Abstract
ABSTRACT
Suitable animal models are necessary for testing new vaccination strategies and evaluate their protective efficacy in vivo against human pathogens.
Feline immunodeficiency virus (FIV) is a lentivirus that causes an immune deficiency syndrome in cats similar to that induced by HIV in humans and is therefore regarded as a valuable model for understanding the mechanisms of HIV pathogenesis and developing new therapeutic and vaccination approaches.
In this context we are currently developing a vaccination project based on a prime-boost protocol. The priming will be accomplished by using a plasmid DNA expressing FIV Env in combination with the gene coding for feline granulocyte macrophage colony stimulating factor (fGM-CSF), a cytokine involved in proliferation and differentiation of dendritic cells, which are specialized in antigen presentation to the effectors of the immune system. This phase will be followed by a boost with autologous lymphocytes expressing Env on their cellular surface and feline interferon-γ (fIFN-γ ), an immunoadjuvant that is capable of recruiting effector cells in vivo and potentiating cell-mediated responses against the viral antigen. The latter phase will be performed by transducing ex vivo lymphocytes with a replication-defective FIV-derived vector. Cells will be then reinfused into cats, previously immunized with plasmid DNA.
The aim of this study is to develop and functionally characterize priming and boost constructs and evaluate their efficiency in the delivery of the immunogens.
The priming construct was characterized by setting up specific assays to evaluate the biological activity of fGM-CSF and monitor Env expression in transfected cells.
For boosting we produced and characterized different versions of FIV- derived vectors, in order to optimize the transduction efficiency. A two-plasmid strategy was used for producing the vector: a packaging construct expressing Gag- Pol and a vector construct encoding Env and fIFN-γ . In vitro assays were carried out by using prototype vectors containing green fluorescent protein (GFP) as reporter gene in place of fIFN-γ to easily evaluate the transduction efficiency by flow
III
Abstract
cytometry. Vectors were tested in fibroblast and lymphoid cell lines by using two different methods for concentrating vector particles. We also characterized and compared vector constructs for the ability to express Env by Western Blot analysis in transfected and transduced cells.
IV
