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Identification fo Surface Proteins of Streptococcus equi With Potential in Vaccine Development
Department of Veterinary Sciences
Existing vaccines against equine strangles are either of low efficacy in the field or are associated with adverse side effects including abcessation. The recently available genomic DNA sequences of S. equi and S. zooepidemicus give access to the genes for proteins potentially expressed on the bacterial surface and which may be involved in stimulating protective immune responses in the horse.
The project will evaluate these proteins following their expression in E. coli by screening them with antibodies from immune resistant horses and then testing pools of reactive candidate proteins by vaccinating groups of experimental ponies. These ponies will then be challenged by commingling with an infected pony and their resistance to strangles evaluated.
Proteins with protective ability will be candidates for inclusion in new generation strangles vaccines that are likely to be effective and safe. These proteins will also be valuable in development of in vitro tests that correlate with protection.
2011 Project Description
During this reporting period, the focus was on Streptococcus zooepidemicus (Sz), the ancestral archetype from which the clonal S. equi (Se) was derived. Sz and Se share at least 97% DNA homology and therefore express many immunoreative proteins in common. An expression gene library of Sz NC 78 from an epizootic of equine pneumonia yielded E. coli clones expressing 20 different proteins. Many of these proteins were predicted to be surface exposed based on the presence of LPxTG sortase processed or LXYC, LTAC, LAAC lipid motifs. Others were predicted to be secreted and some had transmembrane domains with possible surface exposure. This finding is in agreement with observation from studies on other bacterial pathogens which showed that surface exposed/secreted proteins are many times more likely than cytoplasmic proteins to stimulate acquired immune responses.
Eight surface exposed proteins (SZO 16070, 1430, 14810, 02770, 01820, 16930, 11840) and 2 secreted proteins (SZO 01930 and 06450) that included an iron siderophore receptor, a chemokine protease, a internalin A-like protein, enolase and streptokinase were selected for mouse vaccine trials. Selection was based on predicted functions that might affect virulence or account for features of pathology documented in pneumonic lungs.
Proteins with peptidase/protease activity or which bound a protease proenzyme were included, because of their potential to degrade host protein with resulting necrosis and thrombus formation. These enzymatic activities might also enhance nutrient availability to the proliferating pathogen. Fibrinogen binding could also be linked with nutrient acquisition and evasion of immune response.
The very large populations of S. zooepidemicus visible in lung lesions and in infected lymph nodes imply a requirement for iron and so the putative Fe3+ siderophore receptor (SZO 16070) was included. This protein reacted strongly with equine convalescent sera. Streptokinase (SZO 01930) and enolase (SZP 11840) are involved in activation and binding of plasminogen.
Hi-titer antisera to each recombinant protein have been produced in goats and will be used in passive protection trials in mice. The EHV-a vaccine strain RacH, engineered to express peptides of the SeM , Se18.9 and IdeE proteins, elicited peptide specific serum antibodies in yearling ponies. Protective immunity elicited will be evaluated by co-mingling challenge early in 2012.
Information about the immunoreactive proteins of S. equi and S. zooepidemicus will be valuable in design of new generation vaccines and diagnostics.
Merant, C., Sheoran, A., and Timoney, J.F. (2011). Association of Streptococcus equi with equine monocytes, Veterinary Immunology and Immunopathology, 143: 83-86.
Ijaz, M., Velineni, S., and Timoney, J.F. (2011). Selective pressure for allelic diversity in SEM of Streptococcus equi does not affect immunoreactive proteins SzPSe or Se18.9, Infection, Genetics and Evolution, 11: 1159-1163.
Artiushin, S., Tong, Y., Timoney, J., Lemieux, B., Schlegel, A., Kong, H. (2011). Thermophilic helicase-dependent DNA amplification using the IsoAmp SE experimental kit for rapid detection of Streptococcus equi subspecies equi in clinical samples, Journal of Veterinary Diagnostic Investigation, 23: 909-14.
Waller, A.S., Paillot, R., and Timoney, J.F. (2011). Streptococcus equi: a pathogen restricted to one host, Journal of Medical Microbiology, 60: 1231-40.