Research

Streamlined design of a self-inactivating feline immunodeficiency virus vector for transducing ex vivo dendritic cells and T lymphocytes

Mauro Pistello , Laura Vannucci , Alessia Ravani , Francesca Bonci , Flavia Chiuppesi , Barbara Del Santo , Giulia Freer and Mauro Bendinelli

Retrovirus Center and Virology Section, Department of Experimental Pathology, University of Pisa, Pisa, Italy

author email corresponding author email

Genetic Vaccines and Therapy 2007, 5:8doi:10.1186/1479-0556-5-8

Published:	19 September 2007

Abstract

Background

Safe and efficient vector systems for delivering antigens or immunomodulatory molecules to dendritic cells (DCs), T lymphocytes or both are considered effective means of eliciting adaptive immune responses and modulating their type, extent, and duration. As a possible tool toward this end, we have developed a self-inactivating vector derived from feline immunodeficiency virus (FIV) showing performance characteristics similar to human immunodeficiency virus-derived vectors but devoid of the safety concerns these vectors have raised.

Methods

The pseudotyped FIV particles were generated with a three-plasmid system consisting of: the packaging construct, providing Gag, Pol and the accessory proteins; the vector(s), basically containing FIV packaging signal (ψ), Rev responsive element, R-U5 region at both ends, and the green fluorescent protein as reporter gene; and the Env plasmid, encoding the G protein of vesicular stomatitis virus (VSV-G) or the chimeric RD114 protein. Both packaging and vector constructs were derived from p34TF10, a replication competent molecular clone of FIV. The pseudotyped particles were produced by transient transfection in the Crandell feline fibroblast kidney (CrFK) or the human epithelial (293T) cell line.

Results

To broaden its species tropism, the final vector construct was achieved through a series of intermediate constructs bearing a longer ψ, the FIV central polypurin tract sequence (cPPT), or the woodchuck hepatitis post-regulatory element (WPRE). These constructs were compared for efficiency and duration of transduction in CrFK or 293T cells and in the murine fibroblast cell line NIH-3T3. Whereas ψ elongation and cPPT addition did not bring any obvious benefit, insertion of WPRE downstream GFP greatly improved vector performances. To maximize the efficiency of transduction for ex-vivo murine DCs and T-lymphocytes, this construct was tested with VSV-G or RD114 and using different transduction protocols. The results indicated that the FIV construct derived herein stably transduced both cell types, provided that appropriate vector makeup and transduction protocol were used. Further, transduced DCs underwent changes suggestive of an induced maturation.

Conclusion

In contrast to previously described FIV vectors that were poorly efficient in delivering genetic material to DCs and T lymphocytes, the vector developed herein has potential for use in experimental immunization strategies.