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Recombinant surface proteomics as a tool to analyze humoral immune responses in bovines infected by Mycoplasma mycoides subsp. mycoides SC
KTH, School of Biotechnology (BIO), Proteomics. (Proteomik)
KTH, School of Biotechnology (BIO), Proteomics. (Proteomik)ORCID iD: 0000-0002-1855-703X
KTH, School of Biotechnology (BIO), Proteomics. (Proteomics)ORCID iD: 0000-0001-8141-8449
KTH, School of Biotechnology (BIO), Proteomics. (Proteomik)
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2009 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 8, no 11, 2544-2554 p.Article in journal (Refereed) Published
Abstract [en]

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causing agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP affected cattle and controls were monitored against one third of the surface proteins of M. mycoides SC in a high-throughput magnetic bead based assay. First, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in E. coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP positive and negative sera. Signals were proven to be protein-specific by inhibition experiments and results agreed with western blot experiments. The assay's potential to monitor IgG, IgM and IgA responses over time was shown in a proof-of-concept study with 116 sera from 8 animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M mycoides SC, has been created.

Place, publisher, year, edition, pages
Stanford: HighWire Press , 2009. Vol. 8, no 11, 2544-2554 p.
Keyword [en]
pleuropneumonia cbpp; genetic-characterization; control strategies; lipoprotein lppq; signal peptides; sc; eradication; vaccines; africa; serodiagnosis
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11089DOI: 10.1074/mcp.M900009-MCP200ISI: 000271615200011Scopus ID: 2-s2.0-72149128953OAI: oai:DiVA.org:kth-11089DiVA: diva2:235809
Note
QC 20100719Available from: 2009-09-18 Created: 2009-09-17 Last updated: 2010-12-06Bibliographically approved
In thesis
1. Protein based approaches to understand and prevent contagious bovine pleuropneumonia
Open this publication in new window or tab >>Protein based approaches to understand and prevent contagious bovine pleuropneumonia
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test.The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial.Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. x, 77 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:17
Keyword
CBPP, Mycoplasma mycoides subsp. mycoides small colony type, humoral immune responses, recombinant surface proteins, ELISA, subunit vaccine, suspension bead array
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11108 (URN)978-91-7415-417-7 (ISBN)
Public defence
2009-10-16, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
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Note
QC 20100719Available from: 2009-09-25 Created: 2009-09-18 Last updated: 2010-07-19Bibliographically approved

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Neiman, MajaSchwenk, Jochen M.

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