Research And Development Of Targeted Dendritic Cells Vaccine For Brucellosis

Brucella is facultative intracellular bacteria that can cause abortion in pregnant cattle and undulant fever in humans in a wide range of mammals, including domestic animals and humans. Brucella infects its host through mucosa and wounds. As frequently described, whatever the species considered, Brucella species have strongly proliferated inside macro- hages by inhibiting phagosome-lysosome fusion. Once inside macrophages, pathogens can diminish or abrogate their antigen presentation capacity, thus reducing the T cell medi- ated immune responses, and chronic brucellosis develops. So far, the prevention and contr- ol of brucellosis has been mainly based on application of preventive vaccines with adverse effect.Dendritic cell is the most powerful antigen presenting cell in vivo. Adaptive immune response is conditional upon angtigen presentation of DC. DC not only activates TCs, but also stimulates TCs proliferation significantly. Because of one DC could activates about 100~3000 TCs, it is called"initiator"to immunological reaction. In this study, we develop- ed new generation vaccine based on DC and detected the immunological effect in animal experiments.It was discovried that OMPs from Brucella was possessed of immunogenicity. Given the defect of classic vaccine and the peculiar pathogenic mechanism of brucellosis, we developed the new targeting dendritic cell vaccine with utility of Brucella outer membrane protein as antigens. The lipopolysaccharides and outer membrane proteins (OMPs) in Brucella cellular membrane are the main virulent factors and show appreciated antigeneici- ty. 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. Consequently, the two proteins were selected as the potential candidate antigens.The mannose receptors (MR) are the pattern recognition 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 modified the two kinds of protein antigens withα-D-mannopyranosyl-phenylisothiocyanate. Resorcinol-sulfuric acid analy- sis showed that two protein antigens have been glycosylated successfully.Tweenty mice were randomLy divided into 4 groups marked with A, B, C and D. The 4 groups were administrated vaccine Brucella S2, OMPs, glycosylation-modified OMPs, and PBS, respectively. We took a completely random ANOVA to analyse the immune effect. ELISA results showed that the level of serum antibody from A, B, and C group were remarkly higher than that of D group (P<0.01) at 4d, 7d, 10d after twice administration. The result suggested that OMPs and glycosylation-modified OMPs could significantly induce humoral immune response. Lymphopoiesis test was carried out at 10d after twice administration. The multiplication capacity of T lymphocyte from C group was significant- ly higher than it from A, B and D group. The result indicated that mannose modification of the brucella outer membrane protein paly a important role in induction of cellular immune responses.Adjuvant could influence the immune effect of vaccine. In the research, we compared 4 types of adjuvant: Montanide ISA 71 VG, Montanide IMS 251C VG, ESSAI ISA 206 CELL, Montanide Gel 01 PR by the orthogonal test. The results of Orthogonal test: antigen using Montanide IMS 251C VG nanoparticle adjuvant, immunization dose of mannose glycosylation of the outer membrane of Brucella protein X1μg, intramuscular injection and immunization three times, the trend in terms of antibody production or serum Concentra- Tion levels of IFN-γproduction, it was the best combination.Completely randomized and peritoneal macrophages experiments tested the targeted DCs vaccine immune effects, the results show that the targeted DCs vaccine induced a stronger immune response in the mice body. Safety results showed that: targeted DCs vaccine were safe to BALB / c mice which 6 to 8 weeks old and pregnant mice. The results in this study might pave the way for the further study on preventon of brucellosis based on vaccine strategies.