Adjuvant Efficacy of the ECMS-Oil on Immune Responses against Bordetella bronchiseptica in Mice through the TLR2/MyD88/NF-κB Pathway.

Bordetella infection can be efficiently prevented through vaccination. The current study investigated the effects of an extract of Cochinchina momordica seed (ECMS) combined with oil on the immune responses to the inactivated Bordetella vaccine in mice. Serum IgG and IgG1 level was significantly increased in ECMS-oil group compared to any other group (P < 0.05) 2 weeks after immunization, while groups ECMS200 µg/400 µg-oil had a markedly higher level of serum IgG2b and IgG3 than any other groups (P < 0.05). Moreover, lipopolysaccharide/ConA-stimulated proliferation of splenocytes was significantly enhanced in ECMS 400 µg-oil immunized mice in comparison with mice in any other group (P < 0.05). RT-PCR assay revealed that while ECMS800 µg-oil group had significantly higher levels of serum IL-4, IL-10, Toll-like receptor (TLR)2, and IL-1 beta than any other group (P < 0.05), the levels of serum IL-2, IL-4, and IL-10 were markedly increased in ECMS 400 µg-oil group as compared to any other groups (P < 0.05). Blood analysis showed that ECMS800 µg-oil and oil groups had a significantly higher number of immunocytes than any other groups (P < 0.05). There were significant differences in the number of IgG+, IgG2b+, and IgA+ cells in the lung between ECMS800 µg-oil group and any other groups (P < 0.05). Western blot analysis demonstrated that stimulation with ECMS 25 µg/mL or 50 ng/mL led to a significant increase in the expression of TLR2, MyD88, and NF-κB in Raw264.7 cells (P < 0.05). Compared with any other group, the expression of MyD88 was markedly increased in the cells stimulated with ECMS 50 ng/mL, as indicated by the RT-PCR analysis (P < 0.05). Overall, we observed that ECMS-oil efficiently enhanced the humoral or cellular immune responses against Bordetella and suggested that the mechanism of adjuvant activity of ECMS-oil might involve TLR2/MyD88/NF-κB signaling pathway.

Optimum Preparation Method for Self-Assembled PEGylation Nano-Adjuvant Based on Rehmannia glutinosa Polysaccharide and Its Immunological Effect on Macrophages.

BACKGROUND: Rehmannia glutinosa polysaccharide is the main reason that contributes to the immunological function of R. glutinosa. Due to its disadvantages in clinical use, here we designed the PEGylation nano-RGP (pRL) to improve the drug-targeting effect and the immunological function. Our present work aims to establish the optimum condition of preparing the pRL and to investigate its immunological function on macrophages. METHODS: pRL was prepared by thin film hydration method combined with ultra-sonication technique. And its preparation conditions were optimized with response surface methodology. Also, the lyophilization method was optimized. The characteristics of the pRL were evaluated, including particle size, drug loading, encapsulation efficiency and morphology. The immunological function of pRL on macrophage was investigated through CCK-8 test, ELISA and flow cytometry. RESULTS: The lipid-to-cholesterol molar ratio of 8:1, the addition of DSPE-PEG2000 of 9% and the lipid-to-drug ratio of 5.4:1 were the optimum preparation technology for pRL. The encapsulation efficiency (EE) of pRL under this preparation technology was 95.81±1.58%, with a particle size of 31.98 ± 2.6 nm. The lactose-to-lipid ratio (2:1) was the optimal lyophilization method. pRL promoted macrophage proliferation, which is significantly better than that of nano-RGP without PEGylation (RL). pRL-stimulated RAW264.7 cells showed a high secretion of pro-inflammatory cytokines, which is the characteristic indicator of M1 polarization. Enhanced cellular uptake through macropinocytosis-dependent and caveolae-mediated endocytosis was observed in pRL-treated RAW264.7 cells. CONCLUSION: Our study concluded that PEGylation effectively overcame the poor targeting effect of Rehmannia glutinosa polysaccharide (RGP) and significantly improved the immunological profile of its nano-formulation, which suggested that pRL could serve as an immune adjuvant in clinical application.

Greater efficacy of the ECMS-oil adjuvant over other formulations on immune responses against Bordetella bronchiseptica in rabbits and the underlying mechanism.

In this study, the adjuvant effects of the extract of Cochinchina momordica seed ECMS+oil, oil alone, ECMS alone, conventional alum adjuvant on inactivated Bordetella bronchiseptica (Bb) vaccine or control using antigen alone without adjuvant were evaluated along with the underlying mechanism. The results in experiment A demonstrated that antibody levels in Bb whole cell protein in the ECMS800µg+oil group were significantly higher than in the other adjuvant groups (p<0.05) on day 21. The agglutination antibody titer was also higher than the other groups (p<0.05) on day 37. The ECMS800µg+oil group improved cellular immune responses compared to other adjuvant groups, including control using antigen alone without adjuvant and the PBS group (p<0.05). After Bb challenge, the ECMS800µg+oil group showed the highest protection rate, which was significantly higher than ECMS alone or control using antigen alone without adjuvant and the PBS group (p<0.05 and p<0.01). IgA cells in the ECMS800µg+oil group differed significantly from the IgA cells of other groups in the lungs (p<0.01). The results of cell recruitment showed that the number of lymphocytes in the ECMS400µg+oil were higher than the number of cells for other groups except the ECMS(100µg/800µg)+oil groups (p<0.05). Intermediate cells in the ECMS(100µg/400µg)+oil groups were higher than the number of cells for other groups, including the control using antigen alone group (p<0.05). Neutrophils in the ECMS(100µg/400µg/800µg)+oil groups were significantly higher than the ECMS 800µg and control using antigen alone groups (p<0.05). White blood cells in the ECMS100µg+oil group were significantly higher than the oil, ECMS800µg and control using antigen alone groups (p<0.05). IL-2 expression in the ECMS800µg+oil group was significantly higher than other groups, except for the ECMS400µg+oil group (p<0.05). IL-4 expression in the ECMS800µg+oil group was significantly higher than other groups (p<0.05). GATA3 in the ECMS800µg+oil groups was significantly higher than the oil, ECMS800µg and control using antigen alone group (p<0.05). ECMS-oil adjuvant mixture could most effectively protect B. bronchiseptica immunized rabbits and, therefore, could be an alternative way of improving B. bronchiseptica vaccination in rabbits.