Anti-AAV Neutralizing Antibody Assay Platforms, Guidance and Need For Harmonization

In vitro Cell Transduction Assay for anti-Adeno Associated Virus Neutralizing Antibodies

The basic schematics of the cell-based assay for the measurement of relative titers of neutralizing antibodies are shown in the figure below. Briefly, the specific AAVs are engineered with reporter genes (LacZ, Luciferase, or GFP) that allows the relative of  AAV transduction into the cells. The expression of the reported gene is proportional to the AAV transduced into the cell system. 

 In the absence of neutralizing antibodies, the AAV-Reporter vector can transfect the cells and expresses the reporter gene. The color intensity of expressed reporter genes is measured using an appropriate detection method.

In the presence of neutralizing antibodies, reduced transduction of the AAV-Reporter vector into the cells is reflected by the reduced expression of the reporter gene. The % reduction in transduction is calculated from the signal produced with or without the presence of neutralizing antibodies/ 

Selection of Cells

- Based on the review of published literature, a variety of cell lines has been used for the detection of neutralizing antibodies against AAVs in the gene therapy therapeutics

- Cell lines include: Huh cells, Hela cells, 2V6.11cells & HEK

- The suitability & cell lines depend on the detection of the efficiency of the AAV transfection into the cells and expression of reporter genes. 

- For harmonization, the use of HEK cells are recommended when possible

- The master cell bank and the working cell bank should be created and characterized to reduce the lot-to-lot variability, passages, etc. 

Selection of Positive Controls

- Positive controls are generated by immunization of AAVs in the animals including mice, monkeys, etc. 

- For evaluation of neutralizing antibodies against the wildtype AAVs, an industry-wide harmonized approach are necessary. For this type of researches, characterization, and use of the same positive control would enable comparison of incidence and magnitude of anti-AAV specific neutralizing antibody titers across the study.

- Mostly, recombinant AAVs are used as gene therapy vectors. In these instances, proper characterization of polyclonal or monoclonal antibodies for its affinity, neutralization capacity are recommended

Selection of Reporter Genes & Detection System 

- The selection of the reporter genes is critical to achieving the acceptable sensitivity of these cell-based assays. 

- The reporter systems such as LacZ, Luciferase or GFP have been used in the published literature 

- For the practical purpose and harmonization, the selection of one reporter system is highly recommended. 

- The luciferase detection system can be used for harmonization purposes.

Stepwise Protocol for the Cell-Based Neutralizing Antibody Assay

1. Preparation and culture of cells

-Appropriately dilute the cells from the working cell bank and seed the optimized number of cells to the 96 wells culture plate and incubate overnight (16-20 hours) at 37-degree celsius with 5% carbon dioxide. 

2. Dilution of Positive Controls, Negative Controls & Test Samples

- Perform serial dilution of the positive controls, negative controls, and test samples. Choose an appropriate serial dilution 1:2 or 1:3 for the dilution of controls/samples

3. Incubation and Neutralization of the AAV-Luciferase

- Dilute the AAV-Luciferase vector in serum-free medium and mix with the controls/samples to allow the AAV-Luciferase vector with the neutralizing antibodies

- Separately dilute the positive and negative controls with the serum-free medium (without AAV) that serve as negative transduction control

- In addition, dilute AAV-Luciferase Vector with the assay buffer  without serum to measure the maximal transduction

 - Incubate the mixture of 30 minutes to 60 minutes

4. In-vitro transduction

- Transfer the controls/samples + AAV-Luciferase mixture to the 96 well plates cultured with cells

- Incubate the cells in the incubator for 20-24 hours, and allow the AAV transduction into the cells

- After incubation, wash the cells to remove residual AAV-Luciferase vector

5. Cell Lysis and Addition of Detection System

- Add the lysis buffer containing detection system (Bright Glo, Promega) and measure the luciferase activity on the luminometer

- Transduction efficiency is measured as Relative Light Unit per second

- Neutralizing Antibody Titers are expressed as the highest serum dilution that inhibited AAV transduction by greater than 50% compared with control without serum (100% transduction)


Melani et al 2015, Human Gene Therapy Methods

Leborgne et al 2019, Cellular Immunology

Kruizk et al 2019, Hum Gene Ther Methods

Gorovits et al 2020, AAPS Journal