Induction of potent human immunodeficiency virus type 1-specific T-cell-restricted immunity by genetically modified dendritic cells

Abstract

A novel technology combining replication- and integration-defective human immunodeficiency virus type 1 (HIV-1) vectors with genetically modified dendritic cells was developed in order to induce T-cell immunity. We introduced the vector into dendritic cells as a plasmid DNA using polyethylenimine as the gene delivery system, thereby circumventing the problem of obtaining viral vector expression in the absence of integration. Genetically modified dendritic cells (GMDC) presented viral epitopes efficiently, secreted interleukin 12, and primed both CD4(+) and CD8(+) HIV-specific T cells capable of producing gamma interferon and exerting potent HIV-1-specific cytotoxicity in vitro. In nonhuman primates, subcutaneously injected GMDC migrated into the draining lymph node at an unprecedentedly high rate and expressed the plasmid DNA. The animals presented a vigorous HIV-specific effector cytotoxic-T-lymphocyte (CTL) response as early as 3 weeks after a single immunization, which later developed into a memory CTL response. Interestingly, antibodies did not accompany these CTL responses, indicating that GMDC can induce a pure Th1 type of immune response. Successful induction of a broad and long-lasting HIV-specific cellular immunity is expected to control virus replication in infected individuals.

FIG. 1

Characterization of the antigen. (a) Molecular clone encoding the integrase-defective HIV-1 (LW/int) vector. This plasmid can express full-length Tat, Rev, Nef, Vpr, Vpu, Vif, Gag, reverse transcriptase, and envelope proteins derived from the HIV-1 LW primary isolate (22) and a truncated integrase protein. LTR, long terminal repeat. (b through d) Infection of primary human lymphocytes (b), macrophages (c), and DC (d) with wild-type HIV-1, LW (⧫), and the integrase-mutant retrovirus vector LW/int (564). In contrast to the parental wild-type virus (LW), the integrase-mutant virus was unable to induce productive infection in primary human cells.

FIG. 1

Characterization of the antigen. (a) Molecular clone encoding the integrase-defective HIV-1 (LW/int) vector. This plasmid can express full-length Tat, Rev, Nef, Vpr, Vpu, Vif, Gag, reverse transcriptase, and envelope proteins derived from the HIV-1 LW primary isolate (22) and a truncated integrase protein. LTR, long terminal repeat. (b through d) Infection of primary human lymphocytes (b), macrophages (c), and DC (d) with wild-type HIV-1, LW (⧫), and the integrase-mutant retrovirus vector LW/int (564). In contrast to the parental wild-type virus (LW), the integrase-mutant virus was unable to induce productive infection in primary human cells.

FIG. 2

HIV-1 vector expressing GMDC can prime naïve T cells in vitro. (a) Characterization of monocyte-derived DC by isotype control and antibody staining. (b) GMDC expressing the HIV-1 vector. DC were transduced with plasmid DNA containing wild-type HIV-1 (pLW) (⧫), a plasmid carrying the integrase-defective mutant (pLW/int) (■), and a control plasmid encoding the green fluorescent protein (Clontech) (▴) using PEI-mediated gene delivery. (c) Priming of naïve T cells (TC) by DC, GMDC, and DC pulsed with hi-HIV-1. IFN-γ production was analyzed in T cells with a flow cytometer 3 days after priming.

FIG. 3

Characterization of GMDC-primed T cells. Naïve T cells were primed with GMDC transduced with either LW/int or control pGFP plasmid DNA and cultured for 14 days. T cells were restimulated with autologous B-LCL cells primed with either Zn finger-inactivated HIV or microvesicle control (gift from Jeff Lifson, NCI, Frederick, Md.). (Top panel) Gating of CD4⁺ and CD8⁺ T cells on the CD3⁺ population. Labels on the right and the top indicate antigens used for priming and restimulation, respectively.

FIG. 4

Characterization of in vitro-induced HIV-specific CTL. (a) HIV-specific lysis. Naïve autologous lymphocytes were stimulated with GMDC (left) and control (right) autologous DCs. Seven days later, primed T cells were tested for HIV-specific CTL activity against autologous target macrophages (□) and macrophages pulsed with Gag (p55) protein (■). (b) Analysis of in vitro-primed HIV-1-specific CTL. Effector T cells induced by GMDC were tested against autologous target macrophages pulsed with the Gag protein (p55) (effector/target ratio, 50:1) in the presence and absence of CD8-specific antibodies (CD8AB). (c) GMDC activation of T cells specific to the dominant HLA-A*02-restricted Gag CTL epitope. GMDC derived from a naïve HLA-A*02 individual were used to prime autologous T cells. Seven days later the primed T cells were restimulated overnight with either autologous B-LCL cells (left) or B-LCL cells pulsed with HLA-A∗02-restricted p17 Gag_77–85 (SLYNTVATL) peptide (right).

FIG. 5

Increased IL-12 secretion by GMDC activates T-cell priming and CTL response. (a) Increased IL-12 production by GMDC. DC (left panel) and GMDC (right panel) were stained with antibodies, analyzed by flow cytometry, and plotted as IL-12 (20C2; PharMingen) versus Class II (Immu-375; Immunotech). (b) IL-12 augments Th1-type primary immune responses. Naïve peripheral lymphocytes (TC) were primed with GMDC (1:10 ratio) in the presence (right) and absence (left) of 5 ng of IL-12 (R&D) per ml. Seven days later, HIV-1-specific CTL were tested against autologous target DC (17) pulsed with hen egg lysozyme (■) or with hi-HIV-1 (⧫). The panels on the right of the graphs show the percentages of IFN-γ-producing CD8⁺ T cells after 3 days of priming with GMDC in the presence (right) and absence (left) of IL-12.

FIG. 6

GMDC induce a vigorous CTL response in nonhuman primates. (a) Localization of GMDC in the draining lymph node. (Left) DNA-expressing GMDC are stained white with an antisense probe (dark field). (Right) Interdigitating DC (stained brown with p55 antibody [55K-2 Dako]) expressing the DNA (black dots). Control in situ hybridization (sense probe) on parallel sections was negative. (b) HIV-1-specific CTL after immunization with GMDC in two macaques. Effector CTL were measured 3 weeks after immunization in the absence of restimulation. Memory CTL were measured 7 months after immunization (restimulation with Gag). The target cells were autologous B-LCL cells infected with HIV-1 Gag (■), HIV-1 Gag-Pol-Env (▴), or control vaccinia recombinant virus (◊). (c) Kinetics of the CTL responses showing HIV-1 Gag-specific effector CTL and memory CTL of one animal (96280). Solid bars, specific lysis of target autologous B-LCL cells infected with HIV-1-Gag vaccinia virus; open bars specific lysis of target autologous B-LCL cells infected with control vaccinia virus. W., weeks; m., months.

FIG. 6

GMDC induce a vigorous CTL response in nonhuman primates. (a) Localization of GMDC in the draining lymph node. (Left) DNA-expressing GMDC are stained white with an antisense probe (dark field). (Right) Interdigitating DC (stained brown with p55 antibody [55K-2 Dako]) expressing the DNA (black dots). Control in situ hybridization (sense probe) on parallel sections was negative. (b) HIV-1-specific CTL after immunization with GMDC in two macaques. Effector CTL were measured 3 weeks after immunization in the absence of restimulation. Memory CTL were measured 7 months after immunization (restimulation with Gag). The target cells were autologous B-LCL cells infected with HIV-1 Gag (■), HIV-1 Gag-Pol-Env (▴), or control vaccinia recombinant virus (◊). (c) Kinetics of the CTL responses showing HIV-1 Gag-specific effector CTL and memory CTL of one animal (96280). Solid bars, specific lysis of target autologous B-LCL cells infected with HIV-1-Gag vaccinia virus; open bars specific lysis of target autologous B-LCL cells infected with control vaccinia virus. W., weeks; m., months.