High-throughput screening of patient sera for pre-existing AAV vector antibodies

Pre-existing neutralizing antibodies (nAbs) pose a serious barrier to safe and effective gene therapy use. It has been reported that approximately 70% of adults have sera antibodies for AAVs (adeno-associated viruses) and memory T cells [27]. A recent study investigated the prevalence of nAbs against AAV-1, -2, -5, and -8 in blood samples from healthy donors. The results were troubling as they found that 47-74% of samples had AAV-2 nAbs even though low titers can block in vivo transduction [28].

Though viral genomic material has been removed, the capsid carrying the therapeutic payload can trigger an immune response. Prior viral exposure can lead to undesired humoral and/or T cell-mediated immunity that will be reactivated upon vector delivery [29-32].

Figure 1. Wild-type AAV exposure and patient characteristics determine an individual’s response at vector delivery and overtime.

It is critical to assess immune status in patients to monitor gene therapy efficacy [33]. Subjects previously treated with gene therapy may require a second dose as an initial treatment may have activated a patient’s adaptive immune system to eliminate the vector upon reintroduction. This proves that assessing a patient’s immune status is critical to monitoring gene therapy efficacy [33].

To improve the effectiveness of vector delivery, image cytometers can be employed for:

• Rapid screening for nAbs prior to treatment
  • Cell-based in vitro transduction inhibition assays
• Long-term patient monitoring
  • Humoral immune responses for antibodies against the vector and gene therapy protein product
  • T-cell immune responses

Use plate-based imaging to screen patient sera for neutralizing antibodies and monitoring immune responses over time

The Celigo Image Cytometer is a plate-based high-throughput system that simultaneously images and analyzes cells in standard multi-well plates using brightfield and fluorescence technology. The Celigo rapidly screens for sera nAbs that can neutralize AAV vectors for potential pre-existing immunities.

1. High-throughput automated imaging and direct analysis in 96-well plates
2. No trypsinization necessary to count cells
3. Measure antibody neutralization effects in less than ten minutes

This section provides two technical examples of how the Celigo Image Cytometer can be used to develop a robust screening assay for potential pre-existing immunity to AAV vectors.

1. High-throughput neutralization assay of AAV-GFP vectors using sera
2. High-throughput neutralization assay of AAV-GFP vectors using neutralizing antibodies