Immunisation with the BCG and DTPw vaccines induces different programs of trained immunity in mice

Abstract

In addition to providing pathogen-specific immunity, vaccines can also confer nonspecific effects (NSEs) on mortality and morbidity unrelated to the targeted disease. Immunisation with live vaccines, such as the BCG vaccine, has generally been associated with significantly reduced all-cause infant mortality. In contrast, some inactivated vaccines, such as the diphtheria, tetanus, whole-cell pertussis (DTPw) vaccine, have been controversially associated with increased all-cause mortality especially in female infants in high-mortality settings. The NSEs associated with BCG have been attributed, in part, to the induction of trained immunity, an epigenetic and metabolic reprograming of innate immune cells, increasing their responsiveness to subsequent microbial encounters. Whether non-live vaccines such as DTPw induce trained immunity is currently poorly understood. Here, we report that immunisation of mice with DTPw induced a unique program of trained immunity in comparison to BCG immunised mice. Altered monocyte and DC cytokine responses were evident in DTPw immunised mice even months after vaccination. Furthermore, splenic cDCs from DTPw immunised mice had altered chromatin accessibility at loci involved in immunity and metabolism, suggesting that these changes were epigenetically mediated. Interestingly, changing the order in which the BCG and DTPw vaccines were co-administered to mice altered subsequent trained immune responses. Given these differences in trained immunity, we also assessed whether administration of these vaccines altered susceptibility to sepsis in two different mouse models. Immunisation with either BCG or a DTPw-containing vaccine prior to the induction of sepsis did not significantly alter survival. Further studies are now needed to more fully investigate the potential consequences of DTPw induced trained immunity in different contexts and to assess whether other non-live vaccines also induce similar changes.

Keywords: Dendritic cells; Innate immune memory; Trained immunity; Vaccination; Vaccine nonspecific effects.

Fig. 1.. The DTPw and BCG vaccines induce long-lasting alterations in monocyte and DC cytokine responses.

Female 3–4 week old (n = 5) C57BL/6J mice were subcutaneously immunised with BCG (SSI), DTPw (SII) or saline (mock) and boosted two weeks later (A). Splenocytes were harvested 10 weeks following prime immunisation and cultured with media alone (unstim) or with heat-killed C. albicans (1 × 10⁶ cells/mL; HKCA), LPS (10 ng/mL), or resiquimod (1 μg/ml; R848) with Poly I:C (10 μg/ml), for 4 h. TNFα and IL6 were quantified via intracellular cytokine staining (ICS) in monocytes (B-C) and DCs (D-E). (F-G) Blood samples from BCG (SSI), DTPw (SII) or saline (mock) immunised mice were collected at V + 6 weeks, erythrocyte depleted and stimulated with LPS (100 ng/mL; 3 h). TNFα and IL6 production in monocytes (F) and DCs (G) were assessed via ICS. Flow cytometry analysis of spleen (H) and bone marrow (I&J) collected from mice 10 weeks after prime immunisation with BCG (SSI), DTPw (SII) or saline (mock). Data are represented as the mean ± SEM. Data in B-E represent a single independent experiment, and data from F-J represent a separate independent experiment. A one-way ANOVA was used to assess statistical significance. * = P > 0.005, ** = P > 0.05, *** = P > 0.001, ns = not significant.

Fig. 2.. Immunisation with DTPw and BCG differentially alters chromatin accessibility in conventional DCs.

ATAC-seq was performed on 50,000 sorted cDCs from n = 4 DTPw (SII), n = 4 BCG (SSI) or n = 2 mock immunised female C57BL/6J mice. (A) Analysis with the ATACseqQC Bioconductor package revealed a strong enrichment of ATAC-seq reads at transcription start sites (TSS) (enrichment score > 15) indicating high-quality ATAC-seq data. (B) MDS analysis of ATAC-seq data. (C) Gene set enrichment analysis (GSEA) revealed an enrichment of genes involved in key immune and metabolic pathways among loci with altered chromatin accessibility in BCG and DTPw-immunised mice. (D) GSEA comparing chromatin accessibility in DCs from BCG and DTPw-immunised mice. (E) Heatmap of normalised read counts at loci identified in the GSEA analysis to have altered chromatin accessibility. (F) Boxplot of normalised read counts at selected loci relative to GAPDH. CPM = counts per million. (G) ATAC-seq read alignments at selected peaks of interest. Statistical significance was assessed via a one-way ANOVA with multiple comparisons. * = P < 0.05, ** = P < 0.01. Data are from a single experiment.

Fig. 3.. The order in which vaccines are administered alters monocyte and DC trained immune responses.

(A) Female C57BL/6J mice (10 mice/group) were subcutaneously immunised at 3–4 weeks of age with saline (mock), BCG (SSI)), or DTPw (SII) and boosted 2 weeks later or were immunised with both vaccines administered in different orders (DTPw + boost + BCG or BCG + DTPw + boost). Splenocytes collected at 12 weeks post the prime immunisation were either unstimulated (media) or were stimulated ex vivo with LPS (100 ng/mL), HKCA (1 × 10⁶ cells/mL) or R848 (1 μg/ml) + Poly I:C (10 μg/ml) prior to the assessment of the proportion of TNFα⁺ and IL6⁺ monocytes (B-C) and DCs (D-E) by intracellular cytokine staining. (F) Fold-enrichment of selected loci normalised to GAPDH was assessed by ATAC-qPCR. Data are represented as the mean ± SEM and are from a single experiment. Statistical significance was assessed via a one-way ANOVA with multiple comparisons. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Fig. 4.. Immunisation with DTPw alters cytokine responses and acute illness severity in mice 24 h after the induction of polymicrobial sepsis.

(A) Female C57BL/6J mice were subcutaneously immunised with saline (mock), BCG (SSI) or DTPw (SII) and responses were boosted 2 weeks later. 6 weeks after the prime immunisation, sepsis was induced by cecal ligation and puncture. Clinical scores (B-C) and surface temperature (D) were assessed over a 24-hour period following the induction of sepsis, and area under the curve (AUC) was compared between groups. (E) Bacterial load in blood was quantified via aerobic culture. (F) Cytokine concentrations in peritoneal lavage fluid were assessed via multiplex immunoassay. Data are represented as the mean ± SEM and are from a single experiment. Statistical significance was assessed using a Kruskal-Wallis test with multiple comparisons. * = P < 0.05.

Fig. 5.. Immunisation with the pentavalent DTPw-Hep-Hib vaccine does not alter survival in a cecal ligation and puncture model of polymicrobial sepsis.

(A) Female C57BL/6J mice (n = 25) were immunised and with saline (mock), BCG (SSI) or EasyFive® pentavalent DTPw-HepB-HiB vaccine (Penta) and sepsis induced 6 weeks later via cecal ligation and puncture surgery. (B) Clinical scores and (C) temperature were assessed for 7 days and the area under the curve (AUC) was compared between groups. (D) Cytokine concentrations in peripheral blood collected 24 h post-surgery were assessed via multiplex immunoassay. (E) Survival was recorded over 7 days. Data are represented as the mean ± SEM and are from a single experiment. Clinical scores, temperature and cytokine data were compared via Kruskal-Wallis test with multiple comparisons. Survival data were compared via a Mantel-Cox survival test. * = P > 0.05, ** = P > 0.01, *** = P > 0.001.

Fig. 6.. Immunisation with a pentavalent DTPw-HepB-HiB vaccine does not alter survival in a cecal slurry injection model of neonatal sepsis.

(A) Neonatal mice (4–5 days old) were immunised with saline (mock) or Pentavac® SII DTPw-HepB-HiB vaccine (Penta) and similarly boosted 5 and 10 days later. Sepsis was induced via intraperitoneal cecal slurry injection 10 days following the prime immunisation, at 14 days old. (B) Clinical scores were monitored every 6–12 h for 7 days. Survival curves of all mice (C), and male (D) and female (E) mice are shown. Data are represented as the mean ± SEM and are from a single experiment. Survival data were compared via Mantel-Cox survival test. Clinical scores were compared via a Wilcoxon Rank-Sum Test with multiple comparisons. ns = not significant.