Development of a mouse model for post-exposure vaccination

In order to evaluate vaccines for their activity post-exposure in animals infected with M.tb., we developed and characterized an animal model that represents a modification of the previously published Cornell model [4]. Mice were exposed to M.tb. via the aerosol route. At week 6 post-infection (p.i.), the mice were treated with a combination of rifabutin and isoniazide provided in the drinking water for six weeks. Antibiotic treatment quickly resulted in significant reduction in lung bacterial load to levels below 2 log₁₀ CFU and the bacterial numbers remained low for at least two weeks post-cessation of the antibiotic treatment (Figure 1A). After week 14, a spontaneous relapse of the infection was seen and the bacteria grew to between 3 and 4 log₁₀ CFU measured at week 22 p.i. (10 weeks post-cessation of antibiotic treatment). The bacterial load was reflected in the IFN-γ response directed to both ESAT-6 and TB10.4 with responses after completed antibiotic treatment below detection levels and highly increased levels as the bacteria resumed growth (Figure 1B).

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Figure 1. Mouse model of latent TB and relapse.

(A) Groups of BALB/c x C57BL/6 (CB6F1) mice were infected by aerosol route with approximately 50 CFU of M.tb. Erdman. At week 6 post infection (p.i.) mice were given antibiotics in the drinking water ad libitum for six weeks (grey area) and allowed to spontaneously resume growth. Five-six mice (week 32 p.i. n=3) were sacrificed at various time-points during infection and lungs were harvested for determination of bacterial load during primary infection (week 0-6 p.i.), antibiotic treatment/latent phase (week 6-14 p.i.) and during the relapse phase (week 22-32 p.i.) and are expressed as log₁₀ CFU. Data are shown as scatter dot plot and medians are displayed as a horizontal line for each time-point. (B) Lymphocytes were isolated at various time-points from perfused lungs and re-stimulated with ESAT-6 (open circles) and TB10.4 (open squares). Antigen-specific IFN-γ response was measured by ELISA. Data are shown as mean ± SEM of three independent experiments except for week 14 and 27 which are from one experiment. (C) Photomicrographs illustrating typical ‘acute’/primary infection (week 6 p.i.), antibiotic treatment/latent (week 12) and relapse (week 30 p.i.) phase lesions after aerosol infection with M.tb. Haematoxylin and eosin stain. Scale bar = 250 μm.

https://doi.org/10.1371/journal.pone.0080579.g001

We also examined the pathological changes in the animals during the course of infection. Six weeks post-infection when antibiotic treatment was initiated, relatively large granulomas were detected in the lungs (Figure 1C, acute infection week 6 p.i.). Although reduced in size, granulomas could still be identified following six weeks of antibiotic treatment (Figure 1C, antibiotic treatment, weeks 12 p.i.). By week 30 p.i. (Figure 1C, relapse), the increasing bacterial load due to bacterial regrowth was associated with multifocal un-encapsulated granulomas, frequently surrounding the bronchioles, consisting of sheets of closely apposed microvesiculated macrophages with infiltrating dense aggregates of lymphocytes.

ESAT-6 but not TB10.4 containing fusion-protein vaccines prevent relapse of infection

The ESX family members ESAT-6 and TB10.4 have previously been demonstrated to share very similar characteristics regarding immune recognition during infection and protective activity in preventive TB vaccines [26]. To further study the role of these antigens in TB vaccines for preventive and post-exposure administration we compared vaccines that differ only in these two components. The H56 vaccine contains Ag85B, ESAT-6 and Rv2660c [10], where H28, a very similar vaccine, differs only by the substitution of ESAT-6 with TB10.4 [27] (overview of all vaccine constructs and models can be seen in Figure 2A and B).

Pre-exposure vaccination.

To compare the immunogenicity of H28 (Ag85B-TB10.4-Rv2660c) and H56 (Ag85B-ESAT-6-Rv2660c), mice were vaccinated three times subcutaneously (s.c.) with the two fusion-proteins formulated in CAF01 cationic liposomes, previously reported to be an efficient delivery system for TB subunit vaccines [28]. Two weeks after the last immunization, peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with the vaccine antigens and subsequently analyzed by intracellular staining (ICS) for IFN-γ and IL-2 production. Vaccine-specific CD4 T cells were detected in both H28 and H56 vaccinated animals (Figure 3A, left panel). The highest frequency of antigen-specific CD4 T cells was directed to Ag85B comprising 0.5-0.6 % of the CD4 T cell population in both vaccine groups. The TB10.4 response in H28 vaccinated mice and the ESAT-6 response in H56 vaccinated mice was lower compared to the Ag85B response comprising approximately 0.1% TB10.4 specific CD4 T cells in the H28 vaccinated group and about 0.2 % ESAT-6 specific CD4 T cells in the H56 vaccinated group. Six weeks following M.tb. challenge, pronounced immune responses directed towards TB10.4 in H28 vaccinated mice (10 %) and ESAT-6 in H56 vaccinated mice (8 %) were detected in the lung whereas the Ag85B specific responses comprised a smaller proportion of the CD4 T cell population (2-5 %) (Figure 3A, right panel). Although the Rv2660c response was measurable (H28: 0.9%, H56: 0.25%), the level remained much lower than the responses to the two ESX antigens. Hence, both H28 and H56 were highly immunogenic and promoted a response to the corresponding ESX antigen strongly recognized in the lung following infection.

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Figure 2. The vaccine constructs and animal challenge models.

(A) A schematic overview of the multi-component vaccine constructs which include the ESAT-6-containing vaccines: H1 (Ag85B-ESAT-6) and H56 (Ag85B-ESAT-6-Rv2660c), and the TB10.4-containing vaccines: H4 (Ag85B-TB10.4) and H28 (Ag85B-TB10.4-Rv2660c). (B) Experimental setups for the pre-exposure, post-exposure and therapeutic models. Pre-exposure model: Mice receive three immunizations with a two week interval between each immunization. Six weeks after the last immunization, mice were aerosol challenged with 100 CFU of M.tb. Erdman. Six weeks post-infection (p.i.) mice were sacrificed and CFU levels monitored in the lungs. Post-exposure model: Mice receive an aerosol challenge with 50 CFU of M.tb. Erdman. After six weeks of infection, antibiotics are administered through the drinking water for a six week period. Vaccinations are given at week 10, 13, and 16 after infection. CFU levels in the lungs are monitored between week 17 and 49 p.i. Therapeutic model: Mice receive an aerosol challenge with 50 CFU of M.tb. Erdman. The mice receive three immunizations with two weeks interval (starting at week 6 p.i.), and the CFU levels in the lungs are monitored at week 21 p.i.

https://doi.org/10.1371/journal.pone.0080579.g002

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Figure 3. Prophylactic vaccination with H28 and H56 result in similar levels of protection.

Groups of CB6F1 mice were vaccinated three times with two weeks interval with H28 or H56 formulated in CAF01 or with CAF01 alone. (A) Two weeks following vaccination with either H28 or H56, PMBCs (pooled from six mice) were stimulated with Ag85B, TB10.4, ESAT-6 or Rv2660c and antigen specific responses assessed by flow cytometry. The frequency of cytokine producing CD4 T cells expressing IFN-γ, IL-2 or both cytokines was determined by intracellular staining for cytokines (ICS). Lung lymphocytes (pooled from six mice) were obtained after six weeks of infection and the frequency of cytokine producing CD4 T cells determined by ICS. Data are representative of three experiments. (B) Protection was assessed at week 6 post infection by culturing lung homogenates and enumerating bacteria (expressed as log₁₀). Data displayed are box and whiskers of six mice and minimum and maximum CFU levels. As a negative control, a group of mice received CAF01 only. Values marked with an asterisk are significantly different from naïve control animals as assessed by ANOVA and subsequent Tukey’s multiple-comparison test (***, p<0.001). The data are typical for several experiments as illustrated in Table S1.

https://doi.org/10.1371/journal.pone.0080579.g003

The vaccinated animals were tested for protection against M.tb. aerosol challenge by plating lung homogenates and culturing them for 2-3 weeks before counting bacterial colonies. Both H28 and H56 vaccination resulted in a significant reduction in the bacterial load compared to the control group (Figure 3B) and provided approximately 1 log reduction of bacterial numbers which is the level typically obtained by effective TB vaccines in this standardized model of preventive vaccination [7].

To further compare ESAT-6 and TB10.4-bearing vaccines, we expanded the comparison to cover the H1 (Ag85B-ESAT-6) [29] and H4 (Ag85B-TB10.4) [26] vaccines, and performed a series of challenge experiments. H4, H28, H1 and H56 pre-exposure vaccination resulted in similar levels of protection (in the range of 1-1.5 log reduction of CFU) (see Table S1). Thus, in agreement with previous publications, H1 and H56 promoted similar levels of protection in a short term challenge model [10] and this level was comparable to the TB10.4-based vaccines H4 and H28.

Post-exposure vaccination.

We continued by comparing H28 and H56 in the post-exposure animal model described above. Infected mice were vaccinated at week 10, 13 and 16 p.i. and immune responses were assessed one week after the last immunization (week 17 p.i.). At this time-point infection-driven immune responses in CAF01 vaccinated control animals were barely detectable (Figure 4A). This was in contrast to the response measured in the blood of H28 and H56 vaccinated animals, where vaccine-specific CD4 T cells were readily detected against Ag85B (in both groups), ESAT-6 mostly in the H56 vaccinated group and TB10.4 exclusively in the H28 vaccinated group. Rv2660c-specific responses were below the threshold of detection by ICS (0.05%) and did not differ between the H28 and the H56 vaccinated mice (data not shown). The Ag85B-specific CD4 T cells in both the H56 and H28 vaccinated group were primarily poly-functional co-expressing the three cytokines IFN-γ, IL-2 and TNF-α although both IFN-y+TNF-α+, IL-2+TNF-α+ and TNF-α+ CD4 T cell populations were also represented (Figure 4A). The cytokine profile of the ESAT-6-specific CD4 T cells in the H56 group and the TB10.4-specific CD4 T cells in the H28 group were similar and with the same distribution as the vaccine-driven Ag85B response. The small but still detectable ESAT-6 response in the H28 group (which does not contain ESAT-6) was different and dominated by an infection-driven effector IFN-y+TNF-α+ CD4 T cell population whereas TB10.4 responses remained negligible in animals not receiving a TB10.4-containing vaccine (H56 and un-vaccinated control animals).

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Figure 4. H56 but not H28 vaccination provides post-exposure protection against relapse.

CB6F1 mice (n=12-18) were infected by the aerosol route and at week 6 post-infection (p.i.) treated with antibiotics given in the drinking water ad libitum. Mice were vaccinated three times s.c. with CAF01 alone, H28/CAF01 or H56/CAF01 at week 10, 13 and 16 p.i. (A) PBMCs (pooled from 16 mice) were obtained by submandibular bleeding one week after the last vaccination and the frequency and quality of antigen specific CD4 T cell responses were determined by six hour culturing of lung lymphocytes with Ag85B, TB10.4 or ESAT-6 from adjuvant control (black bars), H56/CAF01 (grey bars) and H28/CAF01 (white bars) animals. After culturing, cells were stained for CD4, IFN-γ, IL-2 and TNF-α, analyzed by ICS and subpopulations were divided according to their cytokine profile. Similar patterns of immune responses have been observed in two other experiments. (B) Protection was assessed at week 44 p.i. by culturing lung homogenates (n=12 for test groups and n=18 for control group) for 2-3 weeks at 37°C and enumerating the number of bacteria (expressed as log₁₀). Data displayed are box and whiskers showing median values and minimum and maximum CFU levels. Values marked with an asterisk are significantly different from naïve control animals as assessed by non-parametric Kruskall-Wallis multiple-comparison test (**, p<0.01, *, p<0.05). Data is representative of two independent experiments, although individual H28 and H56 vaccine evaluations have been done in several other experiments as illustrated in Figure 6.

https://doi.org/10.1371/journal.pone.0080579.g004

The vaccines were subsequently tested for their ability to inhibit the growth of M.tb. in the TB post-exposure model. Bacterial numbers was assessed at week 44 p.i. (Figure 4B). In agreement with previously published data, H56 post-exposure vaccination efficiently controlled bacterial growth, resulting in approximately ten times lower numbers of bacteria in these animals compared to the control group. In comparison, H28 vaccination did not prevent regrowth of the bacteria and the bacterial load was similar to the load measured in control animals. H56 vaccinated mice had significantly lower levels of bacteria compared to both control animals and H28 vaccinated animals and only 2 out of 12 animals had relapse of bacterial growth when vaccinated with H56, whereas 8 out of 12 mice in the H28 group had reactivated (defined as a bacterial load above 2 log₁₀).

Comparison of ESAT-6 and TB10.4; vaccine efficacy, T cell quality and gene expression

As the only difference between H56 and H28 is the substitution of ESAT-6 with TB10.4, we decided to focus on the post-exposure vaccine activity of these two antigens individually instead of being part of fusion-proteins. We therefore compared the efficacy of ESAT-6 and TB10.4 post-exposure vaccination in controlling relapse at week 37 and week 49 p.i. (Figure 5A). ESAT-6 vaccinated animals had a lower bacterial load compared to the control group at both time-points (Δlog₁₀: 1.41 and 1.46 at week 37 and week 49 p.i, respectively). Although the difference did not reach statistical significance for the individual time-points, ESAT-6 gave a statistical significant protection in the overall experiment (Mann-Whitney U-test: ESAT-6 vaccinated compared to control group: week 37 p.i. p=0.0541, week 49 p.i. p=0.1893, overall experiment p=0.0168). In contrast, TB10.4 vaccination only resulted in a very modest reduction of bacteria (approximately 0.4 log₁₀ CFU compared to the control group), which was not significantly different from control animals at any time-point or for the experiment in total (TB10.4 vaccinated compared to control group: TB10.4 vs. control: week 37 p=0.3357 week 49 p=0.8665, overall experiment: p=0.4176). To address the possibility that the protective differences exerted by ESAT-6 and TB10.4 were a result of a different level of gene expression at different stages of the infection, we compared the expression of esxA, esxH, acr (alpha-crystallin) and fhpB (Ag85B) at week 3 and week 20 p.i. (Figure 5B). acr and fhpB was included for comparison as these genes have previously been reported to represent genes expressed either during early growth (fhpB) [13] or during hypoxia-induced stress (acr) [30]. The expression of esxA and esxH was found to be high and at comparable levels at week 3 p.i. At week 20 p.i. the onset of adaptive immunity and control of bacterial growth had resulted in a general down-regulation of gene expression but the relative expression of esxA compared to esxH, remained at the same level. The expression of acr was also down regulated at the late time-point but remained high in contrast to the expression level of fhpB which was below the detection level (100 relative RNA copies per chromosome) at week 20 p.i. The failure of TB10.4 post-exposure vaccination is therefore not a consequence of a specific down-regulation of esxH in the later stages of infection. Finally, we investigated if a difference in the quality of the vaccine-induced immune response could explain the protective differences after ESAT-6 and TB10.4 post-exposure vaccination. Lymphocytes from the lungs of vaccinated animals were obtained and re-stimulated with the homologous antigen preparation at two time-points, week 17 p.i. (1 week post last vaccination) and week 49 p.i. (week 33 post last vaccination). Approximately 2 % TB10.4 specific CD4 T cells were detected in TB10.4 vaccinated mice at week 17 p.i. and this frequency increased to approximately 4.5 % at week 49 p.i. (Figure 5C). The TB10.4-specific CD4 T cells consisted of about 35-50 % IFN-γ+IL-2+TNF-α+ CD4 T cells at the two time-points. A similar level of vaccine-induced response of a similar quality was observed in ESAT-6 vaccinated mice at the two time-points. Both the TB10.4 and ESAT-6 specific immune response in control animals (infection-driven) consisted for comparison primarily of IFN-γ+TNF-α+ CD4 T at both time-points (data not shown). Thus, the vaccine-induced response generated by TB10.4 was very similar to the ESAT-6 induced response both in terms of magnitude and quality i.e. the proportion of IFN-γ+IL-2+TNF-α+ CD4 T cells and differed markedly from the infection promoted response to these antigens.

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Figure 5. ESAT-6 and TB10.4; gene expression, immunogenicity and post-exposure vaccine efficacy.

CB6F1 mice were infected, treated with antibiotics as described above and vaccinated with ESAT-6/CAF01 (n=7), TB10.4/CAF01 (n=7) or CAF01 alone (n=8) at week 10, 13 and 16 post infection (p.i.). (A) At week 37 and 49 p.i. mice were sacrificed, lungs harvested for assessment of protection by culturing lung homogenates at 37° C and enumerating the number of bacteria (expressed as log₁₀). Log₁₀ CFUs are shown for CAF01 only control animals (open triangles), TB10.4 (open squares) and ESAT-6 (open circles) vaccinated animals. Data are shown as median ± interquartile range and represent 6-8 individual animals. Evaluations of ESAT-6 and TB10.4 have been done in several experiments with similar outcome (the overall dataset is depicted in Figure 6). (B) M.tb. RNA was extracted from lung lobes of CB6F1 mice and used for real-time PCR to assess the gene expression level of the acr, esxA, esxH and fhpB. Data shown as relative transcript numbers (normalized data) and depicts the mean ± standard deviation (n=4). The dotted line denotes the level of detection at 100 relative RNA copies per bacterial genome. (C) Lymphocytes were extracted at week 17 and 49 p.i. from perfused lungs of six pooled mice, vaccinated with TB10.4 or ESAT-6. The cells were re-stimulated with their respective vaccine antigen and stained for CD4, IFN-γ, IL-2 and TNF-α for ICS analysis. Data is displayed as the total frequency of cytokine producing CD4 T cells (producing IFN-γ, IL-2, TNF-α or any combination of the three). The pie-charts represent the proportion of the subpopulation of responding CD4 T cells color-coded according to their cytokine profile.

https://doi.org/10.1371/journal.pone.0080579.g005

Large scale evaluation of post-exposure activity of ESAT-6 and TB10.4 containing vaccines

Given the surprising difference on the post-exposure activity of the H56 and the H28 vaccines (and the ESAT-6 and TB10.4), we proceeded by comparing the protective efficacy conferred by the individual components in these two constructs (Ag85B, TB10.4, ESAT-6 and Rv2660c), the two closely related fusion-proteins H1 (Ag85B-ESAT-6) and H4 (Ag85B-TB10.4) and the Rv2031c molecule. Rv2031c was included because of its widely accepted role as a latency-associated antigen; up-regulated under hypoxia [31] and preferential recognition in latently infected individuals [32]. To overcome the shortcomings related to intra-and inter-experimental variation, we normalized data for fifteen individual experiments conducted over 3 years within the Gates Grand Challenges GC12 Consortium (see materials and methods for details) and pooled the result into one large analysis based on data from 726 mice (Figure 6). The data from the pooled analysis is depicted as Δlog₁₀ protection (i.e. the difference in bacterial loads in vaccinated animals compared to control animals in each individual experiment). Based on this analysis it was evident that vaccines giving significant levels of protection compared to the control group were the ESAT-6-containing vaccines H1, H56 as well as ESAT-6 administered alone. Post-exposure vaccination with H56 resulted in significantly better protection compared to all the other vaccine candidates included in the analysis, with the exception of H1. H56 in the pooled analysis resulted in a Δlog10 value compared to the control group of 0.9821 log compared to e.g. H28 which had a Δlog₁₀ value of 0.09843. Thus, ESAT-6 containing vaccines have a unique capability to inhibit relapse of bacterial growth when given post-exposure which is not seen with any of the other antigens and vaccines, although these vaccines had prophylactic activity.

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Figure 6. Meta-analysis of post-exposure vaccine efficacy.

The post-exposure activity of various vaccines was tested in fifteen individual experiments including a total number of 726 mice. The data shown is a meta-analysis performed by determining the log₁₀ protection for each individual experiment as follows: log_10protection = median log₁₀CFU_controlgroup −log₁₀CFU_{vaccinated animal} as described in the material and methods section. Data are displayed as single scatter plot with a black line indicating the median values. Values marked with an asterisk above ESAT-6, H1, and H56 are significantly different from the un-vaccinated control groups as assessed by the non-parametric Kruskall-Wallis multiple comparison test (***, p<0.001, **, p<0.01, *, p<0.05).

https://doi.org/10.1371/journal.pone.0080579.g006

ESAT-6 vaccination controls relapse of bacterial growth but does not reduce bacterial load in a chronic steady-state infection

Post-exposure administration of ESAT-6 based vaccines efficiently controlled the relapse of bacterial growth in the modified Cornell model and we continued by investigating if this activity had a broader application e.g. if ESAT-6 could be used therapeutically to influence the course of a chronic infection in mice. For this purpose, vaccination was administered in a model where the bacterial numbers had been stable throughout infection after establishment of adaptive immunity. Thus we wanted to determine if ESAT-6 post-exposure vaccination would perturb the host-pathogen equilibrium, in favor of the host, leading to a reduction in bacterial load in the steady-state scenario.

ESAT-6 was given to mice with a chronic infection characterized by a high bacterial load, approximately 5 log₁₀ CFU, after reaching a steady-state condition following week 6 of infection (Figure 7A). Immunizations with ESAT-6 in CAF01 was given at week 6, 8 and 10 p.i. Therapeutic effect of ESAT-6 was evaluated at week 21 p.i. by plating and culturing lung homogenates. Post-exposure vaccination during a chronic infection did not reduce the bacterial load nor did it exacerbate disease compared to control animals (ESAT-6: 5.01 log₁₀ CFU versus control 4.70 log₁₀ CFU, respectively, p=0.8518). In agreement with the findings above, mice immunized with ESAT-6 in combination with antibiotic treatment in the modified Cornell model had a significant lower bacterial load compared to control mice at all time-points examined ranging from 1-2.3 log₁₀ difference in median values (week 18 p= 0.0411, week 27 p= 0.0303, week 40 p= 0.0129) (Figure 7B).

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Figure 7. Comparison of therapeutic and post-exposure vaccine efficacy.

(A) In the therapeutic model, CB6F1 mice were aerosolly infected and vaccinated three times with two weeks interval starting at week 6 post infection (p.i.) (black arrows) with ESAT-6/CAF01 (open circle) or CAF01 alone (open triangle). The therapeutic efficacy was assessed at week 21 p.i. Data shown are median values ± interquartile range week 0-20 from 3-8 individual animals and at week 21 p.i. n=7 for control animals and n=8 for ESAT-6 vaccinated. The two groups were compared with a Mann Whitney U-test and p<0.05 was considered significant. (B) In the post-exposure model, mice were infected for six weeks and then given antibiotics for six weeks, as described in materials and methods. Groups of mice were vaccinated with ESAT-6/CAF01 or CAF01 alone at week 10, 12 and 14 p.i. (black arrows) and protection assessed at week 18, 27 and 40 p.i. by culturing lung homogenates and enumerating the number of bacteria (expressed as log₁₀). Data shown are median values ± interquartile range for the control group (open triangles, n=6) and ESAT-6 vaccinated group (open circle, n=6). Values marked with an asterisk are significantly different from naïve control animals as assessed by non-parametric Mann-Whitney U-test (**, p<0.01, *, p<0.05).

https://doi.org/10.1371/journal.pone.0080579.g007

Thus, ESAT-6 post-exposure vaccination results in protection against relapse of bacterial growth when administered to mice with a low bacterial load at the time-point for vaccination. ESAT-6 vaccination does, on the other hand, not have a therapeutic effect when administered in mice with an ongoing chronic infection.

Ethics statement

Experiments were conducted in accordance with the regulations set forward by the Danish Ministry of Justice and animal protection committees by Danish Animal Experiments Inspectorate Permits 2004-561-868 and 2009/561-1655, and in compliance with European Community Directive 86/609 and the U.S. Association for Laboratory Animal Care recommendations for the care and use of laboratory animals. The experiments were approved by the Statens Serum Institute animal ethics board headed by DVM Kristin E. Engelhart Illigen.

Animals

Inbred female BALB/c x C57BL/6 (CB6F1) mice was obtained from Harlan Scandinavia (Allerød, Denmark). Mice were kept at the experimental animal facilities at Statens Serum Institut. Infected mice were housed in cages contained within laminar-flow safety enclosures (Scantainer; Scanbur) in a biosafety level 3 facility, and provided with irradiated food and filtered drinking water.

Cloning and purification of fusion proteins

The rv2660c gene was amplified by PCR fromM.tb. H37Rv chromosomal DNA using either 5′-GGATGTTCGCAGTGATAGCGGGCGTCGAC-3′ or 5′-ATGGGGCGGCGTGATAGCGGGCGTCGAC-3′ as sense oligonucleotide and 5′-TTAGTGAAACTGGTTCAATCCCAGTATC-3′ as anti-sense oligonucleotide. Stop codon is underlined, and sequence overlaps to the esxA or esxH sequence are in bold. The amplified rv2660c gene was fused to fhpB-esxA or fhpB-esxH by overlapping PCR and inserted into the pDest17 expression vector (Invitrogen). E. coli BL21-SI cells (Invitrogen) harboring fhpB, esxA, esxH, rv2660c, fhpB-esxA, fhpB-esxH, fhpB-esxH-rv2660c or fhpB-esxA-rv2660c produced all the recombinant proteins as inclusion bodies. All proteins were purified according to the same protocol. Inclusion bodies were washed three times in 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.1% deoxycholic acid and dissolved in 8 M urea, 100 mM Na2PO4, 10 mM Tris-HCl pH 8.0 (buffer A) before being applied to metal affinity columns (Clontech). Bound proteins were washed five times with two column volumes of buffer, alternating between 10 mM Tris-HCl pH 8.0, 60% isopropanol and 50 mM NaH2PO4 pH 8.0, before being eluted in buffer A supplemented with 200 mM imidazole. After inspecting for impurities, relevant fractions were pooled and dialyzed against 3 M urea, 10 mM Tris-HCl pH 8.5 and applied to anion-exchange columns (Pharmacia) washed with 5 column volumes and eluted using a linear NaCl gradient, from 0 to 1 M over 40 column volumes. Based on purity, fractions were pooled and dialyzed against 25 mM NaH2PO4 pH 8.0, 10% glycerol, 150 mM NaCl, 0.05% Tween20. Finally, protein concentrations (NanoOrange™ Protein Quantitation Kit, Life Technologies, Denmark) and protein purities (scanning of stained gels) were determined.

Synthetic peptides

Synthetic overlapping peptides (15 or 18-mers) covering the complete primary structure of Ag85B, ESAT-6 and TB10.4 were synthesized by standard solid-phase methods on a SyRo peptide synthesizer (MultiSynTech, New England) at the JPT Peptide Technologies (Berlin, Germany) or GenScript (New Jersey, USA). Peptides were lyophilized and stored dry at −20°C until reconstitution in PBS and DMSO (1:1 ratio).

Histopathological analyses

Lungs were removed post mortem at week 6, 12, and 30 after aerosol infection with M.tb. (six mice per group per time-point). The right lung lobe of each mouse was fixed by immersion in 10% neutral-buffered formalin and processed for histological examination. Sections were stained using haematoxylin and eosin and by the Ziehl-Neelsen method and were evaluated without prior knowledge of stage of infection or of treatment group. Lesions were quantified using computer-aided histomorphometry (Palm®robo software, version 1.2.3; Palm Microlaser Technologies AG Ltd, Bernried, Germany).

Vaccine evaluations

Cell preparation, ELISA and flow cytometric analyses

Peripheral blood mononuclear cells (PBMCs) and lung lymphocytes from perfused lungs were isolated, cultured and assessed for immune responses as previously described [29,44,53]. PBMCs were purified on a density gradient of mammal Lympholyte® cell separation media (Cedarlane Laboratories Inc., Canada) according to the manufacturer’s protocol, and followed by two washing procedures using RPMI. Lung lymphocytes were obtained by passage of lungs through a 100 µm nylon cell strainer (BD Pharmingen, USA) followed by two washing procedures using RPMI. Cells were cultured in sterile microtiter wells (96-well plates; Nunc, Denmark) containing 2×10⁵ cells/well for ELISA or 1-2×10⁶cells/well for flow analyses. Both ELISA and flow cultures were in 200 µl of RPMI-1640 supplemented with 5 x 10^-5 M 2-mercaptoethnaol, 1% pyruvate, 1% HEPES, 1% (v/v) premixed penicillin-streptomycin solution (Invitrogen Life Technologies), 1 mM glutamine, and 10% (v/v) fetal calve serum (FCS) at 37°C/5% CO₂ and re-stimulated with antigen at 2 µg/ml.IFN-γ levels were measured in supernatants after 72 hours incubation at 37°C by ELISA as previously described [44]. For intracellular staining of cytokines (ICS) cells from blood or lungs of mice were stimulated for 1 hour with 2 µg/ml of antigen and subsequently incubated for 5 h at 37°C with 10 µg/ml brefeldin A (Sigma-Aldrich, Denmark) at 37°C. Cells were washed in FACS buffer (PBS containing 0.1% sodium azide and 1% FCS) before staining with a combination of the following rat anti-mouse antibodies PerCP-Cy5.5-anti-CD8α (53-6.7, RM4-5), APC-Cy7-anti-CD4 (GKI.5), and FITC-anti-CD44 (IM7). Cells were washed with FACS buffer before fixation and permeabilization using the BD Cytofix/Cytoperm™ (BD, San Diego, CA, USA) according to the manufacturer's protocol before staining intracellular with PE-Cy7-Anti-IFN-γ (XMG1.2), PE-anti-TNF-α and APC-anti-IL-2 (JES6-5H4). After washing, cells were suspended in PBS/or formaldehyde solution 4% (w/v) pH 7.0 (Bie & Berntsen, Denmark) and samples were analyzed on a six-color BD FACSCanto flow cytometer (BD Biosciences, USA). Responses were analyzed using Flowjo Software (© Tree Star, Asland, OR, USA) followed by Pestle and Spice software [54] by Boolean gating of IFN-γ, TNF-α and IL-2 (only IFN-γ and IL-2 in Figure 3A) positive CD4 CD44^high (gating sequence: singlets, lymphocytes, CD4 vs FSC-A, CD44 vs cytokine). The cytokine responsive cell populations divided according to their cytokine profile was reported either as total % cytokine positive cells (i.e. the sum of all the subpopulations as in Figure 3A and 5C) or with the representation of each individual sub populations (as in Figure 4A). The background (BG) levels of cytokine production measured in media stimulated cells were deducted (BG<0.5%).

Meta-analysis

The meta-analysis was generated based on fifteen independent experiments with a total number of 726 mice. Each dot in Figure 6 corresponds to a Δlog₁₀ protection value for one individual mouse where differences in the overall CFU level in the different experiments were normalized. This was done by subtracting the CFU value of the individual vaccinated by from the median CFU level for the group of control animals in each experiment. Each data point was therefore calculated as follows: median log₁₀CFU_controlgroup −log₁₀CFU_{vaccinated animal}. To take the variation of individual control animals into consideration for the statistical analysis, the same subtraction was done for each individual control animal (median log₁₀CFU_controlgroup −log₁₀CFU_{control animal} and the overall data set analyzed by a Kruskal-Wallis statistical analysis. The analysis in total represent 520 data points of vaccine efficacies in the animal post-exposure model of which 40 data points, was calculated based on the original experiments previously reported in the publication by Aagaard et al. [10].

Genome Expression Profiling of MTB using Two-Step Multiplex real time RT-PCR

Frozen lungs from TB-infected mice were homogenized in Trizol containing 20 μg/ml acrylamide. After centrifugation, the debris with unbroken M.tb. bacilli was resuspended in Trizol and the M.tb. bacilli were disintegrated by bead-beating for 30 seconds at speed 6, 3x (Fastprep24 bead-beater, MP Biomedicals) using 0.1mm zirconia/silica beads (Biospec). Total RNA was isolated using RNeasy kit (Qiagen) and two off-column RQ1 DNase treatments (Promega), and finally resuspended in 50 μl RNase-free water (Ambion).

Statistical analysis

For the pre-exposure data which follows a normal distribution protective efficacies of the vaccines (log₁₀ CFU´s) were compared by one-way analysis of variance (ANOVA) followed by Tukey´s multiple comparison test of the means. For the post-exposure evaluation of vaccines, we generally found larger variation within the groups and the data points did not follow a normal distribution, hence non-parametric test were used for these sets of experiments. Protective efficacies (log₁₀ CFU´s) were compared by a non-parametric Mann-Whitney U-test when comparisons were done between two groups e.g. between ESAT-6 vaccinated and control group in Figure 7B and a Kruskall-Wallis for comparison of medians between several groups e.g. in Figure 5 and 6. For all tests, p-values <0.05 were considered statistically significant.