The clinical implementation of gene therapy requires large-scale production of viral vector stocks (VS) derived from packaging cell lines. Upon scaling-up, maintenance of high viral titers and filtration of the VS become significantly challenging. Thus, production schemes amenable to straightforward validation must be developed. To this end, we have established a semi-closed process to manufacture batches of 7 l or more of clinical-grade oncoretroviral VS using 10-tray Cell Factories. Using a peristaltic pump, the VS are collected on 3 consecutive days, filtered, pooled and stored frozen. To ensure the absence of viable vectorproducing cells (VPCs) from each VS unit-dose, we undertook an orthogonal log-removal validation study to demonstrate the ability of both the filtration system to remove viable cells and the VS freezing process to inactivate them. We demonstrate a total VPC-reduction of 11.6 log, thus insuring the absence of contaminating VPCs in transduced clinical samples. We also show that this production process generates stable VS that can be stored at À801C for more than 3 years. Importantly, this relatively simple and affordable process can be customized to generating large volume of VS for small animal or nonhuman primate studies. This methodology is not limited to the generation of cell-free clinical oncoretroviral VS, and can be applied to other types of vectors produced in packaging cell lines, such as lentiviral vectors. Gene Therapy (2006) 13, 95-100.

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Oncoretroviral vectors encoding either full-length Ag or a corresponding immunodominant peptide were expressed in Langerhans-type dendritic cells (LCs) differentiated from CD34+ progenitors. We used human CMV as a model Ag restricted by HLA-A*0201 to define parameters for eventual expression of cancer Ags by LCs for active immunization against tumors. Stimulation by CMVpp65495–503-pulsed LCs, CMVpp65495–503-transduced LCs, and full-length CMVpp65-transduced LCs respectively increased tetramer-reactive T cells with an effector memory phenotype by 10 ± 11, 34 ± 21, and 51 ± 24-fold (p < 0.05) from CMV-seropositive donors. CMV-specific CD8+ CTLs achieved respective frequencies of 231 ± 102, 583 ± 219, and 714 ± 281 spot-forming cells per 105 input cells (p < 0.01) in ELISPOT assays for IFN-γ secretion. LCs expressing full-length Ag stimulated greater lytic activity than either peptide-transduced or peptide-pulsed LCs (p < 0.05), all in the absence of exogenous cytokines. pp65-transduced LCs presenting class I and II MHC-restricted epitopes expanded IFN-γ-secreting CD4+ T cells, whereas pp65495–503-transduced LCs did not. CD4+ T cell numbers even declined after stimulation by pp65495–503 peptide-pulsed LCs. CD4+ T cell depletion confirmed their contribution to the more robust CTL responses. LCs, transduced with a retroviral vector encoding full-length Ag, stimulate potent CTLs directed against multiple epitopes in a CD4+ Th cell-dependent manner.

Intranasal application of vesicular stomatitis virus (VSV) results in the initial infection of the olfactory receptor neurons and a rapid progression of the virus through the mouse central nervous system (CNS). Interleukin-18 (IL-18) is an 18.3-kd cytokine that induces interferon gamma (IFN-gamma) production in mice. IL-18 is synthesized as an inactive precursor that is cleaved and activated by caspase-1/interleukin-1beta converting enzyme (ICE). IL-18 shares several biological properties with IL-12, including the ability to induce IFN-gamma production in T lymphocytes and natural killer (NK) cells. In the CNS, microglia and astrocytes produce IL-18 and IL-12. We have previously shown that IL-12 promotes recovery from VSV encephalitis. This led us to examine the potential role of IL-18 in the pathogenesis of VSV encephalitis. We show that both IL-18 and caspase-1 mRNA are consistently present in the CNS of mice. The addition of exogenous IL-18 to cell cultures does not affect the production of VSV, and addition of exogenous IL-18 at the time of infection does not alter the morbidity or mortality of BALB/c mice. In vitro studies with neutralizing monoclonal antibody to IL-18 had no effect. From these results we conclude that in this system and under the experimental conditions used, unlike IL-12 and IFN-gamma, IL-18 does not play a significant role in the host response to VSV infection.

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