Characterization of the Protective Immune Responses Conferred by Recombinant BCG Overexpressing Components of Mycobacterium tuberculosis Sec Protein Export System

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the only approved vaccine against tuberculosis (TB). However, its efficacy in preventing pulmonary TB in adults is limited. Despite its variable efficacy, BCG offers a number of unique and beneficial characteristics, which make it suitable as a vaccine vehicle to express recombinant molecules. In Mycobacterium tuberculosis, the general Sec pathway is an essential cellular process, and it is responsible for exporting the majority of proteins across the cytoplasmic membrane, including potent immune-protective antigens, such as members of the antigen 85 (Ag85) complex. We engineered BCG to overexpress the M. tuberculosis SecDFG proteins in order to improve the efficiency of the Sec-dependent export system and, thus, enhance the secretion of immunogenic proteins. BCGSecDFG displayed increased intracellular survival within macrophages in vitro and greater persistence in the lymphoid organs of vaccinated mice than parental BCG. In addition, vaccination with BCGSecDFG generated higher numbers of IFN-γ-secreting T cells in response to secreted mycobacterial antigens compared to BCG, particularly members of the Ag85 complex. Furthermore, vaccination with BCGSecDFG significantly reduced the bacterial load in the lungs and spleens of M. tuberculosis-infected mice, which was comparable to the protection afforded by parental BCG. Therefore, the modification of protein secretion in BCG can improve antigen-specific immunogenicity.


Introduction
Mycobacterium tuberculosis, the causative agent of TB, continues to spread due to the delay in detecting patients with active pulmonary TB [1] and the existence of a large reservoir of latently infected individuals, whose infection can reactivate over their lifetime. The current TB vaccine BCG is protective against severe forms of TB in children (meningitis and miliary TB), but confers inconsistent and variable protection against pulmonary TB in adolescents and adults, which is the transmissible form of the disease [2,3]. Previous sensitization with environmental mycobacteria or M. tuberculosis infection is considered one of the main factors associated with BCG lower efficacy against pulmonary tuberculosis [4]. This failure of BCG to provide consistent protection against adult pulmonary TB makes new and more effective TB vaccines critically important. Although BCG's protective efficacy is

Generation of BCG SecDFG Strains
The construction of recombinant BCG overexpressing the SecDFG fusion protein was performed using the pMOD12-expression plasmid, which contains two independent and constitutively active promoters from BCG, HSP60, and HSP70, with two disparate multiple-cloning sites (MCSs) [27]. The DNA fragment codings for M. tuberculosis histagged SecD (Rv2587c) and SecF (Rv2586c) were inserted into the MCS1 of pMOD12 under the control of Phsp60, whereas c-Myc-SecG encoding-gene (Rv1440) was ligated into the MCS2 of the plasmid under Phsp70 promoter. The resultant plasmid was used to transform competent BCG and recombinants selected after plating on solid 7H11 media with kanamycin (50 µg/mL). The recombinant strain generated was called BCG SecDFG .

Macrophage Infection
RAW 264.7 murine macrophage cells (ATCC TIB-71) were plated at 2 × 10 5 cells/well and BCG pMOD12 or BCG SecDFG were then added to the cells at a multiplicity of infection (MOI) of 1:1 or 1:5 in RPMI media supplemented with 10% fetal bovine serum (FBS). Cell culture was incubated (37 • C, 5% CO 2 ) for 4 h, extracellular bacteria removed by extensive washing, and colony forming units (CFU) determined by lysis of cells and growth on supplemented 7H11 agar medium.

Vaccination of Mice and M. tuberculosis Challenge
All murine experiments were performed in strict accordance with the approval granted by the University of Sydney Animal Ethics Committee (K75/9-2012/3/5846). Female C57BL/6 mice, aged 6-8 weeks old, were purchased from the Animal Resources Center (Perth, Australia). For the adoptive transfer studies, P25-TCR-transgenic mice with transgenic expression of a TCR specific for I-A b -Ag85B 240-254 peptide generated on a C57BL/6 were utilized. For subcutaneous (s.c.) vaccine administration, mice were anaesthetized with gaseous isofluorane (4%) and injected at the base of tail with either 5 × 10 5 CFU of BCG SecDFG , the same amount of BCG carrying the empty plasmid vector (BCG pMOD12 ), or left unvaccinated as negative control. Eight weeks after vaccination, mice were challenged with M. tuberculosis H37Rv in an inhalation exposure system using a Middlebrook airborne infection apparatus (Glas-Col, Terre Haute, IN, USA) with an infective dose of 100 CFU. Four weeks after challenge, serial dilutions of lungs and spleen homogenates were plated on supplemented Middlebrook 711 agar (Difco™ BD) to enumerate the bacterial loads. All experiments involving M. tuberculosis were performed in a Physical Containment Level 3 laboratory at the Centenary Institute.

Flow Cytometry
For the analysis of antigen-specific cytokine production by T cells, single-cell suspensions (5 × 10 6 cells/mL) were stimulated for 3-4 h (37 • C, 5% CO 2 ) with P25 peptide, CFP, or concentrated BCG SecDFG supernatant (10 µg/mL) in the presence of Brefeldin A (10 µg/mL; Sigma-Aldrich). Cell suspensions were incubated with CD16/CD32 (2.4G2; BD Biosciences) to block Fc receptors and then stained with Fixable Blue Dead Cell stain (Thermo Fisher Scientific) and fluorophore-conjugated monoclonal antibodies against surface markers (Supplementary Table S1). Cells were fixed and permeabilized using BD Cytofix/Cytoperm kit (BD Biosciences). Fluorophore-conjugated monoclonal antibodies against intracellular markers (Supplementary Table S2) were diluted in BD Perm/Wash (BD Biosciences) for intracellular staining. Immunostained cells or beads were fixed in 10% buffered formalin prior to data acquisition using an LSR-Fortessa analyzer (BD Biosciences). Acquired data was analyzed using FlowJo analysis software (Treestar Inc., Ashland, OR, USA) using the gating strategy illustrated in Supplementary Figure S1. A Boolean gate combination was used to calculate the frequency of single-or multiple-cytokine-positive cell subsets.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA, USA). The significance of differences between experimental groups was evaluated by one-or two-way analysis of variance (ANOVA), with pairwise comparison of multi-grouped data sets achieved using Tukey's honest significant difference (HSD) post hoc test. The results were considered significant when the p values were ≤0.05.

Construction and Characterization of rBCG Strains Overexpressing the SecDFG Components from the M. tuberculosis Sec Export System
A dual-promoter expression plasmid, pMOD12, was used to construct the rBCG strain overexpressing the recombinant proteins SecD, SecF, and SecG [27], termed BCG SecDFG ( Figure 1A). Bands of the expected size for SecD (60.23 kDa) and Sec F (47.01 kDa) were detected by immunoblot compared to parental BCG, confirming the overexpression in the recombinant strains ( Figure 1B). However, the expression of SecG (8.16 kDa) was not detected in the isolates tested ( Figure 1B), possibly due to the limitations of detecting low-molecularweight products [30]. To confirm the functional impact of the SecDFG overexpression, an immunoblot was performed to determine the presence of the immunodominant Ag85 proteins in the extracellular milieu. A band with a size of~34 kDa was detected with higher intensity in the BCG SecDFG clones compared to normal BCG ( Figure 1C), suggesting that the overexpression of the SecDFG altered the secretion of Ag85 proteins to the extracellular environment. The BCG SecDFG displayed a reduced growth rate in the culture compared to the wild-type BCG, suggesting the impact of protein overexpression on growth ( Figure 1D).

Construction and Characterization of rBCG Strains Overexpressing the SecDFG Components from the M. tuberculosis Sec Export System
A dual-promoter expression plasmid, pMOD12, was used to construct the rBCG strain overexpressing the recombinant proteins SecD, SecF, and SecG [27], termed BCG S-ecDFG ( Figure 1A). Bands of the expected size for SecD (60.23 kDa) and Sec F (47.01 kDa) were detected by immunoblot compared to parental BCG, confirming the overexpression in the recombinant strains ( Figure 1B). However, the expression of SecG (8.16 kDa) was not detected in the isolates tested ( Figure 1B), possibly due to the limitations of detecting low-molecular-weight products [30]. To confirm the functional impact of the SecDFG overexpression, an immunoblot was performed to determine the presence of the immunodominant Ag85 proteins in the extracellular milieu. A band with a size of ~34 kDa was detected with higher intensity in the BCG SecDFG clones compared to normal BCG ( Figure  1C), suggesting that the overexpression of the SecDFG altered the secretion of Ag85 proteins to the extracellular environment. The BCG SecDFG displayed a reduced growth rate in the culture compared to the wild-type BCG, suggesting the impact of protein overexpression on growth ( Figure 1D).  . Cell lysates (20 µg) were prepared, separated on a 12% SDS-PAGE gel, and transferred to nitrocellulose membrane, and polyhistidine-tagged SecDF was detected using Ni-HRP conjugate for secondary detection of his-tagged proteins (Lane 1: BCG pMOD12 , Lane 2: BCG SecDFG clone 1, Lane 3: BCG SecDFG clone 2). (C). Culture supernatants and cell lysates (20 µg) were examined by immunoblot using anti-Ag85 complex polyclonal antibody. (D). Bacterial growth in 7H9 medium (OD 600 ). Values represent mean ± SD of two independent cultures. E-F. RAW 264.7 murine macrophage cells were infected with rBCG strains a MOI of 1 (E) or 5 (F) and CFU was determined at indicated timepoints. Data are the means ± SEM (n = 6) and are representative of two independent experiments. Statistical significance between groups was determined by ANOVA with Sidak's multiple-comparisons test (* p < 0.05; ** p < 0.01; **** p < 0.0001).

Increased In Vitro Intracellular Persistence and In Vivo Immunity by BCG SecDFG
We hypothesized that the improved secretion of proteins may alter the interaction with host cells. This could be due to increased secretion of virulence/persistence determinants or enhanced cell-association via improved export of receptor-binding molecules, such as Ag85B [31]. RAW264.7 murine macrophage cells were infected with BCG pMOD12 or BCG SecDFG and the bacterial load was determined at various timepoints. Although there was no difference in the initial uptake by the BCG SecDFG s at 4 h post-infection compared to the cells infected with the parental BCG, the BCG SecDFG displayed a significant increase in recovered CFU at 168 h following infection at an MOI of 1 ( Figure 1E) and, at 72 h, at an MOI of 5 ( Figure 1F) compared to the BCG pMOD12 . Thus, the SecDFG expression appeared to alter the capacity of the BCG to survive/replicate long-term within the host cells.
To determine the impact of the SecDFG on the in vivo persistence, the mice were vaccinated with either the BCG pMOD12 or the BCG SecDFG , and the kinetics of the BCG growth were measured by enumerating the bacterial load in the organs. In the draining lymph nodes (DLNs), similar numbers of CFU were observed in both vaccinated groups at day 1 following the vaccination. However, the BCG SecDFG demonstrated greater persistence than the parental strain in the DLNs and spleens at day 7 post-vaccination (Figure 2A). In the lungs and spleens, the numbers of the BCG SecDFG declined to undetectable levels by day 84, but remained elevated in the DLNs, indicating enhanced dissemination to this site.
To determine whether the difference in the expressions of the secretion-system components in the BCG SecDFG had an effect on the immune response generated, a panel of secreted mycobacterial proteins (Ag85A, Ag85B, Ag85C, and MPT32) was used to stimulate the lymphocytes from the BCG pMOD12 and the BCG SecDFG -vaccinated mice, and the frequency of the antigen-specific IFNγ-producing cells was determined by ELISPOT. In the spleens, the increased immunogenicity induced by the BCG SecDFG compared to the parental BCG was apparent, particularly for the three Ag85 antigens, at day 28 post-vaccination ( Figure 2B). The IFN-γ responses to the CFP were highest in the BCG SecDFG -vaccinated mice at 28 days, demonstrating the persistent immune responses to these antigens after the vaccination of the mice. In the DLNs, the BCG SecDFG responses were typically compared to those of the To determine whether the difference in the expressions of the secretion-system components in the BCG SecDFG had an effect on the immune response generated, a panel of secreted mycobacterial proteins (Ag85A, Ag85B, Ag85C, and MPT32) was used to stimulate the lymphocytes from the BCG pMOD12 and the BCG SecDFG -vaccinated mice, and the frequency of the antigen-specific IFNγ-producing cells was determined by ELISPOT. In the spleens, the increased immunogenicity induced by the BCG SecDFG compared to the parental BCG was apparent, particularly for the three Ag85 antigens, at day 28 post-vaccination ( Figure 2B). The IFN-γ responses to the CFP were highest in the BCG SecDFG -vaccinated mice at 28 days, demonstrating the persistent immune responses to these antigens after the vaccination of the mice. In the DLNs, the BCG SecDFG responses were typically compared to those of the control mice, but these differences only reached significance after the stimulation with the M. tuberculosis CFP ( Figure S2).
The increased persistence of the BCG SecDFG in the lymphoid organs, together with the increased IFN-γ responses in the vaccinated mice, suggested the vaccine may induce the increased priming of T cells. To test this, p25 transgenic CD4 + T cells specific for the dominant P25 epitope of Ag85B (CD45.1 + ) were transferred to recipient C57BL/6 mice (CD45.2 + ), and the next day, the mice were vaccinated s.c. with the BCG strains ( Figure  3A). The mice that received the BCG SecDFG had significantly higher numbers of p25 CD4 + T The increased persistence of the BCG SecDFG in the lymphoid organs, together with the increased IFN-γ responses in the vaccinated mice, suggested the vaccine may induce the increased priming of T cells. To test this, p25 transgenic CD4 + T cells specific for the dominant P25 epitope of Ag85B (CD45.1 + ) were transferred to recipient C57BL/6 mice (CD45.2 + ), and the next day, the mice were vaccinated s.c. with the BCG strains ( Figure 3A). The mice that received the BCG SecDFG had significantly higher numbers of p25 CD4 + T cells in the LNs and spleens compared to the mice that received the BCG pMOD12 ( Figure 3B). A similar pattern of cytokine-producing P25 CD4 + T cells was observed from the LN cells, with both BCG strains generating a high proportion of triple-positive IFN-γ + IL-2 + TNF + CD45.1 + T cells at day 6 post-vaccination ( Figure 3C). cells in the LNs and spleens compared to the mice that received the BCG pMOD12 ( Figure 3B). A similar pattern of cytokine-producing P25 CD4 + T cells was observed from the LN cells, with both BCG strains generating a high proportion of triple-positive IFN-γ + IL-2 + TNF + CD45.1 + T cells at day 6 post-vaccination ( Figure 3C). Figure 3. Expansion of transferred p25-specific CD4 + T cells following BCG SecDFG vaccination. Approximately 5 × 10 5 CFSE-labeled p25 splenocytes (CD45.1 + ) were transferred via i.v. injection to C57BL/6 (CD45.2 + ) recipient mice (n = 4) prior to vaccination. The following day, mice were vaccinated s.c. with 5 × 10 5 CFU of BCG pMOD12 or BCG SecDFG , or left unvaccinated. Six days after the vaccination, the numbers of lymphocytes were enumerated in the DLNs and spleens. Representative flow plots are shown (A), together with CD45.1 + P25-specific CD4 + T numbers (B), as measured by flow cytometry. (C). Intracellular cytokine staining was performed on lung cells at 4 weeks postinfection after re-stimulation with CFP (10 μg/mL) and Brefeldin A, and the frequency of IFN-γ, IL-2, and/or TNF positive CD4 + T cells was assessed. Data are representative of two independent experiments. Statistical significance was determined by ANOVA with Tukey's multiple-comparisons test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; gating strategy is shown in Supplementary Materials.

Protective Efficacy of BCG SecDFG against Aerosol M. tuberculosis Infection
Next, we determined whether the modification of the protein secretion by the BCG affected the protective efficacy. Ten weeks following s.c. immunization with BCG pMOD12 or BCG SecDFG , the mice were challenged with a low dose of M. tuberculosis H37Rv and, 4 weeks later, the bacterial numbers in the lungs ( Figure 4A) and spleens were enumerated ( Figure  4B). In the lungs, both the BCG pMOD12 and the BCG SecDFG vaccinations provided significant protection compared to the unvaccinated mice at 4 weeks post-M. tuberculosis challenge ( Figure 4A). There was no significant difference between the BCG SecDFG and the parental strain in the capacity to reduce the M. tuberculosis burden, with a similar reduction in M. tuberculosis in the lungs (1.45 and 1.31 log10, respectively). In the spleens, the BCG SecDFG provided increased protection, with a reduction in M. tuberculosis by approximately 0.20 log10 CFU compared to the control BCG, although this difference was not statistically sig-

Protective Efficacy of BCG SecDFG against Aerosol M. tuberculosis Infection
Next, we determined whether the modification of the protein secretion by the BCG affected the protective efficacy. Ten weeks following s.c. immunization with BCG pMOD12 or BCG SecDFG , the mice were challenged with a low dose of M. tuberculosis H37Rv and, 4 weeks later, the bacterial numbers in the lungs ( Figure 4A) and spleens were enumerated ( Figure 4B). In the lungs, both the BCG pMOD12 and the BCG SecDFG vaccinations provided significant protection compared to the unvaccinated mice at 4 weeks post-M. tuberculosis challenge ( Figure 4A). There was no significant difference between the BCG SecDFG and the parental strain in the capacity to reduce the M. tuberculosis burden, with a similar reduction in M. tuberculosis in the lungs (1.45 and 1.31 log 10 , respectively). In the spleens, the BCG SecDFG provided increased protection, with a reduction in M. tuberculosis by approximately 0.20 log 10 CFU compared to the control BCG, although this difference was not statistically significant ( Figure 4B). However, the BCG SecDFG -vaccinated mice generated an increased percentage of IFN-γ + IL-2 + TNF + lung CD4 + T cells following the M. tuberculosis aerosol challenge compared with the mice that received the parental BCG strain ( Figure 4C). Overall, these results indicate that the BCG SecDFG was protective against M. tuberculosis infection and maintained improved vaccine-specific immunity after the challenge. nificant ( Figure 4B). However, the BCG SecDFG -vaccinated mice generated an increased percentage of IFN-γ + IL-2 + TNF + lung CD4 + T cells following the M. tuberculosis aerosol challenge compared with the mice that received the parental BCG strain ( Figure 4C). Overall, these results indicate that the BCG SecDFG was protective against M. tuberculosis infection and maintained improved vaccine-specific immunity after the challenge.  6) were immunized s.c. with 5 × 10 5 CFU of BCG pMOD12 (blue) or BCG SecDFG (red), or PBS as control (black), and 10 weeks post-immunization, mice were challenged with ~100 CFU of M. tuberculosis H37Rv via aerosol route. The bacterial loads in lungs (A) and spleens (B) were enumerated following culture on Middlebrook 7H11 agar. (C). Intracellular cytokine staining was performed on lung cells at 4 weeks post-infection after re-stimulation with CFP (10 μg/mL) and Brefeldin A, and the frequency of IFN-γ, IL-2, and/or TNF-positive CD4 + T cells was assessed by flow cytometry. The data are the means ± SEM and representative of two independent experiments. The numbers on top of the bar graphs indicate the Log protection compared to the unvaccinated group. Statistical significance was determined by ANOVA with Tukey's multiplecomparisons test (* p < 0.05; ** p < 0.01; **** p < 0.0001).

Discussion
BCG is the only approved vaccine against TB, but in low and middle-income countries, the protection provided by BCG is insufficient to contain transmissions. Hence, there is an urgent need to improve BCG efficacy to make it more effective and long-lasting in controlling and preventing TB infection. In this report, we sought to improve BCG immunity by altering its protein-secretion system, thus potentially increasing the quality and magnitude of the immune responses against secreted proteins [32]. Indeed, we observed

Discussion
BCG is the only approved vaccine against TB, but in low and middle-income countries, the protection provided by BCG is insufficient to contain transmissions. Hence, there is an urgent need to improve BCG efficacy to make it more effective and long-lasting in controlling and preventing TB infection. In this report, we sought to improve BCG immunity by altering its protein-secretion system, thus potentially increasing the quality and magnitude of the immune responses against secreted proteins [32]. Indeed, we observed an increased secretion of Ag85 proteins, which are key immunogenic proteins of mycobacteria [19]. Previous studies investigated the impact of the heterologous expression of proteins from the ESX-1 secretion machinery on the quality and magnitude of the immune responses against M. tuberculosis infection [32,33]. A recombinant BCG-overexpressing ESX-1 was shown to provide superior protection against highly virulent M. tuberculosis relative to parental BCG, stimulating increased proportions of polyfunctional CD4 + T cells and strong CD8 + T-cell-effector responses [32,33]. These findings support the concept of altering the protein secretion systems in BCG to increase the transport of immunogenic proteins to enhance protective efficacy.
In vitro infection studies using a murine macrophage cell line revealed a greater survival for BCG SecDFG over time (Figure 1). The observed increase in the bacterial numbers observed at 72 h post-infection may have been caused by the increased replication of the bacteria inside the host cells. Protein secretion plays a crucial role in modulating the interactions between bacteria by altering host-cell functions, including vesicular trafficking and host immune responses [34]. During intracellular growth, M. tuberculosis produces virulence factors that actively modify the phago-lysosome to create conditions conducive to bacterial proliferation [35]. The proteins exported by the Sec system include several important virulence factors [12]; thus, the overexpression of SecDFG could result in the secretion of proteins that enhance the survival of BCG inside host cells.
We observed the greater persistence of the BCG SecDFG in the DLN and the increased recovery of the BCG SecDFG from the spleens of mice 7 days post-immunization (Figure 2), which was consistent with the enhanced BCG SecDFG persistence observed in in vitro macrophage cell culture. It is known that following s.c. vaccination, the initial BCG replication occurs in the DLN, which results in higher bacterial dissemination to the spleen than to the lungs [36]. It has been proposed that BCGs chronically persist in the draining lymph nodes of mice after s.c. vaccination [37], and may function as a reservoir for dissemination to the spleen and lungs [38]. Some studies have reported that BCG disseminates to the lungs in low numbers around 20 weeks following s.c. vaccination [36]. However, in our study, we were not able to detect BCG or BCG SecDFG in the lungs at any timepoints. Overall, these data suggest that the overexpression of M. tuberculosis SecDFG protein in BCG improved the bacterial persistence in the murine organs, particularly in the DLNs, which correlates with the greater dissemination to the spleen.
The greatest impact on the immune responses induced by the BCG SecDFG vaccination was observed following stimulation with members of the Ag85 complex ( Figure 2). This was also more evident upon recall with M. tuberculosis CFP. CFP contains many different proteins, resulting in various antigen exposures to immune cells, the most predominant of which are members of the Ag85 complex [39]. This also correlates with previous work demonstrating the enhanced immunogenicity of BCG-overexpressing Ag85B [40] and the role played by Ag85B in facilitating host-cell interactions [31]. The enhanced Ag85B secretion was further validated when examining the priming of the Ag85B-specific (p25) transgenic T cells, which demonstrated the increased proliferation of antigen-specific T cells (Figure 3), possibly reflecting a greater secretion of Ag85B in the BCG SecDFG -vaccinated mice. The analysis of the cytokines secreted by the p25 T cells showed that the BCG SecDFG induced a higher proportion of polyfunctional CD4 + T cells to produce multiple cytokines, including IFN-γ, IL-2, and TNF ( Figure 3). We also observed an increase in the frequency of multifunctional Th1-like immunity induced by BCG SecDFG vaccination post-M. tuberculosischallenge ( Figure 4). Th1-type responses are recognized as essential for protection against mycobacterial diseases, including TB [41], and polyfunctional CD4 + T cells secreting IFN-γ, IL-2, and TNF have been proposed as markers of protective immunity [38]. Furthermore, the IL-2 + TNF + CD4 + T-cell population is described as having an effector-memory phenotype with high proliferative capacity [42,43]. Multiple studies have shown that IFNγ and TNF are required for TB immunity, although, by contrast, several other studies have also shown that the magnitude of IFNγ-producing T cells alone does not correlate with the degree of protection against TB [44].
In accordance with the observed immunogenicity induced by the BCG SecDFG , the vaccine reduced the bacterial burden on both the lungs and the spleens of the M. tuberculosisinfected mice at 4 weeks post-challenge (Figure 4). Although the parental strain of the BCG also showed a decreased bacterial burden, the BCG SecDFG displayed a trend toward greater protection in both organs. Previously, we showed that the overexpression of Ag85B in BCG does not improve the protective effect of BCG in mice, despite a marked increase in antigen-specific immunity [45]. Thus, it would be of interest to determine the efficacy of BCG SecDFG in other pre-clinical models, since the improved secretion of mycobacterial proteins appears to have an impact on BCG's protective efficacy [46]. Overall, this study demonstrates that the modification of the protein-secretion apparatus in BCG can serve to enhance protein secretion and influence bacterial persistence and immunogenicity. The assessment of this vaccine in other pre-clinical models, as well as the combination of this strategy with other modifications to BCG, may serve to generate a suitable candidate to improve the efficacy of BCG vaccination in humans.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/vaccines10060945/s1; Figure S1: Gating strategy used for assessing p25-specific CD4 + T cells proliferation following vaccination; Figure S2: Induction of antigen specific IFNγ-secreting cells in mouse lymph nodes following BCG SecDFG vaccination; Table S1: Flow cytometry antibodies used for surface staining; Table S2: Flow cytometry antibodies used for intracellular staining.
Author Contributions: A.N., C.C., R.P. and J.A.T. were responsible for the study's conception and design. A.N. and C.C. contributed to the acquisition of the data. A.N., C.C. and J.A.T. contributed to the analysis and interpretation of the data. A.N. and C.C. drafted the manuscript. J.A.T. and W.J.B. provided critical revisions. All authors have read and agreed to the published version of the manuscript.