Pulmonary MTBVAC vaccination induces immune signatures previously correlated with prevention of tuberculosis infection

Article Pulmonary MTBVAC vaccination induces immune signatures previously correlated with prevention of tuberculosis infection Graphical Abstract Highlights dPulmonary MTBVAC delivery confers immune signature correlating with TB protection dThis signature spreads through the lung without a recall response in the skin dVaccine-induced T cells have increased mucosal homing and tissue residency markers dVaccine-induced antibodies enhance phagocytosis of M. tuberculosis Authors Karin Dijkman, Nacho Aguilo, Charelle Boot, ..., Eugenia Puentes, Carlos Martin, Frank A.W. Verreck Correspondence [email protected] In Brief Dijkman et al. show that pulmonary immunization with the M. tuberculosisderived vaccine candidate MTBVAC confers a local mucosal antigen-specific signature—polyfunctional Th1/Th17 cells exhibiting increased homing and tissue residency marker expression, IL-10, and phagocytosis-promoting immunoglobulins—that has been associated previously with protection from TB infection and disease in rhesus macaques. Dijkman et al., 2021, Cell Reports Medicine 2, 100187 January 19, 2021 ª2020 The Author(s). https://doi.org/10.1016/j.xcrm.2020.100187 ll Article Pulmonary MTBVAC vaccination induces immune signatures previously correlated with prevention of tuberculosis infection Karin Dijkman, 1 Nacho Aguilo, 2,3 Charelle Boot, 1 Sam O. Hofman, 1 Claudia C. Sombroek, 1 Richard A.W. Vervenne, 1 Clemens H.M. Kocken, 1 Dessislava Marinova, 2,3 Jelle Thole, 4 Esteban Rodrı ´guez, 5 Michel P.M. Vierboom, 1 Krista G. Haanstra, 1 Eugenia Puentes, 5 Carlos Martin, 2,3 and Frank A.W. Verreck 1,6, * 1 Biomedical Primate Research Centre (BPRC), Rijswijk, the Netherlands 2 Department of Microbiology, Faculty of Medicine, IIS Aragon, University of Zaragoza, Zaragoza, Spain 3 CIBERES, Instituto de Salud Carlos III, Madrid, Spain 4 TuBerculosis Vaccine Initiative (TBVI), Lelystad, the Netherlands 5 Biofabri, Pontevedra, Spain 6 Lead contact *Correspondence: [email protected] https://doi.org/10.1016/j.xcrm.2020.100187 SUMMARY To fight tuberculosis, better vaccination strategies are needed. Live attenuated Mycobacterium tuberculosisderived vaccine, MTBVAC, is a promising candidate in the pipeline, proven to be safe and immunogenic in humans so far. Independent studies have shown that pulmonary mucosal delivery of Bacillus Calmette-Gue ´- rin (BCG), the only tuberculosis (TB) vaccine available today, confers superior protection over standard intradermal immunization. Here we demonstrate that mucosal MTBVAC is well tolerated, eliciting polyfunctional T helper type 17 cells, interleukin-10, and immunoglobulins in the airway and yielding a broader antigenic profile than BCG in rhesus macaques. Beyond our previous work, we show that local immunoglobulins, induced by MTBVAC and BCG, bind to M. tuberculosis and enhance pathogen uptake. Furthermore, after pulmonary vaccination, but not M. tuberculosis infection, local T cells expressed high levels of mucosal homing and tissue residency markers. Our data show that pulmonary MTBVAC administration has the potential to enhance its efficacy and justifies further exploration of mucosal vaccination strategies in preclinical efficacy studies. INTRODUCTION Despite advances in treatment and care, tuberculosis continues to cause approximately 1.6 million deaths and an additional 10 million cases of active disease annually. 1 Control of this ongoing epidemic is complicated by a lack of accurate diagnostics, lengthy treatment regimens, and an increase in drug-resistant tuberculosis (TB) incidence. An effective vaccination strategy preventing TB infection or disease is therefore of critical importance for controlling the continuing TB epidemic. Unfortunately, the only prophylactic vaccine currently available, Bacillus Calmette-Gue ´rin (BCG), despite preventing dissemination of the disease, is notoriously variable in protecting adults and adolescents from pulmonary TB. Pulmonary disease is the major cause of morbidity and mortality and the driver of TB spread. 2 Geographical location, prior non-tuberculous mycobacterium (NTM) exposure, and over-attenuation of BCG have been implied in the variable BCG efficacy. 2–4 Regardless of the underlying mechanisms of this variation in efficacy, it is evident that a more reliable vaccine strategy is urgently needed. Currently, multiple novel TB vaccines are being developed to replace BCG at birth or to serve as a (heterologous) booster on top of prior BCG vaccination. 5,6 One of these new candidate vaccines is MTBVAC, a live attenuated whole-cell vaccine designed as a potential replacement for neonatal BCG vaccination. MTBVAC was generated by genetic modification of a clinical Mycobacterium tuberculosis (Mtb)isolateofthe lineage 4 Euro-American genotype and harbors deletions in two virulence genes, phoP and fadD26. 7,8 These deletions interfere with transcription, synthesis, and/or secretion of multiple virulence factors, including early secretory antigenic target 6 (ESAT6) and phthiocerol dimycocerosates (PDIMs). 9 Because MTBVAC is Mtb derived, it contains genomic regions of difference (RDs) that are absent from M. bovis,andin particular also RD1, that is lacking from M. bovis-derived BCG. 9 MTBVAC, therefore, has a broader antigenic repertoire that is linked to its enhanced protective capacity. 10 Although RD1 encodes notorious virulence factors, such as ESAT6, secretion is tightly regulated and interrupted by the targeted phoP deletion in MTBVAC. 9 Accordingly, in early-stage clinical evaluation in adults and infants, intradermal MTBVAC immunization has been found to have an acceptable safety profile comparable with BCG, corroborating its attenuated phenotype. 11,12 Cell Reports Medicine 2, 100187, 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 In preclinical studies MTBVAC presented with an ability to protect from experimental TB infection and disease better than BCG. 7,13 It has been shown to confer improved protective efficacy against Mtb in newborn mice at a single dose at birth. 13 In guinea pigs, revaccination with MTBVAC after BCG priming resulted in a further reduction of Mtb burden in the lung compared with BCG alone, 14 and non-human primate data on the efficacy of (re)vaccination with intradermal MTBVAC have been established recently. 15 Although initially designed as a vaccine for newborns, MTBVAC is also considered for revaccination of BCG-primed adolescents and adults. 12 Phase2 dose-finding, safety, and immunogenicity studies in neonates (NCT03536117) and QuantiFERON-negative and -positive adults (NCT02933281) are in progress, and a subsequent phase 3 efficacy study in neonates is scheduled to start in 2021. The indicated route of administration for BCG and MTBVAC is the skin, which induces limited immune responses at the pulmonary mucosa, the primary site of infection with Mtb. A growing body of data generated in preclinical models of TB shows that altering the route of BCG administration to the pulmonary mucosa significantly improves its protective efficacy. 16–19 In previous work, we showed that pulmonary but not intradermal BCG vaccination could protect highly susceptible rhesus macaques (Macaca mulatta) from repeated low-dose Mtb infection and TB-associated pathology. 20 Macaques are considered to be a predictive model for TB vaccine development because of their close phylogenetic relationship to man and highly similar TB disease development. 21,22 In this model, the protection conferred by mucosal BCG statistically correlated with induction of polyfunctional interleukin-17A (IL-17A)+ CD4+ T cells at the pulmonary mucosa and IL-10 production by bronchoalveolar lavage (BAL) cells, whereas elevated levels of antigen-specific immunoglobulins were found in association with mucosal BCG immunization as well. Although the mucosa of the airways can be considered an environment of robust innate host defense to warrant homeostatic balance, potentially resulting in rapid clearance and poor immunogenicity of live attenuated vaccines, our data from mucosal delivery in non-human primates (NHPs) have shown local persistence of BCG and protection-associated immunity in the airways in the absence of overt respiratory adversity. 20 Interestingly, on this note, prior exposure of BCG to alveolar lining fluid from naive animals in vitro has been described to enhance its protective efficacy when administered peripherally to mice. 23 Also, in other NHP studies exploring pulmonary delivery of BCG, no adversity has been reported. 24,25 However, for pulmonary mucosal delivery of MTBVAC, the tolerability and immunogenicity remain to be established. In light of this, here we set out to assess the tolerability and immunogenicity of pulmonary mucosal delivery of MTBVAC in rhesus macaques. Using a two-by-two factorial design strategy, we compared MTBVAC with BCG by standard intradermal injection and endobronchial instillation of a standard human dose. Corroborating our earlier observations regarding alternative BCG delivery, we show that pulmonary MTBVAC administration was well tolerated and induced local IL-17A-producing T cells, IL-10 production, and Mtb-specific immunoglobulin A (IgA). Compared with BCG, vaccination with MTBVAC resulted in more rapid induction of immune responses and broader antigenic specificity. Beyond what we have reported previously for mucosal BCG, we identified increased expression of mucosal homing markers of purified protein derivative (PPD)-specific T cells in lung wash samples of mucosally vaccinated but not Mtb-infected animals. Furthermore, we show that vaccineinduced mucosal antibodies are functional in binding to live Mtb and facilitating pathogen uptake by phagocytes. In our attempt to identify an immune correlate of pulmonary wholecell TB vaccination that is assessable by peripheral sampling, we exploited in vivo recall stimulation by tuberculin skin testing (TST) but could not identify meaningful responses in skin or skin-draining axillary lymph nodes. This study corroborates and extends beyond previous findings and provides a rationale for future exploration of mucosal administration of MTBVAC with the perspective of improving our prophylaxis against TB infection and disease. RESULTS Local immune signatures after mucosal MTBVAC vaccination To interrogate whether the Mtb-derived MTBVAC vaccine candidate, like M. bovis BCG, is tolerated well and induces unique immune features upon pulmonary mucosal administration, we designed a dedicated safety/immunogenicity study (without infectious challenge) in rhesus macaques, represented schematically in Figure 1. We vaccinated with MTBVAC using a single (human) dose similar to BCG (5 310 5 colony-forming units [CFUs]/dose) through the standard intradermal route (M.id) or endobronchial instillation (M.muc) for direct comparison with intradermal or mucosal BCG vaccination (B.id and B.muc, respectively; Figure 1). On a daily basis, animals were monitored for changes in condition and well-being (including but not limited to alertness, appetite, and respiration), but no deviation from normal behavior was observed that would indicate vaccine adversity. Moreover, there were no signals of serological increase in C-reactive protein (CRP) levels during the study that would indicate an adverse systemic inflammatory response related to treatment (Figure S1). Although we aimed to address vaccine persistence by culturing from lung wash samples, our effort failed because of technical error; therefore we could not confirm persistence of MTBVAC in the airway like we have shown previously for BCG. 20 In Figure 1. Study design schematic Shown is a schematic overview of vaccination strategies and post-vaccination sampling (of peripheral blood and BAL). Of note, BALs were harvested bilaterally only for the mucosally vaccinated groups 3 and 8 weeks after vaccination. 2Cell Reports Medicine 2, 100187, January 19, 2021 Article ll OPEN ACCESS A B C D E F H G (legend on next page) Cell Reports Medicine 2, 100187, January 19, 2021 3 Article ll OPEN ACCESS summary, and within limits of observation, live attenuated Mtbderived MTBVAC, also by pulmonary mucosal delivery, was well tolerated by rhesus macaques. Flow cytometry profiling of T cells in BAL revealed robust induction of PPD-specific CD4+ T cells in both mucosally vaccinated groups, with MTBVAC eliciting higher responses than BCG for almost all cytokines, especially early after vaccination (Figures 2A–2D). Although intradermal BCG and MTBVAC showed an increase in interferon g(IFNg)-, tumor necrosis factor alpha (TNFa)-, and IL-2-producing T cells in the airways in the weeks following vaccination (Figures 2A–2C), IL-17A production was uniquely observed in the mucosally vaccinated groups (Figure 2D). These IL-17A+ T cells also produced IFNg,TNF-a, and IL-2, confirming induction of a local, quadruple-positive, CD4+ T cell population (Figure 2E), found previously to be associated in this species with protection from TB infection and disease. 20 Little PPD-specific cytokine production was observed in BAL CD8+ T cells (Figures S2A–S2E). Local lymphocyte proliferation was observed predominantly after mucosal vaccination (Figure S2F). Although, by endobronchial instillation, the vaccine was targeted to the lower right lobe, we investigated whether vaccineinduced immune responses would disseminate or be restrained to the targeted lobe only. 3 and 8 weeks after vaccination, we bilaterally collected BAL for immune profiling and found that PPD-specific, cytokine-producing T cells were present in lower right and lower left lung lobes, albeit at a somewhat lower frequency in the non-targeted lobe (Figure 2F). In either lobe, MTBVAC induced earlier and higher responses compared with BCG, including polyfunctional CD4+ Th17 cells. We also profiled immune responses in lung-draining lymph nodes, the canonical site of T cell priming for respiratory antigenic challenge. PPD-specific IFNg, TNF-a, and IL-2 production by CD4+ T cells was observed most prominently after mucosal vaccination but was also detectable after intradermal vaccination (Figure S2G). Interestingly, antigen-specific IL17A+ CD4+ T cells were not apparent in these lymph nodes (Figure S2G) because the precursor frequency of these cells was too low to be detected or because Th17 priming occurs elsewhere; for instance, in tertiary lymphoid structures in the lung. 26 Because we previously also identified IL10 production by unfractionated BAL cells as a correlate of protection, we investigated, by flow cytometry analysis, whether IL-10 production could be T cell derived. Although we confirmed high levels of PPD-specific IL10 production in stimulated BAL cell supernatants after mucosal vaccination with MTBVAC as well as BCG (Figure 2G), by flow cytometry, only very low frequencies of IL10+ CD4+ T cells were detected in the BAL of mucosally vaccinated animals (Figure 2H). Although the frequencies are low and conclusions therefore little robust , on average, only 2% of IL17+CD4+ BAL T cells obtained from mucosally vaccinated animals were found to be IL-10+ (data not shown). Thus, it seems that the high levels of IL-10 may not be T cell derived but produced by local innate immune cells in response to innate receptor ligation by mycobacterial compounds in the PPD preparation. Pulmonary vaccination with MTBVAC induces typically faster and higher polyfunctional CD4+ T cell responses and IL-10 secretion signals associated previously with protection by pulmonary BCG vaccination. Peripheral immunity after pulmonary vaccination In parallel to the pulmonary immune responses, we profiled peripheral T cell immunity in search of potential correlates of protection with a perspective for translation to clinical settings. However, as before, when assessing adaptive PPD-specific CD4+ and CD8+ T cell cytokine responses by flow cytometry, no discriminating qualitative signals could be identified that distinguished mucosally from intradermally vaccinated animals. CD4+ T cell cytokine production was observed from week 3 post-vaccination onward and was most prominent in intradermally vaccinated animals and the M.muc group (Figure 3A; Figures S3A–S3D). A slight increase in PPD-specific CD8+ T cell cytokine production was only apparent in the intradermally vaccinated groups 6 weeks post-vaccination (Figure 3B; Figures S4A–S4D). No differences in CD4+ and CD8+ T cell polyfunctionality could be detected. We used an IFNgenzyme-linked immune absorbent spot (ELISPOT) assay to assess the breadth of immune responses induced by MTBVAC in comparison with BCG. In line with the flow cytometry data, after stimulation with PPD, which contains antigens shared by BCG and MTBVAC, intradermal MTBVAC was indistinguishable from intradermal BCG by IFNgsecretion (Figure 3C). After stimulation with ESAT6 and CFP10, antigens produced by MTBVAC but absent from BCG, we observed IFNgproduction only by peripheral blood mononuclear cells (PBMCs) of MTBVAC-vaccinated animals regardless of vaccination route (Figure 3D). Of note, mucosal MTBVAC appeared to be equally potent in inducing PPD-specific IFNgsignals (Figure 3C). Although we previously observed comparable peripheral immune responses after mucosal and intradermal BCG vaccination, here mucosal BCG vaccination appeared to be less potent in inducing peripheral cytokine production and proliferation (Figures 3A–3C and 3E, respectively). Figure 2. Pulmonary mucosal vaccination with MTBVAC induces immune signatures associated with protection Shown is an overview of BAL cell immune responses after mucosal or intradermal vaccination with BCG or MTBVAC. (A–D) Flow cytometry analysis over time of (A) IFNg, (B) TNF-a, (C) IL-2, and (D) IL-17A CD4+ T cell responses after vaccination. (E) Stacked bar graphs depicting CD4+ T cell cytokine polyfunctionality over time after PPD recall stimulation (by group median values). (F) PPD-specific cytokine production of CD4+ T cells in the lower right and lower left lung lobes at week 3 and week 8, indicating primary and disseminated vaccine responses. (G) Secretion of IL-10 by unfractionated BAL cells stimulated with PPD at week 8, plotted as culture medium control-corrected values. (H) Flow cytometry analysis of IL-10 production by CD4+ T cells over time after vaccination. All graphs show 6 animals per group. In (A)–(D) and (H), + indicates PPD-stimulated samples, and indicates unstimulated, culture medium-incubated samples as controls. Horizontal lines within bars indicate group medians. Significance of group differences was determined by two-sided Mann-Whitney test adjusted for multiple comparisons. Holms-adjusted p %0.05 is depicted. Color coding per individual is consistent throughout the paper. 4Cell Reports Medicine 2, 100187, January 19, 2021 Article ll OPEN ACCESS A B C D E FG (legend on next page) Cell Reports Medicine 2, 100187, January 19, 2021 5 Article ll OPEN ACCESS As an alternative approach, we investigated the capacity of vaccine-induced T cells by an in vivo recall stimulation in TST. To this end, 8 weeks after vaccination, we intradermally injected saline (Sal) or old tuberculin (Tub) on opposite arms of each animal and took biopsies of the injection sites 3 days later. The skin biopsies were subsequently processed and characterized by flow cytometry to measure the delayed type hypersensitivity (DTH) response. By visual inspection of the local skin reaction, redness and swelling appeared after intradermal but not mucosal vaccination (data not shown). Accordingly, a tuberculin-specific influx of antigen-specific CD3+ T cells was exclusively observed in intradermally vaccinated animals (Figure 3F). These T cells showed higher frequencies of cytokine-producing subsets after intradermal vaccination (Figure 3G). When assessing antigen-specific T cells from axillary lymph nodes that drain the TST-DTH skin site, IFNg-, TNF-a-, and IL-2-producing CD4+ T cells were detectable in intradermally but not mucosally vaccinated animals (Figure S5). So, although mucosal vaccination does result in a peripheral blood response (by flow cytometry and IFNgELISPOT), it does not enable these cells to migrate to the site of a skin challenge and, therefore, rules out their analysis for a potential biomarker assay. The superior induction of local immune responses by mucosal MTBVAC over mucosal BCG was also reflected in the periphery, and these responses appeared to cover a broader range of antigens, including ESAT6/CFP10. However, within the limits of our analyses, no peripheral adaptive responses discriminating between mucosal versus intradermal immunization could be identified. Mucosal homing marker expression after vaccination In our search for peripheral correlates of protection, we also considered the possibility that pulmonary rather than intradermal vaccination would imprint peripheral T cells with a higher expression of pulmonary mucosal homing markers. To this end, we assessed the expression of CD103, CXCR3, and CCR5, all known to be involved in homing to the pulmonary mucosa, 27 on peripheral CD4+ T cells by means of flow cytometry. Prior to vaccination, approximately 25% of circulating CD4+ T cells expressed one or more of these homing markers. After vaccination, either peripherally or mucosally, this percentage did not change, nor was the pattern of homing marker coexpression notably altered in the mucosally vaccinated groups (Figure 4A; Figure S6). Because only a small fraction of all peripheral CD4+ T cells is vaccine specific (Figure 3A), we also assessed homing marker expression of cytokine-producing CD4+ T cells (IFNgand/or IL-17A). However, because of the low number of cytokine-positive events in the periphery, it was not possible to measure robust and reliable percentages of homing marker expression over time. Using a cutoff of a minimum of 100 cytokine-positive events, we only found robust frequencies 8 weeks post-vaccination. The frequency of homing markers expressed by PPD-specific T cells was comparable with that in the total CD4+ T cell population, although cytokine+ CD4+ T cells from all vaccinated groups consisted of more CCR5 single-positive cells (Figure 4B). When comparing homing marker expression between groups, again, no marked differences between the mucosally and intradermally vaccinated groups were apparent. Although the aforementioned homing markers did not reveal a peripheral correlate either, we went on to analyze their expression on cytokine-producing T cells in the airways after mucosal vaccination as well as after experimental pulmonary Mtb infection. For the latter, samples were obtained from another, independent infection study to characterize protective versus pathogenic BAL responses. Again, we analyzed the expression of CD103, CXCR3, and CCR5 of PPD-specific IFNgand/or IL-17A-producing Tcells.AfterMtb infection, a high local CD4+ T cell cytokine response is induced, similar to mucosal MTBVAC and higher than mucosal BCG administration (Figure 4C). However, the expression of homing markers was significantly lower in PPD-specific T cells from Mtb-infected animals compared with animals vaccinated mucosally with BCG or MTBVAC (Figures 4Dand 4E). Although, after Mtb infection, approximately 20% of cytokine-producing cells expressed a combination of CD103, CXCR3, and CCR5, 80% of vaccination-induced T cells were positive for one or more of these markers (Figure 4D). In addition to profiling chemokine receptor expression, we also measured CD69 and PD-1 co-expression on cytokine-positive CD4+ T cells as an indicator of a functional tissue-resident phenotype. 28 Previously, we have found that CD69 expression of BAL CD4+ T cells was higher after mucosal over intradermal BCG vaccination and, separately, that IFNg+TNF-a+IL-2+IL-17A+ T cells expressed higher levels of PD-1. 20 When assessing co-expression of these two markers on IFNg-and/orIL-17A-producingCD4+ T cells induced by mucosal vaccination, a substantial portion (15%–50%) of these cells was found to co-express both markers. Contrarily, after Mtb infection, CD69 and PD1 co-expression on PPD-specific T cells was significantly lower (typically less than 10%) (Figure 4F). Pulmonary vaccination, associated previously with enhanced protection, results in the presence of antigen-specific T cells Figure 3. Peripheral immune responses after vaccination Shown is a characterization of the height and breadth of peripheral immune responses after vaccination. (A and B) Stacked bar graphs depicting (A) CD4+ and (B) CD8+ T cell cytokine polyfunctionality over time (by group median values) after PPD stimulation. (C and D) PBMC IFNgproduction in response to stimulation with (C) PPD or (D) ESAT6-CFP10 fusion protein, measured by ELISPOT over time. (E) PPD-specific proliferation of PBMCs, plotted as a stimulation index (the ratio of antigenover medium control-stimulated values) over time. (F) T cell numbers in skin biopsies taken (3 days) after intradermal injection of saline (Sal) or old tuberculin (Tub) 8 weeks after vaccination. (G) PPD-specific cytokine production by T cells from Tub skin biopsies (right panel). The dotted line in (C) indicates the maximum limit of detection. In (G), + indicates PPD-stimulated samples, and indicates unstimulated, culture mediumincubated samples as controls. All graphs show 6 animals per group, except for (G), where there are 5 animals for the B.muc, M.id, and M.muc groups. Horizontal lines indicate group medians. Significance of group differences was determined by two-sided Mann-Whitney test adjusted for multiple comparisons. Holmsadjusted p %0.05 is depicted. Color coding per individual is consistent throughout. 6Cell Reports Medicine 2, 100187, January 19, 2021 Article ll OPEN ACCESS with a distinct tissue residency and mucosal homing phenotype. The observation that Mtb infection does not elicit this phenotype suggests that these cells could be involved in protection. Mucosal antibody levels and functionality In our previously reported vaccination and RLD Mtb infection study, mucosal BCG vaccination resulted in pulmonary PPDspecific Ig responses. 20 This observation, in combination with the recent interest in the role of Igs in protection from TB, 29,30 prompted us to investigate the humoral immune response in this study in more detail. As observed previously for BCG, mucosal vaccination with MTBVAC also resulted in a marked increase in Mtb-specific IgA, IgG, and IgM levels in BAL fluid, as detected by ELISA, and in a modest increase in some of the intradermally MTBVAC-vaccinated animals (Figure 5A). Like the cellular immune responses, humoral immunity was found to disseminate from the vaccine-targeted lobe (Figure 5B). A BC E D F Figure 4. Expression of mucosal homing markers after intradermal and pulmonary vaccination (A) Expression of CCR5, CD103, and CXCR3 on ex vivo CD4+ T cells from peripheral blood over time, depicted as group median values. (B) CCR5, CD103, and CXCR3 expression by PPD-specific (IFNg+ and/or IL-17A+) CD4+ T cells from PBMCs at week 8 after vaccination. (C–E) Comparison of PPD-specific T cells from BALs from pulmonary BCG-vaccinated, MTBVAC-vaccinated, and Mtb-infected animals. (C) Frequencies of IFNg+ and/or IL-17A+ CD4+ T cells after stimulation with PPD. (D and E) Expression of CCR5, CD103, and CXCR3 by cytokine+ CD4+ T cells, (D) depicted as group median values (stacked bar graph) or (E) as individual frequencies for separate markers. (F) Percentage of CD69 and PD1 double-positive cells of cytokine+ CD4+ T cells. For all graphs, n = 6 animals per group, with the exception of (B) where n = 5 for the BCG.muc group. Horizontal lines indicate group medians. Significance of group differences was determined by two-sided Mann-Whitney test adjusted for multiple comparisons. Holms-adjusted p %0.05 is depicted. Color coding per individual is consistent throughout. Cell Reports Medicine 2, 100187, January 19, 2021 7 Article ll OPEN ACCESS A B C D (legend on next page) 8Cell Reports Medicine 2, 100187, 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#: 565283; RRID: AB_2739154 anti-CD20 – BV421 (clone 2H7) Biolegend Cat#: 302334; RRID: AB_10965543 anti-CD28 – ECD (clone CD28.2) IOTest Cat#: 6607111; RRID: AB_1575955 anti-CD45 – BV786 (clone D058-1283) BD Biosciences Cat#: 563861; RRID: AB_2738454 anti-CD45RA – PE-CF594 (clone 5H9) BD Biosciences Cat#: 565419; RRID: AB_2739229 anti-CD69 – APC (clone FN-50) Biolegend Cat#: 310910; RRID: AB_314845 anti-CD69 – BV785 (clone FN-50) Biolegend Cat#: 310932; RRID: AB_2563696 anti-CD95 – BV605 (clone DX2) Biolegend Cat#: 305628; RRID: AB_2563825 anti-CD103 – FITC (clone Ber-ACT8) Biolegend Cat#: 350204; RRID: AB_10639865 anti-CCR5 – APC-H7 (clone 3A9) BD Biosciences Cat#: 560748; RRID: AB_1937308 anti-CCR7 – BV650 (clone G043H7) Biolegend Cat#: 353234; RRID: AB_2563867 anti-CXCR3 – PE-Cy7 (clone G025H7) Biolegend Cat#: 353720; RRID: AB_11219383 anti-PD1 – BV510 (clone EH12.2H7) Biolegend Cat#: 329932; RRID: AB_2562256 anti-IFN-g– BV711 (clone 4S.B3) BD Biosciences Cat#: 502540; RRID: AB_2563506 anti-IL-2 – AF488 (clone MQ1-17H12) Biolegend Cat#: 500314; RRID: AB_493368 anti-IL10 – PE (clone JES3-9D7) Biolegend Cat#: 501404; RRID: AB_315170 anti-IL-17A – PE-Cy7 (ebio64DEC17) Biolegend Cat#: 25-7179-42; RRID: AB_11063994 anti-IL-17A – BV605 (clone BL168) Biolegend Cat#: 512326; RRID: AB_2563887 anti-TNF-a– BV650 (clone Mab11) BD Biosciences Cat#: 502938; RRID: AB_2562741 VIVID – BV421 Thermofisher Cat#: L34955 GolgiPlug BD Biosciences Cat#: 555029; RRID: AB_2869014 Cytofix/Cytoperm BD Biosciences Cat#: 554714; RRID: AB_2869014 Goat Anti-Human IgM(mchain) Antibody, Alkaline Phosphatase (AP) Conugate, Affinity purified Invitrogen Cat#: A18838; RRID: AB_2535615 Goat Anti-Human IgA(achain) Antibody, Alkaline Phosphatase (AP) Conugate, Affinity purified Invitrogen Cat#: A18784; RRID: AB_2535561 Anti-MONKEY IgG (gamma chain)(GOAT) Antibody Peroxidase Conjugated Rockland Inc Cat#: 617-103-012; RRID: AB_218715 IgA biotinylated detection Ab (MT57) Mabtech 3860-4; RRID: AB_10736549 IgG biotinylated detection Ab (MT78) Mabtech 3850-6; RRID: AB_10666158 IgM biotinylated detection Ab (MT22) Mabtech 3880-6; RRID: NA Bacterial and virus strains M. tuberculosis strain Erdman K01 BEI Resources Cat#: NR-50781 BCG, strain Sofia (5 310 5 CFU/dose) InterVax Ltd Cat#: N/A MTBVAC (5 310 5 CFU/dose) Biofabri Cat#: N/A Chemicals, peptides, and recombinant proteins Lymphoprep TM Axis-Shield Cat#: AXI-1114547 Perchloric acid Sigma-Aldrich Cat#: 244252-1L Formaldehyde (16%) Thermo Scientific Cat#: 28906 p-Nitrophenyl Phosphate (p-NPP) Alkaline Phosphatase Substrate Merck Millipore Cat#: ES009-500mL (Continued on next page) Cell Reports Medicine 2, 100187, 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, Frank Verreck ([email protected]). Materials availability This study did not generate new unique reagents. However, any remaining biomaterials from this study can be made available and shipped at receiver’s cost upon specific request to the Lead Contact, for which we require completion of a Simple Letter Agreement for Transfer of Materials. Data and code availability All data there is, are presented in this paper (including supplementals) and can be made available in different formats upon reasonable request. This study did not generate any unique code. EXPERIMENTAL MODEL AND SUBJECT DETAILS Ethics & Animal Handling All housing and animal care procedures were performed at the Biomedical Primate Research Centre (BPRC) in Rijswijk, the Netherlands, and in compliance with European directive 2010/63/EU as well as the ‘‘Standard for Humane Care and Use of Laboratory Animals by Foreign Institutions’’ provided by the Department of Health and Human Services of the US National Institutes of Health (NIH, identification number A5539-01). BPRC is accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). An ethical framework approval from the independent, central animal experiments authority in the Netherlands (in Dutch: Centrale Commissie Dierproeven, CCD) was in place, and before start the study plan was approved by BPRC’s institutional animal welfare body (in Dutch: Instantie voor Dierwelzijn, IvD). The BCG and MTBVAC immunogenicity study was registered under CCD.009.D (while the Mtb-infection samples used for immunological cross-comparison, were taken from a study registered under CCD.009.C; see also further below). Continued REAGENT or RESOURCE SOURCE IDENTIFIER Streptavidine-FITC Biolegend Cat#: 405202 TMB, ELISA substrate MT Diagnostics Cat#: SB04/B TMB, ELISPOT substrate Mabtech Cat#: 3651-10 Critical commercial assays NHP specific IFN-gamma ELISPOT antibody pairs U-CyTech Cat#: 610-10 BOVIGAM TM Tuberculin PPD stimulating antigen, Bovine; Purified Protein Derivative (M. bovis) Life Technol. NV Cat#: 760060 BOVIGAM TM Tuberculin PPD stimulating antigen, Avian; Purified Protein Derivative (M. avium) Life Technol. NV Cat#: 760065 Milliplex NHP Cytokine Magnetic Bead Panel Merck Millipore Cat#: PRCYTOMAG 40K Experimental models: organisms/strains purpose-bred Macaca mulatta (rhesus macaques); adult (> 4 years of age) males and females; Indian-genotype BPRC N/A Software and algorithms Eli.Analyze (ELISPOT; v6.1) A.EL.VIS GmbH N/A 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 N/A GraphPad Prism v 8.4.2 GraphPad Software https://www.graphpad.com:443/ Other Old Tuberculin Synbiotics, Inc N/A Mycobacterium Tuberculosis - tuberculine PPD for in vitro use; Purified Protein Derivative (M.tuberculosis) AJ Vaccines Cat#: 2391 M. tuberculosis strain HN878 Whole Cell Lysate BEI Resources Cat#: NR-14824 e2 Cell Reports Medicine 2, 100187, January 19, 2021 Article ll OPEN ACCESS Twelve female and twelve male Indian-type rhesus macaques (Macaca mulatta) were selected from BPRC’s breeding colonies and stratified by gender, age, body weight and social indicators for pairwise housing into 4 groups of 6 animals. Treatment was randomly assigned to each group. Selected animals were negative for prior exposure to mycobacteria, as assessed by tuberculin skin testing with Old Tuberculin (Synbiotics Corporation, San Diego, CA) and an IFNgELISPOT using Purified Protein Derivative (PPD) from Mycobacterium bovis, Mycobacterium avium (both Life Technologies NV) or Mycobacterium tuberculosis (AJ Vaccines, Copenhagen, Denmark) for in vitro recall stimulation of PBMC. Animals were housed pairwise at biosafety level 3 throughout the experiment and provided with enrichment in the form of food and non-food items on a daily basis. Animal welfare was monitored daily. Animal weight was recorded prior to each blood collection event. All animal handling and bio-sampling was performed under ketamine sedation (10 mg/kg, by intra-muscular injection). For endobronchial instillation ketamine sedation (5mg/kg) was supplemented with intramuscular medetomidine (0.04 mg/kg) and an analgesic applied to the larynx. Eight weeks after vaccine administration animals reached study endpoint by protocol and were euthanized by intravenous injection of pentobarbital (200 mg/kg) under ketamine sedation. All animal care and veterinary personnel were blinded to experimental treatment. Vaccines, Vaccine Preparation & Administration Animals were vaccinated either with Bacillus Calmette Gue ´rin strain Sofia (InterVax Ltd., Ontario) or with MTBVAC (Biofabri, Spain), and either via the skin or the pulmonary mucosa. The intradermally vaccinated groups received a standard, adult human dose of 1.56.0 x10 5 CFU BCG or 3.0-17.0 x10 5 CFU MTBVAC in 0.1 mL reconstituted vaccine in the skin (abbreviated as BCG.id or MVAC.id). The mucosally vaccinated groups were administered the same dose, but in 10 mL of sterile saline solution by endobronchial instillation into the lower right lung lobe (abbreviated as BCG.muc/B.muc or MTBVAC.muc/M.muc). The vaccines, regardless of administration route, were prepared from a single, pooled mix of freshly reconstituted vials, immediately prior to administration. Vaccination was executed for all animals in a single session in random order within 2-3 hours from vaccine preparation. Mtb Infection For the comparison of homing marker expression by cytokine positive pulmonary mucosal T cells, BALs from Mtb infected animals were obtained 11 weeks after endobronchial instillation of 3 to 15 CFU of Mtb Erdman (NR-50781, BEIResources). Infection was confirmed for all animals by the induction of Mtb-specific IFNgproduction by PBMCs and by post-mortem pathology assessment (data not shown). Biosample Collection & Processing Cells from the pulmonary mucosa were recovered at specific time points by broncho-alveolar lavage (BAL), targeting either the lower right or lower 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 minutes 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 RPMI supplemented with 10% fetal bovine serum (FBS), glutamax and penicillin/streptomycin (from hereon referred to as R10) and used in downstream assays. BAL fluid was filter-sterilized by centrifugation through 0.2 mm PVDF membrane plates (Fisher Scientific) before analysis. Peripheral blood mononuclear cells (PBMC) were isolated from heparinised blood collected by venepuncture. Isolation of PBMCs was performed by density gradient centrifugation with Lymphoprep lymphocyte separation medium (Axis-Shield, UK), and PBMCs were subsequently resuspended in R10 for downstream immunological assays. METHOD DETAILS Flow cytometry T cell cytokine production and homing marker expression was assessed by flow cytometry. Freshly isolated PBMC were incubated overnight with Mtb PPD (5 ug/mL) in the presence of GolgiPlug transport inhibitor (BD Biosciences). PMA/ionomycin stimulated samples were taken along as technical/positive controls. The next day, cells were washed and incubated with the panels listed in the Key Resources Table. To facilitate intracellular cytokine staining, cells were permeabilized with Cytofix/Cytoperm (BD Biosciences) before addition of cytokine antibodies. After overnight fixation with 2% paraformaldehyde, 66 samples were acquired on a 3-laser, 14-color LSR-II flow cytometer (BD Biosciences). Analyses were performed in FlowJo version 10 (Treestar). T cells were selected as Singlets/Lymphocytes/Viable/CD14-CD20-/CD45+/CD3+ events, after which further CD4 and CD8 gating was applied. Any anomalies indicative of unstable signal acquisition were excluded using the ‘‘Time’’ parameter. Cytokine positivity was determined by placement of cytokine gates on the medium control samples and subsequently applying the gates to the corresponding PPD stimulated samples. Luminex Cytokine production by BAL cells stimulated for 72 hours with PPD (5 ug/mL, final concentration), was assessed by customised Milliplex Luminex kits (Merck Millipore, USA). Assays were performed according to manufacturer’s protocol. In short: supernatants of Cell Reports Medicine 2, 100187, January 19, 2021 e3 Article ll OPEN ACCESS stimulated BAL cells were incubated with beads coated with cytokine-specific antibodies. Bound cytokines were visualized using biotin-coupled detector antibodies and PE-labeled streptavidin. Beads were acquired on a Bioplex 200 system and cytokine levels were calculated with Bioplex Manager software version 6.1 (both Biorad, CA, USA). IFNgELISPOT Non-human primate specific IFNgELISPOT (U-CyTech, the Netherlands) was performed on PBMC according to manufacturer’s protocol on. Briefly, 200,000 PBMC were incubated in triplicate for 24 hours with Mtb-derived PPD (AJ Vaccines, Denmark) or recombinant ESAT6-CFP10 fusion protein (provided by Kees Franken from the Ottenhoff lab, Leiden University Medical Centre). The next day cells were washed and transferred to anti-IFNgcoated membrane plates (Millipore). After 24 hours, cells were discarded and membrane-bound IFNgwas visualized using a biotinylated anti-IFNgdetector antibody, streptavidin-horseradish peroxidase conjugate and tetramethylbenzidine substrate. Spots were quantified using an automated reader (AELVIS, Hannover). Immunoglobulin ELISA Antibody levels in BAL were determined by Enzyme Linked ImmunoSorbent Assay (ELISA). In brief, 96-well plates were coated with either 5 mg/mL Mtb strain HN828 Whole Cell lysate (BEI Resources, VA, USA) in PBS. After overnight blocking with 1% BSA, samples were added to the wells. Bound antibodies were subsequently detected either with horse radish peroxidase-conjugated anti-IgG (Rockland, PA, USA), alkaline phosphatase-conjugated anti-IgA (Fisher Scientific) or alkaline phosphatase-conjugated IgM (Sigma), and the subsequent addition of para-nitrophenylphosphate substrate for ELISA color development. All samples were normalized to arbitrary units (AU) against a serial dilution of a positive reference sample included in all assays. Mtb binding and phagocytosis assay To assess Mtb binding of immunoglobulins, 0.05 mL aliquots of BAL fluid obtained prior to and 8 weeks after vaccination, were incubated at 37C for 1 hour with 10 7 CFU of Mtb H37Rv-dsRed. (Korbee et al., 2018) Subsequently, samples were equally divided over three vials and biotinylated detection antibodies specific for IgA, IgG or IgM (all from Mabtech) were added (final dilution 1:500). After 30 minutes of incubation at room temperature, streptavidin-FITC (final dilution 1:400) was added, and samples were incubated for a further 60 minutes toward detection of bound antibody. Samples were fixed overnight with 2% PFA before analysis. For the phagocytosis assay, 5x10 5 THP1 cells were seeded in a 24-wells plate and activated by overnight incubation with 10ng/mL PMA at 37C. The next day, 10 7 CFU of Mtb H37Rv-GFP was incubated with BAL fluid as described above, and subsequently added to the activated THP-1 cells. After 4 hours cells were dissociated with trypsin-EDTA and resuspended in 2% PFA for overnight fixation. The binding assay samples were subsequently acquired on a 4 laser, FACSAriaIII system; samples from the phagocytosis assay were acquired on a 3 laser, Beckman Coulter Gallios system. For both assays, Mtb H37Rv-dsRed or -GFP incubated in PBS only was taken along as a negative control. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analysis was performed with Graphpad Prism version 8 and R version 3.5.1. 67 Significance of differences between groups was calculated by two-sided Mann-Whitney testing of which Holm’s adjusted p values are reported. Correlation statistics were generated by Spearman’s rank analysis. e4 Cell Reports Medicine 2, 100187, January 19, 2021 Article ll OPEN ACCESS