#04-175

Complete

Date Full Report Received

07/19/2006

Date Abstract Report Received

07/19/2006

Investigation

Institution:
Primary Investigator:

The goal of the study was to examine the potential application of a novel herpes simplex virus-1-based vector system for porcine reproductive and respiratory syndrome virus vaccine development. The herpes simplex virus-1 vector is a defective virus particle that cannot reproduce in host. It contains a plasmid DNA with inserted foreign genes such as porcine reproductive and respiratory syndrome virus glycoprotein 5 gene and a limited genetic sequence from herpes simplex virus-1. Since the plasmid DNA is packaged in a capsid structure formed by herpes simplex virus-1 structural proteins, it can infect the same type of cells as herpes simplex virus-1 virus particles. The herpes simplex virus-1 vector has been investigated as a vector for human immunodeficiency virus –1 vaccine development in the previous studies. Potent T cell and antibody immune response were induced by herpes simplex virus-1 containing human immunodeficiency virus-1 glycoprotein 120 gene. The advantages of the herpes simplex virus-1 vector system include 1) enhances immunogenicity of antigens by immunomodulation of dendritic cells, 2) infects mucosal surfaces and accommodates a large amount of DNA up to a 150-kilobase insert, 3) contains as many as 15-20 copies of packaged plasmid and poses little risk to humans and animals due to its defective replication property. We intended to gather key preliminary data to determine if herpes simplex virus-1 vector could also be used in porcine reproductive and respiratory syndrome vaccine development. We chose to use glycoprotein 5 of porcine reproductive and respiratory syndrome virus as a model antigen due to the extensive previous studies showing the protective efficacy of glycoprotein 5. Additionally, we intended to optimize the glycoprotein 5 gene expression by adapting viral codon usage to the codon bias of Sus Scrofa (swine) genes to generate a codon-optimized glycoprotein 5 gene for enhanced expression in mammalian cells. The rational for gene optimization is that virus encoded genes usually utilize a rare codon usage that is not common in mammalian genes. Therefore, direct expression of viral genes in mammalian systems often fails to achieve optimal protein expression in mammalian cells, which hampers the efficient delivery of protein to the immune system in animals. To examine the immunity generated by herpes simplex virus-1 containing the optimized glycoprotein 5 gene, animals were immunized with plasmid DNA containing the optimized glycoprotein 5 gene first, followed by boosting immunization with herpes simplex virus-1 vector carrying the optimized glycoprotein 5 gene. Our results showed that herpes simplex virus-1 containing the optimized glycoprotein 5 was immunogenic and induced both antibody and T cell immunity in pigs. However, we did not detect any virus neutralization antibody after immunization. At 21 days after challenge of animals with porcine reproductive and respiratory syndrome virus, a subset of animals developed virus neutralization antibody ranging from 1:4 to 1:16. To examine the protection efficacy of herpes simplex virus-1 vector containing glycoprotein 5 gene against challenge with porcine reproductive and respiratory syndrome virus, we collected sera at 10 and 21 days after virus challenge and collected tissues including tonsils, lymph nodes, and lungs at 21 days after virus challenge (at the time of euthanasia). Real-time RT-PCR was used to quantify the viral RNA copies in each sample. We did not see an obvious reduction of viral RNA copies in sera in herpes simplex virus-1 carrying glycoprotein 5 gene immunized animals compared to the control groups at 10 and 21 days after virus challenge. Similar results were observed in collected tissue samples. There was no correlation between virus neutralization antibody titer and viral RNA copies in sera and tissues. It is possible that modification of glycoprotein 5 by the herpes simplex virus-1 vector system influences the immunogenicity of the expressed protein, which contributes to the poor antibody response and the poor protection efficiency against virus challenge. Alternatively, the poor antibody response could be due to the extra peptide sequences added to the optimized glycoprotein 5 for detection and purification purpose, which may alter the original viral protein conformation and therefore, antibody epitope specificity. Overall, we provided proof-of-concept information for the utility of herpes simplex virus-1 vector as a vaccine delivery system for porcine reproductive and respiratory syndrome virus. Future efforts should be focused on the understanding of the immunogenic properties of porcine reproductive and respiratory syndrome viral structural and non-structural proteins and their biological and immunological properties in herpes simplex virus-1 vector system.