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Seven-week-old female C57BL/6 mice, purchased from Charles River (Lecco, Italy), were housed under specific pathogen-free conditions in the animal facility of the Laboratory of Molecular Microbiology and Biotechnology (LA.M.M.B.), Department of Medical Biotechnologies at University of Siena, and treated according to national guidelines (Decreto Legislativo 26/2014). All animal studies were approved by the Italian Ministry of Health with authorization no. 1004/2015-PR on September 22 2015.

Mice were subcutaneously primed with the chimeric TB vaccine antigen H56 administered alone or in combination with the liposome system CAF01, and boosted after 4 weeks with H56 antigen alone (Figure 1). Both the innate and adaptive immune responses elicited by the two vaccine formulations were explored. T and B cells responses were characterized 10 days after boosting (day 38) within the local dLN and spleen, while the induction of H56-specific IgG serum response was followed at different time points, up to 7 weeks post boost (day 77). In parallel, other groups of mice were immunized with the same vaccine schedule described earlier, and the molecular signature of the vaccine formulations was explored by sequencing RNA from blood at early time points (1, 2, and 7 days) after primary and secondary immunizations, and thus following the transcriptomic profile over time without sacrificing the animal (Figure 1).

Groups of four to five mice were immunized by the subcutaneous route at the base of the tail. Vaccine formulations consisted of the chimeric TB vaccine antigen H56 (2 μg/mouse for priming, 0.5 μg/mouse for boosting; Statens Serum Institut, Denmark), administered alone or combined with the adjuvant CAF01 (250 µg DDA and 50 µg TDB) per mouse (Statens Serum Institut, Denmark). Vaccine formulations containing CAF01 were injected in a volume of 150 μl/mouse of 10 mM Tris buffer, while formulations containing H56 alone in a volume of 100 μl/mouse of PBS. Mice primed with both H56 + CAF01 or H56 alone were boosted with H56 antigen 4 weeks later.

For antibody analysis, blood samples were taken by temporal plexus bleed on days 0, 14, 28, 38, 56, and 77 after primary immunization. Blood was incubated for 30 min at 37°C, centrifuged at 1,200 × g at 4°C for 15 min, and then serum was collected and stored at −80°C until further analysis.

For transcriptomic analysis, blood samples were collected from individual mice on days 1, 2, 7, 28, 29, 30, and 35 after priming. Blood (200 µl) was mixed in a 1:2.8 ratio with PaxGene reagent, in a 2 ml Biosphere Eppendorf tube (39, 40). The tube was inverted five times and incubated at room temperature for 2 h before freezing at −80°C. Total RNA was extracted using the PaxGene blood RNA kit (Qiagen, Germany) following manufacturer’s instructions and resuspended in 60 µl of BR5 buffer. RNA was quantified using (i) the total RNA nano kit on a 2100 Bioanalyzer (Agilent Technologies, USA), (ii) the RNA broad range kit on a Qubit 2.0 (Thermo Fisher, USA), and (iii) the Nanophotometer (Implen, Germany).

Libraries were prepared using TruSeq stranded RNA reagents (Illumina, USA) according to manufacturer’s instructions, using 500 ng of total RNA input per sample with dual indexing to enable all libraries to be sequenced in the same run. Pooled libraries were sequenced on four runs of an Illumina NextSeq 500 instrument with 75 bp single end reads. Image analysis and base calling were performed using Illumina’s RTA software version 2.4.11 and bcl2fastq version 2.18.0.12. Reads were filtered to remove those with low base call quality using Illumina’s default chastity criteria. Raw sequence data were deposited in Sequence Read Archive under Bioproject number PRJNA437839.

B and T responses were analyzed in dLN (sub iliac, medial, and external). Samples were mashed onto 70 µm nylon screens (Sefar Italia, Italy) and washed two times in complete RPMI medium (cRPMI, Lonza, Belgium) containing with 100 U/ml penicillin/streptomycin and 10% fetal bovine serum (Gibco, USA). Samples were treated with red blood cells lysis buffer according to manufacturer instruction (eBioscience, USA). Cells were incubated for 30 min at 4°C in Fc-blocking solution [cRPMI medium with 5 µg/ml of CD16/CD32 mAb (clone 93; eBioscience, USA)]. To evaluate germinal center B lymphocytes from dLN, cells were stained with AF700-conjugated anti-CD45R (anti-B220, clone RA3-6B2; BD Biosciences), BV421-conjugated anti-GL7 (clone GL7; BD Biosciences), and PE-Cy7-conjugated anti-CD95 (clone Jo2; eBioscience). To evaluate H56-specific CD4+ T lymphocytes, cells from dLN were stained for 1 h at RT with PE-conjugated I-A(b) M. tuberculosis Ag85B precursor 280–294 (FQDAYNAAGGHNAVF) tetramer (kindly provided by NIH MHC Tetramer Core Facility, Emory University, Atlanta, GA, USA), washed and surface stained with HV500-conjugated anti-CD4 (clone RM4-5; BD Biosciences), and BV786-conjugated anti-CD44 (clone IM-7; BD Biosciences). Samples were labeled with Live/Dead Fixable Near-IR Stain Kit according to manufacturer’s instructions (Invitrogen, USA). All antibodies and tetramers were titrated for optimal dilution. Approximately 5–10 × 10⁵ cells were acquired on a LSRFortessa_TM X-20 flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v10 (TreeStar, USA).

Antibody-secreting cells (ASC) were evaluated in the spleen by ELISpot PLUS for mouse IgG kit (Mabtech) 10 days post boost. Multiscreen filter (PVDF) plates (Millipore, USA) were pre-wet for 2 min with EtOH 70%, washed with sterile dH₂O and coated with H56 (5 µg/ml) diluted in PBS (Sigma-Aldrich) for the detection of antigen-specific IgG. After incubation overnight at 4°C, the coated wells were washed with sterile PBS and blocked for 30 min with serum free CTL-Test B culture medium (CTL, USA) supplemented with 1% l-glutamine (Sigma-Aldrich, USA). After removal of the blocking medium, 1 × 10⁶ cells/well were added in a volume of 100 µl of CTL-Test B medium for the analysis of H56-specific IgG ASC. Each sample was assayed in triplicate, and the plates were incubated overnight for 20 h at 37°C in a humidified atmosphere with 5% CO₂. After incubation, cells were removed by washing with PBS and 100 µl/well of anti-IgG biotinylated detection antibody (Mabtech, Sweden), diluted to 1 µg/ml in PBS containing 0.5% fetal calf serum (FCS, Gibco), was added. After incubation for 2 h at room temperature followed by washing steps, ELISpot plates were incubated with 50 µl/well of streptavidin–horseradish peroxidase (Mabtech) diluted 1:1,000 for 1 h at room temperature. Plates were washed again with PBS, and then 100 µl/well TMB substrate solution (Mabtech) was added for approximately 10 min. The reaction was stopped by extensive washing in dH₂O, and plates were then dried in the dark at room temperature. The number of spots was determined by plate scanning and analysis services performed at Cellular Technology Limited (Germany).

Serum H56-specific IgG were quantified by ELISA at different time points. Flat bottomed Maxisorp microtiter plates (Nunc, Denmark) were coated with H56 (0.5 µg/ml) for 3 h at 37°C and overnight at 4°C in a volume of 100 μl/well. Plates were washed and blocked with 200 µl/well of PBS containing 1% BSA (Sigma-Aldrich) for 2 h at 37°C. Serum samples were added (100 μl/well) and titrated in twofold dilution in duplicate in PBS supplemented with 0.05% Tween 20 and 0.1% BSA (diluent buffer). After incubation for 2 h at 37°C, samples were incubated with the alkaline phosphatase-conjugate goat anti-mouse IgG (100 μl/well diluted 1:1,000 in diluent buffer, Southern Biotechnology, USA) for 2 h at 37°C and developed by adding 200 μl/well of 1 mg/ml of alkaline phosphatase substrate (Sigma-Aldrich). The optical density was recorded using a Multiskan FC Microplate Photometer (Thermo Scientific). Antibody titers were expressed as the reciprocal of the highest dilution with an OD value ≥ 0.2 after background subtraction.

Raw reads were aligned to mouse genome (UCSD mm10 annotation), and counts were generated using STAR (41). Differential gene expression was determined using edgeR (42) with the generalized linear model (GLM) fitting approach for both longitudinal analysis and pairwise adjuvanted versus non-adjuvanted vaccine comparisons at each time point. For each comparison, edgeR generated a list of genes associated with a log fold-change and to a false discovery rate (FDR, i.e., the P value after multiple test correction). Genes were considered significantly differentially expressed when FDR was <0.05. For enrichment analysis, lists of genes were sorted by FDR, then mouse gene identifiers were converted to human gene identifiers using an in house script utilizing the BiomaRt Bioconductor package. Enrichment analysis was performed using the tmod R package (43) with blood transcription modules (BTMs) developed by Li and coworkers (19). Significance of module enrichment was assessed using the CERNO statistical test (a modification of Fisher’s combined probability test) and corrected for multiple testing using the Benjamini–Hochberg correction.

Analysis was performed on individual samples, and data were reported in box plots encompassing minimum and maximum values. The Kruskal–Wallis test, followed by Dunn’s post test for multiple comparison, was used to assess statistical differences among the number of (i) Ag85B-specific CD4+ T cells, (ii) germinal center B cells, and (iii) H56-specific ASC in different groups of mice (naïve, primed with antigen H56 alone or primed with H56 + CAF01). H56-specific IgG were reported in graphs as log-transformed geometric mean titers with 95% confidence intervals and compared using t-test. Statistical significance was defined as P < 0.05, and analyses were performed using Graph Pad Prism version 7 (GraphPad Software, USA).

Antigen-specific CD4+ T cell responses were analyzed in mice primed with the chimeric TB vaccine antigen H56 administered alone or in combination with the liposome system CAF01, and boosted after 4 weeks with H56 antigen alone (Figure 1). T cell responses were characterized 10 days after boosting (day 38) within the local dLN. CD4+ T cells specific for the immunodominant epitope Ag85B, part of the H56 fusion protein, were identified using Ag85B_280–294-complexed MHC class II tetramers. Staining specificity of Ag85B_280–294-complexed MHC class II tetramers was determined using a control tetramer complexed with an unrelated antigen, which showed a level of staining below 0.02% (data not shown). Ten days after booster immunization with H56 antigen, the number of tetramer-binding CD4+ T cells in dLN was about 66,000 (±39,000) in mice primed with H56 + CAF01. This was significantly higher than the amount of antigen-specific CD4+ T cells present in mice primed with the H56 antigen alone (8,260 ± 6,000, P < 0.01, Figure 2A). The percentage of antigen-specific CD4+ T cells in respect to total activated CD4+ T cells was 0.55% and 0.07% in mice primed with H56 + CAF01 and H56 antigen alone, respectively (P < 0.05, data not shown). These data show that inclusion of CAF01 in the priming formulation induces an antigen-specific proliferation of CD4+ T cells which is not observed with the antigen alone.

B cells responses were characterized 10 days after boosting (day 38) within the local dLN and spleen. Similar to the CD4+ T recall response, the reactivation of the B cell response was significantly higher in mice that had been primed with H56 + CAF01 compared with H56-primed mice (Figure 2B). Indeed, a significant expansion of the B220+ GL7+ CD95+ germinal center B cells (GC-B cells) 10 days following booster immunization was measured in mice primed with the CAF01 adjuvant (P < 0.05 versus H56-primed mice, P < 0.01 versus naïve mice), while the amount of cells detected in mice primed with H56 alone was not significantly higher compared with naïve mice (Figure 2B). The observed GC-B cells response is clearly a recall response of cells elicited by the primary immunization, since we have previously demonstrated that primary immunization with H56 alone does not stimulate the germinal center reaction, while the CAF01 adjuvant is a strong promoter of this reaction (4).

The induction of antigen-specific IgG antibody responses was also assessed at different time points after priming and boosting (Figure 2C). The IgG response elicited by both H56 alone or combined with CAF01 was very similar at 12 days after priming, while a significantly higher response was observed at day 28 and maintained 10 days after boosting in mice primed with antigen and adjuvant compared with animals primed with antigen alone (day 38, P < 0.001). This was further confirmed by assessing antigen-specific ASC in the spleen collected 10 days after booster immunization (Figure 2D). The number of H56-specific ASC was higher in mice primed with H56 + CAF01 compared with animals primed with H56 alone with mean values of 51 and 20, respectively.

The molecular signature of the vaccine formulations was analyzed in mice primed with H56 alone or in combination with CAF01, and boosted with H56 antigen alone (Figure 1). The transcriptomic profile was followed over time (without sacrificing the animals), sequencing blood RNA samples collected at early time points (1, 2, and 7 days) after primary and secondary immunizations (4 samples/each time point). An average of 7.2 µg total RNA/sample (range 1.473–60.552 μg/sample, as quantified by fluorimetry) was obtained. The isolated RNA was generally of high quality, with an average RNA Integrity number of 9.05 (range 5.8–9.9), as measured by the Bioanalyzer, and a mean 260/280 absorbance ratio of 2.17.

Gene expression in the blood of mice primed either with H56 + CAF01 or H56 alone was first compared with gene expression in blood collected before immunization. In Figure 3 the left column reports bivariate plots showing differences in gene expression between the H56 + CAF01 and H56 alone immunized mice at each time point. Differentially expressed genes were subjected to functional analysis using the Reactome database.¹

At day 1 after priming, 39 genes were found to be upregulated, while 1,147 genes were downregulated by both formulations. The gene modulation was essentially limited to pathways of platelet function adhesion, activation and degranulation, and clotting, as expected for injection and repeated bleeding of mice. At day 2 after priming, 143 genes were upregulated, while 1,031 genes were downregulated. Among the upregulated genes there was an activation of pathways related to innate immunity, neutrophils degranulation and IL4-IL13 signaling. At day 7, 124 genes were upregulated, and 588 were downregulated by both vaccines. Overall, the transcriptomic response was very consistent between the two vaccine formulations.

Four weeks after priming, both groups of mice received a booster immunization with H56 antigen alone. The day before booster immunization, blood was collected from both groups of mice, and gene expression analysis performed at this time point was then used as a baseline. Gene expression in the blood of mice primed with H56 + CAF01 or H56 alone and boosted with H56 was indeed compared with gene expression in blood collected in the same group of mice before booster immunization. In the right column of Figure 3 bivariate plots show differential gene expression at 1, 2, and 7 days after H56 boosting.

Among the genes upregulated 1 day after H56 boost in mice primed with H56 + CAF01, we observed an over representation of genes relevant to innate immune response, centered primarily on the interferon response, as well as IL4 and IL13 signaling. Importantly, these genes were regulated almost exclusively in the H56 + CAF01 primed group, but not in the group that received H56 alone as priming. Two days after boost, only a few genes were found to be differentially expressed, and, again, as at day 1 post boost, gene regulation was observed primarily in the H56 + CAF01 primed group, but not in the group of mice primed with the antigen alone. Interestingly, according to the Reactome analysis, the upregulated genes were enriched in M. tuberculosis response genes; however, due to the very small number of genes regulated at this time point, these results should be taken with caution. By 7 days after the boost, the two groups displayed a similar gene expression pattern, mainly related to genes involved in cell cycle and proliferation, suggesting the activation of an adaptive response and clonal expansion of responding antigen-specific cells (Figure 3, right column).

To identify the CAF01-specific transcriptomic response, the blood gene expression in mice primed with H56 + CAF01 was compared with that of mice primed with H56 alone. All comparisons were performed at 1, 2, and 7 days after priming and after H56 boost. Table 1 reports the number of differentially expressed genes identified in edgeR by the genewise Negative Binomial Generalized Linear Models algorithm. Lists of genes, sorted by FDR, were converted to human IDs and tested for the significance in the enrichment of BTMs (19). BTMs include 346 sets of genes which are coordinately expressed and exert a specific function, described by the module title, including innate and adaptive immunity or general biological processes such as cell cycle, transcription or signal transduction. Figure 4 reports modules that were found to be significantly enriched (FDR < 0.001) in at least one tested time point. Data analysis at days 1, 2, and 7 after priming indicated that the major differences between the two vaccine formulations were detected at day 2, with 2.2% of differentially expressed genes and upregulation of modules related to innate immunity by the CAF01 adjuvanted vaccine formulation. In particular, modules related to antigen-presenting cells (monocytes and DCs) were found to be enriched, together with neutrophils and TLR inflammatory signaling. Differential gene expression analysis found no significant difference between H56 + CAF01 and H56 alone vaccinated groups at day 1, and only 17 significant genes at day 7 (Table 1). Nevertheless, module enrichment analysis identified seven enriched modules at day 1 and one module at day 7. These modules were also related to innate immune response, indicating that CAF01 modulates the transcriptomic response also at those time points.

The most significant differences between the two vaccine formulations could be appreciated after antigen boost, thus demonstrating the key role of the boost in highlighting the skewing of the immune system induced by the adjuvant used for priming. In fact, at day 1 after boost, 34 out of the 44 significant modules were modulated by CAF01, with 8.1% of genes being differentially expressed (Figure 4; Table 1). In particular, the innate immunity compartment was found to be affected: 10 modules regarding monocytes and antigen presentation, three modules associated with neutrophil recruitment and six modules related to interferon response were upregulated, while one module associated with natural killer (NK) cells was downregulated. No enrichment of modules related to B- and T-cell populations was observed at this early time point. At day 2 after boost, the overall difference between the two formulations was lower: 13 modules were significantly enriched, mainly related to innate response with a minor modulation of B cells. Seven days after the boost, despite the relatively high number of differentially expressed genes (Table 1), only eight modules were affected (Figure 4). In particular, these were regarding adaptive immune response with three upregulated modules regarding T cell activation and one downregulated relevant to B cells activities.