Analyzing Cellular Immunity to AAV in a Canine Model Using ELISPOT Assay
Adeno-associated viral (AAV) vector-mediated gene transfer represents a promising gene replacement strategy for treating various genetic diseases. One obstacle in using viral-derived vectors for in vivo gene delivery is the development of host immune responses to the vector. Recent studies have demonstrated cellular immune responses specific to capsid proteins of various AAV serotypes in animal models and in human trials for different diseases. We developed a canine-specific ELISPOT assay to detect such immunity in dogs received AAV treatment. Here, we describe in detail the use of a constructed panel of overlapping peptides spanning the entire VP1 sequence of AAV capsid protein to detect specific T-cell responses in peripheral blood in dogs following intramuscular injection of AAV. This high-throughput method allows the identification of T-cell epitopes without the need for large cell numbers and the need for major histocompatibility complex molecule-matched cell lines.
Key wordsAdeno-associated virus AAV Peptide library Dog ELISPOT assay
We thank Drs. Jeffery Chamberlain and Dusty Miller for providing vectors used in the protocols and Dr. Kathy High for providing the cFIX plasmid. We thank Drs. Christine Halbert and Carolina Berger for technical advice, E. Zellmer and E. Finn for technical assistance, A. Joslyn, and the canine team, and M. Spector, DVM, and J Duncan, DVM, for their care of the dogs. We further thank S. Carbonneau, H. Crawford, B. Larson, K. Carbonneau, and D. Gayle for administrative assistance and manuscript preparation.
This work was supported by NIH R01 AR056949-01A1, NIH CA15704, and by Career Development Award for Z. Wang from the Muscular Dystrophy Association (MDA 114979).
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