Production And Immune Characterization Of Brucella Ghosts

Brucellosis is one of the most widely popular and harmful zoonosis, with the pathological manifestations of arthritis, endocarditis and meningitis in humans and cattle. It leads to abortion and infertility resulting in serious economic losses, which caused a loss of600million a year in Latin American, and an average of150million in the American in90’s. The prevalence of Brucellosis was rising over the past decade in China. Compared to0.09human in10million in1996, there were2.77persons in10million got sick in2011, according to incomplete statistics. The incidence rate increased30times in ten years and about4million people infected. Brucella is an important gram-negative and facultative intracellular pathogen, so it is important to define the nature of a protective immune response to the virulent strains of Brucella in order to design new vaccines for brucellosis, methods to evaluate potential vaccines and innovative treatments of infected animals and people. However the vaccine against Brucella infection has a certain virulence, side effects, and can lead to confuse the natural infection with immunity, which limit the application of Brucella vaccine.Bacterial ghost (BG) is an empty bacterial envelope of Gram-negative bacteria produced by the controlled expression of cloned PhiX174gene E which leads to the formation of40-200nm transmembrane tunnels with the release of cytoplasmic contents. BGs possess the same surface properties as their living counterparts and are able to induce both cellular and humeral responses, which are new vaccine in bacterial diseases. So, BGs have abroad application in disease immune and prevention.To explore the rapid detection of Brucella, multiplex PCR assay was developed. Four pairs of primers were designed based on the specific IS711element, biovar-specific gene of B. melitensis, B. abortus and B. suis. The results showed that sensitivity and specificity of the assay was high accurate. The PCR could provided a reliable detection assay for clinical identification of Brucella. Thus, it has an important significance for early detection and epidemiological survey of Brucellosis.In this study, antimicrobial peptide LL-37and lysis gene E were coexpressed to inactivate the bacteria. The results showed that antimicrobial peptide LL-37had no significant effect for promotion of the cell lysis rate of E.coli. The plasmid pBBRlMCS::E was electrically transformed into B. melitensis strain M111and grown at28℃for24h and then incubated at42℃to induce the expression of gene E to generate the bacterial ghosts (BGs). The morphological characteristics, the lysis rate and immunological properties of BGs were studied. The results showed that the lysis rate of BGs can reach100%. BGs have the surface structure of normal bacterial, but the surface of BGs significantly shrank and cell morphology changed. Mice were injected intraperitoneally or subcutaneous with live Brucella melitensis M111, Brucella melitensis ghosts (BMG), Brucella melitensis ghosts with polyinosinic-polycytidylic acid (BGPA), and formalin-killed Brucella (FKB). The antibody level, IFN-y and IL-4level, CD4+and CD8+numbers in mice with intraperitoneal injection were significantly higher than that of the group with the subcutaneous injection, suggesting that intraperitoneal injection was better than subcutaneous to induce humoral and cell immunity levels. Meanwhile, mice immunized with BGPA by intraperitoneal showed significantly higher IgG than the BMG, M111and FKB group. However the antibody titers showed no significant difference between BMG and M111group. IFN-y levels of the BMG group were significantly higher than that of the other groups,and reached48.207pg/ml. IL-4levels of M111group were significantly higher than that of the other groups by intraperitoneal inoculation and reached88.211pg/ml. There is no statistically significant difference the number of CD4+and CD8+of the M111, BMG and BGPA group were significantly higher than that of the FKB group. Thus, vaccine of BMGs produced equivalent humoral and cell immunity with live vaccine.In conclusion, the Brucella ghost showed good safety and immunogenicity, which can be the new candidate vaccine against Brucella infection. Consequently, the development of this new bacterial ghost vaccine provides a new idea for the development of other intracellular bacterial vaccine.