Title: Expression of chicken parvovirus VP2 in chicken embryo fibroblasts requires codon optimization for production of naked DNA and vectored Meleagrid herpesvirus type 1 vaccines Authors
Submitted to: Virus Genes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 3, 2013
Publication Date: July 17, 2013
Citation: Spatz, S.J., Volkening, J.D., Mullis, R.A., Li, F., Mercado, J.C., Zsak, L. 2013. Expression of chicken parvovirus VP2 in chicken embryo fibroblasts requires codon optimization for production of naked DNA and vectored meleagrid herpesvirus type 1 vaccines. Virus Genes. 47:259-267. DOI: 10.1007/s11262-013-0944-9. Interpretive Summary: The generation of a novel vaccine against the recently discovered chicken parvovirus (ChPV), one of the major causative agent of Runting-Stunting Syndrome (RSS) of chickens was achieved. Transient expression of the major antigen virus protein 2 (VP2) of chicken parvovirus in cell culture from eukaryotic expression vectors has been problematic. Although the cloning of the VP2 gene within the expression vectors and the correctness of the gene’s nucleotide sequence could be confirmed, no evidence of protein expression using immunohistochemical methods could be demonstrated. To investigate why expression vectors containing the VP2 gene fail to generate VP2 protein bicistronic vectors and synthesized a variant of the VP2 gene was generated. This variant contained codons that were optimized for usage by the chicken translational machinery. The ability to express VP2 of chicken parvovirus in chicken cell cultures and in birds was a monumental achievement.
Technical Abstract: Meleagrid herpesvirus type 1 (MeHV-1) is an ideal vector for the expression of antigens from pathogenic avian organisms in order to generate vaccines. Chicken parvovirus (ChPV) is a widespread infectious virus that causes serious disease in chickens. It is one of the etiological agents largely suspected in causing renting and stunting syndrome in chickens. Attempts to express the wild type gene encoding for the capsid protein VP2 of ChPV by insertion into the thymidine kinase gene of MeHV-1 were unsuccessful. However transient expression of a codon-optimized synthetic VP2 gene cloned into the bicistronic vector pIRES2-DS-Red2, could be demonstrated by immunohistochemical staining of transfected chick embryo fibroblasts (CEFs). Red fluorescence could also be detected in these transfected cells since the red fluorescent protein (RFP) gene is downstream from the internal ribosome entry site (IRES). Strikingly, fluorescence could not be demonstrated in cells transiently transfected with the bicistronic vector containing the wild type or non-codon-optimized VP 2 gene. Immunohistochemical staining of these cells also failed to demonstrate expression of wild type VP2 indicating the lack of expression was at the ribonucleic acid (RNA) level and the VP2 protein was not toxic to CEFs. Chickens vaccinated with a DNA vaccine consisting of the bicistronic vector containing the codon-optimized VP2 elicited a humoral immune response as measured by a VP2 specific antibody assay. This VP2 codon-optimized bicistronic cassette was rescued into the MeHV-1 genome generating a vectored vaccine against chicken parvovirus disease.