A Genotype of Modified Vaccinia Ankara (MVA) that Facilitates Replication in Suspension Cultures in Chemically Defined Medium

Abstract

While vectored vaccines, based on hyperattenuated viruses, may lead to new treatment options against infectious diseases and certain cancers, they are also complex products and sometimes difficult to provide in sufficient amount and purity. To facilitate vaccine programs utilizing host-restricted poxviruses, we established avian suspension cell lines (CR and CR.pIX) and developed a robust, chemically defined, culturing process for production of this class of vectors. For one prominent member, modified vaccinia Ankara (MVA), we now describe a new strain that appears to replicate to greater yields of infectious units, especially in the cell-free supernatant of cultures in chemically defined media. The new strain was obtained by repeated passaging in CR suspension cultures and, consistent with reports on the exceptional genetic stability of MVA, sequencing of 135 kb of the viral genomic DNA revealed that only three structural proteins (A3L, A9L and A34R) each carry a single amino acid exchange (H639Y, K75E and D86Y, respectively). Host restriction in a plaque-purified isolate of the new genotype appears to be maintained in cell culture. Processing towards an injectable vaccine preparation may be simplified with this strain as a complete lysate, containing the main burden of host cell contaminants, may not be required anymore to obtain adequate yields.

High-cell-density cultivations to increase MVA virus production

“…Because MVA spreads preferably via cell-to-cell contact a culture format was developed where cell agglomerates of infected and non-infected cells are being induced for production of vaccine preparations. A MVA strain (MVA-CR19) was recently obtained with this system that propagates also in nonagglomerated CR.pIX suspension cells [16]. With this new isolate that represents a different genotype of MVA, titers in the order of 10 8 IU/mL can be obtained at conventional-cell-densities (CCD) of about 2.0 Â 10 6 cells/mL.…”

“…With this new isolate that represents a different genotype of MVA, titers in the order of 10 8 IU/mL can be obtained at conventional-cell-densities (CCD) of about 2.0 Â 10 6 cells/mL. As a higher fraction of MVA-CR19 is released into the supernatant, harvest of infectious units does not require whole-cell lysates anymore, facilitating the subsequent downstream processing [16].…”

“…A seed virus bank of the MVA-CR19 isolate (4.5 Â 10 8 virions/mL) propagated in CR.pIX cells was prepared as described before [16]. Before infection, seed virus aliquots were treated for 1 min in a sonication water bath to break up virus aggregates.…”

“…For quantification of the concentration of infectious virus particles (here lysate), infected cells were treated with three freeze/ thaw cycles (À80°C/RT) and treated for 1 min in a sonication water bath (45 kHz) prior to centrifugation at 1500g, 10 min, RT, to discard cellular debris. For the quantification of virus released by host cells (here supernatant), samples were centrifuged at 200g, 5 min, RT, supernatant was retrieved and treated also with three freeze/ thaw cycles before storage [16]. All virus samples were stored in aliquots of 0.5-1 mL at À80°C.…”

“…Mean and standard deviation of three independent cultivations. [16,30] and a bioreactor ( Table 1, CBwt) [15,30]. Thus, the results obtained were considered as representative for CCD infections.…”