Structural and antigenic characterization of the avian adeno-associated virus capsid.

All adeno-associated virus (AAV) vectors currently used in clinical trials or approved gene therapy biologics are based on human or non-human primate AAVs. A major challenge for AAV gene therapy is the high prevalence of circulating neutralizing antibodies (NAbs) in the general population targeting the virus capsids leading to vector inactivation and a loss of treatment efficacy. A strategy to escape detection by NAbs is the utilization of AAVs that do not disseminate in the primate population and exhibit low or no antigenicity. One such example is avian AAV (AAAV), which was first identified in preparations of the Olson strain of quail bronchitis, an avian adenovirus. AAAV shows very low sequence identities (~54-58%) to the AAV serotypes including to the sequences of the structurally diverse AAV4 and AAV5. In this study, the structure of empty and genome-filled AAAV capsids was determined by cryo-electron microscopy (cryo-EM) at 2.5 and 3.1 Å resolution. Furthermore, AAAV was found to utilize galactose for cell attachment, similar to AAV9 and AAVrh.10. Characterization of AAAV's antigenic properties revealed that 30% of human sera from healthy individuals were capable of neutralizing transduction. This high rate of antigenicity is caused by conserved epitopes around the fivefold channel of the capsid allowing cross-reactivity of NAbs. This was further confirmed by mapping a cross-reactive human anti-AAV9 monoclonal antibody using cryo-EM. This structure-function characterization will be beneficial to further expand the current repertoire of AAV vectors in human gene therapy applications. IMPORTANCE AAVs are extensively studied as promising therapeutic gene delivery vectors. In order to circumvent pre-existing antibodies targeting primate-based AAV capsids, the AAAV capsid was evaluated as an alternative to primate-based therapeutic vectors. Despite the high sequence diversity, the AAAV capsid was found to bind to a common glycan receptor, terminal galactose, which is also utilized by other AAVs already being utilized in gene therapy trials. However, contrary to the initial hypothesis, AAAV was recognized by approximately 30% of human sera tested. Structural and sequence comparisons point to conserved epitopes in the fivefold region of the capsid as the reason determinant for the observed cross-reactivity.