The Tuberculosis Vaccine Candidate Bacillus Calmette-Guérin ΔureC::hly Coexpressing Human Interleukin-7 or -18 Enhances Antigen-Specific T Cell Responses in Mice

Bacillus Calmette–Guérin (BCG), the only approved tuberculosis vaccine, provides only limited protection. Previously, we generated a recombinant derivative (BCG ΔureC::hly), which secretes the pore-forming toxin listeriolysin O (LLO) of Listeria monocytogenes. This vaccine shows superior protection against tuberculosis in preclinical models and is safe in humans. Here we describe two new vaccine strains which express human interleukin-7 (hIL)-7 or hIL-18 in the genetic background of BCG ΔureC::hly to modulate specific T cell immunity. Both strains exhibited an uncompromised in vitro growth pattern, while inducing a proinflammatory cytokine profile in human dendritic cells (DCs). Human DCs harbouring either strain efficiently promoted secretion of IL-2 by autologous T cells in a coculture system, suggesting superior immunogenicity. BALB/c mice vaccinated with BCG ΔureC::hly, BCG ΔureC::hly_hIL7 or BCG ΔureC::hly_hIL18 developed a more robust Th1 response than after vaccination with parental BCG. Both strains provided significantly better protection than BCG in a murine Mycobacterium tuberculosis challenge model but efficacy remained comparable to that afforded by BCG ΔureC::hly. We conclude that expression of hIL-7 or hIL-18 enhanced specific T cell responses but failed to improve protection over BCG ΔureC::hly in mice.


Introduction
An estimated 30% of the world's population is latently infected with Mycobacterium tuberculosis, the aetiological agent of tuberculosis (TB) [1]. Of an estimated 8.7 million new TB cases worldwide in 2011, 1.4 million people died of whom 95% were from low-to middle-income countries [1]. In line with this, 1.1 million clinical TB cases have been reported to account for human immunodeficiency virus (HIV) coinfected individuals, with approximately 500,000 TB-related deaths globally [2,3]. Additionally, the advent of drug-resistant M. tuberculosis strains complicates treatment while limiting chances of survival [4].
Vaccines remain the most cost-effective means to counteract the global challenges related to infectious diseases including TB [5]. Bacillus Calmette-Guérin (BCG) is the only licensed vaccine for TB, and protects children but leaves adults unprotected from the most prevalent form of the disease, pulmonary TB [6]. This calls for better vaccines against TB. Two main strategies are pursued in TB vaccine research -subunit and live vaccines. Subunit vaccines are generally aimed at boosting cellular immunity initially raised by BCG administered as prime vaccination [7]. Live vaccines are developed to replace BCG itself. Principally, a robust CD4 + T helper 1 (Th1) response represented by interferon gamma (IFN-c) and tumour necrosis factor-alpha (TNF-a) expression should be induced by the prime vaccine to eventually form a pool of memory T cells which control TB disease [8,9].
We have previously reported the superior protective efficacy of BCG DureC::hly (VPM1002), a recombinant BCG vaccine candidate that is urease-deficient and heterologously expresses listeriolysin O (LLO) [10,11]. One of the main features of this strain is its ability to perforate the host cell phagosomal membrane, thus releasing antigens into the cytosol of the host macrophage and promoting crosspriming [7,10,12].
Interleukin (IL)-7 and IL-18 have been implicated in immunity to M. tuberculosis infection [13,14,15,16]. More specifically, IL-7 is involved in homeostatic regulation of T-and B-cell proliferation in humans and mice [17]. Administration of purified recombinant IL-7 has been shown to influence recall T cell responses to M. tuberculosis infection with and without prior BCG vaccination [15,16]. IL-18, induces IFN-c secretion jointly with IL-12 as well as expression of TNF-a [18]. Both IFN-c and TNF-a are proinflammatory cytokines critical in shaping Th1-mediated immune responses in TB [19]. Mice lacking expression of IL-18 are susceptible to M. tuberculosis infection [13,20]. Moreover, high levels of IL-18 were detected in sera of patients with advanced pulmonary TB [21].
Expression of recombinant cytokines by BCG has been shown to promote better immunogenicity [22,23,24]. We therefore hypothesized that incorporating the expression of human (h)IL-7 or IL-18 into BCG DureC::hly could improve its immunogenicity. In this study, we describe two newly-derived candidate derivatives of BCG DureC::hly coexpressing either IL-7 or IL-18, namely BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18, respectively. Both strains were evaluated for their intracellular fitness in primary human cells, and their immunomodulatory properties as well as protective efficacies against aerosol challenge with M. tuberculosis.

Ethics Statement
All experimental procedures involving mice were performed in accordance with requirements of, and approval by, the State Office for Health and Social Services (Landesamt für Gesundheit und Soziales), Berlin, Germany under permission number G0307/ 11. Mice were sacrificed by cervical dislocation, and all efforts were made to minimize suffering and pain.

Infection of Primary Human Macrophages and Dendritic Cells
CD14 + primary human monocytes were purified from peripheral blood mononuclear cells (PBMCs, provided by Charité Hospital, Berlin, Germany) by magnetic separation on an LS column using antihuman CD14 MACS positive selection beads (Miltenyi). Cell pellets of purified CD14 + human monocytes were then resuspended in 20 mL of sterile RPMI medium (Gibco) supplemented with 10% fetal calf serum, 1% L-glutamine, 1% HEPES and 0.1% 2-mercaptoethanol (complete RPMI). After determining cell density, the suspension was equally divided in tissue culture flasks. Dendritic cells (DCs) were generated by addition of 50 ng/mL human GM-CSF and 25 ng/mL human IL-4, while macrophages (MWs) were generated by addition of 50 ng/mL, human GM-CSF over 6 days of incubation at 37uC with 5% CO 2 . For infections, 10 mL of bacterial cultures grown to logarithmic phase (OD 600 ,0.6) were pelleted at 4000 rpm for 5 min at RT. Bacteria were washed with PBS and subsequently dislodged in 1 mL PBS. Single cell suspensions were prepared using a 23G needle (Braun, Germany) over 5 syringe-driven plunges. Human cells were infected at a multiplicity of infection (MOI) of 10 in 48-well culture plates and incubated at 37uC with 5% CO 2 for 6 days.

Coculture of Primary Human T Cells with Autologousinfected DCs
Primary human T cells were purified via magnetic separation on an LS column using the pan T cell isolation kit (Miltenyi). Purified T cells were coincubated with infected DCs at a ratio of 20 T cells per infected DC over a 24-h period at 37uC with 5% CO 2 . Cell-free culture supernatants were collected at designated time points by eliminating cellular debris using Costar Spin-X columns (Corning, USA). The flow-through was then stored at -20uC for multiplex or ELISA assays. Infected cells were washed once with PBS (1500 rpm, 5 min, RT) and lysed with 100 mL PBS/1% Triton X-100 for 10 min prior to plating in serial dilutions on Middlebrook 7H11 agar.

ELISA, Multiplex Cytokine Assays and Flow Cytometry
The Bio-Rad Pro Human Cytokine 27-plex Panel was used for primary human MW and DC experiments while the Mouse Cytokine 23-plex Panel was used for analysis of cytokines in sera of vaccinated mice. In all multiplex assays, the volume of the coupled beads, detection antibodies and streptavidin-PE conjugate was halved and topped up with the appropriate buffer. The assays were otherwise performed according to the manufacturer's instructions. Assay plates were read using the Bio-Plex 200 system (Bio-Rad). DuoSet human IL-7 and IL-2 ELISA kits were purchased from R&D Systems. Human IL-18 matched ELISA antibody pairs were purchased from eBioscience. Flow cytometric experiments were performed on a LSRII cytometer (BD Biosciences) and analyses were carried out using the FlowJo software (Tree Star Inc.). Antimouse antibodies for flow cytometry used in this study were as follows: antiCD3e-Alexa Flour 700; clone: 17A2 (eBioscience), antiCD4-V500; clone RM4-5 (BD Horizon), antiCD8a-PerCP; clone: 169 (in-house), antiCD44-Pacific Blue; clone: IM7 (in-house), antiCD154-APC; clone: MR1 (eBioscience), antiIFN-c-PE-Cy7; clone: XMG1.3 (BioLegend) and antiTNF-a-FITC; clone: XT-22 (in-house).

Analysis of CD4 + T Cells from Vaccinated Mice
All in vivo experiments were carried out with 9-to 10-week old female BALB/c mice purchased from Janvier (France). Spleens, lungs and draining (inguinal) lymph nodes (dLNs) of mice were aseptically removed and individually homogenized in 2 mL of complete RPMI medium with penicillin and streptomycin (cRPMI+p/s) using 70-mm cell strainers (BD) and 2-mL syringe plungers to prepare single-cell suspensions (SCS). Cell strainers were washed with 12 mL of cRPMI +p/s, and along with the SCS centrifuged at 1300 rpm for 5 min at 4uC. Lungs were first cut into small pieces, digested with 10 mL of collagenase mix (0.7 mg/mL Collagenase IV (Sigma-Aldrich) and 0.3 mg/mL Collagenase D (Roche) in cRPMI+p/s) and incubated at 37uC with 5% CO 2 for 30 min prior SSC preparation. Erythrocytes in samples were lysed by addition of 2 mL 0.0083% NH 4 Cl/0.001% KHCO 3 /0.037% EDTA/H 2 O at RT for 2 min. Subsequently lysis was stopped by 12 mL PBS/0.2% bovine serum albumin and cells were pelleted at 1300 rpm for 5 min at RT. The dLN and lung cell pellets were resuspended in 500 mL cRMPI+p/s and spleen cell pellets in 3 mL cRPMI+p/s. Five mL of the respective suspensions was added to 200 mL of 1x AccuCheck bead suspension (Invitrogen; 1:5 diluted) for flow cytometric counting. A total of 500 mL of each cell suspension was seeded in a 48-well plate, and stimulated with 10 mg/mL of M. tuberculosis H37Rv whole cell lysate in presence of 5 mg/mL brefeldin A for 6 h at 37uC/5% CO 2 . Cells were then washed, resuspended in 200 mL of PBS-BSA and transferred to a 96-well microtitre filter plate. After centrifugation (1300 rpm, 3 min) cell pellets were resuspended in 90 mL PBS-BSA and stained on ice for 20 min with 10 mL of a 106surface stain master mix containing antiCD4-V500, antiCD44-Pacific Blue, antiCD3-Alexa Fluor 700 and antiCD8-PerCP at 1:20 dilution. Afterwards, cells were washed with 200 mL of PBS-BSA, centrifuged (1300 rpm, 3 min), resuspended in 60 mL of BD Cytofix/Cytoperm solution and incubated on ice for 20 min. Following one wash step with 150 mL perm wash (BD), cell pellets were resuspended in 50 mL of the following antibodies in perm wash for intracellular staining on ice for 30 min (antiCD154-APC; 1:20 dilution), antiTNF-a-FITC and antiIFN-c-PECy7, 1:200 dilution). This was followed by a final wash step with 150 mL perm wash and resuspended in 200 mL PBS-BSA. The stained cell suspension was applied to a 35 mm cell strainer capped onto a 12675 mm tube (BD) for a quick spin followed by flow cytometry analysis.

Safety and Protective Efficacy of rBCG Strains in Mice
Mice were vaccinated subcutaneously in the tail base with 10 6 CFUs of either BCG SSI 1331, BCG DureC::hly, BCG Dure-C::hly_hlL7 or BCG DureC::hly_hIL18. At designated time points post-vaccination, spleens and dLNs of vaccinated mice were aseptically removed for CFU enumeration. For the protective efficacy study, mice were challenged via the aerosol route with 200 CFUs of M. tuberculosis H37Rv 90 days post-vaccination. At designated time points, lungs and spleens were aseptically removed, homogenized in PBS/0.05% Tween 80 and plated in serial dilutions onto 7H11 agar for CFU enumeration.

Statistical Analyses
The GraphPad Prism 6 software was used for data analysis. Results were expressed as mean 6 standard error of the mean (SEM) and analyzed using one-way or two-way analysis of variance (ANOVA) and Bonferroni's or Tukey's multiple comparisons post-test as applicable. Value of p,0.05 was considered statistically significant.

Results
Generation of BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18 We first confirmed presence of the hIL-7 or the hIL-18 gene in the respective BCG DureC::hly transformants by PCR using genomic DNA (gDNA) as template and specific oligonucleotides. A schematic representation of the IL expression cassette is shown in Figure 1A. Cytokine gene and hly transcription was then investigated by RT-PCR. Expression of LLO is an integral feature of the parental strain, BCG DureC::hly and its derivatives. The mRNA level of hIL-7 was lower than that of hIL-18 produced by the respective strain ( Figure 1B). However, the stronger transcription of hIL-18 was associated with reduced amounts of hly mRNA which encode LLO, the integral component of BCG DureC::hly. ELISA analysis of whole cell extracts and culture supernatants revealed that our observation with gene transcription of hIL-7 and hIL-18 correlated with hIL-7 and hIL-18 protein expression by BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18, respectively ( Figure 1C). However, the majority of protein was accumulated in the cytosolic fraction and only minor quantities were secreted.

BCG DureC::hly_hIL18 Exhibits a Proinflammatory Phenotype in vitro
The intracellular growth and survival kinetics of BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18 were studied in primary human MWs and DCs. BCG DureC::hly served as control in these experiments. The growth kinetics of BCG DureC::hly, BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18 were similar in both MWs and DCs, indicating that recombinant cytokine expression did not compromise intracellular fitness of the strains under investigation (Figure 2A). Intriguingly, bacterial growth was more efficiently restricted in DCs than in MW. Secretion of a panel of proinflammatory cytokines, including IL-6, TNF-a and G-CSF was highly upregulated in DCs infected with BCG DureC::hly_-hIL18 (Fig. 2B), indicating that the potential of BCG DureC::hly_-hIL18 to initiate proinflammatory cytokine signalling promoted Th1 responses. However, we did not observe such differences with infected MWs (data not shown). In order to investigate whether BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18 enhance T cell activation, we infected primary human DCs with either strain and cocultured them with autologous T cells over a 24-h period. The secretion of IL-2, a cytokine important for T cell proliferation, was chosen as readout. BCG DureC::hly, BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18 were all significantly better at priming T cells compared to BCG as determined by IL-2 secretion ( Figure 2C). Taken together, expression of IL-7 or IL-18 in the genetic background of BCG DureC::hly did not influence T cell activation in this model.

BCG DureC::hly_hIL18 Induces a Proinflammatory Cytokine Response in Mice
One of the desired features of a live TB vaccine is prompt clearance from the host as a measure of safety. We vaccinated mice with BCG SSI 1331, BCG DureC::hly, BCG DureC::hly_hIL7 or BCG DureC::hly_hIL18 and monitored dissemination to as well as clearance from the spleen and draining (inguinal) lymph nodes (dLNs). All rBCG strains were cleared within 60 days postvaccination from both organs while BCG SSI 1331 was found in the dLNs up to 120 days post-vaccination ( Figure 3A). However, no bacteria were observed in the spleen beyond 90 days postvaccination. Thus, improved clearance and indirectly safety of BCG DureC::hly was preserved in both derivative strains under investigation.
We investigated serum cytokine levels using a multiplex platform for 27 cytokines. All comparisons were made in relation to BCG DureC::hly. In contrast to the derivative strain expressing hIL-7, BCG DureC::hly_hIL18 vaccinated mice showed up-regulation of proinflammatory cytokines IL-6, KC, CCL5, IL-2 and G-CSF ( Figure 3B). In more detail, KC is a murine orthologue of human IL-8, an important neutrophil chemoattractant [28]. In line with this, G-CSF is instrumental in proliferation of neutrophils [29]. IL-6 is involved in T cell proliferation and B cell stimulation [30,31] as well as prostaglandin release [32]. IL-2 also reflects T cell proliferation while CCL5 plays a role in T cell chemotaxis [33,34]. This observation was consistent with the cytokine analysis performed with culture supernatants of infected human DCs, confirming the proinflammatory potential of BCG DureC::hly_-hIL18 as compared to its parental strain ( Figure 2C). BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18 enhance antigen-specific Th1 responses The main objective of this study was to assess the effect of human cytokine coexpression by BCG DureC::hly on T cell responses. Mice vaccinated with BCG SSI 1331, BCG DureC::hly, BCG DureC::hly_hIL7 or BCG DureC::hly_hIL18 were monitored for modulation of M. tuberculosis-specific CD4 + T cells based on their expression of CD40L over time. M. tuberculosis H37Rv whole cell lysate was used as source of T cell antigens.
In addition to CD40L + expression, functionality of the M. tuberculosis-specific CD4 + T cells was also examined. BCG DureC::hly_hIL18 contributed to the largest proportions of CD40L + TNF-a + and CD40L + TNF-a + IFN-c + CD4 + T cells on days 60 and 90 post-vaccination, respectively (Fig. 4B, Table 1). CD40L + IFN-c + CD4 + T cells were also elicited by the vaccine  was made in the lungs and spleen at 60 days post-vaccination. No CD40L + IFN-c + CD4 + T cells were seen in any of the vaccinated mice after day 60 post-vaccination. We conclude that vaccination of mice with either BCG DureC::hly_hIL7 or BCG DureC::hly_hIL18 induced specific CD4 + T cell responses lasting up to at least 90 days. Particularly, BCG DureC::hly_hIL18 was capable of inducing a proinflammatory immune response early on, which triggers enhanced CD4 + T cell responses up to 90 days post-vaccination.
Protective Efficacy of BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18 For validating the protective capacities of BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18 against TB, vaccinated mice were challenged with M. tuberculosis H37Rv via the aerosol route and bacterial burdens in lungs and spleens were monitored over time. A schematic representation of the efficacy study is displayed in Figure 5A. In a vaccination group that received a mix of BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18, we aimed to analyze potential synergies. At 30 days post-aerosol challenge, mice vaccinated with BCG DureC::hly, BCG DureC::hly_hIL7 or BCG DureC::hly_hIL18 showed similar protection as BCG SSI 1331 in lungs, while at day 200 all rBCG strains provided superior protection over BCG SSI, as reported earlier for BCG DureC::hly ( Figure 5B) [10]. In spleens, no differences between BCG strains were observed, but all strains conferred significant protection as compared to an unvaccinated control group. We conclude that expression of hIL-7 or hIL-18 in the genetic background of BCG DureC::hly did not alter protective efficacy in mice.

Discussion
BCG remains the most widely applied human vaccine in current use, with over 4 billion doses administered [6,7]. Over the years, BCG has been cultured and passaged by various laboratories across the globe, giving rise to a range of genetically different BCG substrains [35]. BCG protects against severe forms of TB in children but not against the most prevalent pulmonary form in all age groups [6,36]. Moreover, BCG is only to be given pre-exposure and therefore does not apply to individuals with latent TB infection (LTBI), who do not present with clinical symptoms but nonetheless are at 5-10% risk of developing clinical TB disease during their lifetime [37]. Various efforts are currently underway to improve antitubercular immunity by rBCG vaccines [38].
BCG was first considered as a vehicle for heterologous expression of foreign proteins with the development of mycobacterial expression vectors in the early 1990s [26]. Since then, BCG was used to express proteins from an array of human pathogens such as Plasmodium falciparum [39], the human immunodeficiency virus (HIV) [40], measles virus [41] Streptococcus pneumonia [42] and Borrelia burgdoferi [43], as well as immunomodulatory molecules such as cytokines, i.e. GM-CSF, IL-18, IFN-c [22,24] and others. Taken together, BCG proved to be a useful platform for heterologous antigen expression.
In this study, we designed and constructed experimental rBCG vaccine candidates coexpressing either human IL-7 or IL-18 in the genetic background of BCG DureC::hly, a TB vaccine candidate that is in phase IIa clinical development [11] (ClinicalTrial.gov, Table 1. Evaluation of cytokine expression by antigen-specific CD4 + T cells. #NCT01479972). The BCG DureC::hly strain shows superior protection against TB in preclinical models as compared to canonical BCG [10]. The scientific rationale behind generation of this strain was to increase access of mycobacteria-derived antigens to the host's antigen processing and presentation machinery to ultimately improve protective efficacy against TB. We hypothesized that by capitalising on the characteristics of BCG DureC::hly, better potentiation of cellular immunity could be achieved using soluble mediators such as IL-7 or IL-18.
We expected that perforation of the phagosome by means of the thiol-activated perforin LLO containing either BCG DureC::hly_-hIL7 or BCG DureC::hly_hIL18 could allow release of the recombinant cytokines into the cytosol of host cells thereby modulating T cell responses. IL-18, a highly inflammatory cytokine that is produced by macrophages probably plays a major role in protective immunity to TB [13,20]. IL-7 primarily acts on T cells, notably resting memory T cells which express the IL-7 receptor alpha chain (IL-7Ra) [44]. Biological activity of human IL-7 and IL-18 was demonstrated in mice suggesting reasonable cross activity in both species [45].
Semi-quantitative RT-PCR of BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18 using specific oligonucleotides revealed that gene transcription of hIL-7 by the former was lower than hIL-18 gene transcription by the latter ( Figure 1B). Conversely, the level of hly transcripts in BCG DureC::hly_hIL18 was lower than that observed in all other rBCG strains ( Figure 1B). Although hly and hIL-18 are both controlled by the constitutive groEL2 promoter ( Figure 1A), transcription of hIL-18 was favoured over hly. In contrast, the groEL2 promoter-driven hly transcription remained uncompromised in BCG DureC::hly_hIL7 while hIL-7 transcript levels were reduced ( Figure 1B). Consistent with this, abundance of hIL-7 protein expressed by BCG DureC::hly_hIL7 was lower than that of hIL-18 expressed by BCG DureC::hly_hIL18 ( Figure 1C). The fact that cytokine genes were not translated into GC-rich mycobacterial codon usage [46] prior introduction into the vaccine strain might have affected expression of hIL7 and hIL18.
BCG DureC::hly_hIL18 seemed to induce more profound proinflammatory cytokine release compared to BCG DureC::hly in human DCs although the number of intracellular bacteria did not significantly differ between the strains (Figures 2A and B). This may be due to the physiological effects of vaccine-derived human IL-18 present in the culture on human DCs. Up-regulation of chemokines such as CXCL10, CCL2 and CCL5 indicates that BCG DureC::hly_hIL18 could enhance recruitment of monocytes and T cells to the site of vaccination, thus amplifying early immunological events in shaping T cell responses [47,48,49,50]. G-CSF contributes to development of neutrophils, which have been implicated in shuttling BCG to dLNs for T cell priming [51]. Collectively, these features could facilitate generation of long-lived mycobacterial antigen-specific T cells shortly after vaccination. We also observed enhanced IL-2 secretion triggered by all three rBCG strains, indicating T cell activation and proliferation ( Figure 2C). We did not expect to observe similar cytokine profiles in culture supernatants of DCs infected with BCG DureC::hly_hIL7 since IL-7 is not involved in proinflammatory immune responses but rather in T cell homeostasis [52].
The improved clearance patterns of all our recombinant vaccines from mice suggests a better safety profile compared to BCG ( Figure 3A), in line with the requirements of a live TB vaccine candidate aiming to replace BCG, especially since BCG can cause disseminated disease termed BCGosis in HIV-positive patients [53]. Urease deletion in mycobacteria has been shown to prevent microbe-driven deacidification of the phagosome in antigen-presenting cells (APCs) [54]. Since this feature complements the optimal biological activity of LLO at pH 5.5, BCG DureC::hly facilitates higher cytosolic antigen turnover and thus, enhanced apoptosis of the APCs, which in turn allows for efficient cell-mediated immune attack and clearance of the BCG DureC::hly strains [10]. Moreover, we found higher levels of proinflammatory cytokines such as CCL5, G-CSF and IL-6 (among others) in sera of BCG DureC::hly_hIL18-vaccinated mice compared to those vaccinated with BCG DureC::hly, confirming the cytokine profile we observed with infected human DCs ( Figure 3B).
Protective immunity to TB is largely orchestrated by antigenspecific Th1 CD4 + T cells [5]. Upon establishing a successful immune synapse with an epitope-loaded major histocompatibility complex class II (MHC-II) molecule on the surface of an antigen presenting cell (APC), CD4 + T cells stably express the activation marker CD40L (or CD154), thereby indicating antigen-specific stimulation [55]. In vitro re-stimulation with M. tuberculosis H37Rv whole cell lysate revealed that mice vaccinated with BCG DureC::hly_hIL7 or BCG DureC::hly_hIL18 responded to specific antigenic challenge (measured by CD40L expression) by a magnitude of 10-fold compared to unvaccinated mice ( Figure 4A). In addition, the modulation of IFN-c-and TNF-aproducing Th1 CD4 + T cells in dLNs, spleens and lungs of BCG DureC::hly_hIL7or BCG DureC::hly_hIL18-vaccinated mice hint to favourable immunity elicited by both strains ( Figure 4B). Yet, the protective efficacy afforded by BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18 was not superior to that of BCG DureC::hly in the mouse model ( Figure 5B).
Analysis of BCG DureC::hly_hIL7 and BCG DureC::hly_hIL18 in non-murine model systems may be considered in the future. Other reasons for the lack of improved efficacy of cytokine-expressing vaccine candidates might be: (i) Low abundance of hIL-7 and hIL-18 as seen in the culture supernatant analysis in Figure 1C. Stronger secretion capacity may have resulted in higher cytokine content in the host MW cytosol and hence, enhanced cytokine export via the endogenous secretion system. (ii) Overload of the mycobacterial protein export machinery due to use of the same export system (P groEL2 -Ag85BSS) both for LLO and cytokines. This was already reflected in gene transcription efficiency (semiquantitative RT-PCR data; Figure 1B) as a direct effect of compromised groEL2 promoter activity.
Taken together, our data suggest that expression of human IL-7 or IL-18 in BCG allows modulation of immune reactions in response to vaccination. More work is required in order to elucidate mechanisms which contribute to protective immunity against M. tuberculosis. Efforts are currently underway in our laboratory to better understand the molecular basis of immunomodulation driven by BCG DureC::hly. This may lead to development of alternative strategies to improve the already potent protection elicited by BCG DureC::hly.