Mycobacterium bovis BCG Pasteur ATCC 35734 (hereafter referred to as BCG), or its isogenic derivative, second generation M. bovis BCGΔBCG1419c11 were cultured in Middlebrook 7H9 broth supplemented with 0.2% glycerol, 10% OADC, and 0.05% Tween 80, at 37 °C, 5% CO2, 100 rpm, up to an OD600nm ≈ 0.8, and used for safety studies in mice and guinea pigs, evaluation of antibody response in guinea pigs, RNA isolation as described10 and real time qPCR assays to determine relative expression of the sigA and BCG1419c genes to that of rrs as recently reported33. For evaluation of efficacy in guinea pigs, BCG and BCGΔBCG1419c were cultured in Proskauer and Beck (P&B) medium with 0.1% Tween 80 to mid-log phase. Aliquots were stored at − 80 °C and thawed before use. M. tuberculosis H37Rv (TMCC 102) was initially grown for three passages as a pellicle on P&B medium to produce seed stocks. Working stocks with a maximum of six passages were expanded from the seed stocks in P&B medium with 0.1% Tween 80. Working stocks were prepared at the mid-log phase, and aliquots were stored at − 80 °C and thawed before use.
Macrophage infection and assessment of replication of the BCG strains
This was performed essentially as described12. Briefly, 10,000 RAW 264.7 cells per well were grown in 96-well plates in 200 μL DMEM 2% FBS and antibiotic–antimycotic solution 1×. Once removed this media, cells were infected with 100 μL of BCG Pasteur or BCGΔBCG1419c, suspended in DMEM 2% FBS (MOI = 10). The infected macrophages were incubated at 35 °C with 5% CO2 for 6 h (considered as time zero post-infection). Then, the macrophages were washed three times with PBS solution, and 200 μL per well of fresh DMEM 2% FBS with antibiotic–antimycotic solution 1× were added to further incubate the cells. Samples were taken at 24 and 72 h post-infection. The assay was repeated with 3 replicates by infection/sample, in two independent experiments (n = 6 samples/BCG strain). To determine CFUs at each time point, we added 100 μL per well of 0.1% Triton X-100 and incubated at 37 °C in 5% CO2 for 15 min. The lysates were serially diluted into 0.05% PBS-Tween 20 solution, and 10 μL were inoculated onto 7H10 OADC agar plates, in duplicate. The plates were incubated at 37 °C in 5% CO2 for 4 weeks to perform the corresponding colonies enumeration.
Safety studies in mice
Pathogen-free, 8–9 weeks old, female BALB/c, C57BL/6, and athymic (CAnN.Cg-Foxn1nu/Crl) nude mice were obtained from Bioterio Morelos (Mexico). Mice were maintained in vented cages with high-efficiency air-filters, with food and water ad libitum. Mice were randomly allocated to receive either BCG or BCGΔBCG1419c. BALB/c and C57BL/6 mice (n = 10/group) were immunized subcutaneously in the base of the tail with 2.5–2.7 × 107 Colony Forming Units (CFU) of either BCG or BCGΔBCG1419c, suspended in 100 μL of sterile saline. The dose recommended for BCG in humans is 1–8 × 105 CFU, then we used between 30-250 times the equivalent of a human dose. Weight was registered on a weekly basis up to 2 months post-vaccination, where CFU were determined in lungs and spleens, after euthanasia by cervical dislocation. For CFU enumeration, whole lungs or spleens from 5 mice per group were lysed and serial dilutions of the lysates were used for culture onto 7H9 OADC plates with 0.5% glycerol, incubated for 4 weeks at 37 °C, 5% CO2.
For safety studies in immunocompromised hosts, athymic mice (n = 10) received a single intravenous dose of 1 × 106 CFU of either BCG or BCGΔBCG1419c, suspended in 100 μL of sterile saline. The primary endpoint of the experiment was defined as survival up to 180 days post-inoculation of animals vaccinated with BCG compared with those receiving BCGΔBCG1419c. At this time, surviving animals were humanely euthanized and bacterial load in lungs and spleen was quantified, as secondary endpoint. As a measure of health, weight was registered on a weekly basis. When any individual mouse reached a weight loss ≥ 20% of its maximum value, this was used as humane endpoint for euthanasia, by cervical dislocation. Whole lungs and spleens were lysed with Polytron (Kinematica, Luzern, Switzerland) in isotonic saline, and four dilutions of each homogenate were spread onto duplicate plates containing Middlebrook 7H10 agar (Difco Labs, Detroit MI, USA) enriched with 10% OADC. Plates were incubated for 4 weeks at 37 °C, 5% CO2. Weight registry and CFU enumeration were performed in a blind manner with respect to what BCG was applied to each animal.
Excessive dermal reactivity in Guinea pigs
This assay was conducted as reported by Fitzpatrick et al.34. Briefly, for each of the BCG strains tested, a group of six pathogen-free, female outbred Hartley guinea pigs (200–250 g) was used (12 animals total), which were obtained from Bioterio Morelos (Mexico). Animals were maintained in vented cages with high-efficiency air-filters, with food and water ad libitum. Each guinea pig was injected intradermally with 104, 105, and 106 CFU of BCG or BCGΔBCG1419c. A randomization plan was used to determine the injection sites for the doses (front, middle, or hind on either left or right flank of each guinea pig). The endpoint of the experiment was defined as the size of the lesions formed at the site of injection with BCGΔBCG1419c compared with that of BCG application, which were observed for 30 days and registered at regular intervals as indicated in “Results”. For each animal, the papule sizes induced by the BCG strains were compared at each dose applied. At the end of the experiment (30 days post-vaccination), animals were euthanized with sodium pentobarbital (150 mg/kg, intravenous, i.v.), The operator registering papule sizes was without prior knowledge of the BCG strain being applied at each site.
Absence of virulent mycobacteria in Guinea pigs
This assay was conducted as reported by Fitzpatrick et al.34. Briefly, groups of six pathogen-free, female outbred Hartley guinea pigs (weight range 200–250 g) were used, which were obtained from Bioterio Morelos (Mexico). Animals were maintained in vented cages with high-efficiency air-filters, with food and water ad libitum. Each guinea pig was injected intramuscularly with 1.5 × 107 CFU of either BCG or BCGΔBCG1419c (appromiately 100X the dose used in humans). Animals were weighed weekly and observed daily for 42 days for any TB symptoms. The primary endpoint of the experiment was defined as signs of TB symptoms, which will result in weight loss. As secondary endpoint, we determined lung pathology at 42 days post-vaccination in animals that received these BCG strains. At the end of this time, animals were euthanized with sodium pentobarbital (150 mg/kg, i.v.), and examined by necropsy. For pathological evaluation, both lungs were obtained from 4 guinea pigs and were perfused with 10% formalin in PBS pH 7.2. Once fixed, lungs were included in paraffin blocks and histological sections of 4 μM were obtained for both lungs and stained with Hematoxylin and Eosin (H&E). Histological and morphometric analysis was performed on these preparations by determining the percentage of pneumonic involvement using the Leica Application Suite v4.3 software. Pneumonic involvement is calculated as follows: the total area of the histological section is measured; this gives us a total area in square microns (μM2). Then, the area covered by the large blood vessels, bronchi and bronchioles is measured, these are considered spaces where there is no lung tissue (that is, the space of those vessels and bronchi), the result of this measurement is subtracted from the area of all the tissue previously measured, and this gives us a lung area that we consider as 100%.
Then, the area occupied by pneumonia is measured, defined as the cluster of proinflammatory cells (macrophages, neutrophils, lymphocytes, monocytes, epithelioid cells, and protein secretions) with undefined borders, which affect the lung structure, preventing adequate gas exchange. The percentage of pneumonia is then calculated with respect to the total area, per sample. Weight registry, histological and morphometric analysis were performed in a blind manner with respect to what BCG was applied to each animal.
Recombinant protein expression and purification
E. coli BL21(DE3) harboring pMRLB.41 was cultured in 500 mL of LB /Amp (100 μg/mL) at 37 °C with shaking at 260 rpm until reaching an OD600 = 0.5, to add sterile IPTG (Sigma, USA) at a final concentration of 0.5 mM, followed by further incubation at 37 °C, 300 rpm for 4–6 h. Cells were pelleted by centrifugation at 4 °C, 10,000 rpm for 20 min, to store the pellet at − 20 °C until lysis. For bacterial lysis, 5 mL of binding buffer (20 mM Tris–HCl, 500 mM NaCl, 5 mM Imidazole, pH 7.9) were added per each gram of cell pellet, which was resuspended and sonicated with a probe (Sonics, USA) at level 5 for 15 s, with 40 s rest, for 15 cycles. After lysis, the material was centrifuged at 10,000 rpm for 20 min, recovering the supernatant and transferring it to a new 50 mL tube for a second round of centrifugation to remove debris and produce the soluble fraction, to which Protease inhibitor cocktail (Sigma, EU) was added at a final concentration of 0.1× and store at − 20 °C until use.
For purification of His-tagged, recombinant Ag85A, 1 mL of HisPur ™ Ni-NTA Resin (Thermo Fisher Scientific, USA, Catalog 88221) was placed in a column (BioRad, USA), equilibrated with 10 column volumes (CV) of binding buffer and the resin was incubated with the soluble fraction at 4 °C with gentle shaking overnight. The resin with bound protein was washed with 6 CV of washing buffer (20 mM Tris–HCl, 500 mM NaCl, 60 mM Imidazole, pH 7.9), followed by 10 CV of 10 mM Tris–HCl. Next, 10 CV of 0.5% of ASB-14 in 10 mM Tris–HCl were used to remove endotoxins, followed by 10 CV of 10 mM Tris–HCl to remove residual ASB-14. To recover the purified recombinant proteins, five steps of elution were performed, each one where 1 mL of elution buffer (10 mM Tris–HCl, 1 M Imidazole, pH 8) was incubated with the resin for 5 min, to elute and recover in 1.5 mL tubes. Presence and apparent purity of the recombinant proteins within the eluted fractions were confirmed by SDS-PAGE in 15% gels stained with brilliant blue G (Sigma, USA). Fractions presenting the highest abundance and purity of the recombinant protein were combined and dialyzed using a membrane (SnakeSkin™ Dialysis Tubing, 10 K MWCO, 22 mm) (Thermo Scientific, EU), immersed in 1 L of 1X PBS (in MilliQ water) and incubated overnight at 4 °C with gentle shaking, with two buffer exchanges every 2 h. Aliquots of dialyzed proteins were quantified in triplicate using the micro BCA kit (BioVision, EU, Catalog K813), with samples read at 595 nm in a 680 XR microplate reader (BioRad, EU), by comparison with a standard curve of BSA (Sigma, USA, from 3 to 1500 μg/mL). LPS content was verified with the Pierce Chromogenic Endotoxin Quantitation Kit (Thermo Fisher, Cat A39553) according to the manufacturer’s instructions, and found to be 0.7 EU for Ag85A in 50 ng of its His-tagged recombinant forms. 100 μL aliquots of purified and dialyzed proteins were stored at − 20 °C until further use, to avoid repeated freeze–thaw cycles.
Evaluation of antibody response in vaccinated guinea pigs
Three pathogen-free, female outbred Hartley guinea pigs (weight range 200–250 g) were used per group, which were obtained from Bioterio Morelos (Mexico). Animals were maintained in vented cages with high-efficiency air-filters, with food and water ad libitum. Each guinea pig was injected intradermally with 103 CFU of either BCG or BCGΔBCG1419c. After 70 days, blood was drawn to obtain sera. The primary endpoint of the experiment was defined as anti-PPD IgG and anti-Ag85A levels in guinea pigs vaccinated with BCG compared with those receiving BCGΔBCG1419c. For this, IgG antibodies were detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). For this, 50 ng of either bovine PPD (PRONABIVE, obtained from Mycobacterium bovis AN5) or His-tagged Ag85A, were added to each well of Costar 96 well plates (Corning, USA, catalog 3590) and incubated at 37 °C for 1 h. Then, 150 µL of 3% BSA in PBS 1× were placed for incubation at 37ºC for 1 h. This blocking solution was discarded and then 50 µL of guinea pig sera (1:5 in PBS 1×) were added and incubated at 37 °C for 1 h. Each well was washed three times with 200 µL of PBST (PBS 1×, 0.05% Tween 20), to add 50 µL of an HRP-conjugated, rabbit anti-guinea pig IgG secondary antibody (Invitrogen, USA, catalog 61-4620) diluted 1:2000 in 1% BSA-PBS 1×, to further incubate at 37 °C for 1 h. After washing three times, 50 µL of 1-Step TM Ultra TMB-ELISA (Thermo Scientific, USA, catalog 34028) were added. Five minutes after adding the substrate, 50 µL of sulfuric acid 0.5 M were added to stop the reaction, and the optical density (OD) at 450 nm was determined using a microplate spectrophotometer (xMark Biorad, USA). ELISA analysis was performed in a blind manner with respect to what BCG was applied to each animal. Values reported are after subtracting the OD450 of control wells with no sera added.
Protection in the Guinea pig aerosol challenge model
Outbred Hartley guinea pigs were maintained in an ABSL-3 facility at Colorado State University, with sterile chow and water ad libitum. Five or ten animals per group, depending on the group (approximately 450–500 g, Charles River Laboratory) were injected subcutaneously with a microchip for identification and assessment of body temperature, which was measured to track the clinical progression of the disease. The microchip implant (IPT- 300 Bio Medic Data Systems [BMDS], Inc., Seaford, DE) allowed daily measurement of body temperature and also carried information about experiment number and animal number. The body temperature of individual guinea pigs was assessed each weekday at approximately the same time using a DAS-6006/7 scanner transponder (BMDS). Guinea pigs were placed into groups of five or ten, with control group receiving sterile pyrogen-free saline, while experimental groups received 103 CFU via the intradermal route of BCG or BCGΔBCG1419c.). At this time post-vaccination, guinea pigs were infected with a low dose aerosol (10–20 CFU) of virulent M. tuberculosis H37Rv using the Madison Aerosol Exposure Chamber (University of Wisconsin, Madison, WI). The body weight of each guinea pig was assessed weekly. The endpoint of the experiment was defined as protection against M. tuberculosis challenge conferred in vaccinated versus non-vaccinated animals. For this, at day 40 post-infection and upon necropsy, CFU, pathology (lung, spleen, and liver), and cytokine ELISA were determined.
CFU, pathology assessment in infected organs, and cytokine ELISA in lungs
The right caudal lobe of the lung and a piece of the spleen and liver from the guinea pig was utilized to analyze pathological lesions. The excised lung lobe was inflated with formalin and placed in total into formalin. For processing, the lung lobe, spleen, and liver were embedded in paraffin and sections cut and stained with H & E. A pathologist examined the sections, without prior knowledge of the groups, and provided a score based on the extent of lung involvement, fibrosis, and lesion type. The right cranial lobe of the lung and half of the spleen were sampled to assess CFU numbers. Bacterial load was determined by plating serial dilutions of organ homogenates onto nutrient 7H11 agar supplemented with OADC. Colonies were enumerated after 3 weeks of incubation at 37 °C and are expressed as log10 transformed data. For lung, spleen, and liver pathology, we used a score based on the extent of organ involvement, inflammation, granuloma formation, necrosis, mineralization, and fibrosis and lesion type as previously described in detail35. Lung homogenates that were used for determining CFU were also used to assess IFN-γ, TNF-α, IL-17 and IL-10. IL-17 and TNF-α kits were purchased form Kingfisher Biotech (Saint Paul, MN) and the IL-10 kit was purchased from Reddot (Kelowna, BC, Canada).
For safety experiments in mice and guinea pigs, the local animal ethics committee approved all experiments, which were performed following Mexican guidelines regarding ethical and safe handling of experimental animals such as: NOM-07- SEMARNAT-SSA1-2002, NOM-033-ZOO-1995, and NOM-062- ZOO-1999. Experiments with nu/nu mice and Hartley guinea pigs were conducted under permit 2020-008A. Experiments with BALB/c and C57BL/6 mice were conducted under permit 2020-001C. For experiments with guinea pigs at Colorado State University, the Institutional Animal Care and Use Committee (IACUC) approved all experimental procedures under permit IACUC: #1206.
Adherence to ARRIVE guidelines
All protocols involving animals were performed according to the ARRIVE guidelines 2.0 (https://arriveguidelines.org/arrive-guidelines), where the essential 10 and recommended set of details were indicated per specific experimental approach.
Unless indicated otherwise, data are presented as means with standard deviations, median and ranges, or median with standard deviation. Distribution of data was determined with the Shapiro–Wilk test. Comparison among groups were performed in Prism v.8, according to their distribution (normal or non-normal) and number of groups being compared. For comparison of CFU evaluated in RAW macrophages, in vitro replication, and safety experiments performed in mice and guinea pigs, an unpaired, two-tailed Student’s t test was used to compare data from 2 groups, with Welch’s correction when standard deviation from the groups was different. A Mann–Whitney test was used to compare 2 groups with a non-normal distribution, as it was for the real time qPCR comparison between BCG and BCGΔBCG1419c. For comparison of M. tuberculosis CFU obtained in vaccinated versus non-vaccinated guinea pigs, CFU were log10 transformed and compared with a Brown-Forsythe and Welch ANOVA test followed by Dunnett’s multiple comparison test, or a Kruskal–Wallis test. To compare weight gain/loss and papule sizes, in animals receiving BCG or BCGΔBCG1419c, as well as for OD600nm comparison, a multiple t tests corrected for multiple comparison with Holm–Sidak method at α = 0.05 was used. For comparison of survival curves, the Log-rank (Mantel-Cox) test was used. Group comparisons where p < 0.05 were considered different. Categorized data (histological analyses) were analyzed with a H Kruskal–Wallis test using SPSS version 25 (α = 0.05). In all cases, p value was adjusted for multiple comparisons with Bonferroni correction.