The antitumor immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70/SEA complex protein vaccine following peroral administration route

Abstract

Previous studies have shown that there are profuse lymphatic tissues under the intestinal mucous membrane. Moreover, vaccine administered orally can elicit both mucous membrane and system immune response simultaneously, accordingly induce tumor-specific cytotoxic T lymphocyte. As a result, the oral route is constituted the preferred immune route for vaccine delivery theoretically. However, numerous vaccines especially protein/peptide vaccines remain poorly available when administered by this route. Nanoemulsion has been shown as a useful vehicle can be developed to enhance the antitumor immune response against antigens encapsulated in it and it is good for the different administration routes. Of particular interest is whether the protein vaccine following peroral route using nanoemulsion as delivery carrier can induce the same, so much as stronger antitumor immune response to following conventional ways such as subcutaneous (sc.) or not. Hence, in the present study, we encapsulated the MAGE1-HSP70 and SEA complex protein in nanoemulsion as nanovaccine NE (MHS) using magnetic ultrasound method. We then immuned C57BL/6 mice with NE (MHS), MHS alone or NE (-) via po. or sc. route and detected the cellular immunocompetence by using ELISpot assay and LDH release assay. The therapeutic and tumor challenge assay were examined then. The results showed that compared with vaccination with MHS or NE (-), the cellular immune responses against MAGE-1 could be elicited fiercely by vaccination with NE (MHS) nanoemulsion. Furthermore, encapsulating MHS in nanoemulsion could delay tumor growth and defer tumor occurrence of mice challenged with B16-MAGE-1 tumor cells. Especially, the peroral administration of NE (MHS) could induce approximately similar antitumor immune responses to the sc. administration, but the MHS unencapsulated with nanoemulsion via po. could induce significantly weaker antitumor immune responses than that via sc., suggesting nanoemulsion as a promising carrier can exert potent antitumor immunity against antigen encapsulated in it and make the tumor protein vaccine immunizing via po. route feasible and effective. It may have a broad application in tumor protein vaccine.

Fig. 1

The photo of nanoemulsion taken by transmission electron microscope (100,000×). One drop of diluted NE (MHS) nanovaccine (1:100) was dropped onto copper sieve, stained by 0.3% tungsten phosphate, and then observed under TEM when it dried. The vesicles in nanoemulsion showed approximately global shape with similar diameter, ranging from 15 to 25 nm

Fig. 2

ELISpot assay of MAGE-1-specific T-cell precursors from the splenocytes of vaccinated mice. C57BL/6 mice were vaccinated with NE (-), MHS or NE (MHS) via sc. or po., respectively. Each mouse was immunized three times every 10 days. Mice were terminated on tenth day after the last immunization and splenocytes were isolated. Data shown represent averaged results obtained from six mice ± SEM. All analyses were done in duplicates. One-way ANOVA was performed for statistical analysis. ^ns denotes no significantly difference between the two groups. ^ns1 denotes no significantly difference from NE (-)-sc. group. ^ns2 denotes no significantly difference from NE (-)-po. group. A value P < 0.05 was considered significant. ^# denotes significantly different from NE (-)-sc. group, ^Δdenotes significantly different from NE (-)-po. group, and * denotes significantly difference between the two groups

Fig. 3

MAGE-1-specific lysis against B16-MAGE-1 cells by CTLs induced by vaccination with NE (MHS) via peroral immunization route. Mice were vaccinated as described in the Fig. 2. The splenocytes of mice were harvested and restimulated with irradiated B16-MAGE-1 cell. The percentage of specific lysis of CTLs on B16-MAGE-1 target cells was determined by a cytotoxicity assays. The percentage of MAGE-1-specific lysis was calculated by subtracting the percentage lysis of CTLs on B16 from that on B16-MAGE-1 target cells. Data shown represent average results obtained from six mice ± SEM. All analyses were done in duplicates. The MAGE-1-specific lyses of CTLs from mice sc. and po. vaccinated with NE (MHS) was the highest two in six groups, and those sc. vaccinated with MHS protein alone were higher than NE (-)-sc., NE (-)-po., and MHS-po. group. Moreover, there was no statistical difference of the MAGE-1-specific lysis of CTLs between NE (MHS)-sc. and NE (MHS)-po. group

Fig. 4

The immunotherapy effect of challenged B16-MAGE-1 melanoma with the tumor vaccine. Mice were sc. inoculated with B16-MAGE-1 tumor cells (1 × 10⁵ cells/mouse, respectively). Seven days later, they were randomly divided into six groups (n = 6 mice/group) and vaccinated as described in the Fig. 2. Data presented are mean ± SEM. Vaccination with NE (MHS), whether via sc. route or po. route, significantly delayed tumor growth compared with vaccination using MHS or NE (-), and there were no statistical differences between NE (MHS)-sc. and NE (MHS)-po. group at the observation points

Fig. 5

The preventive effect of challenged B16-MAGE-1 melanoma with the tumor vaccine (n = 10 mice/group). The tumor-free rates of C57BL/6 mice inoculated with NE (MHS) vaccine via different routes showed no statistical differences at anytime of observation after tumor cells. Compared with the mice of the other groups, the tumor occurrence time was significantly suspended in the mice sc. or po. vaccinated with NE (MHS) nanovaccine