Preparation of nanoliposomes containing HER2/neu (P5+435) peptide and evaluation of their immune responses and anti-tumoral effects as a prophylactic vaccine against breast cancer

Abstract

HER2/neu is an immunogenic protein inducing both humoral and cell-mediated immune responses. The antigen-specific cytotoxic T lymphocytes (CTLs) are the main effector immune cells in the anti-tumor immunity. To induce an effective CTL specific response against P5+435 single peptide derived from rat HER2/neu oncogene, we used a liposome delivery vehicle. In vivo enhancement of liposome stability and intracytoplasmic delivery of peptides are the main strategies which elevate the liposome-mediated drug delivery. Liposomes containing high transition temperature phospholipids, such as DSPC, are stable with prolonged in vivo circulation and more accessibility to the immune system. Incorporation of DOPE phospholipid results in the effective delivery of peptide into the cytoplasm via the endocytotic pathway. To this end, the P5+435 peptide was linked to Maleimide-PEG2000-DSPE and coupled on the surface of nanoliposomes containing DSPC: DSPG: Cholesterol with/without DOPE. We observed that mice vaccinated with Lip-DOPE-P5+435 formulation had the highest number of IFN-γ- producing CTLs with the highest cytotoxic activity that consequently led to significantly smallest tumor size and prolonged survival rate in the TUBO mice model. In conclusion, our study indicated that the liposomal form of P5+435 peptide containing DOPE can be regarded as a promising prophylactic anti-cancer vaccine to generate potent antigen-specific immunity.

Fig 1

Confirmation of conjugation between P5+435 peptide and Maleimide-PEG-DSPE by using TLC (A) and HPLC (B) analyses.

Fig 2. Tricine SDS page analysis.

Lane 1: Ladder, Lane 2: Pure P5, Lane 3: Pure P435, Lane 4: P5+435, and Lane 5: Lip-DOPE-P5+435. The peptide concentration of sample was 2 μg/μl.

Fig 3. The In vitro cytokine production using ELISpot assay.

The evaluation of IFN-γ (A) and IL-4 (B) production in P5+435 peptide-stimulated splenocytes isolated from immunized and non-immunized (buffer) BALB/c mice. ****, ** and * denote P<0.0001, P<0.01 and P<0.05, respectively. The data are shown as Mean ± SD (N = 3).

Fig 4. Cytokine analysis by flow cytometric assay.

The percentage of cytokine-producing cells in splenocytes of immunized mice was determined by using the depicted strategy of gating (A). The percentage of CD8⁺ and CD4⁺ T cells (B and C, respectively) and IFN-γ in the gated CD8⁺ and CD4⁺ T cells, and IL-4 in the gated CD4⁺ T cells were measured (D, E, and F, respectively). Data represent Mean ± SD (N = 3). **P<0.01, ***P<0.001 and ****P<0.0001.

Fig 5. In vitro cytotoxic activity of CD8⁺ T lymphocytes.

CTLs activity of spleen cells isolated from the immunized mice against rHER2/neu-expressing TUBO cells. Data are shown as Mean ± SD (N = 3). **** Sign shows the significance of difference (P<0.0001).

Fig 6. Protective effect of immunization with liposomal formulations in BALB/c mice against rHER2/neu-expressing TUBO cells.

(A) Nine mice per group were immunized three times. Two weeks following the last immunization Six mice were subcutaneously challenged with 5×10⁵ TUBO cells. (B) Tumor volume of each mouse per group compared to the buffer group. Dot line shows the last day in which all mice in the buffer group died. (C) Tumor growth was measured two times a week for 107 days. (D) The survival of mice was followed during 107 days. The values are shown as Mean ± SEM (N = 6) *P<0.05.