Effect of controlled human Plasmodium falciparum infection on B cell subsets in individuals with different levels of malaria immunity

Abstract

Continuous exposure to Plasmodium falciparum (Pf) has been associated with alterations in B cells. We investigated the effect of controlled human malaria infection (CHMI) on B cell phenotypes in individuals with different Pf immunity status: malaria-naïve, immunized with PfSPZ-CVac and semi-immune (lifelong-exposed) volunteers. Compared to naïve, semi-immune but not vaccinated individuals, had increased baseline frequencies of immature B cells (CD19⁺CD10⁺), active naive (IgD⁺CD27^-CD21^-) B cells, active atypical (IgD^-CD27^-CD21^-) memory B cells (MBCs), active classical (IgD^-CD27⁺CD21^-) MBCs and CD1c⁺-B cells but lower frequencies of some IgG⁺-B cells. The frequencies of CD1c⁺ active atypical MBCs correlated positively with anti-Pf antibodies and negatively with circulating eotaxin levels, while the opposite was observed for IgG⁺ resting atypical MBCs. During early blood-stage infection (day 11 after CHMI), there was an expansion of resting classical (IgD^-CD27⁺CD21⁺) MBCs in all three groups. Vaccination, compared to placebo, altered the effect of CHMI on B cells, showing a positive association with resting classical MBCs (β = 0.190, 95%CI 0.011-0.368) and active naïve-PD1⁺ (β = 0.637, 95%CI 0.058-1.217) frequencies, and a negative one with CD1c⁺ resting atypical MBCs (β=-0.328, 95%CI -0.621--0.032). In addition, the sickle cell trait in semi-immune subjects altered the effect of CHMI on several B cells. In conclusion, lifelong but not vaccine exposure to malaria was associated with increased frequencies of multiple B cell subsets, with higher and lower percentages of CD1c and IgG expressing-cells, respectively. A single infection (CHMI) induces changes in B cell frequencies and is modulated by sickle cell trait and malaria-immunity status.

Keywords: B cells; Controlled human malaria infection; Plasmodium falciparum; cytokines.

Figure 1

Scheme of clinical trials. A) Life-long exposed individuals trial (LACHMI-001). B) Malaria naïve individuals clinical trial (TÜCHMI-001). C) Malaria naïve PfSPZ-CVac vaccinated individuals trial (TÜCHMI-002). Days of blood sample collection for PBMC cryopreservation and subsequent B cell analysis are indicated.

Figure 2. Gating strategy.

Excluded “DUMP” population (CD3⁺CD14⁺CD16⁺); viable B cells (VBC) (CD19⁺ and not excluded); immature B cells (VBC and CD10⁺); plasmablasts and germinal center cells (VBC, but not Immature VBCs, IgD⁻ and CD38⁺⁺); switched (VBC, but not immature VBCs and CD38⁺ (not high) and IgD⁻); acMBCs abbreviates active classical memory B cells (MBCs) (Switched: CD21⁻ CD27⁺); rcMBCs, resting classical MBCs (switched: CD21⁺CD27⁺); aaMBC, active atypical MBCs (switched: CD21⁻CD27⁻); raMBCs, resting atypical MBCs (switched: CD21⁺CD27⁻); unswitched (VBC not Immature VBCs, not CD38⁺⁺ and IgD⁺); resting naïve (unswitched: CD21⁺CD27⁻); active naïve (unswitched: CD21⁻CD27⁻).

Figure 3. Effect of CHMI on B cell subsets distribution.

Boxplots show the percentages of selected B cell populations before (Day 0) and at different timepoints after the CHMI, in the categorized groups: A) naive, B) vaccinated, and C) semi-immune individuals. Median, and 25th and 75th percentiles (lower and upper hinge respectively) are represented as boxes. Outside values are not displayed in the graphs. Differences between timepoints were assessed by Friedman’s test, followed by two-by-two comparisons corrected with Bonferroni’s test for multiple comparisons.

Figure 4. Effect of vaccination status in the B cell distribution at different follow-up periods after CHMI.

Mean cell subset frequencies plus standard error of the mean are represented for the different arms of the TÜCHMI-2 cohort over time. Only B cell populations with statistically significant interactions with vaccination are shown.

Figure 5. Significant correlations of B cell frequencies, P. falciparum IgG levels (surrogates of Pf exposure) and cytokine concentrations at baseline.

Scatter plots of immune responses measured in semi-immune (LACHMI-001, red), vaccinated (TÜCHMI-002, blue dots) and naïve (TÜHMI-001, green) individuals together, with Spearman’s coefficients and raw p-values. A) IgG to AMA-1 correlated negatively with IgG⁺ MBC and positively with CD1c⁺ MBC. B) Negative correlation of IgG to MSP1₄₂ vs. eotaxin, IFN-g or MCP-1. C) Eotaxin correlated negatively with CD1c⁺ MBC and positively with IgG⁺ MBC.

Figure 6. Significant correlations of B cell frequencies, P. falciparum IgG levels (surrogates of Pf exposure) and cytokine concentrations at baseline in semi-immune individuals.

A) Positive correlations of IgG⁺ MBC vs. pro-inflammatory cytokines in semi-immune volunteers. B) CD1c⁺ aaMBC correlated positively with anti-PfMSP1₁₉ and anti-PfMSP1₄₂ IgG levels C) PD1⁺ acMBC correlated positively with pro-inflammatory cytokines. Full correlograms of all immune markers are shown in supplementary materials.