Principal Scientist Altasciences Laval, Quebec, Canada
Purpose: Fit-for purpose assays for Total Antibodies (TAbs) and Neutralizing Antibodies (NAbs) to AAV have been developed in a way to be standardized, qualified or validated depending on the context of use. Designed with adaptability in mind, these assays can be transferred to other AAV serotypes or species and have demonstrated successful application across multiple serotypes and preclinical models.Gene therapy efficacy depends on the efficient delivery of therapeutic genes, often hindered by pre-existing anti-AAV antibodies (TAbs), including neutralizing subsets (NAbs). These antibodies, whether from natural exposure or prior treatment, can drastically reduce vector transduction. Therefore, screening for such antibodies in preclinical models is essential, as pre-existing immunity may limit therapeutic success.To support this, standardized TAb and NAb were developed to be compatible across serotypes and animal models, ensuring broad use in preclinical settings. The original AAV8 TAb assay served as a foundation for adapting the method to an AAV9 derived serotype requiring only minor adjustments. . Similarly, key parameters from the AAV8 NAb assay supported rapid development of the AAV9 NAb assay. Both are currently undergoing validation. Additionally, the AAV8 NAb assay was qualified at an external site, confirming its reproducibility and robustness.
Methods: The TAb assay uses an indirect Electrochemiluminescence (ECLIA) format. Biotinylated AAV8 empty capsid and biotinylated monkey IgG are captured on a Streptavidin SECTOR plate. The assay detects anti-AAV8 antibodies in NHP serum with a sensitivity of 1 ng/mL. For other serotypes, the same format is used with ruthenylated capsids replacing anti-species detection reagents. The NAb assay follows a “thaw and go” design that provides results within 24 hours. HEK293T cells are thawed, plated in 96-well plates, and transduced with an AAV8-luciferase vector after a 1-hour pre-incubation with samples. Adding test samples within 3 hours of cell plating minimizes proliferation effects, supporting standardized MOI when applicable. Due to batch variability in vector quality, MOI adjustments may be needed. For some serotypes, assay duration was extended to 48-72 hours to optimize performance. Transduction is enhanced using a cellular energy homeostasis inhibitor prior to sample addition. Both assays use a mouse anti-AAV8 monoclonal antibody in surrogate serum as a positive control. The surrogate serum also serves as the negative control and dilution buffer, ensuring long-term lot consistency. A shared dilution scheme across both assays supports direct result comparison.
Results: Assay Adaptability Both assays were successfully adapted for an AAV9-derived vector. The same anti-AAV9 monoclonal antibody was used across formats to ensure performance alignment. Vector substitution and titration optimization were required for NAbs, while key assay conditions: cell number, inhibitor usage, reading time were maintained. For the TAb assay, ruthenylation of the AAV9 capsid enabled detection. Surrogate matrix optimization was performed to better reflect NHP serum responses to this serotype. The AAV8 NAb assay was applied to serum from a nanopig model, yielding a 75% positivity rate, consistent with NHP data. No gender-related differences were observed across models. These outcomes support the assays’ transferability across species and serotypes for preclinical immunogenicity assessments. Anti-AAV Antibody Prevalence In NHP samples, both AAV8 assays showed high seropositivity: 75% for TAbs and 64% for NAbs, with no gender bias. The higher TAb rate reflects: Detection of all binding antibodies, not just functional NAbs, Higher analytical sensitivity of the TAb assay for low-abundance antibodies. These results are consistent with previously established prevalence data for AAV8, obtained using assays from other vendors. With AAV9, prevalence was 60% across assays in NHPs. Reproducibility of the AAV8 assay was confirmed via inter-site comparison of NHP serum lots. Key observations: All high-NAb sera were also TAb-positive (100%). 75% of low-NAb sera were TAb-positive. 75% of NAb-negative sera were still TAb-positive. For the NAb assay, inter-site analysis showed 67% concordance across 12 NHP lots. These findings illustrate the complementary nature of the assays: TAb detects all binding antibodies with greater sensitivity, including NAb, while NAb captures only functional inhibitors of transduction. Thus, low-level antibodies missed by the NAb assay but detected by the TAb assay still contribute valuable immunogenicity data. These distinctions underscore the importance of interpreting assay results in the context of biological variability and assay-specific mechanisms.
Conclusion: The TAb and NAb assays described offer robust, rapid, and standardized tools for evaluating pre-existing anti-AAV immunity essential for advancing AAV-mediated gene therapies. Their core strengths include high reproducibility, shared dilution schemes for results comparison, and fast turnaround (as little as 24 hours), enabling swift decision-making in preclinical programs. Their adaptability to multiple AAV serotypes and animal models supports broad preclinical application. The complementary nature of the TAb and NAb assays, in detecting both binding and functional antibodies, allows for a comprehensive immunogenicity assessment. Together, these attributes make the assays powerful tools for managing pre-existing immunity and guiding vector strategy in early development. Future work will assess transferability to human serum, supporting clinical readiness.
