Stronger induction of trained immunity by mucosal BCG or MTBVAC vaccination compared to standard intradermal vaccination

Article Stronger induction of trained immunity by mucosal BCG or MTBVAC vaccination compared to standard intradermal vaccination Graphical Abstract Highlights dNonhuman primates recapitulate trained immunity upon live attenuated TB vaccination dIntravenous BCG induces changes in H3K27 acetylation and enhances cytokine production dMucosal BCG improves induction of trained immunity of monocytes over intradermal BCG dThe M. tuberculosis-derived candidate vaccine MTBVAC appears equally potent as BCG Authors Michel P.M. Vierboom, Karin Dijkman, Claudia C. Sombroek, ..., Nacho Aguilo, Carlos Martin, Frank A.W. Verreck Correspondence [email protected] In Brief Vierboom et al. demonstrate the induction of trained immunity in blood and bone marrow monocytes after vaccination with live attenuated TB vaccines in nonhuman primates. Mucosal respiratory delivery of BCG or MTBVAC induces trained immunity more efficiently compared to standard intradermal vaccination. Vierboom et al., 2021, Cell Reports Medicine 2, 100185 January 19, 2021 ª2020 The Author(s). https://doi.org/10.1016/j.xcrm.2020.100185 ll Article Stronger induction of trained immunity by mucosal BCG or MTBVAC vaccination compared to standard intradermal vaccination Michel P.M. Vierboom, 1,8, *Karin Dijkman, 1 Claudia C. Sombroek, 1 Sam O. Hofman, 1 Charelle Boot, 1 Richard A.W. Vervenne, 1 Krista G. Haanstra, 1 Maarten van der Sande, 2 Liesbeth van Emst, 3 Jorge Domı ´nguez-Andre ´s, 3 Simone J.C.F.M. Moorlag, 3 Clemens H.M. Kocken, 1 Jelle Thole, 4 Esteban Rodrı ´guez, 5 Eugenia Puentes, 5 Joost H.A. Martens, 2 Reinout van Crevel, 3 Mihai G. Netea, 3 Nacho Aguilo, 6,7 Carlos Martin, 6,7 and Frank A.W. Verreck 1 1 Biomedical Primate Research Centre, Rijswijk, the Netherlands 2 Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University, Nijmegen, the Netherlands 3 Radboud University Medical Centre, Nijmegen, the Netherlands 4 TuBerculosis Vaccine Initiative, Lelystad, the Netherlands 5 Biofabri, Pontevedra, Spain 6 Department of Microbiology, Faculty of Medicine, IIS Arago ´n, University of Zaragoza, Zaragoza, Spain 7 CIBERES, Instituto de Salud Carlos III, Madrid, Spain 8 Lead contact *Correspondence: [email protected] https://doi.org/10.1016/j.xcrm.2020.100185 SUMMARY BCG vaccination can strengthen protection against pathogens through the induction of epigenetic and metabolic reprogramming of innate immune cells, a process called trained immunity. We and others recently demonstrated that mucosal or intravenous BCG better protects rhesus macaques from Mycobacterium tuberculosis infection and TB disease than standard intradermal vaccination, correlating with local adaptive immune signatures. In line with prior mouse data, here, we show in rhesus macaques that intravenous BCG enhances innate cytokine production associated with changes in H3K27 acetylation typical of trained immunity. Alternative delivery of BCG does not alter the cytokine production of unfractionated bronchial lavage cells. However, mucosal but not intradermal vaccination, either with BCG or the M. tuberculosis-derived candidate MTBVAC, enhances innate cytokine production by bloodand bone marrow-derived monocytes associated with metabolic rewiring, typical of trained immunity. These results provide support to strategies for improving TB vaccination and, more broadly, modulating innate immunity via mucosal surfaces. INTRODUCTION Canonical vaccinology aims at the induction of long-lived adaptive memory in antigen-specific lymphocyte populations and T and/or B cells. Innate immunity in vaccinology is primarily acknowledged in the context of vaccine formulation and the quintessential innate immune activation of professional antigen-presenting cells (APCs) that will prime and skew the adaptive response of lymphocytes. 1 For years, innate immune responses and myeloid monocyte/macrophage function were considered constant and invariable over time, but it is now widely recognized that also innate immune cells can adapt and display memory-like phenotypes. This innate immune ‘‘memory’’ is typically referred to as trained immunity. 2 Induction of trained immunity is defined by a functional adaptation of innate immunity after a primary insult resulting in a more effective response upon a ‘‘secondary’’ encounter with an unrelated pathogen. 2 It is characterized by increased cytokine and/or chemokine production, 3 metabolic rewiring, 4 and epigenetic reprogramming of innate immune cells. 5 Trained immunity is not associated with the characteristic immune receptor gene rearrangements that are associated with the antigen specificity of adaptive memory, but rather is driven by the epigenetic control of specific immune gene clusters resulting in the upregulation of particular pro-inflammatory cytokines. 6 An epigenetic mark in particular associated with trained immunity in monocytes, and used in the present study, is the acetylation of histone 3 at the position of lysine 27 (H3K27ac). 7 Recent work in mice has shown that Bacillus Calmette-Gue ´rin (BCG)-mediated training of the myeloid monocyte/macrophage lineage is sustained by the epigenetic reprogramming of hematopoietic stem cells (HSCs), the myeloid precursors in bone marrow (BM). 8 Mycobacterium bovis-derived BCG, currently the only available vaccine to fight tuberculosis (TB), 9 is the prototypical biological agent for which trained immunity has been demonstrated. Trained immunity has been linked to the beneficial heterologous off-target effect of infant BCG vaccination, which from epidemiological analyses appears associated with reduced childhood Cell Reports Medicine 2, 100185, January 19, 2021 ª2020 The Author(s). 1 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ll OPEN ACCESS mortality from causes other than TB. 10–13 Emerging evidence supports the hypothesis that not only adaptive memory but also the memory-like plasticity in innate immunity positively contributes to immune protection against and early clearance of M. tuberculosis (Mtb) infection. 14,15 Although not established as features of innate training, a comparative analysis in macaque species suggests that markers of innate immunity and myeloid monocyte function are associated with differential TB disease susceptibility. 16 Despite the widespread use of live attenuated BCG as prophylaxis against TB, it is only partially efficacious and by and large fails to protect adolescents and adults from the infectious pulmonary manifestation of Mtb infection. 17 Thus, TB continues to be a major threat to global human health, and a better vaccination strategy is urgently needed to relieve the burden of TB. 18,19 While intradermal injection is the standard route for BCG vaccination, historical data in rhesus macaques have shown that mucosal or intravenous administration provided superior signals of protection against experimental infection. 20 We have recently demonstrated that pulmonary mucosal BCG vaccination reduced disease in a cohort of rhesus macaques in which intradermal BCG failed to show any protective effect. 21 In a subsequent study, exploiting repeated limiting dose (RLD) rather than single dose Mtb challenge, we have shown that mucosal but not intradermal BCG provides signals of prevention of infection. 22 Most recently, Darrah and colleagues 23 have corroborated the superior protective capacity of BCG when given intravenously. Both studies did not identify an immune biomarker in the blood that correlated with the protective efficacy of either mucosal or intravenous BCG vaccination. Locally, however, antigen-specific interleukin-17A-positive (IL-17A + ) polyfunctional T helper (Th) cells and IL-10 secretion in the airways did correlate with protection, 22 and the article by Darrah et al. 23 suggests that tissue-resident lymphocytes may be pivotal in conferring protection. While from these non-human primate (NHP) studies unconventional administration of BCG appears to be a most interesting lead for improving our current TB prophylaxis, it remains to be established, however, if and how alternative routing of BCG affects innate and trained immunity. Various candidate TB vaccines, ranging from subunit formulations to live attenuated and recombinant mycobacteria, are at various stages of clinical testing in the current vaccine-development pipeline (www.tbvi.eu/what-we-do/pipeline-of-vaccines/). Of the live attenuated candidates, MTBVAC is unique in that it is derived from a clinical isolate of Mtb (Mt103) rather than from M. bovis (which is the parental strain of BCG and recombinant BCG candidates). 24,25 MTBVAC is attenuated by deletion of the phoP and the fadD26 gene. Thereby, the expression of virulence factors is critically disrupted, while an intrinsically broader antigen repertoire in comparison to BCG is preserved. 26 Aguilo et al. 27 demonstrated that the expression of ESAT6 and CFP10 by MTBVAC is key to its protective capacity in a mouse models of TB. MTBVAC has shown preclinical efficacy better than BCG in animal models. 24,27 MTBVAC vaccination protects rhesus macaques against aerosol challenge with Mtb and induces immune signatures analogous to those observed in clinical studies. 28 It has tested safe and immunogenic in adult and infant human populations so far. 9,29 Similar to BCG, MTBVAC is able to generate trained immunity through the induction of glycolysis and glutaminolysis, and the accumulation of histone methylation marks at the promoters of proinflammatory genes. Recently, Taranco ´n et al. 30 showed that MTBVAC-induced heterologous protection against a lethal challenge with Streptococcus pneumoniae in an experimental murine model of pneumonia. In the present study, we sought to investigate the impact of live attenuated mycobacterial vaccination via the respiratory mucosa on trained innate immunity in NHPs. To this end, we followed a 2 32 factorial design strategy for an immunogenicity analysis in adult rhesus macaques, vaccinating animals by intradermal injection or by endobronchial instillation either with BCG or with MTBVAC. We confirmed the distinctive adaptive response profile in the airways upon pulmonary mucosal delivery of BCG and MTBVAC, and analyzed the production of innate and adaptive cytokines after in vitro stimulation of cells obtained from lung, blood, and BM. We piloted the induction of trained immunity after intravenous BCG injection, demonstrating the increased acquisition of H3K27ac marks and resulting in prominent increased cytokine production after heterologous stimulation in monocytes isolated from blood and BM. We subsequently demonstrated that respiratory mucosal administration of live attenuated mycobacterial vaccines more efficiently induced trained immunity in blood and BM monocytes, compared to intradermal immunization, whereby MTBVAC was equally potent as BCG. Our findings underpin the innate immune stimulatory potential of the candidate TB vaccine, MTBVAC. For live attenuated mycobacterial vaccines in general, they suggest the enhancement of innate immune training via respiratory mucosal vaccine administration. RESULTS In the present study, we set out to investigate whether mucosal administration of live attenuated TB vaccines in rhesus macaques could modulate innate immune responses and induce trained immunity. To this end, we selected healthy, purpose-bred adult rhesus macaques (Macaca mulatta), both males and females, and stratified them into comparable groups of 6 animals each (Table S1). By random assignment in a 2 32 factorial design strategy, these groups received either a standard human dose of BCG or the equivalent dose of the live attenuated candidate vaccine, MTBVAC, by intradermal injection (BCG.id; MTBVAC.id) or by endobronchial instillation into the lower right lung lobe (BCG.muc; MTBVAC.muc). For the assessment of adaptive and/or innate immunity, relevant samples were collected before and after vaccination from the airways by bronchoalveolar lavage (BAL), from the blood by venipuncture, and from the BM by needle aspiration. Before analyzing the intradermal and mucosal vaccination groups, and since it had been reported as an effective research strategy for measuring trained immunity in mice, 8 a separate group of 3 rhesus macaques were vaccinated by intravenous BCG injection (BCG.iv) to pilottrained immunity profiling of monocytes (2 weeks after vaccination). A schematic diagram of vaccination and sampling over time is displayed in Figure 1. 2Cell Reports Medicine 2, 100185, January 19, 2021 Article ll OPEN ACCESS BCG and MTBVAC are immunogenic and both display an adaptive immune profile specific to the route of vaccination To confirm the efficiency of vaccination, we performed a specific interferon-g(IFN-g) enzyme-linked immunosorbent spot (ELISpot) assay after in vitro recall stimulation of peripheral blood mononuclear cells (PBMCs) with the protein-purified derivative (PPD) of Mtb (or culture medium as a negative control). For both BCG and MTBVAC and by either route of delivery, we found an increase in antigen-specific IFN-grelease after primary vaccination. In line with previous observations, 21,22 the vaccineinduced IFN-gresponse was higher after intradermal over mucosal BCG delivery, and a comparable response pattern was observed with MTBVAC (Figures 2A and 2B). When stimulating with a recombinant fusion protein of ESAT6 and CFP10, two antigens encoded in the region of deletion (RD)1 that is absent from M. bovis BCG but present in MTBVAC, positive IFN-grelease signals were obtained only from MTBVAC-vaccinated animals, as expected (Figure 2C). To further confirm the adaptive response profile that we registered previously after pulmonary mucosal BCG as a correlate of the prevention of Mtb infection, we assessed the cytokine production profile of CD4 + T lymphocytes from the airways by flow cytometry 22 after ex vivo stimulation with PPD. As expected, we detected the most prominent levels of polyfunctional Th17 cells (producing IL-17A, IFN-g, tumor necrosis factor a [TNF-a], and IL-2) in BALs that were collected 8 weeks after primary vaccination with BCG via the pulmonary mucosal rather than the intradermal route (Figure 2D). Mtb-derived MTBVAC was found to induce a similar response pattern and to be at least as potent as BCG. Regardless of the vaccine, significant numbers of polyfunctional Th17 cells were also recovered from the lower left lung lobe (i.e., opposite of the site targeted by endobronchial immunization), suggesting that the polyfunctional Th17 response is not contained to the site of vaccination but disseminates locally. Establishing trained immunity in monocytes after intravenous immunization with BCG To evaluate the induction of trained immunity after standard intradermal versus mucosal vaccination with live attenuated TB vaccines, we established the feasibility of the detection of trained immunity in the nonhuman primate model. For this, we immunized three animals with BCG intravenously, a protocol that was successfully explored in mice by Kaufmann et al. 8 To minimize the interference of a developing adaptive immune response while still being able to detect robust innate immune responses, we took samples from different compartments at week 2 after vaccination. Next to the analysis of BAL cells from the lung, we performed a more selective analysis of myeloid CD14 + monocytes purified from fresh PBMCs (PBMC.mo) and BM (BM.mo), the prototypic cells in which the trained immunity phenotype has been described. CD14 + monocyte populations were obtained by positive selection (using magnetic bead-cell sorting) and typically reached a purity of ±90% (Figure S1). Both unfractionated BAL cells and enriched monocytes from blood and BM were subsequently analyzed for signals of trained immunity after in vitro restimulation with Mtb-derived whole-cell lysate (WCL) or the heterologous Escherichia coli derived Tolllike receptor (TLR)-4 agonist lipopolysaccharide (LPS). After 24 h, supernatants were harvested, frozen, and analyzed later for cytokine production, indicative of trained innate immunity, including TNF-a, IL-6, and IL-1b. After intravenous BCG vaccination, we found marginal suppression of cytokine secretion, if any effect at all, after comparing Mtb or LPS stimulation of BAL (Figures 3A and 3B). Stimulation of PBMC.mo and BM.mo with Mtb revealed markedly increased levels of TNF-a2 weeks after BCG.iv (Figure 3A), but no apparent modulation of IL-6 and IL-1bsecretion levels. However, heterologous stimulation with LPS demonstrated prominent increased production of all three cytokines by both monocytes from the blood and BM (with the exception of a single animal with high baseline levels in BM.mo; Figure 3B, PBMC.mo and BM.mo). Metabolic rewiring, specifically a switch from oxidative phosphorylation to glycolysis under aerobic conditions, 4 has been described as an initiating event underlying trained immunity. Therefore, we measured lactate, a metabolite of glycolysis, levels in the supernatants of stimulated bloodand BM-derived monocytes as a surrogate marker of metabolic rewiring, and thus trained immunity. Lactate production was observed for 2 of 3 LPS-stimulated BM.mo, but undetectable for PBMC.mo (Figure 3C). Ultimately, we determined whether BCG.iv vaccination resulted in epigenetic reprogramming, another hallmark of trained innate immunity, in blood-derived monocytes (PBMC.mo). Therefore, we examined the dynamics of histone modification H3K27ac (an epigenetic mark associated with active chromatin in the context of trained immunity 7 ) after BCG.iv by chromatin immune precipitation, followed by high-throughput sequencing (chromatin immunoprecipitation sequencing [ChIP-seq]) to assess the changes associated with BCG.iv vaccination. H3K27ac changes were detected at many loci (Figures 3D–3F). In total, we detected 792 regions increased and 646 regions decreased in H3K27ac (Figure 3D). While the regions showing increased acetylation were associated with genes related to the cell cycle, signaling, and activation of the immune response, Figure 1. Schematic representation of the timeline of vaccination in the different vaccination strategies The BCG.iv group (n = 3) was analyzed at weeks 1 and 2. The groups that were immunized with BCG.id (n = 6), BCG.muc (n = 6), MTBVAC.id (n = 6), and MTBVAC.muc (n = 6) were analyzed at weeks 1 and 8. Only from mucosally vaccinated animals BAL was collected from the right lung as well as the left lung. See also Table S1. Cell Reports Medicine 2, 100185, January 19, 2021 3 Article ll OPEN ACCESS regions decreased in acetylation were associated with genes involved in cellular differentiation (Figure 3E). Examples for both an active region, IFN regulatory factor 3 (IRF3), and suppressed region, syndecan 2 (SDC2) are given (Figure 3F). These data are in line with vaccination-induced changes in the acetylome and the cellular program related to the induction of trained immunity. Pro-inflammatory cytokine production after stimulation of BAL cells with Mtb or LPS We then sought to compare the induction of trained immunity after standard intradermal versus mucosal vaccination with the live attenuated TB vaccines BCG and MTBVAC. We analyzed cytokine/chemokine production in BAL cells after ex vivo stimulation with homologous Mtb WCL or heterologous LPS. Using a tailored multiplex kit for cytokine and chemokine measurement, we assessed the production of molecules that have previously been associated with lymphocyte activation and function (IFN-g, IL-2, IL-17A), inflammation, leukocyte differentiation, migration, and/or with trained immunity (TNF-a, IL-6, IL-1b, granulocyte-macrophage-colony-stimulating factor [GM-CSF], macrophage inflammatory protein 1a[MIP-1a; CCL3], and IFNg-induced protein 10 [IP-10; CXCL10]). 3,11,31 In line with the flow cytometric detection of polyfunctional Th17 cells, we found significantly elevated levels of IL-17A, IFN-g, IL-2, and TNF-a secretion from Mtb WCL-stimulated BAL cells—in particular after mucosal vaccination with BCG or MTBVAC (Figure 4A). A significant increase in the secretion of IL-2 and TNF-a(but not IL-17A and IFN-g) was also measured after intradermal vaccination. As the response levels of BCG and MTBVAC in this regard were indistinguishable, we have depicted results and performed statistical analyses on the 12 animals of both BCG and MTBVAC ABC D Figure 2. Mucosal vaccination establishes a unique local adaptive signature defined by polyfunctional Th17 cells (A–C) Individual IFN-gELISpot responses after in vitro recall stimulation of PBMCs against PPD (A) before vaccination or (B) 8 weeks after vaccination or with (C) ESAT6-CFP10 8 weeks after vaccination. Horizontal lines in bars indicate group medians; n = 6 animals/group. ANOVA adjusted for multiple comparisons; Dunn’s multiple comparison test. (D) The percentages of polyfunctional IL-17A + PPD-specific CD4 + T cells (also producing IFN-g, TNF-a, and IL-2) were determined by flow cytometric analysis of BAL cells before (PRE) and 8 weeks after intradermal (.id) or mucosal (.muc) vaccination. Cells were typically collected from the lower right lung lobe (R); targeted for vaccination) and for mucosally vaccinated animals also from the left lung (L) to establish dissemination of the immune response. The data are presented as medium control corrected (cc); n = 6 animals/group; Wilcoxon matched-pairs signed rank test. ULD, upper limit of detection; LLD, lower limit of detection. See also Figure S7. 4Cell Reports Medicine 2, 100185, January 19, 2021 Article ll OPEN ACCESS AB D C E F (legend on next page) Cell Reports Medicine 2, 100185, January 19, 2021 5 Article ll OPEN ACCESS arms together, per route of administration. The release of pro-inflammatory IL-6, after in vitro stimulation with Mtb WCL, comparing preand post-vaccination time points, was rather heterogeneous. In contrast, IL-1b, GM-CSF, and MIP-1a (CCL3) secretion was, by trend or with statistical significance, decreased in mucosally vaccinated animals (Figure 4A). CXCL10 (IP-10), however, was markedly and significantly increased upon Mtb WCL stimulation, regardless of the nature of the vaccine or the immunization route. This cytokine/chemokine secretion profile of BAL cells after stimulation with a preparation as crude as the WCL of Mtb, containing both protein antigens and (non-protein) innate receptor ligands, is the net result of adaptive and innate activation of all subsets that are present in the unfractionated BAL cell population. In addition, it must be noted that the relative abundance of immune cell subsets in BAL is affected by pulmonary mucosal but not by intradermal BCG vaccination toward a relative increase in T lymphocytes over alveolar macrophage (Figure S2). To at least circumvent the adaptive component of this response profile and toward the assessment of trained immunity by heterologous innate stimulation, we also stimulated BAL cells with LPS to measure the ensuing cytokine production capacity. Upon LPS, and in contrast to Mtb WCL stimulation, IL-17A, IFN-g, and TNF-aconcentrations after vaccination were no different from what was found at baseline (Figure 4B). Most likely as a result of the bystander activation of T lymphocytes specifically recruited into the airways after mucosal vaccination, IL-2 release upon LPS stimulation was increased with BAL cells from mucosally vaccinated animals (Figure 4B), but to a lesser extent compared to Mtb WCL stimulation (Figure 4A). Also compared to Mtb WCL stimulation, the vaccination-modulated GM-CSF secretion of BAL cells was lost with LPS, and that of CXCL-10 was much lower and heterogeneous (Figure 4B). LPS-stimulated IL-6 levels were comparably heterogeneous as with Mtb WCL stimulation. IL-1band MIP-1a, as with Mtb WCL stimulation, showed diminished secretion upon the LPS stimulation of BAL cells after vaccination, regardless of the route of vaccine delivery (Figure 4B). This cytokine/chemokine secretion analysis of unfractionated BAL cells upon heterologous LPS (or homologous Mtb WCL) stimulation did not reveal a signal of trained immunity in the airways after (mucosal) live attenuated mycobacterial vaccination. Induction of trained immunity in monocytes after mucosal vaccination with BCG and MTBVAC Since BAL cells did not provide a signal indicative of trained immunity, we sought to compare the induction of trained immunity through ex vivo heterologous LPS stimulation of bloodand BM-derived monocytes, as was shown for BCG.iv between intradermal and mucosal immunized animals, with live attenuated BCG or MTBVAC. To demonstrate that the cytokine production is due to ex vivo heterologous LPS stimulation and exclude the possibility of continued innate activation by the (persisting) vaccine, unstimulated medium controls were included that showed minimal to no production of cytokines (Figure S3). While T cell-derived cytokines IL-17A, IFN-g, and IL-2 were not detected upon monocyte stimulation (not shown), the enhancement of LPS-stimulated secretion of TNF-a, IL-6, and IL-1bwas evident in some but not all animals that were vaccinated by intradermal injection (Figure 5A). Specifically, 4 of 11 animals (2 from each of the BCGand the MTBVAC-vaccinated groups) showed elevated TNF-aproduction with an effect size (independent of baseline production) of >2-fold increase after BCG.id/ MTBVAC.id (Figure 5B; due to a technical error, we missed the baseline values of a single animal of the MTBVAC.id group). Similarly, 4 of 11 showed the opposite effect of decreased TNF-alevels after BCG.id/MTBVAC.id, while 3 of 11 showed no considerable modulation of LPS-stimulated TNF-aafter intradermal vaccination (Figure 5B). Of note, Mtb-derived MTBVAC appeared equally as potent as BCG in the induction of trained immunity in PBMC.mo (Figure S4A). Notably, the secretion levels of pro-inflammatory IL-6 and IL-1bshowed a pattern similar to that of TNF-a. The ratio of the production for the 3 signature cytokines (TNF-a, IL-6, and IL-1b) did not deviate over a period of 8 weeks in a cohort of unvaccinated animals (Figure S4C). In contrast to intradermal BCG/MTBVAC vaccination, the mucosal delivery of these live attenuated vaccines resulted in a robust and significant enhancement of TNF-a, IL-6, and IL-1b production after LPS stimulation in 10 of 12 animals (Figures 5A and 5B; 5 animals from the BCG and the MTBVAC vaccinated group each). The median effect size was 15.1, 6.2, and 5.9 for the fold increase in TNF-a, IL-6, and IL-1bproduction, respectively (Figure 5B). For GM-CSF and MIP-1a, 2 cytokines that have been associated with trained immunity, a similar pattern of enhanced secretion after prior mucosal rather than intradermal vaccination was obtained (Figures 5A and 5B). This enhancement, however, was less prominent than for TNF-a, IL-6, and IL-1b, with a median fold-increase of GM-CSF and MIP-1aafter BCG.muc/MTBVAC.muc of 2.1 and 2.9, respectively (Figure 5B). CXCL-10 was not detected from these monocyte stimulation analyses (Figure 5A). Comparing BCG to MTBVAC within each route of delivery (id versus muc) strategy demonstrated that both vaccines were equally potent in mediating trained immunity (Figure S5A). We explored the mycobactericidal activity with Figure 3. Trained immunity after intravenous BCG vaccination (A and B) Freshly isolated BAL cells (from the right lung) and monocytes from blood (PBMC.mo) and bone marrow (BM.mo) were stimulated for 24 h with (A) Mtb whole-cell lysate (25 mg/mL) or (B) LPS (0.1 mg/mL) before and 2 weeks after intravenous vaccination with BCG. The data are presented as medium control corrected. (C) Lactate production as an indicator of metabolic rewiring was measured in 24-h supernatant from LPS-stimulated monocytes purified from blood (PBMC.mo) or BM (BM.mo). See also Figure S1. (D) Heatmap of H3K27ac reads (red) over BCG-specific peaks. The intensity over the center of the peak ±12 kb is depicted. (E) The top GO pathways associated with the nearest genes to dynamic H3K27ac, with adjusted p values. (F) H3K27ac dynamics at interferon regulatory factor 3 (IRF3) and syndecan 2 (SDC2) locus for PBMC.mo preand post-vaccination. The complete list of genes and values can be found at GEO: GSE159046. 6Cell Reports Medicine 2, 100185, January 19, 2021 Article ll OPEN ACCESS A B (legend on next page) Cell Reports Medicine 2, 100185, January 19, 2021 7 Article ll OPEN ACCESS PBMC.mo isolated from residual frozen PBMCs, but within the limits of our efforts, we did not detect mycobactericidal activity by trained monocytes (Figure S6). Next to PBMC.mo, we addressed the LPS-stimulated cytokine/chemokine secretion potential of BM-derived CD14 + monocytes (BM.mo) before and after BCG or MTBVAC Figure 4. Cytokine production by BAL cells Freshly isolated BAL cells were stimulated (A) with Mtb whole cell lysate or (B) LPS, before (PRE) and 8 weeks after intradermal (.id) or mucosal (.muc) vaccination (with either BCG, in circles, or MTBVAC, in triangles). Cells were typically collected from the lower right lung lobe (R), the lobe targeted by vaccination, and for mucosally vaccinated animals also from the left lung (L), to establish dissemination of the immune response. The data are presented as medium control corrected (mcc); n = 12 animals/group; Wilcoxon matched-pairs signed rank test. p values are indicated at the top of each graph. See also Figure S2. A B Figure 5. Trained immunity after mucosal vaccination in peripheral blood monocytes Cytokine production was measured after 24 h of stimulation with LPS (0.1 mg/mL). (A) Paired display of cytokine levels before and 8 weeks after vaccination. The data are presented as medium control corrected (mcc; BCG in circles; MTBVAC in triangles). Statistical significance was calculated by Wilcoxon’s non-parametric paired analysis test (n = 12 animals/group; PRE versus 8 weeks post-vaccination). p values are indicated at the top of each graph. (B) Median fold increase in cytokine production is indicated in italics; significant p < 0.05 is indicated in bold; n = 12 animals/group; Wilcoxon signed rank test. See also Figures S1,S3,S4,S5, and S6. 8Cell Reports Medicine 2, 100185, January 19, 2021 Article ll OPEN ACCESS STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-CD3 – AF700 (clone SP34-2) BD Biosciences Cat#: 557917; RRID: AB_396938 anti-CD4 – PerCP.Cy5.5 (clone L200) BD Biosciences Cat#: 552838; RRID: AB_394488 anti-CD8a– APC-H7 (clone SK1) BD Biosciences Cat#: 641400; RRID: AB_164536 anti-CD14 – BV421 (clone M5E2) BD Biosciences Cat#: 301830; RRID: AB_10959324 anti-CD20 – BV421 (clone 2H7) Biolegend Cat#: 302330; RRID: AB_10965543 anti-CD28 – ECD (clone CD28.2) IOTest Cat#: 6607111; RRID: AB_1575955 anti-CD45RA – PE-CF594 (clone 5H9) BD Biosciences Cat#: 565419; RRID: AB_2739229 anti-CD95 – BV605 (clone DX2) Biolegend Cat#: 305628; RRID: AB_2563825 anti-IL-2 – AF488 (clone MQ1-17H12) Biolegend Cat#: 500314; RRID: AB_493368 anti-IL-17A – PE-Cy7 (clone ebio64DEC17) Biolegend Cat#: 25-7179-42; RRID: AB_11063994 anti-TNF-a– BV650 (clone Mab11) BD Biosciences Cat#: 502938; RRID: AB_2562741 anti-IFN-g– BV711 (clone 4S.B3) BD Biosciences Cat#: 502540; RRID: AB_2563506 anti-CD3ε– FITC (clone SP34) BD Biosciences Cat#: 556611; RRID: AB_396484 anti-CD14 – V450 (T€ uk4) Milteny Biotec Cat#: 130-113-152; RRID: AB_10831023 anti-CD20 – BV605 (clone 2H7) Biolegend Cat#: 302334; RRID: AB_2563398 anti-CD45 – BV786 (clone D058-1283) BD Biosciences Cat#: 563861; RRID: AB_2738454 anti-CD3 – BV421 (clone SP34-2) BD Biosciences Cat#: 562877; RRID: AB_2737860 anti-CD14 – BV786 (clone M5E2) BD Biosciences Cat#: 301840; RRID: AB_2563425 anti-CD16 – PE-CY7 (clone 3G8) BD Biosciences Cat#: 560716; RRID: AB_1727433 anti-CD20 – BV605 (clone 2H7) Biolegend Cat#: 302334; RRID: AB_2563398 anti-CD45 – AF700 (clone D058-1283) BD Biosciences Cat#: 561288; RRID: AB_10613813 anti-CD66 – PE (clone TET2) Milteny Biotec Cat#: 130-093-133; RRID: AB_871699 anti-CD206 – PerCP (clone 15-2) BioLegend Cat#: 321122; RRID: AB_10899411 Bvbuffer BioLegend Cat#: 563794; RRID: AB_2869750 VIVID – BV421 Thermofisher Cat#: L34955 Life/Death – eFluor 506 eBioscience Cat#: 65-0866-14 anti-H3K27ac Diagenode Cat#: pab-196-050; RRID: AB_2637079 Bacterial and virus strains BCG (strain Sofia; 5 310 5 CFU) InterVax Ltd Cat#: not applicable MTBVAC (M.tub strain; 5 310 5 CFU) Biofabri Cat#: not applicable Mtb Erdmann BEI Resources Cat#: NR-50781 Chemicals, peptides, and recombinant proteins Purified Protein Derivate (PPD; M.tub) AJ Vaccines Cat#: 2391 Purified Protein Derivate (PPD; M.bov) Life Technologies NV Cat#: 760060 Purified Protein Derivate (PPD; M.av) Life Technologies NV Cat#: 760065 Whole Cell Lysate (WCL; Mtb HN878) BEI Resources Cat#: NR-14824 Lipopolysaccharide (0111:B4 strain; Ultrapure) Invivogen Cat#: tlrl-3pelps PMA Sigma-Aldrich Cat#: P8139 Ionomycine Sigma-Aldrich Cat#: I0634 Roswell Park Memorial Institute (RPMI) Life Technologies NV Cat#: 52400041 LymphoprepTM Axis-Shield Cat#: AXI-1114547 Fetal Calve Serum (FCS) Life Technologies NV Cat#: 10270106 (Continued on next page) Cell Reports Medicine 2, 100185, January 19, 2021 e1 Article ll OPEN ACCESS RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Michel PM Vierboom ([email protected]). Materials availability This study did not generate new unique reagents. Data and code availability Raw data files of the ChIPseq analysis on BCG trained monocytes begore and after vaccination have been deposited in the NCBI Gene Expression Omnibus under accession number GEO: GSE159046. EXPERIMENTAL MODEL AND SUBJECT DETAILS Animals and ethics All animal experiments were in accordance with Dutch Law on animal experimentation, which is in accordance with the EU Directive 2010/63/EU on the protection of animals used for scientific purposes. The study was approved by the institutional animal welfare body (in Dutch: Instantie voor Dierwelzijn, IvD; accession number CCD009D) of BPRC. A cohort of 8 nonvaccinated control animals was used for Figure S4C. The study in which these animals were used was approved by the institutional animal Continued REAGENT or RESOURCE SOURCE IDENTIFIER Perchloric acid Sigma-Aldrich Cat#: 244252-1L Formaldehyde (16%) Thermo Scientific Cat#: 28906 Protease Inhibitor Cocktail Sigma-Aldrich Cat#: P8465 Dynabeads protein A Invitrogen Cat#: 10002D Dynabeads protein G Invitrogen Cat#: 10004D BSA Sigma-Aldrich Cat#: A7030 Tagment DNA buffer Nextera Cat#: 20034197 AMPureXP beads Beckman Coulter Cat#: A63880 KAPA HiFi Hotstart Ready Mix KAPA Biosystems Cat#: KK2601 Nextera DNA Library Prep Kit Illumina Cat#: FC-121-1031 KAPA Library Preparation Kit KAPA Biosystems Cat#: KK8400 TMB (ELISPOT substrate) Mabtech Cat#: 3651-10 Critical commercial assays CD14 microbeads Milteny Biotec Cat#: 130-091-097 Custom NHP Legendplex (11-plex) Biolegend Cat#: 92919 Monkey IFN-gELISPOT U-CyTech Cat#: CT610-10 Lactate-ELISA Promokine Cat#: PK-CA577-K627 Deposited data ChIP sequencing data before and after BCGiv vaccination This paper GEO: GSE159046 Experimental models: organism/strain Rhesus monkeys (Indian); male and female; adult (> 4 years); purpose bred BPRC breeding colony not applicable Software and algorithms Eli.Analyze (ELISPOT; v6.1) A.EL.VIS GmbH not applicable FACSDiva Software v 8.0.1 (BD LSRII) BD Biosciences SCR_001456 Flowjo software v 10 Treestar SCR_000410 LEGENDplexTM Data Analysis Software (V8.0) Biolegend/Vigene Tech not applicable GraphPad Prism v 8.4.2 GraphPad Software https://www.graphpad.com e2 Cell Reports Medicine 2, 100185, January 19, 2021 Article ll OPEN ACCESS welfare body (Accession number CCD009F). The BPRC is accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC) and has an approved Assurance (#A5509-1) for the care and use of animals on file at the National Institutes of Health (NIH). The rhesus monkeys were housed in socially compatible pairs at the Biomedical Primate Research Centre (BPRC; animal biosafety level ABSL-3). The animals were offered a daily diet consisting of monkey food pellets (Hope Farms, Woerden, the Netherlands), fruit and vegetables of the season, and bread. Drinking water was available ad libitum via automatic water systems. The study was carried out in 27 healthy purpose-bred, pedigreed, male and female rhesus monkeys (Macaca mulatta) of Indian origin (5.99 – 13,50 kg; see Table S1). All animals were screened to be negative for pre-existing immunity against mycobacterial antigens as determined by interferon gamma (IFN-g) ELISpot assay after in vitro recall stimulation with Purified Protein Derivate (PPD) of M.tuberculosis,M.avium and M.bovis. All animal handling and biosampling was performed under ketamine sedation (10 mg kg1, by intramuscular injection). For bronchoalveolar lavage (BAL) and intrabronchial vaccination inoculation with BCG or MTBVAC ketamine (5 mg kg1) was supplemented with intramuscular medetomidine (0.04 mg kg1) and an analgesic sprayed into the larynx. Vaccines and vaccination BCG standard dose & MTBVAC standard dose. Animals immunized with Bacillus Calmette–Guerin (BCG) strain Sofia (InterVax Ltd.) received a single standard adult human dose of 5 310 5 CFU. Animals immunized with MTBVAC received an equivalent human dose of 5 310 5 CFU. Vaccines were delivered in a volume of 0.1 mL intradermally and 10 mL intravenously or intrabronchially. Mucosal vaccination in the lung was performed by intrabronchial delivery of the same dose in 10mL of sterile saline solution to a segmental bronchus in the lower right lung lobe using a bronchoscope. Vaccination was executed for all animals in a single session in random order within 2-3 hours from vaccine preparation METHOD DETAILS Biological sample collection and processing Cells from the pulmonary mucosa were recovered by BAL at week 1 for all groups from the lower right lung lobe; at week 2 after intravenous BCG and week 8 after intradermal BCG from lower right lung lobe and week 8 after mucosal vaccination from both the lower right and left lung lobe. Three volumes of 20 mL of prewarmed 0.9% saline solution were consecutively instilled and recovered. BAL fluid was harvested by centrifugation of BAL samples for 10 min at 400 g after 100 mm filtration. Supernatant was subsequently decanted and stored at 80C pending further analysis. The BAL cell pellet was taken up in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FCS, glutaMAX, and penicillin/streptomycin (from now on referred to as R10) and used freshly in downstream assays. Heparinized blood for immune monitoring, was collected by venipuncture. Bone marrow was obtained through needle extraction of the caput humerus. PBMCs and BM cells were obtained by density gradient centrifugation with Lymphoprep lymphocyte separation medium (Axis-Shield) and resuspended in R10 for further analysis. Mycobacterium uptake and killing assay To assess mycobacterium uptake, PBMC.mo (30.000 cells/well) were incubated for 1h at 37C with Mtb Erdmann at a MOI of 5. Non-phagocytosed mycobacterium was washed away and monocytes were incubated with high dose (30 mg/mL) gentamycin for 10-minutes. PBMC.mo were subsequently lysed with 0.05% SDS in water, titrated and plated on to 7H10 agar for bacterial enumeration. To asses killing, PBMC.mo were incubated with Mtb Erdmann at a MOI of 5 for 1h at 37C and subsequently cultured with low gentamycin (5 mg/mL) for 24 hr. Bacteria were enumerated as described before. As a control, bacteria were incubated without PBMC.mo. Data are presented as % of control (Figure S5). Monocyte isolation CD14+ cells (monocytes) were labeled with magnetic CD14 MicroBeads. The cell suspension was loaded onto a MACSColumn which is placed in the magnetic field of a MACS Separator. The magnetically labeled CD14+ cells are retained in the column. The unlabeled cells run through, this cell fraction is depleted of CD14+ cells. After removal of the column from the magnetic field, the magnetically retained CD14+ cells can be eluted as the positively selected cell fraction. Purity was analyzed by flowcytometry (Figure S1). IFN-gELISPOT A NHP specific IFN-gELISpot assay was used on PBMC, according to the manufacturer protocol (U-CyTech, Utrecht), to determine the frequency of antigen specific IFN-gproducing cells. In brief, 200.000 freshly isolated PBMC were incubated in triplicate for 24 h with specified antigens or control stimuli. Subsequently, supernatant was collected and stored (80C), and cells were transferred to specific anti-IFN-gcoated filter plates (PVDF, Millipore) for an additional overnight (18 h) incubation. Cells were discarded and membrane bound IFN-gwas detected using biotinylated anti-IFN-gantibody, streptavidin-horseradish peroxidase conjugate Cell Reports Medicine 2, 100185, January 19, 2021 e3 Article ll OPEN ACCESS and tetramethylbenzidine (TMB) substrate (the latter from MAbTech, Stockholm). Spots were quantified using an automated reader (AELVIS, Hannover). Flow cytometry Antigen-specific T cell responses were determined at WK-1 (PRE) and week 8 indicated time points by flow cytometry locally using BAL cells. Cells were stimulated overnight in the presence of Golgiplug transport inhibitor. The following mAb staining T cell panel was used: Intracellular staining: CD3 AF700 (SP34-2; BD), TNF-a PE-CY7 (Mab11; BD), IL-2 BV510 (MQ1-17H12; BioLegend), IFN-g APC (4S.B3; BD); IL-17A PE-Cy7 (ebio64DEC17; Thermofisher). Surface staining: CD4 PerCP-Cy5.5 (L200; BD), CD8a APC-H7 (SK1; BD), CD14 BV421 (M5E2; BD), CD20 BV421 (2H7; BioLegend), CD45 BV786 (D058-1283; BD). Gating strategy for T cells (Figure S7; Related to Figure 2D) T cells were gated as Singlets/Lymphocytes/Viable/ CD14-CD20-/CD45+ /CD3+ before CD4 and CD8 gating was applied. After doublet exclusion, lymphocytes were gated based on size and granularity. Any anomaly indicative of unstable signal acquisition was excluded using the time parameter. Events from the combined time-gates were plotted against the dump channel containing the viability, CD14 and C20 markers and subsequently gated as viable, CD14and CD20-. Cells were further selected for CD45 and CD3 positivity before CD4 and CD8 gating was applied. Boolean gating of any cytokine expression of IL-2/IFN-g/TNFa/IL-17A. BAL cell composition analysis Cells were stimulated overnight with WCL. The following mAb staining panel was used to analyze BAL cell composition: CD45 AF700 (D058-1283; BD); CD66 PE (TET2; Miltenyi); CD206 PerCP (15-2;Biolegend); CD16 PE-CY7 (3G8; BD); CD3 BV421 (SP34-2;BD); CD20 BV605 (2H7; Biolegend); CD14 BV786 (M5E2; BD). Gating strategy for BAL cell composition (Figure S2B) Singlets/ Leucocytes (CD45)/Viable/Time gate: CD206+/CD14+ (Alveolar Macrophages); CD3 (T cells); CD20 (B cells); CD16+ (NK-cells) and CD66+ (Neutrophils). BAL cell composition is expressed as % from the CD45+/viable fraction. Isolation of CD14+ monocytes PBMC or BM cells were incubated with CD14 magnetically labeled Microbeads (Miltenyi Biotec; Netherlands). The cell suspension is loaded onto a MACSColumn which is placed in the magnetic field of a MACS Separator. The magnetically labeled CD14+ cells are retained in the column. The column is washed once. The column was removed from the magnetic field and the magnetically retained CD14+ cells were then eluted as the positively selected cell fraction. Purity of CD14+ fraction was subsequently tested by flow cytometry. Gating strategy for CD14+ monocytes (Figure S1B) Singlets/CD45+/ Viable/CD14+. CD14+ cells are expressed as % from the CD45+/viable fraction. Multiplex cytokine assay Cells from different sources (PBMC.mo, BM.mo and BAL were stimulated with WCL and titrated amount of LPS (E.coli; Ultrapure). Freshly isolated cells 25000 cells were incubated ex-vivo for 24 hr with each stimulus after which the supernatant was collected. Medium alone was taken along for control. Cytokine production was assessed with multi-analyte flow assay kit using a custom nonhuman primate Legendplex TM (Biolegend; USA). Assays were performed according to the manufacturer’s instructions. Briefly, the supernatants of stimulated cells were incubated with beads coated with cytokine-specific antibodies. Bound cytokines were visualized using biotin-coupled detection antibodies and R-phycoerythrin-conjugated streptavidin. Beads were acquired on 3 laser, 14 color LSR-II flow cytometer (BD Biosciences). A NHP custom made 11-plex consisted of TNF-a, IL-6, IL-1bcharacterizing trained immunity, GM-CSF, CCL3 (MIP-1a), CXCL10 (IP-10) and cytokines associated with adaptive immunity IFN-g, IL-2, IL-17A. Lactate measurements Supernatant collected from the 24 h innate stimulation was analyzed for the production of lactate as a surrogate marker for the metabolic rewiring (Warburg-effect) underlying trained immunity. Proteins in the supernatant, interfering with the lactate analysis, were denatured precipitated with percholoric acid (PCA) and removed by high speed centrifugation. The clear deproteinized supernatant was collected and neutralized with NaOH. Samples were subsequently diluted 15-fold and analyzed in a L-Lactate Assay Kit I (Promokine; PromoCell GmbH; Germany) according to manufacturer’s instruction. Chromatin immunoprecipitation Cell pellets were dissolved in PBS and crosslinked in solution by using formaldehyde (1% final volume, shaking 10 min at room temperature). Glycine (0.125 M) was added to quench the reaction. Cells were then washed two times with cold PBS. Cell pellets were lysed in a volume of 110ml using lysis buffer (20 mM HEPES pH 7.6, 1% SDS, 1 3Protease Inhibitor Cocktails). Samples e4 Cell Reports Medicine 2, 100185, January 19, 2021 Article ll OPEN ACCESS were sonicated using the Biorupter Pico (Diagenode) with eight cycles (30 s on/30 s off). Afterward, the samples were spun at 16,000 3g for 5 min at room temperature and the supernatant was stored at 80C. Chromatin from 100,000 cells was used for ChIPmentation. ChIPmentation was performed as described by Schmidl et al., 46 with several modifications. In short, the chromatin was incubated overnight at 4C rotating in dilution buffer (1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8, 167 mM NaCl), 1 3Protease Inhibitor Cocktail 47 and 1mg of antibody [H3K27ac (C15410196, Diagenode)] in a total volume of 300 ml. The next day per ChIP 10ml protein A Dynabeads and 10ml protein G Dynabeads (both Invitrogen) were added. Beads were washed before use with dilution buffer (+0.15% SDS, +0.1% BSA) and incubated with the chromatin and antibody mix for 60 min at 4C rotating. Afterward, the beads were washed at 4C once with ChIP wash buffer 1 (2 mM EDTA, 20 mM Tris pH 8, 1%Triton X-100, 0.1% SDS, 150 mM NaCl), twice with ChIP washbuffer 2 (2 mM EDTA, 20 mM Tris pH 8, 1% Triton X-100, 0.1%SDS, 500 mM NaCl), and twice with TE (1 mM EDTA, 10 mM TrispH 8). Beads were resuspended in 24 ml Tagment DNA buffer (Nextera) and 1ml Tn5 enzyme (produced in-house) and incubated for 10 min at 37C with 550 rpm shaking. Afterward, the beads were washed twice with WBI (20 mM HEPES, 150 mM NaCl, 0.1% SDS, 0.1% DOC,1% Triton X-100, 1 mM EDTA, 0.5 mM EGTA) and twice with WBIV (20 mM HEPES, 1 mM EDTA, 0.5 mM EGTA) with 5 min rotating at room temperature in between. Samples were decrosslinked for 1 h at 55C 1,000 rpm shaking followed by an overnight incubation at 65C using elution buffer (0.5% SDS, 300 mM NaCl,5 mM EDTA, 10 mM Tris pH 8) and proteinase K. Samples were incubated one additional hour the next day with elution buffer and proteinase K for 1 h at 55C. The samples were purified using 2 3SPRI AMPureXP beads. qPCR was used to determine the sufficient amount of PCR cycles needed to amplify the library. The libraries were amplified using the KAPA HiFi Hotstart Ready Mix (KAPA Biosystems) and Nextera Index Kit 1 (i7) and 2 (i5) primers (Illumina). Amplified libraries were purified using a 0.65 3SPRI AMPureXP beads incubation followed by a 1.8 3SPRI AMPureXP beads incubation. Library concentration was measured using the KAPA Library Quantification Kit (KAPA Biosystems); library size was determined using the BioAnalyzer High Sensitivity DNA Kit (Agilent). Sequencing was performed using an Illumina HiSeq 2000, and 50-bp paired-end reads were generated. ChIP-seq analysis Sequenced reads were quality and adaptor trimmed with trim galore and cutadapt (Krueger, Felix, Trim galore. ‘‘A wrapper tool around Cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files 516 (2015): 517. ‘‘Picard Toolkit.’’ 2020. Broad Institute, GitHub; Marin, Marcel. Cutadapt removes adaptor sequences from high-throughput sequencing reads. EMBnet.journal). These reads were aligned against the UCSC Rhesus reference genome (RheMac10) with Burrows-Wheeler Aligner (BWA) program 48 with default parameters. Duplicate reads were removed with picard MarkDuplicates. Peaks were called with MACS2 and default settings 49 and used to detect the binding sites for ChIP-seq tracks. Tags within a given region were counted and adjusted to represent the number of tags within a 1 kb region. Subsequently the percentage of these tags as a measure of the total number of sequenced tags of the sample was calculated and displayed as heatmaps using fluff. 50 To determine genomic locations of binding sites, the peak file was analyzed using a script that annotates binding sites according to all RefSeq genes. Necropsy and post-mortem evaluation Rhesus monkeys were scheduled by protocol for euthanasia and necropsy at 2 weeks (3 animals for BCG IV ) or 8 weeks (BCG ID , BCG MUC , MTBVAC ID , MTBVAC MUC ) after vaccination. At the end of the experiment blood (< 45 ml) and bone marrow was collected before euthanasia with sodium pentobarbital (> 50 mg/kg). Samples were stored at 80C for analysis at later time point. QUANTIFICATION AND STATISTICAL ANALYSIS Data handling of measurements of cytokine production by Multiplex The Legendplex TM is a bead-based assay for flow cytometry allowing for the measurement of multiple cytokines in the same sample. Bead count per analyte was ±300/sample. Data obtained with a bead count arbitrarily set at < 25 was excluded. Standard curve (pg/mL) taken along on each plate: 10000, 2500, 625, 156, 39.1, 9.8, 2.4 pg/mL. PRE and WK2/WK8 samples from individual animals were measured on the same plate to avoid inter-plate variation. Samples from animals from different treatment groups were randomly distributed over plates. If lower limit of quantification (LLOQ) was < 2.4 pg/mL, the lowest detectable concentration was set at 2.4 pg/mL. If LLOQ was > 2.4 pg/mL, the lowest detectable concentration was set at LLOQ. This data procedure ensured a conservative estimation of the fold change between PRE and WK2/WK8 post vaccination. Statistics comparing routes of vaccination, cytokine, and lactate production Statistical parameters including number of animals, percentage of cells levels of cytokine production, fold increase of cytokine production and absolute increase in lactate production are reported in the Figures and Figure legends. Data was analyzed by ANOVA adjusted for multiple comparisons, Wilcoxon’s non-parametric paired analysis test, Wilcoxon signed rank test to where applicable. Statistical analyses were conducted using GraphPad Prism 7 software (version 7.0d; GraphPad Software, La Jolla, CA). Cell Reports Medicine 2, 100185, January 19, 2021 e5 Article ll OPEN ACCESS Statistics epigenetics Peaks were called with MACS2 and default settings 49 and used to detect the binding sites for ChIP-seq tracks. All peaks were merged using bedtools and for each sample tags per peak were counted. Differential peaks were called by comparing the two sets of samples using these criteria: the sum of counted tags within one peak using all samples should be > 200 and the fold difference should be > median plus 2xSD. The testing for gene set/pathway enrichments was done using Fisher’s exact testing. e6 Cell Reports Medicine 2, 100185, January 19, 2021 Article ll OPEN ACCESS