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. 2015 Jun 3;181(2):286–296. doi: 10.1111/cei.12634

Prime-boost vaccination strategy with bacillus Calmette–Guérin (BCG) and liposomized alpha-crystalline protein 1 reinvigorates BCG potency

K F Siddiqui 1, M Amir 1, N Khan 1, G Rama Krishna 1, J A Sheikh 1, K Rajagopal 1,1, J N Agrewala 1,✉
PMCID: PMC4516444  PMID: 25845290

Abstract

Bacillus Calmette–Guérin (BCG) remains the only available and most widely administered vaccine against Mycobacterium tuberculosis (Mtb), yet it fails to protect vaccinated individuals either from primary infection or reactivation of latent tuberculosis (TB). Despite BCG’s variable efficacy against TB, the fact remains that BCG imparts protection in children against the disease, indicating that BCG possesses a wide protective antigenic repertoire. However, its failure to impart protection in adulthood can be linked to its failure to generate long-lived memory response and elicitation of an inadequate immune response against latency-associated antigens. Therefore, to improve the protective efficacy of BCG, a novel vaccination strategy is required. Consequently, in the present study, we have exploited the vaccination potential of liposomized α-crystalline 1 (Acr1L), a latency-associated antigen to induce enduring protective immunity against Mtb in BCG-primed animals. It is noteworthy that an increase in the multi-functional [interferon (IFN)-γhi/tumour necrosis factor (TNF)-αhi] CD4 and CD8 T cells were observed in BCG-primed and Acr1L-boosted (BCG-Acr1L) animals, compared to BCG alone. Further, substantial expansion of both central memory (CD44hi/CD62Lhi) and effector memory (CD44hi/CD62Llo) populations of CD4 and CD8 T cells was noted. Importantly, BCG-Acr1L exhibited significantly better protection than BCG, as evidenced by a reduction in the bacterial burden and histopathological data of the lungs. In essence, BCG-Acr1L could be a potent future vaccination strategy to reinvigorate BCG potency.

Keywords: Acr1, BCG, CD4 cells, CD8 cells, prime boost, tuberculosis, vaccine

Introduction

Bacillus Calmette–Guérin (BCG) is the only vaccine approved against tuberculosis (TB). Unfortunately, it fails to generate an enduring memory T cell response against Mycobacterium tuberculosis (Mtb), as indicated by the fact that it protects childhood but not the adult manifestation of the disease 1–3. Further, it does not protect immunized individuals either from Mtb primary infection or reactivation of latent infection 4–7. This failure may be due to BCG being cleared by the host immunity before its antigens could optimally prime the immune system to generate an enduring memory T cell response against latency-associated antigens. These antigens are crucial in imparting protection against Mtb 8,9. Several proteins of Mtb are expressed during latency; one such antigen is alpha-crystalline protein 1 (Acr1) (16 kDa antigen; HspX; α-crystallin1; Rv2031c) 10–12. This antigen is considered to be a potent vaccine candidate against dormant Mtb. Interestingly, Acr1 elicits higher interferon (IFN)-γ release in individuals with latent TB compared to those with active disease 8. This signifies that Acr1 helps in the maintenance of a disease-free state in such subjects, thus making Acr1 an attractive target for the development of vaccine against TB 12. Recently, substantial progress has been made in developing vaccines against TB 13. However, most of the vaccines are based on immunodominant antigens that are recognized during the early stages of Mtb infection 14–17. In addition, there are very few vaccine studies based on latency-associated antigens 9,18–20.

A potentially successful vaccine should have the ability to induce and maintain long-term antigen-specific enduring memory T cells, which should be expanded easily on re-exposure to the pathogen. Antigen-specific memory T cells express cytokines which activate the cells of the immune system, thereby helping in rapid clearance of the invading bacterium and protecting the host from subsequent infections. Apart from the well-documented role of CD4 T cells, several reports have indicated the protective role of CD8 T cells in TB 21–23. The antigens delivered to antigen-presenting cells (APCs) normally undergo an exogenous pathway for processing and presentation to CD4 T cells, but not to CD8 T cells. However, fusogenic liposomes prepared from the yeast lipids can deliver antigen into the cytosol of APCs, leading to the generation of not only antigen-specific CD4 but also CD8 T cells 24. In addition, liposomes are not only effective adjuvants or vehicles to deliver antigens, but can successfully evoke CD4 T helper type 1 (Th1) and Th2 immunity and enhance memory T cell responses 25,26.

Following the above-mentioned approaches, we adopted a prime-boost vaccination strategy using BCG and liposomized-Acr1 (BCG-Acr1L) antigen to invigorate the protective efficacy of BCG against Mtb. BCG-Acr1L vaccination proved advantageous in significantly improving BCG potency, as evidenced by augmentation in the immune response and decline in the mycobacterial burden in animals exposed to Mtb.

Materials and methods

Mice

Female C3H/HeN mice (6–8 weeks) were procured from the CSIR Institute of Microbial Technology (CSIR-IMTECH) animal facility after approval from the Institute’s Animal Ethics Committee. For immunization and infection experiments, animals were housed in the Biosafety level-3 facility of CSIR-IMTECH. Animals were offered commercial diet and water ad libitum.

Mycobacterial strains

Mtb (H37Rv) and M. bovis (BCG, Danish strain) were a kind gift from Dr V. M. Katoch, NJIL and OMD, Agra. Mycobacteria were cultured in 7H9 medium containing 0·05% Tween-80 and supplemented with 10% oleic albumin dextrose catalase (OADC).

Protection studies

Mice (six to eight per group) were primed subcutaneously with BCG (1 × 106 CFU/animal) and 21 days later were administered a booster dose of Acr1L (50 μg/mice) (BCG-Acr1L). Similarly, the control groups were primed with either Acr1L, Acr1 or BCG and boosted with Acr1L (Acr1L-Acr1L) or Acr1 (Acr1-Acr1) or Acr1 (BCG-Acr1), respectively. In addition, groups were kept inoculated with BCG and placebo [phosphate-buffered saline (PBS)]. A dose of 50 μg/mouse free Acr1 or entrapped in liposomes was used. The animals were rested for 160 days and then aerosol-challenged with a low dose of H37Rv using an inhalation exposure system (Glas-Col, Terre Haute, IN, USA) to deposit approximately 100 live bacteria in the lungs [as checked by colony-forming unit (CFU) plating after 24 h of exposure]. After 35 days, animals were killed and lungs were harvested. Serially diluted lung homogenates were plated on Middlebrook 7H11 medium supplemented with 2-thiophene carboxylic hydrazide (TCH, 2 µg/ml) and OADC. Colonies were counted after 3–4 weeks of incubation at 37°C.

Entrapment of Acr1 in liposomes (Acr1L)

The yeast lipids were isolated from Saccharomyces cerevisiae and liposomes were prepared as described earlier 27. Briefly, yeast lipids were reduced to thin dry film. The film was hydrated followed by sonication in a bath-type sonicator for 15–30 min. The liposomes formed were mixed at this stage with an equal volume of Acr1. This mixture was flash-frozen, thawed and then lyophilized. The lyophilized powder was reconstituted in PBS. It was washed a further three times with PBS to remove the traces of the unentrapped solute. The protein entrapped in the liposomes was estimated by lysing with 1% Triton X-100 solution followed by the addition of bicinchoninic acid (BCA) reagent to lysed liposomes and then incubating at 37°C for 45 min. Finally, the absorbance was measured at 570 nm wavelength.

Isolation of lymphocytes from spleen and lungs

Mice immunized as indicated in protection studies were killed 35 days after aerosol Mtb challenge. Spleens and lymph nodes were removed aseptically and a single-cell suspension was prepared. Red blood cells (RBCs) were lysed using an ACK lysis buffer, washed three times with PBS and resuspended in complete medium [RPMI-1640 + 10% fetal bovine serum (FBS)]. Viable cells were counted using the trypan blue dye-exclusion method. Lung cells were prepared as described elsewhere 28. Briefly, the lungs were perfused through the right ventricle with chilled PBS. Once lungs became white, they were removed, chopped and incubated with digestion mixture collagenase (0·7 mg/ml) and DNase (30 µg/ml) in media at 37°C for 1 h. Digested tissues were disrupted and passed through 70-µm pore size nylon cell strainers. The RBCs were lysed by ACK lysis buffer. The resultant single-cell suspension was washed three times, resuspended in complete media and used for cultures.

Proliferation assays

Cell proliferation assays were set as described previously 28–30. Briefly, lymphocytes (2 × 105 cells/well) isolated from spleen and lymph nodes were cultured in triplicate in 200 µl of complete RPMI-1640 10% FCS with optimal concentrations of Acr1L, Acr1 and purified protein derivative (PPD) in 96-well U-bottomed plates. After 72 h, the cells were pulsed with [methyl-3H]-thymidine (0·5 μCi/well) and plates were harvested 16 h later using the Tomtec-Harvester-96 (Tomtec, Hamden, CT, USA). The incorporated radioactivity was measured using the Wallac 1450 Microbeta Trilux β-scintillation counter (Perkin Elmer, Waltham, MA, USA).

Immunophenotyping

Cells were stimulated with Acr1, Acr1L and PPD, as described earlier, for the proliferation assay. After 48 h, cells were harvested and incubated with Fc block and then stained for CD4, CD8, CD44, CD62L and their isotype-matched controls for 30 min on ice. After washing three times, cells were fixed with paraformaldehyde and acquired using the fluorescence activated cell sorter (FACS) Aria II cell sorter (BD Biosciences, San Jose, CA, USA). Data were analysed with Diva software (version 6·1·2).

Cytokines enzyme-linked immunosorbent assay (ELISA)

Cultures were set as described for the proliferation assay. The supernatants (SNs) for interleukin (IL)-4 and IFN-γ were collected after 48 h. Cytokine levels were estimated by sandwich ELISA, as per the manufacturer’s instructions, and the results were expressed in pg/ml.

Intracellular staining

Lymphocytes (2 × 105 cells/ml) were cultured with Acr1, Acr1L (25 μg/ml) and PPD (25 μg/ml) in 96-well U-bottomed plates for 48 h. Cells were pooled and washed three times with buffer (PBS–FBS 1%). Cells were restimulated with phorbol myristate acetate (PMA) (50 ng/ml) and ionomycin (1 μg/ml) for 6 h and brefeldin A (10 μg/ml) was added in cultures for the last 4 h. After stimulation, cells were washed three times with staining buffer [bovine serum albumin (BSA) 1%, NaN3 0.01% in PBS]. Fc receptors were blocked and then stained with fluorochrome-labelled anti-CD4 and CD8 antibodies. Cells were washed three times with staining buffer and fixed in 2% paraformaldehyde. Cells were then permeabilized with buffer (0·01% saponin PBS–FCS 1%). Further, cells were incubated with fluorochrome-labelled anti-cytokine monoclonal antibodies (mAbs) (or isotype-matched control antibodies) in permeabilization buffer. The incubation period for each step was 30 min at 4°C, and after every incubation the usual washing steps were followed. Later, cells were fixed in paraformaldehyde and acquired on a FACS Aria II, followed by data analysis using FACS Diva (BD Biosciences, San Jose, CA, USA).

Immunoglobulin (Ig)G1 and IgG2a isotype ELISA

Serum samples were collected from Mtb aerosol-challenged mice after 35 days. Acr1-specific antibodies were determined in the serum samples, as described elsewhere 28. Briefly, diluted serum samples (×100) were added on Acr1 (10 μg/ml) precoated plates. Acr1-specific IgG1 and IgG2a were detected using biotinylated anti-mouse IgG1 or IgG2a antibodies, followed by the addition of avidin-horseradish peroxidase (HRP). Colour was developed by adding o-phenylenediamine (OPD)-H2O2 and reaction was stopped using 7% H2SO4. Plates were read at 492 nm. The usual procedures of incubation and washing were followed after each step. Results are expressed as the ratio of IgG2a to IgG1.

Histopathological analysis

Mice were killed and lung tissues were fixed in 10% buffered formalin. Histological sections were stained using haematoxylin and eosin, as described elsewhere 28. Photomicrographs were captured on Olympus IX71 microscope at either ×10 or ×20 magnifications.

Results

Characterization of Acr1 antigen of Mtb entrapped in yeast liposomes

To elicit both CD4 and CD8 T cell responses, we entrapped Acr1 antigens of Mtb in liposomes (Acr1L), prepared from fusogenic lipids isolated from yeast. The size of the liposomes was determined by differential light scattering (DLS) to be of an average diameter of 227·4 nm. Little difference was observed between the size of Acr1L and the empty liposomes. Further, we also measured the poly disparity index and diffusion coefficient (Fig. 1a,b). The shape of the liposomes was spherical, as determined by scanning electron microscopy (SEM); (Fig. 1c). The majority of the liposomes were much smaller at the edges of detection under the magnification used. The transmission electron microscope (TEM) images indicated that the liposomes were spherical and unilamellar (Fig. 1d,e). The entrapment efficiency was approximately 50%, as examined by protein estimation (data not shown). Further, efficacy of entrapment was substantiated by fluorescence microscopy data using fluorescein isothiocyanate (FITC)-labelled antigen (Fig. 1f,g). This preparation of Acr1L was used in the prime-boost vaccination study with BCG.

Figure 1.

Figure 1

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Characterization of liposomes for shape, size and entrapment of alpha-crystalline protein 1 (Acr1) antigen. Liposomes prepared from fusogenic yeast lipids were characterized by (a) intensity distribution curve indicating the size of empty liposomes by differential light scattering (DLS); (b) intensity distribution curve of protein entrapped in the liposomes; (c) scanning electron microscopy (SEM) images at magnification 20X; (d,e) transmission electron microscope (TEM) images of empty and protein entrapped liposomes, respectively; (f,g) differential interference contrast (DIC) and fluorescence images of fluorescein isothiocyanate (FITC)-tagged Acr1 entrapped in liposomes. Arrows indicate FITC-Acr1 entrapped in liposomes. The data represented are of three independent experiments.

Immunization with BCG-Acr1L induces long-lasting memory Th1 response

The mice primed with BCG were boosted with liposomized Acr1 (BCG-Acr1L). The animals were rested for 160 days to generate a bona fide memory T cell response. Later, the animals were sacrificed and the Mtb-specific T cell response was monitored. Compared to BCG, BCG-Acr1L exhibited significantly better T cell proliferation on in-vitro priming cells with either PPD (P < 0·001) or Acr1 (P < 0·01) (Fig. 2a,b). Control groups immunized with placebo (PBS) or Acr1 alone failed to improve the T cell recall response (Fig. 2a). Among the subsets of CD4 T cells, Th1 cells play an important role in the protection against Mtb infection. Therefore, we monitored the release of IFN-γ, a Th1 cytokine. A significantly (P < 0·001) higher production of IFN-γ was observed in BCG-Acr1L-vaccinated animals compared to BCG alone on in-vitro exposure of cells with either Acr1L or PPD (Fig. 2c,d). The IFN-γ release was comparatively higher in cultures exposed in vitro to Acr1L than controls. It has been reported that Acr1 and its epitopes predominantly induces secretion of IFN-γ 12,31–33. This was suggestive of the fact that BCG-Acr1L immunization induced a robust Th1 memory response.

Figure 2.

Figure 2

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Elicitation of immune responses after prime boost with bacillus Calmette–Guérin (BCG)-liposomized alpha-crystalline protein 1 (Acr1L). Mice (six to eight per group) were primed subcutaneously (s.c.) with BCG [1 × 106 colony-forming units (CFU)/animal] and 21 days later were administered a booster dose of Acr1L (BCG-Acr1L). Similarly, the control groups were primed with Acr1L and boosted with Acr1L (Acr1L-Acr1L) or Acr1-Acr1 or BCG-Acr1 or BCG-phosphate-buffered saline (PBS) or PBS-PBS (placebo). Animals were rested for 160 days before aerosol challenge with Mycobacterium tuberculosis (Mtb). Thirty-five days after challenge, mice were killed and in-vitro cell cultures were set. T cell proliferation was studied after in-vitro stimulation of cultures with (a) purified protein derivative (PPD) or (b) Acr1L. The proliferation was measured by [methyl-3H]-thymidine incorporation. The results are expressed as a stimulation index (SI), calculated by dividing counts per minute (cpm) of antigen-stimulated cultures with unstimulated cells. Interferon (IFN)-γ was estimated by enzyme-linked immunosorbent assay (ELISA) in the supernatants (SNs) of the cells cultured for 48 h in the presence of (c) Acr1L or (d) PPD. The data are expressed as pg/ml; (e) Acr1-specific immunoglobulin (Ig)G1 and IgG2a isotypes were detected in the serum and expressed as ratio of IgG2a/IgG1. Data represented as mean ± standard error of the mean (s.e.m.) are of two independent experiments, with six to eight mice per group. Statistical analysis was performed by Tukey–Kramer multiple comparison tests. *P < 0·05; **P < 0·01; ***P < 0·001.

It has been well established that when B cells interact with Th1 cells they produce mainly IgG2a, while Th2 cells secrete primarily IgG1. The significant (P < 0·001) increase in the ratio of Acr1-specific IgG2a/IgG1 further substantiated the predominance of Th1 cells upon BCG-Acr1L immunization (Fig. 2e). The proliferation data for cytokine secretion signify that vaccination with BCG-Acr1L significantly evokes long-lasting (160 days) Th1 immunity against Mtb.

BCG-Acr1L induces enduring CD4 and CD8 T cell memory response

Generation of long-lasting memory T cell response is a hallmark of a successful vaccine. Compared to BCG, BCG-Acr1L considerably expanded the pool of both central memory (CD44hi/CD62Lhi) (BCG versus BCG-Acr1L: 18% versus 31% and 28 versus 35%, when challenged in vitro with PPD and Acr1L, respectively) and effector memory (CD44hi/CD62Llo) (BCG versus BCG-Acr1L: 10 versus 16% and 10 versus 15%, when challenged in vitro with PPD and Acr1L, respectively) CD4 T cells (Fig. 3a). Importantly, in-vitro challenge of the cells with Acr1L showed better expansion of central memory pool of CD4 T cells than PPD. A similar trend was apparent in the case of effector memory CD8 T cells (Fig. 3b). Further, the cells isolated from the lungs showed a sizeable increase in the total number of central memory CD4 and CD8 T cells expressing CD44hi/CD62Lhi (Fig. 3c). These findings suggest that BCG-Acr1L can effectively evoke the generation of enduring memory CD4 and CD8 T cells.

Figure 3.

Figure 3

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Vaccination with bacillus Calmette–Guérin (BCG)-liposomized alpha-crystalline protein 1 (Acr1L) induces enduring memory CD4 and CD8 T cells. Lymphocytes were obtained from BCG-Acr1L-administered mice, which were challenged with Mycobacterium tuberculosis (Mtb). The lymphocytes were isolated from spleens and lymph nodes and cultured with purified protein derivative (PPD) and Acr1L. After 48 h, cells were harvested, stained for the expression of memory markers CD44 and CD62L and analysed by flow cytometry on (a) CD4 T cells and (b) CD8 T cells. (c) Lymphocytes isolated from the lungs were stimulated with PPD and stained for memory T cell markers CD44 and CD62L. Numbers in the inset indicate percentage of cells expressing CD44hi/CD62Lhi. Data are representative of two independent experiments with six to seven mice in each group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Immunization with BCG-Acr1L augments lung immunity

The adaptive immune response to Mtb is initiated in the draining lymph nodes and effector T cells migrate subsequently to the site of infection 34–36. The adaptive immunity in the lungs plays a decisive role in imparting protection against Mtb. CD4 T cells isolated from the lungs of mice immunized with BCG-Acr1L exhibited substantial (P < 0·01) release of IFN-γ (Fig. 4a). In contrast, a significant (P < 0·05) decrease in the secretion of IL-4 was observed (Fig. 4b) and an increase in the frequency of polyfunctional [IFN-γhi/tumour necrosis factor (TNF)-αhi] CD4 (Fig. 4c,e) and CD8 T (Fig. 4d,f) cells was noted in the lungs (Fig. 4c,d) and spleen cells (Fig. 4e,f) of BCG-Acr1L-vaccinated mice. Furthermore, TNF-αhi expressing CD4 and CD8 T cells were seen to predominate over IFN-γ+ cells (Fig. 4c,d). Little difference was observed in the control groups of animals immunized with placebo (PBS), Acr1 alone or BCG. These results (Fig. 4a–f) demonstrate the predominance of multi-functional (IFN-γhi/TNF-αhi) CD4 and CD8 T cells in both the lungs and spleen in the BCG-Acr1-immunized mice. Both IFN-γ and TNF-α are considered to play potent roles in conferring immunity against Mtb.

Figure 4.

Figure 4

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Bacillus Calmette–Guérin (BCG)-liposomized alpha-crystalline protein 1 (Acr1L) vaccination induces multi-functional T helper type 1 (Th1) cells. Lymphocytes were isolated from the lungs of animals immunized with BCG-Acr1L, BCG-Acr1, Acr1L, Acr1, BCG and phosphate-buffered saline (PBS), which were later challenged with Mycobacterium tuberculosis (Mtb). The cells were cultured with purified protein derivative (PPD) for 48 h. Later, supernatants (SNs) were harvested and enzyme-linked immunosorbent assay (ELISA) for the estimation of (a) interferon (IFN)-γ and (b) interleukin (IL)-4. Data expressed as pg/ml in bar diagrams were analysed with the Tukey–Kramer test. **P < 0·01; ***P < 0·001. Intracellular expression of tumour necrosis factor (TNF)-α and IFN-γ was monitored by flow cytometry in (c,e) CD4 T cells and (d,f) CD8 T cells in (c,d) lung cells; (e,f) splenocytes. Figures in the contour plots indicate percentage of IFN-γ and TNF-α-expressing T cells. The results are representative of two independent experiments with six to eight mice per group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

BCG-Acr1L vaccination significantly protects mice from Mtb

The ultimate protective efficacy of any vaccine against TB is established by enumerating the mycobacterial burden in the lungs of the vaccinated animals. Compared to BCG alone, BCG-Acr1L prime-boost vaccination resulted in a significant (P < 0·05) decline in the mycobacterial load in the lungs. Further, compared to placebo or Acr1, a considerably higher decline in CFU was perceived (P < 0·001) (Fig. 5a), and a substantial amelioration in the histopathological changes was noted in the lungs. This was evidenced by a reduction in the size and number of granulomas and less consolidated and comparatively normal alveolar structure of the lungs (Fig. 5b), thus establishing the potent role of BCG-AcrL1 in eliciting protection against TB.

Figure 5.

Figure 5

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Bacillus Calmette–Guérin (BCG)-liposomized alpha-crystalline protein 1 (Acr1L) provides significantly better protection than BCG. Mice (six to eight per group) were immunized with BCG-Acr1L, BCG-Acr1, Acr1L, Acr1, BCG and placebo, which were later aerosol-challenged with Mycobacterium tuberculosis (Mtb). After 35 days, animals were killed. (a) Mtb load is represented as mean ± standard error of log10 colony-forming units (CFU)/g of the lung. Statistical analysis was performed by Student–Newman–Keuls multiple-comparisons post-test to compare the significance between the two groups. *P < 0·05; ***P < 0·001. (b) Lungs were fixed in formalin and sections were stained with haematoxylin and eosin. Photomicrographs (×20) display the lung sections. Arrows indicate small or large developing follicular granulomas.

Discussion

The failure to protect adulthood TB signifies the inability of BCG to elicit enduring memory T cells and therefore long-lasting immunity 1. Recently, BCG vaccination has been demonstrated to evoke a weak central memory T cell response, which is hypothesized as a fundamental basis for the failure of the BCG vaccine in humans 37. In addition to this, BCG is also incapable of providing sterilizing immunity against primary Mtb infection due to an inadequate immune response against latency-associated antigens 8. Latency-associated antigens have been projected to be potential candidates for vaccine development against TB 4,9,38. Acr1 protein of Mtb is one of the most immunogenic antigens, which is expressed predominantly at the time of latency 10. We recently reported that this protein inhibits the maturation and differentiation of immature dendritic cells (DCs) by inducing a tolerogenic phenotype 39. Once DCs mature, this protein activates the DCs and induces the release of potent proinflammatory cytokines (unpublished data). Thus, introduction of a prime-boost vaccination strategy using BCG and Acr1 could emerge as a successful vaccination approach. A DNA-based booster vaccine expressing 16 kDa has been tested effectively 40 but, considering the clinical relevance and ethical issues of DNA immunization, we evaluated a protein-based booster approach. We observed Acr1 to be ineffective in evoking a T cell response or secretion of IFN-γ (Fig. 2). However, liposomes are known to assist the immunogenic potential of antigens 24–27. Hence, we encapsulated Acr1 in the fusogenic liposomes prepared from yeast lipids. There is a distinct advantage of making liposomes from fusogenic lipids, as they can induce and enhance the generation of both CD4 and CD8 T cells. Consequently, we used liposomes prepared from fusogenic lipids to entrap Acr1 (Acr1L). In a prime-boost regimen, Acr1L significantly bolstered the vaccination potential of BCG.

Besides the importance of CD4 T cells, a recent study also demonstrated a major role for CD8 T cells in anti-TB immunity 23, indicating that CD8 T cells should be included in strategies for the development of new TB vaccines. Antigen-entrapped liposomes have been used to generate antigen-specific memory T cells 24,41; hence, in the current study, mice were primed with BCG and boosted with Acr1L. Further, to study the bona fide long-term memory T cells, mice were rested for 160 days after vaccination before being challenged with Mtb. The major findings arising from this study were that BCG-Acr1L vaccination augmented: (i) the generation of both CD4 and CD8 T cells; (ii) the pool of mainly Th1 cells; (iii) the frequency of multi-functional (IFN-γ+/TNF-α+) CD4 and CD8 T cells; (iv) the proportion of enduring memory CD4 and CD8 T cells; (v) robust immunity in the lung cells; and (vi) clearance of the mycobacterial burden in the lungs and reduced pathology.

One of the fundamental features of a successful vaccine is its ability to elicit long-lasting T cell memory. We have demonstrated that vaccination with BCG-Acr1L induces a better memory T cells response than BCG alone, both in the lungs and spleen. The memory T cell response generated was bona fide, as evidenced by the fact that mice prime-boosted with BCG-Acr1L were rested for 160 days before the memory response was monitored. BCG-Acr1L generated a vast pool of effector and central memory T cells in CD4 and CD8 T cells. We also observed that Mtb-specific T cells were principally of the Th1 phenotype, as seen by the predominant production of IFN-γ. To further validate the generation of the Th1 response, we also monitored the levels of IgG1 and IgG2a isotypes. A predominant secretion of Mtb-specific IgG2a was noted in the mice vaccinated with BCG-Acr1L, a hallmark of the Th1 phenotype 42. The CFU and pathology data further supported the protective potential of BCG-Acr1L vaccination. Reports in the literature indicate that predominance of the Th2 response is detrimental in achieving protection against TB. Thus, to gain protective immunity against TB, it is not only essential to have a predominant Th1 response but also a limited Th2 response 43. We observed a significant decrease in IL-4 secretion in BCG-Acr1L-vaccinated animals compared to BCG alone. However, a decline in the Th2 response could alleviate pathology during Mtb infection 44,45, but histological analysis of infected lungs in animals immunized with BCG-Acr1L showed minimal consolidation and infiltration with a comparatively normal alveolar structure.

BCG vaccine alone was unable to induce optimal CD8 T cells necessary for the clearance of Mtb 46,47. In contrast, vaccination with BCG-Acr1L results in an enhanced pool of multi-functional (IFN-γ+/TNF-α+) CD4 and CD8 T cells in both lungs and secondary lymphoid organs. Recent reports have shown that T cells producing multiple cytokines, such as the concomitant release of IFN-γ and TNF-α, are functionally superior to their single-positive counterparts 48. We observed a markedly higher frequency of Acr1-specific multi-functional CD4 and CD8 T cells in BCG-Acr1L-immunized animals. Multi-functional T cells form a reservoir of effector and central memory CD4 and CD8 T cells and therefore may be mediating an efficient and sustained protection against Mtb.

We found that BCG-Acr1L was more efficient than BCG in reducing the bacterial burden in the lungs even after 160 days of immunization. This establishes an important role for BCG-Acr1L vaccination in imparting enduring protective memory CD4 and CD8 T cells response against Mtb. Our approach of encapsulating Acr1 antigen of Mtb in fusogenic liposomes prepared from yeast lipids bolstered the induction and enhancement of memory CD4 and CD8 T cells. Currently, it is difficult to elucidate precisely the mechanism involved in enhancing the memory T cell response by BCG-Acr1L. However, it is known that liposomes boost the formation of memory T cells by releasing memory-enhancing cytokines IL-1, IL-6, IL-7 and IL-15 49. Moreover, it is important to mention here that this strategy is beneficial in reinvigorating the potency of the BCG vaccine in enhancing long-lasting immunity, because it is known that BCG fails to generate long-lasting immunity, as shown by the fact that it can protect only children, but not adults, from TB 1–3.

In conclusion, a prime-boost regimen employing BCG and Acr1 entrapped in fusogenic liposomes has overcome the snags associated with BCG failure to generate enduring memory T cell immunity. This is evidenced by a significant improvement in inducing the generation of the long-lasting protective efficacy of BCG by BCG-Acr1L immunization. Therefore, vaccination with BCG-Acr1 may be an important future strategy to reinvigorate BCG efficacy as a vaccine to control TB.

Acknowledgments

The authors are thankful to Dr Manoj Raje and Mr Anil Theophillus for electron microscopy and Council of Scientific and Industrial Research and Department of Biotechnology, India for financial support. K. F. S., M. A. and N. K. are the recipients of fellowship of the Department of Biotechnology and G. R. K. and J. A. S. of the Council of Scientific and Industrial Research, India.

Disclosure

The authors declare that they have no conflicts of interest.

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