| ObjectiveAccording to WHO's report on Tuberculosis (TB) early this year the infected people with TB are over 2 billion tenth of which are existing patients globally and there are 0.55 billion people infected with TB already 4.5 million of which are active TB patients in China. Among the patients about half million patients are showing broad resistance to medicines available. BCG is an only vaccine marketing and clinically available however with some instability in quite some clinical application. It is an urgent work for us to develop both safe and effective vaccine to TB.BCG as an attenuated vaccine has also been applied as immune adjuvant besides its major function and in some cases is used to treat patients with cancer such as carcinoma of urinary bladder cancer. There are data to support that BCG, animal and human being share some antigen. In many cases while treating patients with carcinoma of urinary bladder cancer with BCG by the way of filling obvious side effects usually result in discontinuity of such treatment.Ag85 complex is consist of A,B and C components, existing in their cell wall and culture filtrate of Bacillus tuberculosis as well as similar bacteria. Ag85A is predominant in total three components and it has been proved that Ag85A antigen is a critical antigen with immune protection. The evidence from recent studies reveals that Ag85A may enhance CD8~+ CTL activity, secretion of Th1 related cytokines such as: IL-2,TNF-αand IFN-γ, killing effect of monocyte and NK to tumor cells. Due to the long culture period, complicated growing condition of Bacillus tuberculosis, Ag85A fragile attributes of degrading and agglutination as well as technical bottle neck in extraction and purification to obtain it, also for some potential hypersensitivity accompany long term application in 1996 Huygen first time reported that in mouse model through the way of intramuscular injection of plasmid carrying gene fragment encoding Ag85A antigen, cellular and humeral immunity to TB attack had been successfully established.Since 1990's numerous research results have showed the great potential either in efficacy or application to DNA vaccine. Especially in most recent years the studies are broadening into aspects involved in anti-infection, antitumor, hypersensitivity and organ graft with hope to mankind. DNA vaccine may be delivered through mucosal, skin and intramuscular ways and may be prepared in the formulations of spraying, oral product or injection fitting various target genes expressing vaccines for either up regulating or down regulating immunity, even modifying types of immune response. Oral delivery for DNA vaccine is an easy way with advantage to be accepted however to Ag85A so far it is not clear that while taken orally, how to cause changing in subpopulations and biological activity of IEL as well as mechanism of this changing in local mucosal immune system. Based on our previous study to prove oral Ag85A vaccine may establish immunity in mouse model the present study here is to investigate further and mechanism of inducing immune response, furthermore to provide data to evaluate potential clinical application to treatment or prophylaxis.Methods1. Constructing recombinant plasmid pcDNA3.1/myc-hisA-Ag85AAmplifying Ag85A gene with PCR and ligate with pcDNA3.1/myc-hisA forming pcDNA3.1/myc-hisA-Ag85A, transforming into DH5α, store at-70℃for study.2. Preparation of Ag85A DNA vaccinePurify plasmid pcDNA3.1/myc-hisA-Ag85A with V-gene, endotoxin free, ultra pure plasmid DNA kit, transform the plasmid into attenuated Salmonella typhi with electroporation technique. Grow the transformed attenuated Salmonella typhi in quantity.Simultaneously prepare liposome encapsulated plasmid pcDNA3.1/myc-hisA-Ag85A.3. Animal groups and immunization(1) Determining subpopulations of IEL in local intestine mucosal system after immunization: Divide C57BL/6 mice into three groups that is: testing group, empty plasmid group and normal control, Three times immunization for each mouse totally 10 days between first time and second time, 14days between second time and third time. In the first and second time immunization filling animals in testing group with liposome encapsulated Ag85A DNA vaccine, at last time filling animals in testing group with attenuated recombinant Salmonella typhi, filling animals in control with Ag85A DNA vaccine free liposome. On 21 days post last filling sacrifice mice, isolate IEL and determine subpopulations.(2) Testing biological activity of IEL induced with Ag85A DNA vaccine: Divide C57BL/6 mice into three groups that is: testing group, empty plasmid group and normal control, Immunization twice for each mouse between 14days.Filling animals in testing group with recombinant Salmonella typhi, filling animals in control group with attenuated V1Jns.tPA.On the 1,3,6,9 and 12day post last filling Isolate IEL and test biological activity.4. Isolation of IELTaking whole small intestine, cut it into pieces, digest with enzyme on the shaker for moment follow by isolating lymphocytes with percoll.5. IEL subpopulationsIEL subpopulations are analyised with FLCM.6. IEL culture in vitroAfter separation of IEL adjust IEL conc. Add 5μg/ml Ag85A protein, incubate 48, and take supernatant for cytokine test.7. Ctokine assaySeparately IL-2 level in IEL supernatant is tested with routine cell line dependent method and double sandwich ELISA for testing IFN-γ,IL-4,IL-10 and TGF-β。8. FasL assayExtracting total RNA in IEL test expression of FasL mRNA with RT-PCR. Result is given in ratio by FasL density in electrophoresis band to density ofβ-actin.9. Determination of cytotoxityStimulate IEL with mitomycin treated transfected Ag85A cDNA p815 cells, mix the cells with 51Cr labeled P815 cells. After incubation measure cpm in supernatant withγcounter.Culculate cytotoxity. Cytotoxity (%) = (IEL cpm - base cpm/max.cpm -base cpm) x100%10. IEL proliferation testAdd 5μg/mlAg85A protein and [~3H]thymidine to IEL culture media, incubate and measure cpm withβliquid scintillation counter. Proliferation index=testing cpm-base cpm/base cpm.11. SIgA determinationTake 5cm middle part of small intestine, dissect it, homogenize , centrifuge, take supernatant, and then SIgA level in intestine is determined with immunoassay testing kit.Results1. pcDNA3.1/myc-hisA-Ag85A and pcDNA3.1/myc-hisA transformation are successful and recombinant bacteria is established.2. Changing of IEL subpopulationsThe data from FLCM detection show that number of CD3~+IEL in testing group are (5.89±1.15) x 106, (5.55±0.97) x 106 in control group and (5.32±1.10) x 106 in normal control group, giving no significance(p>0.05); The percentage of CD3~+ CD4~+IEL in testing group are 39.65±5.46%, 39.78±4.32% in control group and 38.924±3.46% in normal control group giving no significance(p>0.05). The percentages of CD3~+CD8~+αβIEL and CD3~+CD8~+ααIEL in testing group are 27.25.65±1.98% and 50.75±4.82% respectively, 21.37±3.82% and 41.08±3.98% in testing control group respectively; 19.80+0.82% and 39, 35±4.32% in normal control group respectively giving significance(p<0.05). The percentages of TCRαβIEL in testing group are 42.72±2.25%, 44.24±2.01% in testing control group and 42.38±1.34% in normal control group, giving no significance(p>0.05). The percentages of TCRγδIEL in testing group are 38.21.±2.28%, 32.08±0.98% in testing control group and 31.35±4.32% in normal control group, giving no significance(p>0.05). There is also no significance between the result of detection for subpopulation with CD40L IEL and that with CD45~+IEL. The same tendency was found in IEL with CD19~+. However there is lower expression of FasL in IEL in testing group giving great significance(p<0.01) against one in testing control. The same tendency was found in both IEL with CD25~+ and IEL with Foxp3~+. Contrastly we have detected out great significant increasing in IEL with CD103~+ in testing group as compared to testing control group(p<0.01).3. IL-2 level in testing group is increased markedly (6679±793cpm) on the day 6 post last immunization and to the max. on the day 9 (10,806±1,293cpm) with great significance against testing control (1,075±186) ,normal control (308±59) (P<0.01) . IFN-γlevel in testing group is increased markedly (198.78±12.35 pg/ml) on the day 6 post last immunization with significance against testing control ( 75.88±6.03 pg/ml),normal control(53.56±4.85)(P<0.01). IL-10 level in testing group is increased(35.76±2.78pg/ml) on the day 6 post last immunization, with significance against testing control (13.98±1.12 pg/ml) (P<0.05). TGF-P level in testing group is increased markedly (116.56±2.33 pg/ml) on the day 6 post last immunization with great significance against testing control (19.54±2.05 pg/ml) ,normal control (21.66±0.88 pg/ml) (P<0.01) . There is no significance to IL-4 level.4. FasL mRNA expression on the day 8 post last immunization in recombinant plasmid is 0.61±0.25 significantly increased as compared with testing control,0.28±0.087) and normal control 0.19±0.13) (P<0.05) .5. IEL cytotoxityTesting IEL cytotoxity on the day 9 post last immunization result shoes cytotoxity in testing group is 67.35±5.56%, 14.89±3.73% in testing control and 10.26±3.31% in normal control, giving significance(P<0.05).6. IEL proliferationDetermine [~3H]thymidine inserting cpm on the day 6 and day 9 post last immunization separately. Proliferation test result shows 3608±372 in testing group on day 6 and 3536±298 on day 9 which are significant to 598±79 in testing control and 675±67 in normal control (P<0.01) ,reveals Ag85A exerts a marked proliferating stimulation on IEL growth.7. SIgA generationSIgA level is 0.38±0.05 ng/ml in testing group giving significance against 0.24±0.04 ng/ml in testing control and 0.2±0.09 ng/ml in normal control (P<0.05) . ConclusionpcDNA3.1/myc-hisA-Ag85A and pcDNA3.1/myc-hisA transformation are successful and recombinant bacteria is established.Via oral delivery of Ag85A DNA vaccine It may be attained to the positive induction of CD3~+CD8~+αβIEL, CD3~+CD8~+ααIEL , TCRγδIEL and IEL with CD103~+ while resulting in negative induction to IEL with CD25~+ and IEL with Foxp3~+. Above oral way to deliver Ag85 A DNA vaccine doesn't affect total CD3 IEL and CD3~+TCRαβIEL. This provides very important clue that Ag85A DNA vaccine, while delivery via oral way may predominantly induce cellular immune response mediated by CD8~+CTL, not immune tolerance.Via oral delivery of Ag85A DNA vaccine it may stimulate increasing levels of cytokines IL-2, IFN-γ, IL-10 and TGF-βwhile doesn't affect level of IL-4. Also results in upregulating CTL cytotoxicty and generation of SIgA, which means cytokines secreted by Th1 are involved in this process.
|
PDF Full Text Request