Fig 2: Antibody responses upon immunization with Control, HER2ic, and HER2ma VLPs. Mice were immunized with Control, HER2ic, or HER2ma VLPs in a prime-boost regimen, in a nonadjuvant, or in combination with Poly (I:C) or AddaVax. (A) Serum antibodies of vaccinated mice were investigated in an ELISA against recombinant HER2 protein. Results are shown as mean ± SEM (standard error of mean) of each vaccination group (n = 5). In (B), IgG1:IgG2a:IgG2b subtype ratios of induced anti-HER2 antibodies elicited by the indicated vaccinations are shown.

Fig 3: Effector functions of induced anti-HER2 antibodies. In (A), the antibody-dependent cellular cytotoxicity (ADCC) activities of induced anti-HER2 antibodies pre- and post-tumor implantation are shown. The results are given as the area under the curve (AUC). Every symbol represents the AUC of a single mouse (n = 5). Differences between Control VLP and HER2ic/HER2ma VLP vaccinations were assessed using an unpaired nonparametric t-test. In (B), the correlations between tumor volumes in vivo and ADCC activity in vitro are shown. Tumor volumes are indicated from the day of serum sample collection (21 days post-implantation). Every dot represents the correlation of a single mouse. The Pearson correlation coefficient (r2) and significance values are indicated on the graph (* p < 0.05, ** p < 0.01, *** p < 0.001).

Fig 4: Infection schematic and characterization of Control, HER2ic, and HER2ma virus-like particle (VLP) vaccines. In (A), the schematic of recombinant baculoviruses (rBVs) used for the generation of VLPs is shown. In (B–D), sucrose-density purified VLPs were negatively stained and investigated using a transmission electron microscope (FEI Tecnai G2 200 kV, FEI, Hillsboro, OR, USA). All types of VLPs ((B) the control, (C) HER2ic, (D) HER2ma) appeared spherical. In (E), the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis results of the VLP preparations are shown. Trastuzumab, a monoclonal anti-HER2 antibody (3B5), and a monoclonal rabbit antihuman HIV-1 Gag antibody were used to probe truncated HER2, full-length human HER2 protein, and the HIV-1 Gag matrix protein, respectively.

Fig 5: In vivo antitumor efficacy of prophylactic administration of HER2 VLPs. In (A), the study design is shown. Six to eight-week-old female BALB/c mice were vaccinated intramuscularly (IM) in a prime-boost regimen. Each experimental cohort (no adjuvant (I), polyinosinic–polycytidylic acid (Poly (I:C)) (II), and AddaVax (III)) was further divided into three subgroups (n = 5): Control VLPs, HER2ic VLPs, and HER2ma VLPs. Four weeks after boosting, HER2+ mammary carcinoma cells were implanted intradermally (ID) into the flank of the right posterior leg of immunized mice. Blood samples were taken submandibularly just before tumor implantation and three weeks after. (B) Tumor progression (I): individual tumor growth curves, with each dot representing tumor volume per mouse at the indicated time point. The dotted line on the tumor volume axis represents the experimental endpoint (1000 mm3). (C) Tumor progression (II): statistical analysis of tumor progression based in tumor volume measurements by day 20 post implantation; two-way Anova–Tukey multiple comparison results summary for the HER2ic and HER2ma VLP groups are shown. (D) Long-term survival: overall survival of each experimental group (** p = 0.0061). (E) Histopathology: immunohistochemistry revealed the presence and distribution of CD8+ T-lymphocytes in 5-µm-thick tumor sections. Black arrows note individual CD8+ T-cells. Squares highlight areas of CD8+ T-cell aggregates. Rounded lines define areas with an abundance of CD8+ T-cells and myeloid cells. Magnification 200×; scale bar 100 µm.