pharmaceutics Article Protective Efficacy of Inhaled BCG Vaccination Against Ultra-Low Dose Aerosol M. tuberculosis Challenge in Rhesus Macaques Andrew D. White 1, * , Charlotte Sarfas 1 , Laura S. Sibley 1 , Jennie Gullick 1 , Simon Clark 1 , Emma Rayner 1 , Fergus Gleeson 2 , Martí Català 3 , Isabel Nogueira 4 , Pere-Joan Cardona 5,6 , Cristina Vilaplana 5,6 , Mike J. Dennis 1 , Ann Williams 1 and Sally A. Sharpe 1 1 Public Health England, National Infection Service, Porton Down, Salisbury SP4 0JG, UK; charlotte.sarfas@phe.gov.uk (C.S.); laura.sibley@phe.gov.uk (L.S.S.); J.Gullick@soton.ac.uk (J.G.); simon.clark@phe.gov.uk (S.C.); emma.rayner@phe.gov.uk (E.R.); mike.dennis@phe.gov.uk (M.J.D.); ann.rawkins@phe.gov.uk (A.W.); sally.sharpe@phe.gov.uk (S.A.S.) 2 The Churchill Hospital, Headington, Oxford OX3 7LE, UK; fgleeson@icloud.com 3 Comparative Medicine and Bioimage Centre of Catalonia (CMCiB), Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Badalona, 08916 Catalonia, Spain; marticatalasabate@gmail.com 4 Servei de Radiodiagnòstic, Hospital Universitari Germans Trias i Pujol, Badalona, 08916 Catalonia, Spain; inogueiram@gmail.com 5 Unitat de Tuberculosi Experimental, Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Universitat Autònoma de Barcelona, CIBERES, 28029 Madrid, Spain; pj.cardona@gmail.com (P.-J.C.); cvilaplana@gmail.com (C.V.) 6 Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES). Av. Monforte de Lemos, 3-5. Pabellón 11. Planta 0. 28029 Madrid, Spain * Correspondence: Andrew.white@phe.gov.uk; Tel.: +44-198-061-2100  Received: 7 April 2020; Accepted: 22 April 2020; Published: 25 April 2020  Abstract: Ten million cases of tuberculosis (TB) were reported in 2018 with a further 1.5 million deaths attributed to the disease. Improved vaccination strategies are urgently required to tackle the ongoing global TB epidemic. In the absence of a validated correlate of protection, highly characterised pre-clinical models are required to assess the protective efficacy of new vaccination strategies. In this study, we demonstrate the application of a rhesus macaque ultra-low dose (ULD) aerosol M. tuberculosis challenge model for the evaluation of TB vaccination strategies by directly comparing the immunogenicity and efficacy of intradermal (ID) and aerosol BCG vaccination delivered using a portable vibrating mesh nebulizer (VMN). Aerosol- and ID-delivered Bacille Calmette-Guérin (BCG) induced comparable frequencies of IFN-γ spot forming units (SFU) measured in peripheral blood mononuclear cells (PBMCs) by ELISpot, although the induction of IFN-γ SFU was significantly delayed following aerosol immunisation. This delayed response was also apparent in an array of secreted pro-inflammatory and chemokine markers, as well as in the frequency of antigen-specific cytokine producing CD4 and CD8 T-cells measured by multi-parameter flow cytometry. Interrogation of antigen-specific memory T-cell phenotypes revealed that vaccination-induced CD4 and CD8 T-cell populations primarily occupied the central memory (TCM) and transitional effector memory (TransEM) phenotype, and that the frequency of CD8 TCM and TransEM populations was significantly higher in aerosol BCG-vaccinated animals in the week prior to M. tuberculosis infection. The total and lung pathology measured following M. tuberculosis challenge was significantly lower in vaccinated animals relative to the unvaccinated control group and pathology measured in extra-pulmonary tissues was significantly reduced in aerosol BCG-vaccinated animals, relative to the ID-immunised group. Similarly, significantly fewer viable M. tuberculosis CFU were recovered from the extra-pulmonary tissues of aerosol BCG-vaccinated macaques relative to unvaccinated animals. In this study, a rhesus macaque ULD M. tuberculosis aerosol challenge model was applied as a refined and sensitive system for the evaluation of TB vaccine efficacy and to confirm that aerosol BCG vaccination delivered by Pharmaceutics 2020, 12, 394; doi:10.3390/pharmaceutics12050394 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 394 2 of 25 portable VMN can confer a significant level of protection that is equivalent, and by some measures superior, to intradermal BCG vaccination. Keywords: tuberculosis; BCG; vaccine; non-human primate; macaque; aerosol vaccination; immunogenicity; T-cell memory 1. Introduction An estimated 10 million incident cases of Mycobacterium tuberculosis infection were reported in 2018, and 1.5 million deaths were attributed to tuberculosis (TB) disease [1]. Ten percent of new TB cases occurred in people with HIV and drug-resistant strains of M. tuberculosis were responsible for approximately 500,000 infections during this time; hence, there remains an urgent need for improved vaccination strategies to tackle the ongoing global TB epidemic. In the absence of a validated correlate of protection, highly characterised pre-clinical models are required to assess the protective efficacy of new vaccination strategies. The disease that develops in non-human primates (NHPs) following M. tuberculosis exposure closely resembles the tuberculous disease observed in humans, and the similarities between NHP and human anatomy, physiology, and immune response make primate models the most relevant for the assessment of TB vaccines [2–5]. However, the choice of M. tuberculosis challenge strain and dose can influence the level of disease that develops, posing the risk that promising new vaccines may be disregarded due to a failure to protect against a challenge inoculum that is unrepresentative of natural infection [4,5]. We have recently established an ultra-low dose (ULD) aerosol challenge system using the M. tuberculosis Erdman strain that produces consistent and measurable levels of disease in both rhesus and cynomolgus macaques [6]. In this study, we demonstrate the application of the ULD M. tuberculosis challenge model in rhesus macaques for the evaluation of vaccine efficacy and use the measurement of bacterial burden, qualitative gross pathology, histopathology and advanced in vivo imaging as readouts of vaccination mediated modulation of disease burden. The attenuated strain of Mycobacterium bovis, Bacille Calmette-Guérin (BCG), was first used in the 1920s and remains the only licensed prophylactic TB vaccine [1]. Delivered as an intradermal (ID) injection, BCG is known to reduce the occurrence of severe childhood forms of TB [7] but provides limited and geographically variable protection against the infectious pulmonary manifestation of TB disease in adults [8,9]. In addition to a portfolio of novel TB vaccine candidates that are progressing through clinical trials [1], alternative BCG delivery strategies such as revaccination [10,11] and vaccination via alternative delivery routes are currently under investigation [12]. The protection imparted by BCG vaccination against experimental M. tuberculosis infection has been shown to be enhanced when the vaccine is delivered by aerosol to mice [13], guinea pigs [14,15], and rhesus macaques [16]. Historically, the size and complexity of the apparatus required to generate and deliver viable bacterial aerosols in a controlled and consistent manner have hindered the deployment of aerosol BCG vaccination strategies. However, advances in nebuliser technology, and, specifically, the advent of portable vibrating mesh nebulisers (VMN) [17,18], has made the prospect of mass aerosol BCG vaccination campaigns a more practical proposition. We have previously demonstrated the safety of aerosolised BCG vaccination delivered using the Omron U22 VMN and the capacity of this approach to generate robust mucosal and systemic immune responses in a dose-dependent manner [19]. In this study, we directly compare the immunogenicity and efficacy of BCG vaccination delivered by aerosol or ID injection in the rhesus macaque ULD M. tuberculosis challenge model and demonstrate the value of the model as a platform for the assessment of novel TB vaccine candidates and strategies. Pharmaceutics 2020, 12, 394 3 of 25 Pharmaceutics 2020, 12, x 2 of 25 2.2. Materials Materials and and Methods Methods 2.1. 2.1. Experimental Experimental Animals Animals Nineteen Nineteenrhesus rhesusmacaques (Macaca mulatta) macaques (Macaca mulatta) of of Indian Indian origin origin aged aged between between 3.7 3.7 and and3.93.9years yearsofof age age were sourced from an established, closed UK breeding colony. Absence of previous exposureto were sourced from an established, closed UK breeding colony. Absence of previous exposure to mycobacterial antigens was confirmed by tuberculin skin test as part of colony mycobacterial antigens was confirmed by tuberculin skin test as part of colony management management procedures and by screening procedures and byforscreening IFN-γ ELISpot (MabTech, for IFN-γ ELISpot Nacka. Sweden) (MabTech, responses Nacka. Sweden) to tuberculin-PPD (purified responses to tuberculin- protein derivative) (SSI, Copenhagen, Denmark), and pooled 15-mer PPD (purified protein derivative) (SSI, Copenhagen, Denmark), and pooled 15-mer peptidespeptides of ESAT6 and CFP10of (Peptide Protein Research LTD, Fareham, UK). ESAT6 and CFP10 (Peptide Protein Research LTD, Fareham, UK). Compatible Compatiblesocialsocialgroups groups were housed were in accordance housed in accordancewith the withHome Office (UK) the Home OfficeCode (UK) of Practice Code of for the Housing and Care of Animals Used in Scientific Procedures (1989), and the Practice for the Housing and Care of Animals Used in Scientific Procedures (1989), and the National National Committee for Refinement, Committee forReduction and Replacement Refinement, Reduction (NC3Rs) Guidelines on(NC3Rs) and Replacement Primate Accommodation, Guidelines on Care and Primate Use, August 2006 (NC3Rs, 2006). Cages were approximately 2.5 m high Accommodation, Care and Use, August 2006 (NC3Rs, 2006). Cages were approximately 2.5 m high by 4 m long by 2 m deep, constructed by 4 m long with by 2 m high-level observation deep, constructed withbalconies andobservation high-level with a floorbalconies of deep and litterwith to allow foraging. a floor of deep Additional environmental litter to allow enrichment foraging. Additional was affordedenrichment environmental by the provision of toys, swings, was afforded feeding puzzles, by the provision of toys, and DVDs swings, for visual feeding stimulation. puzzles, and DVDs In for addition visualto standard old-world stimulation. In addition primate pellets, to standard diet was primate old-world further supplemented pellets, diet waswithfurther a selection of fresh vegetables supplemented with a and fruit. For selection each procedure, of fresh vegetablessedation and fruit.wasForapplied each by intramuscular injection with ketamine hydrochloride (10 mg/kg) (Ketaset, procedure, sedation was applied by intramuscular injection with ketamine hydrochloride (10 mg/kg) Fort Dodge Animal Health (Ketaset,Ltd, Southampton, Fort Dodge Animal UK). NoneLtd, Health of the animals hadUK). Southampton, beenNone usedofpreviously the animals forhad experimental been used procedures, previously for experimental procedures, and each socially compatible group was randomlytreatment. and each socially compatible group was randomly assigned to a particular study assigned The to astudy design particular andtreatment. study all procedures were approved The study design andbyallthe Public Health procedures were England, approvedPortonby the Down Public Animal Welfare and Ethical Review Body, and authorised under an appropriate Health England, Porton Down Animal Welfare and Ethical Review Body, and authorised under an UK Home Office project license. appropriate UK Home Office project license. The Theexperimental experimental design designand andschedule scheduleof ofprocedures, procedures,including includingthe theapplication applicationof ofvaccination vaccination and experimental challenge, are summarized and experimental challenge, are summarized in Figure 1. in Figure 1. Figure1.1.Study Figure Studytimeline timelinerelative relativeto toaerosol aerosolororIDIDBCG BCGvaccination. vaccination.Rhesus Rhesusmacaques macaquesreceived receivedBCG BCG vaccinationdelivered vaccination deliveredby aerosol(n(n==6)6)or byaerosol orintradermal injection(n(n==6) intradermalinjection 6)at atstudy studyweek weekzero zeroororwere wereleft left asunvaccinated as unvaccinatedcontrols controls(n(n== 7). 7). All animals received received ultra-low ultra-low dose dose (ULD) (ULD) aerosol aerosol challenge challenge with with M. M. tuberculosisErdman tuberculosis Erdmanstrain strain(ERD (ERD≤≤ 10 10 CFU) CFU) atat study study week week 2121and andwere weremonitored monitoredforforup upto to16 16weeks weeks followinginfection following infection(study (studyweek week37).37).Blue Blue shaded shaded circles circles represent representprocedures proceduresinvolving involvingblood bloodsample sample collectionand collection andapplication applicationof ofimmunological immunological analyses, analyses, large large open open circles circles represent represent key key study study events: events: vaccination vaccinationand andULD aerosolM. ULDaerosol M.tuberculosis tuberculosischallenge, challenge,open opencircles circlesindicate indicateapplication applicationofofin invivo vivoCT CT scanning. scanning.All animals All werewere animals euthanized, and post-mortem euthanized, (PM) necropsies and post-mortem conducted upon (PM) necropsies completion conducted upon of the study schedule completion (shaded of the study circles). schedule (shaded circles). 2.2. 2.2. Vaccination Vaccination BCG BCGvaccinations vaccinationswereweredelivered deliveredtotosedated sedatedanimals animalseither eitheras asaa100-µL 100-μLintradermal intradermal(ID) (ID)injection injection using using Danish strain 1331 (SSI, Copenhagen, Denmark) delivered to the upper left arm, or arm, Danish strain 1331 (SSI, Copenhagen, Denmark) delivered to the upper left or by by exposure exposure to aerosolised to aerosolised BCG Danish BCGstrain Danish strain 1331 1331 using created createdanusing Omron an MicroAir Omron MicroAir mesh nebuliser mesh nebuliser (Omron (Omron Healthcare UK Ltd., Milton Keynes, UK) and a modified paediatric anaesthesia mask.mask. Healthcare UK Ltd., Milton Keynes, UK) and a modified paediatric anaesthesia The The vaccination dose was selected to be equivalent to a standard adult intradermal dose, vaccination dose was selected to be equivalent to a standard adult intradermal dose, after taking into after taking into account account the expected the expected losses losses in viable in viable BCGBCGtitre titre associated associated withwith the aerosol the aerosol delivery delivery processprocess [19]. [19]. BCG BCG vaccine was prepared by adding 1 mL PBS to each vaccine vial to give an estimated vaccine was prepared by adding 1 mL PBS to each vaccine vial to give an estimated concentration of concentration 6 6 CFU/mL. Multiple vials were pooled together to ensure standardisation between 2 ×2 × of 1010 6 toto8 8× × 1010 6 CFU/mL. Multiple vials were pooled together to ensure standardisation between vaccinations, before delivering one ml of the BCG preparation to each animal. The BCG titre in aerosol and ID BCG vaccine preparations was confirmed to be within the range specified by the manufacturer Pharmaceutics 2020, 12, 394 4 of 25 vaccinations, before delivering one ml of the BCG preparation to each animal. The BCG titre in aerosol and ID BCG vaccine preparations was confirmed to be within the range specified by the manufacturer by bacterial culture on Middlebrook 7H11 selective agar containing oleic acid, bovine albumin, dextrose and catalase (OADC) (Biomerieux, Basingstoke, UK), for enumeration of viable colony forming units (CFU). 2.3. M. Tuberculosis Challenge Strain The Erdman K01 stock (HPA-Sept 2011) used for challenge was prepared from stocks of the M. tuberculosis Erdman strain K 01 (BEI Resources). A stock suspension was initially prepared from a 5-mL bacterial starter culture originally generated from colonies grown on Middlebrook 7H11 supplemented with oleic acid, albumin, dextrose and catalase (OADC) selective agar (BioMerieux, Basingstoke, UK). A liquid batch culture was then grown to logarithmic growth phase in 7H9 medium (Sigma-Aldrich, Gillingham, UK) supplemented with 0.05% (v/v) Tween 80 (Sigma-Aldrich, Gillingham, UK). Aliquots were stored at −80 ◦ C. The titre (viable bacilli per mL) of the stock suspension was determined from thawed aliquots by enumeration of colony-forming units following a culture of serial dilutions on Middlebrook 7H11 OADC selective agar. 2.4. Aerosol Exposure 2.4.1. Apparatus and Procedure The methodology and apparatus used to deliver M. tuberculosis via the aerosol route were as previously described [3,20,21]. In brief, aerosols were generated from a suspension of M. tuberculosis at a pre-determined concentration (see below) using a 3-jet Collison nebuliser (BGI, Butler, New Jersey, US) and delivered, using a modified Henderson apparatus [22] controlled by an AeroMP (Biaera Technologies, Hagerstown, Maryland, USA) control unit [23], to the nares of each sedated animal via a modified veterinary anaesthesia mask. A “head-out”, plethysmography chamber (Buxco, Wilmington, North Carolina, USA) enabled the aerosol to be delivered simultaneously with the measurement of the respiration rate. 2.4.2. Quantification of Ultra-Low Aerosol Dose The number of bacilli deposited and retained in the lungs of macaques cannot be measured directly, and, therefore, the dose is derived from a calculation based upon the concentration of viable organisms in the circulating aerosol and the volume of aerosol inhaled by the animal. This is termed the presented dose, and the derivation of this and the retained dose (the number of organisms assumed to be retained in the lung) have been described previously for high, medium and ultra-low aerosol doses [3,6,21]. Aerosol challenge data from previous experiments were used to predict the concentration of bacteria in the nebuliser required to result in a retained dose of approximately five viable bacilli [3,6,20,24]. 2.5. Computed Tomography (CT) Imaging CT scans were collected from animals using a 16-slice Lightspeed CT scanner (General Electric Healthcare, Milwaukee, WI, USA) at 3, 8, 11 and 16 weeks after aerosol exposure to M. tuberculosis (Figure 1), as described previously [25,26]. To enhance visualisation of lesions and lymph nodes, Niopam 300 (Bracco, Milan, Italy), a non-ionic, iodinated contrast medium, was administered intravenously (IV) at 2 mL/kg body weight. Scans were evaluated for the number and distribution of pulmonary lesions across lung lobes and the presence of nodule cavitation, conglomeration, consolidation as an indicator of alveolar pneumonia and a “tree-in–bud” pattern as an indicator of bronchocentric pneumonia. The lung-associated lymph nodes were assessed for enlargement and the presence of necrosis. Total lung volumes were calculated for each macaque using the COPD application of Philips IntelliSpace Portal software 9.0 (Phillips Healthcare, UK). Tuberculous lesions were identified and categorised as micronodules (spherical lesions smaller than 0.3 cm3 ) or regions of consolidation Pharmaceutics 2020, 12, 394 5 of 25 (pneumonic TB lesions bigger than 0.3 cm3 ). The volume of the consolidated regions was obtained using the Philips IntelliSpace Portal, Tumor Tracking application, and micronodule diameter was approximated as 1, 2, 3 or 4 mm. The total volume of damaged tissue was quantified by summing consolidated and micronodule volumes, and this was expressed as a percentage of the total lung volume measured for each macaque. 2.6. Clinical Assessment Animals were sedated at two weekly intervals for blood sample collection and to measure body weight and temperature, red blood cell (RBC) haemoglobin levels and erythrocyte sedimentation rate (ESR). RBC haemoglobin levels monitored as an indication of anaemia, measured using a HaemaCue haemoglobinometer (Haemacue Ltd., Dronfield, UK). ESR was monitored using the Sediplast system (Guest Medical, Edenbridge, UK) as a general measure of M. tuberculosis-induced inflammation. Animal behaviour was observed throughout the study for contra-indicators, and the time of necropsy, if prior to the end of the planned study period, was determined by experienced primatology staff based on a combination of the following adverse indicators: depression or withdrawn behaviour, abnormal respiration (dyspnoea), loss of 20% of peak post-challenge weight, ESR levels elevated above normal (>20 mm), haemoglobin level below normal limits (<100 g/dL), increased temperature (>41 ◦ C) and abnormal thoracic radiograph. 2.7. Immune Response Analysis 2.7.1. Interferon-Gamma (IFN-γ) ELISpot Interferon-gamma (IFN-γ) ELISpot assays were performed on peripheral blood mononuclear cells isolated from heparin anti-coagulated blood using standard methods, as previously described [24]. 2.7.2. Quantification of Secreted Biomarkers in Mycobacterial Antigen Stimulated Blood Cultures Antigen-specific secretion of a range of cytokines, chemokines and growth factors was assessed by dilution of heparinised blood samples 1:10 with serum-free Roswell Park Memorial Institute (RPMI; R0) medium, before six day culture at 37 ◦ C, 5% CO2 , with 10 µg/mL tuberculin PPD (Statens Serum Institute, Copenhagen, Denmark), or 10 µg/mL phytohaemmagglutinin (PHA) from Phaseolus vulgaris (Sigma Aldrich, Gillingham, UK) as a positive control, or R0 alone as a negative control [27]. Following incubation, culture supernatants were aspirated using a 1-mL syringe (BD Biosciences, Oxford, UK) and passed through two 0.2-µm PES filters (GE Life Sciences, Amersham, UK), before storage at −80 ◦ C. Secreted biomarkers in filtered culture supernatants were quantified using a 37-plex Procartaplex bead array assay (Thermo Fischer Scientific, Loughborough, UK), applied according to the manufacturer’s instructions, for detection of the following analytes: Eotaxin, G-CSF, GM-CSF, IFN-alpha, IFN-gamma, TNF-alpha, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-18, IL-1b, IL-1RA, IL-2, IL-23, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, I-TAC (CXCL11), MCP-1 (CCL2), MIP-1alpha, MIP-1beta (CCL4), SDF-1alpha (CXCL12), MIG (CXCL9), CD40-Ligand, BLC (CXCL13), BDNF, SCF, VEGF-D, bNGF, FGF-2, PDGF-BB, VEGF-A. Culture filtrates were analysed using a Luminex Magpix instrument (Luminex Corporation, Hertogenbosch, Netherlands) equipped with the xPONENT 4.2 software package. Standard curves were generated in duplicate and used to interpolate the concentration of each analyte. All data below the limit of detection specified by the kit manufacturer was assigned a zero value. The analyte concentration measured in negative control cultures was subtracted from antigen-stimulated samples to obtain a measure of antigen-specific biomarker secretion, and the analyte concentration was multiplied by ten to account for the initial dilution of the sample and provide a value per ml of blood. Pharmaceutics 2020, 12, 394 6 of 25 2.8. Intracellular Cytokine Staining and Memory T-cell Analysis 2.8.1. Polyfunctional Intracellular Cytokine Staining and Antigen-Specific Memory T-cell Assay Intracellular cytokine staining (ICS) was performed using 1 × 106 PBMC in medium (R10) consisting of RPMI 1640 supplemented with L-glutamine (2 mM), penicillin (50 U/mL) and streptomycin (50 µg/mL) (all from Sigma Aldrich, Gillingham, UK) and 10% heat-inactivated foetal bovine serum (Labtech Ltd., Uckfield, UK). These cells were stimulated with a 10-µg/mL solution of CD28 and CD49d co-stimulatory antibodies (both from BD Biosciences, Oxford, UK) and either 10 µg/mL PPD (SSI, Copenhagen, Denmark), or 5 µg/mL staphylococcal enterotoxin b (SEB) (Sigma Aldrich, Gillingham, UK), or R10 medium alone as a negative control for a total of six hours at 37 ◦ C in a 5% CO2 supplemented incubator. Following the initial two hours of incubation, the protein transport inhibitor Brefeldin-A (Sigma Aldrich, Gillingham, UK) was added to the incubation mixture at a final concentration of 10 µg/mL. Following incubation, cells were washed with FACS buffer consisting of PBS + 1% FCS and incubated for 30 min at room temperature with optimal dilutions of the amine-reactive Live/Dead Fixable Red viability cell stain (Life Technologies, Renfrew, UK) and the antibodies CD4 APC-H7, CD8 PerCP-Cy5.5, CD95 Pe-Cy7 (all from BD Biosciences, Oxford, UK), CD28 BV-421, CD45RA-PeCy7 (Biolegend, London, UK), CCR7-PE (eBioscience, Hatfield, UK), CD14-ECD and CD20-ECD (both from Beckman Coutler, High Wycombe, UK). Following surface marker staining, the cells were washed and then permeabilised by incubation at room temperature for 15 min with Fix/Perm reagent (BD Biosciences, Oxford, UK). Further cell washes were applied using Permwash buffer (BD Biosciences, Oxford, UK), before staining for intracellular antigens by incubation at room temperature for 30 min with the antibodies CD3-AF700, IFN-γ-FITC, TNF-α-BUV395 (all from BD Biosciences, Oxford, UK), IL-2-APC (Miltenyi Biotech Ltd., Bisley, UK), IL-17-BV711 (Biolegend, London, UK). BD Compbeads (BD Biosciences, Oxford, UK) were labelled with the above fluorochromes for use as compensation controls. Following antibody labelling, cells and beads were washed by centrifugation and fixed in 4% paraformaldehyde solution (Sigma Aldrich, Gillingham, UK) prior to flow cytometric acquisition. 2.8.2. Flow Cytometric Acquisition and Analysis Cells were analysed using a five laser LSRII Fortessa instrument (BD Biosciences, Oxford, UK), and data were analysed using FlowJo (version 9.7.6, Treestar, Ashland, US). Cytokine-producing T-cells were identified using a forward scatter-height (FSC-H) versus side scatter-area (SSC-A) dot plot to identify the lymphocyte population, to which appropriate gating strategies were applied to exclude doublet events, non-viable cells, monocytes (CD14+ ) and B cells (CD20+ ). For ICS analysis, sequential gating through CD3+ , followed by CD4+ or CD8+ gates, were used before individual cytokine gates to identify IFN-γ-, IL-2-, TNF-α- and IL-17-producing populations. Polyfunctional cells were identified using Boolean gating combinations of individual cytokine-producing CD4 or CD8 T-cells. Antigen-specific T-cell memory profiles were identified by applying a summed CD4 or CD8 cytokine Boolean combination, followed by gating for CD95 surface staining. Differentiation of effector, transitional effector, central memory, and stem cell memory T-cell populations was established by CD45RA, CD28 and CCR7 expression patterns (Section 3.9.3). The software package PESTLE version 1.7 (Mario Roederer, Vaccine Research Centre, NIAID, NIH) was used for background subtraction to obtain antigen-specific polyfunctional ICS and memory T-cell cytokine responses. Graphpad Prism (version 8.0.1) was used to generate graphical representations of flow cytometry data. 2.9. Necropsy Animals were anaesthetised and clinical data collected. Blood samples were taken prior to euthanasia by intracardiac injection of a lethal dose of anaesthetic (Dolelethal, Vétoquinol UK Ltd., Towcester, UK 140 mg/kg). A post-mortem examination was performed immediately and gross pathological changes were scored using an established system based on the number and extent of lesions present in the lungs, spleen, liver, kidney and lymph nodes, as described previously [3]. Samples Pharmaceutics 2020, 12, 394 7 of 25 of spleen, liver, kidneys and tracheobronchial, inguinal and axillary lymph nodes were removed and sampled for quantitative bacteriology. The lungs, including the heart and lung-associated lymph nodes, were removed intact. The lymph nodes were measured and examined for lesions. The whole lung was fixed by intra-tracheal infusion with 10% neutral buffered formalin (NBF) using a syringe and 13CH Nelaton catheter (J.A.K. Marketing, York, UK). To ensure complete and rapid fixation of the tissue, the catheter tip was inserted through the trachea into each main-stem bronchus in turn and lobes on each side were infused until they were expanded to a size consistent with normal inspiratory dimensions; the trachea was then ligated to retain the fluid. The infused lung was immersed in 10% NBF. In addition, samples of kidneys, liver, spleen, and sub-clavicular, hepatic inguinal and axillary lymph nodes were fixed in 10% NBF. 2.10. Pathology Studies The fixed lungs were sliced serially, and each lung lobe evaluated separately and scored based on the number and extent of lesions present by application of the pathology scoring system applied at necropsy [25]. Discrete lesions were counted in the parenchyma, and where lesions had coalesced, these were measured and recorded. Areas of consolidation in each lobe were recorded and quantified. The remaining tissues were examined during trimming. 2.11. Histological Examination Representative samples from each lung lobe and other organs were processed to paraffin wax, sectioned at 3–5 µm and stained with haematoxylin and eosin (HE). For each lung lobe, tissue slices containing macroscopically visible lesions were chosen for histological examination. Where gross lesions were not visible, a sample was taken from a pre-defined anatomical location from each lobe to establish consistency between animals. The nature and severity of the microscopic tuberculous lesions were evaluated subjectively by a pathologist who was blinded to prevent bias, and lesions were graded according to organisation state and severity as previously described [28]. Furthermore, additional features of disease were recorded as present or absent; these included multi-nucleated giant cells, airway invasion, fulminating pneumonia (the latter defined as representing inflammatory changes with the parenchyma which extended between granulomas) and the presence of lesions that appeared to originate directly from within the bronchus-associated lymphoid tissue (BALT). 2.12. Bacteriology The spleen, kidneys, liver and tracheobronchial lymph nodes were sampled for the presence of viable M. tuberculosis post-mortem [20]. Where available, tissue sections with and without visible tuberculous lesions were collected for analysis. Weighed tissue samples were homogenised in 2 mL of sterile water, and either serially diluted in sterile water prior to being plated, or plated without dilution onto Middlebrook 7H11 OADC selective agar. Plates were incubated for three weeks at 37 ◦ C, and resultant colonies were confirmed as M. tuberculosis and counted. Mean colony-forming units (CFU) per gram from each tissue sample were determined. 2.13. Statistical Analyses Differences in measures of the immune response, including IFN-γ ELISpot and biomarker secretion profiles, T-cell functional and memory population frequencies measured by polyfunctional flow cytometry, as well as pathology scores, pulmonary disease measures and clinical measures of disease burden recorded at the end of the study, were conducted using the non-parametric Mann–Whitney U-test function in GraphPad Prism version 6.05 (GraphPad Software Inc, La Jolla, California, USA). Similarly, GraphPad was used to calculate the area under the curve (AUC) of each animal’s response for further comparison of the cell-mediated response measured by IFN-γ ELISpot between the groups. Negative values in antigen-specific ICS and IFN-γ ELISpot data generated Pharmaceutics 2020, 12, 394 8 of 25 by background subtraction were replaced by a zero value [29]. Differences in the rate of disease progression (survival rates) of animals in each test group were compared with a Mantel–Cox log rank test and the Gehan–Breslow–Wilcoxon test in GraphPad Prism, version 6.05. 3. Results 3.1. Safety of ID and Aerosol Delivered BCG In agreement with previous reports [3,19], BCG vaccinations were well tolerated by all animals. Mild local reactions, including induration and erythema at the site of immunization, were observed following ID vaccination; adverse indicators were not apparent in animals that received the aerosol BCG vaccination regimen. Clinical measures, including body weight, temperature, peripheral lymph node size, red cell haemoglobin concentration (Hb), and erythrocyte sedimentation rate, remained within normal ranges following aerosol or ID BCG vaccination. 3.2. M. Tuberculosis Challenge Dose and Disease Progression Animals were challenged with a median estimated retained dose of three CFU aerosolised M. tuberculosis Erdman Strain K01 (Table 1). Changes in clinical parameters, including weight loss and perturbations in temperature and Hb concentration, were observed across all vaccination groups within four weeks of challenge. Four animals were euthanised prior to completion of the 16-week post-M. tuberculosis challenge schedule due to weight and behavioural changes indicative of progressive disease which met humane endpoint criteria; these included three unvaccinated control animals and one aerosol BCG-vaccinated animal (Figure 2). One ID BCG-vaccinated animal was removed from the study three weeks following M. tuberculosis challenge due to a non-disease related injury. Clinical and disease parameters recorded from this animal were excluded from the analysis to avoid biasing of efficacy readouts. The time at which animals progressed to meet predefined humane end-point criteria was compared between the vaccination groups using a log-rank or Gehan–Breslow–Wilcoxon test (Figure 2); these analyses did not indicate significant differences between the vaccination regimens (p = 0.18 and 0.20). Table 1. Aerosol challenge doses of M. tuberculosis delivered to rhesus macaques. Vaccine Animal Identification Presented Dose Estimated Retained Group and Route Number (cfu) Dose (cfu) U12 23 3 U26 22 3 U41 23 3 Group A BCG Aerosol U46 25 4 U58 23 3 U66 25 4 U18 22 3 U28 27 4 Group B BCG ID U31 21 3 U56 24 3 U72 23 3 U15 19 3 U22 18 3 U36 23 3 Group C No vaccine U49 23 3 U57 26 4 U60 23 3 U73 21 3 Study median 23 3 Pharmaceutics 2020, Pharmaceutics 12,12, 2020, 394x 9 8of of 25 25 Disease progression (%) Figure 2. Kaplan–Meier plot indicating proportions of vaccinated and unvaccinated non-human Figure 2. Kaplan–Meier plot indicating proportions of vaccinated and unvaccinated non-human primates (NHPs) able to control disease progression following ultra-low dose aerosol challenge with primates (NHPs) able to control disease progression following ultra-low dose aerosol challenge with M. tuberculosis. M. tuberculosis. 3.3. In Life CT Evaluation of Disease Pathology 3.3. In Life CT Evaluation of Disease Pathology CT imaging was applied at regular intervals following exposure to low dose aerosols of CT imaging M. tuberculosis wasmonitoring for the applied at of regular diseaseintervals burdenfollowing exposure and assessment of to low dose features aerosols ofofM. characteristic TBtuberculosis for the monitoring disease pathology. Three weeks ofafter disease burden aerosol and assessment challenge of featuresmore with M. tuberculosis, characteristic nodules wereof TB disease pathology. Three weeks after aerosol challenge with M. tuberculosis, counted in the lungs of unvaccinated macaques (group median 12) in comparison to the groups that more nodules were counted received BCGin the lungs of unvaccinated intradermally (median 8) or macaques by aerosol(group (medianmedian 12) in3A). 7) (Figure comparison to theofgroups A comparison that the early lesions quantified in unvaccinated animals to the reduced number of lesions measured in the vaccinatedthe received BCG intradermally (median 8) or by aerosol (median 7) (Figure 3A). A comparison of early islesions groups quantified indicative in unvaccinated of an initial vaccine effectanimals to the in both the reduced aerosol- number of lesions and ID-vaccinated measured animals. in the This initial vaccinated groups vaccine-induced is indicative reduction of an of disease wasinitial vaccine effect also apparent whenin theboth the aerosol- volume and ID-vaccinated of lung tissue affected by animals. This initial vaccine-induced reduction of disease was also apparent when tuberculous pathology was quantified in relation to total pulmonary volume. This revealed a significantthe volume of lung tissue affected by tuberculous pathology was quantified in relation to total pulmonary reduction in the proportion of the lung affected by the disease in the ID and aerosol BCG-vaccinated volume. This revealed groups three a significant weeks afterreduction in the infection M. tuberculosis proportion of the3B,C). (Figure lung affected by the disease The occurrence in the of further ID and features aerosol BCG-vaccinated groups three weeks after M. tuberculosis infection typical of M. tuberculosis-related pulmonary disease, such as areas of pneumonia associated with the (Figure 3B,C). The occurrence lung parenchyma of further features typical (consolidation) of M. tuberculosis-related and airways pulmonary (tree-in-bud morphology), weredisease, apparentsuchinas areas of animals pneumonia associated with the lung parenchyma (consolidation) and airways from all treatment groups from three weeks post-M. tuberculosis, progressing to include cavitation from (tree-in-bud morphology), seven weeks afterwere apparent exposure M. tuberculosis in animals from3A). (Figure all treatment groups from three weeks post-M. tuberculosis, progressing to include cavitation from seven weeks after M. tuberculosis exposure (Figure 3.4. Disease Pathology Recorded in Pulmonary and Extra-Pulmonary Tissues 3A). The extent of tuberculosis disease pathology was recorded at necropsy in a range of pulmonary, lymphoid and extra-thoracic organs using a scoring system based on the number of lesions and extent of disease [20]. Lung pathology was assessed in formalin-fixed tissue sections prepared from each lung lobe. Total pathology scores were established by summing scores recorded in each tissue, whereas pulmonary and dissemination scores were derived from summed lung lobe scores or summed scores recorded from extra-thoracic tissues excluding lymph nodes (Figure 4A–F). A comparison of group median total pathology scores revealed a significant reduction in tuberculosis pathology in both aerosol and ID BCG vaccination groups relative to the unvaccinated control group (p ≤ 0.017). Lung pathology was also reduced, reaching significance in ID BCG-vaccinated animals (p = 0.005), and disease pathology in extra-thoracic tissues was significantly lower in aerosol-vaccinated animals relative to both unvaccinated (p = 0.005) and ID BCG-vaccinated animals (p = 0.02), indicating that aerosol BCG vaccination had reduced the occurrence of M. tuberculosis-attributable disease pathology in sites typically associated with extrapulmonary M. tuberculosis dissemination. Pharmaceutics 2020, 12, 394 10 of 25 Pharmaceutics 2020, 12, x 9 of 25 Figure 3. Figure 3. Development Development of of thoracic thoracic pathology pathology measured measured by by computed tomography (CT) computed tomography X-ray (CT) X-ray scanning at regular intervals following low dose aerosol challenge with M. tuberculosis. scanning at regular intervals following low dose aerosol challenge with M. tuberculosis. (A) Pulmonary (A) Pulmonary nodule counts recorded from thoracic CT scans of individual animals nodule counts recorded from thoracic CT scans of individual animals at weeks 3, 7, 11 and 16 at weeks 3, 7, 11 and 16 following following aerosolaerosol challenge challenge with M. withtuberculosis M. tuberculosis are shown; are shown; UMUM = unmeasurabledue = unmeasurable duetoto lesion lesion conglomerationand conglomeration andlung lung lobe lobe consolidation. consolidation. Colour Colour gradients gradients in heatinmap heatplots maprepresent plots represent the presencethe presence or absenceor absencepulmonary of typical of typical pulmonary features featuresdisease, of tuberculous of tuberculous disease,conglomeration including nodule including nodule and conglomeration cavitation, and cavitation, consolidation consolidation and tree-in-bud and tree-in-bud morphology morphology due to pulmonary due to and inflammation, pulmonary lymph node enlargement or necrosis. (B,C) Relative volume of tuberculous lung lesions quantified bylung inflammation, and lymph node enlargement or necrosis. (B,C) Relative volume of tuberculous CT lesions quantified analysis. Data pointsbyshow CT analysis. Data area the percentage points show thelung of damaged percentage area of damaged tissue measured lung in individual tissue animals, measured with in individual group median, animals, interquartile range with and group minimum median, interquartile and maximum valuesrange shownand minimum by box and whiskerand maximum plots. valuesdenote Asterisks shownsignificant by box anddifferences whisker plots. Asterisks measured denote within significant (Wilcoxon, differences Figure B) andmeasured between within (Wilcoxon, treatment Figure B) and between groups (Mann–Whitney, Figure treatment groups (Mann–Whitney, Figure C), p ≤ 0.05. C), * p ≤ 0.05. 3.5. Histopathology 3.4. Disease pathology recorded in pulmonary and extra-pulmonary tissues. The occurrence The extent of granulomatous, of tuberculosis microscopic disease pathology waslesions recordedin at tissues examined necropsy is summarised in a range of pulmonary, in Table 2. In the lung, a range of lesion types was present in each animal from lymphoid and extra-thoracic organs using a scoring system based on the number of lesions and extentall groups (Figure 5A–C), with the most of disease [20].frequently observed Lung pathology was lesions being assessed in types 4 to 6. There formalin-fixed didsections tissue not appear to be prominent prepared from each differences in either lesion type or their frequency of occurrence lung lobe. Total pathology scores were established by summing scores recorded in each between the groups. The presence tissue, of lesions appearing to originate in BALT was observed with similar whereas pulmonary and dissemination scores were derived from summed lung lobe scores frequency between all groupsor (Figure 5D); this was also true for the presence of multi-nucleated giant summed scores recorded from extra-thoracic tissues excluding lymph nodes (Figure 4A–F). A cells within lesions. Fulminating pneumonia comparison(Figure of group5E)median was notedtotalinpathology a proportion of animals scores revealedin all groups, and a significant features in reduction such as airway tuberculosis invasion and cavitation were noted in all groups but with a greater frequency pathology in both aerosol and ID BCG vaccination groups relative to the unvaccinated control in unvaccinated animals. group In addition, the local spread of granulomatous lesions to the pleura covering (p ≤ 0.017). Lung pathology was also reduced, reaching significance in ID BCG-vaccinated animals (p the rib cage was present in all animals = 0.005), of the pathology and disease unvaccinated group, compared in extra-thoracic tissues towas three aerosol BCG-vaccinated significantly and three ID lower in aerosol-vaccinated BCG-vaccinated animals relative to animals. The lung-associated both unvaccinated (p = 0.005)lymph and IDnodes of all animals BCG-vaccinated in all groups animals were (p = 0.02), similar, indicating with large areas of parenchyma replaced by granulomatous inflammation that aerosol BCG vaccination had reduced the occurrence of M. tuberculosis-attributable disease including widespread caseous pathology necrosis in sites(Figure 5F),associated typically whereas, microscopic evidence ofM. with extrapulmonary tuberculous tuberculosis pathology that was present dissemination. in the peripheral lymph nodes (axillary, inguinal, hepatic and sub-clavicular) of unvaccinated and ID BCG-vaccinated animals, was not observed in the aerosol-vaccinated group. Pharmaceutics 2020, 12, 394 11 of 25 Pharmaceutics 2020, 12, x 10 of 25 Total pathology score U15 U22 U36 U49 U57 U60 U73 U12 U26 U41 U46 U58 U66 U18 U28 U31 U56 U72 Lung pathology score U15 U22 U36 U49 U57 U60 U73 U12 U26 U41 U46 U58 U66 U18 U28 U31 U56 U72 20 p = 0.005 p = 0.02 pathology score Extra-thoracic 15 10 5 0 U15 U22 U36 U49 U57 U60 U73 U12 U26 U41 U46 U58 U66 U18 U28 U31 U56 U72 ol ID ol os tr on er C A Figure 4. Gross-pathology assessed at necropsy. The occurrence and extent of tuberculous disease Figure 4. Gross-pathology assessed at necropsy. The occurrence and extent of tuberculous disease pathology were recorded at necropsy using a standardised scoring system. This approach was used pathology were recorded at necropsy using a standardised scoring system. This approach was used to determine total pathology scores from a range of pulmonary and extra-pulmonary tissues (A,B), to determine total pathology scores from a range of pulmonary and extra-pulmonary tissues (A,B), lung pathology derived from scores assessed in each lung lobe (C,D), and an extra-thoracic score lung pathology derived from scores assessed in each lung lobe (C,D), and an extra-thoracic score comprised of scores recorded from tissues typically associated with bacterial dissemination (E,F). comprised of scores recorded from tissues typically associated with bacterial dissemination (E,F). Plots A, C and E show scores recorded from specific tissues in each individual animal, whereas plots B, Plots A, C and E show scores recorded from specific tissues in each individual animal, whereas plots D and F display summed tissue scores with group median values indicated. Animals that met humane B, D and F display summed tissue scores with group median values indicated. Animals that met end-point criteria are indicated with red data points. Significant differences between groups determined humane by end-pointU-test Mann–Whitney criteriaare areshown. indicated with red data points. Significant differences between groups determined by Mann–Whitney U-test are shown. 3.5. Histopathology The occurrence of granulomatous, microscopic lesions in tissues examined is summarised in Table 2. In the lung, a range of lesion types was present in each animal from all groups (Figure 5A– C), with the most frequently observed lesions being types 4 to 6. There did not appear to be prominent differences in either lesion type or their frequency of occurrence between the groups. The presence of lesions appearing to originate in BALT was observed with similar frequency between all groups (Figure 5D); this was also true for the presence of multi-nucleated giant cells within lesions. Fulminating pneumonia (Figure 5E) was noted in a proportion of animals in all groups, and features Pharmaceutics 2020, 12, 394 12 of 25 Table 2. Occurrence of microscopic changes associated with M. tuberculosis infection. Pulmonary Extra-Pulmonary Tissues Lymph Nodes Vaccine Bronchus Associated Multi-Nucleated Lung Group and Route Animal ID Lung Lesion Type Lymphoid Tissue Spleen Liver Kidney Peripheral Giant Cells Associated (1–6) Involvement (n of 7 Lung Lobes) Present (+) or Absent (-) U12 1, 2, 4, 5 0 5 - - - + - U26 1, 2, 4, 5, 6 0 6 + + - + - U41 1, 2, 4, 5, 6 0 7 + + + + - Group A BCG Aerosol U46 1, 4, 5, 6 1 7 - - - + - U58 1, 2, 4, 5 3 7 - - - + - U66 2, 3, 4, 5, 6 0 6 - + + + - U18 1, 2, 4, 5, 6 1 5 - + + + + U28 1, 2, 3, 4, 5, 6 3 7 + + + + - BCG Group B U31 1, 2, 4, 5, 6 1 6 + + + + + ID U56 1, 4, 5, 6 0 3 + + - + + U72 1, 4, 5, 6 0 4 + + - + - U15 1, 4, 5, 6 0 5 + + + + + U22 1, 2, 4, 5, 6 0 6 + + + + + U36 1, 2, 4, 5, 6 1 6 + + + + - No Group C U49 1, 2, 3, 4, 5, 6 0 7 + + + + - vaccine U57 4, 5, 6 0 4 - - + + + U60 1, 2, 4, 5, 6 0 5 + + + + + U73 1, 4, 5, 6 0 7 + + + + + vaccinated and three ID BCG-vaccinated animals. The lung-associated lymph nodes of all animals in all groups were similar, with large areas of parenchyma replaced by granulomatous inflammation including widespread caseous necrosis (Figure 5F), whereas, microscopic evidence of tuberculous pathology that was present in the peripheral lymph nodes (axillary, inguinal, hepatic and sub- Pharmaceutics of clavicular) 2020, 12, 394 unvaccinated 13 of 25 and ID BCG-vaccinated animals, was not observed in the aerosol- vaccinated group. Figure Figure5.5.Microscopic Microscopic changes changes in the lung in the lungandandassociated associatedlymph lymphnode. node.(A) (A)Lung, Lung,Animal Animal U41 U41 (Group (Group A). A). (B) Lung, Animal U18 (Group B). (C) Lung, Animal U36 (Group C). Multifocal, tuberculous (B) Lung, Animal U18 (Group B). (C) Lung, Animal U36 (Group C). Multifocal, tuberculous lesions lesions of of varying varying types types scattered scattered throughout throughout the parenchyma. the parenchyma. (HE, (HE, 2× objective). 2× objective). (D) Lung, (D) Lung, AnimalAnimal U58 U58 (Group (Group A). Bronchus-associated A). Bronchus-associated lymphoid lymphoid tissue tissue invaded invaded bygranuloma, by tuberculous tuberculouswithgranuloma, with a peripheral a rim of peripheral rim of lymphoid tissue remaining (arrows) (HE, 10× objective). (E) Lung, Animal lymphoid tissue remaining (arrows) (HE, 10× objective). (E) Lung, Animal U46 (Group A). Fulminating U46 (Group A). Fulminating pneumonia. pneumonia. (HE, 5× objective). (HE, 5× objective). (F) Lung-associated (F) Lung-associated lymph node, Animal U26 lymph node, (Group A).Animal U26 Effacement (Group A). Effacement of the lymphoid tissue by caseous necrosis and granulomatous of the lymphoid tissue by caseous necrosis and granulomatous inflammation. (HE, 2× objective). inflammation. (HE, 2× objective). 3.6. Viable M. Tuberculosis Recovered from Extra-Pulmonary Tissues Tissue samples were collected from a range of extra-pulmonary tissues, including the spleen, kidneys, liver and tracheobronchial (hilar) lymph nodes, for the quantification of viable M. tuberculosis by bacterial culture (Figure 6). Extra-pulmonary dissemination of M. tuberculosis was detected in all animals with an equivalent bacterial load measured in the tracheobronchial lymph nodes, a primary site of dissemination from the lung, regardless of vaccination status (Figure 6D). However, significant differences in the quantity of viable bacteria recovered from extra-thoracic tissues were observed Tissue samples were collected from a range of extra-pulmonary tissues, including the spleen, kidneys, liver and tracheobronchial (hilar) lymph nodes, for the quantification of viable M. tuberculosis by bacterial culture (Figure 6). Extra-pulmonary dissemination of M. tuberculosis was detected in all animals with an equivalent bacterial load measured in the tracheobronchial lymph nodes, a primary site of dissemination from the lung, regardless of vaccination status (Figure 6D). Pharmaceutics 2020, 12, 394 14 of 25 However, significant differences in the quantity of viable bacteria recovered from extra-thoracic tissues were observed between the treatment groups, with M. tuberculosis CFU/g of spleen, liver and kidneys betweensignificantly the treatment lower in aerosol groups, with M.BCG-vaccinated animals tuberculosis CFU/g in comparison of spleen, to the unvaccinated liver and kidneys significantly group. lower In contrast,BCG-vaccinated in aerosol the bacterial burden animalsmeasured in tissue in comparison to samples collected from the unvaccinated group.theInIDcontrast, BCG- vaccinated group did not differ significantly from that measured in unvaccinated animals the bacterial burden measured in tissue samples collected from the ID BCG-vaccinated group did not (Figure 6B–D). differ significantly from that measured in unvaccinated animals (Figure 6B–D). Viable M.tuberculosis (Log10 CFU/g tissue) Viable M.tuberculosis (Log10 CFU/g tissue) ol ID ol ol ol ID tr os os tr on on er er C C A A Viable M.tuberculosis (Log10 CFU/g tissue) ol ID ol tr os on er C A Figure 6. Viable M. tuberculosis recovered from Hilar lymph nodes and extra-thoracic tissues. Figure 6. Viable M. tuberculosis recovered from Hilar lymph nodes and extra-thoracic tissues. Representative tissue samples collected from spleen (A), kidney (B), liver (C), and tracheobronchial Representative tissue samples collected from spleen (A), kidney (B), liver (C), and tracheobronchial lymph nodes (D) were homogenised and cultured on selective agarose for assessment of M. tuberculosis lymph nodes (D) were homogenised and cultured on selective agarose for assessment of M. bacterial load in extrapulmonary and extra-thoracic tissues. Individual points represent log10 CFU/g tuberculosis bacterial load in extrapulmonary and extra-thoracic tissues. Individual points represent tissue recovered from each animal with the group median indicated by horizontal bar. Animals in log 10CFU/g tissue recovered from each animal with the group median indicated by horizontal bar. which disease met humane end-point criteria are indicated with red data points. Significant differences Animals between in which groups disease met determined humane end-point by Mann–Whitney U-testcriteria are indicated with red data points. are shown. Significant differences between groups determined by Mann–Whitney U-test are shown. 3.7. Frequency of Antigen-Specific IFN-γ Secreting Cells Measured by ELISpot Systemic immune responses induced by aerosol or ID BCG vaccination and ULD M. tuberculosis challenge were profiled using an ex vivo IFN-γ ELISpot assay. Vaccination-induced PPD-specific IFN-γ spot forming units (SFU) were detected four weeks following ID and six weeks after aerosol BCG vaccination (Figure 7), indicating delayed initiation of the systemic adaptive immune response following aerosol vaccination, as has previously been described [19]. Comparison of IFN-γ SFU’s between vaccination groups revealed that the frequency of IFN-γ secreting cells was significantly lower in the aerosol BCG-vaccinated group at the four-week time point (p = 0.009; Figure 7C). Nevertheless, challenge were profiled using an ex vivo IFN-γ ELISpot assay. Vaccination-induced PPD-specific IFN-γ spot forming units (SFU) were detected four weeks following ID and six weeks after aerosol BCG vaccination (Figure 7), indicating delayed initiation of the systemic adaptive immune response following aerosol vaccination, as has previously been described [19]. Comparison of IFN-γ SFU’s Pharmaceutics 2020, 12, 394 15 of 25 between vaccination groups revealed that the frequency of IFN-γ secreting cells was significantly lower in the aerosol BCG-vaccinated group at the four-week time point (p = 0.009; Figure 7C). Nevertheless, IFN-γ IFN-γ SFU SFU frequencies frequencies peaked peaked levels at comparable at comparable ten weekslevels ten weeks post-vaccination post-vaccination in both in both the aerosol and the aerosol and ID BCG-vaccinated groups and remained comparable for the rest ID BCG-vaccinated groups and remained comparable for the rest of the pre-M. tuberculosis challenge of the pre-M. tuberculosis period of thechallenge period of study. Indeed, thethe study. area Indeed, under the area the curve under(AUC) analysis the curve analysis of the IFN-γ(AUC) of the IFN- SFU vaccination γphase SFU indicated vaccination phase indicated equivalence between the aerosol and ID BCG-induced equivalence between the aerosol and ID BCG-induced response (p = 0.49; Figure 7D), response (p = 0.49; and Figure 7D), comparative and comparative analysis analysis applied one week priorapplied one weekchallenge to M. tuberculosis prior to (study M. tuberculosis week 20), challenge indicated (study week 20), indicated that there was no significant difference between the IFN-γ that there was no significant difference between the IFN-γ SFU frequency measured in the vaccination SFU frequency measured groups in the at the vaccination final groups pre-challenge timeatpoint. the final pre-challenge time point. Figure 7. Systemic purified protein derivative (PPD)-specific IFN-γ spot forming unit (SFU) frequency Figure 7. Systemic purified protein derivative (PPD)-specific IFN-γ spot forming unit (SFU) frequency measured by ELISpot assay following aerosol or ID Bacille Calmette-Guérin (BCG) vaccination. measured by ELISpot assay following aerosol or ID Bacille Calmette-Guérin (BCG) vaccination. PPD- PPD-specific IFN-γ SFU measured in individual aerosol (A) or ID (B) BCG-vaccinated animals are specific IFN-γ SFU measured in individual aerosol (A) or ID (B) BCG-vaccinated animals are shown shown as points with vaccination group median line. Vaccination with aerosol or intradermal BCG is as points with vaccination group median line. Vaccination with aerosol or intradermal BCG is indicated by a dotted line at study week zero. Pre-vaccination SFU frequency is shown as the median of indicated by a dotted line at study week zero. Pre-vaccination SFU frequency is shown as the median at least 3 replicate values for each animal. Plot C shows a dot plot comparison of IFN-γ SFU frequency of at least 3 replicate values for each animal. Plot C shows a dot plot comparison of IFN-γ SFU four weeks after vaccination, and plot D shows area under the curve (AUC) values calculated from each animal’s SFU frequency profile between weeks 0–20. The group median is indicated by horizontal lines and Mann–Whitney U-test significance values are shown. Ultra-low dose M. tuberculosis challenge led to increases in M. tuberculosis-specific IFN-γ SFU frequencies, measured in response to stimulation with PPD or peptides spanning the CFP-10 or ESAT-6 sequence, in all animals (Figure 8A–I). Comparison of IFN-γ SFU frequencies between the unvaccinated control and vaccinated groups revealed significantly greater PPD-specific IFN-γ cell frequencies in indicated by horizontal lines and Mann–Whitney U-test significance values are shown. Ultra-low dose M. tuberculosis challenge led to increases in M. tuberculosis-specific IFN-γ SFU frequencies, measured in response to stimulation with PPD or peptides spanning the CFP-10 or ESAT-6 sequence, in all animals (Figure 8A–I). Comparison of IFN-γ SFU frequencies between the Pharmaceutics 2020, 12, 394 16 of 25 unvaccinated control and vaccinated groups revealed significantly greater PPD-specific IFN-γ cell frequencies in unvaccinated animals, 4 and 10 weeks following M. tuberculosis challenge (p = 0.002 and p = 0.003),animals, unvaccinated and in CFP-10-specific 4 and 10 weeksIFN-γ SFU frequencies following at weeks M. tuberculosis challenge (p = 12 8, 10 and (p =and 0.002 p =p 0.003), 0.015, = 0.015 andinp CFP-10-specific and = 0.004) post-challenge IFN-γ SFU(Figure 8J,K). at frequencies Significant differences weeks 8, 10 = 0.015, and 12 (p were notp = detected p = 0.004) between 0.015 and the experimental groups in terms of ESAT-6–specific IFN-γ SFU profiles when responses were post-challenge (Figure 8J,K). Significant differences were not detected between the experimental groups compared at terms in individual study time points of ESAT-6–specific or across IFN-γ the post-M. SFU profiles whentuberculosis challenge responses were phase at compared of individual the study (Figure study 8L). points or across the post-M. tuberculosis challenge phase of the study (Figure 8L). time M.tb 8000 U12 6000 U26 4000 U41 U46 3000 U58 U66 2000 1000 0 n 21 23 25 27 29 30 33 35 37 n 20 21 23 25 27 29 30 33 35 37 ia ia ed ed Weeks M M 8000 U18 6000 U28 4000 U31 U43 3000 U56 U72 2000 1000 0 20 21 23 25 27 29 30 33 35 37 n n 21 23 25 27 29 30 33 35 37 n 21 23 25 27 29 30 33 35 37 ia ia ia ed ed ed M M M n 20 21 23 25 27 29 30 33 35 37 n 21 23 25 27 29 30 33 35 37 n 21 23 25 27 29 30 33 35 37 ia ia ia ed er l ol Co ID Ae rol l Co ID l o ID ed ed ro ro so er M os M M nt nt nt ro A Co A Figure 8. Systemic PPD-, CFP10- and ESAT-6-specific IFN-γ spot forming unit frequency measured by Figure 8.assay ELISpot Systemic PPD-,aerosol following CFP10-challenge and ESAT-6-specific IFN-γ spot with M. tuberculosis. forming unit Mycobacterial frequency measured antigen-specific IFN-γ by ELISpot assay following aerosol challenge with M. tuberculosis. Mycobacterial SFU measured in individual aerosol (A,D,G), or ID (B,E,H) BCG-vaccinated and unvaccinated antigen-specific control IFN-γ animals (C,F,I) SFU measured during in theindividual aerosol post-challenge (A,D,G), phase of theorstudy ID (B,E,F) (weeks BCG-vaccinated 21–37) are shown andasunvaccinated points with control the (C,F,I) group vaccination animals duringline median theplotted. post-challenge Ultra-lowphase dose of the study (ULD) aerosol(weeks 21–37) are M. tuberculosis shown is challenge as points with the vaccination group median line plotted. Ultra-low dose (ULD) aerosol indicated by a dotted line at study week 21. Plots J and K show PPD- (J) and CFP-10-specific (K) IFN-γ M. tuberculosis challenge SFU is indicated frequencies betweenby vaccinated a dotted line andatunvaccinated study week 21. PlotsatJ specific groups and K show weeksPPD- (J) and post-M. CFP-10- tuberculosis specific (K) exposure, IFN-γ where SFU frequencies significant between differences were vaccinated measured by and unvaccinated groups Mann–Whitney U-test (pat≤specific weeks 0.05). Plot L post-M. shows tuberculosis AUC exposure,for values calculated where eachsignificant vaccinationdifferences group from were themeasured by Mann–Whitney PPD-, CFP-10- U-test and ESAT-6-specific (p ≤ 0.05).profiles response Plot Lfollowing shows AUC values calculated M. tuberculosis for each challenge. Group vaccination medians are group from by indicated thehorizontal PPD-, CFP-10- lines and ESAT-6-specific response profiles following M. and Mann–Whitney U-test significance is indicated by floating bars.tuberculosis challenge. Group medians are indicated by horizontal lines and Mann–Whitney U-test significance is indicated by floating bars. 3.8. Mycobacterial Antigen-Specific Secretion of Biomarkers Measured by Cytokine Bead Array 3.8. Mycobacterial The secretionAntigen-Specific of 37 cytokinesSecretion of Biomarkers and chemokines wasMeasured byin quantified Cytokine Bead Arraywhole blood PPD-stimulated cultures Thecollected secretionprior of 37to, and at weeks cytokines 2, 6, 10 and and chemokines 20 quantified was following aerosol or ID BCG whole in PPD-stimulated vaccination, blood to explore potential differences in immunomodulatory signalling associated with the route cultures collected prior to, and at weeks 2, 6, 10 and 20 following aerosol or ID BCG vaccination, of vaccine to delivery. Of the 37 analytes measured, the concentration of 24 (Eotaxin, GM-CSF, explore potential differences in immunomodulatory signalling associated with the route of vaccine IFN-alpha, IFN-gamma, TNF-alpha, delivery. Of the 37 analytesIL-10, IL-12p70, measured, the IL-13, IL-15, IL-17A, concentration IL-18, IL-1b, of 24 (Eotaxin, IL-1RA, GM-CSF, IL-2, IL-23, IFN-alpha, IFN- IL-6, CD40-Ligand, gamma, TNF-alpha,BDNF, IL-10, SCF, VEGF-D, IL-12p70, bNGF, IL-13, IL-15,FGF-2, IL-17A,PDGF-BB, VEGF-A) IL-18, IL-1b, IL-1RA,was either IL-2, undetectable IL-23, IL-6, CD40- (below Ligand,the limitSCF, BDNF, of detection VEGF-D, of the assay bNGF, FGF-2,system), PDGF-BB,or VEGF-A) did not differ significantly was either following undetectable BCG (below the vaccination by either route and was excluded from further analyses. Mycobacterial antigen-specific secretion of eight chemotactic signalling molecules (chemokines) IL-8 (CXCL8), IP-10 (CXCL10), I-TAC (CXCL11), MCP-1 (CCL2), MIP-1beta (CCL4), SDF-1alpha (CXCL12), MIG (CXCL9) and BLC (CXCL13), as well as the cytokines and growth factors IL-4, IL-5, IL-7, MIP-alpha and G-CSF, was found to increase significantly following vaccination by either aerosol or ID injection relative to pre-vaccination levels (p ≤ 0.05; Figure 9). In most cases, analyte concentrations measured in ID BCG-vaccinated animals were significantly higher than in the aerosol-vaccinated group six weeks after vaccination, although titres chemotactic signalling molecules (chemokines) IL-8 (CXCL8), IP-10 (CXCL10), I-TAC (CXCL11), MCP-1 (CCL2), MIP-1beta (CCL4), SDF-1alpha (CXCL12), MIG (CXCL9) and BLC (CXCL13), as well as the cytokines and growth factors IL-4, IL-5, IL-7, MIP-alpha and G-CSF, was found to increase significantly following vaccination by either aerosol or ID injection relative to pre-vaccination levels (p ≤ 0.05; Figure 9). In most cases, analyte concentrations measured in ID BCG-vaccinated animals Pharmaceutics 2020, 12, 394 17 of 25 were significantly higher than in the aerosol-vaccinated group six weeks after vaccination, although titres reached equivalence at later study weeks, indicating that it was the kinetics of the response that reached equivalence differed between theatgroups later study weeks, rather thanindicating its overallthat it was the kinetics composition. Notably,of the response that differed concentration of the between the groups rather than its overall composition. Notably, the concentration chemokine IP-10, a known pro-inflammatory biomarker involved in the recruitment of pro- of the chemokine IP-10, a knowncells inflammatory pro-inflammatory biomarker during mycobacterial involved infection in theand [30,31], recruitment of pro-inflammatory the cytokine IL-5, which is morecells during associated closely mycobacterial withinfection [30,31], and theTh2 an anti-inflammatory cytokine IL-5,profile response which[32]is more and closely associated negatively withM. modulates an anti-inflammatory Th2 response profile [32] and negatively modulates M. tuberculosis-specific tuberculosis-specific CD4 T-cell TNF-α production in vitro [33], were detected at significantly (or close CD4 T-cell to TNF-α production significance, p = 0.06, forinIL-5) vitrohigher [33], were detected atin concentrations significantly (or closeanimals aerosol-vaccinated to significance, two weeksp =after 0.06, for IL-5) higher concentrations in aerosol-vaccinated animals two weeks after vaccination. vaccination. Figure 9. PPD-specific Figure PPD-specificsecretion secretionofof cytokine cytokineandand chemokine biomarkers chemokine measured biomarkers in whole measured blood culture in whole blood supernatant. culture Box plots supernatant. Boxdisplay plots the groupthe display median grouptitre of each median cytokine titre or cytokine of each chemokine or+/- interquartile chemokine +/- range measured interquartile rangeprior to (-1) and measured at to prior weeks 2, 4, at (-1) and 10 weeks and 20 2, following 4, 10 andvaccination. 20 followingDots represent Dots vaccination. titres measuredtitres represent in individual measured animals with the animals in individual group minimum with theand maximum group minimum values andshown maximumby whiskers. values Significant differences measured by Mann–Whitney U-test between the groups are shown by whiskers. Significant differences measured by Mann–Whitney U-test between the groupsindicated, * p ≤ 0.05; ** p ≤ 0.01. are indicated. 3.9. Cellular Immune Responses Measured by Flow Cytometry 3.9. Cellular immune responses measured by flow cytometry 3.9.1. CD4 and CD8 T-cell Functional Profiles Following Aerosol or ID BCG Vaccination 3.9.1. CD4 and CD8 T-cell Functional Profiles Following Aerosol or ID BCG Vaccination The frequency and functional profile of CD4 and CD8 T-cell populations were assessed by The frequency multi-parameter andstaining ICS functional profile ofthe to measure CD4production and CD8 T-cell populations of the cytokines were assessed IFN-γ, TNFα, by multi- IL-2 and parameter ICS staining to measure the production of the cytokines IFN-γ, TNFα, IL-2 and IL-17 IL-17 at regular intervals during the vaccination phase of the study. Aerosol and ID BCG vaccination at regular intervals during the vaccination phase of the study. Aerosol and ID BCG vaccination induced CD4 and CD8 T-cells with functional profiles typical of the Th1 (producing IFN-γ or TNFα)induced and Th17 (producing IL-17) phenotypes, as well as IL-2 producing populations (Figure S1). Despite these similarities in the functional repertoire, comparison of the total (summed) cytokine response revealed that vaccine-induced cytokine production was delayed following aerosol, relative to ID, BCG vaccination with peak frequencies of cytokine-producing CD4 and CD8 T-cells measured at the four-week post-vaccination time point in ID-vaccinated animals, whereas aerosol BCG vaccination led to peaks in CD8 T-cell cytokine production at week 10 and at week 20 for CD4 subsets (Figure 10). similarities in the functional repertoire, comparison of the total (summed) cytokine response revealed that vaccine-induced cytokine production was delayed following aerosol, relative to ID, BCG vaccination with peak frequencies of cytokine-producing CD4 and CD8 T-cells measured at the four- week post-vaccination time point in ID-vaccinated animals, whereas aerosol BCG vaccination led to peaks in CD8 Pharmaceutics T-cell 2020, cytokine production at week 10 and at week 20 for CD4 subsets (Figure 10).18 of 25 12, 394 Figure 10. Total PPD specific-cytokine production by CD4 and CD8 T-cells. Dots represent the summed Figure 10. ofTotal frequency CD4 PPD specific-cytokine (A) and productionIFN-γ, CD8 (B) T-cells producing by CD4IL-2,and CD8 TNF-α T-cells. or IL-17 Dots represent measured the in peripheral summed frequency of blood mononuclear CD4 cells (A) andcollected (PBMCs) CD8 (B) from T-cells producing individual IFN-γ,prior animals IL-2, to TNF-α (weekor-1) IL-17 andmeasured following in peripheral aerosol (blueblood mononuclear bars) or intradermalcells (PBMCs) (green collected bars) BCG. Barsfrom show individual the groupanimals medians prior withtosignificant (week -1) differences and following determined by Mann–Whitney aerosol (blue U-test bars) or intradermal indicated (green bars)by an asterisk, BCG. Bars showp ≤the 0.05. group medians with significant differences determined by Mann–Whitney U-test indicated by an asterisk, p ≤ 0.05. 3.9.2. Multifunctional T-cell Populations Induced by Aerosol BCG or ID BCG Vaccination 3.9.2. The Multifunctional T-cell Populations functional profile Induced of T-cell subsets was by Aerosol explored inBCG or ID greater BCGby detail Vaccination identification of cells producing multiple profile The functional combinations of subsets of T-cell the cytokines IFN-γ, TNFα was explored and IL-2 in greater simultaneously detail in response by identification of cells to stimulation with tuberculin PPD. BCG vaccination delivered by aerosol or by ID producing multiple combinations of the cytokines IFN-γ, TNFα and IL-2 simultaneously in response injection led to stimulation to significant increases in IFN-γ-, with tuberculin PPD.IL-2- BCG and TNF-α-producing vaccination deliveredpolyfunctional by aerosol or byCD4 ID T-cells (p = injection led0.02, to p = 0.05); and trends for increased IFN-γ and IL-2, and TNF-α and IL-2 multifunctional populations significant increases in IFN-γ-, IL-2- and TNF-α-producing polyfunctional CD4 T-cells (p = 0.02, p = (Figure 0.05); 11).trends and CD4 T-cell populations for increased IFN-γproducing and IL-2,IFN-γ alone, typically and TNF-α and IL-2considered to indicate multifunctional a more populations terminally differentiated phenotype [34], were more prevalent in the ID BCG-vaccinated group, whereas IL-2-producing monofunctional CD4 populations were more prevalent in aerosol BCG-vaccinated animals, potentially indicating greater proliferative capacity [34]. The frequency of polyfunctional CD8 T-cells increased significantly at 10 weeks following aerosol BCG vaccination (p = 0.01), whereas other cytokine-producing CD8 T-cell populations primarily consisted of cells producing IFN-γ or TNF-α alone (Figure 11C,D). Despite the reoccurring trend for the cellular immune response to be delayed when BCG was delivered as an aerosol, with peak cytokine production from CD4 and CD8 T-cell terminally differentiated phenotype [34], were more prevalent in the ID BCG-vaccinated group, whereas IL-2-producing monofunctional CD4 populations were more prevalent in aerosol BCG- vaccinated animals, potentially indicating greater proliferative capacity [34]. The frequency of polyfunctional CD8 T-cells increased significantly at 10 weeks following aerosol BCG vaccination (p = 0.01), whereas other cytokine-producing CD8 T-cell populations primarily consisted of cells Pharmaceutics 2020, 12, 394 19 of 25 producing IFN-γ or TNF-α alone (Figure 11C,D). Despite the reoccurring trend for the cellular immune response to be delayed when BCG was delivered as an aerosol, with peak cytokine production occurring atfromweekCD4 10 asand CD8 to opposed T-cell weekoccurring at week 4 following 10 as opposed ID vaccination, to week significant 4 following differences ID were not vaccination, significant measured between differences were the vaccination not measured between the vaccination groups. groups. A. B. C. Frequency (%) of cytokine producung CD8 T-cells D. Figure 11. Figure PPD-specificpolyfunctional 11. PPD-specific polyfunctionalCD4 CD4andandCD8CD8T-cell T-cellprofiles profiles measured measured inin PBMC. PBMC. BarBar charts charts show vaccination group median frequencies of cytokine-producing CD4 (A,B) and CD8 show vaccination group median frequencies of cytokine-producing CD4 (A,B) and CD8 (C,D) T-cells (C,D) T-cells measured in measured in PBMCs PBMCs at at specific specific weeks weeks prior prior to to (week (week -1) -1) and and following following (weeks (weeks 4,4, 10 10 and and 20) 20) BCG BCG vaccination. Significant vaccination. Significantdifferences differencesbetween betweenpre- pre-and and post-vaccination post-vaccinationvalues valuesare are indicated indicated by by bars bars vaccination with p-values shown. Cytokine frequencies measured in individual animals vaccination with p-values shown. Cytokine frequencies measured in individual animals are are represented by dots. represented by dots. 3.9.3. Antigen-Specific Memory T-cell Profiles Following Aerosol or ID BCG Vaccination To assess the memory status of cytokine-producing T-cell populations, central and effector memory T-cells were identified by expression of the cell activation markers CD95 and CD45RA, as well as differential expression patterns of the co-stimulatory receptor CD28 and lymph node homing marker CCR7. Therefore, T-cell memory phenotype was determined on CD95+ cells as CD28+ CCR7+ assessed and compared at regular intervals following aerosol and ID BCG vaccination (Figure 12A– G). Cytokine-producing CD4 T-cells primarily occupied TCM and TranEM phenotypes, with only low frequencies of cytokine-producing TEM cells detected. Antigen-specific CD8 Tscm populations were not detected, whereas cytokine-producing CD4 Tscm populations were detected at low frequency and Pharmaceutics remained 2020, 12, 394 unchanged from pre-vaccination levels, indicating that variation in20the of 25 functional parameters measured is not induced by BCG vaccination in this cell population (Figure 12D). central memory Similar patterns(TCM), CD28+ CCR7 were observed − transitional effector memory (TranEM), effector memory in the phenotype of+the wider CD8 T-cell populations, although (TEM) CD28 - CCR7 − , and stem cell memory CD45RA CD28 + CCR7+ (Tscm) (Figure 12H,I) [35–37]. a trend toward greater frequencies of cytokine-producing TranEM cells in comparison to TCM cells Using was alsothis classification, observed (Figureantigen-specific cytokine-producing 12E–G). Comparison of CD8 memory memory CD4 and populations CD8 T-cell between profiles were the vaccination groups revealed that significantly greater frequencies of antigen-specific TranEM (p = 0.05) and12A–G). assessed and compared at regular intervals following aerosol and ID BCG vaccination (Figure TCM Cytokine-producing CD4 T-cells primarily occupied TCM and TranEM phenotypes, (p = 0.03) populations were measured in PBMCs isolated from aerosol BCG-vaccinated animals 20 with only low frequencies weeks followingof cytokine-producing TEM cells vaccination, indicating thatdetected. Antigen-specific aerosol BCG CD8 Tscm populations induced antigen-specific CD8 T-cellwere populations that remained in circulation within a week of the M. tuberculosis challenge delivered and not detected, whereas cytokine-producing CD4 Tscm populations were detected at low frequency at remained unchanged study week 21 (Figure 12E,F). from pre-vaccination levels, indicating that variation in the functional parameters measured is not induced by BCG vaccination in this cell population (Figure 12D). Figure Figure 12.12. Antigen-specific Antigen-specific CD4 CD4 and and CD8CD8 memory memory T-cell T-cell profiles profiles measured measured inin PBMCs. PBMCs. Plots Plots HH and and I I show show representative representative bivariate bivariate density density plots plots of of central central to to effector effector memory memory T-cell T-cell populations populations defined defined by the pattern of CD28 and CCR7 staining on CD95 expressing CD4 (H) and CD8 by the pattern of CD28 and CCR7 staining on CD95 expressing CD4 (H) and CD8 (I) T-cells, overlaid (I) T-cells, overlaid withcytokine-producing with cytokine-producing cells cells represented representedasascoloured coloured dots. Plots dots. A–GA–G Plots showshow the frequency of memory the frequency of memory CD4 (Plots A–D) and CD8 (plots E–G) T-cell populations producing IFN-γ, IL-2 ormeasured CD4 (Plots A–D) and CD8 (plots E–G) T-cell populations producing IFN-γ, IL-2 or TNF-α, TNF-α, prior to (week measured prior to-1) and following (week (weeks (weeks -1) and following 4, 10 and 20)and 4, 10 BCG 20)vaccination. Box plots BCG vaccination. showshow Box plots the group the median and interquartile range with minimum and maximum values shown by whiskers. Significant group median and interquartile range with minimum and maximum values shown by whiskers. differences measured by Mann–Whitney U-test are indicated by an asterisk (*), p ≤ 0.05. Significant differences measured by Mann–Whitney U-test are indicated by an asterisk (*), p ≤ 0.05. Similar patterns were observed in the phenotype of the wider CD8 T-cell populations, although a 4. Discussion trend toward greater frequencies of cytokine-producing TranEM cells in comparison to TCM cells wasTB remains also a leading observed cause (Figure of global 12E–G). mortality Comparison of and CD8ismemory responsible for morebetween populations deaths than any other the vaccination infectious disease [38]. groups revealed that The continuedgreater significantly emergence of multi-drug frequencies resistant strains of antigen-specific = 0.05) and of M.(ptuberculosis, TranEM and TCM = 0.03) populations the(poverlap between thewere HIVmeasured and TB epidemics, necessitates in PBMCs isolated fromaaerosol more efficacious vaccination BCG-vaccinated animals 20 weeks following vaccination, indicating that aerosol BCG induced antigen-specific CD8 T-cell populations that remained in circulation within a week of the M. tuberculosis challenge delivered at study week 21 (Figure 12E,F). 4. Discussion TB remains a leading cause of global mortality and is responsible for more deaths than any other infectious disease [38]. The continued emergence of multi-drug resistant strains of M. tuberculosis, and the overlap between the HIV and TB epidemics, necessitates a more efficacious vaccination regimen for the control of TB. In the absence of a validated correlate of protection against TB, highly characterised animal models that can be used to differentiate promising vaccine candidates and refinements to the current vaccination regimen are required. We have previously described the development of an ultra-low dose aerosol challenge model in rhesus and cynomolgus macaques using an inoculum of the M. tuberculosis Erdman strain that is representative of natural infection [6]. The aerosol M. tuberculosis Pharmaceutics 2020, 12, 394 21 of 25 challenge dose applied in this study was comparable to these prior experiments, with a median presented dose quantified as 23 CFU, which equates to an estimated 3 CFU retained within the lung [21], demonstrating the reproducibility of the ULD aerosol challenge system we have developed. The selection of an appropriate M. tuberculosis challenge strain and dose is critical to pre-clinical TB vaccine evaluation strategies. Our goal in developing the ULD aerosol M. tuberculosis challenge model was to establish a system that can reproducibly deliver a challenge inoculum that is sufficient to cause disease in all animals, but not so overwhelming that a promising vaccine may be rejected because it is unable to provide immunity against a level of disease that is not typical of natural infection. In this study, we report the application of this model for the assessment of TB vaccination regimens and demonstrate the capacity to measure vaccination mediated differences in disease pathology and M. tuberculosis bacterial load afforded by BCG vaccination applied by aerosol or intradermal injection. Furthermore, variation in the pattern of extra-thoracic disease was measured between the groups that received BCG vaccination delivered parenterally or to the mucosal surfaces of the lung as an aerosol, demonstrating the sensitivity of the model to detect subtle differences in disease parameters and differentiate between similar vaccination strategies. In comparison to higher dose M. tuberculosis challenge systems [3,24,39,40], the disease that develops in rhesus macaques following ULD aerosol M. tuberculosis exposure follows a slower pattern of progression [6]. This feature offers distinct advantages for evaluation of vaccine efficacy, including the reduced occurrence or delayed onset of progressive disease that can lead to a requirement for animals to be euthanised on welfare grounds. As well a notable refinement in terms of animal welfare, harmonisation of the timespan available for the development of infection and features of tuberculous disease leads to improved comparability of vaccine efficacy readouts between treated and untreated control groups and is consequently a valuable improvement to in vivo efficacy study design. These key features of the rhesus macaque ultra-low dose aerosol challenge model combine to provide a reproducible and sensitive system for assessment of the protective efficacy of novel TB vaccine candidates and regimens. We have recently described the mucosal and systemic cellular immune response induced by BCG delivered as an aerosol using a portable VMN [19], and, in this study, we set out to directly compare the protective efficacy against ULD M. tuberculosis challenge imparted by aerosol and ID BCG vaccination. Consequently, mucosal immune responses were not interrogated to avoid potential interference with lung immunity prior to the aerosol M. tuberculosis challenge, and comparative immunology was focused on systemic response profiles. It was evident from analysis of the frequency of antigen-specific cytokine-producing cells measured by ELISpot and flow cytometry, as well as the secretion of cytokine and chemokine markers quantified by multiplex bead array, that the onset of the systemic adaptive cellular immune response was significantly delayed following aerosol BCG vaccination, although, the peak and duration of these responses were comparable between the vaccination groups. For example, IFN-γ SFU frequencies were statistically equivalent immediately prior to aerosol challenge with M. tuberculosis, indicating that the delayed onset of vaccine-induced systemic immunity is unlikely to have interfered with a subsequent anamnestic response upon exposure to M. tuberculosis. Indeed, the frequency of mycobacteria-specific IFN-γ secreting cells increased concurrently in the aerosol- and ID-vaccinated groups following M. tuberculosis infection, albeit at a significantly slower rate than was seen in unvaccinated animals, which is likely to be indicative of the improved control of infection imparted by BCG vaccination delivered by either route [3,24]. Similarly, the functional profile of CD4 and CD8 T-cell subsets induced by aerosol or ID BCG vaccination were comparable when measured by multiparameter ICS assay, where a trend for delayed detection of antigen-specific cytokine-producing T-cell populations in aerosol BCG vaccinated animals was also apparent. However, interrogation of the memory phenotype of antigen-specific T-cell populations revealed that whilst cytokine-producing CD4 and CD8 populations were detected across the memory T-cell axis (encompassing TCM–TEM phenotypes) in both vaccination groups, TCM and TransEM CD8 T-cell populations induced by aerosol BCG vaccination were significantly Pharmaceutics 2020, 12, 394 22 of 25 higher than in ID BCG-vaccinated animals twenty weeks after vaccination. The continued presence of circulating antigen-specific TCM and TransEM CD8 T-cells within a week of aerosol M. tuberculosis challenge suggests that these cells may have contributed to the disease control observed in aerosol BCG-vaccinated macaques. The variable efficacy of BCG vaccination, when delivered as an intradermal injection, is well documented in humans [8] and in preclinical models, including primates [24,41,42]. In common with previous reports, our experiment corroborates that there is variability in the degree of protection afforded to rhesus macaques by ID BCG vaccination, although a comparison between groups revealed that levels of disease pathology were significantly reduced in both ID and aerosol BCG-vaccinated groups relative to unvaccinated animals, providing a valuable and relevant benchmark against which novel vaccination regimens can be assessed. Aerosol BCG vaccination delivered in the laboratory setting has been shown previously to be immunogenic and to afford enhanced protection against inhaled M. tuberculosis infection in the rhesus macaque [16,19]. The availability of portable vibrating mesh nebuliser (VMN) technology has made the proposition of a widespread aerosol BCG vaccination campaign a viable alternative to needle-based vaccine delivery. With this comparison of the immunogenicity and efficacy of VMN delivered aerosol- and parenterally-delivered BCG, we have shown that the total level of disease pathology that developed in ID and aerosol BCG-vaccinated animals was equivalent following ULD M. tuberculosis challenge, although pathology measured in extrapulmonary tissues was significantly reduced by aerosol-delivered BCG vaccination. Similarly, the level of viable M. tuberculosis quantified in extra-thoracic (secondary) sites of infection was significantly reduced by aerosol BCG vaccination relative to unvaccinated animals, whereas the reduction in bacterial load measured in ID BCG-vaccinated animals failed to reach significance. Although these levels of efficacy appear relatively modest in comparison to those reported from historical studies where shorter vaccination to challenge intervals and the less virulent H37Rv strain of M. tuberculosis challenge inoculum were employed [16], our findings provide verification that aerosolised BCG delivered by VMN can provide a significant level of protection to rhesus macaques against M. tuberculosis challenge which is at least equivalent, and by some measures (such as reduced extrapulmonary dissemination and disease) superior, to intradermally delivered vaccination. Similarly, we note that the levels of protection demonstrated herein are comparable to those recently reported by Darrah et al. who used an alternative VMN device, dose and preparation of BCG, and M. tuberculosis challenge system, although their experiments were not powered to demonstrate vaccine efficacy relative to unvaccinated animals [43]. Therefore, we believe that there is a collective body of evidence supporting the further investigation of aerosol delivered BCG vaccination as an alternative to the current needle-based vaccination policy, although further optimisation of vaccination dose and refinement of the aerosol delivery apparatus to ensure efficient deposition and targeting of the vaccine should be investigated [44]. We note with interest the recent phase I clinical trial applications centred on VMN-delivered aerosol BCG [45,46], which may produce the requisite safety data to take this approach forward into further clinical studies. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/12/5/394/s1, Figure S1: PPD-specific production of individual cytokines measured by intra-cellular cytokine staining. Plots A, B, C and D show the frequency of IFN-γ, IL-2, TNF-α and IL-17 measured in CD4 T-cells; plots E, F, G and H show cytokine production measured in CD8 T-cells. Box plots display the group median frequency of cytokine-producing cells +/- interquartile range with minimum and maximum values shown by whiskers. Dots represent the frequency of cytokine-producing cells measured in individual animals. BCG vaccination is indicated by a dotted line at study week zero. Author Contributions: conceptualization, A.D.W., S.A.S., and A.W.; methodology, A.D.W., M.J.D., C.S., L.S.S., J.G., S.C., E.R., F.G., M.C., I.N., P.-J.C., and C.V.; formal analysis, A.D.W., C.S., and L.S.S.; investigation, A.D.W., M.J.D., C.S., L.S.S., J.G., S.C., E.R., F.G., M.C., and I.N.; resources, M.J.D. and A.D.W.; writing—original draft preparation, A.D.W.; writing—review and editing, A.D.W., S.A.S., A.W., M.J.D., C.S., L.S.S., S.C., E.R., and M.C.; visualization, A.D.W., L.S.S., C.S., and E.R.; supervision, A.D.W., S.A.S., A.W., P.-J.C., and C.V.; project administration, A.D.W.; funding acquisition, S.A.S. and A.W. 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