Use of novel insertion sites for the development of polyvalent ORFV D1701-V vectored vaccines

Abstract

Viral vector-based vaccines are promising to elicit strong cellular and humoral immune responses. Within the genus Parapoxvirus of the Poxviridae, the Orf Virus (ORFV) strain D1701-V comprises various properties particularly favorable for the development of a vector platform technology and was shown to facilitate various vaccination approaches. To enhance viral vectors’ immunogenicity, current strategies implement the use of immunomodulatory elements and the elimination of viral immunomodulatory genes still present in the viral genome. To allow the insertion of multiple transgenes into a single vector, the present work examines the suitability of novel insertion sites for transgene expression by simultaneous deletion of ORFV D1701-V encoded genes non-essential for viral replication. Novel D1701-V deletion mutants were subjected to detailed characterization of the genetic stability of inserted transgenes, their growth behavior and capability to induce transgene expression in different target cells in vitro. Additionally, the D1701-V mutants’ immunogenicity was analyzed by their ability to activate pro-inflammatory pathways, peripheral blood mononuclear and antigen presenting cells, or to induce antigen-specific immune responses in vitro and in vivo. Additionally, proof-of-concept studies were performed to evaluate the suitability of ORFV D1701-V recombinants for the expression of immune checkpoint inhibitors (ICIs).