Abstract Deciphering protection mechanisms against Mycobacterium tuberculosis (Mtb) remains a critical challenge for the development of new vaccines and therapies. We analyze the phenotypic and transcriptomic profile in lung of a novel tuberculosis (TB) nanoparticle-based boosting mucosal vaccine Nano-FP1, which combined to BCG priming conferred enhanced protection in mice challenged with low-dose Mtb. We analyzed the vaccine profile and efficacy at short (2 weeks), medium (7 weeks) and long term (11 weeks) post-vaccination, and compared it to ineffective Nano-FP2 vaccine. We observed several changes in the mouse lung environment by both nanovaccines, which are lost shortly after boosting. Additional boosting at long-term (14 weeks) recovered partially cell populations and transcriptomic profile, but not enough to enhance protection to infection. An increase in both total and resident memory CD4 and CD8 T cells, but no pro-inflammatory cytokine levels, were correlated with better protection. A unique gene expression pattern with differentially expressed genes revealed potential pathways associated to the immune defense against Mtb. Our findings provide an insight into the critical immune responses that need to be considered when assessing the effectiveness of a novel TB vaccine. Keywords: Mycobacterium tuberculosis, nanovaccines, immune protection, lung infection, transcriptomic analysis Introduction Despite being well-known and treated for years, tuberculosis (TB) is the leading cause of death from a single infectious pathogen worldwide. The World Health Organization estimates that one third of the world’s population carries the bacillus in a latent form, with a 10% probability of those infected to develop TB during their lifetime, and that contributes to 10 million new cases of TB occur yearly ([47]1). Control of the global TB epidemic has been challenged by the lack of an effective vaccine. Bacille Calmette–Guérin (BCG) remains the only licensed TB vaccine, although its efficacy against the pulmonary form of TB in adulthood is highly variable ([48]2, [49]3). Unfortunately, the development of novel effective vaccines is hampered by the limited knowledge we have of the mechanisms that provide protection against Mycobacterium tuberculosis (Mtb). The immune response against Mtb is complex and incompletely characterized. Although evidence supports the fundamental role of CD4^+ T cells and cytokines (such as interferon gamma (IFNγ) ([50]4, [51]5), tumor necrosis factor alfa (TNFα), interleukins 2 (IL-2) ([52]6–[53]9), and 12 (IL-12) ([54]10, [55]11)) in TB, there are still no reliable correlates of protection. In this scenario, it becomes difficult to predict the outcome of the disease or to monitor the efficacy of novel vaccines. Mucosal vaccination ([56]12–[57]18) and mucosal boosting of BCG, combining the overall protection conferred by BCG with the reinforcement of the mucosal immunity in the lungs ([58]19–[59]25), have been considered attractive strategies against pulmonary TB. Hart et al. ([60]26) demonstrated that the combination of subcutaneous BCG plus two intranasal boosts of a novel nanovaccine, Nano-FP1, significantly reduced the bacterial burden in mouse lungs after TB infection. Nano-FP1 is based on nanoparticles produced by the emulsification of yellow carnauba (YC) palm wax with sodium myristate (NaMA), coated with a fusion protein made of three different antigens of Mtb: the secreted protein Ag85B, the 16-kDa latency induced protein alpha crystalline (Acr) and the heparin-binding hemagglutinin (HBHA). Similar boosting nanovaccines based on other antigen combinations were also tested, but they did not show improved efficacy against Mtb (R. Reljic, unpublished). Among them is the Nano-FP2 vaccine, which displayed one single antigen replacement in its fusion protein compared to Nano-FP1, with antigen Ag85b replaced by the Mannose Binding Protein 64 (MPT64). We intend to identify a phenotypic and/or transcriptomic profile of the efficacy of the Nano-FP1 TB vaccine in mice. Herein, we performed a systematic analysis of the novel Nano-FP1 prototype, analyzing the cellular signature and gene expression profile triggered in the pulmonary environment. Nano-FP2 was also tested as an example on a non-protective vaccine candidate. The effect produced by the intranasal boost with Nano-FP1 on previously BCG-immunized mice was evaluated at short-term (2 weeks), medium-term (7 weeks), and long-term (11 weeks) intervals. We found a unique cellular and transcriptional profile at short-term, characterized by alterations in CD4^+ T cell populations and marked changes in gene expression. Nonetheless, we observed that the boosting effect was transient and it did not trigger an effective immunological memory against TB long term. Our findings suggest a critical role for the long-lived CD4+ T cell immunity that should be mandatory when assessing the effectiveness of a novel TB vaccine. Materials and Methods Mice Six-week-old female specific pathogen-free C57BL/6 mice were purchased from Envigo (Spain). The mice were maintained under barrier conditions in a BL-3 biohazard animal facility at the University of Minho, Braga, Portugal, with constant temperature (24 ± 1°C) and humidity (50 ± 5%). The animals were fed a sterile commercial mouse diet and provided with water ad libitum under standardized light-controlled conditions (12 h light and dark periods). The mice were monitored daily, and none of the mice exhibited any clinical symptoms or illness during this experiment. For the early response experiment, 6-week-old female specific pathogen-free C57BL/6 mice were purchased from Scanbur (Denmark), and housed in pathogen- free conditions at the Animal Department of MBW, Stockholm University, Sweden. Mice were acclimatized for at least 1 week before use and supervised daily. The specified pathogen free condition of the facility was confirmed by continuous use of sentinel mice. All animal experiments were performed with ethical approval from the hosting institutions and according the national regulations and legislation of that country. The study was approved by and performed in accordance with guidelines of the CEEA Xunta de Galicia, code ES-360570215601/17/INV. MED.02.OUTROS04/AGF/02. Early response experiments were approved by and performed in accordance with guidelines of the Stockholm North Ethical Committee on Animal Experiments, permit number N170/15. Nanovaccines Formulation Two different vaccine candidates were used as intranasal (in) boost to BCG, hereafter referred to as Nano-FP1 and Nano-FP2. Both candidates consist of a combination of yellow carnauba palm wax with sodium myristate (YC-NaMA) nanoparticles (NPs) (Bethlehem, PA, USA) and a fusion protein (FP) composed of an N-terminal histidine tag and the Mtb antigens Acr (Rv2031c), Ag85B (Rv1886c), and the heparin-binding domain of HBHA (Rv0475) (FP1) or MPT64 (Rv1980c), Acr and HBHA (FP2). The vaccine formulation included 0.1% Yc-NaMA NPs, 200 µg/ml of the corresponding FP and 400 µg/ml PolyI:C (Sigma Aldrich) in saline solution, with 50 µl delivered to the each mouse. Nano-FP1 was used as the prototype of interest, taking into account previous studies reporting its protective effect ([61]26) and Nano-FP2 was used as a representative of a nonprotective vaccine candidate (data not shown). Study Groups and Immunization Protocol The vaccination groups and schedules are shown in [62]Figures 1A and [63]2A . For subcutaneous (s.c.) priming vaccination, mice received 0.5 million CFUs of BCG strain Pasteur. Twelve and 14 weeks later, mice from the Nano-FP1 and Nano-FP2 groups were anesthetized with 100 µl of ketamine–xylazine and 50 µl of the corresponding nanovaccine was intranasally administered. A group of mice were also administered a 3rd boost of the corresponding nanovaccine 25 weeks after sc. BCG. Animals were divided in four experimental groups: Unvaccinated mice (henceforth referred to as Naive group); mice vaccinated with subcutaneous (s.c.) BCG alone (BCG group); mice vaccinated with BCG s.c. and 12 weeks later an intranasal boost with Nano-FP1 (BCG/Nano-FP1 group) or Nano-FP2 (BCG/Nano-FP2 group). Animals were studied at different time points (2, 7, and 11 weeks (after two intranasal challenges), and 14 weeks (after an additional third intranasal challenge). Figure 1. [64]Figure 1 [65]Open in a new tab CFUs measurement in lung and spleen of Mtb infected mice. (A) Mice were left unvaccinated or vaccinated either with BCG alone (BCG) or BCG followed by intranasal Nano-FP1 (BCG/Nano-FP1) or Nano-FP2 (BCG/Nano-FP2). At the points indicated after the second boost, mice were infected with Mtb through the aerosol route. Lungs and spleen were collected 30 days after infection and plated to assess bacterial burdens in all groups, as described in Material and Methods (n = 9–10 mice per group). Mtb colony-forming units (CFUs) were determined in the lungs (B) and spleen (C). Mann–Whitney-Wilcoxon test was used for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Figure 2. [66]Figure 2 [67]Open in a new tab Immune cell populations in lung. (A) Groups of mice were vaccinated as described in [68]Figure 1 . (B) Lung immune cell populations at 2 weeks. (C) Percentages of selected immune populations CD45+ CD4^+ T cells and (D) neutrophils in lungs analyzed at different time points (2, 7, 11 and 14 weeks). Data represent percentages of each cell population referred to the total of immune CD45-positive cells. *1 and *3: The percentage (%) of CD4^+ T in the BCG/Nano-FP1 group was significantly higher than those in Naive, BCG and BCG/Nano-FP2 groups at 2 weeks. *2: The % of interstitial macrophages was significantly higher than that one in Naive group at 2 weeks. *4: The % CD4^+ T cells was significantly higher than that one in Naive group at 7 weeks.*5: The % of neutrophils was significantly higher than that one in BCG group at 11 weeks. Kruskal–Wallis test and Dunn’s multiple comparisons test were used for statistical analysis. *p < 0.05. For the early immune experiment (24 h), mice were subcutaneously vaccinated with either 0.5 million CFUs of BCG or PBS. Twelve weeks later, they were administered intranasally one dose of either Nano-FP1 or PBS. 24 h later, mice were sacrificed. Animals were divided in four experimental groups (1): mice receiving s.c and intranasal PBS as control (herein referred as PBS/PBS-24h); (2) only vaccinated with BCG s.c. (BCG/PBS-24h), (3) only with an intranasal boost of Nano-FP1 (PBS/Nano-FP1-24h), and (4) animals received BCG s.c. and twelve weeks later one intranasal dose of Nano-FP1 (BCG/Nano-FP1-24h). Bacteria The H37Rv strain of M. tuberculosis was grown in Middlebrook 7H9 liquid medium (BD Biosciences, San Diego, CA) for 7–10 days and then sub-cultured in Proskauer Beck (PB) medium supplemented with 0.05% Tween 80 and 2% glycerol, until the mid-log phase. Bacterial stocks were aliquoted and stored at −80°C. Bacterial frozen stocks were used to infect mice via aerosol route, using a Glas-Col inhalation exposure system. Bacterial clumps were disrupted by forcing them through a 26G needle before diluting the bacterial suspension in water (Aqua B. Braun) to a concentration of 2 × 10^6 CFUs/ml to deliver 100 CFUs into the lungs. Infection and Sample Collection Mice were challenged via the aerosol route with the H37Rv strain at different time points (2–3, 7, and 11 weeks) following the last boost of the intranasal nanovaccines. Sample collection was conducted both pre- and post-challenge for each experimental group. Mice from the “pre-challenge” group were euthanized with CO[2] and lung parenchyma, bronchoalveolar lavage (BAL) and spleen were collected for analysis. The remaining animals (“post-challenge” group) were sacrificed four weeks after infection on each of the corresponding time points for organ CFU count. Lung parenchyma and spleen were collected from these mice for immunological assays. Sample Processing BAL was collected by irrigating lungs via trachea with a syringe containing 1 ml of cold PBS. Lungs and spleen were aseptically removed after BAL lavage and were homogenized and processed for immunological assays. Prior to homogenization, lungs were incubated in digestion medium DMEM (Dulbecco’s Modified Eagle Medium, High glucose NEAA, no glutamine, Gibco) supplemented with collagenase 0.15 mg/ml (Sigma Aldrich) at 37°C for 30 min. Spleens and collagenase-incubated lungs were homogenized and filtered through a 40 μm nylon mesh cell strainer (BD Biosciences, San Diego, CA) to obtain a homogenous cell suspension. BAL, lung and spleen cells were treated with red blood cell (RBC) lysing buffer (0.87% of NH[4]Cl solution and 5% of PBS in water) for 5 min, and washed twice with DMEM supplemented with 10% of heat-inactivated fetal bovine serum. Leukocyte fraction was isolated by density gradient centrifugation on an 80%/40% Percoll (GE Healthcare, Sigma Aldrich) gradient. In mice sacrificed post-challenge, the left lung and half spleen were reserved for organ CFU count. Bacterial Counts The number of viable bacteria in lung and spleen from infected mice was determined by plating serial dilutions of the organ (left lung or half spleen) homogenates onto Middlebrook 7H11 agar (Difco Laboratories) supplemented with 10% OADC (Difco Laboratories). Colonies were counted after 3 weeks of incubation at 37°C with 5% CO[2] atmosphere. Antibodies and Surface Staining For analysis of general immune populations and lymphocyte composition in parenchyma lung and BAL, cell pools from 8 to 12 mice per group were used for the 2-week analysis and four mice per group for the 7, 12, and 14 weeks analysis. Cells were incubated for 30 min with antibodies at 4°C, washed with FACs buffer (PBS with 3% FBS and 0.1% of 10 mM sodium azide) and kept at 4°C until flow cytometry analysis. List of antibodies used and references can be found in [69]Table S1 .