Masayuki Fukumura

Human Parainfluenza Virus Type 2 Vector Induces Dendritic Cell Maturation Without Viral RNA Replication/Transcription

The dendritic cell (DC), a most potent antigen-presenting cell, plays a key role in vaccine therapy against infectious diseases and malignant tumors. Although advantages of viral vectors for vaccine therapy have been reported, potential risks for adverse effects prevent them from being licensed for clinical use. Human parainfluenza virus type 2 (hPIV2), one of the members of the Paramyxoviridae family, is a nonsegmented and negative-stranded RNA virus. We have developed a reverse genetics system for the production of infectious hPIV2 lacking the F gene (hPIV2ΔF), wherein various advantages for vaccine therapy exist, such as cytoplasmic replication/transcription, nontransmissible infectivity, and extremely high transduction efficacy in various types of target cells. Here we demonstrate that hPIV2ΔF shows high transduction efficiency in human DCs, while not so high in mouse DCs. In addition, hPIV2ΔF sufficiently induces maturation of both human and murine DCs, and the maturation state of both human and murine DCs is almost equivalent to that induced by lipopolysaccharide. Moreover, alkylating agent β-propiolactone-inactivated hPIV2ΔF (BPL-hPIV2ΔF) elicits DC maturation without viral replication/transcription. These results suggest that hPIV2ΔF may be a useful tool for vaccine therapy as a novel type of paramyxoviral vector, which is single-round infectious vector and has potential adjuvant activity.

Vero/BC-F: an efficient packaging cell line stably expressing F protein to generate single round-infectious human parainfluenza virus type 2 vector

A stable packaging cell line (Vero/BC-F) constitutively expressing fusion (F) protein of the human parainfluenza virus type 2 (hPIV2) was established for production of the F-defective and single round-infectious hPIV2 vector in a strategy for recombinant vaccine development. The F gene expression has not evoked cytostatic or cytotoxic effects on the Vero/BC-F cells and the F protein was physiologically active to induce syncytial formation with giant polykaryocytes when transfected with a plasmid expressing hPIV2 hemagglutinin-neuraminidase (HN). Transduction of the F-defective replicon RNA into the Vero/BC-F cells led to the release of the infectious particles that packaged the replicon RNA (named as hPIV2ΔF) without detectable mutations, limiting the infectivity to a single round. The maximal titer of the hPIV2ΔF was 6.0 × 108 median tissue culture infections dose per ml. The influenza A virus M2 gene was inserted into hPIV2ΔF, and the M2 protein was found to be highly expressed in a human lung cancer cell line after transduction. Furthermore, in vivo airway infection experiments revealed that the hPIV2ΔF was capable of delivering transgenes to hamster tracheal cells. Thus, non-transmissible or single round-infectious hPIV2 vector will be potentially applicable to human gene therapy or recombinant vaccine development.