(M1330-02-08) Analysis of Pre-existing Anti-AAV Antibodies Using a Novel Nanoliter Total Antibody Assay: A Serotype-Independent Approach

Purpose: In clinical trials with adeno-associated virus (AAV) vectors, testing for participants lacking pre-existing neutralizing antibodies is crucial for effective gene therapies. Similar to humans, non-human primates (NHPs) serve as natural hosts for AAVs and frequently harbour neutralizing antibodies against various AAV serotypes commonly employed in pre-clinical investigations. Therefore, pre-testing NHP cohorts for their antibody status is essential in gene therapy studies prior to vector administration. Various methods, such as Neutralizing Antibody (NAb) assays and Total Antibody (TAb) assays, are used to test for anti-AAV antibodies. NAb assays assess how NAbs and other non-antibody factors hinder AAV transduction while TAb assays, with their simpler design and operational ease, typically require serotype-specific assays or AAV capsid labeling (e.g bridging format), introducing time and variability.

Methods: A generic anti-AAV TAb immunoassay was developed on an automated immunoassay platform for the detection of anti-AAV antibodies in human and cynomolgus monkey samples, eliminating the need for serotype-specific assays or AAV capsid labeling. Genericity, i.e. the ability of the assay to accurately detect AAV vectors regardless of serotype, was assessed by testing for pre-existing TAbs against seven of the most commonly used AAV serotypes. In this automated assay, the biotinylated capture reagent is introduced into a microfluidic disc and captured on streptavidin-coated beads in the microstructure flow-through affinity column. A second capture reagent, the AAV capsid of choice, is introduced and interacts with the first capture reagent forming a complex. Subsequently, samples are introduced into individual microstructures where anti-AAV antibodies bind to the immobilized AAV capsid. Finally, a fluorescently-labeled detection reagent is added. The integrated laser-induced fluorescent signal represents the response from the bound anti-AAV antibody to the AAV capsid. Genericity of the assay was assessed by testing for pre-existing TAbs against seven AAV serotypes (AAV1, AAV2, AAV3, AAV5, AAV6, AAV8 and AAV9) for two sample cohorts: 30 human serum samples and 14 cynomolgus monkey serum sample.

Results: In the human cohort, 4/30 samples exhibited indiscriminate positivity for all tested serotypes, while 15/30 samples showed indiscriminate negativity. AAV1, AAV2, and AAV3 had the highest number of positive individuals, whereas AAV5 and AAV9 had the lowest. These findings align with established data on the prevalence of AAV serotypes in humans. For the cynomolgus monkey cohort, 1/14 samples were indiscriminately positive for all serotypes, while 1/14 samples were indiscriminately negative. AAV8 and AAV9 had the greatest number of positive individuals. This finding aligns with existing data regarding the prevalence of AAV serotypes in non-human primates.

Conclusion: The distribution of positive qualification for various serotypes among different individuals, without uniform readings for all serotypes in all samples, suggests that the assay is indeed generic and capable of successfully discriminating between serotypes.

Human and NHP serum samples genericity. Strong positives are highlighted in green, weaker positive samples are marked in yellow, and negative samples remain uncolored.


Comparative results of human serum samples analyzed using Gyrolab Generic Anti-AAV kit and orthogonal methods. Strong positives are highlighted in green, weaker positive samples are marked in yellow, and negative samples remain uncolored.


Performance of positive control spiked samples analyzed using Gyrolab Generic Anti-AAV kit.