Construction Of An Oral Recombinant DNA Vaccine Encoding Tuberculosis Ag85A Gene And Research On The Induction Of T Cell Subpopulation Response

ObjectiveTuberculosis (TB) is a chronic respiratory infectious disease that has afflicted humans for thousands of years. For 80 years, the bacillus Calmette-Gue'rin (BCG) vaccine has been the only licensed TB vaccine given to humans covering 86% of the world population in 2001. However, despite the use of BCG, TB remains a global epidemic with one-third of the world population being infected and an annual rate of 8 million new cases and 2-2.5 million deaths. Regardless of its protection from severe forms of childhood TB, BCG fails to confer protection from adult TB. Furthermore, BCG vaccine may cause severe complications in immunocompromised hosts. Thus, there is an urgent need for developing safe and effective TB vaccines that are able to confer potent protection at the mucosal site.Presently Tuberculosis appears an increasing trend not only in developing countries but also in advanced countries. It is still a severe disease imperiling human health in the 21st century. Migration, HIV infection and multidrug resistants are the major dificulties for Tuberculosis control work.. The protective effect of BCG ranges from 0% to 85%, especially has a very low efficacy to adults and cannot be used for therpay. Protective ability of BCG varies in areas and populations, especially in developing countries such as in Africa areas, BCG is virtually usefuless. Since the global situation of Tuberculosis has become so severely and BCG is far from being an ideal vaccine against Tuberculosis, and there is no suitable vaccine available at moment, it is necessary to search for an efficient and safe and vaccine for us. As a new vaccine, DNA vaccine has been achieved inspiring progress in other infective diseases and tumor research. DNA vaccine can induce both of humoral and cellular immunity. Inoculation of DNA vaccines into muscle or skin by a syringe or a propulsion device such as a gene gun results in uptake of the DNA into cells, followed by transcription and translation of the pathogen's gene and, consequently, an immune response composed of antibodies, T helper cells and cytotoxic T lymphocytes (CTL). DNA vaccines have several advantages over traditional vaccines: they stimulate a full spectrum of immuneresponses including CTLs generally not induced by protein vaccines and they generate exceptionally long lasting immune responses. They provide their own adjuvant in the form of unmethylated bacterial CpG sequences that induce an innate immune response which in turn sponsors activation of an antigen-specific immune response. DNA vaccines, which were first described in 1992, have been shown to induce immune responses to a variety of viral, bacterial and parasitic antigens. In addition, they have shown efficacy in treatment of allergic diseases, autoimmunity and tumor models.DNA vaccines, based on plasmid vectors ex-response to DNA vaccines can be enhanced by genetic engineering of the antigen to facilitate its presentation to pressing an antigen under the control of a strong promoter, which have been shown to induce protective immune B and T cells. Furthermore, the immune response can be modulated by genetic adjuvants in the form of vectors responses to a number of pathogens, including viruses, expressing biologically active determinants or by more bacteria and parasites. They have also displayed efficacy in treatment or prevention of cancer, allergic diseases and traditional adjuvants that facilitate uptake of DNA into autoimmunity. Immunologically, DNA vaccines induce cells. The ease of genetic manipulation of DNA vaccines a full spectrum of immune responses that invites their use not only as vaccines but also as research T cells, T helper cells and antibodies.Vectors employed for the construction of DNA vaccines are bacterial plasmids which are otherwise commonly used for in vitro expression of proteins in Most DNA vaccine vectors contain an intron, which is an element that can increase expression of genes. This 'cassette' is followed by the gene encoding the antigen of interest flanked by the SV40 or bovine growth hormone 3%-untranslated region (BGH 3%-UTR) transcrip termination: polyadenylation sequences. This part of the vector is often referred to as the transcriptional unit responsible for antigen synthesis. The other part is the plasmid backbone that contains an origin of replication (ori) enabling high-yield production in Escherichia coli along with an antibiotic resistance gene, such as ampicillin (not approved by federal agencies for use in humans) or kanamycin (a resistance marker suitable for human vaccines), to confer antibiotic-selected growth in bacteria. This part of the plasmid backbone contains unmethylated CpG sequences that possess important immunomodulatory properties and provides an intrinsic adjuvant effect for DNA vaccines. It is thought that the magnitude of the immune response to DNA vaccines directly correlates with the level of antigen expression, measured in vitro upon transient transfection of cells.Antigen 85A is one of the components of the Antigen 85 complex. This protein is expressed by a wide range of bacteria of genus Mycobacterium. The Antigen 85A protein is coded by the gene called fibronectin-binding protein-A (fbpA) gene. In Mycobacterium tuberculosis, this fibronectin-binding protein-A (fbpA) gene is 1014bp long and has a molecular weight of 32KD.The fbpA gene codes for 338 amino acids which characterize the Antigen 85A protein. Antigen 85A has been applied differently in a wide range of research because of its role in the immunogenicity of the bacteria of genus Mycobacterium. The gene expressing the antigen 85A protein, fbpA, has been identified in different species of the mycobacteria such as M. leprae, M. gordonae, M. bovis, M. avium and they all show close homology to that of M. tuberculosis. Recent animal studies have shown that vaccination with Recombinant Antigen 85A DNA or the protein has powerful immunological properties, resulting in significant secretion of Th1 cells cytokines, particularly interleukins and interferon-γwhich are important in regulating the activities of a number of immune cells, especially the cytotoxic cells. These cytotoxic cells play significant roles in the elimination of viral infected and transformed cells.Increasing evidence suggests that vaccination at the mucosal site is superior to vaccination at other sites in eliciting protection from mucosal infectious diseases. This is partially explained by the observation that memory T and B cells generated upon mucosal vaccination acquire mucosa-homing receptors and preferentially accumulate at the mucosal site of induction. Thus, it is believed that greater immune protection may be achieved if TB vaccine is given mucosally via oral route.The cationic liposome acting as an adjuvant can greatly enhance the expression of recombinant plasmiddue to the function of preventing delivered DNA from digested by Dnase and can also promote the absorbance of cellular level. In addition, liposome has several advantages over traditional adjuvant., such as, easy preparated and ready to use, most important, with the high efficiency in gene transfection.Materials and methods Materials1. MiceMale C57B1/6, 6-8 weeks old, were purchased from Animal Laboratories (Chinese Medical University). C57BL/6 mice were immunized and randomly separated 30 mice into 5 groups: (NS)saline, (JO) plasmid vector, (L+JO) liposomal plasmid vector, (JA)recombinant plasmid, (L+JA)liposomal recombinant plasmid.2. ReagentLipofectamine^TM 200 were purchased from Invitrogen Co.(USA). Mammalian Cell Lysis Kit were purchased from Bio Basic Inc. (Canada) Anti-Chicken IgY, HRP Conjugate and Anti-Chicken IgY-FITC, PureYield Plasmid extraction kit were purchased from Promega Co.(USA). chichen anti-TB Ag85A IgY Monoclonal antibody were purchased from Prosci Co. Mouse CD4—FITC, CD8—PE, CD8—PE-CY5 antibodies were purchased from BD Co. (USA). IFN-r and IL-4 ELISA kit were purchased from R&D Co. (USA). Mouse IFN-r ELISPOT Kit, Mouse IL-4 ELISPOT Kit were purchased from U-CyTech Co. (Netherlands).Method1. The preparation of recombinant pCDNA3.1⁺/Ag85A plasmidThe gene encoding Ag85A mature protein was amplified by polymerase chain reaction (PCR) using forward primer 5'-CAGGATCCGCGCGCGCAGTCTGACCTAGTTGAGGATGC-3', containing BamHI cloning site; reverse primer 5'-GTCTCGAGAGGGCCGCCGCCGTTAATCGCT-3' containing Xho I cloning site, while genome of mycobacterium tuberculosis H37Rv strain as template, and PCR product treated with DNA gel extraction was inserted into cloning vector pUCm-T After transformation into competent DH5α,. the pUCm-Ag85A plasmid was extracted and digested with restriction enzyme BamHI and XhoI, then was subcloned to the same sites of eukaryotic expressing vector pcDNA3.1. after transformation into competent DH5α, the clone growing in SOB agar with amp were selected then the plasmid was extracted. and determine the fragment was correctly inserted into the vector, which was confirmed by partial nucleotide sequencing and restriction endonuclease digestion with restriction enzyme BamHI and XhoI.2.Screening of stably expressed K562 cells(1) The preparation of endotoxin-free plasmid by Pure Yield Plasmid with extraction kit(2) Lipofectamine^TM 2000 mediated transfection into K562 cellsJust prior to preparing complexes, plate 4×10⁵ cells in 500ul of growth medium without antibiotics. Then dilute 0.2μg DNA in 50ul DMEM without serum and mix Lipofectamine^TM 2000 gently before use, dilute 0.5μl in 50μl DMEM without serum. Incubate for 5 minutes at room temperature. Combine the diluted DNA with diluted Lipofectamine^TM 2000. Mix gently and incubate foy 20 minutes at room temperature. Then add 100μl of complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth. Incubate cells at 37℃in a CO₂ incubator for 36 hours for testing transgene expression. For stable cell lines, passage cells at a 1:10 into fresh growth medium 24 hours after transfection. Add G418 into DMEM as selective medium the following day.For K562 cells transfected afer 36 hours, centrifuge for minutes at 420×g, decant the supernatant. Wash the cells twice by resuspending the pellets with 1×PBS; centrifuge and discard the supernatant; Re-suspend the pellet in 1×cell lysis buffer, incubate for 5 minutes; Centrifuge the lysed cells for 45 minutes at 100000×g to prepare a protein solution. The extracted protein was used for SDS-PAGE and Western blotting analysis.; after the preliminary SDS-PAGE, the Recombinant Antigen 85A protein was transferred on to Nitrocellulose membrane and later incubated with chichen anti-TB Ag85A IgY Monoclonal antibody. For this technique, preliminary SDS-PAGE was run on two gels and one was stained using Coomasie stain while the other was used for the immunoblotting. After the transfer of the bands from the gel onto the nitrocellulose paper, the paper was properly blocked with BSA. This blocking step ensured that the binding potentials of non-specific antigens were eliminated. After the preliminary washing and the addition of the secondary antibody-horse radish peroxidase, a band corresponding to that on the Coomasie-stained gel was obtained.(3) Screening of stably expressed K562 cellspassage cells at a 1:10 into fresh growth medium 24 hours after transfection. Add G418 into DMEM as selective medium the following day. The concentration of G418 in medium are 800μg/ml, 400μg/ml, 200μg/ml, 100μg/ml, 50μg/ml respectively. The medium were changed every 3-4 days. The screening pressure continues for 4 weeks, then we determine the lowest concentration of G418 to kill the non-transfection K562 cells as the appropriate pressure to culture another 6 weeks so that stably transfected K562 cells could be screened. (4) Intracellular anti-chicken IgY-FITC staining were used to determine the expression efficacy in vitro.3. The effect on the T cells subset of the oral recombinant plasmid pCDNA3.1⁺/Ag85A mediated with LipofectamineTM 2000(1) Recombinant plasmid containing Ag85A using liposome as a vector was constructed and administered to C57BL/6 mice via oral route.. C57BL/6 mice were immunized three times at 14 days intervals, 30 mice were randomly separated into 5 groups: (NS)saline, (JO)plasmid vector,(L+JO)liposomal plasmid vector, (JA)recombinant plasmid, (L+JA)liposomal recombinant plasmid.(2) Determination of the contents of IFN-r, IL-4 in serum of C57BL/6 mice by double antibody sandwich ELISA, furthermore, the ability of splenocytes secreting IFN-γand IL-4 was tested by ELISPOT method..(3) When the mice were vaccinated with recombinant eukaryotic expressing vector 5 weeks later, titers of serum antibody against Ag85A were detected by ELISA.(4) Determination by flow cytometry of the percentage of the CD4⁺ and CD8⁺ T cell subsets in the splenocytes.(5) Intracellular anti-chicken IgY-FITC staining were used to determine the expression efficacy in vitro.Results1. The nucleotide sequencing of the gene encoding Ag85A mature form of mycobacterium tuberculosis H37Rv strain was identical and there were no undesired mutations, the cloning gene was correctly inserted into the vector pcDNA3.1⁺ as confirmed by restriction endonuclease digestion of Bam HI and XhoI and sequence analysis.2. We obtain a stable cell line which can express the protein with molecular mass about 32KD by using SDS, and showed immunogenicity with monoclonal antibodies of Ag85A, as detected with western blot. 3. The effect on the T cells subset of the oral recombinant plasmid pCDNA3.1⁺/Ag85A mediated with Lipofectamine^TM 2000(1) Lymphocytes obtained from the spleen of pcDNA3.1⁺/Ag85A vaccine mediated with lipofectamine^TM 2000 exhibited lower IFN-γproduction and higher IL-4 production than that for pcDNA3.1⁺ vector immunized mice.(2) The number of spleen MNC secreting IFN-γstimulated by Ag85A protein in vitro Ag85A protein were significantly lower than those of plasmid vector group.(3) L+JA immunized mice elicited higher Ag85A-specific antibodies titres than that for JA immunized mice. The plasmid vector group and recombinant plasmid group both mediated with Lipofectamine^TM 2000, the titres are 1:80 and 1:160 respectively by EL ISA.(4) Determination by flow cytometry of the percentage of the CD4⁺ and CD8⁺ T cell subsets in the splenocytes. The subset both shows the decrease of percentage compared with control group.(5) Fluorescence-activated cell sorter (FACS) analysis after intracellular staining displayed that 41.3% pcDNA3.1⁺/Ag85Atransfected stable K562 cells was FITC positive; whereas the controls only showed 1.5% positive cells.DiscussionIn this study, pCDNA3.1⁺ was chosen as the expression vector of recombinant DNA vaccine due to the CMV promoter and BGH gene. CpG seguence can induce the immune activity, furthermore, this vector can shuttle between the DH5αand K562 cells, the Laz gene can supply another screening marker in addition to the Amp resistence. The sequence analysis of cloning Ag85A showed the complete same amino acid sequence coding.The purity of DNA can greatly affect transfection efficiency. We chose the Vitagene kit to extract endotoxin-free plasmid to avoid the interference of the detection result. G418 can kill the growing cells, so we use it to screen the stably transfected K562 cells through dose selection as screening pressure.CD4⁺T and CD8⁺T cell subsets can directly exhibit the celluar immune level. In ELISPOT assay, we choose serum-free medium and endotoxin-free plasmid to assure the accuracy of the result, Ag85A protein which was isolated from K562 cells can lower the influence of endotoxin because the purity had been detected through SDS-PAGE and western-blot. ELISPOT assay has three typical advantanges. First, high sensitivity; second, single cell detection level; Last, easy operation.The oral recombinant plasmid pCDNA3.1⁺/ Ag85A. mediated with Lipofectamine^TM 2000 showed down-regulation effect of the subsets of CD4⁺ T cells and CD8⁺ T cells could induce Th2 type humoral response and suppressed the secretion of IFN-γin C57BL/6 mice by ELISPOT. In addition, the pCDNA3.1⁺/Ag85A DNA vaccine can greatly enhance the titres of Ag85A-specific antibodies by ELISA. The down-regulation effect of the subsets of CD4⁺ T cells and CD8⁺ T cells reflect the oral immune tolerance. Because the immune system is a network, CD4⁺CD25⁺Treg and TGF-βtogether with apoptosis factors can lead to suppression of CD8⁺T subsets, iDC and mDC can induce tolerance due to the secretion of chromosome. According to our result, liposome can act as adjunvant without toxicity to cells.Conclusion1. DNA vaccine encoding Ag85A mature protein ofmycobacterium tuberculosis was constructed and identified successfully.2. The Ag85A protein was stably expressed after G418 screening transfected K562 cells.3. The oral recombinant plasmid pCDNA3.1⁺/Ag85A. mediated with Lipofectamine^TM 2000 shows down-regulation effect of the subsets of CD4⁺ T cells and D8⁺ T cells.4. The oral recombinant plasmid pCDNA3.1⁺/Ag85A has good immunogenecity and could induce Th2 type humoral response in C57BL/6 mice by ELISPOT.5. The oral recombinant plasmid pCDNA3.1⁺/Ag85A mediated with Lipofectamine^TM 2000 group suppressed the secretion of IFN-γ, while the plasmid pCDNA3.1^+W with Lipofectamine^TM 2000 group increased the IFN-γlevel due to the CpG sequences.6. The oral recombinant plasmid pCDNA3.1⁺/Ag85A mediated with Lipofectamine^TM 2000 can greatly enhance the titres of Ag85A-specific antibodies by ELISA.7. Lipofectamine^TM 2000 can act as an adjuvant through comparation with negative control group.