ProtocolsA flow cytometry-based immuno-titration assay for rapid and accurate titer determination of modified vaccinia Ankara virus vectors
Introduction
Poxviruses are immunogenic vectors capable of carrying multiple large transgenes that can be expressed in host cells. This feature and the fact that they do not integrate into host genomes render poxviruses attractive vectors for vaccine applications (Gulley et al., 2010, Kantoff et al., 2010, von Krempelhuber et al., 2010). Poxvirus-based product development relies on infectious titers to monitor viral production/purification processes and standardize dosage. A novel FACS-based assay was developed to determine rapidly and accurately infectious titers using vectors derived from Modified Vaccinia Ankara (MVA), a highly attenuated strain of vaccinia virus.
MVA is a large, double-stranded DNA poxvirus that infects a wide variety of cell types. It was generated following nearly 600 passages on primary chicken embryo fibroblasts (CEF) during which more than 30 kilobases of vaccinia DNA comprising genes involved in host-range restriction and immunomodulation were deleted (Meyer et al., 1991, Antoine et al., 1998). As a result, MVA can replicate fully in only limited cell types, such as CEF and BHK-21 cells (Carroll and Moss, 1997, Drexler et al., 1998). Otherwise, the replication of MVA aborts at the late stage of the virus life cycle for most mammalian cells (Sutter and Moss, 1992; Chahroudi et al., 2006).
The traditional methods used widely for titer determination are plaque assay and endpoint titration assay (Hierholzer and Killington, 1996). Both plaque assay for plaque forming unit (PFU) and endpoint titration assay for 50% tissue culture infectious dose (TCID50) are time consuming and labor intensive. Multiple replicates or repeats are required to achieve reliable titer values. It is also challenging to determine both infectious and transgene transducing titers simultaneously using these methods. To address these drawbacks, alternative fluorescent-activated cell sorter (FACS)-based methods were recently evaluated to determine titer or cell infection for several viruses including influenza virus (Lonsdale et al., 2003), vaccinia virus (Domínguez et al., 1998), Epstein-Barr virus (Stowe et al., 1998), rabies virus (Bordignon et al., 2002), adenovirus (Gueret et al., 2002, Weaver and Kadan, 2000), baculovirus (Chan et al., 2006) and Dengue virus (Lambeth et al., 2005).
Titer determination of MVA vector was assessed by FACS-based immuno-titration where infected cells were detected after staining with a fluorochrome-conjugated anti-vaccinia antibody. IU titers were derived from the numbers of cells positively stained following infection with serial dilution of the vector. Assay optimization was performed to achieve acceptable IU titer accuracy and robustness. Parameters including the cell type used for infection, the blockage of progeny virion release, and time window post-infection were found to be critical for this assay. Importantly, IU titers derived from the FACS-based assay were similar to TCID50 titers for all MVA vectors tested when using MVA-permissive cells. In addition to IU titers, the FACS-based assay also enabled the simultaneous determination of transducing units (TU) that relates to the expression of transgene products of recombinant virus. TU titers were evaluated using a recombinant virus, MVA-BN®-HER2 that encodes a fragment of the human oncogene for epidermal growth factor receptor 2 (HER-2). Titer determination using an anti-HER-2 antibody instead of the anti-vaccinia probe resulted in equivalent TU and IU values when using permissive host cells in the assay. Hence this assay provides a novel means to characterize concurrently multiple features of viral-based vectors. It compares favorably to standard titer assays, which warrants its evaluation for the testing of products in clinical development.
Section snippets
Cell, virus and antibodies
BHK-21 cell line was obtained from ATCC (Catalog number: CCL-10). The BHK-21 cell line was established from the kidneys of baby Syrian hamsters and previously shown to fully support MVA infection and replication (Carroll and Moss, 1997, Drexler et al., 1998). BHK-21 cells were grown at 37 °C and 5% CO2 in minimum essential medium (MEM), containing 10% fetal calf serum (FCS), penicillin, streptomycin, l-glutamine and nonessential amino acids.
MVA-BN®-HER2 is derived from MVA-BN® strain, developed
Detection of MVA-infected cells by FACS analysis
Commercially available anti-vaccinia antibodies were evaluated for the detection of virion proteins in individual cells treated with MVA-ATI-HER2. The panel of antibodies tested included two FITC-conjugated polyclonal as well as two non-conjugated monoclonal anti-vaccinia antibodies. FACS detection of MVA vector infected BHK-21 cell populations after staining with FITC-conjugated anti-vaccinia and anti-Her2 antibody was shown in Fig. 1. FITC-stained BHK-21 cells were detected following
Discussion
A simple, rapid, and accurate poxvirus titer assay was established using a FACS-based immune-titration method that detects and enumerates individual virus-infected cells. Using MVA as a model, the titers of poxvirus preparations were derived from the percentage of cells infected in vitro immune-stained with a fluorophore-conjugated anti-vaccinia antibody (IU) and /or anti-transgene Her2 antibody (TU). Unlike standard virus titer methods measuring PFU and TCID50, IU/TU titer determination does
Acknowledgements
We thank Drs Dirmeier and Steigerwald (Bavarian Nordic, Martinsried, Germany) for generating and purifying viruses as well as determining TCID50 titers.
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