Bacille Calmette-Guérin Vaccine Strain Modulates the Ontogeny of Both Mycobacterial-Specific and Heterologous T Cell Immunity to Vaccination in Infants

Abstract

Differences in Bacille Calmette-Guérin (BCG) immunogenicity and efficacy have been reported, but various strains of BCG are administered worldwide. Since BCG immunization may also provide protection against off-target antigens, we sought to identify the impact of different BCG strains on the ontogeny of vaccine-specific and heterologous vaccine immunogenicity in the first 9 months of life, utilizing two African birth cohorts. A total of 270 infants were studied: 84 from Jos, Nigeria (vaccinated with BCG-Bulgaria) and 187 from Cape Town, South Africa (154 vaccinated with BCG-Denmark and 33 with BCG-Russia). Infant whole blood was taken at birth, 7, 15, and 36 weeks and short-term stimulated (12 h) in vitro with BCG, Tetanus and Pertussis antigens. Using multiparameter flow cytometry, CD4+ T cell memory subset polyfunctionality was measured by analyzing permutations of TNF-α, IL-2, and IFN-γ expression at each time point. Data was analyzed using FlowJo, SPICE, R, and COMPASS. We found that infants vaccinated with BCG-Denmark mounted significantly higher frequencies of BCG-stimulated CD4+ T cell responses, peaking at week 7 after immunization, and possessed durable polyfunctional CD4+ T cells that were in a more early differentiated memory stage when compared with either BCG-Bulgaria and BCG-Russia strains. The latter responses had lower polyfunctional scores and tended to accumulate in a CD4+ T cell naïve-like state (CD45RA+CD27+). Notably, BCG-Denmark immunization resulted in higher magnitudes and polyfunctional cytokine responses to heterologous vaccine antigens (Tetanus and Pertussis). Collectively, our data show that BCG strain was the strongest determinant of both BCG-stimulated and heterologous vaccine stimulated T cell magnitude and polyfunctionality. These findings have implications for vaccine policy makers, manufacturers and programs worldwide and also suggest that BCG-Denmark, the first vaccine received in many African infants, has both specific and off-target effects in the first few months of life, which may provide an immune priming benefit to other EPI vaccines.

Keywords: Africa; BCG; Pertussis vaccine; T cells; Tetanus vaccine; immunogenicity; ontogeny; vaccine strain.

Figure 1

Magnitude of mycobacterial-specific CD4+ T cell cytokine responses differ between strains. (A) Cross-sectional analysis of mycobacterial-specific CD4 cytokine responses stratified by BCG immunizing strain. Weeks indicate the time point post vaccination with week 0 the pre-vaccination time point (birth for CT cohort and days 4–7 for Jos cohort). Y axes show the frequencies (%) of CD4+ cells producing total cytokine (any combination of IFN-γ, IL-2 or TNF-α) shown on a Log₁₀ scale. Jitter point colors: Green (BCG-Bulgaria), blue (BCG-Denmark), orange (BCG-Russia), with shaded bars showing median value of unstimulated samples. Boxes with mid-line show interquartile ranges and median. Sample sizes by time point: week 0 (Bulgarian: 15, Danish: 35, Russian 14), week 7 (Bulgarian: 32, Danish: 83, Russian 35), week 15 (Bulgarian: 35, Danish: 66, Russian 17), week 36 (Bulgarian: 22, Danish: 60, Russian 17) Mann-Whitney U test was used to compare strains. Adjusted P-values are reported with P < 0.05 were considered significant after multiple comparisons correction. (B) Kinetics of BCG response stratified by BCG immunizing strain. Y axes show the frequencies (%) of CD4+ cells producing total cytokine (any combination of IFN-γ, IL-2 or TNF-α shown on a Log₁₀ scale) for infants matched at least three time points between week 0 and week 36 (Bulgarian: 5, Danish: 21 Russian: 15). A Kruskal-Wallis test was applied to test differences by BCG strain across time (P < 0.05 were considered significant).

Figure 2

Polyfunction of mycobacterial-specific CD4+ T cell cytokine responses differ between strains. (A) Representative flow cytometry plots showing mycobacterial-specific cytokine expression by CD4+ cells stratified by BCG immunizing strain. Axes show IFN-γ vs. TNF-α expression with IL-2+ cells overlaid and represented by blue dots. Numbers indicate frequency of polyfunctional cells as a percentage of total CD4. (B) Pie charts show median proportions of cytokine combinations (G = IFN-γ, 2 = IL-2, T = TNF-α) as a fraction of the total response among antigen responders in each BCG strain group by SPICE analysis. Pie charts are compared using the SPICE permutations test with P < 0.05 considered significant. (C) Week 7 mycobacterial-specific cytokine profile by BCG immunizing strain. Jitter points show the median proportion of each cytokine combination (G = IFN-γ, 2 = IL-2, T = TNF-α) per infant as a fraction of total cytokine+ cells in infants who were responding to BCG and are color-coded by BCG immunizing strain. Shaded bars show interquartile ranges with line showing median. A Wilcoxon Rank Sum Test was used to compare cytokine combinations by strain with P < 0.01 considered significant after multiple comparison correction. (D) Heatmap of posterior probabilities of mycobacterial-specific responses estimated by COMPASS analysis. Rows represent individual infants and grouped by BCG immunizing strain and time post BCG vaccination (color-coded annotations are shown on right-hand side of heat map). Columns represent cytokine subsets (combinations) ordered by degree of functionality (single to polyfunctional; right to left). The color of each cell corresponds to the probability (range 0–1) of a mycobacterial-specific response for a particular cytokine cell subset per infant, with 1 indicating an antigen-specific response and white indicating background.

Figure 3

BCG-Denmark induces a more differentiated T cell memory phenotype compared to BCG-Russia or BCG-Bulgaria strains. (A) Representative flow cytometry plots showing the memory profile of mycobacterial-specific (cytokine positive) CD4 cytokine responses stratified by BCG immunizing strain. Axes show CD27 vs. CD45RA expression and blue dots are cytokine+ cells responding to BCG overlaid against a background of total CD4+ cells. CD45RA+CD27+ represent naïve-like, CD45RA–CD27+ early differentiated (ED), CD45RA–CD27– late differentiated (LD) and CD45RA+CD27– terminally differentiated (TD) phenotypes. (B) Frequency of mycobacterial-specific memory subsets stratified by BCG immunizing strain. Jitter point colors: Green (BCG-Bulgaria), blue (BCG-Denmark), orange (BCG-Russia), show the proportion of cytokine+ cells co-expressing combinations of memory markers CD45RA and CD27 that define memory subsets (as listed in (A). Boxes with mid-line show interquartile ranges and median. Mann-Whitney U test was used to compare strains with P < 0.01 were considered significant after multiple comparisons correction.

Figure 4

BCG strain impacts CD4+ T cells responses to heterologous antigens. Cross-sectional CD4 cytokine responses to heterologous antigens TT, BP, and PHA stratified by BCG immunizing strain. Y axes show the frequencies (%) of CD4+ cells producing total cytokine (any combination of IFN-γ, IL-2, or TNF-α) shown on a Log₁₀ scale. Jitter point colors: blue (BCG-Denmark), orange (BCG-Russia), with shaded bars showing median value of unstimulated samples. Boxes with mid-line show interquartile ranges and median. Mann-Whitney U test was used to compare strains. Adjusted P-values are reported with P < 0.05 were considered significant after multiple comparisons correction.

Figure 5

BCG strain impacts CD4+ T cells cytokine profile to heterologous antigens. (A) Representative flow cytometry plots showing TT, BP, and PHA-specific cytokine expression by CD4+ cells stratified by BCG immunizing strain at week 7. Numbers indicate frequency of polyfunctional cells as a percentage of total CD4. Axes show IFN-γ vs. TNF-α expression with IL-2+ cells overlaid and represented by blue dots. (B) Pie charts show median proportions of cytokine combinations (G = IFN-γ, 2 = IL-2, T = TNF-α) as a fraction of the total response among antigen responders in each BCG strain group by SPICE analysis. Pie charts are compared using the SPICE permutations test with P < 0.05 considered significant. (C) Week 7 heterologous antigens TT, BP, and PHA cytokine profile by BCG immunizing strain. Jitter points show the median proportion of each cytokine combination per infant as a fraction of total cytokine+ cells in infants who were responding to antigen and are color-coded by BCG immunizing strain [Green (BCG-Bulgaria), blue (BCG-Denmark), orange (BCG-Russia)]. Shaded bars show interquartile ranges with line showing median. A Wilcoxon Rank Sum Test was used to compare cytokine combinations by strain with P < 0.01 considered significant after multiple comparison correction.