Nonintegrating lentiviral vectors can effectively deliver ovalbumin antigen for induction of antitumor immunity

Abstract

It has been demonstrated that nonintegrating lentiviral vectors (NILVs) are efficient in maintaining transgene expression in vitro and in vivo. Gene delivery by NILVs can significantly reduce nonspecific vector integration, which has been shown to cause malignant transformation in patients receiving gene therapy for X-linked severe combined immunodeficiency. Strong and sustained immune responses were observed after a single immunization with NILVs carrying viral antigens. However, there is no report to date that evaluates the efficacy of NILVs in inducing antigen-specific antitumor immunity. Using a well-characterized tumor model, we tested in vivo immunization with a self-inactivating lentiviral vector harboring a defective integrase. A high frequency of ovalbumin peptide (OVAp1)-specific CD8(+) T cells and a substantial antibody response were detected in naive mice immunized with an NILV encoding an OVA transgene. Furthermore, this immunization method completely protected the mice against the growth of E.G7 tumor cells expressing the OVA antigen. Thus, this study provides evidence that immunization using NILVs can be a safe and promising approach for exploring cancer immunotherapy.

FIG. 1.

The D64V point mutation in HIV-1 integrase prohibits vector integration on transduction. (A) A schematic diagram of the lentiviral transfer vector encoding a green fluorescent protein (GFP) reporter gene (FUGW) or encoding ovalbumin antigen (FKOVA). R, U5, and ΔU3 are components of the long terminal repeat (LTR) and ΔU3 contains the self-inactivating deletion; SD, splicing donor; SA, splicing acceptor; ψ and Δgag, the encapsulation sequence; RRE, the Rev-responsive element; Ubi, human ubiquitin-C promoter; WPRE, woodchuck hepatitis virus posttransductional regulatory element. (B) HEK293T cells (1.5 × 10⁵ cells were transduced with 1.5 ml of fresh viral supernatant of FUGW/VSVG(IN⁺) or FUGW/VSVG(IN^–). Transduced cells were cultured for 2 weeks and genomic DNA was extracted for quantitative PCR analysis of the integration of the viral vector. Untransduced 293T cells were included as a control. The respective transduction titers for FUGW/VSVG(IN⁺) and FUGW/VSVG(IN^–) were estimated to be 8 × 10⁷ and 1.8 × 10⁷ TU/ml, respectively. The p24 concentration of fresh viral supernatants of FUGW/VSVG(IN⁺) and FUGW/VSVG(IN^–) was 260.9 and 179.1 ng/ml, respectively.

FIG. 2.

Nonintegrating lentivector encoding a GFP reporter gene mediated efficient transduction of dendritic cells (DCs) in vitro and in vivo. (A) Bone marrow cells harvested from naive B6 mice were cultured in the presence of GM-CSF and IL-4 for 6 days to generate bone marrow-derived dendritic cells (BMDCs). The BMDCs then received two spin transductions in the following 2 days with the fresh supernatant of FUGW/VSVG(IN⁺) or FUGW/VSVG(IN^–). GFP expression was monitored by flow cytometric analysis and results are shown as the percentage of GFP⁺ BMDCs (left) and its mean fluorescence intensity (MFI; right). (B) DC2.4 cells were transduced with the fresh supernatant of FUGW/VSVG(IN⁺) or FUGW/VSVG(IN^–) and GFP expression was monitored. (C) Fresh supernatant of FUW/VSVG(IN⁺) (mock), FUGW/VSVG(IN⁺), or FUGW/VSVG(IN^–) was concentrated in 200 μl of sterile PBS and subcutaneously injected into B6 mice. Three days later, the corresponding right inguinal lymph nodes, close to the injection sites, were collected and analyzed to measure the frequency and MFI of GFP⁺CD11c⁺ DCs.

FIG. 3.

Stimulation of OVA-specific CD8⁺ and CD4⁺ T cell responses in vitro by murine bone marrow-derived dendritic cells (BMDCs) modified by nonintegrating lentiviral vector encoding OVA antigen. (A–D) CD8⁺ and CD4⁺ T cells specific for OVA were collected from the spleens of OT1 and OT2 TCR transgenic mice and cocultured in vitro with FKOVA/VSVG(IN⁺)- or FKOVA/VSVG(IN^–)-modified BMDCs for 3 days. FUW/VSVG(IN⁺)-transduced BMDCs served as the negative control. BMDCs pulsed with OVAp1 (SIINFEKL) or OVAp2 (ISQAVHAAHAEINEAGR), which are recognized by OT1 T cells and OT2 T cells, respectively, were included as positive controls. (A and B) OT1 and OT2 T cells were cocultured with various ratios of BMDCs to OT1 or OT2 T cells in vitro for 3 days. The culture supernatant was measured for the secretion of IFN-γ by ELISA. (C and D) An ³H incorporation assay was used to measure the proliferative activities of (A) treated OT1 T cells and (B) OT2 T cells.

FIG. 4.

Footpad injection of nonintegrating lentiviral vector encoding OVA antigen could generate a substantial antigen-specific CD8⁺ T cell response in wild-type B6 mice. (A and B) B6 mice received immunizations with various doses of FKOVA/VSVG(IN^–) or FKOVA/VSVG(IN⁺) via footpad injections. The amount of viral particles injected was quantified by p24 capture ELISA. Two weeks later, T cells were harvested from the treated mice and analyzed by flow cytometry. Mice without immunization were included as a negative control. (A) T cells harvested from spleens were restimulated with OVAp1 for 6 hr and analyzed for IFN-γ secretion by intracellular cytokine staining. (B) The quantity of OVAp1-specific CD8⁺ T cells was estimated with H-2K^b-SIINFEKL–PE tetramer. (C) Kinetic study of immune responses in mice immunized with 300 ng of FKOVA/VSVG(IN^–) or FKOVA/VSVG(IN⁺). Peripheral blood was collected by retro-orbital breeding at the indicated time postimmunization and analyzed by tetramer staining.

FIG. 5.

Nonintegrating lentiviral vector expressing OVA antigen was comparable to its integrating counterpart in inducing antigen-specific CTLs and humoral responses in vivo. (A–G) B6 mice were immunized with either FKOVA/VSVG(IN⁺) or FKOVA/VSVG(IN^–) via footpad injections. The amount of viral particles used for each immunization was quantified to be 900 ng of p24. Mice without immunization (No LV) were included as a control. Mice were analyzed 2 weeks postimmunization. (A) T cells were collected from spleens and examined for the presence of OVAp1-specific CD8⁺ T cells by tetramer staining. (B) Spleen cells from (A) were restimulated with OVAp1 for 6 hr and IFN-γ secretion was evaluated by intracellular staining. (C) IgG serum specific for OVA was assessed by ELISA. (D–G) On restimulation with OVAp1 or OVAp2, spleen cells from (A) were analyzed by ELISPOT to measure IFN-γ or IL-2 spot-forming cells.

FIG. 6.

Protection against tumor growth and durable OVAp1-specific CD8⁺ T cells were observed after a single administration of nonintegrating lentiviral vector encoding OVA antigen. (A and B) B6 mice were immunized with mock vector FUW/VSVG(IN⁺) (Mock), FKOVA/VSVG(IN⁺), or FKOVA/VSVG(IN^–) via footpad injections. The amount of viral particles used for each immunization was quantified to be 900 ng of p24. Mice without immunization were included as a control. (A and B) Two weeks after immunization, each mouse was implanted with either 5 × 10⁶ EG.7 tumor cells, which express OVA antigen, or EL4 tumor cells, which lack OVA antigen. Tumor progression was monitored with fine calipers and is represented as the product of the two largest perpendicular diameters (mm²). Each group consisted of four mice. (C) On day 15 after tumor challenge, spleen cells were harvested from the treated mice from (A) and analyzed by tetramer staining. (D) On day 41 after tumor challenge, peripheral blood mononuclear cells (PBMCs) obtained by eye bleeding of treated mice from (A) were also analyzed by tetramer staining.