Recombinant Surface Proteomics as a Tool to Analyze Humoral Immune Responses in Bovines Infected by Mycoplasma mycoides Subsp. mycoides Small Colony Type

Abstract

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against onethird of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia 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 potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study.

Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia

“…Several studies employing different experimental approaches, e.g., in silico analysis, proteomics, cloning, etc., have described the identification of immunogenic Mycoplasma mycoides subsp. mycoides proteins (15)(16)(17)(18)20), yet as expected, each methodology had its limitation. This study is based on a thorough comparison of recombinant immunogens, using a standardized expression system employing synthetic, codon-optimized genes.…”

“…Nine of the proteins were previously identified as immunogenic with diagnostic potential in collaborative studies (16,17). Eight additional proteins displaying strong serological responses and high disease specificity were also included (15,(18)(19)(20). Among those were lipoprotein Q (LppQ), a highly immunogenic surface protein, previously used for diagnosis of CBPP (15,21).…”

Identification of Mycoplasma mycoides subsp. mycoides Small Colony Genes Coding for T-Cell Antigens

“…It should, however, be pointed out that the protein used in this study represents only 62% of the full ABC transporter protein, and the possibility that the remaining sequence may also contain T-cell epitopes cannot be excluded. This applies to all polypeptides used in this study, except for LppA and GlpO, and warrants further studies using full-length proteins, which can be obtained only after all mycoplasmal tryptophan codons have been mutated to TGG (9). Also, the search for T-cell antigens should not be restricted to genes previously shown to code for proteins recognized by B cells but should be extended to all membrane or transmembrane proteins of the agent.…”

Protein-Specific Analysis of Humoral Immune Responses in a Clinical Trial for Vaccines against Contagious Bovine Pleuropneumonia

“…Serum samples were transported on dry ice and kept at Ϫ80°C until analysis with the bead-based M. mycoides SC recombinant surface protein array (14). During these experiments, the array consisted of 66 recombinant proteins with predicted surface localization and mostly full-length size, including the control protein and the fusion tag His 6 -ABP, which is present in all recombinant proteins (for details, see Table S1 in the supplemental material).…”

“…Monitoring of protein-specific humoral responses was accomplished with the recently developed M. mycoides SC surface protein bead-based array (14). The assay is based on a platform from Luminex Corp. using spectrally distinguishable microspheres (13) to form an array in suspension.…”