Viral Pathogenesis, Recombinant Vaccines, and Oncolytic Virotherapy: Applications of the Canine Distemper Virus Reverse Genetics System

Abstract

Canine distemper virus (CDV) is a highly contagious pathogen transmissible to a broad range of terrestrial and aquatic carnivores. Despite the availability of attenuated vaccines against CDV, the virus remains responsible for outbreaks of canine distemper (CD) with significant morbidity and mortality in domesticated and wild carnivores worldwide. CDV uses the signaling lymphocytic activation molecule (SLAM, or CD150) and nectin-4 (PVRL4) as entry receptors, well-known tumor-associated markers for several lymphadenomas and adenocarcinomas, which are also responsible for the lysis of tumor cells and apparent tumor regression. Thus, CDV vaccine strains have emerged as a promising platform of oncolytic viruses for use in animal cancer therapy. Recent advances have revealed that use of the CDV reverse genetic system (RGS) has helped increase the understanding of viral pathogenesis and explore the development of recombinant CDV vaccines. In addition, genetic engineering of CDV based on RGS approaches also has the potential of enhancing oncolytic activity and selectively targeting tumors. Here, we reviewed the host tropism and pathogenesis of CDV, and current development of recombinant CDV-based vaccines as well as their use as oncolytic viruses against cancers.

Keywords: canine distemper virus; pathogenesis; recombinant vaccine; reverse genetic system; virotherapy.

Figure 1

Reverse genetic system for the recovery of a recombinant canine distemper virus (CDV) wild-type strain. The five recombinant plasmids used for the recovery of a recombinant CDV strain, including one T7-promoter driven full-length cDNA expressing plasmid containing the enhanced green fluorescence protein (EGFP) gene inserted between the fusion (F) and hemagglutinin (H) genes, four cytomegalovirus (CMV) promoter-governed supporting plasmids expressing the nucleocapsid (N), phospo (P), and large (L) proteins respectively, and the T7 polymerase plasmid (a), which could be replaced by baby hamster kidney cells stably expressing T7 RNA polymerase (BSR-T7) or the modified vaccinia Ankara (MVA-T7) virus (b). The recovery process for the generation of the recombinant CDV is as follows: All plasmids are used for the simultaneous cotransfection of 293 cells (a), or BSR-T7 cells or MVA-T7 infected cells (b), followed by identification of the expression of EGFP in infected foci, and the overlay of B95a or Vero-SLAM (Vero cells expressing the signaling lymphocytic activation molecule [SLAM] receptor) cells for subsequent viral propagation.

Figure 2

Schematic of the entry cells and tissues, dissemination, and shedding process of CDV in infected dogs. (a) CDV infects macrophages or dendritic cells of the respiratory epithelium by binding with SLAM; (b) immune cells of the lymph nodes are infected and then the CDV disseminates to the spleen, thymus, etc.; (c) CDV further infects epithelial tissues by binding to the receptor nectin-4 of epithelial cells of the basolateral surface; (d) CDV is released from the apical surface of epithelial cells.

Figure 3

Possible mechanisms of the lysis of tumor cells upon infection with an oncolytic virus. (a) Binding to receptors initiates the internalization of the virus, which then release the viral nucleic acid, starting viral proliferation and release, leading to the apoptosis of tumor cells. (b) A pathogen-associated molecular pattern stimulates the production of antiviral cytokines (IFN-γ, IL-1, IL-6, IL-12, tumor necrosis factor [TNF]), which leads to the activation of immune cells, mediating cytotoxicity and phagocytosis. Meanwhile, major histocompatibility complex (MHC)-I-mediated presentation of viral proteins activates CD8+ cytotoxic T-cells, triggering lysis of the oncolytic virally-infected tumor cells. (c) Secretion of IFN-γ and IL-12 might initiate the complex interplay between IFN-γ, IL-12, natural killer (NK)-cells, and IFN-γ-inducible protein (IP-10), leading to the eventual limitation of tumor angiogenesis. Moreover, externalization of vasostatin is also involved in the inhibition of tumor angiogenesis. The arrows indicate the various steps and the components of an oncolytic virus during exerting the lysis of tumor cells, with the bar indicating their participation inhabiting the tumor angiogenesis.