Hazard Characterization of Modified Vaccinia Virus Ankara Vector: What Are the Knowledge Gaps?

Okeke, Malachy I.; Okoli, Arinze S.; Diaz, Diana; Offor, Collins; Oludotun, Taiwo G.; Tryland, Morten; Bøhn, Thomas; Moens, Ugo

The results from these studies will shed light to the host restriction defect of MVA in human cells and will be of relevance to the ERA of the MVA vector if the results of the in vitro studies are replicated in vivo. Research on identifying the molecular determinants of the MVA host cell defect in human and other mammalian cells have stagnated presumably because of the belief/assumptions that MVA cannot undergo productive infection in human cells. At present, the role of insertions/deletions (indels) and single nucleotide polymorphism (SNP) in MVA non-productive infection of human cells either singly or in combination with the six major deletions remain uninvestigated. There is also no attempt to re-adapt MVA to multiply in human cells as a way of understanding the virus and host cell determinants of host cell tropism. Since MVA host cell restriction in human and other mammalian cells was achieved by multiple serial passage in CEF, we hypothesized that MVA can be re-adapted to multiply in human cells by multiple serial passaging in human cell lines. We infected nonpermissive human Caco-2 (ATCC HTB-37) cells [52] with MVA (ATCC VR-1508) at a low multiplicity of infection (m.o.i.) of 0.01 and blindly passaged it for 40 times. We were able to show 2 to 3 log fold increase in virus titer from p28 to p40 ( Figure 1 ) and production of mature virions ( Figure 2 ). We were able to isolate plaque purified MVA variants from p28 to p40 that were able to undergo efficient productive infection in Caco-2 cells. These observations demonstrate that at least in Caco-2 cells, the host defect of MVA may be reversible. Alternatively, it may indicate that in spite of over 572 passages and extensive plaque purification, variants capable of productive infection are still present within the MVA vaccine stock (ATCC VR-1508). Currently, we are investigating the productive infection potential of these MVA variants in other human cell lines and primary human cells as well as sequencing their genomes and comparing it to the non-passaged MVA (ATCC VR 1508). Future work will extend these experiments with MVA-VR1508 to MVA-BN and examine the attenuation/virulence potential of these Caco-2 productive MVA variants in immunocompetent and immunocompromised in vivo infection models. The results from these studies will shed light to the host restriction defect of MVA in human cells and will be of relevance to the ERA of the MVA vector if the results of the in vitro studies are replicated in vivo.