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Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system
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Contents lists available at SciVerse ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system
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Jomkhwan Meerak a,b , Supason Wanichweacharungruang c , Tanapat Palaga a,∗
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Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand Division of Microbiology, Department of Biology, Faculty of Science, Chiang Mai University, Huaykaew Road, Chiangmai 50200, Thailand Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand
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Article history: Received 22 August 2012 Received in revised form 20 November 2012 Accepted 26 November 2012 Available online xxx
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DNA vaccines are a promising new generation of vaccines that can elicit an immune response using DNA encoding the antigen of interest. The efficacy of these vaccines, however, still needs to be improved. In this study, we investigated the effect of autophagy on increasing the efficacy of a candidate DNA vaccine against Mycobacterium tuberculosis (MTB), a causative agent of tuberculosis. Low molecular weight chitosan was used to encapsulate plasmid DNA containing a gene encoding MTB Antigen 85B (Ag85B), a secreted fibronectin-binding protein. To induce autophagy upon DNA vaccination, the kinase defective mTOR (mTOR-KD) was transfected into cells, and autophagy was detected based on the presence of LC3II. To investigate whether autophagy enhances an immune response upon DNA vaccination, we coencapsualted the Ag85B-containing plasmid with a plasmid encoding mTOR-KD. Plasmids encapsulated by chitosan particles were used for primary subcutaneous immunization and for intranasal boost in mice. After the boost vaccination, sera from the mice were measured for humoral immune response. The DNA vaccine with the autophagy-inducing construct elicited significantly higher Ag85B-specific antibody levels than the control group treated with the Ag85B plasmid alone or with the Ag85B plasmid plus the wild type mTOR construct. Upon in vitro stimulation of splenocytes from mice immunized with recombinant Ag85B, the highest levels of secreted IFN-␥ and IL-2 were detected in mice immunized with the autophagy-inducing plasmid, while no differences in IL-4 levels were detected between the groups, suggesting that the DNA vaccine regimen with autophagy induction induced primarily a Th1 immune response. Furthermore, the enhanced proliferation of CD4+ T cells from mice receiving the autophagyinducing vaccine was observed in vitro. Based on the evidence presented, we conclude that incorporating an autophagy-inducing element into a DNA vaccine may help to improve immune response. © 2012 Published by Elsevier Ltd.
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Keywords: DNA vaccine mTOR Autophagy Mycobacterium tuberculosis Chitosan
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1. Introduction
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Immunization with DNA vaccines has become a highly investigated topic beginning with the first report that naked plasmid DNA is taken up by muscle cells, resulting in the expression of beta-galactosidase [1]. Since then, DNA vaccines aiming to elicit protective immune responses against various infectious diseases have been investigated and have demonstrated the effectiveness of DNA in triggering both humoral and cell-mediated immune responses [2]. Even though there are no reports on the side effects of DNA vaccine administration, the development of DNA vaccines has yet to move beyond PhaseI/II clinical trials in humans, mainly because of low immunogenicity in humans [3]. Autophagy is a mechanism that is essential for cell survival and for maintaining cellular homeostasis during stress through the
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∗ Corresponding author. Tel.: +66 2 2185070; fax: +66 2 2527576. E-mail address: [email protected] (T. Palaga).
break down of cytosolic components and damaged organelles. The process is initiated by the formation of a double layer membrane surrounding the cellular contents to be degraded and involves multiple products of the autophagy-related genes (ATG), such as Atg12, Atg5 and Atg16L [4]. These proteins help conjugate phosphatidylethanolamine to a protein called LC3 (Atg8), which forms part of the double layer membrane. After the double layer membrane closes, the proteins inside, including the Atg proteins, are degraded upon fusion with the phagolysosome [5]. Autophagy also plays an important role in cellular defense against intracellular pathogens such as viruses and intracellular bacteria by surrounding and targeting them for clearance [6]. Furthermore, autophagy plays a key role in feeding cytoplasmic peptide antigens for antigen loading onto MHC II [7]. Increases in antigen presentation and acquired immune responses were reported when autophagy was induced upon antigen exposure [8]. Inhibition of the mammalian target of rapamycin (mTOR) by disruption of the gene encoding mTOR or by rapamycin treatment activates autophagy and has been used widely for studying autophagic phenomena in vitro and in vivo [5].
0264-410X/$ – see front matter © 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.vaccine.2012.11.075
Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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Tuberculosis (TB), a condition caused by aerosol infection with Mycobacterium tuberculosis (MTB), is a major global health problem that kills millions of people worldwide each year [9]. The currently available vaccine, Mycobacterium bovis BCG, is protective only against a severe form of childhood TB, but does not decrease the global TB burden in adults [9]. Therefore, novel vaccines for TB are seriously needed, and many types of new TB vaccines are either in the early stages of development or in the clinical trial pipeline [9]. Currently, DNA vaccines are one of the many types of new TB vaccine candidates under investigation. Previous reports have revealed the potency of plasmid DNAs carrying various MTB antigen-encoding genes in the induction of a protective immune response in mice and non-human primates [10–13]. After the recent completion of MTB whole genome sequencing, several MTB antigens such as Hsp65, Hsp70, Ag85A, Ag85B and ESAT-6 were tested for efficacy as DNA vaccines [14]. Antigen 85B (Ag85B), an abundant 30 kDa secreted MTB protein, was cloned reported to elicit a protective immune response in mice similar to that seen in those who had received the BCG vaccination in combination with bovine herpesvirus 1 VP22 [12]. However, the immunogenicity of most DNA vaccines under development for TB needs to be improved. Autophagy plays an important role in controlling TB infection in macrophages. Induction of autophagy in MTB-infected macrophages overcomes the block in phagosome maturation by the bacteria, and delivers the bacilli for degradation and elimination [15–17]. Interestingly, the efficacy of the BCG vaccine is increased in murine dendritic cells (DCs) in vitro by inducing autophagy by treatment with rapamycin, an inhibitor of mTOR [18]. This effect is due to enhanced antigen presentation of an immunodominant Ag85B. For this reason, we tested whether induction of the autophagic pathway would enhance the presentation of the MTB antigen to the antigen presenting cells (APCs) leading to increased immunogenicity of candidate DNA vaccines. Because MTB is transmitted by aerosolized droplets containing the bacilli, airway and lung mucosal immunity play a critical role in defense against TB infection. In order to enhance mucosal immunity, the DNA vaccine needs to be formulated for intranasal administration. Chitosan, an abundant natural biopolymer, is generally used as a drug and DNA carrier for cellular delivery both in vitro and in vivo [19–21]. It is suggested that both chitosan nanoand micro-particles act as effective immunological adjuvants and safe carriers for the delivery of vaccines [22]. In this study, we present evidence that incorporating an autophagy-inducing plasmid into a DNA vaccine enhances host immune responses to a DNA vaccine against the MTB antigen delivered by chitosan particles in mice.
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2. Materials and methods
For in vitro transfection studies, CN/DNA nanoparticles were transfected into HEK293T cells as previously described [20]. Expression of Ag85B and mTOR was detected by immunofluorescence staining.
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2.1. Reagents
2.7. Vaccine preparation and immunization
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2.2. Mice
CN/DNA nanoparticles were prepared as described above. Female BALB/c (8 weeks old) mice were randomly assigned into four groups, each composed of four mice, for four different immunization regimens (pVITRO-fbpB alone, pVITRO-fbpB with pmTOR-KD, pVITRO-fbpB with pmTOR or pVITRO1 empty vector with pmTOR-KD). Immunization was carried out by priming with 50 g of plasmid DNA encapsulated by chitosan particles delivered by subcutaneous injection, followed by two booster doses at 2-week intervals. The two boosts were delivered intranasally by slow repeated dropping of a total of 30 l of CN/DNA nanoparticle solution (equivalent to 50 g DNA) into the nasal cavity. For immunization with coencapsulation of pVITRO-fbpB with pmTOR-KD or pmTOR, 50 g of each plasmid was used for each mouse.
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Low molecular weight chitosan, 30 kDa with approximately 80–85% deacetylation, was used for plasmid DNA encapsulation (Department of Chemistry, Chulalongkorn University). The plasmids encoding myc-mTOR (pmTOR) and myc-mTORKD (pmTOR-KD) were obtained from Addgene (MA, USA) (Dr. D. Sabatini, Massachusetts Institute of Technology). The plasmid pmTOR-KD contained two point mutations in the mTOR gene resulting in the amino acid substitutions D2357E and V2364I.
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Eight-week-old female BALB/c inbred mice were obtained from the National Laboratory Animal Center (Mahidol University, Salaya,
Thailand). All mice were maintained under SPF conditions and used in accordance with the policies and regulations for the care and use of laboratory animals of the Institute of Biotechnology and Genetic Engineering, Chulalongkorn University.
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2.3. Construction of plasmids and production of recombinant Ag85B
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For construction of the plasmid harboring Ag85B, the full sequence of the fbpB gene encoding Ag85B was amplified from the genomic DNA of the virulent MTB strain H37Rv (a kind gift from Professor Angkana Chaiprasert, Faculty of Medicine Siriraj Hospital, Mahidol University) by PCR. The PCR product was subsequently cloned into the eukaryotic expression vector pVITRO1-neo-mcs (Invivogen, CA, USA). For production of recombinant Ag85B, the PCR product was subcloned into the plasmid pET15b (Invitrogen). The Ag85B protein was purified using HIS-Select® Nickel Affinity Gel (Sigma–Aldrich, MO, USA) according to the manufacturer’s instructions.
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2.4. Transfection and Western blot
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To confirm in vitro expression of Ag85B, myc-mTOR and mycmTOR-KD, the plasmids were separately transfected into HEK293T cells (American Type Culture Collection (Manassas, VA, USA)) using FuGENE® HD Transfection Reagent following the manufacturer’s instructions (Roche, Germany). Protein concentrations were determined using BCATM Protein Assay Reagent (Thermo Scientific, IL, USA). For detection of Ag85B, a rabbit anti-Ag85B polyclonal antibody was used (a kind gift from Prof. Watchara Kasinrerk, Faculty of Associated Medical Sciences, Chiangmai University, Thailand).
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2.5. Preparation of chitosan-loaded DNA
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Chitosan/DNA (CN/DNA) particles were prepared by the coacervation method described previously by Mao et al. [23]. For coencapsulation of two plasmids, the plasmids were mixed well before encapsulation. To test the encapsulation efficiency, DNAloaded CN particles containing DNA equivalent to 1 g were analyzed by electrophoresis on 1% agarose gel to identify any unencapsulated plasmid DNA.
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2.6. In vitro transfection with CN/DNA nanoparticles
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2.8. Blood sampling and detection of a specific antibody response
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Blood samples were collected by tail bleeding each week following immunization. Sera were used for detection of Ag85B-specific antibodies by indirect ELISA. The antibody titers were calculated by end point titration. IgG subtype-specific antibody was examined by ELISA as described above using peroxidase-conjugated rabbit anti-mouse IgG1 and rabbit anti-mouse IgG2a (Invitrogen).
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2.9. Cytokine production and cell proliferation assay
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Cytokine production in response to in vitro stimulation was measured 2 weeks after the final immunization using ELISA. Immunized mice were scarified and the spleens removed to make a single cell suspension. Cells were stimulated with purified recombinant Ag85B (5 g/ml). The culture media was collected for assay of secreted cytokines (IFN-␥, IL-2 and IL-4) using an ELISA kit (ELISA MAXTM Deluxe set, Biolegend) according to the manufacturer’s instructions. To assay cell proliferation, bulk splenocytes were labeled with Cell Proliferation Dye eFluor® 670 (eBioscience) and stimulated with purified recombinant Ag85B as described previously. After incubation for 3 days, cells were harvested and
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immediately analyzed by flow cytometry (FACS Calibur, BD Becton Dickinson). The acquired data were analyzed using Cell Quest Pro flow cytometry analysis software.
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2.10. Statistical analyses
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All experiments were repeated three times. For statistical analyses, all data were analyzed by one way ANOVA using SPSS and p < 0.05 was considered to be statistically significant.
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3. Results
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3.1. Expression of Ag85B and autophagy induction by mTOR-KD
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Expression of Ag85B, mTOR and mTOR-KD was first confirmed by transfection of the recombinant plasmids pVITRO-fbpB, pmTOR and pmTOR-KD into HEK293T cells using a commercial transfection reagent followed by examination by Western blot (Fig. 1A and B). To test whether mTOR-KD induces autophagy in transfected cells, the presence of an autophagy marker, LC3II was determined. As a positive control for autophagy induction, rapamycin treated cells (100 nM for 4 h) were used (Fig. 2B). Rapamycin treatment resulted in a greater expression of LC3II (16 kDa) than in the
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Fig. 1. Autophagy induction by mTOR-KD expression and optimization of plasmid encapsulation by chitosan. (A) Expression of Ag85B (pVITRO-fbpB) in HEK293T cells. Cells were transfected with the indicated plasmid for 72 h and expression of Ag85B was detected by Western blot. The results shown represent two independent experiments. (B) Expression of mTOR and autophagy. Cells were transfected with the indicated plasmids and expression of mTOR was detected by Western blot. Autophagy was detected by the presence of LC3 II (16 kDa). Rapamycin-treated cells were used as a positive control. The results shown represent two independent experiments. (C) Encapsulation efficiency of antigen-encoding plasmids by chitosan. Single and double plasmids were encapsulated with chitosan at N/P ratios 6:1 and 8:1. Encapsulation efficiency was determined by agarose gel electrophoresis. (D) SEM image of representative CN/pmTORKD and pVITRO-fbpB nanoparticles. Two plasmids were encapsulated by chitosan at the N/P ratio of 6:1 and the particles were visualized by SEM (30,000×).
Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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Q3 Fig. 2. Transfection efficiency of coencapsulated CN/plasmid. HEK293T cells were transfected as described in Section 2 using CN/plasmid DNA or a transfection reagent. Expression of Ag85B (green) or mTOR (red) was detected by immunofluorescence staining. Cells were visualized using an inverted florescent microscope. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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control untreated cells. Cells transfected with the pmTOR-KD construct showed greater expression of LC3II than the positive control cells, while mTOR expression did not change the level of LC3II (Fig. 1B). Therefore, overexpression of mTOR-KD, but not of wild type mTOR, is effective in inducing autophagy.
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3.2. Preparation of CN/DNA particles
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Encapsulation was optimized by varying the N/P ratio, the ratio of chitosan to DNA, using the model plasmid pMax-GFP (data not shown). The optimized N/P ratio was applied to the coencapsulation of pVITRO-fbpB and pmTOR-KD into CN particles. Efficiency of encapsulation was determined by agarose gel electrophoresis. As shown in Fig. 1C, N/P ratios of 6:1 and 8:1 gave similar results for complete encapsulation of plasmids but an N/P ratio of 8:1 for coencapsulation of two plasmids resulted in detectable exclusion of DNA. Therefore, the size and shape of the particles encapsulated at an N/P ratio of 6:1 were examined under SEM. The image revealed homogeneous particle size and shape with an average size of about 100–200 nm (Fig. 1D). Therefore, this condition was used to prepare CN/DNA nanoparticles for vaccination for further study.
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3.3. Expression of proteins from plasmid coencapsulated nanoparticles In order to confirm that the plasmids encapsulated with nanoparticles were delivered into cells and expressed proteins,
single or double plasmid encapsulation was carried out at an N/P ratio of 6:1 followed by transfection into HEK293T cells. Protein expression was determined by immunofluorescence staining. Compared to cells transfected using the commercial transfection reagent, the expression level in cells receiving coencapsulated plasmids was lower, but when two plasmids were introduced, both proteins were equally expressed (Fig. 2). Therefore, we determined that the nanoparticles prepared from chitosan could be used effectively as a DNA carrier leading to protein expression.
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3.4. Immune response to DNA immunization
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Mice were immunized with CN/DNA nanoparticles as described in Section 2 and according to the immunization schedule summarized in Fig. 3A. To assess the humoral immune response, the production of Ag85B-specific total IgG and IgG2a after the final immunization was measured by ELISA. The antibody titers were low during the first two weeks after priming and for the following weeks after two subcutaneous boosts at two-week intervals (antibody titer was less than 300, data not shown) in all groups of mice. After the first intranasal boost, the IgG antibody titer slowly increased with the sera taken 2 weeks after the final intranasal boost dramatically increased for specific IgG with a significant difference between the group immunized with plasmid antigen alone and the group immunized with the coencapsulated autophagyinducing plasmid. Furthermore, when the responses from the group
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Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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Fig. 3. Immunization, antibody titer and cytokine production. (A) The vaccination schedule is shown. CN/DNA nanoparticles (pVITRO-fbpB, pmTORKD + pVITRO fbpB, pmTORKD + pVITRO or pmTOR + pVITRO-fbpB) were used for immunization as indicated. Each immunization was carried out using 50 g of plasmid DNA and sera were taken at one and two weeks after each immunization. (B) The level of total anti-Ag85B IgG in sera was determined by ELISA at 2 weeks after the final boost. Sera from preimmunized mice were used as a negative control. (C) For the determination of subtype specific IgG2a, sera collected as above were analyzed by ELISA. (D–E) Bulk splenocytes from immunized mice were stimulated in vitro with recombinant Ag85B (5 g/ml) for 72 h and levels of IFN␥ and IL-4 secreted were analyzed by ELISA. Concanavalin A (10 g/ml) was used as a positive control. The results are shown as the mean ± SD. Two animals per group were used and the results represent two independent experiments performed in triplicate. Statistical significance was analyzed using one-way ANOVA. (An asterisk indicates statistical significance, p < 0.05.)
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immunized with pmTOR-KD and pVITRO-fbpB were compared with those immunized with pmTOR and pVITRO-fbpB, a significant increase in total IgG was evident, suggesting that co-expression of wild type mTOR is not sufficient to induce a higher humoral response. The antibody titer from mice that received pmTOR-KD with empty vector was undetectable. When the titer of specific IgG isotype was investigated, the group that received pmTOR-KD with antigen showed the highest IgG2a titer, suggesting that the immune response is skewed toward a Th1 type immune response. The IgG1 titer from all vaccinated groups was low to undetectable (data not shown). Next, we examined the in vitro response of splenocytes stimulated with recombinant Ag85B. As shown in Fig. 3D and E, mice that received pmTOR-KD together with pVITRO-fbpB responded to antigen stimulation by producing significantly higher levels of IFN␥ than the other groups, while no differences were found in the levels of IL-4. This result and the humoral response result obtained above confirmed that pmTOR-KD helped to increase the Th1 type immune response. To further explore the effectiveness of the DNA vaccine in this study, IL-2 production and T cell proliferation were investigated. As shown in Fig. 4A, splenocytes from mice receiving pmTORKD together with the antigen-encoding DNA showed significantly higher IL-2 production. The proliferation of CD4+ T cells correlated with the level of IL-2 and lymphocytes from mice vaccinated with
the autophagy-inducing plasmid proliferated more vigorously than the other groups. Taken together, these results strongly support that vaccination with the autophagy-inducing plasmid enhances immune response and that the responses are skewed toward Th1 type.
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4. Discussion
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DNA vaccine for TB has long been considered to be an alternative candidate vaccine that strongly induces an immune response in murine models, but the efficacy of the DNA vaccine in humans has been variable [24]. The main purpose of our study was to investigate the effect of autophagy induction in enhancing the immune response in mice vaccinated with a DNA vaccine against the MTB antigen. Autophagy has recently been shown to be a critical cellular defense against intracellular pathogens, including MTB [25]. In addition, it has been demonstrated that this process is involved in providing peptide antigens for MHC class II loading and presentation to CD4+ helper T cells [8]. In fact, targeting an influenza antigen to autophagosomes enhances MHC class II presentation to CD4+ helper T cells [7]. Furthermore, inducing autophagy by treating murine antigen presenting cells with rapamycin enhances antigen processing and presentation and increases BCG vaccine efficacy against MTB infection [18]. Since inhibiting the mTOR pathway is
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Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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Fig. 4. T cell proliferation and IL-2 production. (A) Splenocytes from immunized mice were stimulated with Ag85B (5 g/ml) in vitro as described in Fig. 3. Culture media was analyzed for IL-2 by ELISA. The results are shown as mean ± SD and statistical significance was determined by one-way ANOVA using SPSS. (An asterisk indicates statistical significance, p < 0.05.) (B) Representative CD4+ T-cell proliferation assays were performed by labeling splenocytes from immunized mice with Cell Proliferation eFluor® 670 Dye. Labeled cells (2 × 106 cells) were activated with rAg85B as described above and cultured for 72 h. Cells were harvested and analyzed by flow cytometry. The result represents two independent experiments. The average mean fluorescent intensities (MFIs) for cell population with fast cell division (P1) are 769 and 701 for pVITRO-fbpB and pVITRO-fbpB/pmTORKD, respectively. The MFIs for cell population with slow cell division (P2) are 3638.5 and 3329 for pVITRO-fbpB/pmTORKD and pVITRO-fbpB, respectively. The overall proliferation index for pVITRO-fbpB and pVITRO-fbpB/pmTORKD and are 1.10 and 1.15, respectively.
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a well-known mechanism for inducing autophagy, our study took advantage of a mutant mTOR construct harboring two point mutations reported to act as a dominant negative [26]. It had not been shown previously that overexpression of this construct induced autophagy; however, in this study we clearly demonstrated that overexpression of mTOR-KD induced an increase in autophagy. Since TB is mainly transmitted by inhalation of bacillicontaining droplets, a vaccine administered intranasally is suitable for mimicking the natural route of infection. In order to administer the DNA vaccine intranasally, chitosan was used to encapsulate the DNA. Chitosan has been previously used as a DNA vaccine carrier in oral and nasal delivery systems not only because the polymer is mucoadhesive, but because chitosan may also act as an adjuvant for stimulating the immune response [27,28]. Using only naked DNA gave rise to a minimal immune response in comparison to use of encapsulated DNA. The final intranasal immunization elicited a strong systemic immune response as judged by the rise in antibody titer, especially in the group receiving mTOR-KD plasmid together with the fbpB-containing plasmid. The Ag85B encoding gene from the virulent MTB strain H37Rv was selected as a candidate vaccine antigen in our research because this recombinant protein has been widely used previously [12,18,29–31]. We found that inducing autophagy together with the administration of Ag85B induced a stronger Th1 immune response because IFN␥ was produced at greater levels than IL-4 by splenocytes upon in vitro stimulation. The Th1 type immune response is considered to be the major host response used to contain an MTB infection. When the proliferation of splenocytes was investigated, splenocytes from mice receiving the autophagyinducing DNA vaccine proliferated more than those from the other groups. Moreover, the amount of IL-2 produced was higher in this group of mice. Therefore, the increased T cell proliferation in this study may be due to higher IL-2 production, which is necessary for T cell proliferation. Interestingly, we detected two distinct sets of cell population with difference in rate of cell division. Both cell
population proliferates more in CD4+ T cells from mice receiving pfbpB with pmTORKD. The two populations may represent cells with different affinity of TCR for Ag85 peptide antigen. An immune response by B lymphocytes has been long considered to play a less critical role in containing TB infection, but recent evidence suggests that B lymphocytes and antibodies may play a more significant role in dictating the outcome of the disease [32]. Our study found that the autophagy-inducing DNA vaccine format also significantly increased sera titer of total IgG and IgG2a, which is consistent with Th1 type immune responses. The long-term fate of cells overexpressing mTOR-KD is currently unknown, but excessive autophagy may eventually lead to cell death. The apoptotic body generated during apoptosis is taken up by surrounding antigen-presenting cells and may be involved in cross priming of CD8+ T cells. The cross priming of CD8+ T cells by apoptotic body-engulfing DCs leads to protective immune responses against MTB infection [33]. In conclusion, our study reveals a novel way to enhance the immune response to a DNA vaccine through incorporation of an autophagy-inducing plasmid into the vaccine. This type of vaccine may be useful for immunization to other infectious diseases, such as those caused by viral infections.
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Acknowledgments
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This work was supported by the Grand Challenges Explorations program of the Bill & Melinda Gates Foundation (Grant ID#OPP1007246), the Special Task Force for Activating Research (STAR) from the Centenary Academic Development Project (Chulalongkorn University), the Higher Education Research Promotion and the National Research University Project of Thailand, Office of the Higher Education Commission (HR1164A2). The authors would like to acknowledge financial support from the Research, Development and Engineering (RD&E) fund through The National
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Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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Nanotechnology Center (NANOTEC), The National Science and Technology Development Agency (NSTDA), Thailand (Project No. P-10-10454) to Chulalongkorn University. The equipment used in this study was purchased with funds from the Thai Government Stimulus Package 2 (TKK2555) under the Project for the Establishment of Innovative Food and Health Products and Agriculture. The first author was financially supported by the Ratchadapiseksomphot Endowment Fund for Postdoctoral Research, Chulalongkorn University. Conflict of interest: None declared.
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Appendix A. Supplementary data
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Supplementary data associated with cle can be found, in the online http://dx.doi.org/10.1016/j.vaccine.2012.11.075.
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[14] Okada M, Kita Y. Tuberculosis vaccine development: the development of novel (preclinical) DNA vaccine. Hum Vaccin 2012;6(April (4)):297–308. [15] Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 2004;119(December (6)):753–66. [16] Vergne I, Singh S, Roberts E, Kyei G, Master S, Harris J, et al. Autophagy in immune defense against Mycobacterium tuberculosis. Autophagy 2006;2(July–September (3)):175–8. [17] Alonso S, Pethe K, Russell DG, Purdy GE. Lysosomal killing of Mycobacterium mediated by ubiquitin-derived peptides is enhanced by autophagy. Proc Natl Acad Sci USA 2007;104(April (14)):6031–6. [18] Jagannath C, Lindsey DR, Dhandayuthapani S, Xu Y, Hunter Jr RL, Eissa NT. Autophagy enhances the efficacy of BCG vaccine by increasing peptide presentation in mouse dendritic cells. Nat Med 2009;15(March (3)):267–76. [19] Sato T, Ishii T, Okahata Y. In vitro gene delivery mediated by chitosan. Effect of pH, serum, and molecular mass of chitosan on the transfection efficiency. Biomaterials 2001;22(August (15)):2075–80. [20] Khatri K, Goyal AK, Gupta PN, Mishra N, Vyas SP. Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B. Int J Pharm 2008;354(April (1/2)):235–41. [21] Chew JL, Wolfowicz CB, Mao HQ, Leong KW, Chua KY. Chitosan nanoparticles containing plasmid DNA encoding house dust mite allergen, Der p 1 for oral vaccination in mice. Vaccine 2003;21(June (21/22)):2720–9. [22] Bozkir A, Saka OM. Chitosan nanoparticles for plasmid DNA delivery: effect of chitosan molecular structure on formulation and release characteristics. Drug Deliv 2004;11(March–April (2)):107–12. [23] Mao HQ, Roy K, Troung-Le VL, Janes KA, Lin KY, Wang Y, et al. Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. J Control Release 2001;70(February (3)):399–421. [24] Huygen K, Content J, Denis O, Montgomery DL, Yawman AM, Deck RR, et al. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat Med 1996;2(August (8)):893–8. [25] Deretic V, Levine B. Autophagy, immunity, and microbial adaptations. Cell Host Microbe 2009;5(June (6)):527–49. [26] Huang J, Dibble CC, Matsuzaki M, Manning BD. The TSC1–TSC2 complex is required for proper activation of mTOR complex 2. Mol Cell Biol 2008;28(June (12)):4104–15. [27] Leong KW, Mao HQ, Truong-Le VL, Roy K, Walsh SM, August JT. DNApolycation nanospheres as non-viral gene delivery vehicles. J Control Release 1998;53(April (1–3)):183–93. [28] Kumar M, Behera AK, Lockey RF, Zhang J, Bhullar G, De La Cruz CP, et al. Intranasal gene transfer by chitosan-DNA nanospheres protects BALB/c mice against acute respiratory syncytial virus infection. Hum Gene Ther 2002;13(August (12)):1415–25. [29] Lozes E, Huygen K, Content J, Denis O, Montgomery DL, Yawman AM, et al. Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine 1997;15(June (8)):830–3. [30] Miki K, Nagata T, Tanaka T, Kim YH, Uchijima M, Ohara N, et al. Induction of protective cellular immunity against Mycobacterium tuberculosis by recombinant attenuated self-destructing Listeria monocytogenes strains harboring eukaryotic expression plasmids for antigen 85 complex and MPB/MPT51. Infect Immun 2004;72(April (4)):2014–21. [31] Zhu B, Qie Y, Wang J, Zhang Y, Wang Q, Xu Y, et al. Chitosan microspheres enhance the immunogenicity of an Ag85B-based fusion protein containing multiple T-cell epitopes of Mycobacterium tuberculosis. Eur J Pharm Biopharm 2007;66(June (3)):318–26. [32] Maglione PJ, How Chan J. B cells shape the immune response against Mycobacterium tuberculosis. Eur J Immunol 2009;39(March (3)):676–86. [33] Winau F, Weber S, Sad S, de Diego J, Hoops SL, Breiden B, et al. Apoptotic vesicles crossprime CD8T cells and protect against tuberculosis. Immunity 2006;24(January (1)):105–17.
Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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Contents lists available at SciVerse ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system
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Jomkhwan Meerak a,b , Supason Wanichweacharungruang c , Tanapat Palaga a,∗
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Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand Division of Microbiology, Department of Biology, Faculty of Science, Chiang Mai University, Huaykaew Road, Chiangmai 50200, Thailand Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Bangkok 10330, Thailand
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Article history: Received 22 August 2012 Received in revised form 20 November 2012 Accepted 26 November 2012 Available online xxx
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DNA vaccines are a promising new generation of vaccines that can elicit an immune response using DNA encoding the antigen of interest. The efficacy of these vaccines, however, still needs to be improved. In this study, we investigated the effect of autophagy on increasing the efficacy of a candidate DNA vaccine against Mycobacterium tuberculosis (MTB), a causative agent of tuberculosis. Low molecular weight chitosan was used to encapsulate plasmid DNA containing a gene encoding MTB Antigen 85B (Ag85B), a secreted fibronectin-binding protein. To induce autophagy upon DNA vaccination, the kinase defective mTOR (mTOR-KD) was transfected into cells, and autophagy was detected based on the presence of LC3II. To investigate whether autophagy enhances an immune response upon DNA vaccination, we coencapsualted the Ag85B-containing plasmid with a plasmid encoding mTOR-KD. Plasmids encapsulated by chitosan particles were used for primary subcutaneous immunization and for intranasal boost in mice. After the boost vaccination, sera from the mice were measured for humoral immune response. The DNA vaccine with the autophagy-inducing construct elicited significantly higher Ag85B-specific antibody levels than the control group treated with the Ag85B plasmid alone or with the Ag85B plasmid plus the wild type mTOR construct. Upon in vitro stimulation of splenocytes from mice immunized with recombinant Ag85B, the highest levels of secreted IFN-␥ and IL-2 were detected in mice immunized with the autophagy-inducing plasmid, while no differences in IL-4 levels were detected between the groups, suggesting that the DNA vaccine regimen with autophagy induction induced primarily a Th1 immune response. Furthermore, the enhanced proliferation of CD4+ T cells from mice receiving the autophagyinducing vaccine was observed in vitro. Based on the evidence presented, we conclude that incorporating an autophagy-inducing element into a DNA vaccine may help to improve immune response. © 2012 Published by Elsevier Ltd.
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Keywords: DNA vaccine mTOR Autophagy Mycobacterium tuberculosis Chitosan
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1. Introduction
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Immunization with DNA vaccines has become a highly investigated topic beginning with the first report that naked plasmid DNA is taken up by muscle cells, resulting in the expression of beta-galactosidase [1]. Since then, DNA vaccines aiming to elicit protective immune responses against various infectious diseases have been investigated and have demonstrated the effectiveness of DNA in triggering both humoral and cell-mediated immune responses [2]. Even though there are no reports on the side effects of DNA vaccine administration, the development of DNA vaccines has yet to move beyond PhaseI/II clinical trials in humans, mainly because of low immunogenicity in humans [3]. Autophagy is a mechanism that is essential for cell survival and for maintaining cellular homeostasis during stress through the
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∗ Corresponding author. Tel.: +66 2 2185070; fax: +66 2 2527576. E-mail address: [email protected] (T. Palaga).
break down of cytosolic components and damaged organelles. The process is initiated by the formation of a double layer membrane surrounding the cellular contents to be degraded and involves multiple products of the autophagy-related genes (ATG), such as Atg12, Atg5 and Atg16L [4]. These proteins help conjugate phosphatidylethanolamine to a protein called LC3 (Atg8), which forms part of the double layer membrane. After the double layer membrane closes, the proteins inside, including the Atg proteins, are degraded upon fusion with the phagolysosome [5]. Autophagy also plays an important role in cellular defense against intracellular pathogens such as viruses and intracellular bacteria by surrounding and targeting them for clearance [6]. Furthermore, autophagy plays a key role in feeding cytoplasmic peptide antigens for antigen loading onto MHC II [7]. Increases in antigen presentation and acquired immune responses were reported when autophagy was induced upon antigen exposure [8]. Inhibition of the mammalian target of rapamycin (mTOR) by disruption of the gene encoding mTOR or by rapamycin treatment activates autophagy and has been used widely for studying autophagic phenomena in vitro and in vivo [5].
0264-410X/$ – see front matter © 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.vaccine.2012.11.075
Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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Tuberculosis (TB), a condition caused by aerosol infection with Mycobacterium tuberculosis (MTB), is a major global health problem that kills millions of people worldwide each year [9]. The currently available vaccine, Mycobacterium bovis BCG, is protective only against a severe form of childhood TB, but does not decrease the global TB burden in adults [9]. Therefore, novel vaccines for TB are seriously needed, and many types of new TB vaccines are either in the early stages of development or in the clinical trial pipeline [9]. Currently, DNA vaccines are one of the many types of new TB vaccine candidates under investigation. Previous reports have revealed the potency of plasmid DNAs carrying various MTB antigen-encoding genes in the induction of a protective immune response in mice and non-human primates [10–13]. After the recent completion of MTB whole genome sequencing, several MTB antigens such as Hsp65, Hsp70, Ag85A, Ag85B and ESAT-6 were tested for efficacy as DNA vaccines [14]. Antigen 85B (Ag85B), an abundant 30 kDa secreted MTB protein, was cloned reported to elicit a protective immune response in mice similar to that seen in those who had received the BCG vaccination in combination with bovine herpesvirus 1 VP22 [12]. However, the immunogenicity of most DNA vaccines under development for TB needs to be improved. Autophagy plays an important role in controlling TB infection in macrophages. Induction of autophagy in MTB-infected macrophages overcomes the block in phagosome maturation by the bacteria, and delivers the bacilli for degradation and elimination [15–17]. Interestingly, the efficacy of the BCG vaccine is increased in murine dendritic cells (DCs) in vitro by inducing autophagy by treatment with rapamycin, an inhibitor of mTOR [18]. This effect is due to enhanced antigen presentation of an immunodominant Ag85B. For this reason, we tested whether induction of the autophagic pathway would enhance the presentation of the MTB antigen to the antigen presenting cells (APCs) leading to increased immunogenicity of candidate DNA vaccines. Because MTB is transmitted by aerosolized droplets containing the bacilli, airway and lung mucosal immunity play a critical role in defense against TB infection. In order to enhance mucosal immunity, the DNA vaccine needs to be formulated for intranasal administration. Chitosan, an abundant natural biopolymer, is generally used as a drug and DNA carrier for cellular delivery both in vitro and in vivo [19–21]. It is suggested that both chitosan nanoand micro-particles act as effective immunological adjuvants and safe carriers for the delivery of vaccines [22]. In this study, we present evidence that incorporating an autophagy-inducing plasmid into a DNA vaccine enhances host immune responses to a DNA vaccine against the MTB antigen delivered by chitosan particles in mice.
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2. Materials and methods
For in vitro transfection studies, CN/DNA nanoparticles were transfected into HEK293T cells as previously described [20]. Expression of Ag85B and mTOR was detected by immunofluorescence staining.
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2.1. Reagents
2.7. Vaccine preparation and immunization
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2.2. Mice
CN/DNA nanoparticles were prepared as described above. Female BALB/c (8 weeks old) mice were randomly assigned into four groups, each composed of four mice, for four different immunization regimens (pVITRO-fbpB alone, pVITRO-fbpB with pmTOR-KD, pVITRO-fbpB with pmTOR or pVITRO1 empty vector with pmTOR-KD). Immunization was carried out by priming with 50 g of plasmid DNA encapsulated by chitosan particles delivered by subcutaneous injection, followed by two booster doses at 2-week intervals. The two boosts were delivered intranasally by slow repeated dropping of a total of 30 l of CN/DNA nanoparticle solution (equivalent to 50 g DNA) into the nasal cavity. For immunization with coencapsulation of pVITRO-fbpB with pmTOR-KD or pmTOR, 50 g of each plasmid was used for each mouse.
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Low molecular weight chitosan, 30 kDa with approximately 80–85% deacetylation, was used for plasmid DNA encapsulation (Department of Chemistry, Chulalongkorn University). The plasmids encoding myc-mTOR (pmTOR) and myc-mTORKD (pmTOR-KD) were obtained from Addgene (MA, USA) (Dr. D. Sabatini, Massachusetts Institute of Technology). The plasmid pmTOR-KD contained two point mutations in the mTOR gene resulting in the amino acid substitutions D2357E and V2364I.
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Eight-week-old female BALB/c inbred mice were obtained from the National Laboratory Animal Center (Mahidol University, Salaya,
Thailand). All mice were maintained under SPF conditions and used in accordance with the policies and regulations for the care and use of laboratory animals of the Institute of Biotechnology and Genetic Engineering, Chulalongkorn University.
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2.3. Construction of plasmids and production of recombinant Ag85B
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For construction of the plasmid harboring Ag85B, the full sequence of the fbpB gene encoding Ag85B was amplified from the genomic DNA of the virulent MTB strain H37Rv (a kind gift from Professor Angkana Chaiprasert, Faculty of Medicine Siriraj Hospital, Mahidol University) by PCR. The PCR product was subsequently cloned into the eukaryotic expression vector pVITRO1-neo-mcs (Invivogen, CA, USA). For production of recombinant Ag85B, the PCR product was subcloned into the plasmid pET15b (Invitrogen). The Ag85B protein was purified using HIS-Select® Nickel Affinity Gel (Sigma–Aldrich, MO, USA) according to the manufacturer’s instructions.
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2.4. Transfection and Western blot
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To confirm in vitro expression of Ag85B, myc-mTOR and mycmTOR-KD, the plasmids were separately transfected into HEK293T cells (American Type Culture Collection (Manassas, VA, USA)) using FuGENE® HD Transfection Reagent following the manufacturer’s instructions (Roche, Germany). Protein concentrations were determined using BCATM Protein Assay Reagent (Thermo Scientific, IL, USA). For detection of Ag85B, a rabbit anti-Ag85B polyclonal antibody was used (a kind gift from Prof. Watchara Kasinrerk, Faculty of Associated Medical Sciences, Chiangmai University, Thailand).
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2.5. Preparation of chitosan-loaded DNA
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Chitosan/DNA (CN/DNA) particles were prepared by the coacervation method described previously by Mao et al. [23]. For coencapsulation of two plasmids, the plasmids were mixed well before encapsulation. To test the encapsulation efficiency, DNAloaded CN particles containing DNA equivalent to 1 g were analyzed by electrophoresis on 1% agarose gel to identify any unencapsulated plasmid DNA.
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2.6. In vitro transfection with CN/DNA nanoparticles
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Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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2.8. Blood sampling and detection of a specific antibody response
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Blood samples were collected by tail bleeding each week following immunization. Sera were used for detection of Ag85B-specific antibodies by indirect ELISA. The antibody titers were calculated by end point titration. IgG subtype-specific antibody was examined by ELISA as described above using peroxidase-conjugated rabbit anti-mouse IgG1 and rabbit anti-mouse IgG2a (Invitrogen).
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2.9. Cytokine production and cell proliferation assay
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Cytokine production in response to in vitro stimulation was measured 2 weeks after the final immunization using ELISA. Immunized mice were scarified and the spleens removed to make a single cell suspension. Cells were stimulated with purified recombinant Ag85B (5 g/ml). The culture media was collected for assay of secreted cytokines (IFN-␥, IL-2 and IL-4) using an ELISA kit (ELISA MAXTM Deluxe set, Biolegend) according to the manufacturer’s instructions. To assay cell proliferation, bulk splenocytes were labeled with Cell Proliferation Dye eFluor® 670 (eBioscience) and stimulated with purified recombinant Ag85B as described previously. After incubation for 3 days, cells were harvested and
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immediately analyzed by flow cytometry (FACS Calibur, BD Becton Dickinson). The acquired data were analyzed using Cell Quest Pro flow cytometry analysis software.
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2.10. Statistical analyses
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All experiments were repeated three times. For statistical analyses, all data were analyzed by one way ANOVA using SPSS and p < 0.05 was considered to be statistically significant.
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3. Results
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3.1. Expression of Ag85B and autophagy induction by mTOR-KD
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Expression of Ag85B, mTOR and mTOR-KD was first confirmed by transfection of the recombinant plasmids pVITRO-fbpB, pmTOR and pmTOR-KD into HEK293T cells using a commercial transfection reagent followed by examination by Western blot (Fig. 1A and B). To test whether mTOR-KD induces autophagy in transfected cells, the presence of an autophagy marker, LC3II was determined. As a positive control for autophagy induction, rapamycin treated cells (100 nM for 4 h) were used (Fig. 2B). Rapamycin treatment resulted in a greater expression of LC3II (16 kDa) than in the
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Fig. 1. Autophagy induction by mTOR-KD expression and optimization of plasmid encapsulation by chitosan. (A) Expression of Ag85B (pVITRO-fbpB) in HEK293T cells. Cells were transfected with the indicated plasmid for 72 h and expression of Ag85B was detected by Western blot. The results shown represent two independent experiments. (B) Expression of mTOR and autophagy. Cells were transfected with the indicated plasmids and expression of mTOR was detected by Western blot. Autophagy was detected by the presence of LC3 II (16 kDa). Rapamycin-treated cells were used as a positive control. The results shown represent two independent experiments. (C) Encapsulation efficiency of antigen-encoding plasmids by chitosan. Single and double plasmids were encapsulated with chitosan at N/P ratios 6:1 and 8:1. Encapsulation efficiency was determined by agarose gel electrophoresis. (D) SEM image of representative CN/pmTORKD and pVITRO-fbpB nanoparticles. Two plasmids were encapsulated by chitosan at the N/P ratio of 6:1 and the particles were visualized by SEM (30,000×).
Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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Q3 Fig. 2. Transfection efficiency of coencapsulated CN/plasmid. HEK293T cells were transfected as described in Section 2 using CN/plasmid DNA or a transfection reagent. Expression of Ag85B (green) or mTOR (red) was detected by immunofluorescence staining. Cells were visualized using an inverted florescent microscope. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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control untreated cells. Cells transfected with the pmTOR-KD construct showed greater expression of LC3II than the positive control cells, while mTOR expression did not change the level of LC3II (Fig. 1B). Therefore, overexpression of mTOR-KD, but not of wild type mTOR, is effective in inducing autophagy.
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3.2. Preparation of CN/DNA particles
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Encapsulation was optimized by varying the N/P ratio, the ratio of chitosan to DNA, using the model plasmid pMax-GFP (data not shown). The optimized N/P ratio was applied to the coencapsulation of pVITRO-fbpB and pmTOR-KD into CN particles. Efficiency of encapsulation was determined by agarose gel electrophoresis. As shown in Fig. 1C, N/P ratios of 6:1 and 8:1 gave similar results for complete encapsulation of plasmids but an N/P ratio of 8:1 for coencapsulation of two plasmids resulted in detectable exclusion of DNA. Therefore, the size and shape of the particles encapsulated at an N/P ratio of 6:1 were examined under SEM. The image revealed homogeneous particle size and shape with an average size of about 100–200 nm (Fig. 1D). Therefore, this condition was used to prepare CN/DNA nanoparticles for vaccination for further study.
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3.3. Expression of proteins from plasmid coencapsulated nanoparticles In order to confirm that the plasmids encapsulated with nanoparticles were delivered into cells and expressed proteins,
single or double plasmid encapsulation was carried out at an N/P ratio of 6:1 followed by transfection into HEK293T cells. Protein expression was determined by immunofluorescence staining. Compared to cells transfected using the commercial transfection reagent, the expression level in cells receiving coencapsulated plasmids was lower, but when two plasmids were introduced, both proteins were equally expressed (Fig. 2). Therefore, we determined that the nanoparticles prepared from chitosan could be used effectively as a DNA carrier leading to protein expression.
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3.4. Immune response to DNA immunization
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Mice were immunized with CN/DNA nanoparticles as described in Section 2 and according to the immunization schedule summarized in Fig. 3A. To assess the humoral immune response, the production of Ag85B-specific total IgG and IgG2a after the final immunization was measured by ELISA. The antibody titers were low during the first two weeks after priming and for the following weeks after two subcutaneous boosts at two-week intervals (antibody titer was less than 300, data not shown) in all groups of mice. After the first intranasal boost, the IgG antibody titer slowly increased with the sera taken 2 weeks after the final intranasal boost dramatically increased for specific IgG with a significant difference between the group immunized with plasmid antigen alone and the group immunized with the coencapsulated autophagyinducing plasmid. Furthermore, when the responses from the group
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Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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Fig. 3. Immunization, antibody titer and cytokine production. (A) The vaccination schedule is shown. CN/DNA nanoparticles (pVITRO-fbpB, pmTORKD + pVITRO fbpB, pmTORKD + pVITRO or pmTOR + pVITRO-fbpB) were used for immunization as indicated. Each immunization was carried out using 50 g of plasmid DNA and sera were taken at one and two weeks after each immunization. (B) The level of total anti-Ag85B IgG in sera was determined by ELISA at 2 weeks after the final boost. Sera from preimmunized mice were used as a negative control. (C) For the determination of subtype specific IgG2a, sera collected as above were analyzed by ELISA. (D–E) Bulk splenocytes from immunized mice were stimulated in vitro with recombinant Ag85B (5 g/ml) for 72 h and levels of IFN␥ and IL-4 secreted were analyzed by ELISA. Concanavalin A (10 g/ml) was used as a positive control. The results are shown as the mean ± SD. Two animals per group were used and the results represent two independent experiments performed in triplicate. Statistical significance was analyzed using one-way ANOVA. (An asterisk indicates statistical significance, p < 0.05.)
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immunized with pmTOR-KD and pVITRO-fbpB were compared with those immunized with pmTOR and pVITRO-fbpB, a significant increase in total IgG was evident, suggesting that co-expression of wild type mTOR is not sufficient to induce a higher humoral response. The antibody titer from mice that received pmTOR-KD with empty vector was undetectable. When the titer of specific IgG isotype was investigated, the group that received pmTOR-KD with antigen showed the highest IgG2a titer, suggesting that the immune response is skewed toward a Th1 type immune response. The IgG1 titer from all vaccinated groups was low to undetectable (data not shown). Next, we examined the in vitro response of splenocytes stimulated with recombinant Ag85B. As shown in Fig. 3D and E, mice that received pmTOR-KD together with pVITRO-fbpB responded to antigen stimulation by producing significantly higher levels of IFN␥ than the other groups, while no differences were found in the levels of IL-4. This result and the humoral response result obtained above confirmed that pmTOR-KD helped to increase the Th1 type immune response. To further explore the effectiveness of the DNA vaccine in this study, IL-2 production and T cell proliferation were investigated. As shown in Fig. 4A, splenocytes from mice receiving pmTORKD together with the antigen-encoding DNA showed significantly higher IL-2 production. The proliferation of CD4+ T cells correlated with the level of IL-2 and lymphocytes from mice vaccinated with
the autophagy-inducing plasmid proliferated more vigorously than the other groups. Taken together, these results strongly support that vaccination with the autophagy-inducing plasmid enhances immune response and that the responses are skewed toward Th1 type.
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4. Discussion
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DNA vaccine for TB has long been considered to be an alternative candidate vaccine that strongly induces an immune response in murine models, but the efficacy of the DNA vaccine in humans has been variable [24]. The main purpose of our study was to investigate the effect of autophagy induction in enhancing the immune response in mice vaccinated with a DNA vaccine against the MTB antigen. Autophagy has recently been shown to be a critical cellular defense against intracellular pathogens, including MTB [25]. In addition, it has been demonstrated that this process is involved in providing peptide antigens for MHC class II loading and presentation to CD4+ helper T cells [8]. In fact, targeting an influenza antigen to autophagosomes enhances MHC class II presentation to CD4+ helper T cells [7]. Furthermore, inducing autophagy by treating murine antigen presenting cells with rapamycin enhances antigen processing and presentation and increases BCG vaccine efficacy against MTB infection [18]. Since inhibiting the mTOR pathway is
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Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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Fig. 4. T cell proliferation and IL-2 production. (A) Splenocytes from immunized mice were stimulated with Ag85B (5 g/ml) in vitro as described in Fig. 3. Culture media was analyzed for IL-2 by ELISA. The results are shown as mean ± SD and statistical significance was determined by one-way ANOVA using SPSS. (An asterisk indicates statistical significance, p < 0.05.) (B) Representative CD4+ T-cell proliferation assays were performed by labeling splenocytes from immunized mice with Cell Proliferation eFluor® 670 Dye. Labeled cells (2 × 106 cells) were activated with rAg85B as described above and cultured for 72 h. Cells were harvested and analyzed by flow cytometry. The result represents two independent experiments. The average mean fluorescent intensities (MFIs) for cell population with fast cell division (P1) are 769 and 701 for pVITRO-fbpB and pVITRO-fbpB/pmTORKD, respectively. The MFIs for cell population with slow cell division (P2) are 3638.5 and 3329 for pVITRO-fbpB/pmTORKD and pVITRO-fbpB, respectively. The overall proliferation index for pVITRO-fbpB and pVITRO-fbpB/pmTORKD and are 1.10 and 1.15, respectively.
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a well-known mechanism for inducing autophagy, our study took advantage of a mutant mTOR construct harboring two point mutations reported to act as a dominant negative [26]. It had not been shown previously that overexpression of this construct induced autophagy; however, in this study we clearly demonstrated that overexpression of mTOR-KD induced an increase in autophagy. Since TB is mainly transmitted by inhalation of bacillicontaining droplets, a vaccine administered intranasally is suitable for mimicking the natural route of infection. In order to administer the DNA vaccine intranasally, chitosan was used to encapsulate the DNA. Chitosan has been previously used as a DNA vaccine carrier in oral and nasal delivery systems not only because the polymer is mucoadhesive, but because chitosan may also act as an adjuvant for stimulating the immune response [27,28]. Using only naked DNA gave rise to a minimal immune response in comparison to use of encapsulated DNA. The final intranasal immunization elicited a strong systemic immune response as judged by the rise in antibody titer, especially in the group receiving mTOR-KD plasmid together with the fbpB-containing plasmid. The Ag85B encoding gene from the virulent MTB strain H37Rv was selected as a candidate vaccine antigen in our research because this recombinant protein has been widely used previously [12,18,29–31]. We found that inducing autophagy together with the administration of Ag85B induced a stronger Th1 immune response because IFN␥ was produced at greater levels than IL-4 by splenocytes upon in vitro stimulation. The Th1 type immune response is considered to be the major host response used to contain an MTB infection. When the proliferation of splenocytes was investigated, splenocytes from mice receiving the autophagyinducing DNA vaccine proliferated more than those from the other groups. Moreover, the amount of IL-2 produced was higher in this group of mice. Therefore, the increased T cell proliferation in this study may be due to higher IL-2 production, which is necessary for T cell proliferation. Interestingly, we detected two distinct sets of cell population with difference in rate of cell division. Both cell
population proliferates more in CD4+ T cells from mice receiving pfbpB with pmTORKD. The two populations may represent cells with different affinity of TCR for Ag85 peptide antigen. An immune response by B lymphocytes has been long considered to play a less critical role in containing TB infection, but recent evidence suggests that B lymphocytes and antibodies may play a more significant role in dictating the outcome of the disease [32]. Our study found that the autophagy-inducing DNA vaccine format also significantly increased sera titer of total IgG and IgG2a, which is consistent with Th1 type immune responses. The long-term fate of cells overexpressing mTOR-KD is currently unknown, but excessive autophagy may eventually lead to cell death. The apoptotic body generated during apoptosis is taken up by surrounding antigen-presenting cells and may be involved in cross priming of CD8+ T cells. The cross priming of CD8+ T cells by apoptotic body-engulfing DCs leads to protective immune responses against MTB infection [33]. In conclusion, our study reveals a novel way to enhance the immune response to a DNA vaccine through incorporation of an autophagy-inducing plasmid into the vaccine. This type of vaccine may be useful for immunization to other infectious diseases, such as those caused by viral infections.
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Acknowledgments
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This work was supported by the Grand Challenges Explorations program of the Bill & Melinda Gates Foundation (Grant ID#OPP1007246), the Special Task Force for Activating Research (STAR) from the Centenary Academic Development Project (Chulalongkorn University), the Higher Education Research Promotion and the National Research University Project of Thailand, Office of the Higher Education Commission (HR1164A2). The authors would like to acknowledge financial support from the Research, Development and Engineering (RD&E) fund through The National
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Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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Nanotechnology Center (NANOTEC), The National Science and Technology Development Agency (NSTDA), Thailand (Project No. P-10-10454) to Chulalongkorn University. The equipment used in this study was purchased with funds from the Thai Government Stimulus Package 2 (TKK2555) under the Project for the Establishment of Innovative Food and Health Products and Agriculture. The first author was financially supported by the Ratchadapiseksomphot Endowment Fund for Postdoctoral Research, Chulalongkorn University. Conflict of interest: None declared.
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Appendix A. Supplementary data
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Supplementary data associated with cle can be found, in the online http://dx.doi.org/10.1016/j.vaccine.2012.11.075.
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References
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Please cite this article in press as: Meerak J, et al. Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine (2012), http://dx.doi.org/10.1016/j.vaccine.2012.11.075
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