How is viral titer determined in VectorBuilder?
After harvesting viral particles, if the viral vector carries a fluorescent reporter gene, we usually first check the quality of virus by transducing the virus into some common cell lines (e.g. 293T or 293A) to observe the expression of fluorescent protein. Different methods are then used to quantify the titer of virus depending on viral type. Occasionally, if there is a major discrepancy between fluorescence observation and quantitative measurement, we will perform re-measurement or additional validation to ensure that viruses manufactured by VectorBuilder are of high quality.
To measure lentivirus titer, we transduce 293T cells with lentivirus diluted from the stock. Then, we use a qPCR-based approach to quantify the average number of integration events of the proviral genome (using the copy number of WPRE as a proxy) per host genome (using the copy number of BMP2 as a proxy) to estimate titer in the original viral stock. This approach measures the functional titer which precisely reflects the number of viral particles with infectious capability. There are other methods for measuring lentivirus titer. One is to measure the physical titer by performing RT-qPCR directly on the viral genomic RNA extracted from the virus. But this method can grossly over-estimate titer (typically by ~10 fold and occasionally up to 100 fold), because it measures any viral genome regardless of whether it is from a live infectious viral particle or a dead particle. This problem is exacerbated by the fact that lentivirus is not stable and can quickly lose infectivity if not frozen at -80°C or if subjected to repeated freeze-thaw cycles. Another method is to measure the number of transduced cells based on the expression of either a fluorescent or drug-selection marker carried on the viral vector. This method could severely under-estimate titer because a fluorescent or drug-selection marker may fail to be expressed at detectable levels in some host cells due to silencing or some other reason, and also because one cell may be infected by multiple viral particles.
For adenovirus, we also measure the functional titer. After transducing serially-diluted adenovirus into 293A cells, we use an immunocytochemistry-based approach to count the number of cells being successfully transduced via the detection of adenovirus-specific hexon protein, and each immunostained cell is considered as one infectious unit. Cells are infected at very low multiplicity of infection (MOI) to ensure that most transduced cells are each infected by a single viral particle. This assay shows good correlation with conventional plaque assay. For ultra-purified adenovirus, we directly measure the optical density (using OD260) of the viral particles to estimate titer, because there is a tight correlation between the optical density of ultra-purified adenovirus and functional titer. Adenovirus has very good stability. In our preparation, the viral particles are essentially all alive and can remain functional at room temperature for many days.
Adeno-associated virus (AAV)
We measure the physical titer of AAV by directly extracting viral genome from lysed viral particles, and then using qPCR to accurately quantify the copy number of viral genome (using the copy number of ITR region as a proxy) in the stock. AAV particles are very stable. In our AAV preparation, viral particles are essentially all alive and can remain functional at room temperature for many days. As such, the physical titer, though not measured in a way involving the transduction of cells, is very close to the functional titer.