pathogens Article Surveillance of Daughter Micronodule Formation Is a Key Factor for Vaccine Evaluation Using Experimental Infection Models of Tuberculosis in Macaques Isabel Nogueira 1,† , Martí Català 2,3,† , Andrew D. White 4 , Sally A Sharpe 4 , Jordi Bechini 1 , Clara Prats 3 , Cristina Vilaplana 5,6,7 and Pere-Joan Cardona 5,6,8,9, * 1 Radiology Department, ‘Germans Trias i Pujol’ University Hospital, 08916 Badalona, Spain 2 Comparative Medicine and Bioimage Centre of Catalonia (CMCiB), Germans Trias i Pujol Research Institute (IGTP), 08916 Badalona, Spain 3 Escola d’Enginyeria Agroalimentària i de Biosistemes de Barcelona Departament de Física, Universitat Politècnica de Catalunya (UPC)-BarcelonaTech, 08860 Castelldefels, Spain 4 UK Health Security Agency, Porton Down, Salisbury SP4 0JG, UK 5 Unitat de Tuberculosi Experimental, Germans Trias i Pujol Research Institute (IGTP), 08916 Badalona, Spain 6 Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain 7 Direcció Clínica Territorial de Malalties Infeccioses i Salut Internacional de Gerència Territorial Metropolitana Nord, 08916 Badalona, Spain 8 Microbiology Department, North Metropolitan Clinical Laboratory, ‘Germans Trias i Pujol’ University Hospital, 08916 Badalona, Spain 9 Genetics and Microbiology Department, Universitat Autònoma de Barcelona, 08913 Cerdanyola del Vallès, Spain * Correspondence: pj.cardona@gmail.com † These authors contributed equally to this work. Abstract: Tuberculosis (TB) is still a major worldwide health problem and models using non-human primates (NHP) provide the most relevant approach for vaccine testing. In this study, we anal- ysed CT images collected from cynomolgus and rhesus macaques following exposure to ultra-low Citation: Nogueira, I.; Català, M.; dose Mycobacterium tuberculosis (Mtb) aerosols, and monitored them for 16 weeks to evaluate the White, A.D.; Sharpe, S.A.; Bechini, J.; impact of prior intradermal or inhaled BCG vaccination on the progression of lung disease. All Prats, C.; Vilaplana, C.; Cardona, P.-J. lesions found (2553) were classified according to their size and we subclassified small micron- Surveillance of Daughter odules (<4.4 mm) as ‘isolated’, or as ‘daughter’, when they were in contact with consolidation Micronodule Formation Is a Key (described as lesions ≥ 4.5 mm). Our data link the higher capacity to contain Mtb infection in Factor for Vaccine Evaluation Using cynomolgus with the reduced incidence of daughter micronodules, thus avoiding the develop- Experimental Infection Models of Tuberculosis in Macaques. Pathogens ment of consolidated lesions and their consequent enlargement and evolution to cavitation. In the 2023, 12, 236. https://doi.org/ case of rhesus, intradermal vaccination has a higher capacity to reduce the formation of daughter 10.3390/pathogens12020236 micronodules. This study supports the ‘Bubble Model’ defined with the C3HBe/FeJ mice and pro- poses a new method to evaluate outcomes in experimental models of TB in NHP based on CT images, Academic Editor: Hwa-Jung Kim which would fit a future machine learning approach to evaluate new vaccines. Received: 27 December 2022 Revised: 29 January 2023 Keywords: tuberculosis; BCG vaccine; aerosol vaccination; non-human primate; macaque; bubble Accepted: 1 February 2023 model; computed tomography scanner Published: 2 February 2023 1. Introduction Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. Understanding the dynamics of tuberculosis (TB) disease is essential for the design This article is an open access article of better strategies against it, as TB is a cause of high morbidity and mortality worldwide. distributed under the terms and Mycobacterium tuberculosis (Mtb) infects about 10 million people every year and a quarter of conditions of the Creative Commons humanity is estimated to be infected. Moreover, it has been the deadliest single infectious Attribution (CC BY) license (https:// disease for decades, killing nearly 1.5 million people annually [1]. Unfortunately, this creativecommons.org/licenses/by/ mortality has been overtaken by SARS-CoV-2 in 2020, a further consequence of which has 4.0/). been a devastating impact on TB care resulting in an estimated additional half a million TB Pathogens 2023, 12, 236. https://doi.org/10.3390/pathogens12020236 https://www.mdpi.com/journal/pathogens Pathogens 2023, 12, 236 2 of 21 deaths, thus going back to a mortality level similar to that of 2010 [2,3]. Defeating TB is not yet a feasible goal in the short term, but endeavours must continue to focus on the three cornerstones of prevention, early diagnosis and treatment, and advance the development of new vaccines, biomarkers and drugs with shortened protocols [4–6]. The granuloma is the hallmark of the host response against Mtb infection, appear- ing as a characteristic round histologic structure and ranging in size from 1–3 mm in diameter [7–12]. There are several studies that describe granuloma subtypes, mainly based on their cell proliferation, matrix quality and pattern distribution under a micro- scope [9–11,13–16]. In the context of image analysis, granulomas are expected to profusely mineralize and thus be identifiable as calcified micronodules on CT scan images collected from macaques mainly from 20 weeks after experimental infection [11]. Vaccination is one of the essentials for controlling the disease. The Bacille Calmette– Guérin (BCG) vaccine is an attenuated strain of Mycobacterium bovis, which was first used in 1921 and still plays a pivotal role in high endemic TB countries, despite low efficacy in some regions [17,18]. BCG has demonstrated protection against TB death and disseminated disease, especially against childhood tuberculous meningitis and miliary disease, but has also shown disparate results in the protection afforded to adults and pulmonary involvement [19–24]. Even though new vaccine candidates are in development [25–27], the centenary vaccine remains as the only licensed vaccine [1] and clinical trials are currently ongoing to explore the efficacy of BCG novel protocols, such as the use of alternative delivery routes such as inhaled (or aerosolized) [28–32], or in revaccination regimens [33,34]. The investigation of prevention strategies and treatments depends on well-characterized experimental animal models. The most clinically relevant experimental model is provided by non-human primates (NHP) as they resemble humans in almost every aspect and mimic human physiological patterns. Macaques are widely reported to manifest the same TB disease as people do [35], also sharing comparable efficacy after BCG vaccination and chemotherapy [36–39]. It is worth emphasizing that the investigation of BCG vaccination in macaques allows the exploration of the whole spectrum of the protection status seen in humans, as the vaccine confers a protective effect in cynomolgus macaques and limited protection in rhesus [28,29,40,41]. The model in rhesus macaques is the one that robustly reproduces active TB lesions [14,36,42–45]. In contrast, cynomolgus macaques reveal a greater similarity with the wide spectrum of outcomes that follow human-Mtb interaction, especially the Asian cynomolgus (with Indonesian and Chinese genotypes), which are better able to contain the progression of the infection, reflecting mainly a latent TB infection, and more limited progression towards active disease [14,15,29]. However, Mauritanian genotype cynomolgus macaques are more susceptible to TB disease, showing outcomes of TB infection more akin to those found in rhesus macaques [45,46]. One important consideration when designing experimental models is to use the route of natural infection and an optimal challenge dose that is sufficient to induce measurable disease, but not at a level that could overwhelm potentially protective effects induced by vaccination and confound the evaluation assessment [14,15,30,36,38,44,47]. The infection after experimental exposure to aerosols containing ultra-low dose inoculum has been demonstrated to closely resemble naturally occurring Mtb exposure, causing disease- induced changes in the lung that can be monitored [14,48]. The evaluation method should be sensitive to allow the detection of subtle differences among prophylactic candidates. TB disease dynamics are not yet entirely understood. In fact, there is still no canonical hypothesis to explain the progression from latent to active disease. Daughter lesions have already been described during active TB development, appearing near larger lesions, and being associated with the advance of local infection. The ‘Bubble Model’, firstly conceived by Prats et al. [49] and inspired by soap bubble formation together with previous concepts [5,49–53], hypothesized that progression to active TB was the consequence of the intensity of the inflammatory response in the initial micronodules, the induction of daughter micronodules around the initial nodule and the coalescence of all these lesions that cause the formation of a consolidated lesion, with cavitation capacity. However, Pathogens 2023, 12, 236 3 of 21 this model was built on data obtained from the murine model with C3HeB/FeJ mice by Marzo et al. [54]. Supportive data obtained from the NHP model would endorse future confirmation in humans. Image analysis plays a fundamental role in TB disease assessment. Chest X-ray was classically used for diagnosis and still remains a basic tool due to its widespread availability and sufficient sensitivity. Recently, high-quality diagnostic images such as computed tomography (CT), positron emission tomography–computed tomography (PET-CT) and magnetic resonance imaging (MRI) have emerged. However, to our knowledge, there have been limited opportunities to characterize the development of Mtb-induced disease, particularly the early events after infection. Scanning enables the collection of high-quality data in a non-invasive way, allowing the examination of features of particular interest such as lung consolidations, nodules, micronodules (daughter and isolated), cavitation, tree-in-bud lesions and their distribution [5,11,46,48,49,54–62]. CT scanning allows the acquisition of high-quality images in a few seconds (notably faster than MRI and PET- CT), thus minimizing interference caused by respiratory motion, and being also the most available and economical three-dimensional imaging technology. A score system for the estimation of TB burden disease in the NHP model based on CT images developed by Sharpe et al. [63] has been reported. The aim of this study was to evaluate the progression of Mtb infection in experimental NHP models through the spatial monitoring of the micronodules. We found that the identification of consolidations associated with daughter micronodules would be a useful tool to evaluate the efficacy of new vaccines. 2. Materials and Methods 2.1. Animals Images were obtained from 35 macaques aged 3–4 years; 13 cynomolgus macaques (Macaca fascicularis, of Indonesian genotype) and 22 rhesus macaques (Macaca mulatta, of Indian genotype). The macaques enrolled in the studies were obtained from established closed breeding colonies in the United Kingdom and absence of previous exposure to my- cobacterial antigens was confirmed by tuberculin skin test as part of colony management procedures and by screening for IFN-γ ELISPOT (MabTech, Nacka, Sweden) responses to tuberculin-PPD (SSI, Copenhagen, Denmark), and pooled 15-mer peptides of ESAT6 and CFP10 (Peptide Protein Research LTD, Fareham, UK). BCG vaccinations were delivered to sedated animals either as a 100 µL intradermal (ID) injection using Danish strain 1331 (SSI, Copenhagen, Denmark) delivered to the upper left arm, or by exposure to aerosolised BCG Danish strain 1331 created using an Omron MicroAir mesh nebuliser (Omron Health- care UK Ltd., Milton Keynes, UK) and a modified paediatric anaesthesia mask. The BCG vaccine was prepared for intradermal administration according to manufacturer’s instruc- tions for administration to humans. One ml of Sautons diluent was added to a vial of BCG vaccine to give a suspension of BCG at an estimated concentration of between 2 and 8 × 106 CFU/mL. The vaccination dose was selected to be equivalent to a standard adult intradermal dose [13,14,28]. The macaques were experimentally infected with Mtb (Erdman strain K 01) ultra-low dose aerosol exposure (presented dose 19–26 CFU) as previously described [14], and following exposure to MTB, macaques were housed in facili- ties compliant with the Advisory Committee for Dangerous Pathogens Level 3 (ACDP3) regulations. Compatible social groups were housed in accordance with the Home Office (UK) Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989), and the National Committee for Refinement, Reduction and Replacement (NC3Rs) Guidelines on Primate Accommodation, Care and Use, August 2006 (NC3Rs, 2006). Cages were approximately 2.5 M high by 4 M long by 2 M deep, constructed with high level observation balconies and with a floor of deep litter to allow foraging. Additional envi- ronmental enrichment was afforded by the provision of toys, swings, feeding puzzles and DVDs for visual stimulation. In addition to standard old-world primate pellets, diet was further supplemented with a selection of fresh vegetables and fruit. For each procedure, Pathogens 2023, 12, 236 4 of 21 sedation was applied by intramuscular injection with ketamine hydrochloride (10 mg/kg) (Ketaset, Fort Dodge Animal Health Ltd., Southampton, UK). None of the animals had been used previously for experimental procedures and each socially compatible group was randomly assigned to a particular study treatment. The study design and all procedures were approved by the UKHSA Porton Down Animal Welfare and Ethical Review Body, and authorised under an appropriate UK Home Office project license. Animals were sedated at regular intervals to measure body weight and temperature, red blood cell (RBC) haemoglobin levels and erythrocyte sedimentation rate (ESR) and for collection of blood samples, X-radiographs or CT scans. Animal behaviour was observed throughout the studies 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. The studies reported describe individuals with active TB that was either controlled during the study period or progressed to levels that met humane endpoint criteria. TB was not latent in any of the subjects. 2.2. Experimental Groups Once CT images analysis was concluded, we defined five experimental groups among the thirty-five macaques, based on the genotype and their vaccination status: cynomolgus unvaccinated (CUV, n = 10), cynomolgus intradermal-BCG-vaccinated (CIDV, n = 3), rhesus unvaccinated (RUV, n = 11), rhesus aerosolized, or inhaled, BCG-vaccinated (RAEV n = 6) and rhesus intradermal-BCG-vaccinated (RIDV, n = 5). These groups together with CT scan schedule are shown in Figure 1 and Supplementary Figure S1. Figure 1. Study schedule. Macaques received BCG vaccination (green circles) delivered by aerosol or intradermal injection, or were left as unvaccinated controls. All animals received ultra-low dose aerosol challenge with Mtb 21 weeks after. Gray triangles indicate application of ‘in vivo’ CT scanning. Light grey triangles indicate the time points where not all animals were scanned and the number between brackets shows in how many of them this was effectively performed. In blue, unvaccinated cynomolgus (CUV). In cyan, intradermal-vaccinated cynomolgus (CIDV). In red, unvaccinated rhesus (RUV). In orange, aerosol-vaccinated rhesus (RAEV). In purple, intradermal-vaccinated rhesus (RIDV). 2.3. CT Images We analysed 123 computed tomography (CT) scans obtained from subjects enrolled in different studies [13,14,28] where sequential images were acquired from each macaque on three or four occasions during the post-infection period from the 3rd to the 16th week (Figure 1 and Supplementary Figure S1). Pathogens 2023, 12, 236 5 of 21 Chest multidetector CT scans were obtained by using a 16 slice Lightspeed CT scanner (General Electric Healthcare, Milwaukee, WI, USA), from sedated subjects while free breathing. Technical parameters applied: tube voltage, 120 kVp; tube current modulation, 100–150 mA. Reconstructions were made using a high spatial frequency algorithm and lung window at a slice thickness of 0.625 mm. These technical parameters are extracted from the original animal experimental studies [13,14,28]. 2.4. TB Lung Lesions Analysis from CT Images CT scans were evaluated by a medical consultant radiologist with expertise in respi- ratory diseases and her supervisor. Both were blinded to vaccination status and clinical data. Data show the consensus between both. Images were then examined in Philips IntelliSpace Portal software (2015 Koninklijke Philips N.V.) by multiplanar reformatting axial, coronal and sagittal, and maximum intensity projection (MIP) to detect the smallest nodules. The Tumor Tracking tool from the mentioned software was used to analyse the volume and main axis of each lesion. We used standard image descriptors, accepted and reproducible concepts from radiologic lexicon detailed on Fleischner Society Glossary as well as in previous studies based on CT images [63]. A. Micronodule is a small solid lung nodule with smooth margins. B. Consolidation (also referred to as consolidated lesion) is a pneumonic patch or necrotiz- ing consolidative process that occupies and even destroys alveoli, and it is described in CT images as a soft tissue lesion within lung parenchyma showing irregular margins. C. Cavitation corresponds to the appearance of gas within lung consolidations. It is de- scribed as a central radiolucency image, which matches with gas density, surrounded by soft tissue [8,57]. D. Pleural distance is defined as the distance between the margin of each consolidation to the closest pleura or fissure. 2.5. Statistical Assessment To compare the mean and variance of the different observations in the two groups we used the one-way ANOVA test and Kruskal–Wallis test to determine the existence of signif- icant differences. To compare the observed distribution of data into discrete categories, we built a contingency table and the Fisher’s test. Implementation was conducted in MATLAB and Prism. We computed the mean with available data. In the plots, we indicated with a dashed line if there were missing data. Recollected data are available in the Supplementary Material (Supplementary File raw data). 3. Results 3.1. Preliminary Images’ Assessment and Anatomy We were provided with high-quality CT scan images, similar to those acquired for medical diagnosis in humans. A first anatomical examination corroborated that cynomol- gus macaques had a smaller lung volume than rhesus macaques (Supplementary Figure S2). Overall, 323 consolidations were identified; 285 were found in rhesus and 38 in cynomolgus, with a median of 14 and 2 lesions per animal, respectively (Table 1). A total of 2228 mi- cronodules were detected, the majority of which (1924) were found in rhesus macaques, while 304 micronodules were detected in cynomolgus (Table 2). Collected data revealed that lesions globally increase over time. Right lung lobes developed more consolidations in number but left lung lobes presented with a higher proportion occupied by disease. A non-significant trend was seen for the major involvement to occur in the upper lobes, which was higher in the left side (Supplementary Figure S3). Further CT scans’ inspection showed the lung lobes to be separated by pleural fissures as well as bronchovascular bundles, thus presenting the same airway anatomy pattern seen in humans. The right lung is the largest and is composed of upper, middle, lower and infracardiac (or accessory or azygous) lobes, and the left lung is composed of upper, middle (or lingula) and lower lobes [64–69]. disease. A non-significant trend was seen for the major involvement to occur in the upper lobes, which was higher in the left side (Supplementary Figure S3). Further CT scans’ in- spection showed the lung lobes to be separated by pleural fissures as well as bronchovas- cular bundles, thus presenting the same airway anatomy pattern seen in humans. The Pathogens 2023, 12, 236 right lung is the largest and is composed of upper, middle, lower and infracardiac (or 6 of 21 accessory or azygous) lobes, and the left lung is composed of upper, middle (or lingula) and lower lobes [64–69]. However, in our study, the fissure for the middle left lobe was frequently However, inincomplete our study, or theeven fissureabsent. for the middle left lobe was frequently incomplete or We even absent. established the following measurements per each lesion: For micronodules, we considered We established the following measurements all lesionsper with eacha maximum lesion: axis ranging from 1 to 4.4 mm (which equateswe For micronodules, toconsidered a maximum allvolume lesions ofwith0.044 cm ). Their 3 a maximum length axis mainly ranging from falls 1 toin themm 4.4 granuloma’s (which equates size (1–3to amm), maximum but we also included volume cm3 ). Their of 0.044 rounded spherical lengthlesions mainlywithfalls a inmaximum axis under the granuloma’s size 4.5 (1–3mm,mm), as but theywetended to be stable. also included In contrast, rounded sphericalalmost lesionsallwith lesionsa above 4.5 mm increased or showed an instable size. We defined maximum axis under 4.5 mm, as they tended to be stable. In contrast, almost all lesions two subgroups: (a) Daughter above 4.5 mm micronodules, increased orfound showed around consolidations, an instable such astwo size. We defined satellites, that are subgroups: (a) presum- Daugh- ably ter generated from micronodules, foundthataround consolidated lesion [5,49,54]. consolidations, such as They are located satellites, thatwithin an annular are presumably zone, where generated fromthethat amplitude of this lesion consolidated circular[5,49,54]. crown corresponds to the consolidation’s They are located within an annular short axis; where zone, they were counted for the amplitude of each CT scancrown this circular (Figure 2). (b) Isolated corresponds to themicronodules, consolidation’s the ones short independent axis; they wereorcounted not associated for eachwith CT a consolidated scan (Figure 2).lesion (Figuremicronodules, (b) Isolated 3). The size and thenumber ones independent of them were ordetermined not associated with awithin together consolidated lesion (Figure 3). The their bronchopulmonary size and number segment. of them were determined Consolidations together are large within enough to their bronchopulmonary be followed segment. up and carefully tracked over con- Consolidations secutive scans, andare large their axisenough (mm) and to bevolume followed (cmup3) and were carefully measured tracked (Figureover 4).consecu- We con- tive scans,that andthese theirkinds axis (mm) and volume 3 ) were measured (Figure 4). We considered sidered of lesions modify (cmtheir size over time and are equal or bigger than that 4.5 these mm. kinds of lesions modify their size over time and are equal or bigger than 4.5 mm. Figure2.2.Consolidation Figure Consolidationwith withdaughter daughter micronodules. micronodules. Left Left picture picture shows shows original original CTCT image image fromfrom a a lesion located in the inferior lingular bronchopulmonary segment at 11th week scan in an unvac- lesion located in the inferior lingular bronchopulmonary segment at 11th week scan in an unvacci- cinated rhesus (RUV10). Right picture exposes that daughter micronodules are those situated within nated rhesus (RUV10). Right picture exposes that daughter micronodules are those situated within a a surrounding area that has the same length as the consolidation’s short axis. At same time, this Pathogens 2023, 12, x FOR PEER REVIEW surrounding areathe that has thecomplex same length as the of consolidation’s short axis. At same 7 of 22 time, this figure figure exhibits lesional consisting a consolidation with daughter micronodules. exhibits the lesional complex consisting of a consolidation with daughter micronodules. Figure 3. Isolated micronodules. Left picture shows several small isolated micronodules distributed Figure 3. Isolated micronodules. Left picture shows several small isolated micronodules distributed on both upper lung lobes in an intradermic-vaccinated rhesus (RIDV02) at 3rd week scan. Right on both upper lung lobes in an intradermic-vaccinated rhesus (RIDV02) at 3rd week scan. Right picture exhibits a large isolated micronodule (4.2 mm) located in the anterior bronchopulmonary picture segmentexhibits in the aleft large isolated upper micronodule lobe at (4.2inmm) 3rd week scan located in the anterior rhesus an intradermic-vaccinated bronchopulmonary (RIDV04). No- segment in the left upper lobe at 3rd week scan in an intradermic-vaccinated tice that nodular images not labelled correspond to pulmonary vessels. rhesus (RIDV04). Notice that nodular images not labelled correspond to pulmonary vessels. Figure 3. Isolated micronodules. Left picture shows several small isolated micronodules distributed on both upper lung lobes in an intradermic-vaccinated rhesus (RIDV02) at 3rd week scan. Right Pathogens 2023, 12, 236 picture exhibits a large isolated micronodule (4.2 mm) located in the anterior bronchopulmonary 7 of 21 segment in the left upper lobe at 3rd week scan in an intradermic-vaccinated rhesus (RIDV04). No- tice that nodular images not labelled correspond to pulmonary vessels. Figure4.4.CT Figure CTtridimensional tridimensionalevaluation. evaluation.Example Exampleofofaacavitated cavitatedconsolidation consolidationininan anintradermic- intradermic- vaccinated rhesus (RIDV02) at 16th week (pictures from left to right: axial, sagittal and coronal CT vaccinated rhesus (RIDV02) at 16th week (pictures from left to right: axial, sagittal and coronal CT scan planes). Its main axis measures 31.4 mm and its volume reaches 2.308 cm3. scan planes). Its main axis measures 31.4 mm and its volume reaches 2.308 cm3 . Cavitationsmust Cavitations musthavehaveaagreater greatersize sizethan thanthethesurrounding surroundingbronchial bronchialcalibre, calibre,for for avoiding misinterpretation with air bronchogram. It was assessed avoiding misinterpretation with air bronchogram. It was assessed qualitatively, being qualitatively, being pre- sent (yes) present oror (yes) absent absent(no). (no). In pleural distance measurementswe In pleural distance measurements wealso alsodetermined determinedthe thelung lungvolume volumefrom fromeach each animal, using the COPD tool from the Philips IntelliSpace Portal software, animal, using the COPD tool from the Philips IntelliSpace Portal software, which allowed which allowed theevaluation the evaluationofofthethemain mainairway airwayand andvolume volumefrom fromboth bothlungs lungsseparately. separately. WeWealso alsoobserved observed foci foci of of bronchocentric bronchocentric pneumonia, pneumonia, detected detected as aas a ‘tree-in-bud’ ‘tree-in-bud’ pat- pattern tern in CT images, which is a common CT finding in active lung in CT images, which is a common CT finding in active lung TB [58–61,70,71]. However, TB [58–61,70,71]. How- ever, we didwenotdid not include include them in them our in our analyses analyses due todue tolack their theiroflack of homogeneity homogeneity and insta- and instability, bility, which made them difficult to demarcate which made them difficult to demarcate and to follow-up. and to follow-up. RUV05presented RUV05 presentedwithwithmiliary miliarytuberculous tuberculousdisease diseaseand anddeveloped developedhundreds hundredsofoflunglung micronodulesdistributed micronodules distributed diffusely diffusely andand homogeneously homogeneously (as (as wellwell as extrapulmonary as extrapulmonary mil- miliary iary disease). disease). Such profuse Such profuse dissemination dissemination is well is well known known and hasand beenhas been described described in other in other studies asstudies as ‘widespread ‘widespread discrete pulmonary discrete pulmonary nodules’ nodules’ [63,72]. It is[63,72]. uncommonIt is uncommon and denotesand denotes extremely extremely uncontrolled infection with hematogenous dissemination. uncontrolled infection with hematogenous dissemination. Our study was focused Our study wasonfo- cused on pulmonary pulmonary pneumoniapneumonia and airwayand airway dissemination; dissemination; hence, we hence, removed wetheremoved the data data collected collected from from this this animal fromanimal from theevaluation the statistical statistical evaluation due to theTBdifferent due to the different disease.TB disease. Table 1. Distribution of the lesions, comprising all time points and experimental groups. Consolidations Daughter Micronodules Isolated Micronodules EG Median Range % Median Range % Median Range % CUV (n = 320) 2 [0 6] 9 5.5 [0 30] 23 5.5 [0 110] 68 CIDV (n = 22) 4 [0 6] 45 0 [0 1] 5 4 [2 5] 50 C (n = 342) 2 [0 6] 11 3 [0 30] 22 4 [0 110] 67 RUV (n = 871) 12.5 [1 25] 14 36 [2 96] 49 30 [1 83] 37 RAEV (n = 643) 14.5 [0 19] 12 54 [0 151] 66 18.5 [14 37] 22 RIDV (n = 697) 15 [12 24] 12 55 [45 138] 53 29 [21 117] 35 R (n = 2211) 14 [0 25] 13 51 [0 151] 55 29 [1 117] 32 TOTAL (n = 2553) 10.5 [0 25] 13 27.5 [0 151] 51 20 [0 117] 37 EG: Experimental group. CUV: unvaccinated cynomolgus; CIDV: intradermal-BCG-vaccinated cynomolgus; C: total cynomolgus; RUV: unvaccinated rhesus; RAEV: aerosol-BCG-vaccinated rhesus; RIDV: intradermal- BCG-vaccinated rhesus; R: total rhesus. n means the total number of lesions per each experimental group. Median shows the value separating the higher half from the lower half of the number of each type of lesion per experimental group. Pathogens 2023, 12, 236 8 of 21 Table 2. Distribution of micronodules, comprising all time points and experimental groups. Number (n) Fraction EG Total DM IM DM/IM CUV 293 75 218 0.34 CIDV 12 1 11 0.09 C 304 75 229 0.33 RUV 752 428 324 1.32 RAEV 564 424 140 3.03 RIDV 611 368 243 1.51 R 1924 1216 708 1.72 DM: Daughter micronodules; IM: isolated micronodules; EG: experimental group. CUV: unvaccinated cynomol- gus; CIDV: intradermal-BCG-vaccinated cynomolgus; C: total cynomolgus; RUV: unvaccinated rhesus; RAEV: aerosol-BCG-vaccinated rhesus; RIDV: intradermal-BCG-vaccinated rhesus; R: total rhesus. 3.2. Mtb-Induced Disease Is Worse in Rhesus Macaques In order to exemplify the complexity of the operational procedure, Figure 5 illustrates a particular evolution in one case (RIDV02). Overall development of the natural infection in control groups is summarized in Figure 6. The temporal evolution of the lesions is shown per animal, based on the size and localization per lobe in a 2D representation. Each pair of lungs is reshaped according to each individual volume (adapted to the image obtained from macaque RUV04). At first sight, this summary highlights the greater severity of the infection in rhesus. In particular, at week 3, cynomolgus developed a low number of lesions, with a small volume, which were usually well controlled and exhibited minimal enlargement with time. Lesions were not identified in one individual (CUV10), although infection was confirmed by other methodologies. Other individuals showed a decline in condition only at the late phase (15th week) of the study, e.g., CUV07, in which an extraordinary growth of consolidations was detected on CT images that was probably due to total main bronchus stenosis (despite it being uncommon in cynomolgus). In contrast, lesions in rhesus were abundant and larger at week 3, usually exhibiting enlargement and dissemination over time. Poor control leading to extreme disease was evident in RUV05 (which developed profuse miliary dissemination, described above) and also in RUV06 and RUV08; these animals had to be euthanized before finishing the monitoring period due to progressive disease that met humane endpoint criteria. However, there was at least one case in a rhesus where a spontaneous weak regression of lesions was observed (RUV10) (Figure 6). Cynomolgus macaques developed significantly fewer lung consolidations and mi- cronodules, resulting in a smaller pulmonary volume occupation than that seen in rhesus macaques, as shown in Table 1 and Figure 7. Cynomolgus also had a significantly lower percentage of daughter micronodules than rhesus (Tables 1 and 2, and Figure 8). The overall ratio of daughter/isolated micronodules for non-vaccinated macaques comprising all time points was 1.32 and 0.34 for rhesus and cynomolgus, respectively. Similarly, the number of consolidated lesions associated with daughter micronodules was also significantly lower in cynomolgus (Table 3). These data reflect a higher containment of lesions’ progression in cynomolgus. The size of consolidated lesions was highly dependent on the number of daughter micronodules. This is reflected in Figure 9 where a clear and significant correlation is noted between the size of the consolidated lesions and the number of daughter micronodules, regardless of the vaccination status. This positive slope is markedly higher in rhesus. In addition, a greater portion of occupied pulmonary volume is correlated to a higher number of isolated micronodules (Supplementary Figure S4), so the higher the infectious involve- Pathogens 2023, 12, 236 9 of 21 Pathogens 2023, 12, x FOR PEER REVIEW 9 of 22 ment there of waslungs, theone at least higher thea rate case in of where rhesus pulmonary dissemination a spontaneous (local and weak regression of endobronchial lesions reinfections, was observedwhich (RUV10)fits(Figure with the 6). Dynamic Hypothesis [52]). Figure 5. Figure 5. Pulmonary Pulmonary TB progression. Lesions from TB progression. 3rd to Lesions 16th 3rd from post-challenge week in an intrader- to 16th post-challenge week in an mic-vaccinated rhesus (RIDV02). (A): A 3rd week CT shows a lesion in the anterior bronchopulmo- intradermic-vaccinated rhesus (RIDV02). (A): A 3rd week CT shows a lesion in the anterior bron- nary segment in the right upper lobe that initially is a 2.1 mm isolated micronodule (light blue ar- chopulmonary segment rowhead). (B): An in the 11th week scanright upper exhibits lobe that initially its progression is a 2.1 mm to a consolidation isolated (dark micronodule blue arrow) and (light blue arrowhead). of the development (B): twoAndaughter 11th week scan exhibits micronodules its progression (yellow arrowhead). to(C): a consolidation CT performed(dark blue arrow) at 16th week and shows the a higher enlargement development of the consolidation, of two daughter micronodules in (yellow which arose a cavitation(C): arrowhead). (redCT arrow). performed at Moreover, some small isolated micronodules (green arrowheads) appeared distributed on both 16th week shows a higher enlargement of the consolidation, in which arose a cavitation (red arrow). lungs. Notice that nodular images not labelled correspond to pulmonary vessels. Moreover, some small isolated micronodules (green arrowheads) appeared distributed on both lungs. Notice that nodular images not labelled correspond to pulmonary vessels. Figure 6. Evolution of lesions in unvaccinated macaques. The size and location of TB lesions de- termined from computed tomography (CT) scan taken at specific times after initial infection are shown in each pair of lungs. Lesions (consolidations and micronodules), in black, are represented as spheres of the measured volume and located in the segment where they are identified. (A) In Figure 6. Evolution of lesions in unvaccinated macaques. The size and location of TB lesions blue, reconstructions of CT scans from each unvaccinated cynomolgus. (B) In red, reconstructions determined from computed of CT scan collected from each tomography (CT) scan unvaccinated rhesus. taken RUV05, Macaques at specific RUV06times andafter RUV08initial infection are marked shown in each with dagger pair of symbol lungs. were Lesionsbefore euthanized (consolidations and the end of the micronodules), in black, are represented as study. spheres of the measured volume and located in the segment where they are identified. (A) In blue, reconstructions of CT scans from each unvaccinated cynomolgus. (B) In red, reconstructions of CT scan collected from each unvaccinated rhesus. Macaques RUV05, RUV06 and RUV08 marked with dagger symbol were euthanized before the end of the study. cronodules, resulting in a smaller pulmonary volume occupation than that seen in rhesus macaques, as shown in Table 1 and Figure 7. Cynomolgus also had a significantly lower percentage of daughter micronodules than rhesus (Tables 1 and 2, and Figure 8). The overall ratio of daughter/isolated micronodules for non-vaccinated macaques comprising all time points was 1.32 and 0.34 for rhesus and cynomolgus, respectively. Similarly, the number of consolidated lesions associated with daughter micronodules was also signifi- Pathogens 2023, 12, 236 10 of 21 cantly lower in cynomolgus (Table 3). These data reflect a higher containment of lesions’ progression in cynomolgus. . Figure7.7.Analysis Figure Analysis of unvaccinated of unvaccinated macaques. macaques. (A) displays (A) displays the number the number of identified of identified consolidations consolidations for foreach eachmacaque macaque at at thethe endend of the of study. In light the study. In blue, light each blue,non-vaccinated cynomolgus each non-vaccinated (CUV) cynomolgus (CUV) macaque is represented. In blue, the mean number of lesions for CUV macaques. In light red, each macaque is represented. In blue, the mean number of lesions for CUV macaques. In light red, non-vaccinated rhesus (RUV) macaque is represented. In red, the mean number for RUV macaques. each non-vaccinated Dotted black line shows rhesus the mean (RUV) macaque value for is represented. all non-vaccinated In red, macaques. the mean (B) shows number for RUV the occupied macaques. lung fraction Dotted for eachblack line (C) macaque. shows showsthethemean number value for all non-vaccinated of isolated micronodules formacaques. (B) shows the each macaque. (D) shows the occupied lungfraction of consolidations fraction that present for each macaque. cavitation (C) shows thein any CT of number scan. E shows isolated the number for each micronodules of daughter micronodules for each macaque. In (B–E) each circle represents an experimental meas- macaque. (D) shows the fraction of consolidations that present cavitation in any CT scan. E shows the urement from computed tomography (CT) scans. Light lines represent the evolution for each ma- number caque. of daughter Darker micronodules lines are the for each mean lines’ value. macaque. Dotted Inused lines are (B–E) eachone when circle represents or more macaquesan are experimental measurement not from computed considered because they weretomography euthanized, or(CT) scans.ended the study Lightbefore lines represent the evolution that time point. Data is for each analysed macaque. through Darker one-way lines are ANOVA the meantest lines’ (** p < value. 0.01). Dotted lines are used when one or more macaques Pathogens 2023, 12, x FOR PEER REVIEW 11 of 22 are not considered because they were euthanized, or the study ended before that time point. Data is analysed through one-way ANOVA test (** p < 0.01). . Figure 8. Fraction of daughter micronodules. Number of total Figure 8. Fraction of daughter micronodules. Number of total isolated micronodulesisolated micronodules (NI) and num- (NI ) and number of total daughter micronodules ber of total daughter micronodules (ND) are shown (N ) are shown in grey. Total number D in grey. Total number of micronodules ofismicronodules com- is puted considering all CT scans independently. Total fractions are depicted in blue for unvaccinated computed considering all CT scans independently. Total fractions are depicted in blue for unvac- cynomolgus (CUV); in cyan, cinated cynomolgus (CUV);for in intradermal-vaccinated cynomolgus (CIDV); cyan, for intradermal-vaccinated in red, for cynomolgus unvac- in red, for (CIDV); cinated rhesus (RUV); in orange, for aerosol-vaccinated rhesus (RAEV); and in purple, for intrader- unvaccinatedrhesus mal-vaccinated rhesus(RIDV). (RUV); in orange, for aerosol-vaccinated rhesus (RAEV); and in purple, for intradermal-vaccinated rhesus (RIDV). Table 3. Distribution of consolidations associated with daughter micronodules, comprising all time points and experimental groups. Consolidations with DM DM per Consolidation EG n % Median Range Median Range CUV (n = 29) 19 66 2 [0 4] 3 [1 15] Pathogens 2023, 12, 236 11 of 21 Table 3. Distribution of consolidations associated with daughter micronodules, comprising all time points and experimental groups. Consolidations with DM DM per Consolidation EG n % Median Range Median Range CUV (n = 29) 19 66 2 [0 4] 3 [1 15] CIDV (n = 10) 1 10 0 [0 1] 1 [1 1] C (n = 39) 20 51 1 [0 4] 3 [1 15] RUV (n = 121) 111 92 11.5 [1 24] 3 [1 15] RAEV (n = 80) 79 99 14.5 [0 19] 3.5 [1 15] RIDV (n = 88) 84 95 14 [11 24] 3 [1 15] R (n = 289) 274 95 14 [0 24] 3 [1 15] Total (n = 328) 294 90 9 [0 24] 3 [1 15] Pathogens 2023, 12, x FOR PEER REVIEW 12 of 22 DM: Daughter micronodules. EG: Experimental group. CUV: unvaccinated cynomolgus; CIDV: intradermal- BCG-vaccinated cynomolgus; C: total cynomolgus; RUV: unvaccinated rhesus; RAEV: aerosol-BCG-vaccinated rhesus; RIDV: intradermal-BCG-vaccinated rhesus; R: total rhesus. n mean number of lesions. . Figure 9. Correlation between the volume of consolidated lesions and the number of daughter Figure 9. Correlation between the volume of consolidated lesions and the number of daughter Light circles micronodules. Light micronodules. circles represent represent aasingle singleconsolidation. consolidation.DarkDarkcircles circlesrepresent representthethe mean volume mean vol- valuevalue ume of allofconsolidations that that all consolidations present the same present the samenumber numberof daughter of daughtermicronodules. micronodules. Correlation Correla- value tion between value both between quantities both is written quantities is written r. r.The asas Thesignificance significanceof ofcorrelation correlation value is determined determined fromthe from p-valuethat thep-value thatisisshown shownunder undercorrelation correlationvalue, value, according according to to the the XXtest. test.In Inblue, blue,unvaccinated unvaccinated cynomolgus cynomolgus (CUV); (CUV); in cyan, intradermal-vaccinated intradermal-vaccinatedcynomolgus cynomolgus(CIDV); (CIDV);ininred, red,unvaccinated unvaccinated rhe- rhesus sus (RUV); in orange, aerosol-vaccinated rhesus (RAEV); in purple, intradermal-vaccinated (RUV); in orange, aerosol-vaccinated rhesus (RAEV); in purple, intradermal-vaccinated rhesus (RIDV); rhesus (RIDV); in grey, in allgrey, all macaques macaques (all). (all). We We also detected detected some sometransitional transitionallesions, lesions,which whichcorrespond correspond to to isolated isolated micronod- micronodules ules that that increased increased to ato a size size of more of more thanthan 4.44.4mm mm from from oneone CTCTto to the the next,thus next, thusbecoming becominga aconsolidation consolidation(Table (Supplementary 1). This enlargement occurred mainly in the early phasethe Table 1). This enlargement occurred mainly in early between phase between the third the third and eighth week and eighth week post-challenge. post-challenge. Remarkably, Remarkably, rhesus developed rhesus developed directly a large directly numberaof large number of consolidations consolidations in the early phase in the early after phase after infection, infection, in contrast toincynomolgus; contrast to cynomolgus; hence,lesions hence, transitional transitional lesions represent represent a higher impacta on higher impact on cynomolgus. cynomolgus. Importantly, Im-is there no significant portantly, theredifference betweendifference is no significant both macaque betweengenotypes considering both macaque the progression genotypes consideringof isolated the micronodules progression towards of isolated consolidated micronodules lesions. towards consolidated lesions. 3.3. BCG Vaccination in Rhesus Macaques Reduces the Number of Lesions, although Intradermal Vaccination Better Controls Daughter Micronodules Initial evaluation of the impact of vaccination in macaques suggested that a protec- tive effect appeared by the 3rd week post-challenge (Figures 6, 8 and 10). However, a Pathogens 2023, 12, 236 12 of 21 3.3. BCG Vaccination in Rhesus Macaques Reduces the Number of Lesions, Although Intradermal Vaccination Better Controls Daughter Micronodules Initial evaluation of the impact of vaccination in macaques suggested that a protective effect appeared by the 3rd week post-challenge (Figures 6, 8 and 10). However, a deeper analysis revealed that protection measured in terms of infection (i.e., occupied lung fraction at week 3), did not appear to provide a reliable indicator of later outcome (Supplementary Figures S5 and S6). When each disease volume fraction was normalized against the thogens 2023, 12, x FOR PEER REVIEW maximum infiltrated lung volume per macaque (Figure 11), it revealed a clear impact of the vaccination in cynomolgus macaques early after challenge, although it did not last until the end of the monitoring time. Figure 10. Lesions’ evolution in vaccinated macaques. Each pair of lungs show the size and location . of TB lesions determined from computed tomography (CT) scan taken after initial infection. Lesions Figure 10. Lesions’ evolution in vaccinated macaques. Each pair of lungs sh (consolidations and micronodules), in black, are represented as spheres of the measured volume and tion of located TBsegment in the lesions determined where from computed they were identified. (A) In cyan, thetomography reconstructions of(CT) lungs scan from taken Lesions (consolidations intradermal-vaccinated cynomolgus and micronodules), (CIDV) inInblack, for each CT scan. (B) orange, are represented the reconstructions of as sph lungs from aerosol-vaccinated rhesus (RAEV) for each CT scan. In purple, volume and located in the segment where they were identified. (A) In cyan, the reconstructions of lungs from intradermal-vaccinated rhesus (RIDV) for each CT scan. Macaque RAEV04 marked with dagger lungs from intradermal-vaccinated cynomolgus (CIDV) for each CT scan. (B symbol was euthanized before the planned end of study, according to the welfare monitoring plan. structions of lungs from aerosol-vaccinated rhesus (RAEV) for each CT scan structions of lungs from intradermal-vaccinated rhesus (RIDV) for each CT sc marked with dagger symbol was euthanized before the planned end of study fare monitoring plan. Lesions (consolidations and micronodules), in black, are represented as spheres of the measured volume and located in the segment where they were identified. (A) In cyan, the reconstructions of lungs from intradermal-vaccinated cynomolgus (CIDV) for each CT scan. (B) In orange, the recon- structions of lungs from aerosol-vaccinated rhesus (RAEV) for each CT scan. In purple, the recon- Pathogens 2023, 12, 236 structions of lungs from intradermal-vaccinated rhesus (RIDV) for each CT scan. Macaque RAEV04 13 of 21 marked with dagger symbol was euthanized before the planned end of study, according to the wel- fare monitoring plan. Figure Figure11.11.Evolution Evolutionof of normalized occupied normalized occupied lung lung volume volume perpereacheach individual individual and experimental and experimental group. group. Volume Volumeof ofeach each involved areaisisdivided involved area divided bybythethe maximum maximum occupied occupied lung lung volume volume reached reached bybyeach each individual. (A) Light blue lines indicate values for unvaccinated cynomolgus (CUV). Blue Blue individual. (A) Light blue lines indicate values for unvaccinated cynomolgus (CUV). line line indicatesthe indicates themean mean value ofall value of alllight lightblue blue lines. lines. (B)(B) shows shows light light red red lineslines for unvaccinated for unvaccinated rhesusrhesus (RUV). (RUV). Red Redline lineisisthe the mean valueofofall mean value alllight lightredred lines. lines. (C)(C) showsshows lightlight cyancyan lines lines for intradermal- for intradermal- vaccinated vaccinatedcynomolgus cynomolgus (CIDV). Cyanline (CIDV). Cyan lineshowsshows thethe meanmeanvaluevalue of alloflight all light blue lines. blue lines. (D) shows(D) shows light orange light orangelines arefor lines are foraerosol-vaccinated aerosol-vaccinated rhesus rhesus (RAEV). (RAEV). OrangeOrange line is theline meanis the mean value of allvalue light of all blue lines. (E) shows light purple lines are for intradermal-vaccinated rhesus (RIDV). Purple line is the mean value of all light blue lines. Dotted line appears for some lesions where data are not available due to study finalization or euthanized macaque. Due to the correlation between the volume of the consolidated lesions and the number of daughter micronodules, we investigated if vaccination had an impact on this fraction of micronodules in relation to the enlargement of consolidated lesions, and this was the case. Looking at Figure 8, vaccination clearly reduced this fraction in all vaccinated macaques, although temporally in rhesus, as it rapidly resumed by week 7 to 8. The reduction in this parameter only prevails in cynomolgus until the 15th week. Evaluation of the route of vaccination in rhesus revealed an interesting paradox. While aerosol vaccination (RAEV) reduced the total number of micronodules, more than intradermal vaccination (RIDV), a significantly greater fraction of daughter micronodules were present (3.03) (Table 4). Thus, the protective effect of RAEV relies on the reduction in the total number of micronodules (Tables 2 and 3) rather than the control of subsequent consolidation development. Even though the reduction in total micronodules was weaker in the RIDV, its effect was noted in both types of micronodules, even when this fraction was slightly higher in RIDV (1.51) than non-vaccinated (RUV) (1.32) (Table 2). At the end, this means that progression towards consolidation at a hypothetical later time point would be faster in the RAEV group, followed by RIDV and RUV. The effect of vaccination in rhesus was also observed as a decrease in the proportion of consolidated lesions that were associated with daughter micronodules, which was more pronounced and significant after intradermal (95%) than after aerosol vaccination (99%) (Tables 3 and 5). This effect was even more pronounced in cynomolgus, although in this case there was no variation in the proportion of isolated and daughter micronodules between vaccinated (intradermal) and unvaccinated groups (Tables 2 and 4). This can be explained because, as indicated above, cynomolgus are able to spontaneously stop the progression of the consolidated lesions soon after the infection (i.e., before week 3 after challenge). Pathogens 2023, 12, 236 14 of 21 Table 4. Differences in the proportion of daughter and isolated micronodules per experimental group. Contingency table. EG CUV CIDV C RUV RAEV RIDV R CUV ND CIDV 0.3057 ND C ND ND ND RUV ND ND ND ND RAEV ND ND ND <0.0001 ND RIDV ND ND ND 0.32431 <0.0001 ND R ND ND <0.0001 ND ND ND ND EG: Experimental group. CUV: unvaccinated cynomolgus; CIDV: intradermal-BCG-vaccinated cynomolgus; C: total cynomolgus; RUV: unvaccinated rhesus; RAEV: aerosol-BCG-vaccinated rhesus; RIDV: intradermal- BCG-vaccinated rhesus; R: total rhesus. Numbers correspond to p-values (Fisher’s test). Table 5. Differences in the proportion of consolidated lesions associated with daughter micronodules per experimental group. Contingency table. EG CUV CIDV C RUV RAEV RIDV R CUV ND CIDV 0.0014 ND C ND ND ND RUV ND ND ND ND RAEV ND ND ND 0.0585 ND RIDV ND ND ND 0.0355 >0.999 ND R ND ND <0.0001 ND ND ND ND EG: Experimental group. CUV: unvaccinated cynomolgus; CIDV: intradermal-BCG-vaccinated cynomolgus; C: total cynomolgus; RUV: unvaccinated rhesus; RAEV: aerosol-BCG-vaccinated rhesus; RIDV: intradermal- BCG-vaccinated rhesus; R: total rhesus. Numbers correspond to p-values (Fisher’s test). 3.4. Cavitation Is More Probable in Larger Lesions and Contact with Pleura Appear to Impact on TB Progression Cavitation occurred mostly in rhesus and especially at the late phase, where 34.4% of consolidated lesions developed cavities in non-vaccinated rhesus (RUV), closely followed by 28.4% of the aerosolized (RAEV) and by 24.3% of the ID (RIDV). Analysis of cavitation in rhesus (Figure 12) revealed a relationship with lesion size (p < 0.0001): the higher the consolidation volume, the higher the rate of cavitation, with cavitation more probable in lesions greater than 2 cm3 , which represents a rate of 57%. In contrast, almost 80% of lesions from 0 to 2 cm3 did not cavitate. The anatomic distribution of lesions was not found to influence cavity formation. We also evaluated the relationship between consolidations and pleura, in order to investigate if the contact with pleura favoured the encapsulation process of the lesions and stopped enlargement. As shown in Supplementary Figure S7, the group of smaller lesions (up to 0.5 cm3 , which accounts for the higher number of consolidations) were mainly not in contact with the pleura, especially in rhesus (which have bigger lungs), and from this size on, consolidations were mainly in contact with the pleura. The next step was to determine if consolidations changed between two consecutive CT scans, with respect to their size and distance from pleura. Thus, we evaluated the change in the volume together with the relationship with the contact with pleura. We explored the change in the relative volume gradient, explained through a boxplot (Figure 13). In this regard, differences are significant (p = 0.04 Kruskal–Wallis) in cynomolgus for those consolidations that make contact with the pleura, as they remain stable in volume. Thus, pleural contact is protective in cynomolgus, preventing the progression of consolidations, and this trend is irrespective of their vaccination status. In contrast, in rhesus, consolidations that enlarge and make contact with pleura do not experience a volume control because the increase in the size is too fast and large to benefit from the encapsulation process activated by the pleura. consolidated lesions developed cavities in non-vaccinated rhesus (RUV), closely followed by 28.4% of the aerosolized (RAEV) and by 24.3% of the ID (RIDV). Analysis of cavitation in rhesus (Figure 12) revealed a relationship with lesion size (p < 0.0001): the higher the consolidation volume, the higher the rate of cavitation, with cavitation more probable in lesions greater than 2 cm3, which represents a rate of 57%. In contrast, almost 80% of le- Pathogens 2023, 12, 236 15 of 21 sions from 0 to 2 cm3 did not cavitate. The anatomic distribution of lesions was not found to influence cavity formation. . Figure 12. Relation between the fraction and the volume of cavitated consolidations 16 Pathogens 2023, 12, x FOR PEER REVIEW in of rhesus 22 Figure 12. Relation macaques. between thevolume (A) Consolidation fraction and the volume distribution of cavitated for non-cavitated consolidations (light in rhesus grey) and cavitated (dark macaques. (A) Consolidation volume distribution for non-cavitated (light grey) and cavitated (dark grey) lesions. Vertical lines show the median values for each of the subsets. (B) Cavitation fraction grey) lesions. Vertical lines show the median values for each of the subsets. (B) Cavitation fraction for thebecause control consolidations that are in a desired the increase is range of volumes: 1 3cm3 , 1 from 0 to benefit to 2 cm3 , 3encapsula- to 4 cm3 , 4 to for the consolidations that are inina the sizerange desired toooffast and large volumes: 0 to 1tocm , 1 to 2 cm3, 3the to 4 cm3, 4 to 7 73 and cm 3 and consolidations bigger than 7 cm3 . The number of non-cavitated consolidations observed is tion cm process activatedbigger consolidations by thethan pleura. 7 cm3. The number of non-cavitated consolidations observed is shown shown in black, in black, at the at the top. top. In white, In white, at the at the bottom, bottom, thethe number number of cavitated of cavitated consolidations consolidations observed. observed. We also evaluated the relationship between consolidations and pleura, in order to investigate if the contact with pleura favoured the encapsulation process of the lesions and stopped enlargement. As shown in Supplementary Figure 7, the group of smaller le- sions (up to 0.5 cm3, which accounts for the higher number of consolidations) were mainly not in contact with the pleura, especially in rhesus (which have bigger lungs), and from this size on, consolidations were mainly in contact with the pleura. The next step was to determine if consolidations changed between two consecutive CT scans, with respect to their size and distance from pleura. Thus, we evaluated the change in the volume together with the relationship with the contact with pleura. We ex- plored the change in the relative volume gradient, explained through a boxplot (Figure 13). In this regard, differences are significant (p = 0.04 Kruskal–Wallis) in cynomolgus for those consolidations that make contact with the pleura, as they remain stable in volume. Thus, pleural contact is protective in cynomolgus, preventing the progression of consoli- dations, and this trend is irrespective of their vaccination status. In contrast, in rhesus, consolidations that enlarge and make contact with pleura do not experience a volume . Figure Figure 13.13. Volume Volume increase increase in in thethe consolidations consolidations in in relation relation withwith thethe contract contract withwith pleura. pleura. TheThe increase in in increase volume volume is expressed is expressed as as relative gradient relative perper gradient daydaybetween between twotwo consecutive consecutive CTCT scans. scans. Consolidations Consolidations areare classified between: classified between: notnot in contact (NC), in contact (NC),if in if both CTCT in both scans it isit not scans in in is not contact contact with withpleura; make pleura; makecontact (MC), contact if itifisit not (MC), in contact is not in the in contact first in the CTCT first butbut it isitin is contact in contact in the second; in the second; and in contact (IC), if in both CT scans it is in contact with pleura. It is indicated how many cases and in contact (IC), if in both CT scans it is in contact with pleura. It is indicated how many cases are identified for each classification. In blue, unvaccinated cynomolgus (CUV); in cyan, intradermal- are identified for each classification. In blue, unvaccinated cynomolgus (CUV); in cyan, intradermal- vaccinated cynomolgus (CIDV); in red, unvaccinated rhesus (RUV); in orange, aerosol-vaccinated vaccinated rhesus (RAEV); cynomolgus (CIDV); in red, unvaccinated in purple, intradermal-vaccinated rhesusrhesus (RIDV); (RUV); in all in grey, orange, aerosol-vaccinated macaques (all). rhesus (RAEV); in purple, intradermal-vaccinated rhesus (RIDV); in grey, all macaques (all). 4. Discussion 4. Discussion TBTBremains remains a major a majorkiller of of killer humankind, humankind, and thethe and lack of of lack a surrogate of of a surrogate protection, protection, means there is a constant need for the field to find experimental models able means there is a constant need for the field to find experimental models able to to elucidate elucidate new biomarkers and correlates to assist the development of new more effective vaccines. Nowadays, the macaque is considered the most clinically relevant experimental infection model of TB given its physiological closeness with humans and the similarity in disease manifestations. However, the macaque does lack the interlobular septa, present in human lungs, which have been demonstrated to provide a protective mechanism in the minipig Pathogens 2023, 12, 236 16 of 21 new biomarkers and correlates to assist the development of new more effective vaccines. Nowadays, the macaque is considered the most clinically relevant experimental infection model of TB given its physiological closeness with humans and the similarity in disease manifestations. However, the macaque does lack the interlobular septa, present in human lungs, which have been demonstrated to provide a protective mechanism in the minipig model [9]. The outcome of experimental infection with Mtb has been widely studied in macaques, and models established in rhesus and cynomolgus have been used to assess the efficacy of new drugs and vaccines. Mtb infection in cynomolgus resembles what occurs in humans, with the display of the whole spectrum of human TB, mainly controlling the infection and becoming latently infected. In contrast, rhesus experience a rapid dissem- ination of the infection in the lung, with the development of active TB in almost all the infected animals, a characteristic that enables the capacity of vaccines to protect against this progression to be assessed using manageable group sizes [73]. There are little data on vaccines showing significant protection against Mtb challenge in the macaque model with reports limited to BCG delivered intravenously (IV), for which the clinical deplorability is debatable, or a CMV-vectored vaccine [74] that is at the preclini- cal stage of development, and MTBVAC [28]. While there are very little data on immune responses induced in NHP and humans after exactly the same immunizations, the immune profiles induced following immunization with MTBVAC were shown to reflect those identi- fied in human clinical trials [75–77]. This concordance between immune profiles measured in clinical trials and a macaque preclinical assessment of a novel TB vaccine candidate, demonstrating significantly improved outcomes after Mtb challenge, is a promising indica- tion that the protection provided by MTBVAC vaccination to macaques will translate to the human population. The ability to demonstrate vaccine efficacy is critical, and the role of the macaque models in vaccine development is underpinned by the ability to measure vaccine-associated impacts on the disease that develops following infectious challenge. Our approach has been to test a new way to interpret data derived from these models based on the ‘Bubble Model’, that defines the progression from latent to active TB sup- ported by the interpretation of disease progression defined in the studies on the C3HeB/FeJ mice [49,54,78,79]. As with humans, the C3HeB/FeJ mouse model establishes that the de- velopment of a large lesion after the Mtb infection requires a strong inflammatory response in the initial lesions, the induction of daughter micronodules around them and the coales- cence of all lesions. To evaluate this concept in macaques, we used CT images obtained previously from studies using an experimental model of low-dose aerosol exposure, thus closely resembling what is thought to happen in the natural infectious process, where CT scans were collected at several time points to monitor the evolution of the infection in each animal [13,14,28]. In line with previous reports, three weeks after infection, more large lesions were identified in rhesus compared to cynomolgus, which has been related to the development of a stronger inflammatory response in the former [14,40]. These lead to an exudative lesion, a hallmark of progression towards active TB [51] and a key issue for its development, according to the bubble model. Thus far, the assessment of new vaccines has been mainly based on the induction of an immune response and a reduction in disease burden measured using pathology scoring, which included a semiquantitative approach of the involvement of lungs and other organs, as well as imaging-based measures through PET-CT [11,45], CT evaluation [28,80], histology-based measures (e.g., granuloma staging and prevalence), together with bacterial burden in organs and changes in clinical parameters. There is quite a lot of experience in using imaging as a tool to evaluate BCG efficacy in animal models, with special interest in NPH. Some studies appeared during the last few years that mainly focused on BCG alter- native routes. White et al. [28] analysed CT images in rhesus and used a quantitative score system for the estimation of TB-induced disease burden [63]; Sharpe et al. [81] and recently Sibley et al. [29] used MRI images in ex vivo lungs from macaques to determine pulmonary disease burden applying stereology [46], as well as counting discrete and coalescent lesions; Pathogens 2023, 12, 236 17 of 21 Darrah et al. [72] and DiFazio et al. [82] performed PET-CT images in macaques and mainly evaluated the grade of tuberculous activity by measuring global lung parenchyma FDG avidity, as well as counting lesions (which were called granulomas). Moreover, there are some experiences including other animal models, as Kraft et al. [83] analysed ex vivo lungs from guinea pigs using MRI, and described their imaging appearance, their anatomical distribution and nodule counting. Previous analysis of these images highlighted the early reduction in micronodules after BGC vaccination [28]. Our work was able to advance deeper in the exploitation of these data by differentiating the nodules related with the consolidated lesions, daughter micronodules, from the isolated ones. According to the ‘Bubble model’, daughter micronodules emerge from an initial isolated micronodule, increase in size, consolidate creating larger nodules, which in turn develop more daughter micronodules, a process that enlarges the lesion to the point that it becomes visible by chest X-ray, the hallmark for classifying an Mtb infection as active TB [51]. Data from the C3HeB/FeJ model suggests this progression mainly develops at the beginning of the infection when there is still a lower impact of the adaptive immune response. This is characterized by the infiltration of neutrophiles, which allows the extracellular growth of Mtb, fuelling enlargement and allowing the spread of close new infectious foci due to the drainage of infected foamy macrophages, which in turn attracts new neutrophils, causing the development of daughter lesions [54]. This led us to draw a parallel with the NHP model. On the one hand, rhesus develop highly inflammatory lesions with a high neutrophilic infiltration, a process that might be related with the progression associated to the development of daughter micronodules. Conversely, cynomolgus develop less inflammatory and more contained lesions [40], which would link to a reduced capacity to generate daughter micronodules. The lower inflammatory response, and thus the lower capacity to generate daughter cells, allows the fibroblasts to encapsulate the lesion, stopping its progression [9], and explains why the vicinity of the pleura, and its encapsulation capacity, has only a protective effect in cynomolgus. Our data provide two new measures of pulmonary disease burden induced following infection with Mtb, namely, the ratio daughter/isolated micronodules and the percentage of consolidated lesions linked to daughter micronodules identified through CT in macaques. These measures provided a new tool to evaluate the protective effect of the licenced vaccine (BCG) and revealed differences in disease progression following either intradermal or mucosal (inhaled) administration, even when both routes were able to reduce the number of consolidated lesions and generated a similar reduction in the number of micronodules. While this mucosal aerosol vaccination was better able to reduce the number of isolated micronodules, potentially because of a better capacity to reach and stimulate the immune response in the whole lung, intradermal administration should have an improved ability to reduce the emergence of daughter micronodules, which are the ones responsible for the progression towards active TB. In summary, we defined a new tool for the evaluation of pulmonary disease induced by Mtb infection, through the description of ‘daughter micronodules’, which provides a better understanding of the progression of TB and which can be applied for testing new vaccines. This is also amenable for development as a machine learning tool, as nodules are easily identifiable in CT images and can be quantified. Enhanced measures of TB disease burden and progression, such as those reported here, will refine the models used in the development of new vaccines and reduce the number of animals required, consequently providing important scientific and welfare benefits. Equally, the use of data across studies increased the power of the analysis applied and obtained more information without requiring the use of further macaques in line with the principles of the 3Rs. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pathogens12020236/s1. Figure S1: Experimental groups and CT scan acquisition schedule; Figure S2: Lung size in macaques; Figure S3: Occupied volume fraction per lung lobe; Figure S4: Correlation between the occupied lung fraction and the number of isolated micronodules; Table S1: Summary lesions found; Figure S5: Cynomolgus vaccine effect analysis; Pathogens 2023, 12, 236 18 of 21 Figure S6: Rhesus vaccine effect analysis; Figure S7: Volume distribution of consolidations in contact and not in contact with pleura. Supplementary File S2 contains the complete raw data in an excel file. Author Contributions: Conceptualization, I.N., M.C., P.-J.C. and C.V.; methodology, P.-J.C., C.P., J.B., I.N., M.C. and C.V.; software, J.B.; validation, S.A.S., A.D.W., J.B., C.V. and P.-J.C.; formal analysis, C.P.; investigation, I.N., M.C. and P.-J.C.; resources, S.A.S. and A.D.W.; data curation, C.P. and M.C.; writing—original draft preparation I.N. and M.C.; writing—review and editing, P.-J.C. and S.A.S.; visualization, J.B.; supervision, P.-J.C. and C.P.; project administration, C.V., P.-J.C. and J.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by ‘La Caixa’ Foundation (ID 100010434), under agreement LCF/PR/GN16/10290002 and AGAUR 2017 SGR 00500, 2021 SGR 00920 and 2021 SGR 00934. Institutional Review Board Statement: The study design and all procedures were approved by the Public Health England, Porton Down Animal Welfare and Ethical Review Body, and authorised under an appropriate UK Home project license. 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