengmeng M Li Yanna Y Shen Li L Xu Xindong X

Parasites & vectors 20191030 1

<h4>Background</h4>The identification and characterization of epitopes facilitate the discovery and development of new therapeutics, vaccines and diagnostics for infectious diseases. In this study, we developed a glutathione S-transferase (GST)-peptide fusion protein microplate array for the identification of linear B-cell epitopes and applied this novel method to the identification of linear B-cell epitopes of SjSP-13, an immunodiagnostic biomarker of schistosomiasis japonica.<h4>Methods</h4>Sj ...[more]

PMID: 31666115

Similar Datasets

Project description:IntroductionImmunoinformatic tools can be used to predict schistosome-specific B-cell epitopes with little sequence identity to human proteins and antigens other than the target. This study reports an approach for identifying schistosome peptides mimicking linear B-cell epitopes using in-silico tools and peptide microarray immunoassay validation.MethodFirstly, a comprehensive literature search was conducted to obtain published schistosome-specific peptides and recombinant proteins with the best overall diagnostic performances. For novel peptides, linear B-cell epitopes were predicted from target recombinant proteins using ABCpred, Bcepred and BepiPred 2.0 in-silico tools. Together with the published peptides, predicted peptides with the highest probability of being B-cell epitopes and the lowest sequence identity with proteins from human and other pathogens were selected. Antibodies against the peptides were measured in sera, using peptide microarray immunoassays. Area under the ROC curve was calculated to assess the overall diagnostic performances of the peptides.ResultsPeptide AA81008-19-30 had excellent and acceptable diagnostic performances for discriminating S. mansoni and S. haematobium positives from healthy controls, with AUC values of 0.8043 and 0.7326 respectively for IgG. Peptides MS3_10186-123-131, MS3_10385-339-354, SmSPI-177-193, SmSPI-379-388, MS3-10186-40-49 and SmS-197-214 had acceptable diagnostic performances for discriminating S. mansoni positives from healthy controls with AUC values ranging from 0.7098 to 0.7763 for IgG. Peptides SmSPI-359-372, Smp126160-438-452 and MS3 10186-25-41 had acceptable diagnostic performances for discriminating S. mansoni positives from S. mansoni negatives with AUC values of 0.7124, 0.7156 and 0.7115 respectively for IgG. Peptide MS3-10186-40-49 had an acceptable diagnostic performance for discriminating S. mansoni positives from healthy controls, with an AUC value of 0.7413 for IgM.ConclusionOne peptide with a good diagnostic performance and nine peptides with acceptable diagnostic performances were identified using the immunoinformatic approach and peptide microarray validation. There is need for evaluation of the peptides with true negatives and a good standard positive reference.

Project description:BACKGROUND:The development of a schistosome vaccine has proved challenging but we have suggested that characterisation of the self-cure mechanism in rhesus macaques might provide a route to an effective product. The schistosome esophagus is a complex structure where blood processing is initiated by secretions from anterior and posterior glands, achieved by a mixture of ~40 unique proteins. The mechanism of self-cure in macaques involves cessation of feeding, after which worms slowly starve to death. Antibody coats the esophagus lumen and disrupts the secretory processes from the glands, potentially making their secretions ideal vaccine targets. METHODOLOGY/PRINCIPAL FINDINGS:We have designed three peptide arrays comprising overlapping 15-mer peptides encompassing 32 esophageal gland proteins, and screened them for reactivity against 22-week infection serum from macaques versus permissive rabbit and mouse hosts. There was considerable intra- and inter-species variation in response and no obvious unique target was associated with self-cure status, which suggests that self-cure is achieved by antibodies reacting with multiple targets. Some immuno-dominant sequences/regions were evident across species, notably including: MEGs 4.1C, 4.2, and 11 (Array 1); MEG-12 and Aspartyl protease (Array 2); a Tetraspanin 1 loop and MEG-n2 (Array 3). Responses to MEGs 8.1C and 8.2C were largely confined to macaques. As proof of principle, three synthetic genes were designed, comprising several key targets from each array. One of these was expressed as a recombinant protein and used to vaccinate rabbits. Higher antibody titres were obtained to the majority of reactive regions than those elicited after prolonged infection. CONCLUSIONS/SIGNIFICANCE:It is feasible to test simultaneously the additive potential of multiple esophageal proteins to induce protection by combining their most reactive regions in artificial constructs that can be used to vaccinate suitable hosts. The efficacy of the approach to disrupt esophageal function now needs to be tested by a parasite challenge.

Project description:BackgroundPrevious studies have shown that insulin receptors in schistosomes, triggered by host insulin, play an important role in parasite growth, development and fecundity by regulating glucose metabolism. However, limited information is available on the recently identified endogenous insulin-like peptide (ILP) in blood flukes.ResultsWe isolated ILPs from Schistosoma japonicum (SjILP) and S. recognised (SmILP) and present results of their molecular and structural analysis. SjILP shares 63% amino acid identity with SmILP, but only 18% identity with human insulin. There is high cross immunological reactivity between the S. japonicum and S. mansoni ILPs as observed in western blots using an anti-SjILP polyclonal antibody. ADP binding/hydrolysis ability was observed in both SjILP and SmILP, but not in human insulin, suggesting a parasite-specific role for ILP compared with host insulin. Protein binding assays using the Octet-RED system showed SjILP binds S. japonicum IRs (SjIR1 and SjIR2) strongly. An anti-phospho antibody against extracellular signal-regulated kinase (Erk) recognised a 44-kDa target band in an extract of adult worms after stimulation by rSjILP in vitro, suggesting an important role for SjILP in activating SjIRs and in regulating downstream signal transduction. Immunolocalisation showed SjILP is located on the tegument and the underlying musculature, similar to that observed for SjIR1, but it is also present throughout the parenchyma of males and in the vitelline cells of females, the same locations as SjIR2 described in an earlier published study of ours. The same localisation of SjILP and the SjIRs is suggestive of an interaction between the insulin-like peptide and the IRs. In addition to binding host insulin, schistosomes also can express their own endogenous ILPs, which can activate the parasite insulin signal pathway, thereby playing a critical role in worm growth, development and fertility.ConclusionsThese findings shed new light on ILPs in schistosomes, providing further insight into the distinct and specialised functions of SjIR1 and 2 in S. japonicum and their interaction with host insulin.

Project description:Identification of immunogenic targets against hepatitis B virus (HBV)-encoded proteins will provide crucial advances in developing potential antibody therapies. In this study, 63 treatment-naïve patients with chronic HBV infection and 46 patients who achieved hepatitis B surface antigen loss (sAg loss) following antiviral treatment were recruited. Moreover, six patients who transitioned from the hepatitis B e antigen-positive chronic infection phase (eAg+CInf) to the hepatitis phase (eAg+CHep) were enrolled from real-life clinical practice. Additionally, telbivudine-treated eAg+CHep patients and relapsers or responders from an off-treatment cohort were longitudinally studied. The frequencies and function of B cells were assessed by flow cytometry. We devised a peptide array composed of 15-mer overlapping peptides of HBV-encoded surface (S), core (C), and polymerase (P) proteins and performed a screening on B-cell linear epitopes with sera. Naïve B cells and plasmablasts were increased, whereas total memory, activated memory (AM), and atypical memory (AtM) B cells were reduced in sAg- patients compared with sAg+ patients. Importantly, longitudinal observations found that AtM B cells were associated with successful treatment withdrawal. Interestingly, we identified six S-specific dominant epitopes (S33, S34, S45, S76, S78, and S89) and one C-specific dominant epitope (C37) that reacted with the majority of sera from sAg- patients. Of note, more B-cell linear epitopes were detected in CHep patients with alanine aminotransferase (ALT) flares than in nonflare CInf patients, and five B-cell linear epitopes (S4, S5, S10, S11, and S68) were overwhelmingly recognized by ALT flare patients. The recognition rates of epitopes on C and P proteins were significantly increased in CHep patients relative to CInf patients. Strikingly, a statistically significant elevation in the number of positive epitopes was observed when ALT nonflare patients shifted into the flare phase. Moreover, S76 identified at baseline was confirmed to be associated with a complete response after 48 weeks of telbivudine therapy. Taken together, we identified several functional cure-related B-cell linear epitopes of chronic HBV infection, and these epitopes may serve as vaccine candidates to elicit neutralizing antibodies to treat HBV infection.

Dataset Information

Identification of linear epitopes in SjSP-13 of Schistosoma japonicum using a GST-peptide fusion protein microplate array.

Background

Methods

Results

Conclusions

Publications

Identification of linear epitopes in SjSP-13 of Schistosoma japonicum using a GST-peptide fusion protein microplate array.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets