Virosomes as new carrier system for cancer vaccines

Abstract

HER-2/neu, a tumor-associated antigen (TAAg), plays a critical role in oncogenesis of various tumor types, and its selective overexpression by malignant tumor cells makes it an ideal target for immunotherapy. A prerequisite for clinical vaccines is the construction of safe and highly immunogenic reagents able to generate efficient immune responses against TAAg. Previous protein vaccines, consisting of the extracellular domain of HER-2/neu (pNeuECD), were shown to elicit an immune response that did not provide protection from transplantable tumors expressing HER-2/neu. Here we showed that virosomes, which consist of reconstituted viral envelopes without viral genetic material, can act as a carrier and an adjuvant for a truncated protein pNeuECD. Mice vaccinated with pNeuECD either encapsulated in virosomes or bound to the virosomal membrane (Vir-pNeuECD), generated rNeu-specific humoral and cytotoxic immune responses. In addition, Vir-p(NeuECD) induced significant tumor rejection and additionally did not lead to delayed tumor formation when compared with free pNeuECD in complete Freund's adjuvant. There was no difference between the virosomal constructs. Taken together these results suggest that virosomes, as clinically approved safe vaccines, can be used to elicit both humoral and cell-mediated responses against TAAg and induce tumor rejection. Our model is providing important preclinical data to design human vaccination trials for patients with tumors overexpressing HER-2/neu, either as a primary vaccination or as a boost in combination with other vaccines in a context of an adjuvant treatment plan.

Fig. 1a–d

Expression of rNeu_ECD on a breast cancer cell line and transfected COS cells. a Neu⁺ breast cancer cell line (NF9006); b COS cells transfected with Vir-DNA-Neu_ECD; c COS cells transfected with empty Vir; d COS transfected with Vir-DNA. (Magnification a, c, and d: ×280, and b: ×560). Bar is 50 μm

Fig. 2

FACs analysis of rNeu-transfected syngeneic IT22 fibroblast cell lines: IT22 were cotransfected with a control neomycin vector and with a vector expressing rNeu (IT22-neu). IT22 and IT22-neu cell lines were analyzed for HER-2/neu cell surface expression by indirect immunostaining using the rNeu-specific mAb (7.16.4) followed by RPE-conjugated goat antimouse IgG. For analysis of MHC class I expression, the biotin antimouse H-2D^q/H2L^q (KH117) and for MHC class II the biotin antimouse I-A^q (KH116) were used, followed by streptavidin-RPE. The dark shaded area indicates control staining with RPE-conjugated goat antimouse IgG or streptavidin-RPE alone

Fig. 3

a rNeu-specific CTL activity in mice vaccinated with different DNA-based vaccines. Mice were vaccinated/boosted with the indicated vaccines: WT-VV i.p. (solid diamond), rVV-Neu_ECD i.p. (solid square), fDNA s.c. (solid circle), fDNA-Neu_ECD s.c. (shaded open square), or Vir-DNA-Neu_ECD i.p. (solid triangle); 2–5 weeks after the second vaccination, spleens were removed, and CTL activity was assessed in an XTT-based assay using IT22 and IT22-neu as target cells. Similar results were seen in four independent experiments. b Humoral immune response in mice vaccinated with different DNA vectors. Mice were vaccinated and boosted with the indicated vaccines, and sera were collected 6 weeks after the first vaccination. NF9006 cells were incubated with a 1:50 dilution of serum, followed by REP-conjugated MAbs specific for mouse IgG and analyzed for fluorescence by FACScan. The mean and standard error of the mean of each group are shown

Fig. 4

a Effect of prophylactic vaccination on time to tumor formation. All mice develop tumors within the same time range after tumor cell injection. FvB/N mice were vaccinated and boosted with rVV-Neu_ECD i.p. (solid circle), Vir-p^NeuECDenc i.p. (open triangle), Vir-p^NeuECDmem i.p. (open square), Vir-p^NeuECDenc/mem i.p. (open diamond), free p^NeuECD + adjuvant s.c.(solid triangle), or empty Vir i.p. (solid square). The time from tumor injection to development of palpable tumors was assessed every 3 days. Nine to 15 mice per group were compared. Statistical analysis using the Mann-Whitney rank test was performed. b The effect of the indicated vaccines on tumor progression. Tumor volume was measured every 3 days with Vernier calibers. Tumor volume was calculated using the formula (π/6) × (largest diameter) × (smallest diameter)². Shown are the combined results of three independent experiments with five mice per group. Bars represent SEM. Statistical analysis using the ANOVA rank test was performed

Fig. 5

a Neu-specific CTL activity in mice vaccinated with different protein-based vaccines. Mice were vaccinated/boosted with the following vaccines: rVV-Neu_ECD i.p. (solid circle), Vir-p^NeuECDenc i.p. (open triangle), Vir-p^NeuECDmem i.p. (open square), free p^NeuECD + CFA s.c.(solid triangle), or empty Vir i.p.(solid square). Two to five weeks after the second vaccination, spleens were removed, and CTL activity was assessed in an XTT-based assay using IT22 and IT22-neu as target cells. The results of one representative experiment are shown. b Humoral immune response in mice vaccinated with different protein vectors. IgG ELISA titers in sera (1:25 dilution) from mice after immunization with rVV-NeuCD, different virosomal preparations containing p^NeuECD, free p^NeuECD, and empty virosomes. Negative control mouse sera showed OD_450 nm values between 0.07 and 0.10. Single points represent the mean values of triplicate determinations. The mean and standard error of the mean of each group are shown