2013 Annual Report
1a.Objectives (from AD-416):
Determine the effectiveness of utilizing subgenomic replicating PRRSV heteroclite RNAs to vector the expression of immunomodulatory cytokines as a component of a modified live vaccine preparation, as a means to increase protection from natural infection.
1b.Approach (from AD-416):
We will use the infectious clone of PRRSV strain Inglevac PRRSV MLV as modified to contain DIVA epitope tags within the nsp2 coding region. In addition, cDNA clones of heteroclite RNAs (a specific type of PRRSV RNAs that are packaged within the virus and expressed when virus infects cells) will be individually modified to contain coding sequences for the immunomodulatory cytokines IL-12, IL-15 and IL-18. From these constructs, RNA transcripts will be generated and transfected into PRRSV permissible cell lines. Both full-length and heteroclite RNAs will be packaged, and the resultant virus preparation will be examined for genetic stability over multiple passages, stable expression of the vectored cytokine, as well as induction of cytokine response from primary alveolar macrophages. Upon completion of initial testing in cell culture, virus preparations will be assessed for their ability to serve as an effective vaccine as compared to the currently used IngleVac MLV strain alone. This will be done by vaccinating pigs using either the above prepared MLV/heteroclite mixture, a matched dose of IngleVac MLV or a sham innoculum, followed by a viral challenge using homologous or heterologous PRRSV strains. Protection will be assessed by monitoring weight, body temperature, viral load in serum and lung lesion scores of experimentally infected animals.
Previously obtained cDNAs of two heteroclite RNAs (S2 & S7) from PRRSV strain VR2332 were cloned into a plasmid vector for ease of amplification and subsequent genetic manipulation. The 3' ends of both constructs were corrected, poly(A) tails lengthened, and appropriate restriction enzyme sites added to allow generation of authentic poly(A) tail terminated RNAs free of non-viral nucleotides upon run-off transcription with T7 RNA polymerase. Site directed mutagenesis was performed on both constructs to allow insertion of cytokine expression cassettes. An autocatalytic 2A peptide was also included in the final construct, so that the 2A peptide will self-cleave following translation, releasing the downstream unmodified cytokine gene product. Porcine cytokine genes encoding interleukin-7 (IL-7), IL-15, IL-18, and CD40L were amplified by RT-PCR from pooled total RNA obtained from thymus and lymph node of non-infected and PRRSV-infected pigs. At this time, only the IL-7 gene has been subsequently cloned into the final S2 and S7 heteroclite constructs. As a reporter control, the nanoluciferase gene was also amplified by PCR, and is currently being cloned into the final S2 and S7 heteroclite constructs. This nanoluciferase reporter will allow us to better characterize translation, RNA stability, packaging, and maintenance of heteroclite RNAs in culture. It can also serve as a transfection/packaging control for all heterolite experiments, as the nanoluciferase assay is a rapid and sensitive method for assessing transgene expression. We are in the process of generating S2 and S7 vectored IL-7 RNAs and attempting to rescue DIVA-MLV virus preparations containing these recombinant heteroclite RNAs.