Figure 1.

Study design and impact of i.v. BCG dose on plasma antibody responses

(A) 34 rhesus macaques were vaccinated with i.v. BCG at doses ranging from 3.88 × 10⁴ to 2.49 × 10⁷ CFUs. Macaques were challenged with 4–17 CFUs of Mtb Erdman 24 weeks following vaccination. Plasma and bronchoalveolar lavage fluid (BAL) were collected at multiple time points during the vaccination and infection phase for analysis. Week 8/9 indicates that samples were collected either 8 or 9 weeks following vaccination. Week 9 is used in future figures for simplicity. Figure created using BioRender.com.

(B) Total Mtb CFU measured at necropsy in each macaque.⁶ Boxes represent the interquartile range. The whiskers extend to the smallest and largest values within 1.5 times of the interquartile range. Spearman correlation between log₁₀(total Mtb CFU) and i.v. BCG dose indicated.

(C) Fold change in IgG1 titers to various antigens in the plasma following i.v. BCG vaccination. Fold changes were calculated as fold change in Luminex median fluorescence intensity (MFI) over the pre-vaccination level for each macaque. Macaques are colored by i.v. BCG dose, and each point represents the duplicate average from a single macaque. Dashed vertical line indicates the time of Mtb challenge. Gray shaded area is the background level set to 2 standard deviations above the mean MFI of the pre-vaccination samples. A mixed-effects model was applied to assess the impact of vaccine dose on antibody titers over time. The model p values, indicating the significance of the dose effect, are displayed in the top right corner.

(D) Spearman correlations between i.v. BCG dose and each plasma antibody measurement collected at peak immunogenicity (week 4). Antibody features with low signal (average fold change less than 1.25) were removed prior to i.v. BCG dose correlation analysis. Black dotted horizontal line indicates unadjusted p value of 0.05. Red and blue dots represent antibody features with a significant positive or negative correlation with i.v. BCG dose, respectively, following multiple testing correction (Benjamini-Hochberg adjusted p value <0.05).⁷

(E) Spearman correlations between selected plasma antibody features at peak immunogenicity (week 4) and i.v. BCG dose. Boxes represent the interquartile range. The whiskers extend to the smallest and largest values within 1.5 times of the interquartile range.