Background
Mycobacterium tuberculosis (
Mtb) kills 1.5 million people annually [
1]. Furthermore, the increasing frequency of
Mtb cases exhibiting drug-resistance warrants the need to develop better vaccines or strategies for the prevention and treatment of TB [
2]. The only available vaccine for TB is an attenuated form of
Mycobacterium bovis named as
Bacillus Calmette–Guérin (BCG) [
3]. The efficacy of BCG is poor in populations with a high TB
-burden [
4]. BCG has proven its efficacy against childhood, but not adulthood manifestation of the disease, depicting an inability to generate enduring memory T cells against
Mtb. BCG has lost the RD1 region from its genome. Although RD1 provides virulence in
Mtb, it also evokes strong protective immunity against the bacterium signifying that BCG requires supplementation with certain
Mtb proteins to improve its protective efficacy [
5,
6]. In this regard, several prime-boost studies were conducted with BCG, such as protein and peptide-based subunit vaccines, live attenuated vaccines, and viral vectors with promising results [
7].
Recently, we developed a lipidated promiscuous peptide vaccine comprising of the immunodominant CD4 and CD8 T cell epitopes of Acr1 and TB10.4 proteins of
Mtb conjugated to TLR-2 ligand Pam2Cys [
8,
9]. These constructs elicited enduring memory T cells response and showed better protection than BCG in mouse and Guinea pig TB models. Several advantages are associated with peptide vaccines, such as the selection of immunodominant moieties and the elimination of suppressive and auto-reactive portions of the antigen. However, there are certain issues associated with peptide vaccines due to its cost-effectiveness and synthesis for mass immunization. Hence, expressing the immunodominant epitopes inside the host could be an effective mode to eliminate the issues. An effective mode of expressing the epitopes would be the DNA vaccine strategy. A major advantage of DNA vaccines is that they are simpler to produce and store compared to conventional vaccines, making them less expensive. DNA vaccines can elicit the generation of both CD4 Th1 cells, CD8 T cells, and long-lasting immunity; the immune response that plays a cardinal role in protection against
Mtb [
10].
This encouraged us to design a DNA vaccine comprising of six CD4 T cells and CD8 T cells epitopes of latency, active and chronic stages of Mtb. To check the efficacy of the vaccine, it is important to use an animal model of TB and mice are very useful as their adaptive immune response is similar to humans. Hence, we immunized mice with DNA vaccine and observed induction of protective immune response that significantly reduced the frequency of bacterium in the animals exposed to Mtb. Furthermore, the vaccine considerably improved the efficacy of BCG to protect against Mtb. This vaccine may have future implications in protecting individuals from TB.
Discussion
The poor performance of BCG in TB-endemic areas can be rationalized with multiple explanations. BCG protects the childhood but not adult manifestation of TB [
27‐
30]. Signifying that it lacks the antigenic repertoire that is required in inducing long-lasting protective memory T cells. Consequently, supplementing
Mtb antigenic epitopes in BCG may bolster its performance. Therefore, in the current study, we selected multiple epitopes from latent, active, and chronic stages of TB and synthesized a DNA vaccine to check its efficiency either alone or in a combination of BCG.
It has been previously reported that the Acr1 protein provides enhanced protective efficacy when overexpressed in BCG [
31]. However, Acr1 impairs the maturation and functionality of DCs and supports the intracellular survival of
Mtb [
22,
32]. Similarly, Rv2626c protein has been shown to protect against
Mtb, as well as modulate the functionality of macrophages and assist in the escape of pathogen [
33,
34]. Hence, the expression of CD4 T cell and CD8 T cell epitopes in a DNA vaccine with BCG may be a better approach to combat TB. Promiscuous T cell epitopes have enough potential to bind diverse HLA alleles and evoke T cell activation without requiring extensive antigen processing by APCs [
8,
35]. However, peptide vaccines are weak immunogens and thus require adjuvants to elicit optimum activation of T cells.
Therefore, we selected and expressed multiple T cell epitopes from the latent, active, and chronic stages of TB in the DNA vector to overcome the limits associated with BCG. DNA vaccines can induce both CD4 T cells and CD8 T cell responses against the expressing antigens [
10]. This ability has led to the development of many veterinary vaccines and human clinical trials involving Zika, HIV, dengue, and cancer diseases [
36‐
40]. In connection with TB, DNA vaccines have shown the potential to combat infection [
41‐
46]. In this light, we selected six promiscuous CD4 T cell and CD8 T cell epitopes from the different
Mtb proteins [
11‐
14] and cloned them into pcDNA3.1(−) plasmid. The novelty of the C6 construct is that it has 2 copies of each epitope linked with protease-sensitive amino acid sequences. This allows for APC-mediated protease cleavage and eventual release from C6 expressing cells with the help of a secretory signal (Fig.
1). Furthermore, the use of a plasmid vector helps in the elicitation of the CD8 T cell response [
47]. We generated the YFP reporter construct with C6 by cloning it into the plasmid pcDNA3.1(−) and pEYFP to evaluate its expression and secretion. We confirmed the expression of C6 along with YFP in vitro into the CHO cells by fluorescence imaging, as well as Western blotting (Fig.
2). Also, it was important for the construct to be expressed in vivo to evoke an immune response. We immunized animals with C6 and observed YFP expressing cells in the mice, thus confirming the expression of C6. Furthermore, we examined if the C6 vaccine could enhance the efficacy of BCG. We evaluated the boosting capacity of C6 in BCG vaccinated mice. We noticed following major outcomes on C6 vaccination: (1) generation of memory CD4 T cell and CD8 T cells; (2) enhancement in Th1 responses, as evidenced by the predominant secretion of IFN-γ and TNF-α; (3) promotion of the activation of APCs; (4) boosting of protective efficacy of BCG against
Mtb.
Immunological memory is an indispensable feature of adaptive immunity that protects organisms from subsequent infections [
48]. Moreover, it is a fundamental feature of a successful vaccine [
49]. The generation of short-term memory T cells is one of the reasons for the failure of BCG to impart protection against
Mtb in the vaccinated adult population [
50]. Remarkably, BCG generates better memory CD4 T cells and CD8 T cells response with the addition of memory response by C6 (Fig.
3). The enhancement in memory response could be observed due to the generation epitope-specific immune response. It has been reported that resting T cell population with naïve phenotype i.e. CD62L
hi/CD44
lo can confer protection against
Mtb [
51,
52]. The increase in the resting population upon C6 administration denotes the enhancement in the generation of resting population, which is important for recall response. Intriguingly, C6 potentiated the capacity BCG in augmenting CD62L
hi/CD44
lo. Decreased the percentage of CD44
hi/CD62L
lo cells in C6 and BCG + C6 indicates the capability of effector cells to transit towards memory cell which is poorly associated with BCG and explain its failure in persistent
Mtb infections [
53‐
55].
CD4 T cell subsets express distinct cytokines and transcription factors, thereby responding to different pathogens. Th1 cells protects against
Mtb by secreting IFN-γ and TNF-α and stimulating macrophages to kill intracellular pathogens [
56,
57]. The importance of IFN-γ is illustrated as its absence enhances
Mtb susceptibility, mortality, and defects in macrophage activation [
58]. For initiation and maintenance of defence against
Mtb, TNF-α plays a crucial role in reactivation of latent tuberculosis of rheumatoid arthritis patients during the neutralization by the anti-TNF antibody [
59]. Therefore, the generation of Th1 immunity by a vaccine is quite crucial to protect against TB. The elicitation of higher yield of IFN-γ and TNF-α by BCG + C6 denotes its potential to generate Th1 response (Fig.
4). IL-10 is produced by Th2 cells and can reciprocally regulate the generation of Th1 cells as well as the macrophage and DCs to activate Th1 cells [
60,
61]. Furthermore, it has been shown that BCG infected dendritic cells generate IL-10 producing T cells [
62]. we observed elevated expression of IL-10 in the BCG immunized group. In contrast, C6 alone and along with BCG immunization declined the IL-10 secretion, indicating its ability to promote Th1 cells.
The importance of antigen-presenting cells (APCs), such as DCs and macrophages, in the protection against TB is well elucidated [
21]. Besides phagocytosing and killing the pathogens, these cells simultaneously process and present the pathogenic components to activate and differentiate T cells into effector and memory T cells. These activated T cells help to bolster the function of APCs to release cytokines like IFN-γ and TNF-α [
49,
56,
57].
Mtb can modulate APCs to restrict the generation of adaptive immunity.
Mtb inhibits the maturation of APCs by preventing antigen presentation through MHC along with costimulatory molecules such as CD86, CD40, and CD80 masks the ability to activate antigen-specific T cells [
63,
64]. Interestingly, DNA vaccines can activate APCs by interacting with TLR9 [
65]. It is noteworthy to mention here that the activation of DCs and macrophages in BCG + C6 immunized animals were higher, as evidenced by the increased percentage of MHC-II
hi, CD86
hi, and CD40
hi costimulatory molecules expressing cells (Fig.
5). CD80 preferably interacts with CTLA-4 molecule of T cells and weakly with CD28 and is linked with the generation of anergy and tolerogenic T cells [
66]. The reduction of CD80
hi percent population of DCs and macrophage in BCG + C6 supports the generation of pathogenic T cells rather than tolerance. To activate T cells and generate effector and memory T cells, DCs and macrophages produce IL-6 and IL-12. Thus, activation and secretion of IL-6 and IL-12 are crucial for the APCs [
18]. Increased production of IL-6 and IL-12 in the BCG + C6 group indicates the enhanced capability of DCs and macrophages to activate T cells. Similarly, the role of IFN-γ and TNF-α has been correlated with the functionality of DCs and macrophages [
67,
68]. Therefore, the production of IFN-γ and TNF-α indicates the activation of DCs and macrophage.
Apart from the generation of optimum activation of the immune system, a cardinal feature of a vaccine is to restrict infection. During the progression of TB, the infiltration of inflammatory mononuclear cells leads to the development of granulomas; a habitable niche for
Mtb [
69]. Furthermore, acute bronchopneumonia and necrotizing granulomas have been correlated with the pathology of pulmonary TB [
70]. Remarkably, we observed a decrease in the
Mtb burden and disease pathology of the lungs of the C6 administered group and augmented the potency of BCG (Fig.
6). C6 prevented the dissemination of
Mtb, as depicted by a decrease in the bacterial burden in the spleen. The reduced bacterial burden in the vaccinated group indicates the protective efficacy of C6 with BCG.
The C6 vaccination along with BCG has improved the protection against the Mtb in the mice model of TB. Its protective efficacy has been achieved by the generation of memory T cells against Mtb. These T cells can activate DCs and macrophages with the help of IFN- and TNF-. Moreover, the DCs and macrophages in immunized animals were not affected by the suppressive ability of Mtb and could produce IL-6 and IL-12 to further activate T cells. All these together declined the Mtb burden in the vaccinated group of animals compared to controls.
Methods
Mice
BALB/c and C57BL/6 female mice (6–8 weeks, 16–18 g) were obtained from the Animal House Facility, CSIR-Institute of Microbial Technology, Chandigarh (IMTECH) and kept in Biosafety level 3 laboratory in CSIR-Institute of Microbial Technology, Chandigarh (IMTECH) for experimental procedures.
Bacteria and cell lines
The Escherichia coli (E. coli) DH5α strain was grown in LB media and used in this study for cloning and purification of plasmids. BCG Danish strain (Serum Institute of India PVT. LTD., India) used for immunization. Mtb H37Rv strain was grown in 7H9 + 10%OADC and preserved as 10% glycerol stock at − 80 °C to be used for infection respectively. CHO cell line was used for the transfection studies.
Reagents
All the reagents and primers were purchased from Sigma (St. Louis, MO) and antibodies from eBiosciences (San Diego, CA), Restriction, and ligase enzymes were from New England Biolabs (Ipswich, MA), further unless and otherwise mentioned. Bacterial media were purchased from Himedia (Mumbai, India).
T cell epitopes selection, cloning, and expression
The promiscuous T cell epitope selection was based on binding to multiple HLA alleles. We selected 6 promiscuous CD4 T cells and CD8 T cell epitope peptides from the literature. The peptide sequences were arranged in duplicates and linked with a protease-sensitive amino acid sequence (AVYAFVH). An N-terminal human growth hormone (HGH) secretory signal was linked for the secretion of the protein from the host cells. The chimera gene (C6) for the protein was synthesized (GenScript, Piscataway, NJ). To use the C6 gene as a vaccine, a suitable vector must be used for the expression. Consequently, to utilize as a DNA vaccine, vector pcDNA3.1- was used. The synthesized gene was cloned into the pcDNA3.1- vector at the site of BamHI and HindIII and transformed into E. coli DH5α for multiplication and purification of the plasmid. The presence of the C6 gene in the plasmid was confirmed through colony PCR and agarose gel electrophoresis.
Later, C6 was cloned into the pEYFP-N1 vector at the site of the NheI and HindIII site to generate a YFP tagged protein for the expression confirmation of gene in the host cells. pEYFP-C6 was transformed into
E. coli and kanamycin-resistant colonies were screened through colony PCR and agarose gel electrophoresis. To further confirm the expression of C6 in pcDNA3.1- vector, the C6YFP gene was amplified and cloned into the NheI and NotI site of pcDNA3.1- and transformed into
E. coli and positive colonies were selected through PCR and agarose gel electrophoresis. All the plasmids for the use of immunization and transfections were isolated through the Triton X-114 method [
72].
The CHO cell line was transfected with plasmids by using lipofectamine 2000 (Invitrogen, Carlsbad, CA). The standard manufacturer protocol was followed for the transfection. Transfected cells were used for direct observation under a fluorescent microscope, western blotting, and FACS analysis.
Western blotting
Transfected CHO cell lysate was prepared by harvesting, washing, and lysis in lysis buffer (RIPA buffer, protease, and phosphatase inhibitor cocktail). The culture supernatants (SN) were precipitated through Acetone precipitation. Briefly, five times a volume of 80% chilled acetone was added to the SN and incubated overnight at − 20 °C. Later, SN was pelleted at 10000 g for 15 min at 4 °C. The pellets were washed twice with 80% chilled acetone and air-dried for 45 min at RT. Pellets were dissolved into PBS. The SNs of the cell lysate and culture SNs were estimated and equal concentration was subjected to SDS-PAGE. After transfer onto nitrocellulose membrane and blocking, the membranes were immunoblotted with Abs against YFP. Blots were developed using a chemiluminescence kit (Thermo Scientific, Waltham, MA). Chemiluminescence was detected by ImageQuant LAS 4000 (GE life sciences, UK).
Animal immunization
To study the in vivo expression of C6, C57BL/6 mice were vaccinated with 100 μg of C6YFP, and inguinal LNs and hind limbs were isolated 3d later to check YFP expressing cells. For the immunological studies, BALB/c mice were immunized subcutaneously (s.c.) at the base of the tail with BCG (106 CFU/animal) along with intramuscularly (i.m.) in the hind limb with 100 μg/animal of C6 and controls (pcDNA3.1-, C6, BCG and negative) in PBS as 3 mice in a group. Two booster doses of DNA vaccine were given at the interval of 2 weeks. Later, mice were euthanized for organ analysis.
Aerosol infection and bacterial burden in the lungs and spleen
Immunized mice were rested for 30d and aerosol challenged with 100 CFU of live Mtb by Inhalation Exposure System (GlasCol, LLC, Terre Haute, IN). Thirty days after the infection, animals were sacrificed and bacterial burden in lungs and spleen were determined by inoculation of tissue homogenates on 7H11 plates. Lungs and spleen sections were also preserved in 1% formalin in PBS for the histopathological analysis by hematoxylin and eosin staining.
Spleen and lung lymphocyte culture
Spleen, lymph nodes (LNs), and lung cells were prepared by crushing of tissues followed by RBC lysis. Lymphocytes (2 × 105/well) isolated from spleens/LNs or lungs were cultured in 96-well U bottom plates and stimulated with PPD (25 μg/ml) and 5 C6 peptides (5 μg/ml each) as Rv0476(1–19) was unable to synthesize. For DCs and macrophages activation status studies, cells were stimulated with LPS (1 μg/ml) for 24 h.
Flowcytometry
For phenotypic analysis of T cells, the PPD and peptides stimulated lungs and spleen/LNs cells were analysed by flow cytometry. Lymphocytes culture were harvested and stained with fluorochrome tagged anti-CD4-PE, CD8-APCCy7, CD62L-FITC, CD44-PerCPCy5.5, CD11c-PECy7, F4/80-APC, CD86-PE, CD80-FITC, CD40-PECy5, and MHC-II-PerCPCy5.5abs (BD Biosciences, San Jose, CA). Briefly, lymphocytes were harvested in tubes and washed with FACS buffer (PBS + 2%FCS). Cells were Fc blocked using anti-mouse CD16/CD32 Ab. Later, stained with fluorochrome-labelled Abs. After staining, cells were fixed by using 1% paraformaldehyde in FACS buffer. Cells were acquired in BD-FACS Aria III and BD-FACS Accuri (BD, Franklin Lakes, NJ). The analysis was performed using BD-FACS DIVA, BD-C6, and Flowjo software (BD, Franklin Lakes, NJ).
Cytokine ELISA
The expression of different cytokines in the culture supernatants from PPD and peptide stimulated lymphocyte cultures were monitored by sandwich ELISA. Briefly, primary anti-cytokine antibodies were coated on 96-well plates at 4 °C overnight. Next, wells were blocked with 2% BSA solution for 2 h and incubated overnight at 4 °C with the culture supernatants. Later, plates were incubated 2 h with biotinylated secondary antibodies and 45 min with streptavidin-HRP conjugates. OPD-H2O2 substrates were used to determine the concentration of cytokines along with standards by obtaining reading at 595 nm.
Statistics
All the statistical analysis was performed as One-way ANOVA with Tukey’s test in Graph Pad Prism (GraphPad Software, La Jolla, CA).
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