Construction of stable packaging cell lines for clinical lentiviral vector production.

Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 10(6) transducing units/ml can be harvested from the final producer clones, which can be increased to 10(8) TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors.

vFLIP from KSHV inhibits anoikis of primary endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) infection of endothelial cells is an early event in the aetiology of the endothelial cell tumour Kaposi's sarcoma (KS). We have examined the effect of the KSHV latent protein viral FLICE-like inhibitory protein (vFLIP) on dermal microvascular endothelial cell (MVEC) survival as vFLIP is expressed in the KSHV-infected cells within KS lesions. To do this, we have used a lentiviral vector to express vFLIP in MVECs in the absence of other KSHV proteins. vFLIP activates the classical NF-kappaB pathway in MVECs and causes nuclear translocation of RelA/p65. This NF-kappaB activation prevents detachment-induced apoptosis (anoikis) of MVECs but does not inhibit apoptosis induced by removal of essential survival factors, including vascular endothelial growth factor (VEGF). vFLIP expression inhibits anoikis in part by inducing the secretion of an additional paracrine survival factor(s). The implications of these results for KS development are discussed.

Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses.

Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2(+) cells after intravenous injection and in CD11c(+) dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8(+) T-cell response in HLA-A2 transgenic mice and stimulated a CD4(+) T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A(b). As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.

Immunization with a lentiviral vector stimulates both CD4 and CD8 T cell responses to an ovalbumin transgene.

Lentiviral vectors encoding antigens are promising vaccine candidates because they transduce dendritic cells (DC) in vivo and prime CTL responses. Here we examine their stimulation of antigen-specific CD4(+) T cells, critical for protective immunity against tumors or infectious disease. We constructed lentiviral vectors (lentivectors) expressing ovalbumin, which was secreted (OVA), cytoplasmic (OVAcyt), or fused to either invariant chain (Ii-OVA) or transferrin receptor (TfR-OVA) sequences, targeting the MHC class II presentation pathway. Murine DC infected with the various lentivectors could stimulate OT-I (CD8(+), OVA TCR transgenic) T cells and all except OVAcyt could also stimulate OT-II (CD4(+), OVA TCR transgenic) T cells in vitro. Direct injection of the OVA-, Ii-OVA-, or TfR-OVA-expressing vectors into mice resulted in a CD4(+) T cell response, as shown by expansion of adoptively transferred OT-II T cells and upregulation of CD44 on these cells. The Ii-OVA vector was the most potent inducer of IFN-gamma-secreting CD4(+) and CD8(+) T cells and was the only vector to protect mice completely from challenge with OVA-expressing tumor cells. Therefore directly injected lentivectors can stimulate CD4(+) T cells; both CD4(+) and CD8(+) responses can be enhanced by targeting the antigen to the MHC class II pathway.

Human immunodeficiency virus type 1 vectors with alphavirus envelope glycoproteins produced from stable packaging cells.

Alphavirus glycoproteins have broad host ranges. Human immunodeficiency virus type 1 (HIV-1) vectors pseudotyped with their glycoproteins could extend the range of tissues that can be transduced in both humans and animal models. Here, we established stable producer cell lines for HIV vectors pseudotyped with alphavirus Ross River virus (RRV) and Semliki Forest virus (SFV) glycoproteins E2E1. RRV E2E1-stable clones could routinely produce high-titer pseudotyped vectors for at least 5 months. SFV E2E1-stable clones, however, produced relatively low titers. We examined the properties of RRV E2E1-pseudotyped vectors [HIV-1(RRV)] and compared them with amphotropic murine leukemia virus Env- and vesicular stomatitis virus glycoprotein G-pseudotyped vectors. HIV-1(RRV) displayed a number of characteristics which would be advantageous in ex vivo and in vivo experiments, including resistance to inactivation by heat-labile components in fresh human sera and thermostability at 37 degrees C. Upon single-step concentration by ultracentrifugation of HIV-1(RRV), we could achieve vector stocks with titers up to 6 x 10(7) IU/ml. HIV-1(RRV) efficiently transduced cells from several different species, including murine primary dendritic cells, but failed to transduce human and murine T cells as well as human hematopoietic stem cells (HSC). These results indicate that HIV-1(RRV) could be used in a number of applications including animal model experiments and suggest that expression of RRV cellular receptors is limited or absent in certain cell types such as T cells and human HSC.