Outcomes of controlled human malaria infection after BCG vaccination

Abstract

Recent evidence suggests that certain vaccines, including Bacillus-Calmette Guérin (BCG), can induce changes in the innate immune system with non-specific memory characteristics, termed 'trained immunity'. Here we present the results of a randomised, controlled phase 1 clinical trial in 20 healthy male and female volunteers to evaluate the induction of immunity and protective efficacy of the anti-tuberculosis BCG vaccine against a controlled human malaria infection. After malaria challenge infection, BCG vaccinated volunteers present with earlier and more severe clinical adverse events, and have significantly earlier expression of NK cell activation markers and a trend towards earlier phenotypic monocyte activation. Furthermore, parasitemia in BCG vaccinated volunteers is inversely correlated with increased phenotypic NK cell and monocyte activation. The combined data demonstrate that BCG vaccination alters the clinical and immunological response to malaria, and form an impetus to further explore its potential in strategies for clinical malaria vaccine development.

Fig. 1

Parasitemia, clinical symptoms and laboratory abnormalities after CHMI. Parasitemia was measured by daily qPCR from day 6 after CHMI until the third day after antimalarial treatment. a The Kaplan–Meier survival curve shows percent of volunteers remaining untreated. 8/9 BCG vaccinated (green) and 10/10 control volunteers (grey) surpassed the treatment threshold of 100 parasites per millilitre, and were treated on day 7 after challenge. 1/9 BCG vaccinated volunteers remained below 100 Pf/mL until day 9. b All volunteers did have parasitemia detectable by qPCR on day 7 after CHMI. The graph shows log parasites per millilitre on day 7 post CHMI for BCG vaccinated (green) and control (grey) volunteers. c Adverse events were collected daily. The Kaplan–Meier curve shows the percentage of volunteers experiencing one or more moderate or severe, solicited, symptoms during follow-up, BCG vaccinated volunteers (green) compared to controls (grey). d–f Absolute platelet, lymphocyte and neutrophil differentiation counts were determined by daily hemocytometry starting on day 6 post-challenge. Graphs show relative change in cell counts compared to pre-challenge values in both BCG vaccinated (n = 9, each coloured dot shows and individual volunteer, colours consistently represent the same volunteers across each graph) and non-BCG vaccinated controls (n = 10, grey dots)

Fig. 2

In vivo activation of lymphocytes, monocytes and neutrophils after CHMI. In vivo leucocyte activation was determined by direct staining of fresh whole blood with fluorescent antibodies every 2 days post-challenge. Lymphocytes were defined based on forward scatter and sideward scatter characteristics, and duplet events were excluded. a NK cell activation was defined as the percentage of CD3^-CD56^dimCD16⁺ live cells expressing CD69. b γδT cell activation was defined as the percentage of CD3⁺γδTCR⁺ live cells expressing CD69. c NKT cell activation was defined as the percentage of CD3⁺γδTCR^-CD56⁺ live cells expressing CD69. d αβT cell activation was defined the as percentage of CD3⁺γδTCR^-CD56⁻ live cells expressing CD69. e Monocytes were defined based on forward and side scatter characteristics, and the as HLA-DR⁺CD14⁺. Within the monocyte population, cells were then divided into CD16^- and CD16⁺ monocytes. f–g Within the CD16^- monocyte population, the relative change in mean fluorescent intensity of HLA-DR and CD86 compared to pre-malaria challenge values was determined. h Neutrophils were defined based on forward and side scatter characteristics, and then defined as HLA-DR^-CD14^-CD16⁺CD11b⁺. Activated neutrophils were defined as CD62L^dimCD11b^high. i–j IFN-γ and granzyme B were measured by Luminex assay in citrate plasma taken ever 2 days. Circulating cytokine levels are corrected for baseline levels (pre-BCG vaccination time point) at each time point. In all graphs the grey dots represent non-BCG vaccinated control group volunteers (n = 10), and each coloured dot shows an individual BCG vaccinated volunteer (n = 9). Statistical analysis are between BCG vaccinated and control volunteers at a single time point, and p-values are the results of Mann–Whitney U-test. *p < 0.05. k Circulating CRP levels were measured in citrate plasma are shown for each BCG vaccinated volunteer (colours consistently represent the same volunteers across each graph)

Fig. 3

Early NK cell and monocyte activation correlates with decreased parasitemia. Correlations between a NK cell CD69 expression and log parasitemia on day 7 after challenge infection, and b increase in monocyte HLA-DR MFI and log parasitemia on day 7 are shown for BCG vaccinated (n = 9, green) and control (n = 10, grey) volunteers. Lines show the result of linear regression analysis for both groups

Fig. 4

BCG vaccination increases NK cell responses against P. falciparum. a Percentage of total NK cells staining positive for IFN-γ after 24 h of stimulation with PfRBC before, and 37 days after malaria challenge infection as compared to baseline (pre-BCG vaccination time point), each dot represents an individual BCG vaccinated (n = 9, green) or control volunteers (n = 10, grey). b Percent NK cells staining positive for the degranulation marker CD107a. c Percent NK cells staining positive for granzyme B. Lines and error bars show median and interquartile range. p-values are the result of Mann–Whitney U-test. *p < 0.05. Stimulation was performed in duplo, with replicates combined for flow cytometry analysis