We analyzed the in vivo tumor regression activity of high molecular mass poly-γ-glutamate (γ-PGA) from Bacillus subtilis sups. chungkookjang. C57BL/6 mice were orally administered 10-, 100-, or 2000-kDa γ-PGA or β-glucan (positive control), and antitumor immunity was examined. Our results revealed higher levels of NK cell-mediated cytotoxicity and IFN-γ secretion in mice treated with higher molecular mass γ-PGA (2000 kDa) vs those treated with lower molecular mass γ-PGA (10 or 100 kDa) or β-glucan. We then examined the effect of oral administration of 10- or 2000-kDa γ-PGA on protection against B16 tumor challenge in C57BL/6 mice. Mice receiving high molecular mass γ-PGA (2000 kDa) showed significantly smaller tumor sizes following challenge with the MHC class I-down-regulated tumor cell lines, B16 and TC-1 P3 (A15), but not with TC-1 cells, which have normal MHC class I expression. Lastly, we found that γ-PGA-induced antitumor effect was decreased by in vivo depletion of NK cells using mAb PK136 or anti-asialo GM1 Ab, and that was completely blocked in NK cell-deficient B6 beige mice or IFN-γ knockout mice. Taken together, we demonstrated that oral administration of high molecular mass γ-PGA (2000 kDa) generated significant NK cell-mediated antitumor activity in mice bearing MHC class I-deficient tumors.

The mounting of a specific immune response against the human papillomavirus type 16 E7 protein (HPV16 E7) is important for eradication of HPV16 E7‐expressing cancer cells from the cervical mucosa. To induce a mucosal immune response by oral delivery of the E7 antigen, we expressed the HPV16 E7 antigen on the surface of Lactobacillus casei by employing a novel display system in which the poly‐γ‐glutamic acid (γ‐PGA) synthetase complex A (PgsA) from Bacillus subtilis (chungkookjang) was used as an anchoring motif. After surface expression of the HPV16 E7 protein was confirmed by Western blot, flow cytometry and immunofluorescence microscopy, mice were orally inoculated with L. casei‐PgsA‐E7. E7‐specific serum IgG and mucosal IgA productions were enhanced after oral administration and significantly enhanced after boosting. Systemic and local cellular immunities were significantly increased after boosting, as shown by increased counts of lymphocytes (SI = 9.7 ± 1.8) and IFN‐γ secreting cells [510 ± 86 spot‐forming cells/10⁶cells] among splenocytes and increased IFN‐γ in supernatants of vaginal lymphocytes. Furthermore, in an E7‐based mouse tumor model, animals receiving orally administered L. casei‐PgsA‐E7 showed reduced tumor size and increased survival rate versus mice receiving control (L. casei‐PgsA) immunization. These results collectively indicate that the oral administration of E7 displayed on lactobacillus induces cellular immunity and antitumor effects in mice.

In 2009, the global outbreak of an influenza pandemic emphasized the need for an effective vaccine adjuvant. In this study, we examined the efficacy of poly-γ-glutamic acid/chitosan (PC) nanogel as an adjuvant for the influenza vaccine. PC nanogel significantly enhanced antigen-specific cross-presentation and cytotoxic T lymphocyte (CTL) activity. Compared with alum, the protective efficacy of the pandemic H1N1 influenza (pH1N1) vaccine was substantially increased by PC nanogel, with increased hemagglutination-inhibition titers, CTL activity, and earlier virus clearance after homologous and heterosubtypic [A/Philippines/2/82 (H3N2)] virus challenges. However, CD8⁺T cell-depleted mice displayed no protection against the heterosubtypic virus challenge after immunization with PC nanogel-adjuvanted pH1N1 vaccine. We also observed that using PC nanogel as a vaccine adjuvant had a dose-sparing effect and significantly enhanced the long-lasting protection of the pH1N1 vaccine. Together, these results suggest that PC nanogel is a promising vaccine adjuvant that could broadly prevent influenza virus infection.