A flow cytometry-based immuno-titration titer assay was established to determine infectious unit (IU) and transducing unit (TU) of modified vaccinia Ankara (MVA) virus vectors. This titration method enumerates infected cells by measuring the expression of viral protein for IU and transgene protein for TU in individual cells after staining with fluorophore-conjugated antibodies. It presents many advantages over standard virus titration approaches, such as TCID(50) or plaque assay, for its convenience, rapidity and accuracy as illustrated by excellent assay linearity and reproducibility. Importantly, the IU and the TCID(50) assays generated similar batch-specific titer values when testing varied MVA-derived virus preparations. Assay development revealed that the post-infection time at which viral protein expression is evaluated, host cell type, and blocking the formation and release of progeny virion with nocodazole, an anti-microtubule agent or rifampin, a specific vaccinia virus assembly inhibitor, are critical parameters for the precision, robustness, and accuracy of IU titer determination. An added advantage of this assay is that it enables the concurrent determination of IU and transducing units (TU) by measuring the expression of a transgene product when testing recombinant viruses. The latter was demonstrated using a MVA vector carrying a human HER-2 gene fragment as model. Hence, this assay is very versatile in that it can be used to determine IU as well as multiple TU titers simultaneously. Furthermore, it can readily be adapted to other poxvirus vectors.
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