The Effects Of Salmonella Ghost As Immune Adjuvant On Melanoma-bearing Mice

The principle of vaccination is to produce protective immune responses against challenge,and the vaccine itself is safe.The traditional live-attenuated vaccine has a potential risk of virulence recovery and the poor immunogenic response of the whole-pathogen preparations,which have limited their application.Compared with the traditional vaccines,the novel vaccine has high safety and specificity,but with poor immunogenicity.Therefore,adjuvants are needed to further trigger the required immune response of the vaccine.Bacterial ghost are structurally empty shell of Gram-negative bacteria induced by bacteriophage phix174 lysis gene E without the cytoplasmic contents such as nucleic acids,ribosomes or other cytoplasmic contents.BG maintain the complete outer membrane structures similar to those of innate bacteria,including native immunostimulant,which can be used as effective adjuvant.Because of the high morbidity and mortality,cancer has always been a threat to human health.Novel therapeutic strategies will be investigated based on deeper understandings of tumorigenesis and immunology.With the progress of tumor immunology,the focus of tumor vaccine research is utlizing the strength of immune system and specific immunotherapy.Effective tumor vaccine should enhance both cellular and humoral immune responses to inhibit tumor growth.As soluble antigens elicit low antibody titers or ineffective CTL responses,tumor vaccines often require the addition of adjuvants to exert effective functions.In this study,we produced Salmonella ghost as adjuvant combined with OVA and evaluated its immune-enhancing effect on antigen-presenting cells,as well as the immune functions in initiating both the humoral and cellular immune responses at the cellular and the animal levels.The results of this study provide ideas for the development and progress of clinical vaccine adjuvants and tumor immunotherapy.The research includes the following three parts:Part I:Study on immune function of Salmonella ghost as adjuvant in vitroThe recombinant Salmonella HA2-EBOX was induced to produce SEG.The morphology of SEG was observed by transmission electron microscope(TEM).The results showed that the cytoplasmic contents flowed out to form an empty shell structure,which suggested that Salmonella ghost was successfully prepared.The mixture of SEG and OVA was used to stimulate BMDC,the expression of CD80,CD86,CD40 and MHC II were detected by flow cytometry and levels of cytokine IL-1β,IL-6,IL-10 and TNF-a in the supernantant were measured by indirect ELISA.For the BMDC uptake experiment,SEG(OVA-FITC)were incubated with BMDC in different period.The results indicated that the expression of CD86,CD80,CD40 and MHC Ⅱ were upregulated on SEG-OVA-treated BMDC and secretion of IL-1β,IL-6,IL-10 and TNF-α increased in the supernantant.In addition,SEG(OVA-FITC)can be efficiently taken up by BMDC and green fluorescence from SEG(OVA-FITC)was greatly increased in BMDC in a-dose-dependent manner.In order to clarify the effect of SEG on the adaptive immune responses and examine whether it could promote the response of CD4+T cells and induce the CD8 cytotoxic T cell(CTL)immune response,CD4+T cells from OT-Ⅱ TCR transgenic mice and spleen cells from OT-I TCR transgenic mice were co-cultured with SEG-stimulated BMDC.The results showed that SEG not only promoted the antigen presentation of BMDC to CD4+T cells,but also improve the cross-presentation of antigen to CD8+T cells.Part II:Study on immunogenicity of OVA combined with different doses of SEG-in miceIn vitro,BMDC and RAW264.7 cells were incubated with different doses of SEG for safety evaluation measured by CCK-8.In vivo,BALB/c mice were immunized with a certain dose of SEG to observe the survival state of mice within two weeks.Mice were then randomly divided into 6 groups:SEG-OVA(containing 105,106 or 107 CFU of SEG and 50μg OVA),OVA,SEG and PBS as controls.All of them were subcutaneously immunized and OVA-specific IgG,IgGl and IgG2a in serum were detected by indirect ELIS A.The frequency of CD3+ CD4+T cells,CD3+CD8+T cells in spleen and peripheral blood were detected by flow cytometry.The results showed that SEG-OVA significantly increased the levels of OVA-specific serum IgG,IgGl and IgG2a,and frequencies of CD3+ CD4+T cells,CD3+ CD8+T cells in the peripheral blood and spleen when compared with OVA alone.After immunization with OVA delivered by SEG as vector,IL-4 and IFN-y production in splenocytes of immunized mice were detected by intracellular cytokine staining.Proliferation of splenocytes re-stimulated with OVA was detected by flow cytometry.The results showed that immunization with SEG(OVA)promoted a higher percentage of CD4+IFN-y+T cells in spleen and robust proliferation in splenocytes after re-stimulated with OVA than those of the OVA.Part Ⅲ:The inhibitory effects of SEG as immune adjuvant on melanoma-bearing miceIn a prophylactic tumor model,6-week-old BALB/c mice were randomly divided into 8 groups:SEG-OVA(containing105,106 or 107 CFU of SEG and 50μg OVA),OVA,SEG(OVA),SEG,PBS and Alum-OVA were utilized as controls.All of them were subcutaneously immunized three times at two weeks interval.Seven days after the third immunization,mice were inoculated subcutaneously in the right axilla with B 16-OVA cells.On day 12,the tumor sizes were measured by Vernier caliper.On day 24,mice were sacrificed and their tumors were separated.Meanwhile,splenocytes were collected and the proportion of CD11b+Gr-1+ cells was detected by flow cytometry.The results showed that SEG as an immunoadjuvant could significantly inhibit tumor growth in tumor model.In the pre-established tumor model,mice were first challenged with B16-OVA cells.At 7 days after inoculation,mice were immunized three times at two weeks intervals with SEG-OVA(containing are 105,106or 107 CFU of SEG and 50μg OVA),OVA,SEG(OVA),SEG,PBS and Alum-OVA were utilized as controls.In order to prove the specificity of SEG-OVA against B 16-OVA,B16F10 cells(5×105 cells per mouse)were inoculated prior to immunizing with SEG(OVA).The results showed that SEG-OVA could significantly delayed the growth of tumor and the protection of SEG-OVA against tumors is OVA specific.