Use of adeno-associated virus (AAV) vectors has revolutionized in vivo gene therapy, but the presence of pre-existing neutralizing antibodies remains a major barrier that can hinder clinical application. While large-animal models such as non-human primates have been used to study anti-AAV immunity, their high cost and limited accessibility present challenges for studying the impact of AAV immunity on AAV-based therapies. Here, we evaluate pigs as an immunologically relevant large-animal model for investigating humoral barriers to AAV-based gene therapy. Using ELISA-based profiling across 11 AAV serotypes, we detected immunoglobulin G (IgG) antibodies against AAV capsids in pigs as early as two weeks of age, with titers increasing with age and displaying serotype-specific dynamics. Animals maintained in standard housing displayed greater inter-individual variations and broader serotype-specific reactivities. Functional assays demonstrated that these antibodies neutralized AAV particles in a dose- and serotype-dependent manner, with IgG depletion restoring transduction in vitro. Sequence analysis indicated that capsid identity can partially predict cross-reactive binding, but only under controlled conditions. These findings establish pigs as a tunable model capable of recapitulating age- and environment-dependent features of anti-AAV immunity, providing a platform for studying humoral barriers and evaluating immune evasion strategies in AAV-based gene therapy.
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