Study On Neisseria Gonorrhoeae PorB DNA Vaccine With Salmonella Ghost As Vector

Bacterial ghost represents empty non-denaturated envelopes derived from gram-negative bacteria devoid of cytoplasmic content, and produced by controlled expression of cloned bacteriophage PhiX174 lysis gene E. Bacterial ghost retain natural bacterial outer membrane structure, such as LPS, flagella and outer membrane proteins, which lead to bacterial ghost adherence to the human or animal cell receptors. So it can be employed as vaccine to induce immune responses and delivery system for a foreign antigen, nucleic acid or other compounds to construct combined vaccine against human or animal infection.Gonorrhea, caused by Neisseria gonorrhoeae, is one of sexually transmitted disease (STD). Due to the increase of antibiotic resistance, the treatment of gonorrhea with antibiotics is becoming more and more difficult. Developing a new vaccine is one of the strategies in preventing and treating gonorrhea. Outer membrane protein B (PorB) is the ideal candidate vaccine of gonorrhea. However, studies showed that DNA vaccine could not induce high efficiency of immune response to protect human or animal due to inefficient transfection.In this study, the Neisseria gonorrhoeae candidate vaccine SE- ghost(PorB) was contructed by loading SE-ghost with Neisseria gonorrhoeae PorB DNA vaccine and its immunogenicity was investigated.The research was divided into two parts.Part I:Evaluation of exogenous substances-loaded SE-ghost in vitroRecombinant Salmonella HA2-EBOX, containing a temperature control system, was induced to produce SE-ghost. SE-ghost was observed under scanning electron microscope (SEM) and transmission electron microscope (TEM). The results indicated that Salmonella ghost was successfully produced and the empty cell envelopes devoid of cytoplasmic contents could be observed significantly under SEM and TEM. SE-ghost loaded with fluorescein isothiocyanate (FITC) was respectively transferred into 3LL cells, HeLa cells and RAW264.7 cells in vitro for 48 h and fluorescence of FITC in cells was observed by microscope and flow cytometry. Mix SE-ghost with eukaryotic expression plasmid pEGFP-N1. Detecting the concentration of free DNA in the samples supernatant after centrifugation by spectrophotometry to analysis of the loading efficiency. The expression of pEGFP-N1 in cells was observed by microscope and flow cytometry after transferring into RAW264.7 cells for 48 h. Studies indicated that FITC-loaded SE-ghost was efficiently expressed in 3LL cells, HeLa cells and RAW264.7 cells. SE-ghost loaded with pEGFP-N1 plasmid was efficiently taken up by RAW264.7 cells and a high transfection rate, as shown by fluorescent microscopy and flow cytometry.Part II:Immunogenicity of Neisseria gonorrhoeae PorB DNA vaccine delivered by SE-ghostMix SE-ghost with eukaryotic expression plasmid pVAXl-PorB to produce Neisseria gonorrhoeae candidate vaccine SE-ghost (PorB). SE-ghost (PorB) was used to stimulate bone marrow dendritic cells (BMDC). The expression of CD86, CD80, CD40, MHC-Ⅱon the surface of BMDCs was detected by flow cytometry and the secretion of cytokine IL-1β, IL-6, IL-10, IL-12p70 in BMDCs supernatant was detected by indirect ELISA. The proliferation of allogeneic CD4+T cells with BMDC stimulated was detected by flow cytometry. The results indicated that Neisseria gonorrhoeae candidate vaccine SE-ghost (PorB) was successfully produced. BMDCs cultured with SE-ghost(PorB) were more effective than with medium in up-regulating the expression of molecules, including CD86, CD80, CD40, MHC II and secretion of IL-1β, IL-6, IL-10, IL-12p70 and allogeneic CD4+T cell activation.6-week-old BALB/c mice were divided into four groups, SE-ghost (PorB), pVAX1-PorB, SE-ghost and PBS. The mice were immunized orally,3 times at two weeks interval. Serum antibody IgG and vaginal lotion IgA was detected by indirect ELISA. Bactericidal activity was detected by antibody-dependent complement mediated cytotoxicity assay with Neisseria gonorrhea WHO-A. The results indicated that compared with PBS group, mice from SE-ghost (PorB) group demonstrated significant increases in antigen-specific sera IgG, vaginal lotion IgA. The sera of mice immunized with SE-ghost (PorB) showed greater Neisseria gonorrhoeae (WHO-A) bacteriolytic activity than other groups.The frequency of CD3+CD4+T, CD3+ CD8+T cells in mouse peripheral blood were detected by flow cytometry. The secretion of IL-4, IFN-y within the cytoplasm of the spleen CD4+T cell subsets were detected by intracellular staining. The proliferation of spleen cells stimulated with WHO-A for 48 h was detected by flow cytometry. The results indicated that the populations of CD3+CD4+T, CD3+CD8+T, CD4+IL-4+T, CD4+IFN-y+T cells in immunized mice were significantly higher than those in the controls. Spleen cells from SE-ghost (PorB) group demonstrated significant proliferation as compared to control groups.