Optimized DNA vaccines to specifically induce therapeutic CD8 T cell responses against autochthonous breast tumors

Abstract

Background: Vaccines capable of inducing CD8 T cell responses to antigens expressed by tumor cells are considered as attractive choices for the treatment and prevention of malignant diseases. Our group has previously reported that immunization with synthetic peptide corresponding to a CD8 T cell epitope derived from the rat neu (rNEU) oncogene administered together with a Toll-like receptor agonist as adjuvant, induced immune responses that translated into prophylactic and therapeutic benefit against autochthonous tumors in an animal model of breast cancer (BALB-neuT mice). DNA-based vaccines offer some advantages over peptide vaccines, such as the possibility of including multiple CD8 T cell epitopes in a single construct.

Materials and methods: Plasmids encoding a fragment of rNEU were designed to elicit CD8 T cell responses but no antibody responses. We evaluated the use of the modified plasmids as DNA vaccines for their ability to generate effective CD8 T cell responses against breast tumors expressing rNEU.

Results: DNA-based vaccines using modified plasmids were very effective in specifically stimulating tumor-reactive CD8 T cell responses. Moreover, vaccination with the modified DNA plasmids resulted in significant anti-tumor effects that were mediated by CD8 T cells without the requirement of generating antibodies to the product of rNEU.

Conclusions: DNA vaccination is a viable alternative to peptide vaccination to induce potent anti-tumor CD8 T cell responses that provide effective therapeutic benefit. These results bear importance for the design of DNA vaccines for the treatment and prevention of cancer.

Fig. 1

Immunization of BALB/c mice with plasmid pEC-TM^neu, encoding the extracellular and transmembrane domains of rNEU, elicits both CD8⁺ T cell and antibody responses to rNEU. a Mice (n = 3 per group) were vaccinated on days 0 and 21, as described in “Materials and methods ,” with pEC-TM^neu followed by electroporation. On days 12 and 29, peripheral blood lymphocyte samples were evaluated for the presence of antigen-specific CD8 T cells. Each data point represents the % of p66-specific cells of CD8 T cells for each individual mouse and horizontal line represents the average value of the group. b The production of anti-rNEU antibodies by the vaccinated mice was evaluated by flow cytometry as described in “Materials and methods.” Eight days after second immunization, serum samples (diluted 1:10) were incubated with rNEU-expressing (3T3-rNEU⁺, clear histogram) or rNEU-negative (3T3-rNEU⁻; shaded histogram) cell lines. Result in left panel is a representative example of a mouse vaccinated with pEC-TM^neu. Middle panel is the result using sera from a nonvaccinated (No Vax) mouse. Right panel represents the positive control obtained by staining the cells with 0.5 μg/ml of mouse monoclonal antibody Ab-4. These are representative results of data obtained from two different experiments

Fig. 2

Immunization of BALB/c mice with plasmids, encoding a fragment of rNEU (residues 1–170), induces epitope-specific CD8⁺ and CD4⁺ T cell responses, without the production of antibodies to rNEU. Mice (n = 3 per group) were vaccinated with plasmids pEC_1–170 ^neu and pEC_1–170 ^neu/Ova, and immune responses were evaluated in the same manner as described in Fig. 1. a Percentage of tetramer-positive CD8 T cells in individual mice (data points) and average per groups (horizontal lines) shown after prime and boost. b Eight days after the boost, CD8 and CD4 T cells were purified from pooled splenocytes and antigen-induced IFN-γ secretion was evaluated in ELiSpot assays. As stimulator, p66-loaded P815 (P815/p66) and rNEU-expressing tumor (A2L2) were used to evaluate CD8 T cell responses. Ova_323–339-loaded A20 (A20/Ova₃₂₃) were used for the CD4 T cell responses. rNEU-negative tumor (66.3) and mock-treated P815 and A20 cells were used as negative controls. Results represent the average number of spots from triplicate wells with SD (error bars) of the means. Representative results of data obtained from two different experiments. c Absence of anti-rNEU antibodies in mice vaccinated with pEC_1–170 ^neu (left panel) and pEC_1–170 ^neu/Ova (right panel) determined as described in the legend of Fig. 1. Positive and negative controls gave identical results as those shown in Fig. 1b

Fig. 3

Immunization of BALB-neuT mice with plasmids, encoding a fragment of rNEU (residues 1–170), elicits CD8⁺ T cell responses to the rNEUp66 epitope. Eight-week-old mice (four per group) were vaccinated with plasmids pEC_1–170 ^neu or pEC_1–170 ^neu/Ova, and immune responses were evaluated in the same manner as described for the previous experiments. a Percentage of tetramer-positive CD8 T cells in individual mice (data points) and average per groups (horizontal lines) shown after prime, after boost and in nonvaccinated mice (No Vax, three animals). b Eight days after the boost immunization, CD8 T cells were purified from pooled splenocytes, and antigen-induced IFN-γ secretion was evaluated in ELiSpot assays in the same manner as described in Fig. 2. Representative results of data obtained from two different experiments

Fig. 4

Therapeutic effects induced by plasmid DNA immunization against established tumors. BALB/c mice (a five per group, b seven per group) were s.c. inoculated with 3 × 10⁵ live TUBO cells, and vaccinated with plasmid pEC_1–170 ^neu/Ova (shown by the vertical arrow) either one time (a) or two times (b). Nonvaccinated mice (No Vax) were used as negative controls for each experiment. Tumor growth was measured (two opposing diameters) and recorded every other day. Lines tumor size in area (mm²) of each individual mouse

Fig. 5

Immunization of BALB-neuT mice with plasmids, encoding a fragment of rNEU, results in delay of autochthonous mammary neoplasms. Mice (n = 13 per group) were vaccinated at 10 and 13 weeks of age, with pEC_1–170 ^neu, pEC_1–170 ^neu/Ova or pOva (negative control). All animals were monitored for tumor appearance by manual examination of the mammary glands every 5 days. Measurable masses >2 mm diameter were regarded as tumors. a Mean number of tumors per mouse in each group (tumor multiplicity) bars, SD. Arrows represent the day when the vaccine was administered. b Kaplan–Mayer survival curves for all three groups of BALB-neuT mice. Log-rank test analyses (Mantel–Cox) between the negative control (pOva) and pEC_1–170 ^neu or pEC_1–170 ^neu/Ova revealed a statistical significance of P < 0.0001. c Some of mice that were repeatedly vaccinated were monitored for antigen-specific CD8 T cell responses in peripheral blood lymphocytes by tetramer analysis

Fig. 6

Role of T cell subsets in the antitumor effects of DNA immunization in BALB-neuT mice. Mice (n = 4 per group) were depleted of CD8 T cells (middle panel), CD4 T cells (right panel) or not depleted of T cells (left panel) using monoclonal antibodies before the vaccination as described in “Materials and methods.” All mice were vaccinated with plasmid pEC_1–170 ^neu/Ova administered at 7, 10, 13, 19, and 23 weeks of age. All animals were monitored for tumor appearance by manual examination of the mammary glands every 5 days. a Tumor growth assessed by the average tumor size in each mouse. Numbers at the end of each line represent the number of tumors per mouse. Arrows day when the vaccine was administered. b Kaplan–Mayer survival curves for this experiment. Log-rank test analyses (Mantel–Cox) between the nondepleted control and CD8 T cell-depleted group revealed a statistical significance of P < 0.0067, and between the nondepleted control and CD4 T cell-depleted group revealed a statistical significance of P < 0.0558