Developing a platform system for gene delivery: amplifying virus-like particles (AVLP) as an influenza vaccine

Huiling Wei,S Mark Tompkins,Sateesh Kristhnamurthy,Paul B Mccray,Kaori Sakamoto,Zhenhai Chen,Steve Stice,Zhuo Li,Seth Andrews,Cheryl Jones,Biao He,Andrew Elson,Shannon Phan,Mathew Abraham

doi:10.1038/s41541-017-0031-7

Abstract

Delivery of a gene of interest to target cells is highly desirable for translational medicine, such as gene therapy, regenerative medicine, vaccine development, and studies of gene function. Parainfluenza virus 5 (PIV5), a paramyxovirus with a negative-sense RNA genome, normally infects cells without causing obvious cytopathic effect, and it can infect many cell types. To exploit these features of PIV5, we established a system generating self-amplifying, virus-like particles (AVLP). Using enhanced green fluorescent protein (EGFP) as a reporter, AVLP encoding EGFP (AVLP–EGFP) successfully delivered and expressed the EGFP gene in primary human cells, including stem cells, airway epithelial cells, monocytes, and T cells. To demonstrate the application of this system for vaccine development, we generated AVLPs to express the HA and M1 antigens from the influenza A virus strain H5N1 (AVLP–H5 and AVLP–M1H5). Immunization of mice with AVLP–H5 and AVLP–M1H5 generated robust antibody and cellular immune responses. Vaccination with a single dose of AVLP–H5 and M1H5 completely protected mice against lethal H5N1 challenge, suggesting that the AVLP-based system is a promising platform for delivery of desirable genes.

Highlights

Advances in understanding diseases have created great opportunities to prevent, treat, and manage them
These results indicate that AVLP–enhanced green fluorescent protein (EGFP) is a versatile system that allows the expression of genes of interest in many primary cells and immortalized cell lines
Protein expression was only detected in the first round of infection, indicating that no infectious particles were produced from AVLP expressing HA of H5N1 (AVLP–H5) cells without transfecting the cells with Parainfluenza virus 5 (PIV5) M, F, and HN

Summary

INTRODUCTION

Advances in understanding diseases have created great opportunities to prevent, treat, and manage them. The V/P gene encodes two mRNA species that translate into the V and the P proteins through a non-templated insertion of two G residues at a specific site of the V/P gene during transcription.[2] The V protein plays important roles in viral pathogenesis, as well as in regulating viral RNA synthesis.[1,3] The fusion (F) protein, a glycoprotein, mediates both cell-to-cell and virus-to-cell fusion in a pH-independent manner. We have generated PIV5 VLPs that can deliver and amplify foreign genes in target cells without producing progeny (Amplifying Virus-Like Particle, AVLP). BHK (AVLP–EGFP) cell clones containing PIV5 AVLP genome were expanded under the selection of Hygromycin. M. Particles carrying the PIV5 AVLP genome packaged by PIV5 HN, F, and M were generated in the supernatants of transfected cells. The infected Vero cells were developed into stable cell lines carrying PIV5 AVLP genome [Vero (AVLP–EGFP)] under selection of Hygromycin.

RESULTS

DISCUSSION

10 ADDITIONAL INFORMATION