A Deleted Deletion Site in a New Vector Strain and Exceptional Genomic Stability of Plaque-Purified Modified Vaccinia Ankara (MVA)

Abstract

Vectored vaccines based on highly attenuated modified vaccinia Ankara (MVA) are reported to be immunogenic, tolerant to pre-existing immunity, and able to accommodate and stably maintain very large transgenes. MVA is usually produced on primary chicken embryo fibroblasts, but production processes based on continuous cell lines emerge as increasingly robust and cost-effective alternatives. An isolate of a hitherto undescribed genotype was recovered by passage of a nonplaque-purified preparation of MVA in a continuous anatine suspension cell line (CR.pIX) in chemically defined medium. The novel isolate (MVA-CR19) replicated to higher infectious titers in the extracellular volume of suspension cultures and induced fewer syncytia in adherent cultures. We now extend previous studies with the investigation of the point mutations in structural genes of MVA-CR19 and describe an additional point mutation in a regulatory gene. We furthermore map and discuss an extensive rearrangement of the left telomer of MVA-CR19 that appears to have occurred by duplication of the right telomer. This event caused deletions and duplications of genes that may modulate immunologic properties of MVA-CR19 as a vaccine vector. Our characterizations also highlight the exceptional genetic stability of plaque-purified MVA: although the phenotype of MVA-CR19 appears to be advantageous for replication, we found that all genetic markers that differentiate wildtype and MVA-CR19 are stably maintained in passages of recombinant viruses based on either wildtype or MVA-CR.

A high cell density perfusion process for Modified Vaccinia virus Ankara production: Process integration with inline DNA digestion and cost analysis

“…An immortalized Muscovy duck retina suspension AGE1.CR.pIX cell line was used as a host for production of MVA‐CR19.GFP (infectious titer: 4.1 × 10 8 TCID 50 /ml), which contains a green‐fluorescent‐protein insertion cassette (Jordan et al, 2020). Chemically‐defined CD‐U5 medium (Biochrom‐Merck) supplemented with 2 mM l ‐glutamine (Sigma‐Aldrich) and 10 ng/ml recombinant insulin‐like growth factor (LONG‐R 3 IGF, 91590C; Sigma‐Aldrich) was used for cell growth.…”

Production of Modified Vaccinia Ankara Virus by Intensified Cell Cultures: A Comparison of Platform Technologies for Viral Vector Production

“…[ 16,17 ] To facilitate process intensification and enable continuous virus harvesting an MVA virus strain with reduced dependency on direct cell‐to‐cell contacts would be beneficial. Such a strain, MVA‐CR19, has been obtained with help of the avian suspension cells [ 18,19 ] that are used in the present study.…”

A Rapid, High Throughput, Viral Infectivity Assay using Automated Brightfield Microscopy with Machine Learning

“…9) were captured on a Cytation 1 37 at 20x magnification. Modified vaccinia ankara (MVA) wildtype (WT) and CR-infected CR.pIX cells 53 were prepared as described in Jordan et al 54 , and the images were captured with a Synentec NyONE 36 at 20x. Human immunodeficiency virus (HIV)-infected TZMbl cells were prepared as described by Wang et al 55 and the images captured with a Cytation 5 38 at 20x.…”