Specific-pathogen-free female BALB/c mice (4–8 weeks old), from Instituto Butantan – Central Animal Facility, were maintained in ABSL-2 racks fitted with a HEPA-filtered air intake and exhaust system. They were kept at the animal care facility of the Laboratório de Desenvolvimento de Vacinas, with water and food provided ad libitum. The temperature was maintained from 20–24°C, relative humidity of 40–70%, and a 12 h light/dark cycle. This study was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the Committee of SBCAL (Sociedade Brasileira de Ciência em Animais de Laboratório) recommendations and was approved by the Animal Research Ethical Committee of Instituto Butantan (number: 3435250619).

The rBCG-LTAK63 strain used in this work was previously described (20). BCG or rBCG-LTAK63 were grown in Middlebrook 7H9 (Difco, Detroit, MI, USA) supplemented with 10% of OADC (oleic acid-albumin-dextrose-catalase; BBL, Cockeysville, MD, USA), 0.5% glycerol and 0.05% Tween 80 (7H9-OADC) or plated on Middlebrook 7H10 agar supplemented with 0.5% glycerol and OADC (7H10-OADC).

To evaluate long-term immune response and protection, groups of mice (n=5) were immunized with BCG or rBCG-LTAK63 (1x10⁶ CFU/100 μL) resuspended in phosphate-buffered saline (PBS- 137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 2 mM KH₂PO₄) and administered subcutaneously in the back of the animals.

The intranasal infection was performed as described by Logan et al. (2008) (21). A frozen vial of Mycobacterium tuberculosis H37Rv (kept at -80°C) was thawed, and the inoculum was adjusted to 1.25x10⁴ CFU/mL with PBS. Ninety and 180 days after immunization, the groups of mice were intranasally challenged with the Mtb suspension (500 CFU/40 µL in one nostril). To confirm the bacterial load used, a single mouse from each group was euthanized at day 1 post-inoculation, and the lung homogenates were plated on 7H11-OADC agar. To determine protection, thirty days after infection, animals were euthanized, and the anterior and mediastinal right-lung lobes were collected, homogenized, and plated on 7H11-OADC agar. The bacterial load was determined by counting the CFU numbers after 14-21 days of incubation at 37°C.

Flow cytometry analysis for specific effector T cell and memory T cell were performed as described in previous protocols (22, 23). Briefly, 90 and 180 days after the immunization, axillar draining lymph nodes and lung lobes were collected. Draining lymph nodes were prepared as single-cell suspensions using 70-µm cell strainers (BD Biosciences), and the cells were resuspended in RPMI-1640 medium supplemented with 10% fetal calf serum, 0.15% sodium bicarbonate, 1% L-glutamine and 1% nonessential amino acids.

Lung lobes were digested with DNAse IV (30 µg/mL) and collagenase III (0.7 mg/mL) for 30 min at 37°C. The digested lungs were prepared as single-cell suspensions using 70-µm cell strainers and erythrocytes lysed using an RBC lysing solution (0.15 M NH₄Cl, 10 mM KHCO₃). For both organs, viable cells were counted in a Neubauer chamber using Tripan Blue (0.2%), and cell concentration was adjusted to 1×10⁶ cells/mL. All reagents were purchased from Sigma-Aldrich^®, Merck KGaA, St. Louis, MO, USA.

Cells were plated in 96-well plates (CellWells™) and stimulated with 10 µg of BCG CFP (“culture filtrate protein”, a proteinaceous supernatant of a BCG grown in Sauton medium for 14 days and concentrated through a 5,000 MWCO filter), ConA (positive control) or left unstimulated and incubated at 37°C and 5% CO₂ for 4 h. Then, monensin (3 µM; eBioscience) was added and cultures were further incubated for another 4 h. Cells were then treated with 0.1% sodium azide (Sigma-Aldrich) in PBS for 30 min at room temperature and centrifuged at 400 x g for 15 min. The cellular phenotype was determined by permeabilization with Perm Fix/Perm Wash (BD Pharmingen) and incubation for 30 min with the following conjugated antibodies: TNF-α-FITC (clone MP6-XT22), IFN-γ-PE (clone XMG1.2), CD4-PerCP (clone RM4-5), CD44-APCcy7 (clone IM7), IL-17-BV421 (clone TC11-18H10), CD62L-FITC (clone MEL-14), PD-1-PE (clone J43), KLRG1-APC (clone 2F1).

Cell acquisition of 70,000 (draining lymph nodes) and 200,000 (lungs) total events per sample was performed using a BD FACS Canto II flow cytometer and data analyzed using FlowJo™ v10 Software (BD Life Sciences).

The CD4⁺ effector T cell population was characterized as to expression of IFN-γ, TNF-α and/or IL-17, either as single, double, or triple-positive cells. Memory T cell population was characterized as: naïve (CD4⁺CD44^-CD62L⁺), central memory (TCM-CD4⁺CD44⁺CD62L⁺), effector memory (TEM-CD4⁺CD44⁺CD62L^-), and tissue resident memory (TRM-CD4⁺PD-1⁺KLRG1^-) cells.

The gating strategy for all memory T cell subsets is shown in Supplementary Materials. Supplementary Figure 1 depicts gating for naïve/TEM/TCM (Supplementary Figure 1A) and an example of analysis in the lymph node for each group in both time points (Figure 1B). Supplementary Figure 2 shows gating for TRM (Supplementary Figure 2A) and an example of analysis in the lungs for each group in both time points (Figure 2B). Supplementary Figure 3 displays an example of lymph nodes analysis, based on FMO of a single functional T cell, producing IFN-γ (Supplementary Figure 3A), TNF-α (Supplementary Figure 3B), or IL-17 (Supplementary Figure 3C). Cytokine events were background corrected based on this FMO.

The number of cells in each organ was quantified by multiplying the percentage of cells in each gate by the number of live cells counted in the Neubauer chamber.

Results were tabulated using the software GraphPad Prism 9 (GraphPad, La Jolla, CA, USA). The violin plot was plotted in Origin (Pro), Version Number (2022b – OriginLab Corporation, Northampton, MA, USA). The differences between groups were assessed using one-way ANOVA. Differences in p values < 0.05 were considered statistically significant. All biological experiments were performed at least twice, repeating the immunization and assessments of immune response and protection”.

Protection against TB is correlated with an increased Th1/Th17 cytokine response observed at the time of challenge (Figure 1A). In agreement, here we show that mice immunized with rBCG-LTAK63 displayed a general increase in the Th1 and Th17 cell populations. At 90 days after immunization, there was an increase of a diverse milieu of CD4⁺ T cells expressing TNF-α, IFN-γ and IL-17 either alone or in combination (double and triple polyfunctional cells) in draining lymph nodes and lungs (Figures 1B, C).

In the lymph nodes, rBCG-LTAK63 immunization induced an increased percentage of CD4⁺ single TNF-α and IL-17-producing cells, and combinations of double TNF-α, IFN-γ, and IL-17-producing cells at 90 days. The most significant differences in terms of percentage and in the difference as compared to BCG were in CD4⁺TNF-α⁺ single positive, CD4⁺IFN-γ⁺IL-17⁺ (double positive), and the triple polyfunctional T cells (Figure 1B). In the lungs as the target organ, the single CD4⁺ T cells producing IFN-γ and TNF-α, and the double polyfunctional T cells were also increased as compared to BCG. In this case, the largest differences were seen with the CD4⁺IFN-γ⁺ and the triple positive CD4⁺TNF-α⁺IFN-γ⁺IL-17⁺ T cells (Figure 1C).

Regarding the numbers of polyfunctional T cells in the lymph node, the triple positive CD4⁺TNF-α⁺IFN-γ⁺IL-17⁺ T cells showed the largest difference compared to BCG (Figure 1C). In the lungs, the double positive CD4⁺TNF-α⁺IL-17⁺ T cells were in larger numbers in the rBCG-LTAK63-immunized animals as compared to the BCG group, with a corresponding decrease in the numbers of CD4⁺IFN-γ⁺IL-17⁺ T cells (Figure 1D).

Since an increased presence of effector CD4⁺ T cells was observed until 90 days after the immunization with rBCG-LTAK63, we assessed vaccine-induced memory T cells in the draining lymph nodes and lungs of immunized mice. In the lymph nodes, mice immunized with rBCG-LTAK63 displayed a tendency to decrease the naïve T cell population and significantly increased TCM and TEM cells as compared to BCG (Figure 2A). In the lungs, the same tendency was observed; in this case, rBCG-LTAK63 immunization displayed significantly larger percentages of the TCM and TEM cell populations. There was a trend to an increase in TRM in rBCG-LTAK63-immunized animals as compared to the saline group; however, this increase was not significant (p value 0.17) (Figure 2B).

We had previously shown that rBCG-LTAK63-immunization induces protection against Mtb challenge in the intratracheal model of infection, 90 days after immunization (20). Hence, we here confirmed protection against Mtb challenge using the intranasal model, 90 days after immunization with rBCG-LTAK63. Animals were administered 500 CFU of M. tuberculosis H37Rv intranasally and the bacterial load in the lungs was measured thirty days after the challenge. Also in the intranasal infection model, rBCG-LTAK63 immunization induces better protection than BCG, reducing the bacillary load by more than two logs as compared to the non-immunized group and one log as compared to BCG (Figure 2C).

To determine if the enhanced TEM and TCM cells at 90 days could increase the duration of protection, mice were immunized subcutaneously with 10⁶ CFU (BCG or rBCG-LTAK63), and we assessed TEM and TCM generation, and protection against challenge 180 days later. At 180 days, the CD4⁺ T cells expressing TNF-α, IFN-γ, or IL-17 remained at higher levels in rBCG-LTAK63-immunized animals in comparison to the BCG group in both organs (Figures 3A, B). In the lymph nodes, only CD4⁺TNF-α⁺IL-17⁺ double positive is present at a higher level (Figure 3A), while in the lungs, CD4⁺TNF-α⁺IL-17⁺, CD4⁺IFN-γ⁺IL-17⁺ double positives are increased, together with the triple-positive T cells (Figure 3B).

Regarding the number of polyfunctional T cells in the lymph node, the double positive CD4⁺IFN-γ⁺IL-17⁺ T cells displayed the largest difference compared to BCG (Figure 3C). In the lungs, the double positive CD4⁺TNF-α⁺IFN-γ⁺ T cells were in larger numbers in both groups, but the CD4⁺IFN-γ⁺IL-17⁺ T cells were higher in the rBCG-LTAK63 group (Figure 3C).

An increase in TEM cell populations occurs in both organs at 180 days after rBCG-LTAK63 immunization, while there is no alteration of TEM in BCG groups (Figures 4A, B). Only rBCG-LTAK63 showed a higher percentage of TCM in the draining lymph node, as compared to BCG (Figure 4A). There is also an increase in TRM cells in the lungs of rBCG-LTAK63-immunized animals (Figure 4B).

In terms of protection, even after 180 days, rBCG-LTAK63 immunization sustained higher protection against intranasal challenge with Mtb, reducing the bacillary load in the animals’ lungs by nearly two logs (Figure 4C).

Mice were immunized with BCG or rBCG-LTAK63, challenged with Mtb 180 days later, and the memory cells (naive, TCM, and TEM) were examined using flow cytometry 30 days later. TEM response in infected animal lymph nodes was higher in animals immunized with rBCG-LTAK63 than in those only infected (Figure 5A). In the lungs, TEM was higher in rBCG-LTAK63 group than in both the BCG and infected groups (Figure 5B). TCM population showed no significant difference between groups in both organs as compared to BCG.

The increase in the TEM population in the infected animal’s lungs indicates a possible differentiation from TCM into TEM and further into effector cells. Therefore, we also evaluated the Th1 and Th17 responses. The infection with Mtb increases CD4⁺TNF-α⁺, CD4⁺IFN-γ⁺, and CD4⁺IL-17⁺ in the lymph nodes of rBCG-LTAK63 immunized animals (Figure 5C), while in the lungs, there was a drastic difference in CD4⁺IFN-γ⁺, and CD4⁺IL-17⁺, when compared with BCG (Figure 5D).

Finally, we compared the cell population dynamics across all time points during a longer period of immunization and infection. Regardless of the vaccine used, we can see that after a long period of immunization (180 dpi - before challenge), there is a tendency to decrease in all populations studied, most notably in the lungs of animals (Figure 6). Following infection, there is a decrease in the population of TCM cells in both organs and a considerable increase in the TEM cells in the lungs of the animals immunized with rBCG-LTAK63 (Figures 6G–J). At the same time, there is an increase in the TNF-α (6B) and IL-17 (6F) producing CD4⁺ T cells in the lungs of the rBCG-LTAK63 group, but they remain stable in the BCG group.