Unknown,Transcriptomics,Genomics,Proteomics

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Protein Microarray Analysis of Antibody Responses of Experimentally Infected Dogs to Borrelia burgdorferi Antigens

ABSTRACT: Lyme borreliosis (LB) is a tick-borne infection caused by Borrelia burgdorferi. Dogs are at high risk of exposure to ticks and tick-borne pathogens, including B. burgdorferi. Immunodiagnostic assays are usually based on whole-cell preparations of B. burgdorferi as substrate and, consequently, interpretation of results is confounded by antibody cross-reactivity between borrelial antigens and other bacterial species, as well as the anti-LB vaccination status of the dog. For this study, we examined sera from 33 dogs that were experimentally infected with B. burgdorferi through tick bite. These sera were compared with sera from uninfected dogs in their reactivities to 72 different recombinant B. burgdorferi polypeptides on a protein microarray. Amongst antigens frequently recognized by infected dogs were several known to be immunogens for humans, such as Decorin-binding protein A (BBA25), BBA64, fibronectin-binding protein (BBK32), VlsE, Erp and Bdr, CRASP proteins, OspC proteins and some flagellar antigens. Of special interest were the novel antigens BBB14 and BB0844, both hypothetical lipoproteins about which very little is currently known, and that were frequently and strongly recognized by infected dog sera. The antibody response of B. burgdorferi-infected dogs presents both similarities and differences from human counterparts, and both can be important for improvement of canine LB diagnosis and vaccine development. Antibody profiling was performed on sera from dogs experimentally-infected with B. burgdorferi and unexposed controls against antigens of B. burgdorferi. Thirty-three serum samples from experimental infections, and 6 unexposed controls were probed on a protein microarray displaying 72 unique proteins of B. burgdorferi .

ORGANISM(S): Canis lupus familiaris

SUBMITTER: Elisabeth Baum

PROVIDER: E-GEOD-50074 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Epitope mapping studies aim to identify the binding sites of antibody-antigen interactions to enhance the development of vaccines, diagnostics and immunotherapeutic compounds. However, mapping is a laborious process employing time- and resource-consuming M-bM-^@M-^Xwet benchM-bM-^@M-^Y techniques or epitope prediction software that are still in their infancy. For polymorphic antigens, another challenge is characterizing cross-reactivity between epitopes, teasing out distinctions between broadly cross-reactive responses, limited cross-reactions among variants and the truly type-specific responses. A refined understanding of cross-reactive antibody binding could guide the selection of the most informative subsets of variants for diagnostics and multivalent subunit vaccines. We explored the antibody binding reactivity of sera from human patients and Peromyscus leucopus rodents infected with Borrelia burgdorferi to the polymorphic outer surface protein C (OspC), an attractive candidate antigen for vaccine and improved diagnostics for Lyme disease. We constructed a protein microarray displaying 23 natural variants of OspC and quantified the degree of cross-reactive antibody binding between all pairs of variants, using Pearson correlation calculated on the reactivity values using three independent transforms of the raw data: (1) logarithmic, (2) rank, and (3) binary indicators. We observed that the global amino acid sequence identity between OspC pairs was a poor predictor of cross-reactive antibody binding. Then we asked if specific regions of the protein would better explain the observed cross-reactive binding and performed in silico screening of the linear sequence and 3-dimensional structure of OspC. This analysis pointed to the C-terminal helix of the structure as a major determinant of type-specific cross-reactive antibody binding. We developed bioinformatics methods to systematically analyze the relationship between local sequence/structure variation and cross-reactive antibody binding patterns among variants of a polymorphic antigen, and this method can be applied to other polymorphic antigens for which immune response data is available for multiple variants. Antibody profiling was performed on sera from Borrelia burgdorferi infected and non-infected humans and Peromyscus leucopus rodents against 23 variants of the surface protein OspC . For infected human serum samples, the OspC type of the infecting B. burgdorferi strain is unknown; for experimentally-infected P. leucopus serum samples, it is known. Of human serum samples, 55 were from infected individuals and 25 from naive controls. Of P. leucopus serum samples, 23 were from infected individuals and 7 were from naive controls.

Project description:Leptospirosis is the most widespread zoonotic disease in the world. The lack of an adequate laboratory test is a major barrier for the diagnosis, especially during the early stages of illness, when antibiotic therapy is most effective. Therefore, there is a critical need for an efficient diagnostic test for this life threatening disease. In order to identify new targets that could be used as diagnostic makers for leptopirosis, we constructed a protein microarray chip comprising 61% of Leptospira interrogans proteome and investigated the IgG response against leptospiral antigens from 274 individuals, including 80 acute-, 80 convalescent-phase patients and 114 healthy control subjects from regions with endemic, high endemic and no endemic transmission of leptospirosis. A nitrocellulose line blot assay was performed to validate the accuracy of the protein microarray results. We found 16 antigens that can discriminate between acute cases and healthy individuals from a region with high endemic transmission of leptospirosis, and 18 antigens that distinguish convalescent cases. Some of the antigens identified in this study, such as LipL32, the non-identical domains of the Lig proteins, GroEL and Loa22 are already known to be recognized by sera from human patients, thus serving as a proof-of-concept for the serodiagnostic antigen discovery approach. Several novel antigens were identified, including the hypothetical protein LIC10215 which showed good sensitivity and specificity rates for both acute- and convalescent-phase patients. Our study is the first large-scale evaluation of immunodominant antigens associated with naturally acquired leptospiral infection and novel as well as known serodiagnostic leptospiral antigens that are recognized by antibodies in the sera of leptospirosis cases were identified. The novel antigens identified here may have potential use in both the development of new tests and the improvement of currently available assays for diagnosing this neglected tropical disease. Further research is needed to assess the accuracy of these antigens in more appropriate diagnostic platforms. Antibody profiling was peformed on sera from infected and non-infected subjects to investigate the IgG response against leptospiral antigens. These samples comprised 80 acute-, 80 convalescent-phase patients and 114 healthy control subjects, including 29 subjects from United States (non-endemic area), 35 blood donors from Salvador/Brazil (endemic area) and 50 healthy individuals from region with high endemicity of leptospirosis.

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Protein Microarray Analysis of Antibody Responses of Experimentally Infected Dogs to Borrelia burgdorferi Antigens

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