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The overall goal of the project is to design, construct, and test a novel PCV-2 vaccine candidate that stimulates improved cross-protective immunity against heterologous virus strains than current products. We are using an immune refocusing approach which entails identifying and then mutating key amino acids in epitopes that stimulate stain-restricted immunity. A successful outcome would be a vaccine that protects against heterologous subtypes such as PCV2d and others. We have used a variety of methods to identify these amino acids which are characterized loosely as (1) evolutionarily variable between strains, (2) surface exposed, (3) not directly participating in receptor binding, (4) hydrophilic, (5) charged, and other features. In prior projects, we have observed that reducing the immunogenicity of strain-restricted epitopes allows the recognition of more highly conserved regions that were previously not immunodominant.The short-term goals are:1. Re-analyze PCV2 immunogens and confirm the amino acids that we planned to target when preparing our application proposal.2. Design cloning strategies to introduce the mutations into plasmid DNAs that bear the parental proviral clone. This clone directs the expression of PCV2 when transfected into mammalian cells and allows rescue of an attenuated marker virus vaccine candidate.3. Produce modified proviral clones. Transfect into mammalian cells and rescue recombinant virus.4. Assess the viruses for successful infection of target cells to ensure that they are functional and can enter cells (i.e., we have not reduced receptor interactions).5. Produce and characterize vaccine lots of the attenuated candidates. Test for neutraliation using sera from animals immunized with a variety of PCV2 vaccines/viruses.6. Immunize pigs with the candidates, characterize their immune responses, and determine level of protection when challenged with heterologous PCV2 strains. Examine pigs for signs of infection and disease after challenge. After euthanasia, titer organs for virus load and examine organs by histopathology to observe cytopathic effects of the virus.7. Report findings in publications. Discuss findings with major manufacturers of current vaccines.8. If warranted, use data to apply for a Phase 2 project.Future studies will likely include expanded testing against additional heterologous PCV2 strains, development of production methods for the attenuated vaccine including the analytical package, and investigation of the regulatory roadmap.Further studies will include pre-commercialization and commercialization activities to include regulatory approvals, manufacturine scale-up, and investigations of potential partnerships with existing PCV2 vaccine suppliers.

Tobin, G.; Ramamoorthy, SH, .; Dollery, ST, .; MacLeod, DA, .; Pillai, VI, .
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