(W1030-02-12) Comparative Detection of Pre-existing Anti‑AAV8 Antibodies in Normal Human Serum by a Total Antibody Assay Versus a Transduction Inhibition Assay

Purpose: Pre-existing antibodies to AAV exist in certain human subjects and these antibodies may impact the effectiveness of AAV gene therapy. Thus, patients are usually screened for pre-existing AAV antibodies with a transduction inhibition assay (TI assay) to detect neutralizing antibodies at entry. However, the TI assay is cell-based and technically challenging to conduct. Using AAV8 as an example in this study, we explore if an immunoassay-based total antibody assay (TAb assay) can be used in lieu of the TI assay for patient screening.

Methods: In the TAb assay, AAV8 is directly coated onto a 96-well microplate (Figure 1). Following incubation of human serum samples, a biotinylated AAV8 is added to form a bridging structure for detection of anti-AAV8 capsid antibodies. The TAb assay is performed in two tiers – screening and confirmation, with the confirmed positives as final positive results. In the TI assay, the AAV8 virus harboring luciferase gene is employed, which expresses luciferase after successful transduction into a host cells (Figure 2). The virus is incubated with serum samples, and then added to a 96-well cell plate pre-seeded with HEK293T host cells. After overnight incubation, the luciferase activity is measured chemiluminescently. Upon assay validation, the same set of human serum samples from 36 normal subjects are analyzed with the two assays and the results are compared for concordance.

Results: The TAb assay was validated with the screening cut point factor of 1.23 and the confirmatory cut point of 17.1%, while the TI assay utilized 50% inhibition as the cut point. Sensitivity of the TAb and TI assays were determined to be 14 and 204 ng/mL, respectively. Sample analysis results are summarized in Table 1. Out of 36 samples, 13 samples tested positive and 23 samples negative with the TI assay. Of the 13 TI-positive samples, 11 samples were positive with the TAb assay, indicating diagnostic sensitivity of 84.6% for the TAb assay. Twenty-two of 23 TI-negative samples were also negative with the TAb assay, indicating diagnostic specificity of 95.7%.

Conclusion: An immunoassay (TAb assay) and a cell-based assay (TI assay) have been developed and validated for detection of pre-existing anti-AAV8 antibodies. Results from the two assays are highly concordant, leading to the higher diagnostic sensitivity (84.6%) and the higher diagnostic specificity (95.7%). Based on these results, it is reasonable to use the TAb assay in lieu of the TI assay for screening patients’ samples for an AAV8 gene therapy.


Figure 1. TAb assay design


Figure 2. TI assay design


Table 1. Test results from TAb and TI assays