Peptide design
We designed the potential peptides from previously reported ones [16], [24]. Briefly, to obtain potential hTERT-derived and viral peptides, we used databases with information on MHC class I & II restricted T cell epitopes. The viral and hTERT sequences were analysed using The Immune Epitope Database (IEDB) and high immunogenic peptides were selected. We then analysed the peptide binding affinities using NetMHC 4.0 (https://services.healthtech.dtu.dk/) and IEDB (http://tools.iedb.org/mhci/). The selected sequences were conjugated to Keyhole limpet hemocyanin (KLH). For this study, because we were going to also use the hTERT-derived peptide vaccines in mice, we also analysed the immunogenicity levels of these peptides against mice MHC.
Animals
Twenty-four female Balb/C mice (age, 6‑8 weeks) were purchased from Pasteur Institute (Tehran, Iran). The mice were housed under pathogen‑free conditions with a 12‑h light/dark cycle and provided with food and water ad libitum. They were then randomly divided into four groups of six (shown in Table 3) and quarantined for at least 1 week before use. Ethical approval for this study was sought and granted by Tehran University Medical Sciences (TUMS) Ethics Committee. Animals were handled in accordance with the animal care guidelines of the Animal Care and Use Committee (ACUC) of Tehran University of Medical Sciences (TUMS). Efforts were made to minimise animal suffering.
Cell lines
The murine breast cancer cell lines (4T1) were purchased from Pasteur Institute (Tehran, Iran). The cells were cultured in DMEM (Sigma, US) containing 100 IU/mL of penicillin, streptomycin, 10% heat-inactivated fetal bovine serum (Gibco, US) and were placed in a humidified incubator with 5% CO2 at 370C.
Tumor establishment
At 85% confluence, the cells were detached using 0.25% trypsin (Sigma, US) and washed thrice with PBS. The cells were counted and resuspended in sterilized PBS. For tumor establishment, 5–6×105 4T1 cells in 100µl sterilized PBS were subcutaneously (S.C.) injected into the right fourth mammary fat pat of the Balb/C mice. Once the tumors were palpable, their sizes were measured using digital callipers (Thermo Fisher Scientific). Tumor volume was then calculated as V = 0.5× (L×W2) (L: longest diameter, W: shortest diameter).
Evaluation of peptide immunogenicity by immunization of Balb/C mice
After the establishment of tumors by inoculating the mice with cell lines, groups 2, 3, and 4 of the mice were then immunized S.C. with 20 µg each of MHC class I and II restricted viral and hTERT peptides conjugated to Keyhole Limpet Hemocyanin (KLH) on days 1, 9 and 18. Different mice groups received different peptides or their combination according to Table 3.
Lymphocyte isolation, culture and treatment
Mice were anesthetized by intraperitoneal injection of 240 mg/kg 1.25% Avertin before getting sacrificed. The tumors then were isolated and soaked in 70% ethanol for 5 min. The spleens were removed by laparotomy and washed using cold PBS pH 7.4 and passed through a 70 µm Strainer (BD, US) using a plunger of a glass syringe. Lymphocytes were isolated by means of Ficoll density gradient centrifugation as previously described [25].
Aliquots of 2 × 105 cells were resuspended in 200 µl of Roswell Park Memorial Institute Media (RPMI 1640) (Gibco, USA) supplemented with 1× streptomycin and 10% fetal bovine serum (FBS). Twenty µg MHC class I & II restricted viral and hTERT derived peptides were added to each well as shown in Table 3. Experiments were repeated in triplicates and cells were maintained in a humidified incubator for 48h at 37°C with 5% CO2.
Survival analysis
The tumours were harvested on day 18 postinoculation. Subsequently, 20 mice (n = 5 mice/group) were randomly selected for observation of survival, weight changes and tumor sizes. The mice were observed until death or until their tumors size reached 1800 mm3.
Cell proliferation analysis
Splenocyte proliferation was assessed using a BrdU colorimetric ELISA (Roche) according to the manufacturer’s instructions. In brief, splenocytes were incubated with 10 mg/ml MUC1 (HGVTSAPDTRPAPGSTAPPA) peptides or the irrelevant peptide OVA (SIINFEKL) as a control for 72 h. 16BrdU was added to the wells for 18 h and BrdU incorporation into newly synthesized DNA strands was observed.
Cell viability and proliferation assay
About 1×105 splenic lymphocytes were seeded in 96-well cell culture plates and maintained in a humidified incubator for 24 h at 37°C with 5% CO2. Two µg/ml MHC class I & II restricted viral, hTERT derived peptides, or irrelevant peptides were added to the wells as shown in Table 3 for 72h. Ten µg/ml (PHA) was added in the last 24 h as a positive control. Finally, on the third day, 0.5 mg/ml MTT solution was added to the wells and incubated at 37°C with 5% CO2. After 3 h, 150 µl DMSO4 was added to each well to solubilize formazan crystals formed by viable cells. Absorbance was then measured at 570 nm using a microplate spectrophotometer (Thermo Fisher Scientific; Waltham, MA, USA). The density of formazan formed in control cells was taken as 100% viability. The percentage proliferation was calculated relative to the optical density (OD) of negative control cells.
Detection of interferon (IFN)-γ cytokine
Splenic lymphocytes were cultured as described above and treated with viral and/or hTERT derived peptides conjugated to KLH. The supernatants were harvested after 48h of stimulation with peptides. The concentration of IFN-γ in the supernatants was determined by ELISA (R & D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions with the OD values measured at 450 nm. The concentrations of the samples were then measured by converting absorbance values using a standard curve prepared with serial dilutions of recombinant IFN- γ standards.
Statistical Analysis
Data were analysed using SPSS 20 software (IBM, USA). All values were expressed as the mean ± SD. The differences were determined using the student T-Test for parametric comparison. Survival curves were analysed by a log-rank test. The p-value < 0.05 was considered statistically significant.