Novel human in vitro system for evaluating antimycobacterial vaccines

Abstract

Major research efforts are directed towards the development of a better antimycobacterial vaccine. But progress in the field of tuberculosis vaccine development has been hampered by the lack of human in vitro models to assess vaccine immunogenicity and efficacy. New candidate vaccines will have to be evaluated against the existing Mycobacterium bovis BCG "gold standard." It is therefore important to understand the type of immune responses elicited by BCG vaccination to enable comparisons with potential new candidates. We used a novel human in vitro whole-blood model, which measures immune responses to mycobacteria by use of reporter gene-tagged BCG (BCG lux), to study immune responses to BCG vaccination in 50 neonates in a setting in Cape Town, Republic of South Africa, where tuberculosis is endemic. BCG vaccination significantly reduced growth of BCG lux in whole blood (prevaccination median growth ratio [GR], 9.6; range, 1.3 to 24; postvaccination median GR, 3.9; range, 0.6 to 12.2 [P < 0.0001]). Growth of BCG lux was better restricted in vaccinated infants than in unvaccinated age-matched controls (n = 4). BCG vaccination induced significantly higher gamma interferon production in response to BCG lux (P < 0.0001) and to purified protein derivative (P = 0.0001). No significant changes in either growth of BCG lux or cytokine production occurred in an adult control group (n = 6) over the study period. The whole-blood luminescence model detects changes in cellular immune responses to mycobacteria induced by BCG vaccination. It is therefore a useful new tool in studying the immunogenicity of newly developed vaccine candidates prior to large field trials assessing efficacy.

FIG. 1.

Growth of BCG lux in whole blood from infants pre- and post-BCG vaccination and from unvaccinated infant controls (a), in vaccinated infants according to age at the time of the second blood sampling (b), and in adult controls over the entire study period (c). Three groups of individuals were studied: infants tested prevaccination (n = 33) (○) and again 3 to 6 months postvaccination (n = 33) (•), unvaccinated control infants (n = 4) (×) tested at time of postvaccination assessment of the vaccinated infants, and adult controls (n = 6) tested at time points T₁ (▴) and T₂ (▾). Triplicate whole-blood samples were infected with BCG lux. Growth of BCG lux was measured at 96 h and expressed as a GR of RLU of BCG lux at 96 h to RLU of BCG lux in inoculum.

FIG. 2.

Production of IFN-γ in whole blood during growth of BCG lux in infants pre- and post-BCG vaccination, unvaccinated infants, and adult controls. Triplicate whole-blood samples of infants pre (○)- and post (•)-BCG vaccination (n = 33), unvaccinated control infants (×) (n = 4), and adult controls (T₁ [▴], n = 6; T₂ [▾], n = 6) were infected with BCG lux, and supernatants were harvested at 96 h. IFN-γ was measured in these supernatants by ELISA.

FIG. 3.

Production of IFN-γ in response to PPD in whole blood of infants pre- and post-BCG vaccination, unvaccinated infants, and adult controls. Triplicate whole-blood samples of infants pre (○)- and post (•)-BCG vaccination (n = 33), unvaccinated control infants (×) (n = 4), and adult controls (T₁ [▴], n = 6; T₂ [▾], n = 6) were diluted 1:10 with RPMI medium and stimulated with PPD (10 μg/ml). Supernatants were harvested at day 6, and IFN-γ was measured by ELISA.