Factors affecting immunogenicity of BCG in infants, a study in Malawi, The Gambia and the UK

Abstract

Background: BCG immunogenicity in infants differs between populations and these differences have been attributed to various factors. In this study, the influence of geographical location, season of birth, timing of vaccination, micronutrient status (zinc) and inflammatory status (C-reactive protein, CRP) were assessed.

Methods: Immunogenicity was assessed by cytokine signature in culture supernatants from diluted whole blood samples stimulated with M. tuberculosis PPD, using a multiplex bead assay. Results were correlated with the plasma zinc and CRP concentrations at the time of sampling, and with interview and household data. BCG vaccinated infants were recruited in Malawi, The Gambia and the UK.

Results: In Malawi, infants vaccinated within the first week after birth showed lower production of most cytokines measured than those vaccinated later. The number of cytokines showing significant differences between Malawian and Gambian infants decreased after adjusting for season of birth. In Malawi, a proportion of infants had zinc deficiency and elevated plasma CRP (>10 mg/L), but neither zinc deficiency nor high CRP was associated with production of any of the cytokines measured.

Conclusions: The cytokine/chemokine signatures observed in response to M. tuberculosis PPD in infants at 3 months post BCG vaccination were affected by geographical location, season of birth, and timing of vaccination but not associated with the concentration of plasma zinc or inflammatory status. These factors should be considered in future trials of new TB vaccines.

Figure 1

Cytokine response to M. tb PPD in Malawian and Gambian infants vaccinated in the first week of life. The production of IFN-γ (P = 0.0024), IL-1α (P = 0.0020), IL-1ra (P = 0.0008), IL-6 (0.0002), TNF-β (P = 0.0032), TGF-α (p = 0.0027), IL-12p70 (P = 0.0145), and RANTES (P = 0.0274) was significantly higher in Gambian infants (n = 24) than Malawian infants (n = 30) while most of the other cytokines measured showed similar responses (P > 0.05). The median levels of each cytokine are indicated in red (*P < 0.05, **P < 0.01, ***P < 0.001, Mann Whitney test).

Figure 2

Effect of season of birth on cytokine/chemokine signatures. A. Comparison of cytokine/chemokine signatures between dry and rainy seasons of birth in 30 Malawian infants. Among the 42 cytokines and chemokines measured, 3 cytokines including IFN-γ, TNF-α and IL-10 were higher in the infants who were born in the dry season compared with those born in the rainy season. Conversely, Gro production was significantly higher in infants born in the rainy season than in those with dry season of birth. B. Further analysis considering season of birth between the Malawian (n = 14) and Gambian infants (n = 24) born in the dry season demonstrated that only IL-1α, IL-1ra and IL-6 responses to M. tb PPD were significantly higher in the Gambian infants (P < 0.05, Mann Whitney test). Other cytokines including IFN-γ responses to M. tb PPD in Malawian and Gambian infants who were born in the dry season were not significantly different (P > 0.05, Mann Whitney test). The median responses to M. tb PPD are indicated in red.

Figure 3

Production of cytokines and growth factors in early vaccinated versus late vaccinated infants. The levels of 6 cytokines (sIL-2Rα, IL-1α, IFN-α2, IL-5, IL-13, IL-10) and two growth factors (IL-3 and IL-7) were significantly higher with more than 9-fold difference in the infants who were given BCG between 3 and 11 weeks of life (vaccinated late) compared with those vaccinated within one week of birth (vaccinated early). The concentrations of IFN-γ and TNF-α were also higher in late vaccinated infants but the fold differences in median responses were moderate compared with the above 6 cytokines and 2 growth factors. The median levels of each cytokine are indicated in red. The data from late vaccinated Malawian infants were obtained from the previous study [18] (*P < 0.05, ***P < 0.001, Mann Whitney test).

Figure 4

Concentrations of plasma zinc (Zn²⁺), CRP and IFN-γ in Malawian infants. Zinc deficiency was not detected in the majority of the Malawian infants although 6 of 63 infants showed zinc deficiency (< 65 μg/dL). Levels of CRP greater than 10 mg/L were detected in 8 Malawian infants. The median IFN-γ responses to M. tb PPD were different between early vaccinated infants with rainy (n = 40) and dry season (n = 23) of birth (P < 0.001, Mann Whitney test) while the plasma zinc and CRP concentrations were unaffected by season of birth (P > 0.05, Mann Whitney test). The median levels of zinc, CRP and IFN-γ are indicated in red.