#15-146

Complete

Date Full Report Received

02/02/2017

Date Abstract Report Received

02/02/2017

Investigation

Institution:
Primary Investigator:
Co-Investigators: Kyoung-Jin Yoon

The goals of this project were to identify methods for the high-level expression of the PEDV S protein receptor-binding domain (RBD) from within the genome of the clinically benign virus, Mammalian orthoreovirus (MRV). The eventual goal is to utilize MRV as a replication competent vaccine vector for the mucosal delivery of PEDV antigens to swine. Targeting the mucosa has multiple immunologic and practical advantages including the induction of a more effective immune response and ease and cheaper cost of delivery via oral administration. The work performed in this funding period represents initial benchmarks necessary for achieving these goals, namely identifying the PEDV RBD, as well as a method to drive expression of the PEDV RBD from within the MRV genome.
The MRV genome is very compact, and each gene segment encodes for a protein that is absolutely required for virus replication and cannot be deleted. In our efforts to design MRV for use as a vector, we have previously identified a mechanism to insert foreign genetic sequences within the MRV genome without substantially disrupting the replication and packaging of the virus. The next step in readying the virus for use as a vaccine vector was the subject of this proposal. In order to deliver antigens to cells via MRV, they must not only be encoded within the genome, they must also be expressed as peptides or proteins that can be recognized by the immune system. We tested two methods appropriated from other viruses, the introduction of a sequence, called an internal ribosome entry site (IRES), that can recruit the cellular translation machinery to the internal portion of a gene, and introduction of a sequence that directs the cellular machinery to start translating a protein directly following the termination of an upstream protein to drive dual production of MRV proteins and foreign genetic material (an HIV gp41 peptide and the small fluorescent protein, UnaG). We found that while both of these approaches were capable of driving the production of two proteins from a single mRNA when expressed alone in cells, they were unable to do so when expressed from the virus genome. It is unclear at this time why we were unsuccessful at achieving these goals, and we continue to troubleshoot the assay. Meanwhile, we also were working to identify the PEDV S RBD. In this portion of the project, we were able to identify a small portion of the S protein that when blocked by antibodies, neutralizes virus infection. Future work will include incorporating this region into the MRV genome to confirm expression and detection of the antigen and testing other strategies for dual protein expression from the MRV genome. Contact Cathy Miller (clm@iastate.edu) for additional details.