Enhanced Immune Responses Against Mycobacterium tuberculosis Through Heat-Killed BCG with Squalene-in-water Emulsion Adjuvant

Abstract

The increasing challenge of drug-resistant tuberculosis (TB) calls for the development of innovative therapeutic strategies, highlighting the potential of adjunctive immunotherapies that are both cost-effective and safe. Host-directed therapy (HDT) using immunomodulators shows promise in enhancing treatment efficacy by modulating immune responses, thereby shortening the duration of therapy and reducing drug resistance risks. This study investigated the immunomodulatory potential of combining Heat-killed Bacillus Calmette-Guérin (hBCG) with a Squalene-based oil-in-Water Emulsion (SWE) adjuvant against TB. The therapeutic efficacy of the hBCG-SWE regimen was assessed in a guinea pig model infected with Mycobacterium tuberculosis (M. tb). Furthermore, the impact of hBCG-SWE on TNF-α and MCP-1 production was evaluated in RAW264.7 macrophages, examining the role of TLR2/4 and MyD88 signaling pathways using ELISA, both with and without specific inhibitors. Our findings revealed that hBCG-SWE significantly enhanced TNF-α and MCP-1 production compared to hBCG alone, indicating activation through TLR2/4 and MyD88-dependent pathways. In guinea pigs, hBCG-SWE administration led to notable reductions in lung pathology and spleen bacterial loads versus control groups. These results highlight the capacity of hBCG-SWE to boost innate immunity and provide robust protection against M. tb. Future research should focus on evaluating the ability of hBCG-SWE to shorten conventional chemotherapy and exploring ways to amplify its immunomodulatory efficacy through advanced formulation techniques.

Introduction

Tuberculosis (TB) is the foremost cause of death from infectious diseases and continues to pose a significant global health challenge. The pathogenesis and progression of TB are closely associated with immunodeficiency and an imbalance in the Th1/Th2 immune response, complicating the treatment and management of the disease [1, 2]. Conventional chemotherapy for TB requires prolonged treatments to effectively reduce Mycobacterium tuberculosis (M. tb) in lesions, often extending to several months. This duration extends further for drug-resistant strains, necessitating additional medications [3]. Immunoadjuvant therapy using immunomodulators shows promise in preventing latent M. tb reactivation and managing active TB by enhancing immune function, reducing harmful immune responses, and improving treatment outcomes [4, 5]. Various immunomodulators for TB, including immune-active substances, immunotherapeutic vaccines, chemical agents, traditional Chinese medicine, and cell therapy, are being explored in clinical trials and practice [5]. The Bacillus Calmette-Guérin (BCG) vaccine has been recognized for providing nonspecific protection against reinfections through trained immunity or innate immune memory, independently of T and B lymphocytes, and inducing heterologous Th1/Th17 responses [6, 7]. The role of BCG as an immune modulator has also been acknowledged, leading to its development as an immunoadjuvant for cancer immunotherapy, notably in bladder cancer [8–11]. Furthermore, the BCG cell wall skeleton, emulsified into water-dispersed nanoparticles, can serve as a systemic adjuvant for cancer immunotherapy [12]. The TB immunotherapeutic vaccine shows potential, aiming to modulate the immune system, restore balance, and bolster immunity against M. tb [13, 14]. However, the potential of BCG as an immunomodulatory therapy for TB, aside from its vaccine use, remains underexplored.

The utilization of heat-killed BCG (hBCG) has gained attention for inducing BCG-responsive biological endpoints while avoiding the infection risks associated with live BCG strains [15]. Although hBCG alone provides limited protection against M. tb infection, when combined with Eurocine L3 adjuvant, it offers protection that is comparable or superior to live BCG [16, 17]. The hBCG-and Mycobacterium kansasii Ag85B vaccine combination has also shown efficacy in reducing atopic dermatitis skin lesions in mouse models [18]. Moreover, M. vaccae immunotherapy has demonstrated effectiveness in treating pulmonary TB [19–21], suggesting the potential of hBCG combined with adjuvants as TB immunomodulators.

Adjuvants enhance the strength and longevity of adaptive immunity. The Squalene-based oil-in-Water Emulsion (SWE) adjuvant, MF59, used in influenza vaccines, has been administered globally to over 100 million people in over 30 countries [22, 23]. MF59 has been shown to boost immune responses across a broad spectrum of antigens [24]. The A1D4 fusion protein, containing five M. tb antigens emulsified in MTO adjuvant (incorporating MF59), has shown greater protection against M. tb infection than controls or MTO alone, although it is less effective than BCG [25]. Our previous research indicated that hBCG-SWE enhances the immunogenicity of M. tb antigens and augments immune cell recruitment at the injection site [26]. However, the anti-TB efficacy of hBCG-SWE and its underlying protective immune response mechanisms are yet to be fully elucidated.

This study aims to investigate how hBCG-SWE modulates host immune responses in macrophages and assess its protective efficacy in a guinea pig model infected with M. tb, offering insights into novel TB immune regulatory treatments.

Materials and Methods

Animals and Ethics

Female guinea pigs, weighing 350 ± 50 g, were used as the animal model, following previous research protocols [27]. These animals were sourced from the Institute of Laboratory Animal Resources and the National Institutes for Food and Drug Control (Beijing, China), and maintained in an Animal Biosafety Level (ABSL)-III laboratory as previously described [28]. The study received approval from the Laboratory Animal Welfare and Ethics Committee of the National Institutes for Food and Drug Control (NIFDC), with the animal approval code 201505. All guinea pigs had unrestricted access to tap water and standard rodent chow.

Bacteria and Cell Line

Mycobacterium bovis BCG vaccine (Shanghai D2PB302 strain) was procured from the Chengdu Institute of Biological Products, Chengdu, China. The virulent M. tb strain CMCC(B)95052 was generously provided by the Division of Tuberculosis Vaccine and Allergen Products of the National Institutes for Food and Drug Control, Beijing, China, and stored at -80˚C in our laboratory. RAW264.7 cells, a mouse mononuclear macrophage leukemia cell line, were acquired from the National Biomedical Experimental Cell Resource Library at the Institute of Basic Medicine, Chinese Academy of Medical Science, Beijing, China. These cells were cultivated in DMEM medium (C11885500BT, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (10099-141C, Gibco, USA).

SWE and hBCG Preparation

The prepared adjuvant buffer was composed of 4.3% squalene (S3626, Sigma-Aldrich, USA), 0.5% Tween-80 (P1754, Sigma-Aldrich, USA), 0.5% Span-85 (S7135, Sigma-Aldrich, USA), and 10 mmol/L citrate buffer (S4641, Sigma-Aldrich, USA). It underwent homogenization at 1500–1700 Bar in a microfluidizer (JN-30C, Guangzhou Juneng Biology Technology Co., Ltd, China) and was subsequently filtered using a 0.22 µm filter (SLGPR33RB, Millipore, Sigma-Aldrich, USA). The average particle size of the SWE droplets, determined by a Mastersizer 3000 (Malvern Instruments, UK), was 187 ± 68 nm. The BCG vaccine strain was cultured on Löwenstein-Jensen (LJ) agar for 3–4 weeks at 37 °C, followed by growth in potato Sauton’s medium at 37 °C for 2–3 weeks [29]. The bacteria were then harvested by centrifugation for 5 min at 6000 rpm, washed three times with saline, and eventually weighed. The hBCG was prepared by heating the collected BCG at 85 °C for one hour and stored at − 80 °C in aliquots.

Cell Culture and TNF-α and MCP-1 Detection

RAW264.7 cells, obtained from the National Biomedical Experimental Cell Resource Library, Chinese Academy of Medical Science (Beijing, China), were cultured in DMEM (C11885500BT, Gibco, USA) supplemented with 10% fetal bovine serum (10099-141C, Gibco, USA), 100 g/ml streptomycin, and 100 IU/ml penicillin (15140122, Gibco, USA). Cells were seeded in 24-well plates (3738, Corning, USA) at 250,000 cells/well and incubated for 2 h at 37 °C in a 5% CO₂ atmosphere. They were then treated with SWE (1:100 dilution), varying doses of hBCG, or their combinations. Low and high doses of hBCG, 50 µg/ml and 250 µg/ml respectively, were designated as hBCG-L and hBCG-H. For pathway inhibition assays, cells were pre-treated with 50 µM MyD88 inhibitor (Pepinh-MYD, InvivoGen, USA) [30], 30 µg/ml TLR2/4 inhibitor (OxPAPC, InvivoGen, USA) [31], or 2 µg/ml STAT1 inhibitor (Fludarabine, S1491, Selleck, USA) [32] for 6 h before adding hBCG-H-SWE. Supernatants were collected and stored at − 80 °C for subsequent analysis. Each assay was conducted in triplicate and repeated at least three times. TNF-α and MCP-1/CCL2 levels in the culture supernatants were quantified using OptEIA™ mouse TNF-α (560478, BD Bioscience, USA) and MCP-1 ELISA kits (555260, BD Bioscience, USA), following the manufacturer’s protocol.

Guinea Pig Infection and Immunization

Guinea pigs were initially challenged subcutaneously with 0.5 ml of M. tb (~ 2 × 10³ CFU). These animals were subsequently immunized subcutaneously with 0.5 ml hBCG-SWE or PBS as a negative control on the third day post-infection, following the protocol described previously [33]. Additional immunizations were administered subcutaneously once after one week (10 days), and twice at two-week intervals (24 and 38 days). Fourteen days after the last immunization, all guinea pigs were euthanized. Post-mortem, spleens and lungs were assessed using a lesion index based on established criteria (Table 1) [28]. Half of each spleen was homogenized in 3 ml sterile normal saline containing 0.05% Tween-80 (v/v) (P1754, Sigma-Aldrich, USA), serially diluted, and the selected dilutions were cultured on Lowenstein-Jensen medium at 37 °C for four weeks. Colony-forming units (CFUs) were counted as previously described [28]

Table 1.

Criterion for the gross pathological score of the spleen and lung

Degree of lesion	Spleen	Lung
None	0	0
Mild	10	10
Moderate	20	20
Severe	35	30

Statistical analysis

Statistical analyses were conducted using GraphPad Prism 7 software (version 7.0, GraphPad Software Inc., USA) and SPSS 18.0 (SPSS Inc., Chicago, USA). The data were analyzed using one-way ANOVA or Student’s t-test, as appropriate, to determine the differences between the groups. A p-value of less than 0.05 was considered statistically significant.

Results

The SWE Enhances hBCG-Induced TNF-α and MCP-1 Production in Macrophages

The SWE adjuvant was found to enhance protection against viruses and bacteria by increasing the immune response of the adjuvanted vaccine [34]. To assess if SWE amplified the hBCG-mediated immune response, we examined the production of the inflammatory cytokines TNF-α and MCP-1 with the treatment of SWE, hBCG, and hBCG-SWE in RAW264.7 macrophages. The findings indicated that TNF-α and MCP-1 production was significantly induced with hBCG treatment in a dose- and time-dependent manner but was not triggered by SWE alone in RAW264.7 macrophages (Fig. 1A, B). Notably, the hBCG-H-SWE treatment further significantly enhanced TNF-α production in macrophages compared with the hBCG treatment group (Fig. 1A). A remarkably increased MCP-1 expression was also triggered in macrophages by both hBCG and hBCG-H-SWE treatment compared with the SWE treatment or blank control group. Additionally, a slightly enhanced MCP-1 production pattern was observed in the hBCG-H-SWE treated macrophages compared with that of the hBCG group, although the difference was not statistically significant (Fig. 1B). These data suggest that the combination of hBCG and SWE can more robustly trigger the host anti-M. tb immune response by enhancing TNF-α and MCP-1 production in macrophages, crucial for intracellular and in vivo bacillus clearance.

Fig. 1.

TNF-a and MCP-1 production in RAW264.7 cells induced by SWE, hBCG and hBCG-SWE. RAW264.7 cells (2.5 × 105/well) were treated with SWE (1:100, v/v), hBCG-L (50 µg/ml), hBCG-H (250 µg/ml, final concentration) or hBCG-H-SWE for different times. The levels of TNF-α (A) and MCP-1 (B) from the culture supernatant were measured by ELISA at the indicated times. The data represent means ± SEM and are representative of results from at least four independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001, ****: P < 0.0001

hBCG-SWE Induced TNF-α and MCP-1 Production Through a TLR2/4 and MyD88 Dependent Pathway

To identify the signaling pathway responsible for TNF-α and MCP-1 production induced by hBCG-SWE, we examined the production of these cytokines in the presence of TLR2/TLR4, MyD88, or SATA1 inhibitor in RAW264.7 cells. The TLR2/TLR4 inhibitor OxPAPC significantly reduced the TNF-α and MCP-1 production induced by hBCG-H-SWE (Fig. 2A, B). The decrease in TNF-α and MCP-1 levels was more significant in the presence of MyD88 inhibitor Pepinh-MYD (Fig. 2A, B). Conversely, the SATA1 inhibitor Fludarabine did not affect TNF-α and MCP-1 production by hBCG-H-SWE (Fig. 2C, D). The most notable inhibitory effect was seen with the combined use of Pepinh-MYD and OxPAPC (Fig. 2A, B). These results indicate that TNF-α and MCP-1 induction by hBCG-SWE in RAW264.7 macrophages is significantly tied to the TLR2-Myd88 signaling pathways.

Fig. 2.

The signaling pathway involved in hBCG-SWE induced TNF-α and MCP-1 production was investigated in RAW264.7 cells. The levels of TNF-α (A, C) and MCP-1 (B, D) in the culture supernatant were quantified at the present of MyD88 inhibitor (pepinh-MYD, 50 μM), TLR4 inhibitor (OxPAPC, 30 μg/mL), STAT1 inhibitor (Fludarabine, 2 μg/mL), or their combinations for 6 h. The presented data shows the mean ± SEM and is based on at least four independent experiments, ensuring reliable and representative results. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ND: not detected

The hBCG-SWE Induced Protection Against M. tb Infection

Owing to its similarity to human TB, the guinea pig model [35] was employed to assess the protective efficacy of hBCG-SWE against M. tb infection. Guinea pigs administered subcutaneously with hBCG-H-SWE or PBS post-M. tb infection showed that hBCG-H-SWE treated animals had significantly less pathological damage in the lungs and spleens compared to the PBS group (Fig. 3A). The pathological score average of the PBS group was nearly threefold higher compared to the hBCG-H-SWE-administered guinea pigs (Fig. 3B, C). Furthermore, a significantly lower bacterial burden was observed in the hBCG-H-SWE treated guinea pigs (3.814 ± 0.594) compared to the PBS group (5.378 ± 0.488) (Fig. 3D). Additionally, spleens were analyzed for gross pathological damage, following prior criteria [12], revealing a significant correlation between the spleen lesion index and bacterial counts (Spearman r = 0.7448, p = 0.0014). These findings suggest that hBCG-SWE treatment can provide effective protection against M. tb infection.

Fig. 3.

The protective efficacy of hBCG-SWE against M. tb was assessed in vivo. (A) The degree of pathological lesions in the lung and spleen were assessed and used to define gross pathological scores in guinea pigs post-treated with hBCG-H-SWE or PBS. The protective efficacy was further assessed by measuring the gross pathological scores (B, C) and the M. tb loads in spleens (D) of the guinea pigs. The presented data is based on the mean ± SEM obtained from at least eight independent guinea pigs, ensuring reliable and representative results. ***P < 0.001

Discussion

In this study, we established that hBCG-SWE treatment markedly ameliorated pathological damage in the lungs and spleens of M. tb infected guinea pigs. Our findings reveal that the combined use of hBCG and SWE can synergistically initiate an innate immune response, notably enhancing the production of TNF-α and MCP-1 through MyD88-dependent mechanisms, or partially through MyD88-dependent TLR2/4 signaling pathways (Fig. 4). Additionally, our previous results indicated that SWE treatment could recruit macrophages to the injection site [26], supporting the synergistic interaction between SWE and hBCG in creating a local immuno-stimulating environment, and reinforcing the potential of hBCG-SWE as a modulator for innate immunity.

Fig. 4.

The model showing how hBCG-SWE enhances host immune responses against M. tb infection

Macrophages play a critical role in anti-TB immunity by generating pro-inflammatory mediators such as TNF-α and MCP-1 [36, 37]. Previous studies demonstrated that BCG-induced MCP-1/CCL2 secretion in whole blood cells from pulmonary TB patients peaked at both 18 h and 48 h post-stimulation [38]. Our study found that SWE significantly enhanced hBCG-stimulated TNF-α and MCP-1 production in macrophages. MCP-1 secretion, induced by hBCG-SWE, peaked at 48 h post-addition, slightly later than TNF-α secretion, corroborating the notion that TNF-α-activated MCP-1/CCL2 is crucial for monocyte and T cell recruitment to mycobacterial infection sites, potentially leading to a more vigorous cellular response [38]. The necessity of TNF-α for TB infection control is well-documented, with increased susceptibility observed in patients receiving anti-TNF-α treatment or in mice lacking TNF-α receptors [39, 40]. Nonetheless, excessive TNF-α may facilitate M. tb dissemination through increased mitochondrial membrane permeability and macrophage necroptosis [20]. Our study noted that hBCG-SWE-stimulated TNF-α production peaked at 16 h post-addition, persisting up to 48 h after treatment. Given that the TNF family member 4-1BB ligand (4-1BBL) can sustain TNF-α production by interacting with TLR signaling components during late-phase signaling [41], SWE might influence late-phase signaling in hBCG-induced TLR4-mediated inflammation. Although mitochondrial membrane permeability and macrophage necroptosis were not directly assessed, the observed reduction in pathological damage post-hBCG-SWE treatment suggests that hBCG may not induce macrophage necrosis. Moreover, elevated TNF-α and MCP-1 levels have been documented in the chronic stage compared to the persistent stage or healthy groups in mouse models [42], highlighting the synergistic effect of SWE and hBCG in activating macrophages to secrete TNF-α and MCP-1, critical for countering M. tb growth.

Among the Toll-like receptor (TLR) family, TLR2, TLR4, and TLR9, along with the adaptor molecule MyD88, play critical roles in initiating immune responses against TB [43]. Mycobacterial antigens can activate TLR signaling, leading to pro-inflammatory cytokine production in macrophages and dendritic cells via MAPK and NF-κB pathways [44]. Our earlier findings established that TNF-α upregulation involved NF-κB/RelA translocation, facilitated by binding to the TNF-α gene promoter in a TLR2-dependent manner [45]. MyD88 signaling, essential for adequate innate and acquired immune responses to M. tb, promotes bacterial containment [46]. Hence, simultaneous TLR and MyD88 signaling activation could enhance the anti-TB immune response. While SWE alone does not activate TLRs, its adjuvant effect necessitates MyD88 function [47]. Our study demonstrated that hBCG-SWE-induced TNF-α and MCP-1 production relies on the TLR2/4-MyD88 signaling pathway, suggesting that combining hBCG with SWE might activate TLR2/4 and MyD88, enhancing the immune response against M. tb.

Notably, hBCG has emerged as a vital immunomodulator for various diseases, including COVID-19 [48]. BCG vaccination has been shown to induce epigenetic reprogramming of monocytes in healthy volunteers, leading to increased cytokine production in response to non-specific pathogen infections and certain malignancies like bladder cancer [7, 49]. Yet, it remains uncertain whether BCG immunotherapy can serve as an intervention in individuals pre-immunized with the BCG vaccine but later infected with M. tb. Previous research indicated that hBCG vaccines, when combined with the Eurocin L3 adjuvant, provide enhanced protection against TB, surpassing the efficacy of hBCG alone [16]. Consistently, our study demonstrated that hBCG combined with SWE adjuvant significantly mitigated spleen pathological damage in M. tb-infected guinea pigs. This aligns with earlier findings in guinea pigs, where splenic tissues exhibited greater pathology and CFUs compared to lung tissues in M. tb-infected animals [50, 51].

Additionally, heat-inactivated vaccines such as hBCG offer a safer alternative to live BCG, reducing infection risks in immunocompromised individuals [52]. Thus, hBCG-SWE emerges as a promising candidate for TB immunoadjuvant therapy. With BCG known to induce trained immunity [53], further research is needed to determine if the protective effects of hBCG and SWE are mediated through BCG-induced trained immunity. Moreover, various therapeutic vaccines, including inactivated, subunit, and DNA vaccines, are being explored in China for TB treatment [54–57], with inactivated therapeutic vaccines showing the most promise [13]. Our study underscores the potential of inactivated BCG with potent adjuvants in enhancing immune regulatory functions, representing a promising approach for therapeutic inactivated TB vaccines.

Future research should focus on determining whether hBCG-SWE can reduce the duration of chemotherapeutic regimens and evaluating its effectiveness in real-world settings where treatment may be incomplete or interrupted. Additionally, enhancing the immunomodulatory effect of hBCG-SWE through advanced preparation methods, such as nanoparticle formulations [58], will be crucial in the ongoing development of effective TB therapeutic vaccines.

Author Contributions

XYH and JLL designed the experiments. CZZ, JLL, QDS, and XRL performed the experiments. CZZ and JLL analyzed the data and prepared the figures. CZZ and JLL wrote the manuscript.

Funding

This work was supported by a grant from Natural Science Foundation of Beijing Municipality (7172212), and the Beijing Hospitals Authority Innovation Studio of Young Staff Funding (202136).

Data Availability

All data supporting the conclusions of the current study are available from the corresponding author on reasonable request.

Declarations

Ethics Approval and Consent to Participate

All the animal experiments were approved by the Laboratory Animal Welfare & Ethics Committee of National Institutes for Food and Drug Control (NIFDC) and the approval code is 201505.

Consent for Publication

Not applicable.

Conflict of interest

All authors declare that they have no competing interests.