Escherichia coli DH5α and BL21 (Thermo Fisher Scientific, Inc.), RAW264.7 macrophages (Mouse; ATCC), THP-1 cells (Human; ATCC), the pET32a plasmid (Novagen; Merck KGaA), and the virulent MTB H37Rv (ATCC 93009) and its genome were preserved. H37Rv was cultured in 7H9 Middlebrook broth (cat. no. 271310; Becton, Dickinson and Company) supplemented with 10% oleic acid, albumin, dextrose and catalase (Becton, Dickinson and Company). THP-1 cells were differentiated with 100 ng/ml phorbol 12-myristate 13-acetate (Sigma-Aldrich; Merck KGaA) for 48 h before the experiments. Female C57BL/6 specific pathogen-free mice at 6-weeks-old were purchased from the Hubei Center for Disease Control and Prevention. The mice were maintained under infection barrier conditions in a negative-pressure animal room and fed a sterile commercial mouse diet. All the studies were reviewed and approved by the Animal Ethics and Welfare Committee of Wuhan Pulmonary Hospital (approval no. 2024006).

The DNA sequences used in the present study were obtained from the GenBank database (accession no. NC_000962.3). All sequences were verified by Sangon Biotech Co., Ltd. sequencing to ensure accuracy. The amplification primers for Rv1768 (F: 5'-AAATGGGATGCCCGGGCCGCTCGAGCACCACCACCAC-3'; R: 5'-AGAAATGACACACCAGCACCGGCGCCC-3') and for Rv1983 (F: 5'-ATGGGTCGCG GATCCGTGTCATTTCTGGTCGTGGTTCC-3'; R: 5'-GCTTCCGCCACCGCCGCTTCCCGCCGGATGATCAAAGACT-3') were synthesized by Sangon Biotech Co., Ltd. PCR was performed using PrimeSTAR Max DNA Polymerase (Takara Bio, Inc.) under the following conditions: Initial denaturation at 95˚C for 5 min; 30 cycles of 95˚C for 10 sec, 55˚C for 15 sec and 72˚C for 1 min; and a final extension at 72˚C for 5 min. The PCR products were analyzed by 1% agarose gel electrophoresis and visualized by GelRed staining under UV light. Using the H37Rv genome as the template, the Rv1768 and Rv1983 genes were amplified, cloned and inserted into the pET32a vector by homologous recombination using a ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd.) according to the manufacturer's instructions. The vector primers used for Rv1768 gene insertion were F: 5'-TGGTGTGTCATTTCTGGTCGTGGT-3'; R: 5'-TGGTGGTGGTGGTGCTCGAGCGGCCCGGGCATCC-3', whereas for Rv1983 gene insertion, the primers were F: 5'-GCCGTGAGAATTCGAGCTCCGTCGA-3'; R:5'-GCTTCCGCCACCGCCGCTTCCGGATCCGCGACCCA-3'. Finally, the expression vector pET32a-Rv1768/Rv1983 was constructed and transformed into E. coli DH5α competent cells. Briefly, 10 µl of the recombination reaction was added to 100 µl of competent cells, incubated on ice for 30 min, heat-shocked at 42˚C for 45 sec and placed on ice for 2 min. Next, 900 µl of LB broth was added, and the cells were recovered at 37˚C with agitation at 220 rpm for 1 h. The cells were plated onto LB agar containing 100 µg/ml ampicillin and incubated at 37˚C overnight.

The amino acid sequences encoded by Rv1768 and Rv1983 were retrieved from the NCBI database (accession no. NC_000962.3). Transmembrane helices were predicted using the TMHMM 2.0 server (DTU Health Tech, Technical University of Denmark) with default parameters (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0).

A single colony of BL21 cells carrying the expression vector was picked from an LB agar plate containing 100 µg/ml ampicillin and inoculated into 5 ml LB broth containing 100 µg/ml ampicillin. The culture was incubated overnight at 37˚C with agitation at 220 rpm. The overnight culture was then diluted 1:100 into fresh LB broth containing 100 µg/ml ampicillin and cultivated at 37˚C with agitation at 220 rpm until the optical density at 600 nm (OD600) reached 0.6. Subsequently, 0.5 mM isopropyl β-D-1-thiogalactopyranoside (Beijing Solarbio Science & Technology Co., Ltd.) was added, and expression was induced at 16˚C with agitation at 220 rpm for 18 h. The bacterial cultures before and after induction were collected, and the bacterial cells were harvested after induction. The bacterial cells were disrupted using an ultrasonicator (JY92-IIN; Ningbo Scientz Biotechnology Co., Ltd.) at a frequency of 20 kHz with 3 sec on/5 sec off cycles for 10 min in an ice bath, and then centrifuged at 12,000 x g for 30 min at 4˚C, after which the supernatant and precipitate were collected. The component containing the fusion protein was collected, and refolding was performed by sequential dialysis against renaturation buffer (50 mM Tris-HCl, pH 8.0, 0.5 M L-arginine, 5% glycerol, 1 mM dithiothreitol and 0.5 mM EDTA) containing decreasing urea concentrations [0.5 M for 6 h, then 0 M for 12 h with one buffer change, using a 10-kDa molecular weight cut-off (MWCO) dialysis membrane at 4˚C]. Afterwards, affinity chromatography on a nickel-nitrilotriacetic acid (Ni-NTA) column (GE Healthcare) was used for purification, and the target protein was eluted with 20, 80, 100, 150 and 300 mM imidazole solutions. Finally, proteins were separated by SDS-PAGE using a 12% separating gel and a 5% stacking gel, and then transferred onto a PVDF membrane (MilliporeSigma) at 100 V for 1 h at 4˚C. The membrane was blocked with 5% skimmed milk powder in TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% Tween-20) for 1 h at room temperature. Next, membranes were incubated with a mouse anti-His tag monoclonal antibody (cat.no. ab18184; 1:2,000 dilution; Abcam) for 3 h at 4˚C, followed by three washes with TBST for 10 min each. Subsequently, the membrane was incubated with an HRP-labelled goat anti-mouse antibody (cat. no. 115-035-003; 1:5,000 dilution; Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature, followed by three washes with TBST for 10 min each. Protein bands were visualized using a DAB substrate kit (cat. no. DA1015; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions.

First, 0.25 g chitosan (Sigma-Aldrich; Merck KGaA) was weighed and placed in a 150 ml beaker and 50 ml 5% acetic acid aqueous solution (pH 5.0) was added to it. The beaker was then placed on a magnetic stirrer and stirred at 400 rpm and 40˚C for 1 h. Subsequently, 0.25 g mannose (Sigma-Aldrich; Merck KGaA) and 0.25 g sodium triacetoxyborohydride (Sigma-Aldrich; Merck KGaA) were weighed, dissolved in 10 ml pure water, and the mixed aqueous solution was added dropwise into the chitosan solution using a dropper. The mixture was stirred at room temperature for 24 h. The resulting liquid was transferred into a 10 kDa dialysis bag (Repligen) and dialyzed against pure water on a magnetic stirrer at room temperature for 3 days to obtain an aqueous solution of MCS. After vacuum freeze-drying, the MCS was ready for use. The fusion protein was mixed with the MCS solution in a beaker (1:10) and stirred at 500 rpm on a magnetic stirrer for 30 min. Then, 1 mg/ml tripolyphosphate sodium (TPP; Sigma-Aldrich; Merck KGaA) was slowly added dropwise, and the mixture was stirred for 1 h to obtain MCS-Rv1768-Rv1983. The optimal mass ratio of MCS to TPP (C/T), optimal pH and optimal stirring time were determined through single-factor experiments (Figs. S1 and S2).

Potassium bromide was removed from the drying oven, placed under a tungsten filament lamp to dry, ground into powder form and mixed evenly with the sample.

For Fourier transform infrared spectroscopy (FTIR) analysis, the samples (MCS, chitosan and mannose) were ground with potassium bromide powder at a ratio of 1:100 and pressed into translucent pellets. FTIR spectra were recorded in the range of 4,000-400 cm^-1 with a resolution of 4 cm^-1 using a Nicolet iS10 spectrometer (Thermo Fisher Scientific, Inc.).

A particle size analyzer (ZS90; Malvern Instruments, Inc) was used to determine the particle size, polydispersity index and zeta potential of the nanoparticles at 25˚C. The nanoparticles were freeze-dried and dispersed into pure water. A pipette was used to drop the dispersed sample onto a sample carrier copper grid and allowed to air dry at room temperature. Then, the sample was placed under a transmission electron microscope (Libra 120; Zeiss AG) to observe the morphology of the nanoparticles.

The prepared MCS-Rv1768-Rv1983 solution was centrifuged at 13,684 x g for 30 min at 4˚C. The supernatant was collected, and the precipitate was resuspended in 1 ml of pure water to ensure complete resuspension. The resuspended precipitate was then placed in a freeze-dryer and dried for 24 h to form nanoparticle powder. The protein concentration of the nanoparticle solution before centrifugation and the concentration of unencapsulated protein in the supernatant were measured using a Nanodrop 8000 spectrophotometer (Thermo Scientific, Ltd.). The supernatant of the blank MCS nanoparticles was used as a control. Drug loading (DL; %) was calculated according to the following formula: DL (%)=[((C_t-C_b)-(C_s-C_bs)) x V/M] x100, where C_t is the protein concentration of the nanoparticle solution before centrifugation, C_b is the protein concentration of the blank nanoparticle solution, C_s is the protein concentration in the supernatant after centrifugation, C_bs is the protein concentration of the blank nanoparticle supernatant, V is the volume of the nanoparticle solution and M is the total mass of the dried nanoparticles.

The prepared nanoparticle solution was centrifuged at 13,684 x g for 30 min, after which the precipitate was collected, resuspended in 15 ml pure water, placed in a dialysis bag (10 kDa MWCO), sealed at both ends and immersed in 100 ml PBS (pH 7.4) on a magnetic stirrer at 37˚C and 100 rpm. Samples were taken at 0, 0.5, 1, 3, 6, 12, 24, 36, 48, 56 and 72 h. Each sample was replaced with an equal volume of fresh dialysis medium. The protein concentration at each time point was determined using the Nanodrop. The cumulative release rate Q was calculated as follows: Qn=C₁V₁ + C₂V₂ +…+ C_nV_n; Q=Qn/M x 100, where C₁ and C_n are the sample concentrations at different time points; V₁ and V_n are the sample volumes taken each time; Q_n is the cumulative release amount; and M is the total mass of protein in the nanoparticles.

The cytotoxic effects of the MCS nanoparticles on THP-1 cells were assessed using the Cell Counting Kit-8 Cell Proliferation and Cytotoxicity Assay Kit (cat. no. CA1210; Beijing Solarbio Science & Technology Co., Ltd.). Cells were cultured in 96-well plates at 5x10⁴ cells/well for 24 h at 37˚C in a 5% CO₂ incubator, containing 200 µl RPMI-1640 medium supplemented with 10% FCS (Gibco; Themo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Themo Fisher Scientific, Inc.). The cells were treated with MCS, Rv1768-Rv1983 and MCS-Rv1768-Rv1983 at 50, 100, 150 and 200 µM for 24 and 48 h, respectively. PBS-treated cells served as controls. Cell viability was determined using an ELISA reader (K6600; Beijing Kaiao Technology Development Co., Ltd.).

A total of 24 female C57BL/6 mice were randomly divided into four groups (n=6 per group) as follows: Control group, injected with 200 µl PBS only; MCS group, 0.50 mg MCS dissolved in 200 µl PBS; Rv1768-Rv1983 protein group, 40 µg Rv1768-Rv1983 dissolved in 200 µl PBS; and MCS-Rv1768-Rv1983 group, 0.50 mg MCS encapsulating 40 µg Rv1768-Rv1983 dissolved in 200 µl PBS. Sample size was determined using a power analysis based on the Schoenfeld formula for the log-rank test, with a two-sided significance level of α=0.05, power=80% and an expected hazard ratio of 0.5. Each group was subcutaneously injected once at weeks 0, 2 and 4. Blood samples (~100 µl per mouse per time point) were collected from the orbital sinus using heparinized capillary tubes under isoflurane anesthesia (3-4% for induction, 1-1.5% for maintenance, by inhalation). The first collection was performed 2 weeks after the first immunization (week 2, for T-cell activity analysis), and the second collection was performed at 2 weeks after the last immunization (week 6, for antibody detection), with an interval of 4 weeks between the two draws. To minimize local tissue damage, the two collections were performed from the contralateral eye (left eye for the first draw, right eye for the second draw). Afterwards, the mice were challenged with 5x10⁷ colony-forming units (CFUs) of H37Rv via tail vein injection. Mice were monitored twice daily for signs of morbidity. The humane endpoints established for the present study included: i) ≥20% loss of initial body weight; ii) persistent lethargy, hunched posture or reluctance to move; iii) respiratory distress (labored breathing); or iv) inability to reach food or water. Any mouse meeting at least one of these criteria was immediately euthanized. During the experiment, none of 24 mice reached these humane endpoints. After all the mice in the control group died, all surviving mice were euthanized by cervical dislocation under deep anesthesia induced by intraperitoneal injection of pentobarbital sodium (150 mg/kg). Lungs and spleens were dissected and ground in 1 ml PBS, serially diluted 10-fold and cultured on Löwenstein-Jensen medium plates (BD Biosciences) at 37˚C for 4 weeks. MTB colonies were counted, and the results were expressed as log₁₀ CFU per organ. Portions of lungs from three mice in each group were fixed with 4% paraformaldehyde for 24 h. The tissues were embedded in paraffin and 4-µm sections were cut. After deparaffinisation in xylene twice for 10 min each and rehydration through a graded ethanol series (100, 95, 80 and 70%; 5 min each), sections were examined via H&E and acid-fast staining (Ziehl-Neelsen method). H&E staining was performed with haematoxylin for 5 min and eosin for 2 min at room temperature. Ziehl-Neelsen staining was performed with carbol fuchsin for 10 min and methylene blue for 1 min at room temperature. Sections were observed under a Leica CW4000 microscope, and images were captured using Leica Microsystems GmbH.

Blood cells were isolated from the mice 2 weeks after the first immunization. After the red blood cells were lysed in ammonium chloride potassium buffer (Gibco; Thermo Fisher Scientific Inc.), 5x10⁵ peripheral blood mononuclear cells (PBMCs) isolated from mouse blood and cultured for 48 h in DMEM (Sigma-Aldrich; Merck KGaA) supplemented with 10% FCS, 100 U/ml penicillin and 100 mg/ml streptomycin. Single-cell suspensions were stimulated for 16 h at 37˚C and 5% CO₂ using the fusion protein (5 µg/ml). The cells were continuously cultured for another 5 h in the presence of GolgiPlug (BD Biosciences) according to the manufacturer's instructions. Non-specific binding was blocked by incubating cells with 5 µl FcR blocking reagent (cat. no. 422302; BioLegend, Inc.) per 10⁶ cells in 100 µl staining buffer for 10 min at room temperature. The activated cells were then surface-stained with an FITC-labelled anti-CD4 antibody (cat. no. 340133; 1:20 dilution; BD Biosciences) and a PerCP-Cy5.5-anti-CD8 antibody (cat. no. 341050; 1:40 dilution; BD Biosciences). After surface staining, cells were fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization Kit (cat. no. 554714; BD Biosciences) according to the manufacturer's instructions. Briefly, cells were resuspended in 250 µl BD Cytofix/Cytoperm solution and incubated for 20 min at 4˚C, followed by two washes with 1 ml BD Perm/Wash buffer (cat. no. 554723; BD Biosciences). The cells were then intracellularly stained with PE-anti-IFN-γ (cat. no. 340452; 1:50 dilution; BD Biosciences) and APC-anti-IL-4 (cat. no. 554436; 1:100 dilution; BD Biosciences) in BD Perm/Wash buffer. The numbers of specific cell types were quantified using a FACSAria flow cytometer (BD Biosciences), and data were analysed using FlowJo^™ software version 10.10 (BD Biosciences).

To measure the antibody levels of IgG1 and IgG2a, ELISA was used 2 weeks after the last vaccination, using recombinant Rv1768-Rv1983 fusion protein as a coating antigen. In brief, 2 µg/ml of the fusion protein was coated onto ELISA plates (Costar; Corning, Inc.) overnight at 4˚C in NaHCO₃ buffer (pH 9.6). Next, 5% BSA (Sigma-Aldrich; Merck KGaA) in PBS was added for 2 h at 37˚C. Afterwards, the plates were incubated for 2 h with serum samples at a 1:8 dilution. Subsequently, the bound antibodies were detected with HRP-conjugated goat anti-mouse IgG1 and IgG2a (cat. nos. A2554 and A2664; 1:3,000; Sigma-Aldrich; Merck KGaA) for 1 h at 37˚C. Between each step, plates were washed three times with PBS containing 0.05% Tween-20 (200 µl/well) for 3 min each. After being washed, the assay was developed in o-phenylenediamine dihydrochloride-H₂O₂ substrate solution (Sigma-Aldrich; Merck KGaA) for 15 min at room temperature in the dark. The reaction was stopped by adding 50 µl of 2 M H₂SO₄ per well,and the absorbance was measured at 492 nm using a microplate ELISA reader (K6600, Beijing Kaiao Technology Development Co., Ltd.).

RAW264.7 macrophages were cultured in RPMI-1640 supplemented with 10% FBS and penicillin/streptomycin. After 48 h, the cells were counted by Trypan blue staining. To evaluate the effects of antibodies against H37Rv invasion of macrophages, 1x10⁶ CFU of H37Rv were first incubated with serum (1:10) from different groups at 37˚C for 15 min. Then, the samples were centrifuged at 13,684 x g for 5 min at 4˚C, after which the supernatant was discarded. RAW264.7 macrophages (1x10⁶) were mixed with opsonized H37Rv in 2 ml medium. Phagocytosis was allowed to occur for 1 h at 37˚C in 6-well plates, each well containing a coverslip pre-treated with polylysine (0.1 mg/ml). RAW264.7 cells were seeded at a density of 1x10⁶ cells/well in 2 ml medium. The coverslip was removed, and the cells were fixed with ice-cold acetone at -20˚C for 10 min. The cells were then stained with auramine O (Sigma-Aldrich; Merck KGaA) for 15 min at room temperature and washed with PBS. Finally, the coverslips were observed under a fluorescence microscope (CW4000; Leica Microsystems GmbH). A total of 100 cells were counted from each coverslip. The phagocytic index was calculated as follows: Phagocytic index=total MTB captured by macrophages/number of macrophages capable of capturing bacteria.

The experimental data were analysed with GraphPad Prism 10.1 (Dotmatics). For normally distributed data, comparisons among multiple groups were performed by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls (SNK) post hoc test. For the cytotoxicity assay, data at each time point (24 and 48 h) were analyzed separately using one-way ANOVA with SNK post hoc test to compare differences among concentration groups; no statistical comparison was performed between the two time points. Survival curves were analysed using the Kaplan-Meier method with the log-rank (Mantel-Cox) test. Mycobacterial counts were log₁₀-transformed and analysed by one-way ANOVA. P<0.05 was considered to indicate a statistically significant difference.

Fragments of the Rv1768 and Rv1983 genes were amplified separately, with sizes of 1,854 and 1,674 bp, respectively (Fig. 1A). Using homologous arm recombination, Rv1768 and Rv1983 were ligated into the pET32a vector. The two genes were connected by a 21 bp flexible linker composed of alternating glycine and serine residues. Successful construction of the fusion expression vector was confirmed by electrophoresis and sequencing (Fig. 1B). After the induction, the Rv1768-Rv1983 fusion protein, with a molecular mass of ~129 kDa, was expressed in the form of inclusion bodies (Fig. 1C). Purification was performed using a Ni-NTA affinity chromatography column, and the target protein was obtained with a relatively high purity under 150 mM imidazole gradient elution (Fig. 1D). Western blot analysis further confirmed the successful expression and purification of the fusion protein (Fig. 1E). Bioinformatics analysis revealed no transmembrane regions in Rv1983 and Rv1768, suggesting that they are both extracellular proteins (Fig. S1).

In Fig. 2A, the synthesis pathway of MSC is illustrated. FTIR analysis revealed characteristic absorption peaks at the hydroxyl stretching vibration at 3,376 cm^-1, C-H stretching vibration at 2,927 cm^-1 and N-H bending vibration at 1,650 cm^-1, confirming the presence of primary amine groups. Additionally, the N-H bending vibration at 1,558 cm^-1 and C-N stretching vibration at 1,380 cm^-1 in the MCS indicated the formation of imine groups through reductive amination between mannose and chitosan, confirming the successful grafting of mannose onto chitosan (Fig. 2B). The optimal conditions for preparing MCS nanoparticles were determined as follows: MCS concentration of 1 mg/ml, TPP concentration of 1 mg/ml, C/T ratio of 5:1, pH of 5.0 and stirring time of 50 min (Figs. S2 and S3). The resulting MCS-Rv1768-Rv1983 nanoparticles had an average particle size of 196.3 nm, a polydispersity index of 0.297 and a zeta potential of -12.7 mV (Fig. 2C). When the concentration of the fusion protein was 0.4 mg/ml, the drug loading capacity was 8.02% (Fig. S4). Transmission electron microscopy revealed that the nanoparticles were elliptical in shape, uniform in morphology and well dispersed (Fig. 2D). The release profile of MCS-Rv1768-Rv1983 revealed an initial rapid release phase, reaching ~80% within 12 h, followed by a sustained release phase entering a plateau, with a cumulative release of 95% over 24 h (Fig. 2E). These data indicate that the encapsulation of the fusion protein by MCS provides a sustained release effect and efficient protein utilization.

Cytotoxicity analysis results revealed that the toxicity of MCS to THP-1 cells was extremely low. Compared with the PBS treatment group, after 24 h of interaction with different concentrations of MCS (50-200 µM), cell viability remained essentially unchanged (Fig. 3A). After 48 h, the cell viability remained >90% for all the tested MCS concentrations. Treatment with Rv1768-Rv1983 or MCS-Rv1768-Rv1983 for 24 and 48 h led to a moderate decrease in cell viability, but it remained >85% (Fig. 3B; P<0.05 for Rv1768-Rv1983 and P<0.01 for MCS-Rv1768-Rv1983 vs. PBS control). According to the ISO 10993-5:2009 guidelines for biological evaluation of medical devices, a cell viability of >70% is considered non-cytotoxic (24). The present results, showing cell viability consistently >85%, are well above this threshold.

To evaluate T-cell activation, the percentages of IFN-γ- and IL-4-producing CD4⁺ and CD8⁺ T cells in PBMCs were measured 2 weeks after the first vaccination. After ex vivo stimulation with the fusion protein for 16 h, the percentages of IFN-γ- and IL-4-producing CD4⁺ T cells were significantly greater in the Rv1768-Rv1983 and MCS-Rv1768-Rv1983 groups than in the control and MCS groups (Fig. 4A and B). One-way ANOVA with an SNK post hoc test revealed significant differences between the two immunization groups and the control groups (both P<0.01). For CD8⁺ T cells, the percentage of IFN-γ-producing cells was also significantly greater in the immunization groups than in the control groups (Fig. 4C; P<0.05), whereas the percentage of IL-4-producing CD8⁺ T cells was not significantly different among the groups (Fig. 4D; P>0.05).

To determine the induced humoral immune response, serum IgG1 and IgG2a subclasses were analysed. Compared with those in the control and MCS groups, the IgG1 titres in both the Rv1768-Rv1983 and MCS-Rv1768-Rv1983 groups were significantly greater; moreover, compared with the Rv1768-Rv1983 group, the MCS-Rv1768-Rv1983 group exhibited higher IgG1 levels (Fig. 5A; P<0.05 for MCS-Rv1768-Rv1983 vs. Rv1768-Rv1983). IgG2a levels were also significantly higher in both the immunization groups than in the control and MCS groups, but no significant difference was found between the Rv1768-Rv1983 and MCS-Rv1768-Rv1983 groups (Fig. 5B; P>0.05). After different anti-sera were incubated with H37Rv, the ability of the bacteria to invade macrophages was notably altered. Compared with the Rv1768-Rv1983 group, the MCS-Rv1768-Rv1983 group had a significantly lower phagocytic index (Fig. 5C; P<0.01), indicating that MSC encapsulation enhances antibody-mediated inhibition of MTB invasion.

Survival analysis revealed that compared with the control group, the MCS-Rv1768-Rv1983 group had significantly longer survival (log-rank test, P<0.05), whereas the survival rate of the Rv1768-Rv1983 group did not significantly change (Fig. 6A; P>0.05). Bacterial loads in murine lungs and spleens were determined at 4 weeks after sacrifice. Compared with the control groups, bacterial counts in the lungs were significantly lower in both the MCS-Rv1768-Rv1983 (P<0.01) and Rv1768-Rv1983 groups (P<0.05), while compared with the MCS group, bacterial counts in the lungs were significantly lower in the MCS-Rv1768-Rv1983 group (P<0.05) (Fig. 6B). Notably, compared with the Rv1768-Rv1983 group, the MCS-Rv1768-Rv1983 group exhibited a stronger inhibitory effect on bacterial proliferation. In the spleen, the bacterial counts did not significantly differ among the groups (Fig. 6C; P>0.05). Histopathological examination of lung sections revealed that the control group had marked inflammatory reactions, including alveolar consolidation and fusion. By contrast, both the MCS-Rv1768-Rv1983 and the Rv1768-Rv1983 groups showed less alveolar fusion, reduced swelling and more prominent air spaces (Fig. 6D). Acid-fast staining also revealed fewer acid-fast bacilli in the lungs of MCS-Rv1768-Rv1983-immunized mice than in those of control mice (Fig. 6D; upper panels).