Bhagwati Khatri

The protective efficacy of the Bacillus Calmette-Guérin (BCG) vaccine against Mycobacterium tuberculosis (Mtb) is variable, and the systemic mechanisms linking cellular immunity with host metabolism remain poorly understood. This study employed high-dimensional mass cytometry (CyTOF) within the splenic compartment and serum lipidomics to map the integrated immuno-metabolic landscape in BCG-vaccinated and non-vaccinated mice following infection with the H37Rv strain. We report that BCG vaccination was associated with a distinct phenotype characterised by the priming of antimicrobial myeloid subsets (CD11b+ Mac2+/Mac3+) and lipidomic pathways away from stress-induced inflammatory responses. In contrast, non-vaccinated hosts exhibited a dysregulated response, characterised by a systemic cortisol surge, a lipidomic profile suggestive of reduced bioactive lipids (including PPARγ ligands such as 13-HODE and 19,20-DiHDPA), and the reactive mobilisation of CD103 + CD4+/CD8 + T cells and activated B cell subsets to the spleen. These findings reveal that protection is not solely cellular but relies on a stable metabolic environment that supports immune function. While infection in naive hosts drives a resource-depleting stress response that likely compromises macrophage efficacy, BCG vaccination establishes an integrated multiparametric defence, preventing pathogen-driven metabolic manipulation and maintaining the necessary lipid mediators for effective inflammation resolution and bacterial control.

This study focuses on harmonising the competition ELISA (cELISA) assay for Plasmodium falciparum ( P. falciparum ), using the 1 ^st WHO reference reagent for anti-malaria ( P. falciparum ) human reference serum (10/198). Antibody-mediated immune responses, against the Apical Membrane Antigen 1 (AMA1), play a significant role in protection against malaria. However, the sequence diversity of AMA1 and cross-reactivity among variants pose challenges in assessing antibody responses. To address this, the cELISA assay was selected to examine cross-reactive antibody responses against different variants. The harmonisation process for cELISA was performed in three laboratories. The 10/198 served as an internal standard for the calculation of IgG concentrations in the cELISA using ADAMSEL software. Additionally, a novel semi-automated analytical tool was developed in the R-statistics environment. This tool is freely available for download and streamlines result in generation while minimising human error. This study demonstrated the effectiveness of the 1 ^st WHO reference reagent as a standard for ELISA. Additionally, the ADAMSEL software and R-platform tool provide a user-friendly and accessible tool for the analysis of cELISA data. Its automation capabilities improve efficiency and ensure global accessibility at no cost, benefitting laboratories with limited resources. Importance: This study focuses on the 1 ^st WHO reference reagent (10/198) in harmonizing the competition ELISA (cELISA) assay for P. falciparum. In addition, the introduction of ADAMSEL software for cELISA data analysis, coupled with a novel R-platform tool, available for free, simplifies analytical processes and contributes to global accessibility. This study demonstrates the 1 ^st WHO reference reagent’s efficacy as a competition ELISA standard.

In this study, we report the development of a tuberculosis meningitis (TBM) mouse model using the HN878 strain via the aerosol route. Three genetically different strains of mice, CB6F1, C57BL/6 and BALB/c mice, were used to determine the dissemination of HN878 in the brain. The 8 x 10 ⁸ CFU/ml of HN878 dose was used to infect CB6F1 mice and deposited approximately 3.8 (0.07, SD) log ₁₀ CFU in the lungs. The burden of HN878 in the brain of the control group (administered saline) after approximately 16/17 days post-infection for CB6F1, C57BL/6 and BALB/c were 4.00 (0.47 SD), 3.79 (0.27, SD) and 2.12 (0.41, SD) log ₁₀ CFU/brain, respectively. The log ₁₀ CFU/brain in the BCG vaccinated CB6F1, C57BL/6 and BALB/c mice were 1.05 (0.61, SD), 2.13 (0.33, SD) and 1.42 (0.38, SD) respectively, which, if compared to the control groups, BCG vaccinated mice inhibited dissemination of HN878 in the brain by an impressive 2.94 (CB6F1), 1.66 (C57BL/6) and 0.69 (BALB/c) log ₁₀ CFU/brain reduction. In conclusion, we have established a relatively inexpensive TBM mouse model using an aerosol, a natural route of infection, which will advance research in understanding TBM dissemination to the brain, and preclinical tuberculosis vaccine/drug discovery/drug regimens against TBM.

Two strains of mice (BALB/c and CB6F1) were vaccinated with a range of Bacille Calmette-Guérin (BCG) Danish doses from 3 × 10⁵ to 30 CFU/mouse, followed by aerosol infection with Mtb (H37Rv or West-Beijing HN878 strain). The results indicated that both strains of mice when infected with HN878 exhibited significant protection in their lungs with BCG doses at 3 × 10⁵—3000 CFU (BALB/c) and 3 × 10⁵—300 CFU (CB6F1). Whereas, a significant protection was seen in both strains of mice with BCG doses at 3 × 10⁵—300 CFU when infected with H37Rv. A significant increase in the frequencies of BCG-specific IFNγ⁺ IL2⁺ TNFα⁺ CD4 T cells in the BCG doses at 3 × 10⁵—3000 CFU (BALB/c) and 3 × 10⁵—300 CFU (CB6F1) was seen. The IFNγ⁺ IL2⁺ TNFα⁺ CD4 T cells correlated with the Mtb burden in the lungs of HN878 infected mice (BALB/c and CB6F1) whereas, IFNγ⁺ TNFα⁺ CD4 T cells correlated with the BALB/c mice infected with H37Rv or HN878. The BCG dose at 3000 CFU (an equivalent single human dose in the mice by body weight) is protective in both strains of mice infected with H37Rv or HN878 and may serve an interesting dose to test new TB vaccine in a preclinical animal model.

In this study, 2 strains of mice (BALB/c and CB6F1) were vaccinated with a range of Bacille Calmette-Guérin (BCG) Danish doses from 3×10 ⁵ to 30 CFU/mouse, followed by either immunogenicity evaluation or aerosol infection with Mycobacterium tuberculosis (a laboratory strain H37Rv or West-Beijing HN878 strain). The results indicated that both strains of mice when infected with HN878 exhibited significant protection in their lungs with BCG doses at 3×10 ⁵ – 3000 CFU (BALB/c) and 3×10 ⁵ -300 CFU (CB6F1). Whereas, both strains of mice when infected with H37Rv, significant protection was seen in BCG doses at 3×10 ⁵ - 300 CFU. Immunological evaluation revealed interesting results; i) both strains of mice demonstrated a significant increase in the frequencies of BCG-specific IFNγ ⁺ IL2 ⁺ TNFα ⁺ CD4 T cells in the BCG doses at 3×10 ⁵ – 3000 CFU (BALB/c) and 3×10 ⁵ - 300 CFU (CB6F1); ii) secretion of IL2 and IFNγ were correlated with the bacterial burden in the lungs of HN878 infected CB6F1 mice. The study demonstrated a BCG dose at 3000 CFU (an equivalent single human dose in the mice by body weight index) is protective in both strains of mice and the use of a virulent clinical isolate in testing new tuberculosis vaccine/advancing research is recommended.