HER2/neu DNA vaccination by intradermal gene delivery in a mouse tumor model: Gene gun is superior to jet injector in inducing CTL responses and protective immunity

Abstract

DNA vaccines are potential tools for the induction of immune responses against both infectious disease and cancer. The dermal application of DNA vaccines is of particular interest since the epidermal and dermal layers of the skin are characterized by an abundance of antigen-presenting cells (APCs). The aim of our study was to compare tumor protection as obtained by two different methods of intradermal DNA delivery (gene gun and jet injector) in a well-established HER2/neu mouse tumor model. BALB/c mice were immunized twice with a HER2/neu-coding plasmid by gene gun or jet injector. Mice were then subcutaneously challenged with HER2/neu(+) syngeneic D2F2/E2 tumor cells. Protection against subsequent challenges with tumor cells as well as humoral and T-cell immune responses induced by the vaccine were monitored. Gene gun immunization was far superior to jet injector both in terms of tumor protection and induction of HER2/neu-specific immune responses. After gene gun immunization, 60% of the mice remained tumor-free until day 140 as compared with 25% after jet injector immunization. Furthermore, gene gun vaccination was able to induce both a strong T(H)1-polarized T-cell response with detectable cytotoxic T-lymphocyte (CTL) activity and a humoral immune response against HER2/neu, whereas the jet injector was not. Although the disadvantages that were associated with the use of the jet injector in our model may be overcome with methodological modifications and/or in larger animals, which exhibit a thicker skin and/or subcutaneous muscle tissue, we conclude that gene gun delivery constitutes the method of choice for intradermal DNA delivery in preclinical mouse models and possibly also for the clinical development of DNA-based vaccines.

Figure 1. Short- and long-term tumor protection by intradermal DNA vaccination using gene gun or jet injector: percentage of tumor-free mice after vaccination and tumor challenge. (A, B) Wild-type (WT) BALB/c mice were immunized by gene gun (GG) or jet injector (JI) delivery with pDNA(HER2/neu), mock vector (pVax) or gold particles/PBS on days 1 and 15. On day 25 tumor challenge was performed with 2 × 10⁵ HER2/neu⁺ syngeneic D2F2/E2 tumor cells. Tumor growth was then monitored thereafter until day 140. n = 10 for each group of mice. (A) Short-term protection (day 50). (B) Long-term protection (day 140). * = statistically significant vs. all other groups (p < 0.05)

Figure 2. Anti-HER2/neu T-cell immune responses after vaccination. (A, B) Splenocytes from wild-type (WT) BALB/c mice intradermally immunized with pDNA(HER2/neu) using gene gun (GG) or jet injector (JI) were stimulated with different peptide combinations (derived from the extracellular domain or the intracellular domain of HER2/neu, or both). Specific T-cell responses were analyzed by a interferon γ (IFNγ)-specific (A) or interleukin-4 (IL-4)-specific ELISpot assays. Mice had been immunized with DNA on days 1 and 15. ELISpot assays were performed 7 d after the last vaccination. For ELISpot assays, splenocytes within the different groups of mice were pooled. ECD, extracellular domain; ICD, intracellular domain. * = statistically significant vs. all control groups (p < 0.05).

Figure 3. Anti-HER2/neu T-cell immune response after vaccination as detected by HER2/neu-p63-specific pentamer staining. Splenocytes from wild-type (WT) BALB/c mice intradermally immunized with pDNA(HER2/neu) using gene gun (GG) or jet injector (JI) were stained with HER2/neu-p63-specific pentamers. For cytofluoromtetric analyses, splenocytes from mice belonging to the same experimental group were pooled. Splenocytes were analyzed within a CD3⁺-restricted gate.

Figure 4. CTL assays after vaccination with gene gun or jet injector. (A-C) Wild-type (WT) BALB/c mice were immunized by gene gun (GG) or jet injector immunization (JI) with pDNA(HER2/neu), mock vector (pVax) or gold particles/PBS on days 1 and 15. On day 22, splenocytes were restimulated in vitro for 5 d with irradiated BALB/c 3T3 cells that were pulsed with a HER2/neu-p63 peptide. CTL activity was measured in a standard ⁵¹Cr release assay using HER2/neu⁺ D2F2/E2 tumor cells (A), HER2/neu^- D2F2 tumor cells, pulsed with the HER2/neu-p63 peptide (B), or HER2/neu^- D2F2 tumor cells only (negative control) (C). * = statistically significant vs. all other groups (p < 0.05).

Figure 5. Humoral anti-HER2/neu immune responses after vaccination. (A-D) Antibody responses (total IgG and IgG isotypes) against HER2/neu were determined by a cytofluorometric assay in wild-type (WT) BALB/c mice intradermally immunized with pDNA(HER2/neu) using gene gun (GG) or jet injector (JI). Mice had been immunized with DNA on days 1 and 15. Cytofluorometric assays were performed 7 d after the last vaccination. (A) Anti-HER2/neu, total IgG. (B) Anti-HER2/neu, IgG1. (C) Anti-HER2/neu, IgG2a. (D) Anti-HER2/neu, IgG2b. * = statistically significant vs. all other groups (p < 0.05).