Development of an efficacious recombinant vaccine for the obligate intracellular salmonid pathogen Piscirickettsia salmonis

by Kuzyk, Michael Allan

Piscirickettsia salmonis is the aetiological agent of salmonid rickettsial septicaernia (SRS). an economicaIly devastating rickettsial disease of fmed salmonids. SRS responds poorly to antibiotic treatrnent and no effective vaccine is available for its control. A molecular biology approach was used to characterize and identify antigens of P salmonis that would be suitable to use as a recombinant subunit vaccine to aid in the control of SRS. A system for routine and reliable growth of ?? safmonis was established using a chinook salmon (Oncorhynchus tshawytscha) embryo ce11 Iine. A purification protocol to separate P salmonis from host ceIl material was devised using a combination of differential and Percoll density gradient centrifbgation. Purified l? salmonis was used to generate polyclonal rabbit antisera. Indirect irnmunofluorescence microscopy, immunogold transmission electron microscopy, and biotin labeling of intact f! salmonis confirrned that !? salmonis was effectively separated fion: host ce11 debris and that immunoreactive antigens identified by rabbit antisera were surface associated. Rabbit anti-P safmonis sera recognized the lipooligosaccharide component of bacterial lipopolysaccharide. and 7 protein antigens with relative mobilities of 27, 24, and 16 kDa and 4 migrating between 50-80 kDa. i? salmonis lipopoiysaccharide was observed to be predorninantly low m-W., but less abundant high m.w. species containing O-antigen were present. Genomic DNA was isolated from punfied II salmonis and used to constnict an expression library in lambda ZAP II. In the absence of preexisting DNA sequence, rabbit polyclonal anti-P salmonis semm was used to identifL immunoreactive clones. A lambda clone encoding an immunoreactive 17 kDa outer surface protein (OspA)of E) salmonis was identified. The 4,983 bp insert contained a high molar percentage of adenine and thymine, encoded four intact ORF's, and represented the first non-ribosomal DNA sequence data from P salmonis. OspA is mociified as a bacterial lipoprotein in Escherichia coli and is most closely homologous to a rickettsial 17 kDa surface lipoprotein previousiy only observed within the genus Rickettria. A codon optimized version of ospA was constructed and the iipoprotein nature of OspA was deteminecl to be a limihng fàctor in its production in E. cofi. High level production of immunoreactive OspA targeted to inclusion bodies was achieved