Adjuvant Effect of Bacille Calmette-Guérin on Hepatitis B Vaccine Immunogenicity in the Preterm and Term Newborn

Abstract

Immunization is key to protecting term and preterm infants from a heightened risk of infection. However, preterm immunity is distinct from that of the term, limiting its ability to effectively respond to vaccines routinely given at birth, such as hepatitis B vaccine (HBV). As part of the Expanded Program on Immunization, HBV is often given together with the live-attenuated vaccine Bacille Calmette-Guérin (BCG), known to activate multiple pattern-recognition receptors. Of note, some clinical studies suggest BCG can enhance efficacy of other vaccines in term newborns. However, little is known about whether BCG can shape Th-polarizing cytokine responses to HBV nor the age-dependency of such effects, including whether they may extend to the preterm. To characterize the effects of BCG on HBV immunogenicity, we studied individual and combined administration of these vaccines to cord newborn and adult human whole blood and mononuclear cells in vitro and to neonatal and adult mice in vivo. Compared to either BCG or HBV alone, (BCG + HBV) synergistically enhanced in vitro whole blood production of IL-1β, while (BCG + HBV) also promoted production of several cytokines/chemokines in all age groups, age-specific enhancement included IL-12p70 in the preterm and GM-CSF in the preterm and term. In human mononuclear cells, (BCG + HBV) enhanced mRNA expression of several genes including CSF2, which contributed to clustering of genes by vaccine treatment via principle component analysis. To assess the impact of BCG on HBV immunization, mice of three different age groups were immunized subcutaneously with, BCG, HBV, (BCG + HBV) into the same site; or BCG and HBV injected into separate sites. Whether injected into a separate site or at the same site, co-administration of BCG with HBV significantly enhanced anti-HBV IgG titers in mice immunized on day of life-0 or -7, respectively, but not in adult mice. In summary, our data demonstrate that innate and adaptive vaccine responses of preterm and term newborns are immunologically distinct. Furthermore, BCG or "BCG-like" adjuvants should be further studied as a promising adjuvantation approach to enhance immunogenicity of vaccines to protect these vulnerable populations.

Keywords: Bacille Calmette–Guérin; HBV-specific antibodies; hepatitis B vaccine; innate cytokine profiles; newborn; preterm.

Figure 1

Concurrent stimulation with [Bacille Calmette–Guérin (BCG) + hepatitis B vaccine (HBV)] synergistically enhances IL-1β production in human preterm, term, and adult whole blood. Preterm (A, D), term (B, E), and adult (C, F) whole blood was stimulated with HBV alone, BCG alone, or (BCG + HBV) at 1:10 v/v. After 6 h, supernatants were analyzed for TNF (A–C) and IL-1β (D–F) cytokine production by ELISA. Statistical significance was determined by Kruskal–Wallis with Dunn post hoc test. *p < 0.05, **p < 0.01. N = 7–8 preterm, N = 10–11 term newborns, and N = 10–11 adults.

Figure 2

Cytokine and chemokine profiles induced by hepatitis B vaccine (HBV) and Bacille Calmette–Guérin (BCG) in human preterm, term, and adult whole blood. Preterm, term, and adult whole blood was stimulated for 6 h with either HBV, BCG, or (BCG + HBV) and supernatants analyzed via Multiplex Cytokine Analysis. Statistical significance was determined by repeated measure or ordinary one-way ANOVA with Holm-Sidak post hoc test (or their non-parametric equivalent Friedman or Kruskal–Wallis with Dunn post hoc test). *p < 0.05, **p < 0.01 of preterm vs. term vs. adult; ^#,+p < 0.05, ^##,++p < 0.01 of groups indicated by the corresponding color, respectively, vs. saline or HBV. N = 7 preterm newborns, N = 6 term newborns, N = 7 adults.

Figure 3

Gene expression profiles induced by hepatitis B vaccine (HBV) and Bacille Calmette–Guérin (BCG) in human preterm, term, and adult mononuclear cells. Preterm and term cord blood mononuclear cells, and adult peripheral blood mononuclear cells was stimulated for 4 h with either HBV, BCG or (BCG + HBV) and cells were harvested for quantitative real-time PCR analysis. Statistical significance was determined by repeated measure or ordinary one-way ANOVA with Holm-Sidak post hoc test (or their non-parametric equivalent Friedman or Kruskal–Wallis with Dunn post hoc test). *p < 0.05, **p < 0.01 of preterm vs. term vs. adult; ^#,+p < 0.05, ^##,++p < 0.01 of groups indicated by the corresponding color, respectively, vs. saline or HBV. N = 4–5/group.

Figure 4

Principal component analysis of gene expression data reveals treatment-specific segregation of different age groups. Gene expression data were generated as outlined in Figure 3. (A) Principal components analysis biplot of mRNA gene expression data where observations (samples) are points and gene expression profiles are arrows shows dominant clustering of profiles by treatment. The distance between points approximates gene expression pattern differences among groupings. Arrows indicate genes that have greater biplot scores and drives the differences between groups. Arrowheads close to a particular group indicate genes are expressed at a greater relative abundance differences in those samples. (B) Unsupervised hierarchical heatmap shows clustering of treatments demonstrating log2-fold changes, expression values of genes in each sample; red to blue scale represent intensity of fold changes per genes (red indicates up, blue indicates down). Each row means individual gene and each column indicates groupings of age and treatment. P, preterm; T, term; A, adult.

Figure 5

Combined immunization of newborn mice with Bacille Calmette–Guérin (BCG) and hepatitis B vaccine (HBV) in vivo enhances anti-recombinant HBV surface antigen (rHBsAg) IgG titers 21 days post-immunization. (A) Schematic representation of the immunization and blood draw schedule for the in vivo experiments: prime immunization was administered either on day of life (DOL) 0, DOL7, or at 6–8 weeks of life with either saline, BCG, HBV vaccine, BCG and HBV in a combined s.c. injection or BCG and HBV vaccine injected in separate sites. Booster immunization was performed with either saline in the mice prime-immunized with saline or BCG, or with HBV vaccine in the mice prime-immunized with either HBV vaccine alone, or BCG and HBV (either in combined or separate injection). Blood draws in the adult mice were obtained prior to prime immunization, prior to booster immunization, at 21 days and at 42 days post-prime immunization. Blood draws in the neonatal mice immunized on DOL7 were performed prior to booster immunization, at 21 days and at 42 days post-prime immunization. Blood draws in the mice immunized on DOL0 were obtained at 21 days and at 42 days post-prime immunization. Some neonatal mice from both the group immunized on DOL0 as well as DOL7 were sacrificed for a baseline blood draw prior to prime immunization. (B) Anti-rHBsAg IgG titers in mice immunized on DOL0, mice immunized on DOL7 and at 6–8 weeks of life. (C) Fold change over the HBV vaccine immunized group of the median anti-rHBsAg IgG titers. N = 9–14/group for the mice immunized on DOL0; N = 14–16/group for the mice immunized on DOL7; N = 15–17/group for the mice immunized at 6–8 weeks of life. Data are representative of two independent experiments each of which included all three age groups and within each age group all five treatment groups. Statistical analysis of differences between the treatment groups was performed via Kruskal–Wallis test with Dunn’s post hoc test. *p < 0.05, **p < 0.01.