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. 2011;6(10):e25582.
doi: 10.1371/journal.pone.0025582. Epub 2011 Oct 4.

The breadth, but not the magnitude, of circulating memory B cell responses to P. falciparum increases with age/exposure in an area of low transmission

Sarah I Nogaro  1 , Julius C Hafalla, Brigitte Walther, Edmond J Remarque, Kevin K A Tetteh, David J Conway, Eleanor M Riley, Michael Walther
Affiliations

The breadth, but not the magnitude, of circulating memory B cell responses to P. falciparum increases with age/exposure in an area of low transmission

Sarah I Nogaro et al. PLoS One. 2011.

Abstract

Background: Malaria caused by Plasmodium falciparum remains a major cause of death in sub-Saharan Africa. Immunity against symptoms of malaria requires repeated exposure, suggesting either that the parasite is poorly immunogenic or that the development of effective immune responses to malaria may be impaired.

Methods: We carried out two age-stratified cross-sectional surveys of anti-malarial humoral immune responses in a Gambian village where P. falciparum malaria transmission is low and sporadic. Circulating antibodies and memory B cells (MBC) to four malarial antigens were measured using ELISA and cultured B cell ELISpot.

Findings and conclusions: The proportion of individuals with malaria-specific MBC and antibodies, and the average number of antigens recognised by each individual, increased with age but the magnitude of these responses did not. Malaria-specific antibody levels did not correlate with either the prevalence or median number of MBC, indicating that these two assays are measuring different aspects of the humoral immune response. Among those with immunological evidence of malaria exposure (defined as a positive response to at least one malarial antigen either by ELISA or ELISPOT), the median number of malaria-specific MBC was similar to median numbers of diphtheria-specific MBC, suggesting that the circulating memory cell pool for malaria antigens is of similar size to that for other antigens.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The proportion of individuals with malaria-specific plasma antibodies increases with age.
The percentage of individuals having Ab levels above the cut-off for AMA-1, MSP-119, MSP-2, MSP-3 or DT are shown for each age group. Data are for samples collected in May–June 2009, prior to the annual malaria transmission season. N is equal to the number of samples available per age group. P values indicate the result for the Chi-squared test for trend.
Figure 2
Figure 2. Accumulation of MBC with age.
The total number of MBC/million PBMC (as measured by their conversion into IgG producing cells after culture) were counted for each individual using the B cell ELISpot technique, and are shown according to age group. The horizontal lines show the median values for each age group. N equals the number of tests that were performed per age group.
Figure 3
Figure 3. Prevalence and antigenic breadth of the malaria-specific MBC response increases with age.
The percentage of individuals in each age group with detectable MBC specific for the different malaria Ags and for DT are shown as (A) number of MBC/PBMC, and as (B) % of all MBC. P-values for a Chi-squared test for trend are shown. N equals the number of samples tested per age group. The average number of Ags for which individuals had specific Ab (open bars) or MBC (hatched bars = MBC/PBMC; black bars = %ASC) are shown, by age group, in (C). For all three parameters, the Chi-squared test for trends indicated a significant increase with age (p<0.0001 for Ab and MBC/PBMC, and p = 0.0083 for %MBC). Positive responses to individual antigens were defined as described in methods.
Figure 4
Figure 4. Plasma Ab levels do not correlate with MBC numbers.
A–E: correlation plots for Ab versus % MBC. The cut-off values for defining responders are shown by dotted lines parallel to each axis. The degree of correlation was assessed by calculating Spearman's correlation coefficient for data points where one or both values were above the cut-off. F–G: Each individual's response to each Ag were classified as: MBC+ IgG+ (that is, a response in both the ELISpot and the ELISA was seen), MBC+ IgG− (ELISpot response but no Ab), MBC− IgG+ (no ELISpot response but Ab) or MBC− IgG− (no response in either of the tests). The frequency with which each category occurred in each age group was then expressed as a proportion and is plotted for F) malaria Ag and G) DT. Responder status for MBC was determined using MBC/PBMC. P test for trend values are indicated.
Figure 5
Figure 5. Evidence of malaria exposure in Brefet increases with age.
The percentage of individuals showing a response above the respective cut off to at least one of the malarial antigens tested either in the ELISA or the ELISpot assays is shown, stratified according to age. Chi-squared test for trends indicated a significant increase with age (p = 0.0157).
Figure 6
Figure 6. The magnitude of the malaria antigen-specific MBC response does not increase with age.
The magnitude of Ag-specific serum IgG (A–E) and of Ag-specific MBC responses [expressed as %MBC (F–J) or expressed as MBC/PBMC (K–O)] are shown for individuals who showed evidence of previous malaria exposure (as defined in Figure 5). Box plots indicate the 25th, 50st and 75th percentile, with whiskers representing the 10th and 90th percentiles. Outliers are denoted by a spot. P values are given for Kruskal-Wallis tests.
Figure 7
Figure 7. Naturally acquired MBC responses to malaria antigens are similar in magnitude to vaccine-induced MBC responses to DT.
Ag-specific ELISpot responses of individuals with immunological evidence of prior malaria exposure are shown and expressed as (A) MBC/PBMC and (B) % MBC, for each Ag. The median for each group is indicated by the horizontal line. The p value (Kruskal Wallis test) for (A) = 0.08, and (B) = 0.178.
Figure 8
Figure 8. Boosting of circulating MBC numbers by recent malaria infection.
(A) Pre- and post-malaria season plasma Ab concentrations to the different malaria Ags are shown for all participants for whom paired samples were available (n = 99), pooled across all age groups. Values below the antigen-specific cut-off (as described in the methods) were given the cut-off value. Box plots indicate the 25th, 50st and 75th percentile, with whiskers representing the 10th and 90th percentiles. Outliers are indicated by spots. (B) Comparisons of the ratio of post-season MBC to pre-season MBC for cases (individuals having a>1.5 fold increased Ab concentration post season for at least one of the malarial Ags) and controls. P values are given for the Mann-Whitney test, adjusted for multiple comparisons using the Bonferroni method.
Figure 9
Figure 9. Atypical and classical memory B cells in children 2 months after a malaria episode.
AMB were defined as being CD19+CD21−CD27−CD10−, classical MBC were defined as CD19+CD21+CD27+ . The gating strategy is shown in (A). Viable PBMC were gated using FSC/SSC (not shown) and then displayed according to CD19+ expression. The CD19+ population was displayed according to their expression of CD21 and CD27. To identify atypical MBCs, CD10− cells were subsequently selected from CD21−CD27− cells, using a CD10 gate defined on the entire PBMC population. (B) Percentages of CD19+ cells expressing the atypical and classical MBC phenotype are shown for children <5 and >5 years of age. P-values are given for the Mann-Whitney test.
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