ORIGINAL RESEARCH published: 30 April 2019 doi: 10.3389/fimmu.2019.00894 RUTI Vaccination Enhances Inhibition of Mycobacterial Growth ex vivo and Induces a Shift of Monocyte Phenotype in Mice Satria A. Prabowo 1,2*, Hannah Painter 1,2 , Andrea Zelmer 1,2 , Steven G. Smith 1,2 , Karin Seifert 1† , Merce Amat 3 , Pere-Joan Cardona 4,5 and Helen A. Fletcher 1,2 1 Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom, 2 Tuberculosis Centre, London School of Hygiene and Tropical Medicine, London, United Kingdom, 3 Archivel Farma S.L., Badalona, Spain, 4 Experimental Tuberculosis Unit (UTE), Fundació Institut Germans Trias i Pujol (IGTP), Universitat Autònoma de Barcelona (UAB), Badalona, Spain, 5 Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Respiratorias, Madrid, Spain Tuberculosis (TB) is a major global health problem and there is a dire need for an improved treatment. A strategy to combine vaccination with drug treatment, termed Edited by: therapeutic vaccination, is expected to provide benefit in shortening treatment duration Fabio Bagnoli, and augmenting treatment success rate. RUTI candidate vaccine has been specifically GlaxoSmithKline, Italy developed as a therapeutic vaccine for TB. The vaccine is shown to reduce bacillary Reviewed by: Angelo Izzo, load when administered after chemotherapy in murine and guinea pig models, and is Colorado State University, also immunogenic when given to healthy adults and individuals with latent TB. In the United States absence of a validated correlate of vaccine-induced protection for TB vaccine testing, Bingdong Zhu, Lanzhou University, China mycobacterial growth inhibition assay (MGIA) has been developed as a comprehensive *Correspondence: tool to evaluate vaccine potency ex vivo. In this study, we investigated the potential of Satria A. Prabowo RUTI vaccine to control mycobacterial growth ex vivo and demonstrated the capacity of satria.prabowo@lshtm.ac.uk MGIA to aid the identification of essential immune mechanism. We found an association † Present Address: between the peak response of vaccine-induced growth inhibition and a shift in monocyte Karin Seifert, Federal Institute for Drugs and phenotype following RUTI vaccination in healthy mice. The vaccination significantly Medical Devices, Bonn, Germany increased the frequency of non-classical Ly6C− monocytes in the spleen after two doses of RUTI. Furthermore, mRNA expressions of Ly6C− -related transcripts (Nr4a1, Itgax, Specialty section: This article was submitted to Pparg, Bcl2) were upregulated at the peak vaccine response. This is the first time the Vaccines and Molecular Therapeutics, impact of RUTI has been assessed on monocyte phenotype. Given that non-classical a section of the journal Ly6C− monocytes are considered to play an anti-inflammatory role, our findings in Frontiers in Immunology conjunction with previous studies have demonstrated that RUTI could induce a balanced Received: 03 December 2018 Accepted: 08 April 2019 immune response, promoting an effective cell-mediated response whilst at the same time Published: 30 April 2019 limiting excessive inflammation. On the other hand, the impact of RUTI on non-classical Citation: monocytes could also reflect its impact on trained innate immunity which warrants Prabowo SA, Painter H, Zelmer A, Smith SG, Seifert K, Amat M, further investigation. In summary, we have demonstrated a novel mechanism of action Cardona P-J and Fletcher HA (2019) of the RUTI vaccine, which suggests the importance of a balanced M1/M2 monocyte RUTI Vaccination Enhances Inhibition function in controlling mycobacterial infection. The MGIA could be used as a screening of Mycobacterial Growth ex vivo and Induces a Shift of Monocyte tool for therapeutic TB vaccine candidates and may aid the development of therapeutic Phenotype in Mice. vaccination regimens for TB in the near future. Front. Immunol. 10:894. doi: 10.3389/fimmu.2019.00894 Keywords: RUTI, vaccine, tuberculosis, monocytes, mycobacteria, growth inhibition assay Frontiers in Immunology | www.frontiersin.org 1 April 2019 | Volume 10 | Article 894 Prabowo et al. RUTI Vaccination Shifts Monocyte Phenotype INTRODUCTION cellular frequencies, phenotypes and cytokines that associate with enhanced growth inhibition (16–18). Several variations of Tuberculosis (TB) remains a leading cause of death from human and murine MGIAs have been described in the literature infectious disease and is responsible for an estimated annual [reviewed in (12)]. Here, we implemented the assay using direct 1.6 million deaths globally (1). With the emergence of drug- co-culture of mouse splenocytes with mycobacteria, based on a resistant TB, there is a dire need for new therapy and for shorter, recent optimization work (15), to investigate the potential of the more effective, safer, and better tolerated treatment regimens. RUTI vaccine to control mycobacterial growth ex vivo. Therapeutic vaccination, which is a strategy to combine Monocytes are highly plastic and heterogeneous circulating vaccination with drug treatment, could help to achieve these cells, which are known to change their functional phenotype in objectives and improve current treatments (2). TB vaccines are response to environmental stimulation (19, 20). Two distinct regarded to be equally effective against drug-sensitive and drug- subpopulations of mouse monocytes have been identified, resistant strains, due to the nature of drug-resistant mutations commonly referred to as Ly6C+ and Ly6C− monocytes (21). which are not considered to change the immunological profile of Ly6C+ monocytes represent classical pro-inflammatory and the organism (3). Therapeutic vaccination was first introduced phagocytic monocytes which could subsequently differentiate by Robert Koch in his initial attempts to administer tuberculin into M1 macrophages, while Ly6C− monocytes are regarded to TB patients (4), and currently several therapeutic TB vaccine as non-classical anti-inflammatory monocytes which could candidates are available in the vaccine pipeline. differentiate into M2 macrophages (19). In addition to the The RUTI vaccine is among the few candidates currently induction of an antigen-specific Th1 response, evidence suggests on the clinical pipeline which is specifically developed as the potential importance of a balanced M1/M2 monocyte a therapeutic TB vaccine. The vaccine is composed of function in controlling mycobacterial infection (20, 22). In purified and liposomal cellular fragments of Mycobacterium a previous murine study, the RUTI vaccine was shown to tuberculosis (Mtb) bacilli cultured under stress (to mimic intra- reduce intragranulomatous infiltration and decrease Tumor granulomatous conditions) to induce latency antigens which Necrosis Factor (TNF)-α expression in Mtb infected mice (6). would typically be hidden from the immune system (5). The We hypothesize that immunization with RUTI would lead to immune response to RUTI has been studied in animal models improved control of mycobacterial growth ex vivo and such and clinical studies and is characterized by a poly-antigenic observation could be used to gain insight into the mechanism of response. Its main immunotherapeutic effect is thought to be immune protection. through induction of a T helper-1 (Th1) response, not only In this study, we investigated the impact of RUTI vaccination against growth-related antigens but also structural antigens as in mice using the ex vivo MGIA assay and found an association shown in the murine model (2, 6). The prophylactic capacity of between peak response of vaccine-induced growth inhibition RUTI vaccine has also been evaluated in a murine model and and a shift in monocyte phenotype. Our study demonstrates the the vaccination significantly reduced bacterial counts in both benefit of the ex vivo MGIA to aid the identification of immune lungs and spleens following challenge with virulent bacilli (7). mechanisms of action for therapeutic TB vaccine candidates. RUTI vaccination generated a poly-antigenic response in healthy The MGIA could be used as a tool for screening such vaccine human volunteers (phase I study), as well as in HIV-positive and candidates and might aid the development of therapeutic vaccine HIV-negative patients with latent TB after isoniazid treatment for regimens for TB patients. 1 month in a phase II clinical trial (8, 9). The lack of an immune correlate of protection has been hampering the development of novel TB vaccines, as lengthy MATERIALS AND METHODS and expensive clinical trials with protracted follow-up periods Animals are needed to demonstrate efficacy and proceed to licensure (10). Six to seven week-old female C57Bl/6 mice (Charles River, UK) For a single vaccine candidate, it generally takes at least a decade acclimatized for at least 5 days were housed and handled in the to reach efficacy trials from discovery (3, 10). If we are going to Biological Services Facility (BSF) at London School of Hygiene achieve the WHO target to eliminate TB by 2050, major progress and Tropical Medicine (LSHTM), UK. Mice were provided is required to overcome the painstakingly slow progress. Such standard sterilized food and water ad libitum. Animals were an immune correlate could also be used to help identify vaccine housed in specific pathogen-free individually vented cages with candidates with the greatest potential efficacy. environmental enrichment, with equal day and light cycle, at Mycobacterial growth inhibition assay (MGIA) has been temperature between 19◦ -23◦ C and relative humidity of 45– developed as a simple and comprehensive tool to evaluate 65%. Mice were allocated to cages as groups of six. All animal vaccine immunogenicity ex vivo (11, 12). As an assay that work was carried out in accordance with the Animals (Scientific measures the summative vaccine-mediated host capacity to Procedures) Act 1986 under a license granted by the UK Home control mycobacterial growth, the MGIA is proposed as a Office (PPL 70/8043), and approved locally by the LSHTM screening tool for TB vaccine candidates (13–15). The nature Animal Welfare and Ethics Review Body. of the ex vivo assay does not require that the vaccine-mediated immune mechanism which underlies growth control to be known Immunization in advance, while in turn the MGIA could help to determine Seven experimental groups were established, with six mice per immune mechanisms of protection through investigation of the group (Figure 1). Mice in the treatment groups were vaccinated Frontiers in Immunology | www.frontiersin.org 2 April 2019 | Volume 10 | Article 894 Prabowo et al. RUTI Vaccination Shifts Monocyte Phenotype FIGURE 1 | Experimental design and vaccination schedule. As indicated in the figure, orange arrows mean RUTI vaccination (dotted if boosting). The purple X represents endpoint (mice sacrifice). Enzyme-linked immunospot assay and mycobacterial growth inhibition assay were performed at each endpoint. In total, 42 mice were sacrificed at all time points (6 mice per group). with RUTI, which is based on purified fragments of Mtb After 4 days, splenocytes-mycobacteria mixtures were cultured under stress conditions and liposomed, manufactured collected from the 48-well plates by pipetting up and down three by Archivel Farma (Badalona, Catalonia, Spain). Vaccination times before transferring to 2 ml screw cap tubes. The tubes with RUTI (batch A14, 204 µg) was performed subcutaneously were centrifuged at 12,000 rpm in a bench top micro centrifuge once or twice (3 weeks apart), as has been performed previously and the supernatants were removed (500 µl) while ensuring (6, 7). Five groups of mice were vaccinated with RUTI at week the pellets remain intact. Five hundred microliters sterile tissue 0, among which three groups were boosted at week 3. Mice were culture grade water was added to the 48-well plates which were sacrificed at week 1, 3, 4, 6, and 9 as the designated time points incubated at room temperature for 5 min, followed by pipetting of this experiment. Two groups of mice sacrificed at week 1 and up and down for five times before transferring to the 2 ml screw 6 served as naïve controls. cap tubes with pellets. The pellets were dissolved by pipetting and lysates containing mycobacteria were transferred to Bactec Mycobacteria and Culture Conditions MGIT tubes supplemented with PANTA antibiotic and oleic Bacillus Calmette-Guérin (BCG) Pasteur strain was obtained acid-albumin-dextrose-catalase (OADC) enrichment broth [all from Aeras (Rockville, MD, USA) as frozen aliquots. These were from Becton Dickinson (BD), Oxford, UK]. The MGIT tubes stored at −80◦ C until needed. Mycobacterial suspensions for were incubated in a Bactec MGIT liquid culture system (BD) infection inoculum and BACTEC MGIT standards were prepared until registered positive. The resulting time to positivity (TTP) in antibiotic-free media (described below). All work with cells was converted to bacterial numbers (log10 CFU) using a standard pre-BCG infection and involving BCG infected samples were curve. The standard curve was obtained by a linear regression performed in Biosafety Level (BSL) 2 laboratory. analysis of TTP values from inoculated BCG in 10-fold dilutions against CFUs obtained from plating aliquots of BCG onto 7H11 Ex vivo Mycobacterial Growth Inhibition agar plates containing 10% OADC supplement (Yorlab, York, Assay (MGIA) UK) and 0.5% glycerol. Direct-to-MGIT controls were included At the determined time points, spleens were removed aseptically at each time point, defined as 90 CFU BCG directly placed into from mice and single splenocyte suspensions were prepared Bactec MGIT system without any pre-incubation (at day 0). To by homogenization through 100 µm cell strainers followed by compare the growth inhibition between time points, log10 CFU lysis of red blood cells and washing. Cells were adjusted to 5 values were normalized using the direct-to-MGIT controls by × 106 splenocytes per 300 µl in antibiotic-free media [RPMI- subtracting or adding the values based on the average TTP of 1640 (Sigma-Aldrich, Dorset, UK) + 10% heat-inactivated FBS direct-to-MGIT controls. (Labtech International Ltd, Uckfield, UK) + 2 mM L-Glutamine (Fisher Scientific, Loughborough, UK)]. Mycobacteria were Interferon (IFN)-γ ELISpot diluted in sufficient volume for all samples in the same media to To measure antigen-specific response toward mycobacterial a concentration of 90 CFU per 300 µl. Three hundred microliters antigen following RUTI vaccination over the time course, IFN- aliquots of bacteria were added to the splenocytes, and the γ ELISpot assay was performed. Single cell suspensions of mouse splenocytes-mycobacteria co-culture (600 µl) was then incubated splenocytes were resuspended in RPMI-1640 media containing in 48-well plates (Sigma-Aldrich, UK) at 37◦ C for 4 days. 10% heat-inactivated FBS and 2 mM L-Glutamine. 96-well Frontiers in Immunology | www.frontiersin.org 3 April 2019 | Volume 10 | Article 894 Prabowo et al. RUTI Vaccination Shifts Monocyte Phenotype microtiter ELISpot plates (MAIPS4510, Millipore, Watford, UK) 105 splenocytes were stimulated overnight with PPD (final were coated with 10 µg/ml rat anti-mouse IFN-γ (clone AN18, concentration 10 µg/ml). Cells were pelleted, lysed in 200 µl Mabtech, Nacka Strand, Sweden). Free binding sites were blocked RLT buffer containing 10 µl/ml β-mercaptoethanol and stored in ◦ with the above mentioned media. 2.5 × 105 of total splenocytes −40 C freezer from each time point. Cells were thawed and RNA were added and incubated in duplicate with 10 µg/ml Purified was extracted using the RNAeasy mini kit (Qiagen, Manchester, protein derivative (PPD) (Oxford Biosystem, Oxfordshire, UK), UK) according to the manufacturer’s instructions. After a RPMI media as a negative control, or phytohemaglutinin DNAse treatment with RNase-free DNAse set (Qiagen, UK), (PHA) (1 µg/ml, Sigma-Aldrich) and phorbol myristate acetate total RNA concentration was determined by spectrophotometry (PMA) (0.1 µg/ml, Sigma-Aldrich) as a positive control. Cells with a Nanodrop (Labtech International, Heathfield, UK). One were incubated overnight at 37◦ C with 5% CO2 . IFN-γ was microgram of each sample of total RNA was reverse-transcribed detected with 1 µg/ml biotin labeled rat anti-mouse antibody into complementary DNA (cDNA) using Omniscript R Reverse (clone R4-6A2, Mabtech) and 1 µg/ml alkaline phosphatase- Transcription kit (Qiagen, UK) according to the manufacturer’s conjugated streptavidin (Mabtech). The enzyme reaction was recommendation, using oligo(dT) (Invitrogen, UK) to obtain developed with BCIP/NBT substrate (5-Bromo-4-chloro-3- cDNA. Each PCR was carried out in a 20 µl volume in the indolyl phosphate/Nitro blue tetrazolium) (MP Biochemicals, presence of 10 µl of 2x QuantiTect SYBR Green PCR Master UK) and stopped by washing the plates with tap water when Mix (Qiagen, UK), 1 µl of cDNA (or water as a negative control), individual spots could be visually detected (up to 3 min). Upon MgCl2 to a final concentration of 2.5 mM and primers to a final completion of the color development stage, spots were quantified concentration of 0.5 µM. PCR was carried out for 10 min at using an automated plate reader with ELISpot 5.0 software. IFN- 95◦ C denaturation, followed by 40 cycles at 95◦ C for 15 s, and γ-specific cells are expressed as number of spot forming cells 60◦ C for 1 min in Applied Biosystems 7500 (Applied Biosystems, (SFC) per million splenocytes after non-specific background was CA, USA). subtracted using negative control wells. Analyses were performed for gene expressions of Nr4a1, Cebpb, Itgax, Pparg, Bcl2 (markers of Ly6C− ), Ccr2, Sell, Ly6C2 Flow Cytometry (markers of Ly6C+ ), and β-actin (housekeeping gene). Primers Single splenocyte suspensions were fixed and red blood cells used were listed in Table S1. mRNA expression of β-actin was were lysed using PhosFlow lyse-fix solution (Becton Dickinson, quantified for every target sample to normalize for efficiency in Oxford, UK) for 30 min at 4◦ C prior to freezing. Fixed cells cDNA synthesis and RNA loading. A ratio based on the β-actin were then re-suspended in freezing media (FBS containing 10% mRNA expression was obtained for each sample. DMSO) at the concentration of 106 cells per ml and stored in a ◦ −40 C freezer from each time point. Frozen cells were thawed by adding FACS buffer (PBS containing 5% FBS) and pipetting Statistical Analysis up and down to encourage thawing. Cells were added to 10 ml Statistical analysis was carried out using GraphPad Prism version of FACS buffer and centrifuged for 10 min at 1,800 rpm. Cells 7 (GraphPad, La Jolla, CA, USA). A p-value of < 0.05 was were re-suspended in FACS buffer (concentration 107 cells/ml) considered statistically significant. The specific tests used for each and were left for 15 min on ice for rehydration. analysis are described in the figure legends. Fc block (anti-mouse CD16/32, eBioscience, Loughborough, UK) was added to cells and left for further 10 min on ice prior to surface staining. Cells were aliquoted in FACS tubes (100 µl RESULTS each, 106 cells) and stained with the following titrated antibody: RUTI Vaccination Did Not Induce 1.25 µl CD3-APC/Cy7 (clone 17A2), 2.5 µl CD45R/B220-BV510 (clone RA3-6B2), 1.25 µl CD11b-PerCP/Cy5.5 (clone M1/70), Antigen-Specific IFN-γ but Did Improve 2.5 µl Ly6G-BV711 (clone 1A8), and 5 µl Ly6C-BV421 (clone Mycobacterial Growth Inhibition in HK1.4). All antibodies were purchased from Biolegend (via Murine Splenocytes Fisher Scientific). To assess the immune response to mycobacterial antigens Cells were incubated for 30 min at RT in the dark and washed from mice vaccinated with RUTI, splenocytes were stimulated prior to analysis. Fluorescence minus one (FMO) controls were with PPD and the number of IFN-γ-producing cells was set using cells for each antibody and used to guide gating. measured using the ELISpot assay (Figure 2, red line). OneComp beads (eBioscience, Loughborough, UK) were used to We found a weak, non-significant response at 1 week calculate compensation by staining with single antibodies as per following the second vaccination with RUTI (week 4, p = manufacturer’s instruction. Cells were acquired on a BD LSR II 0.08). This response appeared to have decreased by week 6. flow cytometer. Data was analyzed with FlowJo software version Significant control of mycobacterial growth was observed 10.4 (Treestar Inc., USA). 1 week after the first vaccination and 3 weeks after the second RUTI vaccination (week 6) when compared to the Real-Time Quantitative PCR baseline control (p = 0.0214, Figure 2, blue line). A trend To quantitatively analyse the mRNA expressions in splenocytes of reduction was still observed 6 weeks after the second following RUTI vaccination, real-time quantitative reverse vaccination (week 9), although it did not reach significance transcriptase PCR (qRT-PCR) assays were performed. 5 × (p = 0.064). Frontiers in Immunology | www.frontiersin.org 4 April 2019 | Volume 10 | Article 894 Prabowo et al. RUTI Vaccination Shifts Monocyte Phenotype FIGURE 2 | ELISpot (Red Line). IFN-γ response in mice receiving vaccination with RUTI was measured. Modest PPD antigen-specific responses were detected in splenocytes of healthy C57BL/6 mice across time points. The splenocytes were stimulated overnight with PPD, and the responses were detected using the IFN-γ ELISpot assay. SFC, spot-forming cells. Dark red line indicates mean response, and shading indicates range. Statistical significance was tested using Mann-Whitney test. MGIA (Blue Line). RUTI vaccination induced mycobacterial growth inhibition in murine splenocytes, performed ex vivo in a 48-well plate. Dark blue line indicates mean mycobacterial growth, and shading indicates range. Time point 0 represents unvaccinated naïve-control mice sacrificed at week 1. One-way ANOVA was used to test for significance, followed by t-test. *p < 0.05; **p < 0.01. In a separate experiment performed in rotating tubes instead Shift of Monocyte Phenotype Following of 48-well plates (Figure S1A in Supplementary Material), RUTI- RUTI Vaccination in Healthy induced control of mycobacterial growth was superior to BCG- Mouse Splenocytes induced control when both vaccines were given 6 weeks prior In this experiment, we investigated the impact of RUTI to sacrifice (p < 0.005, Figure S1B). Therefore, week 6 appeared vaccination on the population of immune cells in the spleen to be the peak response of RUTI in the ex vivo MGIA using flow cytometry (Figure 3 and Figure S3 in Supplementary system. Growth control was also significant when compared Material). RUTI did not appear to alter the percentages of to an age-matched control group (p < 0.05, Figure S2A monocytes/macrophages, T-cells, and B-cells in the spleen in Supplementary Material). of healthy mice (p > 0.05, Figures S3A,C,D). We also RUTI vaccination led to control of mycobacterial growth measured monocyte to lymphocyte (ML) ratio as a factor as measured by the MGIA, despite a weak, non-significant influencing mycobacterial growth inhibition (28, 29), defined antigen-specific IFN-γ response. This was consistent with results as the percentage of monocytes/macrophages divided by the of a separate experiment with RUTI and BCG, in which percentage of T-cells and B-cells. We did not find a significant BCG induced IFN-γ-secreting cells whereas RUTI did not and change of ML ratio following RUTI vaccination across time yet cells from RUTI immunized mice were better able to (Figure S3B). control mycobacterial growth than cells from BCG vaccinated We then further characterized monocyte phenotype based mice (Figures S1C,D in Supplementary Material). The lack of on the Ly6C marker, and observed a significant increase of robust IFN-γ response following two doses of RUTI in healthy Ly6C− monocytes/macrophages (non-classical) at weeks 3, 6, mice has been observed previously during potency testing and 9 following RUTI vaccination (p < 0.05, Figure 3B). for batch release (Archivel Farma, personal communication). The peak increase of Ly6C− cells was observed at week Then, four doses of RUTI were required for induction of 6, with the shift being evident compared to both baseline a detectable IFN-γ response. Although IFN-γ is associated and age-matched naïve control at week 6 (Figure S2B in with control of TB infection in mice and reduces the risk of Supplementary Material). The Ly6C+ monocytes/macrophages TB disease in humans (23–25), some studies suggested IFN- (classical) population appeared to be decreasing following γ alone was not sufficient (26, 27). As the MGIA measures vaccination, although there was an initial significant increase the summative effect of immune responses from all cellular observed at week 1 (p < 0.05, Figure 3). The shift of components, our data implied an alternative mechanism by Ly6C+ /Ly6C− phenotype at week 6 was notably consistent which growth inhibition could be enhanced ex vivo following with the peak response of the ex vivo mycobacterial growth RUTI vaccination. inhibition assay, in which enhanced inhibition was observed Frontiers in Immunology | www.frontiersin.org 5 April 2019 | Volume 10 | Article 894 Prabowo et al. RUTI Vaccination Shifts Monocyte Phenotype FIGURE 3 | The shift of Ly6C+ and Ly6C− monocytes/macrophages populations following RUTI vaccination in healthy mice. (A) Gating strategy for flow cytometric analysis. Splenocytes from C57BL/6 mice were fixed, stained and data acquired as described in Materials and Methods. Cell debris was gated out by use of FSC-SSC gate, followed by gating on single cells (FSC-A and FSC-H). A sequential gating strategy was then applied to determine the frequency of T-cells (CD3+ ), B cells (B220+ ), monocytes/macrophages (CD11b+ Ly6G− ssclow ), and the phenotypes of the monocytes/macrophages (Ly6C+ or Ly6C− ) as a percentage of live cells. Plots shown are from a sample of a C57BL/6 spleen. (B) The frequencies of Ly6C+ and Ly6C− monocytes/macrophages were compared at each time point following RUTI vaccination. Dark brown and dark purple lines represent mean percentages of Ly6C+ and Ly6C− monocytes/macrophages, respectively and shading indicates range. Time point 0 represents unvaccinated naïve-control mice sacrificed at week 1. Statistical significance was tested using unpaired t-test, *p < 0.05; **p < 0.01; ***p < 0.005. following two doses of RUTI vaccination. We found a non- on a recent publication by Mildner et al. (21) regarding genomic significant correlation between higher frequency of Ly6C− characterization of murine monocytes. We chose differentially monocytes/macrophages and lower growth of mycobacteria expressed transcripts between the two monocyte subsets across time points (p = 0.247, Spearman r = −0.20, data from several gene clusters of monocyte development. Three not shown). transcripts, namely Nr4a1, Itgax, and Pparg, were selected from a cluster which was strongly upregulated in Ly6C− compared Gene Expression of Ly6C+ - and Ly6C− - to Ly6C+ monocytes. Cebpb was selected from a cluster that Related Markers Induced by showed a gradual increase of expression from Ly6C+ to Ly6C− RUTI Vaccination monocytes. Bcl2 belongs to a cluster characterized by transcripts To confirm findings from the flow cytometry analysis described associated with a progenitor phenotype of MDP (monocyte- in the previous section, we performed real-time qRT-PCR macrophage DC progenitor), which was highly expressed in looking at transcripts associated with Ly6C+ and Ly6C− Ly6C− monocytes. In addition, transcripts associated with monocytes/macrophages. The selection of transcripts was based Ly6C+ monocytes were selected from clusters involved in Frontiers in Immunology | www.frontiersin.org 6 April 2019 | Volume 10 | Article 894 Prabowo et al. RUTI Vaccination Shifts Monocyte Phenotype cell cycle (Sell and Ly6C2) as well as maturation of Ly6C+ characterize immune cells associated with enhanced growth monocytes (Ccr2). inhibition across time. RNA was also isolated to investigate The mRNA expressions of Ly6C− -related transcripts, gene expression by RT-qPCR. The important finding in our including Nr4a1, Itgax, Pparg, Bcl2, were significantly present study was the impact of RUTI vaccination in shifting upregulated following the second RUTI vaccination at week the phenotype of Ly6C+ /Ly6C− monocytes/macrophages in the 6 (p < 0.05, Figures 4A,C–E), with a trend of upregulation spleen of healthy mice. This was the first time the impact of for Cebpb (p = 0.136, Figure 4B). While we did not observe RUTI on monocyte phenotype has been assessed. In addition, a difference in expression of Ccr2, a Ly6C+ -related gene, while others have shown a lack of correlation between antigen- following RUTI vaccination at the peak time point, we saw specific IFN-γ and mycobacterial growth inhibition, this is the significant upregulations of Sell and Ly6C2 at week 6 (p < 0.05, first demonstration of vaccine induced mycobacterial growth Figures 4G–H). inhibition in the absence of antigen-specific IFN-γ. Our results suggest that the enhanced control of mycobacterial growth ex vivo was associated with the increase of Ly6C− monocytes (non- DISCUSSION classical, anti-inflammatory), which were both observed 3 weeks after the second vaccination with RUTI (week 6). Vaccination with RUTI resulted in enhanced control of We confirmed the flow cytometry finding by demonstrating mycobacterial growth, notably at 3 weeks following the second the upregulation of transcripts associated with Ly6C− cells at vaccination (week 6), which was considered as the peak response the peak time point (week 6), while one of the Ly6C+ gene of the vaccine observed in the ex vivo assay system. In addition, transcript (Ccr2) remained unchanged. Among the significantly there was also a significant reduction of mycobacterial growth upregulated transcripts in our study was Nr4a1, which is at 1 week after the first vaccination. There was no significant obligatory for Ly6C− monocytes development (33). Expression induction of antigen-specific IFN-γ as measured by ELISpot of Nr4a1 as a monocyte survival factor is regulated by Cebpb or ELISA. These observations were replicated in a separate (21, 34), which was also elevated following RUTI vaccination. experiment and support the notion that the MGIA measures In addition, the expressions of all other Ly6C− -associated different aspects of immunity following vaccination as compared transcripts (Itgax, Pparg, Bcl2) were significantly upregulated to the IFN-γ-based assay, with the MGIA being reflective following vaccination in our study. As Ly6C− monocytes are of the summative effect of host immune responses at the known to mature from Ly6C+ (35, 36), the increased expression point of tissue harvest, compared to the IFN-γ-based assay of some Ly6C+ transcripts (Sell and Ly6C2) at the peak time which is an assessment of a T-cell mediated recall response point was regarded as a vestige of the transition from Ly6C+ following vaccination. to Ly6C− . Ly6C− monocytes do not represent a distinct lineage In this context, we argue that the ex vivo assay is more and instead arise from the conversion of Ly6C+ cells (37) and capable of measuring the direct, short-term effect of vaccination, approximately 92% of expressed transcripts are shared between while the IFN-γ response is more representative of the medium- the two monocyte subsets (38). Our results suggest that RUTI to long-term protection conferred by vaccination. This was could enhance Ly6C+ cell frequency and induce maturation of supported by a recent finding of Fletcher et al. in which antigen- Ly6C+ to Ly6C− monocytes. This was evidenced by our results specific IFN-γ response was associated with reduced risk of in which an initial increase of Ly6C+ monocytes was observed developing TB disease in BCG-vaccinated South African infants at week 1 following the first RUTI vaccination, followed by the (25). In testing therapeutic vaccine candidates, it might be more shift toward Ly6C− and the decrease of Ly6C+ monocytes at the relevant to measure the direct effect of vaccination, as it would subsequent time points. represent the immediate and potentially synergistic impact of a Ly6C− monocytes secrete anti-inflammatory cytokine upon vaccine during TB chemotherapy. This was depicted in the ex bacterial infection in vivo and when recruited to tissue, are vivo assay, in which significant mycobacterial growth inhibition more likely to differentiate into M2 macrophages (35). This was observed at 1 week following the first RUTI vaccination, is in contrast to Ly6C+ monocytes, which are more likely when there was no apparent increase of IFN-γ response at this to mature into pro-inflammatory M1 macrophages (36). In time point. relation to our findings, the study by Guirado et al. (6) The ex vivo MGIA was shown to be able to capture the demonstrated a decrease of intragranulomatous infiltration in impact of distinct aspects of protective immunity as a part the lungs upon administration of RUTI in infected mice after of a summative measurement of cellular immune responses. treatment. One of the notable findings in that study was In this study, we used BGC as the immune target of the a significant decrease of TNF-α at the earliest time point growth inhibition assay, as previous studies have shown a good after RUTI administration, measured by mRNA expression in concordance of growth between BCG and Mtb in the MGIA assay the lung. Among the producers of TNF-α during bacterial when both mycobacteria were used to assess responses following infection are Ly6C+ monocytes which will subsequently vaccination and treatment (30–32). In addition, studies by differentiate into M1 macrophages (19, 39). Although our Marsay et al. and Zelmer et al. also showed that the ex vivo growth experiment differs with the one previously performed by inhibition using BCG as immune target was correlated with in Guirado and colleagues in several aspects (including the vivo growth in mouse challenge experiment using Mtb Erdman investigated target organ), the decrease of TNF-α observed in (15, 16). In our experiment, we performed flow cytometry to the previous study could be associated with the impact of Frontiers in Immunology | www.frontiersin.org 7 April 2019 | Volume 10 | Article 894 Prabowo et al. RUTI Vaccination Shifts Monocyte Phenotype FIGURE 4 | mRNA expressions of Ly6C− -related (A–E) and Ly6C+ -related genes (F–H) in mice following vaccination with RUTI at week 6 compared to the age-matched naïve control group. Data are expressed as ratio obtained after dividing every value by the expression of β-actin in each sample and multiplying it by a factor (ranging from 101 to 103 ). The box plots show the minimum and maximum values (ends of the whiskers), the median (band near the middle of the box) and interquartile ranges. Statistical significance was tested using unpaired t-test, *p < 0.05; **p < 0.01; ***p < 0.005. RUTI on monocyte populations which has been discovered in addition, the vaccine was also shown to induce a Th3 response our investigation. as a subset population of regulatory T-cells (42). In another RUTI is a poly-antigenic vaccine made from fragmented mouse study, RUTI administration following drug therapy in Mtb bacilli designed to induce host immune response against infected mice stimulated stronger IFN-γ secretion by CD4+ latency epitopes, which is grown in stress, purified and and CD8+ T-cells compared to BCG against early secretory liposomed. The part of RUTI formulation that could potentially antigen target (ESAT)-6, Ag85B, and PPD and also induced an influence monocyte phenotype remains to be further explored. immune response against structural antigens Ag16 kDa and Ag38 Nevertheless, our study has revealed an interesting mechanism kDa (6). While immune responses in infected and drug-treated of action of RUTI as a therapeutic vaccine for TB. As reviewed mice could be reasonably different in comparison to healthy by Prabowo et al. (40), it is essential to prevent the occurrence mice as was done in our ex vivo study, the fact that RUTI did of exacerbated immune response for a successful therapeutic not induce an exacerbated immune response in various animal vaccination strategy in TB. This was exemplified in a recent study, studies could be linked to its impact on Ly6C− monocytes which in which an excessive inflammation from the T-cell compartment was discovered in our investigation. In the experimental animals could also be deleterious in TB (41). The fact that RUTI immunized with RUTI, no elevated IgE levels were observed (43). vaccination could induce a shift toward an anti-inflammatory In this study, histology also revealed no eosinophilia, necrosis or monocyte phenotype might be considered as an advantage in granulomatous infiltration, as well as allergic or hypersensitivity this context and such approach should be further investigated in reactions. Taken together, these findings and observations suggest future studies. that RUTI could induce a balanced immune response, promoting While this could imply that less inflammation might be an effective cell-mediated response whilst at the same time beneficial for the ex vivo control of mycobacterial growth limiting an excessive inflammation, which could be beneficial for following RUTI vaccination in healthy mice, our results its implementation as a therapeutic vaccine for active TB patients should be interpreted with prudence in relation to previous undergoing treatment. results of RUTI testing in in vivo animal models. The Intriguingly, a recent study by Joosten et al. demonstrated the observed trend of correlation between the frequency of Ly6C− protective effect of human non-classical CD14dim monocytes in monocytes/macrophages and growth of mycobacteria across time inhibiting mycobacterial growth following recent Mtb exposure points hinted that this was only one aspect contributing to the and BCG vaccination through the trained innate immunity enhanced ex vivo growth control and other factors might be mechanism (44). As Ly6C− monocyte is the equivalent of playing roles. In a murine model infected with Mtb, RUTI has CD14dim monocyte in mouse (19), our results could also suggest been shown to trigger a balanced Th1/Th2 response as well an impact of RUTI on trained innate immunity which has not as Immunoglobulin (Ig)G1, IgG2a and IgG3 antibodies against been characterized before. BCG is known to induce trained 13 Mtb antigens, reflecting its broad immunogenicity (42). In immunity on human monocytes and such ability has been Frontiers in Immunology | www.frontiersin.org 8 April 2019 | Volume 10 | Article 894 Prabowo et al. RUTI Vaccination Shifts Monocyte Phenotype attributed to the non-specific protective effect conferred by the took the lead in writing the manuscript. All authors reviewed, vaccine (45, 46). A synthetic mycobacterial structure—muramyl provided critical feedback and approved the final version of tripeptide (MTP)—is also considered to induce trained immunity the manuscript. (47), and interestingly, both MTP and RUTI are delivered in liposome. Further studies are required to elucidate downstream and upstream pathways related to the potential effect of RUTI on ACKNOWLEDGMENTS trained innate immunity. We are very grateful to Archivel Farma S.L. for provision of In summary, we have demonstrated the benefit of the ex RUTI vaccine vials used in this study. We would like to thank vivo MGIA assay to help streamlining and identifying immune Ayad Eddaoudi and Stephanie Canning for expert technical mechanisms of a therapeutic TB vaccine candidate. Our results assistance with flow cytometry. We are grateful to all staff at could be complemented by further experiments, such as by using the Biological Service Facility at London School of Hygiene and cells from infected mice. Depleting Ly6C− monocytes and using Tropical Medicine for their assistance with animal work. We also genetically deficient mice could also provide more insight into thank Erni Marlina for technical advices with the qPCR assay. HF the mechanism of protection by Ly6C− monocytes in the ex has received support for this project from EC HORIZON2020 vivo assay system. Future investigation of the novel immune TBVAC2020 (grant no. 643381). Satria Arief Prabowo (SP) mechanism of RUTI observed in our study is also warranted received a PhD scholarship from the Indonesian Endowment in upcoming clinical trials of the vaccine and could potentially Fund for Education (LPDP). KS was supported by the UK accelerate the development of a therapeutic vaccine regimen for Medical Research Council (MRC) and the UK Department TB patients in the near horizon. for International Development (DFID) under the MRC/DFID Concordat agreement (grant reference MR/J008702/1). AUTHOR CONTRIBUTIONS SUPPLEMENTARY MATERIAL SP and HF conceived and planned the experiments. SP performed the experiments, with HP and AZ participating in The Supplementary Material for this article can be found some of the qPCR and MGIA experiments. SP, HF, SS, KS, MA, online at: https://www.frontiersin.org/articles/10.3389/fimmu. and P-JC contributed to the interpretation of the results. SP 2019.00894/full#supplementary-material REFERENCES placebo-controlled phase II clinical trial in patients with latent tuberculosis infection. PLoS ONE. (2014) 9:e89612. doi: 10.1371/journal.pone.0089612 1. Tiberi S, du Plessis N, Walzl G, Vjecha MJ, Rao M, Ntoumi F, et al. 10. Dockrell HM. Towards new TB vaccines: what are the challenges? Pathog Dis. Tuberculosis: progress and advances in development of new drugs, treatment (2016) 74:ftw016. doi: 10.1093/femspd/ftw016 regimens, and host-directed therapies. Lancet Infect Dis. (2018) 18:e183–98. 11. Zelmer AA, Tanner R, Stylianou E, Morris S, Izzo A, Williams A, et al. doi: 10.1016/s1473-3099(18)30110-5 Ex vivo mycobacterial growth inhibition assay (MGIA) for tuberculosis 2. Cardona PJ. The progress of therapeutic vaccination with regard to vaccine testing - a protocol for mouse splenocytes. bioRxiv. (2015):020560. tuberculosis. Front Microbiol. (2016) 7:1536. doi: 10.3389/fmicb.2016.01536 doi: 10.1101/020560 3. Fletcher HA, Schrager L. TB vaccine development and the End TB Strategy: 12. Tanner R, O’Shea MK, Fletcher HA, McShane H. In vitro mycobacterial importance and current status. Trans R Soc Trop Med Hyg. (2016) 110:212–8. growth inhibition assays: A tool for the assessment of protective immunity doi: 10.1093/trstmh/trw016 and evaluation of tuberculosis vaccine efficacy. Vaccine. (2016) 34:4656–65. 4. Vilaplana C, Cardona PJ. Tuberculin immunotherapy: its history doi: 10.1016/j.vaccine.2016.07.058 and lessons to be learned. Microbes Infect. (2010) 12:99–105. 13. Parra M, Yang AL, Lim J, Kolibab K, Derrick S, Cadieux N, et al. Development doi: 10.1016/j.micinf.2009.10.006 of a murine mycobacterial growth inhibition assay for evaluating vaccines 5. Cardona PJ. RUTI: a new chance to shorten the treatment against Mycobacterium tuberculosis. Clin Vaccine Immunol. (2009) 16:1025– of latent tuberculosis infection. Tuberculosis. (2006) 86:273–89. 32. doi: 10.1128/CVI.00067-09 doi: 10.1016/j.tube.2006.01.024 14. Fletcher HA, Tanner R, Wallis RS, Meyer J, Manjaly ZR, Harris S, 6. Guirado E, Gil O, Caceres N, Singh M, Vilaplana C, Cardona PJ. Induction et al. Inhibition of mycobacterial growth in vitro following primary but of a specific strong polyantigenic cellular immune response after short- not secondary vaccination with Mycobacterium bovis BCG. Clin Vaccine term chemotherapy controls bacillary reactivation in murine and guinea pig Immunol. (2013) 20:1683–9. doi: 10.1128/CVI.00427-13 experimental models of tuberculosis. Clin Vaccine Immunol. (2008) 15:1229– 15. Zelmer A, Tanner R, Stylianou E, Damelang T, Morris S, Izzo A, et al. A 37. doi: 10.1128/CVI.00094-08 new tool for tuberculosis vaccine screening: ex vivo mycobacterial growth 7. Vilaplana C, Gil O, Caceres N, Pinto S, Diaz J, Cardona PJ. Prophylactic effect inhibition assay indicates BCG-mediated protection in a murine model of a therapeutic vaccine against TB based on fragments of Mycobacterium of tuberculosis. BMC Infect Dis. (2016) 16:412. doi: 10.1186/s12879-016-1 tuberculosis. PLoS ONE. (2011) 6:e20404. doi: 10.1371/journal.pone.0020404 751-4 8. Vilaplana C, Montane E, Pinto S, Barriocanal AM, Domenech G, Torres F, 16. Marsay L, Matsumiya M, Tanner R, Poyntz H, Griffiths KL, Stylianou E, et al. et al. Double-blind, randomized, placebo-controlled Phase I Clinical Trial of Mycobacterial growth inhibition in murine splenocytes as a surrogate for the therapeutical antituberculous vaccine RUTI. Vaccine. (2010) 28:1106–16. protection against Mycobacterium tuberculosis (M. tb). Tuberculosis. (2013) doi: 10.1016/j.vaccine.2009.09.134 93:551–7. doi: 10.1016/j.tube.2013.04.007 9. Nell AS, D’Lom E, Bouic P, Sabate M, Bosser R, Picas J, et al. Safety, tolerability, 17. Smith SG, Zelmer A, Blitz R, Fletcher HA, Dockrell HM. Polyfunctional and immunogenicity of the novel antituberculous vaccine RUTI: randomized, CD4 T-cells correlate with in vitro mycobacterial growth inhibition following Frontiers in Immunology | www.frontiersin.org 9 April 2019 | Volume 10 | Article 894 Prabowo et al. RUTI Vaccination Shifts Monocyte Phenotype Mycobacterium bovis BCG-vaccination of infants. Vaccine. (2016) 34:5298– Ly6Clow monocytes while preserving macrophage gene function. Immunity. 305. doi: 10.1016/j.vaccine.2016.09.002 (2016) 45:975–87. doi: 10.1016/j.immuni.2016.10.011 18. Jensen C, Lindebo Holm L, Svensson E, Aagaard C, Ruhwald M. 35. Auffray C, Fogg D, Garfa M, Elain G, Join-Lambert O, Kayal S, et al. Optimisation of a murine splenocyte mycobacterial growth inhibition Monitoring of blood vessels and tissues by a population of monocytes with assay using virulent Mycobacterium tuberculosis. Sci Rep. (2017) 7:2830. patrolling behavior. Science. (2007) 317:666–70. doi: 10.1126/science.1142883 doi: 10.1038/s41598-017-02116-1 36. Serbina NV, Jia T, Hohl TM, Pamer EG. Monocyte-mediated defense 19. Yang J, Zhang L, Yu C, Yang XF, Wang H. Monocyte and macrophage against microbial pathogens. Annu. Rev. Immunol. (2008) 26:421–52. differentiation: circulation inflammatory monocyte as biomarker for doi: 10.1146/annurev.immunol.26.021607.090326 inflammatory diseases. Biomark Res. (2014) 2:1. doi: 10.1186/2050-7771-2-1 37. Yona S, Kim KW, Wolf Y, Mildner A, Varol D, Breker M, et al. Fate mapping 20. Mildner A, Marinkovic G, Jung S. Murine monocytes: origins, reveals origins and dynamics of monocytes and tissue macrophages under subsets, fates, and functions. Microbiol Spectr. (2016) 4:1–11. homeostasis. Immunity. (2013) 38:79–91. doi: 10.1016/j.immuni.2012.12.001 doi: 10.1128/microbiolspec.MCHD-0033-2016 38. Polletti S, Natoli G. Understanding spontaneous conversion: 21. Mildner A, Schonheit J, Giladi A, David E, Lara-Astiaso D, Lorenzo-Vivas the case of the Ly6C- monocyte. Immunity. (2017) 46:764–6. E, et al. Genomic characterization of murine monocytes reveals C/EBPbeta doi: 10.1016/j.immuni.2017.04.010 transcription factor dependence of Ly6C- cells. Immunity. (2017) 46:849–62 39. O’Garra A, Redford PS, McNab FW, Bloom CI, Wilkinson RJ, Berry MP. The e7. doi: 10.1016/j.immuni.2017.04.018 immune response in tuberculosis. Annu Rev Immunol. (2013) 31:475–527. 22. Sampath P, Moideen K, Ranganathan UD, Bethunaickan R. Monocyte subsets: doi: 10.1146/annurev-immunol-032712-095939 phenotypes and function in tuberculosis infection. Front Immunol. (2018) 40. Prabowo SA, Groschel MI, Schmidt ED, Skrahina A, Mihaescu T, 9:1726. doi: 10.3389/fimmu.2018.01726 Hasturk S, et al. Targeting multidrug-resistant tuberculosis (MDR-TB) 23. Cooper AM, Dalton DK, Stewart TA, Griffin JP, Russell DG, Orme IM. by therapeutic vaccines. Med Microbiol Immunol. (2013) 202:95–104. Disseminated tuberculosis in interferon gamma gene-disrupted mice. J Exp doi: 10.1007/s00430-012-0278-6 Med. (1993) 178:2243–7. 41. Tzelepis F, Blagih J, Khan N, Gillard J, Mendonca L, Roy DG, et al. 24. Bustamante J, Boisson-Dupuis S, Abel L, Casanova JL. Mendelian Mitochondrial cyclophilin D regulates T cell metabolic responses susceptibility to mycobacterial disease: genetic, immunological, and clinical and disease tolerance to tuberculosis. Sci Immunol. (2018) 3:1–11. features of inborn errors of IFN-γ immunity. Semin Immunol. (2014) doi: 10.1126/sciimmunol.aar4135 26:454–70. doi: 10.1016/j.smim.2014.09.008 42. Cardona PJ, Amat I, Gordillo S, Arcos V, Guirado E, Diaz J, et al. 25. Fletcher HA, Snowden MA, Landry B, Rida W, Satti I, Harris SA, et al. T- Immunotherapy with fragmented Mycobacterium tuberculosis cells cell activation is an immune correlate of risk in BCG vaccinated infants. Nat increases the effectiveness of chemotherapy against a chronical Commun. (2016) 7:11290. doi: 10.1038/ncomms11290 infection in a murine model of tuberculosis. Vaccine. (2005) 23:1393–8. 26. Kagina BM, Abel B, Scriba TJ, Hughes EJ, Keyser A, Soares A, doi: 10.1016/j.vaccine.2004.09.008 et al. Specific T cell frequency and cytokine expression profile do not 43. Gröschel MI, Prabowo SA, Cardona P-J, Stanford JL, van der Werf TS. correlate with protection against tuberculosis after bacillus Calmette-Guerin Therapeutic vaccines for tuberculosis—A systematic review. Vaccine. (2014) vaccination of newborns. Am J Respir Crit Care Med. (2010) 182:1073–9. 32:3162–8. doi: 10.1016/j.vaccine.2014.03.047 doi: 10.1164/rccm.201003-0334OC 44. Joosten SA, van Meijgaarden KE, Arend SM, Prins C, Oftung F, Korsvold 27. Abebe F. Is interferon-gamma the right marker for bacille Calmette-Guerin- GE, et al. Mycobacterial growth inhibition is associated with trained innate induced immune protection? The missing link in our understanding immunity. J Clin Invest. (2018) 128:1837–51. doi: 10.1172/JCI97508 of tuberculosis immunology. Clin Exp Immunol. (2012) 169:213–9. 45. Kleinnijenhuis J, Quintin J, Preijers F, Joosten LA, Ifrim DC, Saeed S, et al. doi: 10.1111/j.1365-2249.2012.04614.x Bacille Calmette-Guerin induces NOD2-dependent nonspecific protection 28. Naranbhai V, Fletcher HA, Tanner R, O’Shea MK, McShane H, Fairfax BP, et al. from reinfection via epigenetic reprogramming of monocytes. Proc Natl Acad Distinct transcriptional and anti-mycobacterial profiles of peripheral blood Sci USA. (2012) 109:17537–42. doi: 10.1073/pnas.1202870109 monocytes dependent on the ratio of monocytes: lymphocytes. EBiomedicine. 46. de Bree LCJ, Koeken V, Joosten LAB, Aaby P, Benn CS, van Crevel R, et al. (2015) 2:1619–26. doi: 10.1016/j.ebiom.2015.09.027 Non-specific effects of vaccines: current evidence and potential implications. 29. Brennan MJ, Tanner R, Morris S, Scriba TJ, Achkar JM, Zelmer A, et al. Semin Immunol. (2018) 39:35–43. doi: 10.1016/j.smim.2018.06.002 The cross-species mycobacterial growth inhibition assay (MGIA) project, 47. Mourits VP, Wijkmans JC, Joosten LA, Netea MG. Trained immunity 2010-2014. Clin Vaccine Immunol. (2017) 24:1–13. doi: 10.1128/CVI.00 as a novel therapeutic strategy. Curr Opin Pharmacol. (2018) 41:52–8. 142-17 doi: 10.1016/j.coph.2018.04.007 30. Tanner R. Development of Mycobacterial Growth Inhibition Assays for the Early Evaluation and Gating of Novel TB Vaccine Candidates. Doctoral Thesis, Conflict of Interest Statement: MA is an employee of Archivel Farma S.L. P-JC is University of Oxford (2015). a consultant of Archivel Farma S.L. and a co-inventor of the patent of RUTI as a 31. Anwar S. Impact of Helminth Infection on Antimycobacterial Immune therapeutic vaccine. Responses in UK Migrants. Doctoral Thesis, London School of Hygiene & Tropical Medicine (2017). The remaining authors declare that the research was conducted in the absence of 32. O’Shea MK, Tanner R, Muller J, Harris SA, Wright D, Stockdale any commercial or financial relationships that could be construed as a potential L, et al. Immunological correlates of mycobacterial growth inhibition conflict of interest. describe a spectrum of tuberculosis infection. Sci Rep. (2018) 8:14480. doi: 10.1038/s41598-018-32755-x Copyright © 2019 Prabowo, Painter, Zelmer, Smith, Seifert, Amat, Cardona and 33. Hanna RN, Carlin LM, Hubbeling HG, Nackiewicz D, Green AM, Punt Fletcher. This is an open-access article distributed under the terms of the Creative JA, et al. The transcription factor NR4A1 (Nur77) controls bone marrow Commons Attribution License (CC BY). The use, distribution or reproduction in differentiation and the survival of Ly6C- monocytes. Nat Immunol. (2011) other forums is permitted, provided the original author(s) and the copyright owner(s) 12:778–85. doi: 10.1038/ni.2063 are credited and that the original publication in this journal is cited, in accordance 34. Thomas GD, Hanna RN, Vasudevan NT, Hamers AA, Romanoski CE, with accepted academic practice. No use, distribution or reproduction is permitted McArdle S, et al. Deleting an Nr4a1 super-enhancer subdomain ablates which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 10 April 2019 | Volume 10 | Article 894