Stability of the HSV-2 US-6 Gene in the del II, del III, CP77, and I8R-G1L Sites in Modified Vaccinia Virus Ankara After Serial Passage of Recombinant Vectors in Cells.

Vajini N Atukorale,Jerry P Weir,Clement A Meseda

doi:10.3390/vaccines8010137

Abstract

The modified vaccinia virus Ankara (MVA), a severely attenuated strain of vaccinia virus, is a promising vector platform for viral-vectored vaccine development because of its attributes of efficient transgene expression and safety profile, among others. Thus, transgene stability in MVA is important to assure immunogenicity and efficacy. The global GC content of the MVA genome is 33%, and GC-rich sequences containing runs of C or G nucleotides have been reported to be less stable with passage of MVA vectors in cells. The production of recombinant MVA vaccines requires a number of expansion steps in cell culture, depending on production scale. We assessed the effect of extensive passage of four recombinant MVA vectors on the stability of the GC-rich herpes simplex type 2 (HSV-2) US6 gene encoding viral glycoprotein D (gD2) inserted at four different genomic sites, including the deletion (del) II and del III sites, the CP77 gene locus (MVA_009–MVA_013) and the I8R-G1L intergenic region. Our data indicate that after 35 passages, there was a reduction in gD2 expression from del II, del III and CP77 sites. Sequencing analysis implicated US6 deletion and mutational events as responsible for the loss of gD2 expression. By contrast, 85.9% of recombinant plaques expressed gD2 from the I8R-G1L site, suggesting better accommodation of transgenes in this intergenic region. Thus, the I8R-G1L intergenic region may be more useful for transgene insertion for enhanced stability.

Highlights

modified vaccinia virus Ankara (MVA) was derived by more than 570 passages of the chorioallantois vaccinia virus Ankara (CVA)in chicken embryo fibroblast (CEF) cells [1]
As MVA and MVA-vectored vaccines are produced in cell culture, we evaluated the stability of a transgene in MVA after several serial passages in cell culture
We evaluated the stability of US6 inserted in the I8R and G1L intergenic region by a more extensive serial passage of MVA18-gD2

Summary

Introduction

MVA was derived by more than 570 passages of the chorioallantois vaccinia virus Ankara (CVA). In chicken embryo fibroblast (CEF) cells [1]. Genome analysis showed that serial passaging of CVA in cells resulted in deletions and mutations in several regions of the genome, in the terminal regions. Genes located in the central region of the genome remained intact, as many are essential for virus replication. Six major deletions were mapped by restriction enzyme analyses and designated as del I through VI [2,3]. Other less prominent deletions as well as several mutations in the genome have been described [4]. The sum total of the deletions from CVA (GenBank #: AM501482)

Methods

Discussion

Conclusion