Haar Janina, Blazevic Dragica, Strobel Benjamin, Kreuz Sebastian, Michelfelder Stefan
Research Beyond Borders, Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach an der Riss, Germany.
Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach an der Riss, Germany.
Mol Ther Methods Clin Dev. 2022 Apr 20;25:360-369. doi: 10.1016/j.omtm.2022.04.008. eCollection 2022 Jun 9.
Adeno-associated virus (AAV) vector applications are often limited by capsid-directed humoral immune responses, mainly through neutralizing antibodies (NAbs), which are present throughout the human population due to natural AAV infections. Currently, antibody levels are often quantified via ELISA-based protocols or by cellular NAb assays and less frequently by NAb assays in mice. These methods need optimization for each serotype and are often not applicable to AAV variants with poor transduction. To tackle these limitations, we have established Meso Scale Discovery (MSD)-based assays for the quantification of binding antibodies (BAbs) and NAbs against the three most commonly used AAV serotypes, AAV2, AAV8, and AAV9. Both assays detect anti-AAV-IgG with high sensitivity and consistency as shown in a screen of sera from 40 healthy human donors. Subsequently, BAb and NAb titers were determined for identification of seronegative animals in a non-human primate (NHP) cohort. Moreover, the MSD-based BAb assay protocol was extended to a panel of 14 different AAV serotypes. In summary, our platform allows a rapid and quantitative assessment of the immunological properties of any natural or engineered AAV variant irrespective of transduction efficiency and enables high-throughput screens.
腺相关病毒（AAV）载体的应用常常受到衣壳介导的体液免疫反应的限制，主要是通过中和抗体（NAb），由于自然感染AAV，这些抗体在整个人口中都存在。目前，抗体水平通常通过基于酶联免疫吸附测定（ELISA）的方案或细胞NAb测定来定量，而在小鼠中通过NAb测定来定量的情况较少。这些方法需要针对每种血清型进行优化，并且通常不适用于转导效率低的AAV变体。为了解决这些限制，我们建立了基于Meso Scale Discovery（MSD）的测定方法，用于定量针对三种最常用的AAV血清型AAV2、AAV8和AAV9的结合抗体（BAb）和NAb。如在对40名健康人类供体的血清筛查中所示，这两种测定方法都能以高灵敏度和一致性检测抗AAV-IgG。随后，测定了BAb和NAb滴度，以鉴定非人类灵长类动物（NHP）队列中的血清阴性动物。此外，基于MSD的BAb测定方案扩展到了一组14种不同的AAV血清型。总之，我们的平台允许对任何天然或工程化的AAV变体的免疫特性进行快速定量评估，而无需考虑转导效率，并且能够进行高通量筛选。
