All the chemicals in this experiment were obtained from Sigma, Aldrich or Acros without further purification. Amino acids, coupling reagents and resins were purchased from GL Biochem. The synthesis of glycosylated amino acid building block Fmoc-Thr(αAc₃GalNAc)-OH was described previously [19], [53]. The 1H-NMR spectra was recorded by an Jeol-ECA-300 spectrometer using CDCl₃ as solvent with frequency of 300 MHz and the analytic data was reported in the Supplementary Material.

Peptides synthesis was performed by standard protocol on a Liberty CEM microwave peptide synthesizer. The natural amino acid building blocks were coupled to resin with 2-(1H-benzotriazole-1-yl)-1,1,3,3- tetramethylhexafluorophosphate (HBTU, 6.0 equiv)/N-hydroxybenzotriazole (HOBt, 6.0 equiv) as activator, N,N-Diisopropylethylamine (DIEA, 12.0 equiv) as activator base, DMF as solvent. For coupling glycoamino acid building block, 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU, 2.0 equiv)/1-Hydroxy-7-azabenzotriazole (HOAt, 2.0 equiv) were used as activator and N-methylmorpholine(NMM, 5.0 equiv) as activator base and N-Methylpyrrolidone(NMP) as solvent. The peptide was detached from resin by TFA/TIS/H₂O (20/1.2/1.2, v/v/v) for 2 h. After removing TFA, peptide was precipitated in diethyl ester and centrifuged with 6000 r/min for 10 min. The crude peptide was purified and analyzed by RP-HPLC on a Waters-600-2487 with solution A (80% acetonitrile/water with 0.06% trifluoroacetic acid) and solution B (100% water with 0.06% trifluoroacetic acid). Absorption signal of 215 nm were detected by UV detector. The MALDI-TOF MS spectra of peptides were recorded by an Applied Bio-systems 4700 Proteomics Analyzer 283 with the matrix of 2, 5-dihydroxybenzoic acid (DHB) or α-cyano-4-hydroxycinnamic acid (CHCA). The details of analytic data were shown in Supplementary Material.

Ten microgram of the purchased CS (sigma, medium molecular weight, 190~310 kDa, 75–85% deacetylation degree) was dissolved in 9.7 mL 1% acetic acid solution. Then 10 M NaOH solution was added slowly until the pH was 5.5 and the solution was filtrated by 0.2 μm filter membrane. Twenty seven microliter 1.5% (w/v) sodium tripolyphosphate (TPP) solution or 24 μL 1.5% (w/v) γ-poly-L-glutamic acid (γ-PGA) was dropwise mingled with a stirred 970 μL CS solution to form nanoparticles suspension. And under these amounts, the charge ratio of positive and negative charge was 2:1 in both nanoparticles. Keep stirring 2 h without separation.

After the nanoparticle suspensions were stirred for 15 min, 100 μL antigen solution contains 100 μg M3 or 100 μg M3 with 120 μg T21 or 228 μg M3−T21 was dropwise added into the two nanoparticle suspensions, respectively. Keep stirring for another 2 h to form the nanovaccines. Otherwise, peptide antigens in 100 μL solution were also added into 900 μL CS solution as negative control experiments. Moreover, 480 μL CS solution and 600 μL Freund’s adjuvant (FA) were combined with 100 μL peptide antigens solution to form water-in-oil emulsion as positive control (Table 1).

M3, M3/T21, M3-T21 were three kinds of peptide antigens, of which M3 was the MUC1 glycopeptide, M3/T21 was mixtures of M3 and a T-helper cell epitope T21, M3-T21 was covalent peptide antigen of M3 and T21. The CS/TPP NPs and CS/γ-PGA NPs were used as adjuvants to form nanovaccines. FA was Freund’s adjuvant that used as positive control. CS were used as negative control.

Eight microliter CS/TPP or 8 μL CS/γ-PGA suspension solution was applied to copper grids of carbon support films for 1 min. Eight microliter phosphotungstic acid (15 mg/mL, pH 6–7) was added for 1 min for negative-stain. After drying overnight, the copper grids were imaged by Hitachi H-7650B transmission electron microscopy.

Dynamic light scattering and zeta potential of CS/TPP and CS/γ-PGA nanoparticles suspensions were measured by the Malvern ZEN3690 Zetasizer apparatus.

Balb/c mice of 4–6 weeks were purchased and bred in Animal Facility of Center of Biomedical Analysis, Tsinghua University. M3, M3/T21, M3-T21 antigens were dissolved in pure water and then added into CS solution, CS/TPP, CS/γ-PGA, and FA respectively (Table 1). From V1 to V12 group, each group was immunized with 200 μL solution of 10 nmol peptide antigens with or without FA by intraperitoneal injection. The first immunization was combined with Complete Freund’s adjuvant, while boosted with Incomplete Freund’s adjuvant. Each group consists of four Balb/c female mice. Mice were injected at intervals of 2 weeks for five times and the serum was collected 1 week after last immunization. Serum without immunization was negative control.

All animal use protocols performed in this study are according to the Institutional Animal Care and Use Committee(IACUC), and the Laboratory Animal Facility at the Tsinghua University is accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International).

High-binding 96-Well ELISA plates (Costar 3590) were coated with 20 μg/mL glycopeptide M3 (100 μL per well) in 0.1 M NaHCO₃ solution (pH=9.6) overnight at 4°C. Then each well was washed by PBS with 0.05% (v/v) Tween-20 for 3 times, and then blocked by incubating 400 μL PBS with 0.25% (w/v) gelation for 3 h at room temperature [52]. After another 3-times washing, the antiserum was diluted and added to each well for 100 μL and incubated for 1.5 h under 37°C. Washed 3 times, the 1:2000 diluted rabbit anti-mouse IgG-Peroxidase antibodies (Secondary antibody, sigma) was added and incubated for 1 h at 37°C. After washed for 4 times, 100 μL OPD substrate (1 mg/mL o-Phenylenediamine in critic acid buffer (pH=5), 1.5 μL/mL 30% H₂O₂) was added to each well and incubated for 30 min at room temperature. Optical absorption was measured at 450 nm. Titers are defined as the highest dilution with 0.1 optical absorption or greater than the absorbance of negative control sera. Each sample was repeatedly measured for 3 times.

High-binding 96-Well ELISA plates were coated with 20 μg/mL glycopeptide M3 (100 μL per well) in 0.1 M NaHCO₃ solution (pH=9.6) overnight at 4°C. Then each well was washed by the PBS with 0.05% (v/v) Tween-20 for 3 times, and then blocked by incubating 400 μL PBS with 0.25% (w/v) gelation for 3 h at room temperature [50]. After another 3-times washing, the antiserum was diluted to 1/50 and added to each well for 100 μL, then incubated for 1.5 h under 37°C. After washing, 100 μL of the 1:1000 diluted goat anti-mouse isotype antibodies (Secondary antibody, sigma), including goat antimouse IgG1, IgG2a, IgG2b, IgG3, IgM, was added and incubated for 1 h at 37°C. After washed for 3 times, 100 μL of the 1:1000 diluted rabbit anti-goat IgG-Peroxidase antibodies (Third antibody, sigma) was added and incubated for 1 h at 37°C. After washing for 4 times, 100 μL OPD substrate was added to each well and incubated for 30 min at room temperature. Optical absorption was measured at 450 nm. Each sample was repeatedly measured for 3 times.

Human breast cancer cell line MCF-7 purchased from Biodee Inc, Beijing were cultured in dulbecco’s modified eagle medium(DMEM) culture medium supplemented with 10% FBS at 37°C. 1/25 diluted antisera of V10, V11, V12 and control groups were incubated with 2.0×10⁵ cells per sample for 1 h on ice. After washing for 3 times, 1/50 diluted FITC-conjugated rabbit anti-mouse IgG antibody (DAKO) were added and incubated for 1 h on ice. After another 3-times washing, FACS analysis was measured on BD Calibur Flow cytometry in Center of Biomedical Analysis, Tsinghua University.

Data were presented as mean±standard deviation (SD). A one-way ANOVA with Bonferroni’s multiple comparison test was used to perform the statistical analysis between data of antibody isotypes. Significant differences were considered as *p<0.05, **p<0.01, ***p<0.001 and n.s. indicates no significant difference.