AAV neutralization assay

The study procedure included a one-time collection of 5 mL of blood for the AAV neutralization assay during the standard of care blood draw for 25 hydroxy vitamin D levels. Patient serum was separated and stored at −80°C. Deidentified and coded study samples were transferred to the Emory National Primate Center Vaccine Biomarker Laboratory, Emory University, for AAV neutralization assays.

Heat-inactivated serum samples were diluted in complete media (Dulbecco's modified Eagle's medium [DMEM] supplemented with 10% fetal bovine serum [FBS], Pen/Strep, and l-glutamine; VWR) at ratios of 1:5, 1:20, 1:80, and 1:240 and plated in duplicate. Previously known seropositive and seronegative samples were included with each assay. Serum dilutions were combined with 50 μL of ∼10¹⁰–10¹¹ AAV viral genomes/mL encoding firefly luciferase and incubated at 37°C in 5% CO₂ for 1 h. These dilutions were used to produce >1,000 relative luciferase units upon transduction.

During incubation, confluent monolayers of HeLa or HEK293T cells (ATCC) seeded at 25,000 cells/well (AAV multiplicity of infection (MOI) range 20,000–200,000) were infected with viral particles of adenovirus-5 (ATCC) to increase luciferase expression of AAV2 and AAVrh74-transduced cells.

After incubation, the antibody–virus mixtures were aliquoted onto the adenovirus-5 HeLa cell monolayers and incubated at 37°C in 5% CO₂. After 24–48 h, plates were removed from the incubator and washed once with phosphate-buffered saline (PBS) before addition of the firefly luciferase substrate (Bright-Glo assay kit; Bio-Rad). Plates were read on a BioTek luminometer within 15 min of substrate addition. Inhibition was determined by normalizing relative light units values against wells without serum and reported as a percentage.

Neutralizing titers were reported as the estimated dilution at which 50% of AAV transduction has been inhibited (IC₅₀), using the nonlinear regression analysis in GraphPad Prism, v9.4.0, software. Samples with IC₅₀ titers >1:5 were considered seropositive based on previous recommendations¹⁴ and a neutralizing titer that has been previously shown to block transduction of the liver.¹⁵ Low, moderate, and high positive NAb titers were defined as dilution of <1:20, 1:20–1:240, and >1:240, respectively.

Five study subjects had repeat blood draws for AAV neutralization titers during the study period.