Bactericidal effect of BPaL and BPaS in TB mouse models after 4-weeks of treatment

Balb/c and C3HeB/FeJ female mice were chronically infected with a low dose aerosol infection of Mtb Erdman strain to deliver ∼75 and ∼100 bacilli respectively. Post-infection, Balb/c and C3HeB/FeJ mice were rested for 4 and 8-9 weeks respectively until they were randomly assigned to the study groups. The mice were treated with monotherapy of linezolid or spectinamide 1599 or combination therapy of BPaL, BPa or BPaS for 4 weeks. Bedaquiline (B), pretomanid (Pa) and linezolid (L) were administered at 25, 100 and 100 mg/kg respectively by oral gavage for 5/7 a week while spectinamide 1599 at 50 and 100 mg/kg in Balb/c and C3HeB/FeJ TB models respectively for 3/7 a week on the alternate days via intrapulmonary aerosol delivery. On the third day of the last treatment, the mice were euthanized, and their lungs and spleen were collected. The organs were homogenized, serially diluted and plated on 7H11 agar with 4% charcoal (to avoid drug carry-over effect) to determine bacterial burden in the form of colony forming units (CFU) in each sample. CFU were enumerated after 4-6 weeks of incubation at 37 °C and expressed as log10.C3HeB/FeJ and Balb/c TB models showing efficacy of monotherapy (A-D) and combination therapy (E-H). The C3HeB/FeJ graphs (E&G) represent the pooled data from three independent studies (n=3-8 each, Figures S1-3) and two of the three studies contained BPa as a reference control. The Balb/c graphs (F&H) represent the pooled data from two independent studies (n=5 each, Figures S7-8). Statistical significance was calculated using one-way ANOVA with Tukey’s multiple comparison test, p < 0.05 was considered significant and ** = p<0.001, *** = p<0.0001, **** = P<0.0001. UnRx = untreated, L = linezolid, S = spectinamide 1599, LOD: limit of detection.

Effect of therapy on the live body weight and lung histopathology of TB mouse models after 4-weeks of treatment

The live body weight of animals in Figure 1 treated with monotherapy (A&B) or multidrug therapy (C&D) was recorded on a weekly basis. The average weight and SEM of each group is shown. At the end of therapy, the mice were euthanized, and their lungs were collected and processed for histopathology and lesion scoring (E-H). FFPE sections were cut at 5 µm, stained with hematoxylin and eosin (H&E) and imaged at 40x (E&G). Lesion scores (F&H) were calculated as the proportion of infected area over the total lung area per animal. Statistical significance was calculated using mixed-effect model (body weight) and one-way ANOVA (lesion score) with Tukey’s multiple comparison test, and p < 0.05 was considered significant and ** = p<0.001, *** = P<0.0001. UnRx = untreated, L = linezolid, S = spectinamide 1599

Complete blood count profiling and bone marrow histopathology of Mtb infected C3HeB/FeJ TB model during 4-weeks of therapy

During therapy of mice in Figure 1, the blood of live animals was collected at 1- and 2-weeks via the puncture of submandibular vein. At the end of 4-weeks therapy, the blood was collected by cardiac puncture. Blood was collected in K2-EDTA (purple top) tube and immediately analyzed for the complete blood count in VETSCAN® HM5 hematology analyzer (Zoetis). (A) monotherapy, (B) multidrug therapy. The MCHC (mean corpuscular hemoglobin concentration) and RDWs (red blood cell distribution width-standard deviation) along with the HGB (hemoglobin concentration) and MPV (mean platelet volume) are shown. A horizontal dotted line indicates the lower end of the reference interval for C3HeB/FeJ mice.

The sternum, femur and tibias bones from each mouse were collected, fixed in 4% PFA, and processed for histology (C&D). Sections were cut at 5 µm, stained with hematoxylin and eosin (H&E) and imaged at 40x. (C) Representative photos of bone marrow sections showing myeloid (black arrows) and erythroid (white arrows) cells in bone marrow of untreated (UnRx) and treatment (BPa, BPaS and BPaS) groups. (D) The number of myeloid (M) and erythroid (E) among a total of 300 cells in 5 different regions were counted for each group and M:E was calculated. Statistical significance was calculated using one-way ANOVA with Tukey’s test for multiple comparisons. p < 0.05 was considered significant and ** = p<0.001, *** = P<0.0001.

Bone marrow cytokine and chemokine profiling in Mtb infected C3HeB/FeJ TB model after 4-weeks of treatment

Femur bones from selected studies in mice in Figure 1 were collected to harvest the bone marrow. The bone marrow was resuspended in PBS, centrifuged and the supernatant was collected for the evaluation of cytokine’s content. BPaL and BPaS therapy showed profile of 26 cytokines and chemokines in bone marrow and the data were converted to Z score and represented as a heatmap (A) and graphically (B). (C) Spearman’s correlation analysis of bone marrow cytokines and chemokines with the lung bacterial burden (CFU).

Statistical significance was calculated using the t test. p < 0.05 was considered significant and ** = p<0.001*** = p<0.0001, **** = P<0.00001.

Immune cell populations in the bone marrow, lung and blood of C3HeB/FeJ TB model after 4-weeks of treatment

The bone marrow, lung and blood from selected studies from Figure 1 were evaluated by flow cytometry. The samples were processed for a panel of 17-color antibodies and the data were analyzed by FlowJo software using manual gating strategy. The myeloid and lymphoid phenotypes present in the untreated (UnRx) and treatment (BPa, BPaL or BPaS) groups are shown. Statistical significance was calculated using one-way ANOVA with Tukey’s test for multiple comparisons. p < 0.05 was considered significant and ** = p<0.001*** = p<0.0001, **** = P<0.00001.

Immune cell populations in the lungs of Mtb infected C3HeB/FeJ TB model after 4-weeks of treatment

Selected mice from those shown in Figure 1 were processed for multiplex fluorescent immunohistochemistry (mfIHC). The mfIHC was performed for a panel of 6-color antibodies + DAPI using Opal-plex Tyramide Signal Amplification (TSA). Slides were scanned using multispectral automated PhenoImagerTM (Akoya Biosciences) and analyzed for different immune cell populations using the inForm® tissue Finder and Phenochart software (Akoya Biosciences). (A) The lung mfIHC full composite image displays B220, CD4, CD8, F4/80, FoxP3 and Ly6G markers along with DAPI staining for nuclei in the TB granuloma. The central and peripheral regions of a TB granuloma and the parenchyma of lung are also shown. (B) Single color composite image of individual markers with DAPI showing distribution of each immune cell population in the TB granuloma. (C) Cell populations (%) of several immune cells per total number of phenotypes calculated in untreated (UnRx) and treatment (BPaL and BPaS) groups based on a panel of 6-color antibodies + DAPI. (D) Spearman’s correlation matrix for several immune cell populations (B220, CD4, CD8, F4/80, FoxP3, Ly6G) showing all relationships. A coefficient with a value of either +1 (blue), 0 (white), or −1 (red) indicates a perfect association, no association, and a perfect negative association of ranks, respectively. Numbers indicate the correlation coefficient.