Immunological profiles associated with distinct parasitemic states in volunteers undergoing malaria challenge in Gabon

Abstract

Controlled human malaria infection (CHMI) using cryopreserved non-attenuated Plasmodium falciparum sporozoites (PfSPZ) offers a unique opportunity to investigate naturally acquired immunity (NAI). By analyzing blood samples from 5 malaria-naïve European and 20 African adults with lifelong exposure to malaria, before, 5, and 11 days after direct venous inoculation (DVI) with Sanaria^R PfSPZ Challenge, we assessed the immunological patterns associated with control of microscopic and submicroscopic parasitemia. All (5/5) European individuals developed parasitemia as defined by thick blood smear (TBS), but 40% (8/20) of the African individuals controlled their parasitemia, and therefore remained thick blood smear-negative (TBS^- Africans). In the TBS^- Africans, we observed higher baseline frequencies of CD4⁺ T cells producing interferon-gamma (IFNγ) that significantly decreased 5 days after PfSPZ DVI. The TBS^- Africans, which represent individuals with either very strong and rapid blood-stage immunity or with immunity to liver stages, were stratified into subjects with sub-microscopic parasitemia (TBS^-PCR⁺) or those with possibly sterilizing immunity (TBS^-PCR^-). Higher frequencies of IFNγ⁺TNF⁺CD8⁺ γδ T cells at baseline, which later decreased within five days after PfSPZ DVI, were associated with those who remained TBS^-PCR^-. These findings suggest that naturally acquired immunity is characterized by different cell types that show varying strengths of malaria parasite control. While the high frequencies of antigen responsive IFNγ⁺CD4⁺ T cells in peripheral blood keep the blood-stage parasites to a sub-microscopic level, it is the IFNγ⁺TNF⁺CD8⁺ γδ T cells that are associated with either immunity to the liver-stage, or rapid elimination of blood-stage parasites.

Figure 1

Higher baseline and early decrease in the frequencies of IFNγ-producing CD4⁺ T cells are associated with parasitaemia control in lifelong malaria-exposed individuals. (a) The LaCHMI study design and parasitological outcome of PfSPZ DVI with Sanaria^R PfSPZ Challenge. This figure is created with BioRender.com. (b) Frequencies of cells producing IFNγ or TNF upon PfRBC stimulation at baseline (C-1) in Europeans, TBS⁺ and TBS⁻ Africans. The boxplots show the median, 1st, and 3rd quartiles and the whiskers extend to the maximum/minimum of the respective groups, no further than 1.5 × the IQR. (c) Representative flow cytometry plot of IFNγ and TNF gating on CD4⁺ T cells. (d-f) IFNγ⁺TNF^-CD4⁺ T cells at baseline and after PfSPZ DVI. (d) The frequency of IFNγ⁺TNF⁻CD4⁺ T cells at baseline. All data points are added to the box plots. P-values for the pairwise comparisons of the frequencies were performed using Tukey’s honestly significant difference (HSD) test. P-value for testing linear trend (P_trend) was obtained from a linear model with orthogonal polynomial contrast. (e) Survival curve showing time until parasitemia according to the baseline frequency of IFNγ⁺TNF⁻CD4⁺ T cells. The frequency was split on the median to create two groups of individuals, a top > 50% (purple) and a bottom < 50% (green). This grouping was then used to compare the survival distributions of the groups using the log-rank test. (f) Changes in the frequencies of IFNγ⁺TNF⁻CD4⁺ T cells after PfSPZ DVI. The frequencies on five (D + 5) and eleven (D + 11) days after PfSPZ DVI were compared against the baseline using linear mixed models to obtain the estimates and corresponding 95% confidence intervals (CI) as well as P-values. The estimates and intervals were visualized using error bars with either solid lines if statistically significant (P < 0.05) or dashed lines if otherwise. P-values for significant comparisons are shown.

Figure 2

IFNγ-producing CD4⁺ T cells are positively correlated with antibodies against antigens highly expressed in malaria asexual blood stage. (a) Spearman correlations between frequencies of IFNγ⁺TNF⁻CD4⁺ T cells and antibody reactivity to Plasmodium protein microarray. All data points are added and the color of the points indicates the grouping used in Fig. 1. The black line and shaded gray area represent linear fit (y ~ x) on rank-transformed values and 95% confidence intervals, respectively. (b) Log₂ ratio of gene expression (in transcript per million; TPM) encoding Plasmodium protein in malaria asexual blood-stage over malaria mosquito sporozoites. Genes were selected based on the association between levels of antibodies against the encoded protein and NAI.

Figure 3

Early decrease in the frequencies of IFNγ⁺TNF⁺DN γδ T is associated with parasite control. (a) Representative flow cytometry plots of IFNγ and TNF gating on γδ T cells (left panel) and boxplots of frequencies of cytokine-producing γδ T subsets (CD4⁻CD8⁻ or DN, CD4⁺, CD8⁺) relative to total γδ T cells, irrespective of groups or time points (right panel). (b) Frequencies of IFNγ⁺TNF⁺DN γδ T cells at baseline (left panel) and after PfSPZ DVI (right panel). The boxplots show the median, 1st, and 3rd quartiles and the whiskers extend to the maximum/minimum of the respective groups, no further than 1.5 × the IQR. The frequencies on five (D + 5) and eleven (D + 11) days after PfSPZ DVI were compared against the baseline using linear mixed models to obtain the estimates and corresponding 95% confidence intervals (CI) as well as P-values. The estimates and intervals were visualized using error bars with either solid lines if statistically significant (P < 0.05) or dashed lines if otherwise. P-values for significant comparisons are shown.

Figure 4

Cytokine-producing cell subsets associated with parasite control is not significantly different upon further stratification by PCR status. (a) Stratification of TBS⁻ Africans by PCR status to TBS⁻PCR⁺ and TBS⁻PCR⁻. Created with BioRender.com. (b) The frequency of IFNγ⁺TNF⁻CD4⁺ T cells at baseline using the data from Fig. 1d, but only including TBS⁻ Africans, which were stratified by PCR status after PfSPZ DVI. Horizontal lines indicate the median and all data points are added. P-value from Welch’s t-test. (c) The frequency of IFNγ⁺TNF⁺DN⁺ γδ T at baseline using the data from Fig. 1j, but only including TBS⁻ Africans stratified by PCR. Horizontal lines indicate the median and all data points are added. P-value from Welch’s t-test. (d) change in the frequencies of IFNγ⁺TNF⁻CD4⁺ T cells (left panel) and IFNγ⁺TNF⁺DN⁺ γδ T cells (right panel) after PfSPZ DVI. The frequencies on five (D + 5) and eleven (D + 11) days after PfSPZ DVI were compared against the baseline using linear mixed models to obtain the estimates and corresponding 95% confidence intervals (CI). The estimates and intervals were visualized using error bars with either solid lines if statistically significant (P < 0.05) or dashed lines if otherwise. P-values for selected comparisons were shown.

Figure 5

Anti-malarial antibodies associated with sub-microscopic parasitaemic individuals. (a) Antibody reactivity of PCR⁺ (including Europeans, TBS⁺ Africans, and TBS⁻PCR⁺ Africans) and PCR⁻ individuals to Plasmodium protein microarray, showing the reactivities at baseline (C-1) to two statistically significant antigens (P-value < 0.05 and absolute Log2 fold-change > 1, annotated). Statistics are based on two-sided Welch t-tests. (b) Boxplots of PCR⁺ individuals were stratified by both nationality (Europeans or Africans) and TBS status (TBS⁺ or TBS⁻). All data points are added. P-values were obtained from t-tests comparing signal intensities of PCR⁺ and PCR⁻ individuals.

Figure 6

Higher baseline and early decrease in the frequencies of IFNγ⁺TNF⁺CD8⁺ γδ T cells are associated with sterilizing immunity in lifelong malaria-exposed individuals. (a) Profile of IFNγ⁺TNF⁺CD8⁺ γδ T cells at baseline and after PfSPZ DVI. Left panel, baseline frequencies of the subset at baseline. All data points are added, and the horizontal line represents median frequencies for each respective group. P-values from Welch’s t-test. Center panel, survival curve showing time until parasitemia according to the baseline frequency of IFNγ⁺TNF⁺CD8⁺ γδ T cells. The frequency was split on the median to create two groups of individuals, a top > 50% (purple) and a bottom < 50% (green). This grouping was then used to compare the survival distributions of the groups using the log-rank test. Right panel, changes in the frequency of IFNγ⁺TNF⁺CD8⁺ γδ T cells after PfSPZ DVI. The frequencies at D + 5 or D + 11 were compared against the baseline using linear mixed models to obtain the estimates and corresponding 95% confidence intervals (CI). The estimates and intervals were visualized using error bars with either solid lines if statistically significant (P < 0.05) or dashed lines if otherwise. (b) UMAP embedding of all cytokine-producing γδ T cells with an overlay of the clusters in different colors (left panel) and a heatmap summary of median expression values of markers expressed by the five γδ T cell clusters (right panel). (f) The frequency of γδ T cells cluster 2 (IFNγ⁺TNF⁺CD27⁺CD45RA⁺CD8⁺ γδ T cells), which was obtained from (b) at each time point. P-values from Welch’s t-test.