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Protein based approaches to understand and prevent contagious bovine pleuropneumonia
KTH, School of Biotechnology (BIO), Proteomics. (Proteomik)
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 [en]
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: urn:nbn:se:kth:diva-11108ISBN: 978-91-7415-417-7 (print)OAI: oai:DiVA.org:kth-11108DiVA: diva2:235819
Public defence
2009-10-16, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20100719Available from: 2009-09-25 Created: 2009-09-18 Last updated: 2010-07-19Bibliographically approved
List of papers
1. Expression and immunogenicity of six putative variable surface proteins in Mycoplasma mycoides subsp. mycoides SC.
Open this publication in new window or tab >>Expression and immunogenicity of six putative variable surface proteins in Mycoplasma mycoides subsp. mycoides SC.
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2008 (English)In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 154, 539-549 p.Article in journal (Refereed) Published
Abstract [en]

Variable surface protein Vmm and five Vmm-type proteins from Mycoplasma mycoides subsp. mycoides SC were analysed to determine whether these proteins are expressed in vivo in animals affected by contagious bovine pleuropneumonia (CBPP) and in vitro. Recombinant versions of these proteins were constructed and expressed in Escherichia coli after mutation of the TGA Trp codons to TGG. These proteins were then analysed by dot and Western blotting with sera from CBPP-affected cattle. Furthermore, affinity-purified polyclonal antibodies to the recombinant proteins were used in Western and colony blotting to look for expression of the putative Vmm-type proteins in cultured M. mycoides SC. This study demonstrates that immunoglobulins in CBPP sera recognize all putative Vmm-type proteins tested, indicating that these proteins or their homologues are expressed by mycoplasmas during natural infections. Vmm and one of the putative Vmm-type proteins showed variable expression in vitro.

Place, publisher, year, edition, pages
Reading: Society for General Microbiology, 2008
Keyword
CONTAGIOUS BOVINE PLEUROPNEUMONIA, ALBUMIN-BINDING REGION, SMALL COLONY TYPE, GENETIC-CHARACTERIZATION, AFFINITY-CHROMATOGRAPHY, GEL-ELECTROPHORESIS, SIGNAL PEPTIDES, LIPOPROTEIN, ERADICATION, AFRICA
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11088 (URN)10.1099/mic.0.2007/010694-0 (DOI)000253927800020 ()
Note
QC 20100719Available from: 2009-09-17 Created: 2009-09-17 Last updated: 2010-09-14Bibliographically approved
2. Recombinant surface proteomics as a tool to analyze humoral immune responses in bovines infected by Mycoplasma mycoides subsp. mycoides SC
Open this publication in new window or tab >>Recombinant surface proteomics as a tool to analyze humoral immune responses in bovines infected by Mycoplasma mycoides subsp. mycoides SC
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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
Keyword
pleuropneumonia cbpp; genetic-characterization; control strategies; lipoprotein lppq; signal peptides; sc; eradication; vaccines; africa; serodiagnosis
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11089 (URN)10.1074/mcp.M900009-MCP200 (DOI)000271615200011 ()2-s2.0-72149128953 (Scopus ID)
Note
QC 20100719Available from: 2009-09-18 Created: 2009-09-17 Last updated: 2010-12-06Bibliographically approved
3. Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay
Open this publication in new window or tab >>Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay
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2009 (English)In: Clinical and Laboratory Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 16, no 11, 1665-1674 p.Article in journal (Refereed) Published
Abstract [en]

A recombinant antigen cocktail ELISA for diagnosis of contagious bovine pleuropneumonia (CBPP) was developed after careful selection of antigens among one third of the surface proteome of the infectious agent Mycoplasma mycoides subsp. mycoides SC (M. mycoides SC). First, a miniaturized and parallelized assay system employing antigen suspension bead array technology was used to screen 97 bovine sera for their humoral immune response towards 61 recombinant surface proteins from M. mycoides SC. Statistical analysis of the data resulted in selection of eight proteins that showed strong serologic responses in CBPP-affected sera and minimal reactivity in negative control sera, with p-values less than 10-6. Only minor cross reactivity to hyperimmune sera against other mycoplasmas was observed. When applied in an ELISA, the cocktail of eight recombinant antigens allowed a five fold signal separation between 24 CBPP-affected and 23 CBPP-free sera from different geographical origin. No false positives and only two false negatives were obtained. In conclusion, the selected recombinant mycoplasma antigens qualified as highly specific markers for CBPP and could be employed in both a suspension bead array platform and a cocktail ELISA setting. This set of proteins and technologies therefore offer a powerful combination to drive and further improve serological assays towards reliable, simple and cost-effective diagnosis of CBPP.

Place, publisher, year, edition, pages
Washington D.C.: American Society for Microbiology, 2009
Keyword
contagious bovine pleuropneumonia, lipoprotein lppq, genetic-characterization, sc, diagnosis, elisa, cbpp, capricolum, tanzania, cluster
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11105 (URN)10.1128/CVI.00223-09 (DOI)000271373700019 ()2-s2.0-70350702934 (Scopus ID)
Note
QC 20100719. Updated from in press to published. Previous title "Multiplex screening of surface proteins from Mycoplasma mycoides subsp. mycoides SC for an antigen cocktail ELISA"Available from: 2009-09-18 Created: 2009-09-18 Last updated: 2013-02-08Bibliographically approved
4. Protein-Specific Analysis of Humoral Immune Responses in a Clinical Trial for Vaccines against Contagious Bovine Pleuropneumonia
Open this publication in new window or tab >>Protein-Specific Analysis of Humoral Immune Responses in a Clinical Trial for Vaccines against Contagious Bovine Pleuropneumonia
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2010 (English)In: Clinical and Laboratory Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 17, no 5, 853-861 p.Article in journal (Refereed) Published
Abstract [en]

Specific humoral immune responses in a clinical trial on cattle for vaccines against contagious bovine pleuropneumonia (CBPP) were investigated. The trial included a subunit vaccine consisting of five recombinant putative variable surface proteins of the infectious agent Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC) compared to the currently approved attenuated vaccine strain T1/44 and untreated controls. Humoral immune responses to 65 individual recombinant surface proteins of M. mycoides SC were monitored by a recently developed bead based array assay. Responses to the subunit vaccine components were found to be weak. Animals vaccinated with this vaccine were not protected and had CBPP lesions similar to the untreated controls. In correlating protein-specific humoral responses to T1/44 induced immunity, five proteins associated with a protective immune response were identified,namely LppQ and those of ORFs MSC_0271, MSC_0136, MSC_0079 and MSC_0431. The five proteins may be important candidates in the development of a novel subunit vaccine against CBPP.

Keyword
subsp mycoides sc, small colony type, lipoprotein lppq, genetic-characterization, cbpp, cattle, eradication, africa, strategies, infection
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11107 (URN)10.1128/CVI.00019-10 (DOI)000277243200023 ()2-s2.0-77951988744 (Scopus ID)
Note
Uppdaterad från manuskript 20100719 Tidigare titel: Protein-specific analysis of humoral immune responses in a CBPP vaccine trial QC 20100719Available from: 2009-09-18 Created: 2009-09-18 Last updated: 2011-01-19Bibliographically approved

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