ESAT-6-derived lipopeptide-based mucosal vaccine elicits distinct humoral immune responses concomitant with a reduction in mycobacterium infection

ABSTRACT

Respiratory mycobacterial infections remain a major global health threat, yet current vaccine strategies, which focus primarily on cell-mediated immunity, have met with limited success. We previously demonstrated that a vaccine composed of five lipopeptides (LPs) encoding immunodominant ESAT-6 epitopes elicits protective cellular immune responses against Mycobacterium tuberculosis infection. In this study, we enhanced the ESAT-LPs vaccine with a novel mucosal immune modulator, heat-killed Caulobacter crescentus (HKCC), and examined both cellular and humoral immunity in a mouse model (BALB/c) of M. avium (Mav) infection. Our findings revealed that incorporating HKCC significantly increased cellular responses and mucosal IgA and systemic IgG titres in pre- and post-exposure models, respectively. These elevated antibody levels correlated with reduced mycobacterial burden in the lungs, spleens, and livers and with improved lung pathology. Notably, serum samples from vaccinated mice markedly reduced the intracellular Mav burden and activated antimicrobial mechanisms in an ex vivo bone marrow-derived macrophage infection model. Heat-inactivated serum samples also reduced intracellular Mav burden, suggesting a complement-independent intracellular bactericidal function of the tested serum samples. Collectively, the ESAT-LPs-HKCC mucosal vaccine induces a robust, distinct humoral response and a remarkable reduction in bacterial load in both prophylactic and therapeutic settings, offering a promising new strategy for combating mycobacterial infections.

Introduction

Mycobacterial infections pose a considerable health threat globally. Mycobacterium tuberculosis (Mtb) is a highly pathogenic member of the genus Mycobacteria and is responsible for tuberculosis (TB), one of the leading causes of death worldwide. Current estimates suggest that about a quarter of the world's population is infected with TB [1]. Additionally, the prevalence of infectious disease caused by opportunistic nontuberculous mycobacteria (NTM) of the Mycobacterium avium complex (MAC), including M. avium (Mav) and M. intracellulare, has increased worldwide. These bacteria are environmental microorganisms commonly found in soil, household dust, and water sources globally [2]. Members of MAC often cause severe respiratory and disseminated infections, particularly in immunocompromised groups such as those with AIDS, organ transplant recipients, and pre-existing lung conditions like bronchiectasis, cystic fibrosis, silicosis, previous TB, or chronic obstructive pulmonary disease (COPD), as well as the elderly [3]. These two main species of MAC account for approximately 80% of pulmonary diseases caused by NTM in different countries [4].

The BCG (Bacillus Calmette-Guérin) vaccine was developed nearly a century ago and remains the only licensed vaccine for preventing Mtb. BCG offers promising protection in children but not in adults [1]. However, it provides partial immunity to NTM infections and is not currently indicated for use against NTM infections globally [5]. Additionally, relying solely on preventive vaccines for serious non-pandemic infectious diseases, such as mycobacterial infections, is impractical due to the logistical challenges of large-scale population vaccination, costs, vaccine hesitancy, and lack of therapeutic value. Anti-mycobacterial drugs face issues with inconsistent adherence to long-term regimens that are hard to tolerate, resulting in antibiotic resistance and complicating treatment options. Therefore, broadly protective vaccines effective in both preventive and therapeutic regimens are crucial for reducing transmission and shortening treatment duration in individuals with active mycobacterial diseases.

The role of the BCG vaccine in inducing cellular immune responses, heterologous immunity, trained immunity, and adaptive/innate memory responses has been extensively studied [6]. BCG-induced immune responses are primarily cellular [7]. Many experimental vaccines that focus solely on cell-mediated protection are in the pipeline, but they have not yet demonstrated significant efficacy [8]. Therefore, exploring and leveraging additional components of immunity may improve the overall effectiveness of vaccines and/or immunotherapy [9]. Humoral immunity is an underexplored area in TB vaccine development; the contribution of humoral responses to controlling mycobacterial infections is not well established and remains controversial. However, the significance of antibodies is increasingly recognized, even against intracellular pathogens. Distinct antibody profiles during the acute and chronic phases of infection may indicate their potential role in disease progression [10]. Importantly, antibodies can recognize, neutralize, and prevent the pathogen from entering different sites early in infection; present pathogen-specific antigens to immune cells; direct infected cells toward cytotoxicity; and activate FcR-mediated functions, among other roles [11]. Consequently, investigating vaccines or immunotherapies that elicit both cellular and humoral immune responses is a promising approach to addressing mycobacterial infections.

Early secretory antigenic target 6 kDa (ESAT-6) is an immunogenic antigen of Mtb that is absent in BCG. Cellular immunity, i.e. CD4⁺ and CD8⁺ T cell responses against ESAT-6, is considered crucial for an effective TB vaccine candidate [12,13]. ESAT-6 is a highly virulent antigen; its secretion helps create a mycobacterium-friendly environment in the host by disrupting innate macrophage function, polarizing macrophages toward a pro-bacterial state, and compromising ESAT-6-specific cellular protection, thereby contributing to immunopathology [14–16]. A fully functional ESAT-6 gene is found only in Mtb, whereas it is partially present in several other NTMs, including Mav [17]. We previously showed that a mixture of palmitoylated peptide epitopes from ESAT-6 (ESAT-LPs), combined with MPL, polyI:C, or gardiquimod as adjuvants, elicit effective cellular responses against Mtb infection [18,19].

An effective adjuvant system is crucial for improving vaccine efficacy, particularly by enhancing the quantity and quality of induced cellular and/or humoral responses [20,21]. In this context, we aimed to incorporate non-pathogenic heat-killed Caulobacter crescentus (HKCC) as a novel mucosal immune modulator to bolster the effect of the ESAT-LPs vaccine. In our earlier studies, we revealed HKCC as a promising booster for an LP-based vaccine against SARS-CoV-2 [22] and as an innate immune-directed immunotherapy for Mtb and Mav infections [23,24]. HKCC provided balanced stimulation of innate and adaptive responses at both systemic and mucosal sites. Building on these mechanistic foundations, the present study investigates the adjuvant effects of HKCC when combined with ESAT-LPs against Mav infection.

In this study, we have designed an intranasal vaccine for Mav infection that can be effective for both prophylactic and therapeutic use, incorporating HKCC with five ESAT-6 lipopeptides. Mucosal immunization of mice with ESAT-LPs and ESAT-LPs-HKCC elicited both cellular and humoral immune responses and significantly reduced mycobacterial loads in both prophylactic and therapeutic models. Most importantly, the inclusion of HKCC produced distinctive humoral response patterns that were significantly correlated with decreased bacterial burden. Native and heat-inactivated serum samples from immunized mice with high antibody titres against ESAT-6 reduced the intracellular bacterial burden in bone marrow-derived macrophages infected with Mav, suggesting that other serum components, including antibodies, activate intracellular mechanisms. Collectively, ESAT-LPs-HKCC emerged as a promising vaccine candidate capable of inducing both cellular and humoral responses for prophylactic and/or therapeutic strategies against mycobacterial infection.

Materials and methods

Peptide constructs and sequence alignment

Lipopeptides derived from ESAT-6 protein EsxA (Mycobacterium tuberculosis H37Rv), NCBI Reference Sequence: YP_178023.1 (LP1 – aa_1-15 P1 MTEQQWNFAGIEAAAK(palmitate)G, LP2 – aa_31-55 EGKQSLTKLAAAWGGSGSEAYQGVQK(palmitate)G, LP3 – aa_46-70 SGSEAYQGVQQKWDATATELNNALQK(palmitate)G, LP4 – aa_61-85 TATELNNALQNLARTISEAGQAMASK (palmitate)G, and LP5 – aa_76-95 ISEAGQAMASTEGNVTGMFAK (palmitate)G) were custom synthesized by Genscript Inc [18]. All lipopeptides were stored as 10 mg/mL stock solutions in DMSO at −20°C, and before each immunization, the stocks were diluted in phosphate-buffered saline (PBS). To evaluate sequence conservation between the designed ESAT-6 epitopes and Mycobacterium avium proteins, candidate Mav protein sequences were retrieved from the NCBI Protein database. Local pairwise alignments were initially performed using NCBI BLASTp against the M. avium reference proteome. To further assess alignment patterns and calculate exact sequence identity and property similarities, pairwise sequence alignments were performed using ClustalW (v2.1) with default parameters (BLOSUM matrix, open gap penalty: 10, gap extension penalty: 0.1). Pairwise comparisons are shown in Supplementary Table S1.

HKCC preparation

Caulobacter crescentus (ATCC) was cultured in PYE medium. Known amounts of log-phase colony-forming units (CFU) were centrifuged and resuspended in sterile PBS to prepare the stock. Stocks were aseptically stored at 4°C. The required CFU were heat-killed at 60-80°C for 1 h before each immunization, then centrifuged at 5000 rpm for 15 min, and resuspended in the appropriate volume of PBS.

Mouse immunizations

All mouse experiments were approved by the University of Alberta’s Animal Care and Use Committee for Health Sciences and conducted in accordance with the Canadian Council of Animal Care guidelines (AUP 000003746). Experiments used 4–6-week-old female BALB/c mice purchased from Charles River Laboratories. Mice were housed in the University of Alberta's housing facility. After a one-week acclimatization period, animals were randomly assigned to treatment groups and immunized as outlined in the study timelines (Figures 1, 2 and 4(a)). The LP mixtures (LP 1, 2, 3, 4, and 5) were prepared by adding 10 μg of each LP to a total of 30 μL of PBS per mouse (15 μL/nostril). For the ESAT-LPs-HKCC group, 50 × 10⁶ CFU of HKCC were combined with the LP mixture and administered intranasally two weeks apart under isoflurane gas anesthesia, following standard protocols and timelines.

Figure 1.

Induction of cellular and humoral responses following intranasal immunization with ESAT-LPs and ESAT-LPs-HKCC in uninfected mice. (a) Schematic timeline of the vaccine construct and sample collection. (b) Six- to eight-week-old female BALB/c mice (n = 3 per group) were immunized intranasally (i.n.) with ESAT-LPs (10 μg each) alone or combined with 50 × 10⁶ CFU of HKCC (total volume: 30 μL, 15 μL per nostril). Eight days after the second immunization, mice were euthanized, and samples (blood, lung lavage, spleen) were collected. Splenocytes were cocultured with irradiated antigen-presenting cells, with or without ex vivo restimulation with the respective LPs. The BrdU assay was used to quantify proliferation responses to individual lipopeptides (LP1-5); cumulative responses are shown. (c) Serum and (d) lung lavage samples were analyzed to detect systemic IgG and mucosal IgA titres, respectively, against individual lipopeptides (LP1-5) and ESAT whole protein. Three biological replicates were analyzed separately, and results are expressed as the mean ± SEM. Statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001) was determined using one-way ANOVA for ESAT whole protein ELISA analysis or two-way ANOVA for BrdU and LPs-specific ELISA analysis followed by Tukey’s test.

Figure 2.

Efficacy of prophylactic immunization with ESAT-LPs and ESAT-LPs-HKCC in Mav-infected mice. (a) Schematic timeline of prophylactic immunization, infection, and sample collection. (b) Six- to eight-week-old female BALB/c mice (n = 5 per group) were intranasally immunized with ESAT-LPs (10 μg each) alone or in combination with 50 × 10⁶ CFU of HKCC (total volume: 30 μL, 15 μL in each nostril) at two-week intervals. One week after the second immunization, mice were infected intravenously with Mycobacterium avium (Mav) (5 × 10⁵ CFU in 50 μL). Four weeks post-challenge, mice were euthanized, and samples (blood, lung lavage, lung, spleen, liver) were collected. Bacterial loads in lungs, spleens, and livers were quantified by CFU assays. Results are shown as the mean ± SEM of CFU counts from individual mice. (c) Representative hematoxylin and eosin (H&E)-stained lung tissue images. (d) Lung severity score for H&E-stained lung sections, assessed in a double-blind manner using criteria described in the methods section and Supplementary Table S2. (e) The BrdU assay was used to quantify proliferation responses to individual lipopeptides (LP1-5); cumulative responses are shown. Statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001) was determined by one-way ANOVA for CFU and severity score analysis and two-way ANOVA for BrdU analysis with Tukey’s test.

Figure 4.

Efficacy of ESAT-LPs with or without HKCC as a post-exposure immunotherapy in Mav-infected mice. (a) Schematic timeline of the therapeutic regimen for ESAT LP+/-HKCC immunization and sample collection. (b) Six- to eight-week-old female BALB/c mice (n = 5 per group) were infected intravenously with Mycobacterium avium (Mav) (5 × 10⁵ CFU in 50 μL). Two weeks after infection, mice were immunized intranasally with ESAT LPs (10 μg each) with or without 50 × 10⁶ CFU of HKCC (total volume: 30 μL, 15 μL per nostril). One week after the second immunization, mice were euthanized, and blood, lung lavage, lung, spleen, and liver samples were collected. Bacterial loads in lungs, spleens, and livers were measured by CFU assays. Results are presented as the mean ± SEM of CFU counts from individual mice. (c) Representative hematoxylin and eosin (H&E)-stained lung tissue images. (d) The lung severity score of H&E-stained lung sections was assessed in a double-blind manner based on the criteria described in the methods section and Supplementary Table S2. (e) The BrdU assay was used to quantify proliferation responses to individual lipopeptides (LP1-5); cumulative responses are shown. Statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001) was determined using one-way ANOVA for CFU and severity score analysis and two-way ANOVA for BrdU analysis followed by Tukey’s test.

Mycobacterium avium infection

Bacterial culture was prepared as described previously [24]. Briefly, Mav was cultured in 7H9 medium. A 50 μL aliquot containing 5 × 10⁵ CFU of log-phase Mav was centrifuged, resuspended in PBS, and then administered intravenously. For prophylaxis, the challenge occurred one week after the second immunization; in the immunotherapy group, immunization was initiated two weeks after infection. Mice were humanely euthanized (under CO₂) at the end of the desired time period, as indicated in the timelines (Figures 2 and 4(a)), and samples were collected for subsequent analysis.

Colony-forming unit assay

Bacterial load analysis was performed per biological replicate. Lungs, livers, and spleens from mice were harvested, placed in sterile PBS, and homogenized. Serial dilutions ranging from 10⁻¹ to 10⁻³ of these homogenates were cultured on 7H11 Middlebrook agar (Sigma-Aldrich) supplemented with Middlebrook ADC enrichment (BD) and incubated at 37°C for a period of 2 to 3 weeks. Bacterial loads were assessed by colony enumeration.

Histopathology analysis

Hematoxylin and eosin (H&E) staining was performed on randomly chosen mouse lung lobes from each group, following the reported protocol [24]. After being fixed in 10% formalin at room temperature (RT) for 2-3 days, the specimens were moved to 70% ethanol. Tissues were first embedded in paraffin, then carefully sectioned and mounted onto glass slides. Next, the slides stained with H&E were scanned using a Zeiss Axioscan Z.1 whole slide scanner to capture detailed images. A scoring system based on established methods was used to evaluate the pathologies, and a detailed scoring rubric is provided in Supplementary Table S2. The number of granuloma lesions was recorded for each lung tissue specimen. Tissues were assessed blindly, with three criteria: loss of free air space, epithelial hyperplasia, and immune cell infiltration. Each factor received a severity score of 1 to 5 (Increasing severity), enabling a standardized evaluation [24]. The total score and the number of granuloma lesions together determine the cumulative lung severity score, which reflects disease severity.

Enzyme-linked immunosorbent assay (ELISA) and correlation analysis

On the sample collection day, blood samples and lung lavage were collected separately from each mouse. Blood samples were centrifuged at 5000 rpm for 15 min, and the separated serum was collected. Immunosorb plates (Thermo Fisher Scientific) were coated with individual LP and ESAT-6 whole proteins (Abcam, 1 μg/mL) and Mav whole-cell lysate (1 × 10⁶ CFU/50 μL/well) separately, then incubated overnight at 4 °C. The wells were then washed three times with ELISA wash buffer, with each rinse performed between steps. The wells were blocked with 1% bovine fetal albumin in PBS for 1 h at RT. Diluted serum (1:100, 1:200) for IgG evaluations and lung lavage (1:1, 1:2) for IgA evaluations were added to the plates, which were incubated at RT with shaking for 2 h. The washed plates were incubated with a secondary antibody (goat anti-mouse IgG/IgA alkaline phosphatase; SouthernBiotech) at 1:4000 in PBS at RT for 1 h, followed by enzymatic colour development with p-nitrophenyl phosphate (SouthernBiotech). At different time points, absorbance (OD) was measured using the DTX 880 Plate Reader (Beckman Coulter). OD values were later converted into concentrations (pg/mL) using a standard curve. Data are presented as mean ± SEM of three to five biological replicates.

Antigen-specific splenocyte proliferation assay

The previously described protocol was followed to analyze antigen-specific splenocyte proliferation [22]. A Roche Cell Proliferation ELISA, BrdU colorimetric kit (Sigma-Aldrich, MO, USA), was used to assess proliferation according to the manufacturer’s instructions. Harvested mouse spleens were processed into single-cell suspensions, and 4 × 10⁵ splenocytes were co-cultured with 4 × 10⁵ antigen-presenting cells (irradiated naïve splenocytes at 3000 rads) in the presence of the immunized lipopeptide (1μg/mL). At least three replicates (96-well plates) from pooled splenocytes in each experimental group were analyzed. Plates were incubated for 4 days, and the BrdU labelling reagent was added. Finally, on day 5, the manufacturer's protocol was followed and OD values were measured using the DTX 880 Plate Reader (Beckman Coulter, CA, USA) at 450 nm. OD values were corrected by subtracting the OD of splenocyte proliferation under no antigen conditions (APC + splenocytes). Data are presented as mean ± SEM of three to five replicates.

Ex vivo bone-marrow infection model

First, bone marrow (BM) from untreated, uninfected mice was isolated using a double-chamber spin-column tube. Then, 5 × 10⁵ BM cells per well were cultured in a 24-well plate in high-glucose DMEM supplemented with 10% FBS and 50 ng/mL murine Macrophage Colony Stimulating Factor (mM-CSF). The cells were incubated for 5 days to allow differentiation into macrophages. The day before infection, the medium was changed to mM-CSF–unsupplemented medium, and the following day, 2 × 10⁶ CFU of Mav per well were added for infection. Plates were incubated for 24 h at 37 °C with 5% CO₂. Next, the wells were thoroughly washed three times with media to remove free bacteria. Then, 0.1 mL of 1:10-diluted native (non-heat-inactivated) or heat-inactivated (56 °C for 30 min [25,26]) serum samples from immunized mice in mM-CSF–unsupplemented BM medium was added, and the mixture was incubated for 4 h; the volume was then adjusted to 1 mL per well. After a 24 h incubation period, supernatants were collected, and cells were lysed with 0.05% SDS. The diluted samples were then cultured on 7H11 Middlebrook agar plates, which were incubated at 37 °C for 2 to 3 weeks. The remaining lysate was collected. Colonies were counted and plotted.

Analysis of intracellular bacterial clearance mechanisms

To evaluate the different components in the supernatant or lysate that may be related to intracellular bacterial killing, we used the following assays: Reactive Oxygen Species (ROS, H₂O₂) detection assay (AMPLEX RED HYDROGEN assay; CATALOGUE #: A22188; Invitrogen), lysosomal enzyme release assay (BETA HEXO Activity Assay Kit; CATALOGUE #: MET-5095; Encompass), reactive nitrogen species detection assay (GRIESS Reagent Kit; CATALOGUE #: G7921; Thermo Fisher Scientific), mouse IFNγ ELISA (CATALOGUE #: 88-8314-88; Thermo Fisher Scientific), TNFα ELISA (CATALOGUE #: 88-7324-88; Invitrogen), and IL-1β (CATALOGUE #: 88-7013A-88; Invitrogen). Manufacturer’s protocols were followed for each assay.

Flow cytometry

Flow cytometry was performed as previously reported [24]. Briefly, harvested lung and spleen tissues were pooled within each experimental cohort and processed into single-cell suspensions to obtain sufficient cells. Cells were plated at 2 × 10⁶ cells/mL in 24-well culture plates and stimulated overnight at 37 °C in 5% CO₂ with 50 ng/mL PMA and 500 ng/mL ionomycin. During the final 4 h of incubation, brefeldin A (1.5 μg/mL) was added. Subsequently, lung lymphocytes and splenocytes were collected, washed, and stained for surface markers and intracellular targets using a dedicated CD4⁺/CD8⁺ T-cell panel (CD3–Alexa Fluor 488, CD4-BV 786, CD8-APC-efluor 780, TNFα-APC, IFNγ-eFluor 450, IL-10-PerCP-cyanine 5.5, IL-17A-PE-cyanine 7, LAP-PE) according to established flow cytometry protocols. Spectral flow cytometry data were captured on a Cytek Aurora system and analyzed using FlowJo software (v10.10, BD Biosciences). Integrated Mean Fluorescence Intensity (iMFI) was calculated by combining the frequency of the cell population (CD4⁺ or CD8⁺) with the magnitude of its response (MFI). Total fraction analysis was then performed separately for lung CD4⁺ helper T cells and CD8⁺ cytotoxic T cells for each marker, and the results were presented as a heatmap.

Statistical analysis

All data were analyzed using GraphPad Prism software version 10.2.0 (GraphPad Software). For in vivo experiments, the number of biological replicates is indicated in the respective figure legends, and group sizes were kept equal within each experiment. We assessed residual normality using the Shapiro–Wilk test (and/or the Kolmogorov–Smirnov test) and visually confirmed it with Q-Q plots and individual data-point plots to ensure that no outliers influenced the group means [27]. Homogeneity of variance was assessed using the Brown-Forsythe test, which is recommended for small group sizes due to its robustness to slight distributional skewness. Residual normality (p > 0.05) and variance homogeneity (p > 0.05) were established, and parametric ANOVA was conducted with appropriate post hoc tests (Tukey’s post hoc test, otherwise specified). Correlation analyses between colony-forming units and antibody titres in serum and mucosal samples were conducted using Spearman’s rank correlation coefficient. A p-value of less than or equal to 0.05 was considered statistically significant across all analyses.

Results

Intranasal immunization of mice with ESAT-LPs and ESAT-LPs-HKCC induces splenocyte proliferation and triggers systemic and mucosal humoral responses

To assess the immunogenicity of ESAT-LPs with or without HKCC, mice were immunized according to the timeline shown in Figure 1(a). Pooled splenocytes were cultured in media containing individual LP (1μg/mL) separately, and a colorimetric BrdU incorporation assay was performed to evaluate antigen-specific proliferation (Figure 1(b)). ESAT-LPs immunization led to enhanced splenocyte proliferation in response to recall antigens compared with the unvaccinated group; however, inclusion of HKCC significantly bolstered this response (Figure 1(b)). To evaluate the cellular immune response, lymphocytes from the lungs and spleens were gated into CD4⁺ helper and CD8⁺ cytotoxic T-cell subsets for intracellular cytokine profiling (Supplementary Figure S1(a)). Heatmap analysis of total fractional cytokine levels showed that ESAT-LPs immunization selectively increased TNFα and IL-17A production in helper and cytotoxic T cells in the lungs and spleens, and enhanced IFNγ levels were observed in lung Th cells. However, adjuvant formulation shifted the CD4⁺ T cell response toward a robust Th1 phenotype, markedly augmenting IFNγ and TNFα expression relative to LP alone and unimmunized controls in both Lungs and spleens, while maintaining the lowest IL-17A levels (Supplementary Figure S1(b,c)).

Next, systemic IgG (Figure 1(c)) and mucosal IgA (Figure 1(d)) titres were measured against individual LP and recombinant ESAT-6 protein. ESAT-LPs immunization increased serum IgG titres compared to the unimmunized group. Intriguingly, a further significant rise in IgG was observed with ESAT-LPs-HKCC (Figure 1(c)). Importantly, both systemic and mucosal antibodies induced by ESAT-LPs and ESAT-LPs-HKCC immunizations recognized the full-length recombinant ESAT-6 protein. The addition of the HKCC to ESAT-LPs showed a trend toward higher systemic and mucosal antibody levels against the ESAT-6 protein (Figure 1(c,d)).

Prophylactic immunization with ESAT-LPs and ESAT-LPs-HKCC reduced the disseminated mycobacterial burden and lung pathologies upon Mav challenge and induced distinct cellular immune responses

ESAT-LPs, with or without HKCC, were used to immunize mice twice, 14 days apart, one week before the Mav challenge (Figure 2(a)). Both immunization groups significantly reduced bacterial burdens in the lungs, spleens, and livers compared to the untreated-infected control. Notably, inclusion of HKCC in the ESAT-LPs resulted in a much lower bacterial load across all organs compared to the ESAT-LPs group (Figure 2(b)). Randomly selected mouse lungs were prepared for H&E staining, and histopathological features were evaluated and scored in a double-blind manner (Figure 2(c,d)). ESAT-LPs-immunized mice exhibited mild inflammation and fewer granuloma lesions compared to untreated-infected mice. Interestingly, mice immunized with ESAT-LPs-HKCC showed further reductions in granuloma lesions and inflammatory pathology (Figure 2(c,d)). Overall, intranasal immunization with ESAT-LPs-HKCC provided much superior effects in terms of reduced bacterial load, granuloma formation, and pulmonary inflammation.

Consistent with outcomes in mycobacterial loads, splenocytes harvested from immunized mice prior to challenge exhibited robust recall proliferation upon ex vivo re-stimulation with LP antigens (Figure 2(e)). Both ESAT-LPs alone and the HKCC-adjuvanted LP formulation induced significantly higher proliferative responses than controls, though no statistically significant difference was observed between the two immunized groups.

We performed parallel intracellular cytokine analyses, and detailed results are shown in Supplementary Figure S2. Total fractional analysis revealed comparable levels of IFNγ, TNFα, and IL-17A in CD8⁺ cytotoxic T cells across both lung and spleen tissue. However, a distinct phenotypic divergence emerged in regulatory marker expression: the ESAT-LPs-HKCC immunized group exhibited significantly lower levels of IL-10 and LAP than the untreated-infected controls, which showed marked upregulation of these regulatory cytokines. A similar trend was observed in the CD4⁺ helper T-cell compartment, where untreated-infected controls mounted an exaggerated response marked by high levels of both pro-inflammatory and regulatory cytokines. In contrast, ESAT-LPs-HKCC immunization attenuated this hyper-inflammatory profile to a balanced, moderate level while selectively suppressing regulatory cytokine involvement (Supplementary Figure S2 (a,b)).

Mucosal antibody response driven by prophylactic immunization with ESAT-LPs and ESAT LPs-HKCC correlates with reduction in mycobacterial burden

We observed that after Mav infection, unimmunized mice showed significantly higher IgG titres against ESAT-LP, ESAT-6 protein, and Mav lysate (Figure 3(a)). In contrast, mice immunized with ESAT-LP +/- HKCC had lower IgG titres. Notably, mucosal IgA antibody levels against ESAT-LPs, ESAT-6, and Mav were barely detectable in the unimmunized-infected control group. Conversely, high levels of IgA were observed in the lung lavages of the ESAT-LP +/- HKCC immunized groups (Figure 3(b)), with the ESAT-LPs-HKCC group showing significantly higher mucosal IgA levels than the ESAT-LPs group (Figure 3(b)). Overall, ESAT-LPs with HKCC strongly induced pulmonary IgA antibodies when given intranasally before challenge. Spearman correlation analysis showed that mucosal IgA titres were negatively correlated with bacterial loads in the lungs, spleens, and livers (Figure 3(c)). In the lungs, ESAT-LPs- and Mav-specific IgA antibodies showed significant inverse correlations (r = −0.8964 and r = −0.9298, respectively). Similarly, negative correlations between bacterial loads in the spleen and liver and IgA titres were significant (Spleen; ESAT-LPs; r = −0.9143 and Mav-specific; r = −0.8649; Liver; ESAT-LPs; r = −0.8179 and Mav-specific; r = −0.8973). Therefore, in the prophylactic immunization regimen, ESAT-LPs-HKCC induces mucosal IgA antibody titres that may associate with protection against Mav infection.

Figure 3.

Systemic and mucosal antibody responses in Mav-infected mice preemptively immunized with ESAT-LPs, with or without HKCC. Following the experimental outline in Figure 2(a), cardiac blood and lung lavage were collected from each mouse. Serum samples (diluted 1:100) and lung lavage samples (diluted 1:1) were used to quantify antibody levels by ELISA. (a) Systemic IgG antibody titres; (b) mucosal IgA antibody titres specific for ESAT-LPs, ESAT-whole protein, and Mav. (c) Spearman’s correlations were calculated between serum IgA titres and CFU counts in the lungs, spleens, and livers. Data are presented as mean ± SEM, with each data point representing an individual mouse (n = 5 per group). Statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001) was determined by one-way ANOVA for ESAT whole protein and Mav-specific ELISA analysis or two-way ANOVA for LPs-specific ELISA analysis followed by Tukey's test.

Therapeutic vaccination with ESAT-LPs and ESAT-LPs-HKCC effectively reduced disseminated Mav load and disease severity, and induced cellular immune responses

To assess immunotherapeutic potential, we first infected mice with Mav and then administered ESAT-LPs and ESAT-LPs-HKCC intranasally, as shown in Figure 4(a). Notably, both groups significantly reduced bacterial loads across all three organs (Figure 4(b)). Nonetheless, ESAT-LPs-HKCC yielded the lowest bacterial CFU in the lungs, spleens, and livers (Figure 4(b)). Histopathological analyses indicated that Mav infection caused severe pathology in the untreated group, whereas treatment groups alleviated pathological symptoms (Figure 4(c)). Consistent with bacterial loads, ESAT-LPs-HKCC-immunized mice exhibited fewer granuloma lesions, inflammatory tissue areas, and epithelial hyperplasia than the ESAT-LPs group (Figure 4(c)). Quantitative double-blind scoring also showed that intranasal ESAT-LPs therapy, with or without HKCC, significantly lowered the lung severity score compared to the untreated-infected group (Figure 4(d)).

In the post-exposure immunotherapy setting, recall responses in immunized mice mirrored the trajectory observed in the prophylactic setting. Ex vivo antigen re-stimulation showed that splenocyte proliferation was markedly enhanced in both vaccinated groups compared with untreated controls. Interestingly, unlike the prophylaxis experiment, co-formulation with the adjuvant resulted in a significant attenuation of splenocyte proliferation relative to antigen alone (Figure 4(e)).

In-depth flow cytometric analysis revealed that cytotoxic lung lymphocytes in the untreated, infected control group exhibited a “cytokine storm-like” profile characterized by the simultaneous elevation of both pro-inflammatory and regulatory cytokines (Supplementary Figure S3(a)). In contrast, immunization with ESAT-LPs, with or without HKCC, established a more balanced, moderate cytokine environment and reduced IL-10 expression. Lung CD4⁺ T-cell subsets in the HKCC-adjuvanted group were strongly enriched for IFNγ, TNFα, and IL-17A (Supplementary Figure S3(a)), suggesting possible Th1 and Th17 effector functions. In the spleen, ESAT-LPs +/- HKCC predominantly induced IFNγ and TNFα expression within the CD8⁺ cytotoxic T-cell compartment, while regulatory cytokine fractions remained comparable across all cohorts (Supplementary Figure S3(b)). Conversely, within the CD4⁺ helper T-cell compartment, vaccinated mice maintained lower cytokine levels than untreated controls, which exhibited peak levels of IL-17A and IL-10; however, TNFα expression remained markedly elevated in the HKCC-incorporated group (Supplementary Figure S3(b)).

Systemic IgG vs mucosal IgA antibody titres are reciprocally detected in Mav-infected mice upon treatment with ESAT-LPs and ESAT-LPs-HKCC, and are associated with a reduction in mycobacterial load

In the post-exposure therapeutic model, intranasal treatment with ESAT-LPs vs. ESAT-LPs-HKCC showed an interesting trend in detected systemic IgG vs. mucosal IgA levels (Figure 5). Strikingly higher levels of systemic IgG reactive to ESAT-LPs, ESAT-6 protein, and Mav lysate were detected in the HKCC-added group compared to the ESAT-LPs alone and untreated-infected groups (Figure 5(a)). By contrast, the ESAT-LPs-treated group showed significantly higher mucosal IgA antibody levels reactive to ESAT-LPs, whole protein, and Mav lysate. In contrast, the ESAT-LPs-HKCC group had negligible IgA levels (Figure 5(b)). Spearman correlation analyses showed a negative correlation between serum IgG titres and bacterial loads in the lungs, spleens, and livers of the mice in this model (Figure 5(c)). ESAT-LPs- and ESAT-6 protein-specific IgG antibody titres were significantly negatively correlated with CFU in the lungs, spleens, and livers r = −0.7954, −0.5821, −0.7000 (LP); r = −0.8186, −0.6214, −0.7679 (ESAT-6 protein), respectively. Similarly, significant inverse correlations were observed between Mav-specific IgG titres and CFU in the lungs, spleens, and livers (r = −0.7587, −0.6523, and −0.7491, respectively). Interestingly, IgA titres detected in the ESAT-LPs immunization group did not correlate with reductions in bacterial load in the post-exposure therapeutic regimen (data not shown). Thus, ESAT-LPs-HKCC-induced IgGs, at least partially, correlate with bacterial clearance against Mav infection when administered after infection.

Figure 5.

Systemic and mucosal antibody titres in Mav-infected mice immunized with ESAT-LPs with or without HKCC. Following the Mav challenge and immunizations shown in Figure 4(a), cardiac blood and lung lavages were collected from the mice. ELISAs were performed using serum samples (1:100) and lung lavage samples (1:1). (a) Systemic IgG antibody titres; (b) mucosal IgA antibody titres specific for ESAT-LPs, ESAT whole protein, and Mav. (c) Spearman’s correlations between serum IgG titres and CFU counts in the lungs, spleens, and livers. Data are presented as mean ± SEM, with each data point representing an individual mouse (n = 5 per group). Statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001) was determined by one- way ANOVA for ESAT whole protein and Mav-specific ELISA analysis or two-way ANOVA for LPs-specific ELISA analysis followed by Tukey's test.

High IgG-containing serum samples reduce intracellular Mav loads in an ex vivo bone marrow infection model

To determine whether antibodies induced in serum samples after immunization with ESAT-LPs and ESAT-LPs-HKCC contribute to reducing intracellular mycobacteria, differentiated bone marrow macrophages (BMs) infected with Mav were treated with serum samples from individual immunized mice, as shown in Figure 6(a). Interestingly, serum from uninfected mice immunized with ESAT-LPs and ESAT-LPs-HKCC significantly reduced intracellular bacilli compared with serum from untreated, uninfected mice (Figure 6(b)). However, there was no significant difference between the two treatment groups (ESAT-LPs vs. ESAT-LPs-HKCC). Next, serum samples from mice with Mav infection in prophylactic and therapeutic experiments with ESAT-LPs and ESAT-LPs-HKCC were analyzed. Serum from the prophylactically immunized groups significantly reduced intracellular Mav bacilli more than serum from untreated-infected mice (Figure 6(c)), but no significant difference was observed between the two treatment groups. Notably, a significant difference was found between ESAT-LPs and ESAT-LPs-HKCC-immunized mice in the ability of serum samples from the immunotherapeutic regimen to inhibit intracellular Mav (Figure 6(d)). About 60% of bacilli were cleared from BM cells exposed to serum samples from ESAT-LPs-HKCC-treated mice. To assess the role of complement components in serum bactericidal activity, bacterial survival was measured after incubation with heat-inactivated serum. No significant difference was observed between heat-inactivated (HI) and non-heat-inactivated (NHI) serum treatments across all groups (Supplementary Figure S4).

Figure 6.

Role of serum antibodies in reducing intracellular Mav loads and activating intracellular antimicrobial mechanisms in an ex vivo bone marrow infection model. Cultured mouse bone marrow cells were allowed to differentiate into bone marrow derived macrophages, and then infected with Mav for 24 h, then washed to remove extracellular bacteria. Mouse serum samples (1:10 dilution) from the respective immunized and infected groups were added to the cultures and incubated for another 24 h. The cells were lysed, and the diluted samples were plated on 7H11 agar plates. After 2–3 weeks, colonies were counted. (a) Schematic representation of the ex vivo bone marrow infection model. (b) Serum samples from immunized mice according to the scheme in Figure 1(a); (c) serum samples from prophylactic immunization as shown in Figure 2(a); (d) serum samples from mice that received vaccine immunotherapy according to the scheme in Figure 4(a). (e–g) Cell supernatants or lysates were also examined for potential mechanisms of action using assays for cytokines TNFα, IL-1β, and IFNγ, and for activities (h–j) Beta-hexosaminidase, H₂O₂, and Nitrite levels. Data are presented as mean ± SEM (n = 3-5 per group). Statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001) was determined by one-way ANOVA followed by Tukey's test.

Activation of intracellular antimicrobial mechanisms in an ex vivo bone marrow infection model

To understand the intracellular mechanisms underlying the reduction in intracellular Mav burden following treatment with high-IgG-containing serum samples from immunotherapy experiments, we collected cell supernatants and cell lysates at the end of the BM infection assay. ELISAs were used to measure TNFα, IL-1β, and IFNγ (from cell supernatant), β-hexosaminidase activity (from cell lysate), ROS (H₂O₂), and nitric oxide (NO) levels (from cell supernatant) across groups (Figure 6(e–j)). The amount of TNFα secreted was slightly higher in serum-treated samples from ESAT-LPs and ESAT-LPs-HKCC immunized mice compared to untreated-infected samples; however, this difference was not statistically significant (Figure 6(e)). In contrast, IL-1β and IFNγ production were decreased in both treatment groups relative to the control (Figure 6(f,g)). Supernatants from serum-treated infected BM cells from ESAT-LPs-HKCC immunized mice had the lowest IFNγ levels (Figure 6(g)).

The β-hexosaminidase activity assay showed that only serum from ESAT-LP-HKCC-immunized mice had significantly higher β-hexosaminidase activity than serum from ESAT-LPs and untreated-infected mice (Figure 6(h)). Additionally, a higher concentration of H₂O₂ was detected when serum from ESAT-LPs-HKCC-vaccinated mice was applied to infected BM (Figure 6(i)). In contrast, samples treated with serum from ESAT-LPs-vaccinated and untreated-infected mice showed very low levels of H₂O₂ (Figure 6(i)). NO concentrations in the supernatants were significantly higher after treatment with serum from ESAT-LPs- and ESAT-LPs-HKCC-immunized mice than with untreated-infected serum (Figure 6(j)). Overall, these assays revealed that treatment of Mav-infected BM cells with serum from mice immunized with ESAT-LPs and ESAT-LPs-HKCC activated several intracellular mechanisms, potentially contributing to bactericidal activity.

Discussion

Pulmonary infections caused by Mycobacterium species pose a substantial threat to global public health. Specifically, TB remains one of the top ten leading causes of mortality worldwide [28]. Non-tuberculous mycobacterial infections are also common respiratory infections in humans, with incidence rising in North America, Europe, and Asia, and with predictions of a threefold increase by 2040 [29]. The currently available BCG vaccine has limited preventive and therapeutic value in terms of its efficacy and safety against non-tuberculous mycobacteria; therefore, novel approaches are urgently needed to combat mycobacterial infections.

Vaccine development for mycobacteria has primarily focused on eliciting robust cellular immune responses. This strategy was based on the key idea that controlling intracellular pathogens requires cellular immunity. However, none of these newer vaccines have been successful so far [8]. It is understandable that combining different immune responses might be an effective way to combat complex mycobacterial infections [9]. Initially, humoral immune responses against mycobacteria were overlooked because of their intracellular nature and the uniquely waxy cell walls of these organisms, which prevent complement-mediated lysis and opsonization. However, B-cell-mediated antibody responses are now recognized as playing an important role in host defense against mycobacteria, and TB vaccines that induce antibodies are being proposed [8]. One of the earliest clues about the role of antibodies in mycobacterial infection came from the distinctive antibody pattern seen in patients with latent and active TB [8,10]. Additionally, B cells have been identified as components of the cellular community in granulomas and are commonly found in the lungs of TB patients [28]. Moreover, a meta-analysis showed that antibody responses in TB patients account for about 10% of the proteome [29]. Passive transfer of polyclonal antibodies has demonstrated protective effects in mouse models, supporting the idea of antibody-mediated protection against mycobacteria [8]. Interestingly, the induction of B cells and antibody responses by BCG vaccination has been inconsistent across studies [30,31]. The dose and route of BCG vaccination have been shown to influence antibody responses [31,32]. Therefore, exploring effective vaccine strategies that generate a broader range of immune responses against the Mycobacteriaceae is essential.

Selecting immunogenic epitopes is essential for an effective vaccine, and adding a potent adjuvant can trigger long-lasting, comprehensive immune responses, including humoral responses [20]. In earlier studies, we showed that our vaccine construct – a mixture of five ESAT-LPs – effectively elicited cytotoxic (CD8⁺) and helper (CD4⁺) T cell responses, including induction of proinflammatory cytokines and reduction of regulatory cytokines, when given intranasally in two doses before the Mtb challenge [18]. Later, we demonstrated that post-exposure ESAT-LP vaccination enhanced immune responses primed by BCG vaccination by reeducating cell-mediated immunity [19]. Overall, ESAT-LP-driven cellular immune responses were promising, linked to protection against Mtb, and further shown to enhance BCG-primed immunity. To determine the broad efficacy of the ESAT-LP vaccine candidate for other mycobacterial species, we used a Mav (Non-tuberculosis) infection model to evaluate the protective efficacy of ESAT-LPs. Protein sequence alignment revealed that several Mav antigens share modest identity but considerable sequence similarity (approximately 70-85%) with our candidate vaccine epitopes. It has been suggested earlier that partial conservation of key MHC-binding core residues is sufficient to drive robust, cross-reactive cellular and humoral immune responses against the native pathogen [33]. CD4⁺ T-cell receptors require as few as five shared amino acids within an MHC class II-bound nonamer peptide to drive cross-reactive activation [34]. Consequently, the sequence similarity in our epitopes appears to be sufficient to prime cross-protective T-cell responses against Mav infection.

It remains unclear whether targeting secreted or cell wall proteins of Mtb with antibodies would provide protective or therapeutic immunity [35–37]. In this study, we introduce a novel mucosal immune modulator, HKCC. Early work by Gupta et al. demonstrated that intranasal HKCC induces key mucosal cytokines (including GM-CSF, IL-12, IL-17A, TNFα, IL-6, and IL-1β) in pulmonary washes, promotes bone marrow-derived dendritic cell (BMDC) activation, and enhances NK/NKT cell interactions to drive IL-12 and IFNγ secretion. Notably, supernatants from HKCC-treated PBMCs exhibited superior mycobactericidal activity against H37Ra and Mav in THP-1 macrophages compared with LPS. In mice, standalone intranasal HKCC therapy recruited and activated local and systemic innate populations (NK/NKT cells, DCs, macrophages, and B cells), conferring protection against H37Ra infection [23]. Subsequently, Patel et al. established HKCC as an effective mucosal adjuvant in a SARS-CoV-2 lipopeptide vaccine, boosting mucosal antibodies, durable T- and B-cell memory, and protection in hamsters and mice [22,38]. Most recently, we delineated the innate mechanisms of HKCC across Mav, SARS-CoV-2, and influenza models, demonstrating that prophylactic HKCC restricts systemic Mav burden, drives local innate lymphoid cell activation, tempers pulmonary inflammation by modulating neutrophil migration, and elicits robust sIgA and serum IgG responses against Mav but not against HKCC [24]. Building on these findings, the present study is the first to evaluate HKCC as a mucosal adjuvant in an ESAT-lipopeptide vaccine against mycobacterial infection. Because the cellular and humoral immune responses to standalone HKCC have been comprehensively characterized in our previous publications [23,24], an HKCC-alone control group was not included to avoid redundant data publication. We hypothesize that combining highly immunogenic ESAT-6 lipopeptide epitopes with HKCC would elicit strong, protective mycobacterial-specific humoral and cellular responses, providing a promising vaccine candidate effective for both preventive and therapeutic use.

Both ESAT-LPs alone and the ESAT-LPs-HKCC formulation significantly enhanced ex vivo antigen-specific splenocyte proliferation after recall stimulation in both Mav-uninfected and infected models. Comprehensive intracellular cytokine profiling confirmed that ESAT-LPs-HKCC immunization elicited robust Th1 responses and IFNγ and TNFα expression in CD8⁺ cytotoxic T-cell compartments in both lung and splenic tissues. Compared with unimmunized controls, this regimen established a potent immune milieu capable of mounting an effective defense against potential intracellular mycobacterial exposure. Type 1 cytokine-expressing cytotoxic T cells and Th1 cells are often associated with induced immune responses against intracellular pathogens and are effective in clearing them [39].

Following Mav challenge, prior vaccination steered the host response toward a controlled, protective immunophenotype. Untreated infected controls consistently mounted an uncoordinated, hyper-reactive T-cell response characterized by simultaneous, elevated expression of pro-inflammatory (IFNγ, TNFα, IL-17A) and immunosuppressive (IL-10, LAP) cytokines. This concurrent surge mimics an uncontrolled cytokine storm state that fails to contain the pathogen, instead driving severe pulmonary immunopathology and accelerating bacterial burden [24]. Furthermore, the detrimental effects of IL-10 have been well documented. IL-10 inhibits Th1 and cytotoxic T cell functions, key regulatory functions in mycobacterial infections [39]. While ESAT-LPs alone induced robust proinflammatory cytokine expression in cytotoxic lung and spleen lymphocytes, co-administration with the HKCC adjuvant tempered this response, promoting a milder, less damaging inflammatory profile that aligned directly with the reduced lung tissue pathology observed on histopathology. The protective effects of IFNγ, TNFα, and IL-17A against mycobacterial infections have been extensively discussed in previous studies [24,40]. Interestingly, therapeutic intranasal administration of ESAT-LPs +/- HKCC in established Mav infection effectively dampened exaggerated CD8⁺ lung T-cell cytokine output and may contribute to attenuated acute pathological symptoms. The elevated IFNγ and TNFα in the splenic cytotoxic T-cell pool following intranasal delivery reflect the localized and systemic efficacy of mucosal administration, which modulates the pulmonary and systemic immune microenvironment against systemic infection, supported by other studies [41]. Nevertheless, ESAT-LPs-HKCC-treated mice showed robust Th1 and Th17 effector responses in the lungs and maintained moderate, sustained pro-inflammatory cytokine levels in splenic CD4⁺ T cells compared with the overwhelmed, dysregulated profiles of untreated controls. Taken together, these data demonstrate that untreated Mav infection drives a detrimental, hyperinflammatory microenvironment in which concurrent regulatory feedback fails to protect the host. In contrast, our mucosal vaccine formulation primes the host immune system to establish a controlled, mostly Th1- and Th17-driven host-beneficial immune microenvironment that limits tissue damage while maintaining antimicrobial efficacy.

In the present investigation, alongside cellular responses, our primary objective was to extensively characterize the antibody responses elicited by immunization. Mice immunized with ESAT-LPs showed antigen-specific IgG and IgA titres in serum and lung lavage, respectively, which were markedly enhanced by the addition of HKCC (Figure 1(c,d)). Notably, in prophylactic or therapeutic experiments with Mav-infected mice, an interesting pattern of antibody response emerged. In the prophylaxis model, we observed significantly higher mucosal IgA titres against ESAT-LP and Mav, but not systemic IgG, in both ESAT-LPs and ESAT-LPs-HKCC immunized groups. Conversely, IgG titres were reduced after Mav infection in ESAT-LPs and ESAT-LPs-HKCC immunized mice compared with untreated-infected controls. Interestingly, a decrease in IgG levels after mycobacterial infection has been previously reported in another study, where intranasally ESAT6-immunized, uninfected mice showed higher serum IgG levels before infection, but these levels decreased after Mtb infection compared to untreated-infected mice [42]. Although the exact mechanism underlying this observation is unclear, mycobacterial infection may modulate or suppress the specific protective IgG response, thereby favouring its survival. Still, it remains unclear why untreated-infected controls exhibited higher IgG levels; apparently, the naturally induced IgG response upon infection in mice is not protective and does not significantly reduce the mycobacterial burden. In fact, high ESAT-6-specific antibody levels are associated with active TB infection in humans [43]. Detailed analyses of antibody subtypes and their specific functional roles in these two scenarios may shed light on this complex interaction/observation.

The majority of immunocompetent individuals exposed to mycobacteria eliminate them early. Competent innate immune responses at mucosal sites, including mucosal IgA, along with systemic adaptive responses, play a crucial role in controlling and clearing the pathogen. Preexposure to NTM has been reported to reduce the effectiveness of the BCG vaccine; however, it also leads to circulating IgA that benefits the host upon later exposure to Mtb. These observations are essential for vaccine development [44,45]. This is supported by a study evaluating protective antibodies in healthy healthcare workers (both latently infected and highly exposed-uninfected) and active TB patients, which found protective antibodies in healthy healthcare workers but not in active TB patients [37]. We speculate that the distinct antibody levels between the preventive and therapeutic models in the current study are mainly due to the timing of immunization; an immune system primed with ESAT-LPs-HKCC before infection resulted in a surge of pulmonary antigen-specific IgA antibodies to defend against the pathogen at the mucosal site [46]. Therefore, HKCC-bolstered IgA in the lung mucosa may provide early protection during infection. Furthermore, antibody-mediated effector functions, such as activating complement and natural killer cells, could be critical in protection against Mtb [32], indicating that early antibody-driven defenses might be indispensable. Evidently, IgA-deficient mice were highly vulnerable to BCG infection [47]. Mucosal IgA is vital in preventing pulmonary infections by neutralizing pathogens and their toxins, hindering their establishment, trapping and activating immune cells through antigen presentation, and promoting opsonophagocytosis [11]. Ultimately, mucosal ESAT-LPs-HKCC vaccination elicits a key effector IgA response, underscoring its potential as a promising preventive vaccine candidate.

For an endemic infection like TB, there is a dire need for an effective therapeutic vaccine as a long-term, sustainable solution, since mass prophylactic vaccination of the public against mycobacterial infection is not feasible. However, research on therapeutic vaccines for mycobacterial infections remains limited. In this study, we demonstrated that the ESAT-LPs-HKCC vaccine candidate is also therapeutically effective. Nevertheless, post-infection immunization with ESAT-LP-HKCC induced significantly higher IgG levels than sIgA (Figure 5). A review of therapeutic vaccines against TB reported that only a few of these vaccines induced IgG titres [48]. Notably, mucosal immunization with ESAT-LPs alone maintained or increased mucosal IgA antibody levels in both infection models compared to vaccination of uninfected mice (Figures 3 and 5). This underscores the dynamic regulation, facilitated by the HKCC immunomodulator, which caused a significant shift in antibody response between pre- and post-exposure infection models. Most importantly, we demonstrated that all antibody responses elicited by ESAT-LPs and ESAT-LPs-HKCC immunization were similar in their binding to ESAT-LPs, the full ESAT protein, and Mav lysate. These findings strongly support the conclusion that the studied vaccine formulations elicit a polyclonal humoral response, including antibodies targeting the lipopeptide immunogen itself, as well as antibodies recognizing the virulence factor ESAT6 and Mav cell components, establishing them as a relevant vaccine platform for inducing antibody-mediated protective mechanisms against the pathogen.

Vaccination during active infection primarily stimulates systemic IgG production, which is highly beneficial for combating established or disseminated infections. In this context, local IgA effector functions are less effective at reaching various infection sites. Systemic IgG drives potent effector functions – such as cellular degranulation, cytotoxicity, enhanced bactericidal activity, improved antigen presentation, and broad immune cell activation – all of which are crucial for helping the host control and resolve extensive disease [11]. Intriguingly, the dynamic pattern of antibody responses induced by ESAT-LPs and ESAT-LPs-HKCC aligns with protective mechanisms suggested in the literature, highlighting their potential as candidate vaccines against mycobacteria. Overall, prophylactic vaccines should focus on inducing mucosal IgA, whereas therapeutic vaccines aim to elicit systemic IgG to help clear pathogens or modulate disease [45]. How the ESAT-LPs-HKCC vaccine candidate cross-regulates these responses remains unclear and will be explored in future studies. Notably, cell-mediated immunity – encompassing proinflammatory cytokines such as IFNγ, TNFα, and IL-17A – serves a critical role in conferring protection across diverse contexts. It also influences humoral responses and helps sustain a host-directed, less inflammatory environment. Another study by Zeng et al. [21] also demonstrated that a subunit vaccine created by fusing components to stimulate cellular and humoral immune responses offered better protection against Mtb than BCG.

The biggest challenge in investigating vaccines against mycobacterial infection is the poor characterization of correlates of protection. To advance experimental vaccine approaches to clinical success, establishing primary animal-level correlates of protection is crucial [9,49,50]. In this context, we measured bacterial loads in the lungs, spleens, and livers of mice in pre- and post-challenge models. Significantly lower bacterial loads were observed in all organs in both prophylactic and therapeutic vaccine regimens using ESAT-LPs and ESAT-LPs-HKCC vaccination, with the lowest bacterial burden in the HKCC-added groups. Lung severity score analysis further revealed that including HKCC in both preventive and therapeutic vaccines significantly reduced lung pathology caused by Mav infection. Notably, we demonstrated a relationship between antibody titres against ESAT-6 and Mav and protection. Intriguingly, significant negative correlations were observed between CFU and IgA or IgG in the preventive and therapeutic vaccine regimens, respectively. Therefore, our study suggests that reduced lung severity and improved lung pathology might be associated with antibody responses. Antibody-mediated responses influence granuloma formation, tissue-associated pathology, and the ultimate course of disease [9]. For instance, non-human primates (NHPs) vaccinated with the live attenuated Mtb strain (MTBVAC) expressing ESAT-6 and CFP-10 showed higher Mtb-specific IgG antibody titres before the Mtb challenge than BCG-vaccinated NHPs, and this was associated with protection [51].

Antibody-mediated control of intracellular bacteria remains a subject of ongoing debate. To better illustrate the role of the humoral component in clearing intracellular mycobacteria, we developed an ex vivo infection model (Figure 6(a)). Similarly, several in vitro infection models have been used to demonstrate the functions of immune components, such as antibodies [52,53]. Our results showed that serum samples induced by ESAT-LPs and ESAT-LPs-HKCC, which are rich in ESAT-6 and Mav-specific antibodies, effectively reduce intracellular mycobacteria in infected BM (Figure 6(b–d)). In this context, Miles et al. [52] reported that ESAT-6- and CFP-10-specific IgG controls intracellular bacteria in infected macrophages but not extracellular bacteria. In addition, our findings demonstrate that heat inactivation of serum did not significantly reduce bactericidal activity across treatment groups, indicating that heat-labile complement proteins are not the primary drivers of bacterial clearance in this model (Supplementary Figure S4). Instead, the persistence of robust bactericidal activity after thermal inactivation strongly suggests that complement-independent mechanisms, most notably antigen-specific antibodies, play a central role in mediating bactericidal activity. However, to definitively identify the protective role of specific immunoglobulins and rule out residual heat-stable serum factors, targeted antibody depletion experiments, such as selective immunodepletion, are warranted to confirm these observations.

Next, we evaluated supernatants and lysates from BM cells to identify differences in key cytokines and killing intermediates between serum-treated groups from immunized and unimmunized mice. Elevated levels of IFNγ and IL-1β were observed in serum-treated samples from untreated-infected mice compared with serum from ESAT-LPs- and ESAT-LPs-HKCC-vaccinated mice. The literature supports the idea that intracellular viable mycobacteria promote greater IL-1β and IFNγ secretion by macrophages [54,55]. It is plausible that cytokine secretion levels reflect the amount of intracellular Mav. Additionally, the dependence of TNFα secretion on viable mycobacterial stimulation aligns with conflicting findings in mouse macrophages [55,56]; however, we observed no significant differences among groups.

We also explored potential intracellular mechanisms underlying mycobacterial reduction. Reactive oxygen species (H₂O₂)_, nitric oxide (NO), and the pathways that generate them, such as the p38 and JNK signalling pathways, may help eliminate intracellular bacilli [57]. H₂O_2-mediated killing of intracellular Mav in human macrophages has been observed in vitro [58]. Moreover, the bactericidal effect of NO has been demonstrated in mycobacteria-infected bone marrow-derived macrophages [57]. Interestingly, mycobacteria-stimulated mouse bone marrow-derived macrophages showed dose-dependent production of H₂O₂ and NO [56]. Additionally, lysosomal degradation of intracellular pathogens is a common mechanism of pathogen clearance, and mycobacteria have evolved strategies to evade this process. For example, β-hexosaminidase, a lysosomal enzyme, exerts mycobactericidal effects [53,59]. Our data show that serum from ESAT-LPs-HKCC-immunized mice significantly increased β-hexosaminidase activity and H₂O₂ production in Mav-infected macrophages compared with ESAT-LPs alone and with serum from untreated-infected mice. However, both ESAT-LPs and ESAT-LPs-HKCC significantly enhanced NO production. These results demonstrate that mucosal immunization with ESAT-LPs and ESAT-LPs-HKCC elicits antibody responses that contribute to the reduction of intracellular mycobacteria.

While this study provides valuable insights into the protective efficacy of mucosal vaccination against mycobacterial infection, several limitations must be acknowledged. First, the sample size per experimental cohort was modest in the first immunization model. Although sufficient to demonstrate statistically significant differences in primary immunological and microbiological outcomes, future studies using larger, independent animal cohorts will be essential to confirm the broad applicability of these observations. Second, infection was established via the intravenous route rather than an aerosol or intranasal route. Although intravenous administration does not fully recapitulate the natural mucosal transmission pathway of mycobacteria, it provides a well-characterized and robust model of pulmonary as well as systemic dissemination. This model allowed us to rigorously evaluate the protective capacity of mucosal vaccination against severe disease by delivering a precise, consistent bacterial inoculum and resulting infection across treatment groups. Nonetheless, evaluating our vaccine platform in aerosol- or intranasal-challenge models will be a critical next step to validate protection in a clinical scenario. Third, selective antibody-depletion experiments are warranted to demonstrate that antigen-specific antibodies mediate this bactericidal effect. Finally, incorporating BCG comparison arms and evaluating long-term memory maintenance will be pivotal objectives for future studies.

In conclusion, our studies demonstrate that ESAT-LPs combined with the HKCC immune modulator elicit targeted, robust humoral and cellular immune responses and reduce mycobacterial loads and pathology severity in both prophylactic and therapeutic models. Interestingly, our data strongly suggest a crucial role for humoral immunity triggered by the ESAT-LPs-HKCC intranasal vaccine in combating mycobacterial infections in both prophylactic and therapeutic settings, as well as in cross-regulation of mucosal IgA vs. systemic IgG levels. Ultimately, these novel mucosal vaccine approaches will help limit the cycle of infection and transmission and, most importantly, prevent uninfected individuals from becoming infected.

Funding Statement

This work was supported by Canadian Institutes of Health Research [grant number PS173314].

Disclosure statement

BA and RK are coinventors on worldwide patents issued on HKCC, and are cofounders of ImMed Biotechnologies, a spinoff company from the University of Alberta.

Supplemental Material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2026.2735151.