A lipidated peptide of Mycobacterium tuberculosis resuscitates the protective efficacy of BCG vaccine by evoking memory T cell immunity

Background The current BCG vaccine induces only short-term protection against Mycobacterium tuberculosis (Mtb), suggesting its failure to generate long-lasting memory T cells. Previously, we have demonstrated that a self-adjuvanting peptide of Mtb (L91), successfully generated enduring memory Th1 cells. Consequently, we investigated if L91 was able to recuperate BCG potency in perpetuating the generation of memory T cells and protection against Mtb infected mice. Methods In the present study, we evaluated the potency of a self adjuvanting Mtb peptide vaccine L91 in invigorating BCG immune response against Mtb in mice. Female BALB/c mice were immunized with BCG. Later, they were boosted twice with L91 or an antigenically irrelevant lipidated influenza virus hemagglutinin peptide (LH). Further, PBMCs obtained from BCG vaccinated healthy subjects were cultured in vitro with L91. T cell responses were determined by surface markers and intracellular cytokine staining. Secretion of cytokines was estimated in the culture supernatants (SNs) by ELISA. Results Compared to the BCG-vaccinated controls, L91 booster significantly enhanced the percentage of memory Th1 cells and Th17 cells and reduced the mycobacterial burden in BCG primed and L91-boosted (BCG-L91) group, even after 229 days of BCG vaccination. Further, substantial augmentation in the central (CD44hiCD62LhiCD127hi) and effector memory (CD44hiCD62LloCD127lo) CD4 T cells was detected. Furthermore, greater frequency of polyfunctional Th1 cells (IFN-γ+TNF-α+) and Th17 cells (IFN-γ+IL-17A+) was observed. Importantly, BCG-L91 successfully prevented CD4 T cells from exhaustion by decreasing the expression of PD-1 and Tim-3. Additionally, augmentation in the frequency of Th1 cells, Th17 cells and memory CD4 T cells was observed in the PBMCs of the BCG-vaccinated healthy individuals following in vitro stimulation with L91. Conclusions Our study demonstrated that L91 robustly reinvigorate BCG potency to invoke enduring protection against Mtb. This novel vaccination stratagem involving BCG-priming followed by L91-boosting can be a future prophylactic measure to control TB. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1301-x) contains supplementary material, which is available to authorized users.

antigenic repertoire to protect against Mycobacterium tuberculosis (Mtb). In contrast, it fails to safeguard adults from TB; which has been suggested to be due to its failure to generate long-lasting memory T cells [8].
Many studies are in progress to bolster BCG efficiency to impart enduring immunity. Memory inducing cytokines like IL-7 and IL-15 have been shown to sufficiently augment the BCG induced memory T cells [8]. Furthermore, booster dose of Mtb antigen Acr1 entrapped in fusogenic-liposomes generated long-term memory T cells and improved BCG potency [9]. Thus, it implies that the protective efficacy of BCG can be boosted through antigen-priming. Recently, we have synthesized a novel lipopeptide vaccine construct L91, which comprises of a promiscuous-peptide derived from Acr1 and the TLR2 agonist Pam2Cys [5,10]. L91 elicited both innate and adaptive immunity successfully through its Pam2Cys and peptide component, respectively [5,10]. TLR-2 promotes the generation of memory T cells, rescued Th1 cells from exhaustion and protected mice from chronic TB [11]. Intriguingly, L91 elicited long-lasting memory T cells and protected mice and Guinea pigs from Mtb infection [10].
In the current study, we have demonstrated that the memory T cell generation and protection efficacy of BCG vaccine against Mtb could be significantly bolstered with L91 boosting of the BCG-vaccinated population. Specifically we observed improvement in the pool of enduring memory Th1 and Th17 responses, the cells that play crucial role in protection against Mtb. In future, this vaccination strategy may be implemented to protect people from TB.

Study design
Female BALB/c mice (6-8 week) were procured from the Experimental Animal Facility, CSIR-Institute of Microbial Technology, Chandigarh, India. Mice were immunized subcutaneously (sc) at the base of tail with the Danish strain of BCG (10 6 CFU/mouse). Twenty-one days later, BCG-primed mice were boosted twice with L91, at an interval of 14 days apart (BCG-L91). Control groups were immunized with BCG alone (BCG), placebo (PBS) or an antigenically irrelevant lipidated influenza virus hemagglutinin peptide (abbreviated as LH or Pam2Cys). Mice were aerosol challenged with Mtb (~100 CFU/mouse), 90 days after the last booster. Subsequently, animals were sacrificed after 90 days of Mtb challenge. Later, immunological (ex vivo), protection and histopathology studies were performed. To monitor the antigen specific T cell response, mice were sacrificed 30 days after Mtb infection, and cellular responses were examined following in vitro stimulation with L91, Pam-2Cys and short term culture filtrate of H37Rv (ST-CF). In all the experiments, changes in the response on vaccination were compared among BCG-L91 and control BCG and placebo (PBS) groups or otherwise indicated.

Vaccine constructs used in study
Lipidated synthetic peptides used in the study were produced by solid phase synthesis method, as described elsewhere [12]. The lipidated promiscuous peptide of sequence SEFAYGSFVRTVSLPVGADE was from the Acr1 antigen of Mtb (L91). The control, non-mycobacterial, lipidated peptide (LH) sequence ALNNRFQIK-GVELKS was from influenza virus hemagglutinin light chain and was shown to be active in BALB/c mice [13].

Mycobacterial strains and BCG
Mtb H37Rv strain was cultured in 7H9 medium containing Tween-80 (0.05%), supplemented with albumin (10%), dextrose and catalase (ADC). Glycerol stocks of H37Rv were prepared and stored at −80 °C, and later used for infection studies. BCG vaccine (TUBERVAC) used for immunization was purchased from Serum Institute of India, Pune, India. TUBERVAC (Bacillus Calmette-Guerin Vaccine I.P.) is a live freeze-dried vaccine derived from an attenuated strain of Mycobacterium bovis and meets the requirements of WHO and I.P. when tested by the methods outlined in WHO, TRS. 745 (1987), 771 (1988) and I.P.

Isolation of lymphocytes from lymph nodes, spleen and lungs
Spleens and LNs obtained from the immunized mice and exposed to Mtb, were pooled and single cell suspension was prepared by gently pressing through frosted slides. Lungs were perfused with chilled PBS and small pieces Rai et al. J Transl Med (2017) 15:201 were prepared and digested with collagenase (2 mg/ ml) for 30 min/37 °C. Later, cells were passed through a sieve (70 μM). Viability was checked by trypan blue dyeexclusion method and cells (2 × 10 5 /well) were added to 96 well U-bottom culture plates and cultured with L91 (1 nmol), Pam2Cys (50 ng/ml), ST-CF (25 μg/ml) and medium for 72 h.

Intracellular cytokine and surface staining
The cells were stimulated as mentioned in the proliferation assay and then stimulated with PMA (50 ng/ ml) and ionomycin (10 μM) for 4 h followed by incubation with brefeldin A (5 mg/ml) for an additional 2 h. The cells were then harvested, washed twice with buffer (PBS-FCS-2%) and fixed with paraformaldehyde (1X) at 4 °C for 30 min. Fixed cells were perforated with saponin (0.2%) and incubated with fluorochrome tagged anti-IFN-γ, IL-17A and TNF-α Abs at 4 °C for 90 min. The cells were washed with saponin (0.2%), followed by wash buffer. For surface staining, the cells were incubated with either fluorochrome labeled Abs or biotinylated Abs/ streptavidin-fluorochrome conjugates. Standard protocols of washing/incubation were followed at each stage.

Cytokine estimation
The cultures were set as mentioned in T cell proliferation assay. Later, culture supernatants (SNs) were collected and cytokine concentrations were determined using a standard sandwich ELISA [14].

Culture of dendritic cells
Monocytes were isolated from the femurs and tibia of the mice. The cells (2 × 10 6 /well) were cultured in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF; 2 ng/ml) and interleukin-4 (IL-4; 4 ng/ ml) for the generation of DCs. On day 3, the cultures were replenished with fresh medium and supplemented with GM-CSF and IL-4. On day 6, the cells were harvested, washed and added to 24 well plates (2 × 10 5 cells/ well). Bone marrow derived dendritic cells (BMDCs) were stimulated with either L91 (3 nmol), F91 (3 nmol), Pam2Cys (50 ng/ml) or LPS (4 μg/ml) for 16 h. SNs were collected for cytokines estimation by ELISA and the expression of surface markers was assessed by flow cytometry.

Western blotting for iNOS
As mentioned above for the cultures of DCs and macrophages, BMDCs were stimulated with L91, F91, Pam-2Cys and LPS for 16 h in 12 well plate (10 6 cells/well). The cells were harvested and lysed in radioimmunoprecipitation assay buffer (RIPA) containing a protease inhibitor cocktail. Protein was estimated and equal amounts (30 μg) were subjected to SDS-PAGE (10%) followed by transfer to PVDF membrane. The non-specific sites were blocked with BSA (5%) and the blot was probed with anti-iNOS Abs (1:200) (Ab3523) or actin as a control. Later, the blot was probed with anti-rabbit-HRP Ab and finally developed using an enhanced chemiluminescence method (Lumigen, Inc. Southfield, MI). The blot was finally scanned using Image Quant LAS 4000 (GE Healthcare, Pittsburgh, PA).

Demonstration of NF-κB by EMSA
BMDCs were stimulated with L91 (9 nmol), F91 (9 nmol), Pam2Cys (150 ng/ml) or LPS (4 μg/ml) for 30 min in 12 well plates (10 6 cells/well). The cells were harvested and nuclear extract was prepared. Nuclear extracts of each sample were incubated with [P 32 ] labeled oligonucleotides containing the binding site for NF-κB at 37 °C for 20 min to allow the formation of DNA-protein complexes, which was then resolved by native gel electrophoresis using a 6% gel. Later, the gel was dried and exposed to a blank screen at room temperature for 6-10 h and scanned by a phosphorimager (Fujifilm, Tokyo, Japan).

Isolation and culture of human PBMCs
Blood was obtained from BCG vaccinated healthy volunteers in sterile vacutainers. Blood was diluted with PBS in 1:1 ratio and overlaid on histopaque. Peripheral blood mononuclear cells (PBMCs) were separated by centrifugation at 400 g for 30 min at 25 °C. PBMCs were washed 3 times with PBS + 2% FCS and in vitro cultured with L91 (1 nmol), F91 (1 nmol) or Pam2Cys (50 ng/ml) for 96 h. During culture, IL-2 (100U) was added after 24 h.

Statistical analysis
The data are presented as mean ± standard error. Statistical analysis was performed using Graph Pad Prism employing an unpaired Student's 't'test.

L91 administration limits the generation of regulatory T cells
One of the main reasons associated with BCG failure is the generation of regulatory T cells (Tregs) following vaccination, which hampers its protective efficacy [5]. Consequently, we examined ex vivo generation of regulatory T cells (Tregs) in the cells isolated from the lungs of the immunized mice after 90 days of Mtb infection. We observed significantly (p ≤ 0.005) higher percentage of FoxP3 + CD4 Tregs from the BCG vaccinated mice, as compared to the control (placebo) group ( Fig. 1a, b). On the other hand, a significant reduction of the FoxP3 + CD4 Tregs (p ≤ 0.0001) was observed in the group of mice that was primed with BCG followed boosting with L91 (BCG-L91), as compared to BCG. Thus, indicating that L91 boosting restricted BCG-mediated induction of Tregs and thereby may be augmenting immunity against Mtb.

L91 rescues CD4 T cells from exhaustion
Mycobacterium tuberculosis is known to induce exhaustion of T cells [11]. Recently, we have demonstrated that signaling via TLR-2 rescues CD4 T cells from exhaustion [11]. L91 comprises of TLR-2 agonist Pam2Cys and a MHC-II binding promiscuous peptide of Acr1 [10]. Therefore, we monitored whether BCG-L91 vaccination could rescue CD4 T cells from exhaustion against Mtb infection. The results showed a substantial reduction of the exhaustion markers PD-1 (p ≤ 0.05) (Fig. 1c, d) and Tim-3 (p ≤ 0.05) (Fig. 1e, f ) on the CD4 gated T cells obtained from BCG-L91 group, compared to those of BCG group (Fig. 1c-f ). There was no difference in the expression of these two markers on the cells isolated from BCG and the placebo group. These results demonstrate that L91-boosting prevented T cell exhaustion in Mtb infected mice.

BCG-L91 elicits predominantly Th1 and Th17 immunity
Th1 cells and Th17 cells are two major arms of adaptive immunity that play a cardinal role in safeguarding against Mtb [15]. Th1 cells are known to mediate protection against Mtb through the release of IFN-γ and TNF-α [7,10,16]. Th17 cells confer protection by chemokine mediated recruitment of the cells of innate and adaptive immunity [17,18]. BCG has shown to elicit a mixed Th1 and Th2 response and also a weak Th17 response [19,20]. Our results show that L91 booster significantly augmented Th1 and Th17 immunity, as evidenced by a greater release of IFN-γ following in vitro stimulation with L91 (p ≤ 0.005) or ST-CF (p ≤ 0.05) and IL-17A (in vitro stimulation with L91: p ≤ 0.005; ST-CF: p ≤ 0.0005) by lung cells (Fig. 2a, b) than the control groups. Thus, BCG-primed and antigen-boost strategy generated better immunity than BCG alone against Mtb [17,21].

BCG-L91 enhances the frequency of T cell memory response
One of the main reasons for BCG's inability to evoke long-term protection against Mtb is its failure to generate enduring memory T cells [5,10]. The results of this study show that L91 induced significantly greater expansion in the pool of both central (CD44 hi CD62L hi ; L91: p ≤ 0.05; ST-CF: p ≤ 0.005) and effector memory (CD44 hi CD62L lo ; L91: p ≤ 0.05; ST-CF: p ≤ 0.05) CD4 T cells in the BCG-L91 administered mice than the control group following in vivo stimulation of L91 and ST-CF (Fig. 3a-c). These results were further substantiated by significantly (p ≤ 0.005) higher display of CD127, another marker for memory CD4 T cells (Fig. 3d, e). No discernible change was detected in the case of BCG group. The CD127 has been established to be an important marker for the sustenance of memory CD4 T cells [24].

L91 reduces the suppression of antigen presenting cells by enhancing NF-κB and iNOS expression
The Acr1 antigen of Mtb is known to tolerize dendritic cells by upregulating the inhibitory molecule Tim-3 and consequently suppresses the host immunity [25]. L91 is derived from Acr1. Hence, it was worth to examine the influence of L91 on Tim-3 expression on dendritic cells. Our results show that in vitro stimulation with L91 significantly (p ≤ 0.0005) downregulated the expression of Tim-3 compared to unstimulated cultures (Fig. 4a). L91 comprises of the components that are capable of inducing both innate and adaptive immunity [10]. Innate immunity is the first line of defense to combat against any pathogen [26]. Nitric oxide (NO) mediated killing of Mtb is one of the well-established protection mechanism [27]. Hence, we thought it would be worth to check the impact of L91 on DCs in inducing the release of NO. Interestingly, we observed a dose-dependent increase in iNOS, the enzyme responsible for NO synthesis (Fig. 4b) upon stimulation with L91. Additionally, L91 considerably (p ≤ 0.0005) augmented TNF-α secretion (Fig. 4c). TNF-α is a major factor in restricting the intracellular survival of Mtb [7,25]. Further, the augmentation in the expression of NF-κB was also detected with L91 stimulation (Fig. 4d). NF-κB is a key transcription factor responsible for the propagation of cells and pro-inflammatory responses [28]. Besides L91, considerably higher NF-κB expression was detected following stimulation with the Pam2Cys control but not un-lipidated peptide (F91). These results together demonstrate

L91 boosting enhances protective efficacy of BCG vaccine
After establishing the role of L91 in inducing the longlasting memory T cell response, we next evaluated the protective efficacy of BCG-L91 against Mtb. To generate bona fide memory T cell response, BCG-L91 vaccinated mice were aerosol challenged with Mtb 90 days after the last immunization. Bacterial burden in the lungs and spleen was enumerated 90 days following the challenge. The results show that L91 boosting led to a greater reduction in the mycobacterial burden in the lungs (p ≤ 0.005) and spleen (p ≤ 0.005), compared to BCG-vaccinated group, and also the Pam2Cys and placebo control (Fig. 5a, b). The decrease in the CFU in the spleen also signifies the importance of BCG-L91 in restricting the dissemination of Mtb.
Further, we substantiated CFU data by histopathological study of lungs. Animals immunized with BCG-L91 exhibited reduced pathology in the lungs, manifested by decreased macrophage infiltration and lesser and smaller size of granulomas, fewer scattered peribronchiolar lymphocyte-rich areas and better preserved alveolar spaces, when compared to the BCG group, placebo and Pam2Cys controls. The control animals revealed Mtb induced severe pathology that was characterized by the larger size and number of lymphocyte rich compact granulomas with signs of irregular lung architecture due to excessive inflammation. Peribronchiolar cuffs and bigger confluent areas of consolidated mixed lymphocyteshistiocytes were also apparent (Fig. 5c). The quantitative assessment of the granulomas in lung sections revealed a decreased Mtb burden in BCG-L91 immunized animals (Fig. 5d).

L91 augments the expansion and activation of human CD4 T cells
L91 efficiently amplified BCG induced immunity in the experimental model of TB. Hence, we were next curious to know whether L91 was able to activate human CD4 T cells obtained from BCG vaccinated healthy volunteers. PBMCs cultured with L91 exhibited significantly higher (p ≤ 0.0005) proliferation and upregulation of the activation marker CD25 (p ≤ 0.0005) on CD4 T cells, as compared to those cultured with free peptide or Pam2Cys control (Fig. 6a-c).

L91 enhances the generation of enduring polyfunctional Th1 cells and Th17 cells in human PBMC
We next observed that L91 stimulation improved the generation of Th1 cells, as it significantly (p ≤ 0.05) expanded the percentage of IFN-γ + CD4 T cells. Further, we observed increased frequency (p ≤ 0.005) of polyfunctional Th1 cells expressing both IFN-γ and TNF-α ( Fig. 7a-d). The results were obtained using blood from the non-TB endemic Australian population. Furthermore, we also monitored the efficacy of L91 using PBMCs from BCG-vaccinated healthy volunteers of the TB-endemic Indian population. It is worth to mention here that the BCG has failed to protect Indian population from Mtb [4]. We observed a significant augmentation in Th1 and Th17 immunity, as documented by the increased percentage of IFN-γ + (p ≤ 0.0005) and IL-17A + (p ≤ 0.005) CD4 T cells, respectively (Fig. 8a-c). Further, enhanced frequency (p ≤ 0.005) of IFN-γ + and IL-17A + polyfunctional Th17 cells was detected (Fig. 8d). Furthermore, expansion (p ≤ 0.005) of the pool of the memory precursors of CD4 T cells (CD45RA + CD45RO + ) was noted (Fig. 8e). These results suggest that L91 efficiently activated polyfunctional human Th1 cells and Th17 cells and expanded the pool of memory CD4 T cells. Thus, has a great potential to reinvigorate the efficacy of BCG vaccine in TB endemic and non-endemic population.

Discussion
The slow progress of the 'Stop TB Program' and the emergence of drug resistant strains of Mtb poses an urgent challenge for the scientific community to develop an effective vaccine against Mtb. BCG is the only currently available vaccine, which is widely administered worldwide [10]. Nevertheless, TB accounts for 9.6 million new cases and 1.5 million mortality annually [1]. In many ways BCG is a controversial vaccine, since it protects 80% of individuals in non-TB endemic regions and 0% in TB-endemic zones [7,29]. BCG protects children but not adults living in TB-endemic regions, further demonstrating its variable efficacy [5,10,30]. Many approaches to improve BCG vaccine have been tried, which includes recombinant BCG with different antigenic formulations [29][30][31]. However, the introduction of a globally effective vaccine candidate, at least in TB-endemic zones, is yet to be achieved. The high protective efficacy of BCG in the developed world and children of TB-endemic sectors suggest that BCG has the adequate antigenic repertoire to protect against Mtb [23]. Nevertheless, BCG efficacy Prime boosting with BCG-L91 improves the protective efficacy of BCG. Animals were vaccinated with BCG and later boosted twice with L91. Ninety days of last booster, the mice were aerosol challenged with Mtb. After 90 days, bacterial burden was enumerated in the a lungs and b spleen. Control animals received saline, LH and BCG. The bacterial number is expressed as log 10 CFU/g of tissue. Results shown as bar diagram are Mean ± SEM and pooled data from 3 experiments (n = 3 mice/group). c Histopathological analysis of lung was done after staining the sections with hematoxylin and eosin. The magnification is depicted at 20 ×/100 × for lungs. The arrows indicate granulomas or tubercle. The asterisk represents the level of significance. d The quantitative assessment of the Mtb infected lungs was done by counting the granulomas in ten random fields of the gross histopathological sections and the average is expressed as Mean ± SD. LH lipidated hemagglutinin peptide, BCG-L91 mice primed with BCG and boosted with L91. **p ≤ 0.005, ***p ≤ 0.0005, ns not significant Rai et al. J Transl Med (2017) 15:201 wanes with age, which indicates its failure to elicit lifelong immunity [32]. In this context, several approaches are being tried to boost BCG ability to generate enduring memory T cells and protection against TB [8,9].
L91 is a chimeric peptide comprising of MHC-II binding peptide and Pam2Cys and promotes the generation of long-term memory CD4 T cells [10]. Therefore, we were encouraged to test the potential of L91 to bolster BCG efficacy by promoting the persistence of memory CD4 T cells and consequently long lasting protection against Mtb.
We carried out a study in which L91 was used to boost the immune response primed by BCG vaccination. As compared to BCG group, the following major findings have been obtained: (i) significant reduction in the number of CFU in the lungs and diminished pathological changes in the Mtb infected mice; (ii) higher proliferation of the CD4 T cells and upregulated expression of IFN-γ and IL-17A; (iii) robust increase in the pool of multifunctional Th1 cells (IFN-γ + /TNF-α + ) and Th17 cells (IL-17 + / IFN-γ + /CXCR3 + /CCR6 + ); (iv) expansion in the percentage of central and effector memory CD4 T cells; (v) the mechanism involved in the reduction of bacterial burden was through iNOS and TNF-α; (vi) the PBMCs obtained from the BCG vaccinated volunteers showed increase in the frequency of polyfunctional Th1 cells, Th17 cells and memory CD4 T cells on in vitro exposure to L91.
One of the main reasons associated with the weak efficacy of BCG vaccine to protect against Mtb is attributed to the generation of Tregs following immunization, which dampens Th1 immunity through release of IL-10 [33][34][35]. The decrease in the frequency of Tregs in the BCG-L91 group compared to BCG group clearly indicates the importance of L91 boosting in reducing the number of Tregs. The inhibition in the development of Tregs by BCG-L91 may be due to the augmented secretion of IFN-γ by Th1 cells [36,37]. Furthermore, the signaling events stimulated by TGF-β are negatively regulated by IFN-γ [38]. TGF-β is responsible for the differentiation of naïve CD4 T cells to Tregs. IFN-γ-mediated phosphorylation of STAT1 leads to the expression of T-bet and Smad7 [38,39], which is known to suppress the regulatory function of Tregs [40].
It has also been well documented that Mtb drives Th1 cells to exhaustion [11]. Th1 cells play a crucial role in protecting against Mtb [10,11]. Recently, we have demonstrated that triggering of TLR-2 with Pam2Cys can enhance the generation of memory Th1 cells and rescues them from exhaustion by downregulating PD-1 and Tim-3 and amplifying co-stimulatory signals and the secretion of pro-inflammatory cytokines [10,11]. TLR-2 agonist Pam-2Cys is a major component of L91. TLR-2 signaling not only rescues T cells from exhaustion but also stimulates both innate and adaptive immunity. Further, the higher susceptibility of TLR-2 −/− animals to Mtb indicates an important role of TLR-2 in protection [41,42].
The induction of Th1 immunity plays a fundamental role in protecting against intracellular pathogens such as Mtb [7,8,10]. Further, MyD88 −/− animals with partially compromised Th1 immunity are more susceptible to TB [15]. We demonstrated that the L91 booster substantially augmented Th1 immunity, as evidenced by improvement in IFN-γ secretion. Pam2Cys is known to stimulate DCs to release IL-12 through TLR-2 signaling [10]. IL-12 is a differentiating factor for Th1 cells. Thus, L91 preferentially expands Th1 cells. Furthermore, we noted the generation of Th17 cells in the animals vaccinated with BCG-L91. Recently, it was demonstrated that Th17 cells confer protection against Mtb by recruiting Th1 cells to the site of infection. Th17 cells show a robust effect on chemokine-mediated infiltration of macrophages and neutrophils at the site of infection [17,18].
The importance of NO has been well documented in the killing of Mtb by inducing apoptosis of infected cells [27]. L91 activates DCs and augments the expression of iNOS and TNF-α. Both NO and TNF-α cumulatively protect the host from Mtb and the mechanism deciphered is by inducing apoptosis of Mtb-infected cells. Apoptosis releases intracellular bacteria, which provides an opportunity for the activated macrophages to engulf and eliminate them [27]. We have shown that a booster dose of L91 efficiently bolsters the protective efficacy of BCG and significantly constrains bacterial burden in the lungs and spleen even after a lengthy period of 229 days of vaccination. The results of protection study suggest that L91 has a unique capability of generation and maintenance of long-lasting memory CD4 T cells and protection against Mtb [10].
Finally, we validated the efficacy of L91 employing PBMCs of BCG vaccinated healthy adult volunteers from TB-endemic and non-endemic zones. Importantly, CD4 T cells exhibited an enhacement in the expression of IFN-γ and IL-17A on in vitro stimulation with L91. Furthermore, an increase in the frequency of polyfunctional Th1 cells and Th17 cells was detected. Finally, it is worth to mention here that a remarkable expansion in the pool of memory CD4 T cells was found in our study, which illustrates the role of L91 in reinvigorating BCG efficacy to evoke long-lasting immunity responsible for protection against Mtb.

Conclusions
This study indicates that BCG priming followed by L91 boosting could efficiently overcome the low long-term protective efficacy associated with BCG vaccination by generating enduring memory T cells and protection against Mtb. The possible mechanism involved is through the involvement of both innate (NO and TNFα) and adaptive (long-lasting polyfunctional Th1 cells and Th17 cells) immunity. In future, this vaccination strategy of BCG-L91 vaccine may be developed into an effective strategy to control TB in the TB-endemic population.