Antibody responses to HBsAg
For the immunopotentiation of the combined NE+Rg1 and HBsAg adjuvant, the humoural immunity results 3 weeks after 1 dose showed that the S+NE+Rg1 group showed a significantly higher mean serum antibody level than the S+ Al and S+Rg1 groups (Mann-Whitney test, P =0.0128 and P =0.0003, respectively). IgG1 in serum showed similar results, and the serum antibody level of the S+NE +Rg1 group was higher than the S+ Al group (P=0.0013). The IgG2a level was significantly higher in the S+NE +Rg1 group than the S+ Al (P < 0.001) and S+NE (P=0.0081) groups. Therefore, NE and Rg1 showed a certain synergistic immune-enhancing effect for the hepatitis B virus, and the higher IgG2a levels suggested that Rg1 enhanced cellular immunity for HBsAg to a certain extent (Figure 2).
Figure 2. Serum anti-HBs and IgG1 and IgG2a antibody levels of mice immunized with HBsAg combined with different adjuvants. One dose of 0.5 μg HBsAg per mouse was intramuscularly injected, and serum was collected 3 weeks after immunization. Quantitative detection of anti-HBs (mIU/ml) and IgG1 and IgG2a (ng/ml) antibodies was performed using commercially available ELISA kits. Ninety-six well plates were coated with 1 μg/ml HBsAg overnight, and the plate was blocked with 2% bovine serum albumin (BSA) for 1 h then washed 5 times with 1‰ Tween 20-PBS. The initial series of diluted mouse serum was added and incubated at 37℃ for 1 h. After washing, HRP-labelled sheep anti-mouse IgG1 or IgG2a antibodies (1:10,000 dilution) were added, and incubated at 37℃ for 1 h. A chromogenic solution was added and incubated at 37℃ for 10 min and the termination solution was added. The absorbance value at dual wavelengths of 450/630 nm was read. Data are presented as the means ± SEM and are representative of one of two independent experiments with similar results. Note: * indicates P＜0.05, ** indicates P＜0.01 and *** indicates P＜0.001.
Cell-mediated immune response to HBsAg
To assess the ability of NE+Rg1 to elicit a cellular immune response when administered intramuscularly with HBsAg, NE+Rg1-induced IL-2 and IFN-γ (as markers of the Th1 response) secreting cells in the spleen were detected using ELISpot. As shown in Figure 3a, mice vaccinated with S+NE+Rg1 produced higher counts of specific IFN-γ- and IL-2-expressing cells than NE-vaccinated mice (P=0.0286), and the SFCs of the NE+Rg1 group were significantly higher than aluminium hydroxide (P=0.0286). The results of ICS were similar to ELISpot. IFN-γ and IL-2 expression in CD8+ T cells of the NE+Rg1 group showed a slightly higher enhancement than the NE group, but the difference was not statistically significant. However, the effect was stronger than the aluminium hydroxide adjuvant (IFN-γ, P=0.0286; IL-2, P=0.0265, Figure 3b).
Figure 3. Specific T-cell responses measured using ELISpot and ICS in mice immunized with HBsAg combined with different adjuvants. Seven days after the immunization boost, mice were sacrificed. Splenocytes were isolated from the spleen and stimulated with a specific S peptide (S28-39, IPQSLDSWWTSL, H-2d restricted). Briefly, for ICS, 250 μl of flow fluid (2% FBS PBS) was added to each well of the 96-well cell plate, and centrifugation was performed at 200 g for 5 min. Then, 50 μl of FITC-CD8 (BD, USA) and PerCP-CD3 (BD, USA) dyes were added to each well for surface dyeing. After centrifugation at 200 g for 5 min, 250 μl of flow fluid was added to each well, and 100 μl Fixation/Permeabilization solution (BD, USA) was added each well. The reaction was performed at 4℃ for 20 min in the dark. Then, 50 μl of PE-IFN-γ and APC-IL-2 dyes (BD, USA) diluted in Perm/Wash buffer (BD, USA) were added to each well. After washing, 200 μl of flow solution was added to each well, and the results were detected using flow cytometry (BD FACS Canto™ II, USA). Data are presented as the means ± SEM and are representative of one of two independent experiments with similar results.
HAI titre and protection against influenza infection
The HAI results showed that the HA+NE serum inhibitory titre was significantly higher than the single HA (approximately 8 times) group after 1 dose of immunization, and there was no significant difference with NE+Rg1. After immunization with 2 doses, the HA+NE and NE+Rg1 groups had similar HAI titres, which were approximately 16 times higher than the single HA group, but these differences were not statistically significant. However, the HAI titre of the NE+Rg1 group was significantly higher than the HA+Rg1 group (P＜0.001). After the 10 LD50 challenge, the mice were monitored for toxicity signs and body weight changes continuously for 14 days. As shown in Figure 5, the survival rates of the HA, HA+NE, HA+NE+Rg1, HA+Rg1 and NS groups were 0%, 100%, 100%, 30% and 0%, respectively on day 14. The weight change curve revealed that weights in the single HA and HA+Rg1 groups decreased to a very low point around the 7th day then gradually increased, but weights in the NE+Rg1 and NE groups gradually recovered after a slight weight loss. The NE+Rg1 group did not show a significant advantage over the NE group, but the former group showed more drastic fluctuations in body weight. These results showed that NE and Rg1 had no synergistic immune enhancement or protective effect against the influenza HA antigen, which is not consistent with the observed results of the NE+Rg1 in combination with HBsAg. The results indicate that the combination of the same adjuvant with different antigens induces different or opposite results.
Figure 4. The serum HAI titre and challenge and the survival rate post-challenge. The mice were intramuscularly injected at weeks 0 and 4, and serum was collected at weeks 0, 4 and 6. The immune dose of HA was 1.5 μg/dose, and Rg1 was 50 μg/dose(a). Mice were challenged with 10 LD50 of the A/Puerto Rico/8/34 influenza strain after administration of 400 μl of 0.4% sodium pentobarbital on Day 14 after the immunization boost. The mortality and body weight of the challenged mice were monitored for the subsequent 14 days. Mice that lost greater than 35% body weight were humanely euthanized (b).
Safety and preliminary mechanism of action
The results of haemolysis and coagulation experiments showed that NE and Rg1+NE had no haemolytic effect on 2% rabbit red blood cells, which preliminarily supports the safety of NE and Rg1+NE for haemolysis.
To detect the mechanism of action of the adjuvant effect of ginsenoside Rg1+NE, we hypothesized that the structure or functional group of Rg1 was the same or similar to the structural or chemical groups that act on TLR4. Many vaccine adjuvants induce the production of interferon and other inflammatory cytokines via the activation of TLRs.[14] Therefore, we evaluated the activation of TLR4 and TLR7 to determine whether ginsenoside Rg1 and NE had an adjuvant effect via activation of TLR4 and TLR7 receptors. Because NE is milky white in appearance, it may interfere with the absorption value to some extent. Therefore, NE and NE+Rg1 were diluted into different multiples. As shown in Figure 5, the absorbance results showed that the A630 values of NE (500×, 5000×, 50000×), NE+Rg1 (50× and 1 μg/ml) and Rg1 (1 μg/ml, 100 ng/ml, 10 ng/ml) were not different than NS. However, the absorbance value induced by 100 ng/ml LPS was close to 1.0 (hTLR-4), and the absorbance induced by 100 ng/ml R848 was over 2.0 (hTLR7). NE and Rg1 did not activate HEK293 hTLR-4 or hTLR-7 cells to secrete the SEAP enzyme, and the substrate did not turn blue. These results indicate that NE and Rg1 are not ligands of TLR4 or TLR7.
Figure 5. Effects on the surface receptors TLR4 and TLR7. HEK293 hTLR-4 and hTLR-7 cells were added to 96-well cell culture plates at a concentration of 4.0×104/well and 180 μl/well. The samples were added as 20 μl/well, cultured at 37℃ with 5% CO2 for 20 h, and the absorbance at a 630-nm wavelength was detected. The final concentrations were 100 ng/ml R848, 100 ng/ml LPS, and 1 μg/ml, 100 ng/ml or 10 ng/ml for Rg1-1, Rg1-2 and Rg1-3, respectively, and 500-, 5000- and 50000-times dilutions of NE-1, NE-2 and NE-3, respectively. In NE+Rg1, NE was diluted 50 times, and the concentration of Rg1 was 1 μg/ml.
Physiochemical characteristics and stability
The results of particle size, PdI and Zeta potential at 37℃ at different times (3, 8 and 12 weeks) showed that NE+Rg1 displayed relatively stable physicochemical characteristics (Figure 6). These results suggest that the combined adjuvant may be stored stably at 4℃ for a long time.