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Jul 1, 2014 (Vol. 34, No. 13)

Boosting Upstream, Downstream Processing

To Expedite Biomanufacturing, Deploy a New Genotype of Modified Vaccinia Virus Ankara

  • Many recombinant viral vectors for expression of ectopic antigens mimic a natural infection and can induce a robust immune response against certain pathogens and tumors. Host-restricted poxviruses and adenoviruses are among the more prominent vaccine strains considered for such approaches.

    Modified vaccinia virus Ankara (MVA) belongs to the group of the poxviruses and was obtained by serial passaging of a vaccinia virus with a broad host range in chicken embryo fibroblasts. After passage 516, MVA was shown to have stably lost the capacity to replicate in human cells.

    Preference for replication in nonmammalian cells confers a high degree of safety and allows application even in immunocompromised patients. However, as opposed to conventional live vaccine strains, there is no subclinical amplification at the site of injection. For this reason, host-restricted viruses are given in high numbers of infectious units per dose for full efficacy, and large production yields are essential for vaccine programs based on such vectors.

    Currently, avian cells used for vaccine production processes are primary chicken embryo fibroblasts. However, to overcome limitations associated with material that has to be prepared freshly for each manufacturing lot, we have developed a continuous avian cell line, AGE1.CR.pIX, which originates from duck primary cells to avoid contamination with active endogenous retroviruses of chickens. AGE1.CR.pIX cells proliferate in true suspension in chemically defined media and are fully permissive for a spectrum of vaccine viruses. Scalable and transportable processes that yield high titers of MVA have been demonstrated in stirred tank and Wave bioreactors.

  • Click Image To Enlarge +
    Figure 1. A recombinant wild-type MVA that expresses the GFP reporter protein does not propagate efficiently in single-cell suspension. In the same cell culture of CR.pIX cells, the virus spreads more easily if cell aggregates are induced with a chemically defined feed medium. The images, which reflect 0.01 multiplicity of infection, were obtained 72 h post infection.

    One challenge in MVA production with poxviruses is that large proportions of infectious units remain cell-associated. As a result, cell aggregates have to be induced in suspension cell cultures by addition of a special medium at the time of infection. Wild-type MVA appears to spread more easily within such aggregates than across the extracellular space (Figure 1).

    Furthermore, because complete cell lysates are harvested by sonication to obtain the cell-associated virus particles as well, cellular debris is formed and may interfere with purification of virus.

  • Extracellular Infectious Units

    Click Image To Enlarge +
    Figure 2. (A) The geno­type of MVA-CR. The bold line depicts the region covered by NGS; the red boxes show the affected genes. (B) MVA-CR19 replicates to higher titers in single cell suspensions (SCS) and in presence of induced aggregates (AGG). (C) A greater percentage of infectious activity is measured in the supernatant (SN) after infection with MVA-CR19 compared to wild-type MVA. Although with reduced magnitude, this effect is also observed in adherent cultures (ADH). (Modified from Jordan et al., 2002, Viruses 5: 321–339.)

    If MVA is produced in adherent cells, virus can be transmitted by cell-to-cell contacts with low selective pressure on production of extracellular virions. To examine whether MVA would adapt to our production technology, we performed serial passaging of wild-type MVA in the suspension cultures and observed a gradual increase of titers.

    Further characterization revealed that 74% of virions were found in the supernatant when using a passaged virus isolate in suspension cultures. In contrast, only 4% of virions were detected in the supernatant after infection with wild-type MVA. The virus strain obtained by passage on suspension cells was named MVA-CR.

    To investigate possible molecular mechanisms for the observed changes in phenotype, genome sequences of different passages were determined. This analysis revealed three mutations in MVA-CR compared to wild-type MVA (Figure 2A). We observed 1) no silent mutations and 2) only the three nonsynonymous substitutions in a recovered contiguous stretch of 135 kb in a population of viruses that was repeatedly passaged in the novel production system. This result highlights a remarkable stability of MVA.

    The observed mutations affect structural proteins of MVA. The A3L gene product is a major core protein of vaccinia virus and is thus essential for morphogenesis. The A9L gene product may act as a linker between the inner viral envelope and the core structure. The A34R gene encodes a glycoprotein in the outer envelope of cell-associated virions and has been implicated in facilitating release of infectious units into the extracellular space.

    The virus isolate obtained from passage 19 in the suspension cell culture was cloned and designated MVA-CR19 (Figures 2B and 2C). In adherent cell monolayers, infection with MVA-CR19 resulted in plaques with more prominent comet formation (consistent with improved shedding of virus) and decreased propensity for syncitia formation.

  • Click Image To Enlarge +
    Figure 3. Less pronounced pH-induced formation of cell fusion suggests that MVA-CR19 has a greater capacity to escape the host cell than does wild-type MVA (MVA WT). (Modified from Jordan & Sandig, 2013, BMC Proceedings 7 (Suppl. 6): O1.)

    This effect (Figure 3) is also consistent with a model of enhanced release of MVA-CR19. If a certain number of virions remain cell-associated, a shift in pH can expose the viral fusion apparatus in such a way that these forms of infectious units induce merging of neighboring cell membranes.

    If a larger number of virions do not remain cell-associated, the capacity for pH-induced syncitia formation is expected to be reduced.

    In additional studies, adherent cultures of various species were infected. The new strain exhibited a similar host-cell restriction as wild-type MVA, suggesting that the safety profile has been maintained. Other studies, which were conducted in small-scale bioreactors, confirmed that plaque-purified MVA-CR19 produces high titers.

    We have also inserted the mutations of MVA-CR singly and in various combinations back into wild-type MVA to examine a cooperation of the mutations for the observed phenotype.

  • Conclusions

    The MVA-CR phenotype is advantageous for several reasons. Although the current production process with induced aggregates is efficient and robust, fewer manipulations are required if the additional medium feed step can be omitted. Determination of cell concentrations and cell viability for process monitoring is also facilitated in the absence of aggregates.

    Finally, harvesting can be simplified if viruses are isolated only from the supernatant. This leads to additional processing options that consist of fewer sources for contamination and reduces complexity in the material for downstream processing.



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