A Novel Naturally Occurring Tandem Promoter in Modified Vaccinia Virus Ankara Drives Very Early Gene Expression and Potent Immune Responses

Sonia T Wennier,Charlotte Steinhäußer,Paul Chaplin,Jürgen Hausmann,Nicole Mayländer,Ulrike Dirmeier,Ursula Wielert,Claudia Mnich,Robin Steigerwald,Kay Brinkmann,Gunnar F Kaufmann

doi:10.1371/journal.pone.0073511

Abstract

Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines.

Highlights

Replication-competent poxvirus vectors have been employed as vaccine vectors since the 1980s to induce immune responses against encoded foreign genes [1,2]
Construction of novel synthetic hybrid promoters The synthetic early-late hybrid promoter, pHyb, which contains one late A-type inclusion body (ATI) promoter element and five copies of the strong early promoter element derived from the p7.5k promoter, has been shown to induce earlier protein expression and stronger immune responses against an encoded transgene compared to the PrS and p7.5k promoters [23]
Since strong early expression has been shown to correlate with strong CD8 T cell responses from modified vaccinia virus Ankara (MVA), we focused on identifying promoters for genes with reported strong early and continuous expression during vaccinia virus (VACV) infection and which were present in MVA

Summary

Introduction

Replication-competent poxvirus vectors have been employed as vaccine vectors since the 1980s to induce immune responses against encoded foreign genes [1,2]. MVA was derived from chorioallantois vaccinia Ankara (CVA), a Turkish smallpox vaccine strain, by serial passaging (> 570 passages) in primary chicken embryo fibroblast (CEF) cells [6,7]. Host range restriction of this virus in cell culture is partially governed by the six major deletions and is to a large extent based on additional mutations among the multitude of genes with altered amino acid sequence compared to the parental CVA virus [11,12]. MVA is well tolerated and immunogenic when administered to immunocompromised patients infected with human immunodeficiency virus (HIV), highlighting its potential as a safe vector for the development of vaccine and gene therapy candidates [16]

Methods

Results

Conclusion