Integrase-defective lentiviral vectors encoding cytokines induce differentiation of human dendritic cells and stimulate multivalent immune responses in vitro and in vivo

Abstract

Integrase-defective lentiviral vectors (ID-LVs) show several hallmarks of conventional lentiviral vectors such as absence of cytotoxic effects and long-term expression in non-replicating target cells. The integration rate of ID-LVs into the genome of target cells is dramatically reduced, which enhances safety and opens perspectives for their use in vaccine development. ID-LVs have been shown to be effective vaccines in mouse models, but functional studies with human cells in vitro and in vivo are lacking. Here, we evaluated whether ID-LVs expressing combinations of cytokines (GM-CSF/IL-4 or GM-CSF/IFN-α) used to transduce human monocytes would result in functional "induced dendritic cells" (iDCs). Overnight transduction of monocytes with high titer ID-LVs generated highly viable (14 days) and immunophenotypically stable iDCs expressing GM-CSF/IL-4 ("SmartDCs") or GM-CSF/IFN-α ("SmyleDCs"). SmartDCs and SmyleDCs maintained in vitro continuously secreted the transgenic cytokines and showed up-regulation of several endogenously produced inflammatory cytokines (IFN-γ, IL-2, -5, -6, and -8). Both iDC types potently stimulated T cells in mixed lymphocyte reactions at levels comparable to conventional DCs (maintained with exogenous cytokines). A single in vitro stimulation of CD8(+) T cells with autologous SmartDCs or SmyleDCs pulsed with peptide pools of pp65 (a human cytomegalovirus antigen) resulted in high expansion of central memory and effector memory CTLs reactive against different pp65 epitopes. We further evaluated the effects of SmartDCs and SmyleDCs to expand anti-pp65 CTLs in vivo using immune deficient NOD/Rag1((-/-))/IL-2rγ((-/-)) (NRG) mice. NRG mice immunized subcutaneously with SmartDCs or SmyleDCs co-expressing the full-length pp65 were subsequently infused with autologous CD8(+) T cells. Both types of iDCs effectively stimulated human CTLs reactive against different pp65 antigenic determinants in vivo. Due to the simplicity of generation, robust viability and combined capacity to stimulate homeostatic, antigenic and multivalent responses, iDCs are promising vaccines to be explored in immunization of lymphopenic patients in the post-transplantation setting.

Dendritic Cell–Mediated Immune Humanization of Mice: Implications for Allogeneic and Xenogeneic Stem Cell Transplantation

“….IL-2rg 2/2 (NRG) mice (.4 wk), secreted several inflammatory cytokines, and effectively enhanced the expansion of adoptively transferred human PBL (huPBL) for anti-pp65 T cell activation in vivo (13). In this study, we compared the effects of immunization with conventional (mo-DC) versus SmyleDC after autologous G-CSF-mobilized human CD34 + HCT into NRG mice.…”

“…The self-inactivating (SIN) lentiviral backbone vector and the monocistronic vectors expressing the CMV-pp65 were described previously (13,15). Construction of the bicistronic lentiviral vector expressing the human (hu)GM-CSF and of the huIFN-a (LV-G2a) interspaced with a P2A element (RRL-cPPT-CMV-hGMCSF-P2A-huIFN-a) was constructed and extensively characterized as described previously (13).…”

“…Construction of the bicistronic lentiviral vector expressing the human (hu)GM-CSF and of the huIFN-a (LV-G2a) interspaced with a P2A element (RRL-cPPT-CMV-hGMCSF-P2A-huIFN-a) was constructed and extensively characterized as described previously (13). The structural integrity of all constructs was reconfirmed by restriction digestion and sequencing analysis of the promoters and transgenes.…”

“…Self-differentiated myeloid-derived lentivirus-induced DC (SmyleDC) can be rapidly generated after overnight infection of monocytes with integrase-defective lentiviral vectors (ID-LV) coexpressing human GM-CSF, IFN-a, and the CMV pp65 antigenic protein (13). The pp65 tegument protein is known to be efficiently presented by several HLA types to public and private TCRs (14).…”

Advances in homology directed genetic engineering of human pluripotent and adult stem cells

“…Furthermore, after 20-24 mo, the VLCFA levels in patients decreased by up to 39%. Finally, IDLVs encoding for cytokines have been used to induce human monocytes to differentiate into dendritic cells and the resulting induced dendritic cells were functional and capable of stimulating multivalent immune responses in vitro and in vivo [23] . In addition to developing non-integrative vectors, researchers are also pursuing approaches to engineer safer viral vectors by overriding the intrinsic preferences of viral genomic integration for transcriptional start sites (TSS); for example, Lim et al [24] discovered that the addition of DNA binding domains at key sites within retroviral vectors shifted their integration patterns toward regions where TSS are relatively rare.…”