Mannosylation Of Outer Membrane Proteins For The Development Of Dendritic Cell Vaccine

Brucella species are facultative intracellular bacteria. Macrophage is predominant host cell of Brucella in natural infection because macrophages express more pattern recognition receptors on their sufaces. The brucella species can inhibit macrophage apopotosis via impairing the secretion of TNF-αin macrophage. As a result, the bacteria can survive and replicate in macrophages. Consequently, the phagocytic capability, the killing effect and antigen presentation of macrophages are severely impaired. Collectively, these results make the brucella can successfully evade the immune response of host cell and lead to chronic infection. On the other hand, the macrophages can activate the na?ve T cells weakly and can not express CD1 molecule, therefore, failing to present lipid antigens to T cells and initiate the cellular killing effection.Dendritic cells (DCs) are the professional antigen-presenting cells (APCs) that can activate the na?ve T cells. DCs can present antigens through MHC-Ⅰand MHC-Ⅱmolecules. Furthermore, DCs can also present lipid antigen via CD1 molecule. However, the DCs are weakly capable of captureing Brucella, as DCs express lower level pattern recognition receptors than macrophages.To observe the morphorlogic changes of macrophages and DCs, the peritoneal macrophages and monocyte-derived DCs (MoDCs) were respectively infected with B. suis and stained with Giemmsa in vitro at different time points. At 4h, certain bacteria were phagocytosed by macrophages. Thereafter, the number of infected macrophages obviously increased. By 6h, the cytoplasm of macrophages began to drop off. At 8h, the majority of macrophage population ingested bacteria, and synchronously, the integrity of plasma membrane disappeared. Moreover, the nuclear of macrophages became shrunk. Strikingly, some membrane vesicles were released from the damaged macrophages at 12h of infection, indicating that the B. suis subverted macrophage function. Next, MoDCs were infected with B. suis in vitro. After 4h of infection, Brucella adhered onto MoDCs and only few were ingested by MoDCs. From 6h to 12h, the number of MoDCs that captured B. suis progressively increased. In sharp contrast to macrophages, the mophorlogy of MoDCs remained intact. In order to make DCs exert powerful capability of antigen presentation and avoid immune limitation of macrophages in Brucella infection, the new Brucella antigen-targeted DC vaccines were designed. The lipopolysaccharides and outer membrane proteins (OMPs) in Brucella cellular membrane are the main virulent factors and show appreciated antigeneicity. Therefore, the consecutive extraction with eradicator and electric elution was employed to isolate these molecules. The result indicated that the two outer membrane proteins, 37.5 kD and 47.2 kD were successfully obtained after SDS-PAGE analysis. Subsequently, the mice were hypodermically injected with the two proteins mixed and Freund adjuvant, with the Brucella vaccine as control. The serum was prepared and analyzed after the fourth booster.The result showed that the two proteins own better immunogenicity. Furthermore, the antibody titer (1:320) was higher than the Brucella vaccine (1:160). Consequently, the two proteins were selected as the potential candidate antigens.The mannose receptors (MR) are the intrinsic membrane receptors on DCs and macrophages, belonging to the lectin receptors. The immature DCs express high level of MR that can effectively capture the biological macromolecules and microorganism that contain mannose residues. However, the Brucella OMPs contain possess a small quantity of mannose residues. Hence, in order to enhance the capability of DCs in our research, we make sure of the glycation ofεamino groups of lysines as the glycosylation site of two proteins through the O-GlycBase and the glycosylation site prediction tool. The proteins were glycosylated withα-D-mannopyranosyl-phenylisothiocyanate, and the production was identified with the resorcinol-sulfuric acid reagents analysis. The result indicated that the two proteins were glycosylated efficiently.To test the immune efficiency of glycosylated proteins, the glycosylated proteins were mixed with Freund adjuvant and hypodermically injected in mice, the purified proteins, PBS and the burcella vaccine as control at the same time, the serum was prepared after the fourth immunity. The result indicated that the glycosylated proteins induced obviously lower level of antibody (1:10) than the pure proteins (1:240) and the brucella vaccine (1:120) after the serum was analyzed by tube agglutination test. On the other hand, the ELISA results suggested that the levels of IFN-γin the pure proteins group, the glycosylated proteins and the brucella vaccine group were remarkably higher than the control group (P<0.01). Furthermore, the level of IFN-γin the glycosylated proteins group was remarkably higher than the pure proteins (P<0.01)and the brucella vaccine(P<0.05). But the levels of IL-4 in the three groups were not markedly higer than the PBS control group (P>0.05), except the purified proteins group. All the resultes indicated that all the three antigens induced the Th1 cellular immune response effectively, and the immune efficiency of the glycosylated proteins was higher than the pure proteins and the Brucella vaccine. Collectively, the brucella glycosylated OMPs enhance the engulf and the antigen presentation of DCs, that induce more efficiently cellular immune response.To enhance the immune efficiency and the target-capability of the glycosylated proteins, avoiding the side-effect of Freund adjuvant, the glycosylated proteins were emulsied with the adjuvant-MONTANIDETM ISA 50V (Seppic System), and the mice were hypodemically injected. The mice were subscribed on 4d after the first inoculation and on 3d, 5d and 7d respectively after the second booster. The serum was prepared for the analysis of IFN-γ. The results suggested that the response could not retain a long time, though the Brucella vaccine induced the higher level of IFN-γimmune response in a short time. However, the level of IFN-γconstantly increased and was notably higher than the Brucella vaccine and purified proteins from 5d to 7d after the second inoculation (P<0.05). These results suggested that the glycosylated proteins induced the efficient Th1 immune response. Furthermore, the most important was that the glycosylated proteins elicited stronger and longer immunological memory than the brucella vaccine.Our research suggests that the 37.5 kD and 47.2 kD OMPs have good immunogenicity. The glycosylated proteins enhance the engulf capability and antigen presentation of DCs that promote the Th1 cellular immune response. Therefore, the Brucella protein antigen- targeted DCs vaccine is absolutely feasible.