Differences in phenotype between long-lived memory B cells against Plasmodium falciparum merozoite antigens and variant surface antigens

Abstract

Plasmodium falciparum infections elicit strong humoral immune responses to two main groups of antigens expressed by blood-stage parasites: merozoite antigens that are involved in the erythrocyte invasion process and variant surface antigens that mediate endothelial sequestration of infected erythrocytes. Long-lived B cells against both antigen classes can be detected in the circulation for years after exposure, but have not been directly compared. Here, we studied the phenotype of long-lived memory and atypical B cells to merozoite antigens (MSP1 and AMA1) and variant surface antigens (the CIDRα1 domain of PfEMP1) in Ugandan adults before and after local reduction of P. falciparum transmission. After a median of 1.7 years without P. falciparum infections, the percentage of antigen-specific activated B cells declined, but long-lived antigen-specific B cells were still detectable in all individuals. The majority of MSP1/AMA1-specific B cells were CD95⁺CD11c⁺ memory B cells, which are primed for rapid differentiation into antibody-secreting cells, and FcRL5^-T-bet^- atypical B cells. On the other hand, most CIDRα1-specific B cells were CD95^-CD11c^- memory B cells. CIDRα1-specific B cells were also enriched among a subset of atypical B cells that seem poised for antigen presentation. These results point to differences in how these antigens are recognized or processed by the immune system and how P. falciparum-specific B cells will respond upon re-infection.

Figure 1:. Timing of sample collection.

Cohort participants (n = 10) were sampled during routine clinic visits roughly every three months and when they visited a study clinic due to illness. For each visit, the outcome of screening for parasitemia is indicated (open circle for negative, closed red circle for positive). Samples used in this study were collected in late 2013 through late 2014 when P. falciparum transmission was high (green squares), and during the second half of 2016 after four rounds of indoor residual spraying (IRS) had reduced P. falciparum prevalence by 80% (orange squares). The median time between the last known P. falciparum infection and collection of the second sample was 1.7 years.

Figure 2:. Abundance of total and antigen-specific B cell subsets in the circulation during high parasite transmission and in the absence of P. falciparum exposure.

The percentage of B cell subsets among circulating B cells is shown for total B cells (A), MSP1/AMA1-specific B cells (B), and CIDRα1-specific B cells (C). For MSP1/AMA1-specific B cells and CIDRα1-specific B cells, the total percentage among all circulating B cells is also shown (right most graphs in each panel). In panels B and C, no antigen-specific DN1 cells were detected pre- and post-IRS for four individuals. These data points therefore overlap and are not clearly visible. Differences between groups were evaluated using a Wilcoxon matched-pairs signed-rank test. P values < 0.05 are shown in blue. Ns, not significant.

Figure 3:. Differences in phenotype between long-lived MSP1/AMA1-specific and CIDRα1-specific B cells.

A) Composite UMAP of antigen-specific B cells from samples collected at two time points from 10 individuals, with B cells colored by antigen-specificity. B) The same UMAP as shown in panel A with antigen-specific B cells colored by subset. C) Median fluorescence intensity of 18 surface and intracellular markers in the six B cell subsets, calculated using flow cytometry data from all 20 samples (two time points for 10 individuals) and color-coded relative to the maximum intensity observed among all B cell populations, including naïve B cells not shown in the heatmap (see Table S3). D) Contour plot overlay of MSP1/AMA1-specific and CIDRα1-specific B cells onto the composite UMAP, shown separately for samples collected pre-IRS and post-IRS. E) Distribution of all MSP1/AMA1-specific and CIDRα1-specific B cells over the six B cell subsets at the pre-IRS and post-IRS time points. The number above the bar represents the total number of cells. Colors represent the different subsets shown in panel D. PB, plasmablasts; actMBC, activated memory B cells; atBC, atypical B cells; MBC, memory B cells.

Figure 4:. Expression of CD95, CD11c, T-bet, and FcRL5 among long-lived switched memory B cells.

The percentage of CD95⁺, CD11c⁺, T-bet⁺, and FcRL5⁺ cells are shown pre-IRS and post-IRS for all switched memory B cells, as well as MSP1/AMA1-specific and CIDRα1-specific switched memory B cells. Differences between groups were tested using a Kruskal-Wallis test, followed by comparisons between all pairs of groups using Dunn’s post-hoc test, which reports P values that have been corrected for multiple comparisons. Only P values < 0.05 are shown. swM, switched memory.

Figure 5:. Distribution of MSP1/AMA1-specific and CIDRα1-specific B cells among atypical B cell subsets.

A) Composite UMAP of atypical B cells from samples collected at two time points from 10 individuals, separated by time point (left column). UMAPs for each of the three atypical B cell subsets are also shown (right three columns in green, orange, and blue). MSP1/AMA1-specific and CIDRα1-specific B cells were projected onto these UMAPs. B) Distribution of MSP1/AMA1-specific and CIDRα1-specific B cells over the three atypical B cell subsets in samples collected during times of frequent P. falciparum infection (pre-IRS) and in absence of parasite infection (post-IRS). C) The normalized percentage of MSP1/AMA1-specific and CIDRα1-specific B cells among different B cell subsets. swM, switched memory B cell; atBCi, atypical B cell subset i.