Silvia Pellicer. WorldPathol S.L. (Spain)

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“Use of ORF-1 product Rep in prevention and diagnosis of Porcine circovirosis” Silvia Pellicer. WorldPathol S.L. (Spain) 4th International Conference on Vaccines and Vaccination 24-26 September 2014. Valencia (Spain)

WorldPathol biotech develops new drugs and biological products on request www.worldpathol.com

PORCINE CIRCOVIRUS DISEASES (PCVD) PMWS (Postweaning multisystemic wasting sindrome) is one of the main causes of economical losses in swine production market. Diagnosis: detection of PCV2 in the lesions (Porcine circovirus type 2). PCV2 infection is wide spread in farms. Only pigs with a high PCV2 load develop the illness due to defective humoral response.

PCV2 Small non-enveloped virus single stranded DNA belonging to Circoviridae family. Genomic organization (ss DNA): ORF-1: Rep ORF-2: Cap main immunogenic antigen of the virus

CURRENT VACCINES Based on inactivated PCV2 Based on recombinant subunit ORF-2 (Cap) Based on non pathogenic strain PCV1 displaying pathogenic ORF-2 (Cap)

WHAT DO WE WANT? Produce a vaccine to prevent PCVD based on subunits Produce a diagnosis kit to differenciate vaccinated from infected animals (DIVA)

PMWS affected animals had lower anti-Cap and anti-Rep antibodies. WHY REP? Pérez-Martín, E. et al., 2008. “Development of two Trichoplusia ni larvae-derived ELISAs for the detection of antibodies against replicase and capsid proteins of porcine circovirus type 2 in domestic pigs”. J Virol Methods. 154(1-2), 167-74 107 pigs sera analysed PMWS affected animals had lower anti-Cap and anti-Rep antibodies.

REP features Product of ORF-1 Essential for virus replication Vega-Rocha et al. 2007 Aminoacids number 314 Molecular Weight 35,78 kDa Isoelectric point 8,41

STRATEGY Optimization of nucleotide sequence of Rep (ORF-1) for expression in E. coli. Construction of a plasmid for Rep overexpression in E. coli. Optimization of Rep overexpression and purification.

E.COLI OVEREXPRESSION OPTIMIZATION STRAIN 1 BEST CONDITION Inductor (mM) Induction time Strain 1 Overnight 1mM inductor 37ºC STRAIN 2 Inductor (mM) Induction time STRAIN 3 Inductor (mM) Induction time

E.COLI PURIFICATION OPTIMIZATION WESTERN BLOT COOMASSIE STAIN Affinity cromatography Good efficiency Purity about 90%

NEXT STEPS: Purification of Rep in medium-large scale. Development of ELISA for Product Quality Assurance. In vivo assays to test immunogenicity of obtained Rep.

THANK YOU! silvia.pellicer@worldpathol.com This work has been partly funded by Project PTQ-12-05797 from Spanish Government