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Chimeric peptide constructs comprising linear B-cell epitopes: application to the serodiagnosis of infectious diseases.

ABSTRACT: Linear B-cell epitopes are ideal biomarkers for the serodiagnosis of infectious diseases. However, the long-predicted diagnostic value of epitopes has not been realized. Here, we demonstrated a method, diagnostic epitopes in four steps (DEIFS), that delivers a combination of epitopes for the serodiagnosis of infectious diseases with a high success rate. Using DEIFS for malaria, we identified 6 epitopes from 8 peptides and combined them into 3 chimeric peptide constructs. Along with 4 other peptides, we developed a rapid diagnostic test (RDT), which is able to differentiate Plasmodium falciparum (P. falciparum) from Plasmodium vivax (P. vivax) infections with 95.6% overall sensitivity and 99.1% overall specificity. In addition to applications in diagnosis, DEIFS could also be used in the diagnosis of virus and bacterium infections, discovery of vaccine candidates, evaluation of vaccine potency, and study of disease progression.

SUBMITTER: Lu Y

PROVIDER: S-EPMC4543967 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Chimeric peptide constructs comprising linear B-cell epitopes: application to the serodiagnosis of infectious diseases.

Lu Yudong Y Li Zhong Z Teng Huan H Xu Hongke H Qi Songnan S He Jian'an J Gu Dayong D Chen Qijun Q Ma Hongwei H

Scientific reports 20150821

Linear B-cell epitopes are ideal biomarkers for the serodiagnosis of infectious diseases. However, the long-predicted diagnostic value of epitopes has not been realized. Here, we demonstrated a method, diagnostic epitopes in four steps (DEIFS), that delivers a combination of epitopes for the serodiagnosis of infectious diseases with a high success rate. Using DEIFS for malaria, we identified 6 epitopes from 8 peptides and combined them into 3 chimeric peptide constructs. Along with 4 other pepti ...[more]

PMID: 26293607

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Project description:IntroductionPeptides (B-cell epitopes) have broad applications in disease diagnosis and surveillance of pathogen exposure. In this framework, we present a pilot study to design and produce a peptide microarray for the integrated surveillance of neglected tropical diseases. The peptide microarray was evaluated against peptides derived from Ascaris lumbricoides, Necator americanus, Schistosoma haematobium, Schistosoma mansoni, Trichuris trichiura, Bacillus anthracis, Mycobacterium leprae, Wuchereria bancrofti, Rabies lyssavirus, Chlamydia trachomatis and Trypanosoma brucei.MethodsS. haematobium was diagnosed using the urine filtration technique. S. mansoni, A. lumbricoides, N. americanus and T. trichiura were diagnosed using the Kato Katz and formal ether concentration techniques. Immunogenic peptides were retrieved from the Tackling Infection to Benefit Africa infectious diseases epitope microarray. Further peptides were predicted using ABCpred. IgG and IgM reactivity against the derived peptides were evaluated using peptide microarray multiplex immunoassays. Positive response was defined as fluorescence intensity ≥ 500 fluorescence units. Immunodominant peptides were identified using color-coded heat maps and bar graphs reflecting the obtained fluorescence signal intensities. Receiver Operating Characteristic analysis and Mann-Whitney-U test were performed to determine the diagnostic validity of the peptides.ResultsSpecies-specific responses with at least one peptide derived from each NTD pathogen were observed. The reactive peptides included; for S. haematobium, XP_035588858.1-206-220 and XP_035588858.1-206-220 immunodominant for IgG and IgM respectively, for S. mansoni, P20287.1-58-72 immunodominant for both antibodies and for T. trichiura, CDW52482.1-326-340 immunodominant for IgG and CDW57769.1-2017-2031 and CDW57769.1-1518-1532 immunodominant for IgM. According to ROC analysis most of the peptides selected were inaccurate; with AUC < 0.5. Some peptides had AUC values ranging from 0.5 to 0.5875 for both IgM and IgG suggesting no discrimination.ConclusionMultiplex peptide microarrays are a valuable tool for integrated NTDs surveillance and for screening parasites exposure in endemic areas. Species sero-reactivity observed in the study maybe indicative of exposure to the different NTDs parasites. However, although peptides with the least cross reactivity were selected there is need to validate the sero-reactivity with recombinant antigens and immune-blotting techniques such as western blotting.

Project description:Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3'-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n = 180) and canine (n = 343) control groups, while sensitivity was higher in canine VL (96%, n = 213) than in human VL (82%, n = 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k = 0.95, 95% confidence interval = 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k = 0.81, 95% confidence interval = 0.76 to 0.87). Results suggest the potential use of this new antigen for routine serodiagnosis of VL in both human and canine hosts.

Project description:Lyme disease, a zoonotic infection caused by the bacterium Borrelia burgdorferi, is transmitted to humans through the bites of infected ticks. Its diagnosis primarily relies on serological methods; however, the existing borreliosis techniques have shown a variable sensitivity and specificity. Our study aimed to map IgG epitopes from five outer membrane proteins (Omp) from B. burgdorferi [Filament flagellar 41kD (PI1089), flagellar hook-associated protein (Q44767), Flagellar hook k2 protein (O51173), Putative Omp BURGA03 (Q44849), and 31 kDa OspA (P0CL66)] lipoprotein to find specific epitopes for the development of accurate diagnosis methods. Using the spot synthesis technique, a library of 380 peptides was constructed to identify linear B cell epitopes recognized by human IgG in response to specific B. burgdorferi-associated proteins. The reactivity of this epitope when chemically synthesized was then evaluated using ELISA with a panel of the patient's sera. Cross-reactivity was assessed through data bank access and in vitro analysis. Among the 19 epitopes identified, four were selected for further investigation based on their signal intensity, secondary structure, and peptide matching. Validation was performed using ELISA, and ROC curve analysis demonstrated a sensitivity of ≥85.71%, specificity of ≥92.31, accuracy of ≥90.7, and AUC value of ≥0.91 for all peptides. Our cross-reactivity analysis demonstrated that the Burg/02/huG, Burg/03/huG, and Burg/12/huG peptides were not reactive to antibodies from patients with Leptospirosis and syphilis compared to those from the B. burgdorferi group. These peptides indicated an excellent performance in distinguishing between B. burgdorferi-infected and non-infected individuals and exhibited a neglected reactivity to antibodies in sera from patients with Leptospirosis and syphilis. These peptides are promising targets for recombinant development, potentially leading to more accurate serological tests and vaccines.

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Chimeric peptide constructs comprising linear B-cell epitopes: application to the serodiagnosis of infectious diseases.

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Chimeric peptide constructs comprising linear B-cell epitopes: application to the serodiagnosis of infectious diseases.

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