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. Nogaro1,2, Julius C. Hafalla2, Brigitte Walther1, Edmond J. Remarque3, Kevin K. A. Tetteh4,
David J. Conway1,4, Eleanor M. Riley2., Michael Walther1*.¤
1Medical Research Council Laboratories, Fajara, Banjul, The Gambia, 2Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London
School of Hygiene and Tropical Medicine, London, United Kingdom, 3Department of Parasitology, Biomedical Primate Research Centre, GJ Rijswijk, The Netherlands,
4Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom
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.
Citation: Nogaro SI, Hafalla JC, Walther B, Remarque EJ, Tetteh KKA, et al. (2011) 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. PLoS ONE 6(10): e25582. doi:10.1371/journal.pone.0025582
Editor: Lisa Ng Fong Poh, Agency for Science, Technology and Research - Singapore Immunology Network, Singapore
Received June 7, 2011; Accepted September 6, 2011; Published October 4, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was funded by the Medical Research Council (UK), The Gambia. J.C.H. was a recipient of a Wellcome Trust Visiting Fellowship (grant reference
number 079920) and is currently supported by a University Research Fellowship from The Royal Society (UK). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Michael.Walther@nih.gov
. These authors contributed equally to this work.
¤ Current address: National Institutes of Health, NIAID, LMIV, Rockville, Maryland, United States of America
The immune system’s ability to mount an accelerated humoral
immune response upon repeated encounter of the same pathogen
allows for rapid reduction in disease severity or even complete
sterile immunity . However, in the case of malaria, sterile
immunity that would prevent re-infection is rare; clinical immunity
is thought to be species and strain-specific and repeated infections
are required to develop immune responses specific for the
prevalent antigenic types in the area of residence . The rapidity
with which effective immunity is acquired thus depends on the rate
of transmission .
The immune control of malaria infection is multi-factorial, and
there is growing consensus that the synergistic action of antibodies
(Ab) and cell mediated effector mechanisms is required for both
anti-parasitic as well as clinical immunity [2,4]. The paramount
importance of Ab in clearing parasitized red blood cells and
reducing clinical symptoms was highlighted several decades ago,
by passive immunoglobulin (Ig) transfer experiments [5,6]. A
number of studies have subsequently demonstrated an association
between protection from clinical symptoms of uncomplicated
disease and levels of anti-malarial Ab . However, the frequency
of repeated malaria infections in some children in highly endemic
areas, together with anecdotal accounts of apparent loss of
immunity in the absence of continuing exposure and experimental
evidence of dysfunctional T cell responses, has raised legitimate
questions regarding the duration of immune memory to malaria
Longitudinal studies showing that titres of many anti-malarial
Ab decline rapidly in children once parasitaemia is cleared after
acute infection [8,10,11,12,13] have contributed to the belief that
humoral memory to malaria may be defective. On the other hand,
there is a growing body of evidence to indicate that Ab responses
become increasingly stable with increasing age  and can be
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long-lived in adults [14,15,16,17]. Technological advances -
particularly the development of the B cell ELISpot assay to
quantify antibody secreting cells (ASC) as a surrogate of circulating
memory B cells (MBC) [1,18,19] – are now allowing the cellular
basis of humoral immune memory in malaria to be investigated. In
this assay, peripheral blood mononuclear cells (PBMC) are
stimulated with a cocktail of B cell mitogens in order to stimulate
the differentiation of MBC into Ab producing plasma cells (PC) 
and the secreted Ig is detected by enzyme immunoassay. Using an
early version of this assay, Dorfman et al. , attempted to
identify malaria-specific MBC among PBMC sampled from
malaria-exposed Kenyan children but only very few cells were
detected . Recent refinements of the technique , have
enhanced its sensitivity allowing detection of malaria-specific MBC
in children from a high transmission area in Mali . Findings
from that study demonstrated that accumulation of MBC was both
gradual and occurred in a stepwise fashion over many years of
repeated exposure . Using a similar method, malaria-specific
MBC were also detected in adults from a low endemicity setting in
Thailand  and these MBC were found to persist - in the
absence of re-exposure - for more than 7 years . Despite the
enhanced sensitivity of the current ELISpot assay, in all of these
studies antigen-specific MBCs could not be detected in a
significant proportion of seropositive individuals. Whether this
reflects a real absence of MBC or insufficient sensitivity of the
assay is currently not clear.
In this study, in an area in The Gambia where malaria
transmission has declined substantially over the last decade
[23,24], to unprecedentedly low prevalence , we find that
the magnitude of the malaria-specific memory B cell response in
children is very similar to that in older individuals and that, in all
age groups, malaria-specific MBC responses are of similar
magnitude to vaccine antigen-specific responses. However, the
prevalence of malaria-specific humoral responses and the breadth
of the response (as judged by the number of antigens recognised by
an individual) increases with age indicating that acquisition of
humoral immunity requires repeated exposure to malaria
Study procedures and baseline characteristics of the
118 healthy volunteers from Brefet, Foni District, The Gambia,
were selected using age-stratified randomisation into six different
age categories (Table S1). Venous blood samples were collected as
part of a cross-sectional survey carried out at the end of the dry
season (May–June 2009) over a six-week period. At the time of
sample collections, study participants were healthy and afebrile.
Using qualitative P.falciparum PCR, one individual in each of the
1–4, 15–24 and 25–39 year age groups and three individuals in the
10–14 year age group carried parasites, none of which were
detected by slide microscopy.
Stool analysis to assess carriage of intestinal helminths and
pathogenic gut parasites was carried out on 46 samples, evenly
spread across the different age groups. Two (4.3%) had Giardia
lamblia cysts (individuals stemming from 0–4 and .39 years age
groups) and one (2.2%) had hookworm ova (13 years old).
Morbidity surveillance was performed during the transmission
season and study participants suspecting they had malaria, or
experiencing symptoms compatible with malaria were asked to
report to the village health worker who performed a rapid
diagnostic test (RDT). Out of eight participants presenting with
symptoms, only one had a positive RDT.
At the end of the transmission season, 8 individuals had shown
evidence of exposure based on a$1.5 fold increase to at least one
of the malaria specific Ab tested for (see methods section); one of
those individuals presented with clinical symptoms compatible
with malaria and tested positive by RDT.
The proportion of individuals with malaria specific Ab
increases with age
Samples collected in May 2009 from Brefet were assayed for Ab
specific to MSP-119, MSP-2, MSP-3, AMA-1 and diphtheria
toxoid (DT), and the percentages of individuals having Ab to the
different Ag were plotted for each age group (Figure 1). The
geometric mean anti-diphtheria IgG concentration of the study
population was 26.9 IU/ml (CI 95%:22.2 to 32.6) with all study
participants having .1.4 IU/ml. Since antibody titres .0.1 IU/
ml are regarded by the test manufacturer as a sign of ‘‘good
immunity’’, all participants were classified as responders with
regard to anti-diphtheria Ab. For all four malarial Ags the
proportion of individuals having malaria-specific Ab increased
significantly with age (Chi square test for trend for all Ags:
p,0.0001). AMA-1 was the only Ag for which by the age of $40
years, all individuals had Abs, confirming that AMA-1 is amongst
the most immunogenic of the malarial Ags [16,26]. In comparison,
for MSP-119, considered to be less immunogenic [16,27,28], the
increase with age was less pronounced with Ab prevalence
reaching 60% amongst adults aged $40 years.
Inter-individual variation in the frequency of circulating
Surprisingly little is known about how the total population of
circulating MBCs - as measured by their conversion into IgG ASC
in the B cell ELISpot assay - develops with increasing age and,
thus, cumulative Ag experience. We therefore plotted the number
of ASC per million cultured PBMC for each age group and
examined the data for any change in ASC numbers with
increasing age (Figure 2). Assuming that the number of ASC
detected after in vitro culture is a reliable reflection of the
precursor frequency of circulating MBCs, we find considerable
inter-individual variation in the frequency of MBC in all age
groups (Kruskal Wallis test: p=0.0024). Although the 1–4 year
olds had significantly lower frequencies of MBC than the 25–39
year olds (p,0.05, Dunn’s post test), logistic regression across all
age groups failed to detect a significant increase with age
(p=0.086); one reason for this may be the anomalous and
unexplained drop in total MBC numbers in the 15–24 year age
group. However, when data for children above 15 years of age
were pooled and the 10–14 year old age group was used as the
baseline group, the model suggests that the MBC frequency in 10–
14 year olds may be higher than in 1–4 year olds (p=0.051) but
that there is no difference in MBC frequency between 10–14 year
olds and those aged 15 years or more (p=0.802).
The proportion of individuals with malaria-specific MBC
increases with age
Previous studies that have looked at the frequency of malaria
Ag-specific MBC measured by ELISpot have reported their results
either as the number of malaria-specific spots per million PBMC
seeded into the well (MBC/PBMC) [20,22], or the proportion of
all IgG ASC (%MBC) that are malaria antigen-specific [15,29,30].
The considerable inter-individual variation we observed in overall
MBC frequencies and the possibility that the population of MBC
might increase during childhood, clearly indicates that the choice
of the denominator may affect the interpretation of the data. In
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this study, we therefore analysed the ELISpot data using both
methods of calculation. Unfortunately, due to a temporary
shortage of IgG detection reagents, we were only able to
enumerate the total number of IgG producing cells (and therefore
calculate the % MBC) for 86 of the 118 participants.
When the data were expressed as MBC/PBMC, the proportion
of individuals with malaria specific MBC above the cut-off
(hereafter referred to as responders) increased significantly with
age for all Ags (Figure 3A). Responder status was defined as
malaria Ag-specific MBC frequencies above the upper limit of the
99% CI of the median MBC/PBMC frequency for the negative
control Keyhole Limpet Hemocyanin (KLH), being 1.88 spots/
million PBMC. A similar trend was observed when data were
expressed as % MBC (Figure 3B; here responder status was
defined as values above the upper limit of the 99% CI of the
median % MBC determined for KLH, being 0.004%), but, apart
from AMA-1, the trends were not statistically significant.
Importantly, the two methods of analysis showed a substantial
level of agreement with regards to identifying individual
responders, with kappa values ranging from 0.55 (MSP-2), 0.64
(MSP-119), 0.72 (DT), 0.78 (AMA-1), to 0.81 (MSP-3).
The breadth of the malaria-specific immune response
increases with age
To assess how the breadth of the immune response to malaria
Ags develops with age, the average number of malaria Ags
recognised by each individual’s plasma or cells was stratified by
age. As shown in Figure 3C, the number of Ags against which
specific plasma IgG or MBC were detected increases significantly
with age (Chi-square test for trend p,0.0001 for ELISA and
MBC/PBMC; and p=0.0083 for %MBC). Interestingly, from the
age of 10 years, the repertoire of Ags recognised by plasma IgG
was significantly broader than the repertoire recognized by MBC
(expressed as MBC/PBMC; p value for Chi square for each age
strata: ,0.012). When the same comparison was carried out
between ELISA data and ELISpot data expressed as %MBC, the
repertoire of Ags recognized by plasma IgG became significantly
broader from 25 years onwards (p value for Chi square for each
age strata: ,0.0015).
Lack of correlation between plasma IgG responses and
MBCs detectable by ELISpot
IgG measured in plasma by ELISA is an indicator of the
presence of antibody-secreting effector cells (plasma cells) in
peripheral tissues or bone marrow whereas ASC detected by
cultured ELISpot assay indicate the presence of circulating MBC.
To explore the relationship between these two very different
measures of the humoral immune response, IgG concentrations
were plotted against MBC numbers for all subjects for each Ag
(Figure 4A–E) and a correlation coefficient was calculated using
data points for which one or both variables were above the
respective cut-offs. No significant positive correlation was detected
between MBC and Ab for any of the Ags tested, regardless
whether ELISpot results were expressed as %MBC (Figure 4) or as
MBC/PBMC (data not shown).
To explore this data further, we classified each individual’s
responses to all 4 malaria Ags into the following categories: MBC+
IgG+ (that is, a response in both the ELISpot and the ELISA was
seen), MBC+ IgG2 (ELISpot response but no Ab), MBC2 IgG+
(no ELISpot response but Ab) or MBC2 IgG2 (no response in
either of the tests). The frequency with which each category
occurred in each age group was then expressed as a proportion
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. 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.
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and is plotted in Figure 4F for all the malaria Ags combined and
Figure 4G for DT, using MBC/PBMC for the ELISpot data.
The proportion of responses to malaria Ags categorized as
MBC+ IgG+ increases with age, while the proportion of responses
classified as MBC2 IgG2 decreases (Chi squared test for trend,
p,0.0001 for both), which presumably reflects cumulative
exposure to malaria over time. Interestingly however, the
proportion of responses classified as MBC2 IgG+ also increased
with increasing age (Chi squared test for trend: ,0.0001)
suggesting that antibody secreting plasma cells persist even though
the frequency of circulating MBC remains below the threshold of
Similar analysis was performed for responses to DT (Figure 4G).
Since all participants had anti-diphtheria Ab, only two categories
(MBC+ IgG+ and MBC2 IgG+) existed. With increasing age,
significantly more responses were classified as MBC+ IgG+ (Chi
squared test for trend: 0.009). Since diphtheria vaccination is only
given during childhood and natural exposure to diphtheria is now
extremely rare in The Gambia , we speculate that this change
reflects either age-dependent qualitative changes in the immune
response or recurrent bystander B cell activation (driving DT-
specific MBC into periodic clonal expansion) rather than repeated
exposure to diphtheria Ag.
The magnitude of MBC responses expressed as % MBC were
only poorly correlated with numbers of MBC/PBMC (Table 1),
reflecting the considerable inter-individual variation in total MBC
Similar frequencies of malaria-specific and diphtheria-
We were interested to explore i) whether the magnitude of the
malaria-specific humoral and B cell ELISpot responses increases
with age, and ii) whether the magnitude of malaria specific B cell
ELISpot responses differs from DT specific responses. This
requires defining each participant’s prior exposure to the antigens.
Based on the 100% prevalence of IgG against diphtheria in the
study group, it can be assumed that every participant has
encountered diphtheria antigen in the past. Determining prior
exposure to malaria is less straightforward, especially in this
community where the very low levels of transmission over the last
10 years mean that the younger individuals may have had very few
(if any) previous malaria infections. Extensive polymorphism of
malarial blood stage antigens means that even among exposed
individuals, prior exposure to the precise antigenic variants used in
our assays cannot be assumed. Also, different individuals may
respond differently to a given antigen  and, as we have shown,
circulating IgG antibody may not be detectable despite the
presence of corresponding MBCs, and vice versa. Given the very
low levels of malaria transmission in Brefet (23) we believe it is
justifiable to consider individuals who are seronegative to all four
malaria antigens, and who lack detectable MBC to any of these
four antigens, to be malaria unexposed; conversely individuals who
have IgG or MBC to one or more malaria antigens were
considered to have been exposed. By this definition, 60–80% of
children and .90% of adults had evidence of prior malaria
exposure (Figure 5). Figure 6 shows the magnitude of malaria
specific IgG and MBC responses for all participants considered as
malaria exposed across age groups, and diphtheria responses for
all participants. Consistent with the high coverage of the childhood
immunisation programme in The Gambia , anti-diphtheria
plasma IgG concentrations declined during childhood from a peak
in children aged 1–4 years (Kruskal Wallis test, p=0.004, with
significant p value for post test between 1–4 years and 10–14 years,
Figure 6A), but were relatively stable from age 10 years onwards.
However, there was no significant difference in diphtheria-specific
MBC numbers between different age groups (Figure 6F and 6K).
By contrast, and as expected from previous studies, the
concentration of all malaria-specific antibodies tested increased
with age (Figure 6B–E).
For all the Ags tested, Ag-specific MBC frequencies (expressed
as %MBC) did not vary significantly across age groups (Figure 6F–
J). With the exception of MSP-119 (Figure 6M), for which a
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.
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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 shownby dottedlinesparalleltoeachaxis. The degreeofcorrelation was assessedby calculatingSpearman’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+ IgG2 (ELISpot response but no Ab), MBC2 IgG+ (no ELISpot response but Ab) or MBC2 IgG2 (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.
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significant decrease in MBC numbers was observed between age
groups 10–14 and 15–24 years, the same result was obtained when
MBC were expressed as MBC/PBMC (Figure 6K, L, N and O).
Of note, the magnitude of malarial Ag -specific MBC responses
amongst those with evidence of previous malaria exposure was not
different from that for diphtheria, irrespective of the chosen
denominator (Kruskal Wallis test p=0.08 [MBC/PBMC], or
p=0.178 [%MBC]; Figure 7A, B).
Effect of recent malaria exposure on malaria-specific
plasma IgG antibodies and MBCs
To capture evidence of malaria exposure during the transmis-
sion season we performed active morbidity surveillance of our
study population from August 2009 till December 2009. Only one
symptomatic case with a positive RDT was detected (in a 5 year
old child). In addition, we repeated the serological survey in
December 2009 and tested these samples together with those
collected in May 2009. Taking the study population as a whole we
observed small, but significant, decreases in the median concen-
trations of Abs to AMA-1, MSP-119, MSP-2 and MSP-3
(Figure 8A), confirming the lack of recent re-exposure to malaria
in the majority of subjects.
However, 8 (including the 1 RDT positive case) out of 99
participants that were tested showed a$1.5 fold increase in plasma
IgG levels to at least one malaria Ag suggesting that they may have
recently experienced a malaria infection; 7 out of 8 of these
infections are assumed to have been asymptomatic. PCR carried
out at the end of the transmission period detected P. falciparum
DNA in only one of these 8 subjects. A ‘‘rising titre’’ of serum Ab is
widely used as a diagnostic tool and suggests that these 8
individuals were indeed infected with malaria. However, despite
the fact that (apart from the one RDT positive case) none of them
presented with any signs of illness during the study period, we
cannot entirely rule out that the increased malaria Ab titres may
be the result of bystander activation of malaria-specific B cells by
another asymptomatic infection.
Nevertheless, when we compared the change in frequency (pre-
and post-malaria season) of malaria-specific MBCs among the 8
subjects who had evidence of a recent malaria infection (cases)
with the change in MBC frequency in 8 age-matched controls with
no evidence of recent infection, the post/pre-season ratio for
malaria-specific MBCs (calculated as a % of all MBC) tended to be
higher in cases than in controls (Figure 8B). While the low
numbers of cases precludes the drawing of any firm conclusions,
this analysis does suggest that MBC numbers can be boosted in the
same manner as serum IgG concentrations. Interestingly, the
post/pre-season ratio for diphtheria-specific MBCs was also
significantly higher in cases than in controls, suggesting that
recent infection may have resulted in polyclonal, antigen-
independent, ‘‘bystander’’ activation of diphtheria-specific MBCs.
Atypical memory B cells
A recent study reported an increased frequency of dysfunctional
and exhausted MBCs (designated atypical memory B cells, AMB)
in HIV-infected patients compared to healthy individuals . B
cells with a similar surface phenotype (CD19+CD272CD212and
CD102) have also been reported to be more prevalent in adults
from an area with high malaria transmission (Mali) than in
malaria-naı ¨ve volunteers (from the USA), and their proportion was
higher among asymptomatic parasite carriers than among non-
parasitized individuals . This prompted us to examine the
frequency of AMB with this phenotype in 16 children aged 1–15
years, who were followed up as part of an ongoing malaria case-
control study [33,34]. All children had a confirmed episode of
malaria 2 months previously, but were parasite free at the time of
blood collection, and live in an area of low malaria endemicity at
the Gambian coast [24,25]. The median proportion of B cells
expressing the AMB phenotype (CD19+CD272CD212and
CD102; Figure 9A) was 4.71% (CI 95%: 2.48–6.41%) with no
significant difference between children below and above the age of
5 years (Figure 9B). This compares to AMB frequencies of 15.5%
in Malian adults, 9.8% in Malian children (aged 2–10 years) and
1.6% in U.S. adults , supporting the hypothesis that malaria
induces AMB in an exposure-dependent manner. Interestingly,
the proportion of classical MBCs (CD19+CD27+CD21+) amongst
all B cells was twice as high in children aged more than 5 years
than in children aged less than 5 years (Figure 9B), consistent with
the age-related increase in the repertoire of antigens recognised by
the MBC population.
The advent of methods to enumerate circulating MBC  has
enabled researchers to begin to explore the induction and
maintenance of humoral immune responses to malaria in humans
[15,20,22]. While the B cell ELISpot may still benefit from further
improvements to its sensitivity, the assay is sensitive enough to
detect durable MBC responses to both P.falciparum and P.vivax in
infrequently exposed Thai adults  and, in a recent report from
a rural village in Mali where people are exposed to ,50 infective
Table 1. Correlation between the two methods of calculating
The magnitude of the MBC response, expressed either as MBC/PBMC or % MBC
was assessed for correlation. n: number of pairs tested for each Ag; r2:
Spearman correlation coefficient.
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
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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, 50stand 75thpercentile, with whiskers representing the
10th and 90th percentiles. Outliers are denoted by a spot. P values are given for Kruskal-Wallis tests.
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mosquito bites per month during the peak of the transmission
season, to demonstrate that the P. falciparum-specific MBC
compartment expands in a step-wise fashion with increasing age
and/or repeated malaria infections . Using a similar assay, we
have now described the acquisition of humoral immunity to four
malaria blood stage antigens (selected on the basis of their
immunogenicity and potential functional relevance) in a rural
community in The Gambia where malaria transmission has
declined sharply in the last 15 years [23,35] and where only ,8%
of study subjects appear to have been exposed to malaria infection
in the 2009 rainy season. The absence of anti-MSP-119specific
IgG in children ,10 years in December 2009 , the high
coverage with insecticide treated nets (ITNs), and the virtual
absence of clinical infections in the study cohort all support the
assumption that malaria transmission in this community has been
extremely low for several years.
Rather surprisingly, given the huge differences in malaria
endemicity between the two sites, the age-specific prevalence of
both Ab and MBC in The Gambia was remarkably similar to the
prevalence reported in Mali . Although there are some slight
differences in methodology between the studies (age grouping,
definition of cut off values, use of fresh or cryopreserved cells) the
similarity in the results is striking. Given the findings of Weiss et al.
 that acute malaria infections lead to only transient expansion
of the malaria-specific MBC pool and that the subsequent
contraction of the MBC pool results in very inefficient accumu-
lation of humoral immunity, and the findings of Wipasa et al. 
of very long-lived B cell memory in areas of exceptionally low
transmission, it may be that relatively infrequent exposure to
malaria is as effective (or more effective) at inducing long-lived
humoral immunity than is persistent re-infection. If so, we perhaps
need to consider the possibility that repeated exposure to antigen
gradually drives MBC to differentiate into tissue resident cells
(either ASCs or non-circulating memory cells) or – possibly –
causes clonal exhaustion. On the other hand, the clear increase
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. 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, 50stand 75thpercentile, 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.
Malaria-Specific MBC in Low Transmission Areas
PLoS ONE | www.plosone.org8 October 2011 | Volume 6 | Issue 10 | e25582
Malaria-Specific MBC in Low Transmission Areas
PLoS ONE | www.plosone.org9October 2011 | Volume 6 | Issue 10 | e25582
that we observed in the number of Ags recognised with increasing
age indicates that the antigenic breadth (and likely, therefore, the
functional relevance) of the B cell response requires at least some
degree of repeated exposure to malaria. The underlying biology of
these observations clearly deserves further investigation.
A caveat to these interpretations, however, is that the similarity
of MBC responses in the different studies may reflect some
limitation of the assay system rather than a true biological
phenomenon. For example, we find for all Ags, including
diphtheria, that although the prevalence of responses varies, the
magnitude of the MBC responses (in those classified as exposed) is
similar in all age groups. One potential explanation that deserves
investigation is that, even in individuals with long-lived humoral
immune memory, the maximum precursor frequency of circulat-
ing MBCs for any individual Ag is in the order of 1 MBC per
million PBMCs and that the spot frequency of 5–10 spots per
million PBMC simply reflects the differentiation of these very few
MBCs during the 6 days of mitogenic stimulation into MBC. In
other words, it may be necessary to seed ELISpot cultures with
many more PBMCs in order to detect the true frequency and
prevalence of circulating Ag-specific MBCs. Nevertheless, the
similar magnitude of the MBC response to malaria Ags and to DT,
a protein of fairly similar length to the malaria Ags used, implies
that mounting a B cell response to malaria Ags is not necessarily
any more difficult than mounting a B cell response to other Ags.
Although it is possible that numbers of DT-specific MBC may
have been much higher immediately after vaccination in infancy,
the lack of any detectable decline in DT-specific MBC frequency
with increasing age tends to argue against this possibility.
Another caveat of the B cell ELISpot assay is that we do not
know whether the stimulation medium differentiates MBC into
ASC at a fixed ratio or whether this is a true reflection of what
happens in vivo upon encounter with an Ag. Moreover, for different
types of Ags the efficiency of the differentiation of MBC into ASC
may be different. A direct comparison of MBC frequencies derived
from the ELISpot assay with MBC precursor frequencies derived
from limiting dilution assays or from antigen-specific flow
cytometry (using chromophore-labled antigen) is urgently needed
to validate the accuracy and sensitivity of the B cell ELISpot assay.
Related to this is the question of whether B cell ELISpot data
should be presented as the proportion of all MBCs that are specific
for the Ag of interest (% MBC) [15,29,30] or as the number of
MBCs among all the PBMCs seeded into the plate (MBC/PBMC)
[20,22]. Our data indicate that for a given number of PBMC,
there is considerable inter-individual variation in the total number
of MBC. Further, the total number of MBC may increase with age
during childhood, presumably as a result of exposure to an ever-
increasing number of Ags. Indeed, the total number of MBCs
detected in the ELISpot assay tended to be higher in slightly older
children than in very young children and, by flow cytometry, the
proportion of all B cells that are classical MBCs was also higher in
older children than in younger children. Although we could not
detect any further expansion of total MBC numbers from
adolescence into adulthood, suggesting that MBC numbers may
reach an equilibrium in late childhood, our power to detect such a
trend was limited by the anomalous drop in MBC numbers in the
15–24 year age group. The reason for this drop is unclear but is
highly unlikely to be assay or operator dependent since a random
selection of participants across all age groups were tested on each
day of the study and the range of values on plates that included
low-responding 15–24 years olds did not differ systematically from
the ranges on other plates. Given the very low prevalence of HIV
infection in this community (,2%)  this drop is unlikely to be
related to undiagnosed HIV infection. Further studies in this age
group are required. In any event, increasing total MBC numbers
are likely to lead to an underestimation of Ag-specific responses
when expressed as %MBC. This may be of particular relevance in
diseases where the compartment of IgG-producing cells is
expanded by means of polyclonal stimulation as has been
suggested for malaria [37,38]. For instance, in the high
transmission area in Mali, an extremely marked increase in total
MBC numbers with age was observed , and reporting results
from such a setting as % MBC would further distort the
relationship between age and malaria specific memory responses.
The number of PBMC per ml of blood is more stable. For this
reason, we suspect that MBC/PBMC may better reflect the
precursor frequency of malaria specific MBC in the peripheral
blood, but further studies are required to be certain that this is the
As in most studies of human immune responses, access to the
tissues of real interest - in this case spleen and bone marrow - is
restricted and observations from studies of PBMC need to be
interpreted with caution. In our study, the presence of detectable
Abs and MBC did not correlate for any of the Ags tested, and a
significant proportion of individuals lacked detectable circulating
MBCs despite having significant titres of the corresponding plasma
Ab. While this may reflect insufficient sensitivity of the assay, it is
also highly plausible that ASCs present in tissues (either as short-
lived plasma cells (SLPC) in spleen or as SLPC or long-lived
plasma cells (LLPC) in bone marrow) continue to secrete Abs even
though the frequency of circulating MBCs has fallen below the
lower limit of detection of the ELISpot assay. A similar lack of
correlation between MBC and serum Abs was observed in the
Thai study , where Ag exposure is infrequent, but not in Mali
, where re-exposure to Ag is very common. These observa-
tions, together with the evidence from our comparison of malaria
cases and controls in The Gambia, suggest that both Abs and
circulating MBC numbers are boosted by re-infection but that, in
the absence of boosting, circulating MBC numbers decline to
undetectable levels even though tissue resident LLPC continue to
secrete Ab. Our observation that the proportion of individuals
with Abs but no detectable circulating MBC increases with age - at
least for the blood stage malaria Ags tested here - suggests that
older people are less likely to have been recently infected, perhaps
indicating a degree of pre-erythrocytic immunity in these
individuals. However, age-dependent differences in immune
responses may also contribute to this effect. Furthermore, due to
the marked reduction in malaria endemicity in The Gambia in the
last decade, the malaria exposure of children in this community is
not only of shorter duration than in adults but is also qualitatively
very different from that experienced in childhood by those who are
Figure 9. Atypical and classical memory B cells in children 2 months after a malaria episode. AMB were defined as being
CD19+CD212CD272CD102, 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, CD102cells were subsequently selected from CD212CD272cells, 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.
Malaria-Specific MBC in Low Transmission Areas
PLoS ONE | www.plosone.org 10October 2011 | Volume 6 | Issue 10 | e25582
B cell mitogenic activity has long been ascribed to P. falciparum
 and has been invoked as the underlying cause of bystander
activation of unrelated B cells  and of the hypergammaglob-
ulinaemia commonly described in highly malaria endemic areas
[39–40]. While both the Malian study by Weiss et al.  and the
present study confirm a bystander effect of acute malaria (boosting
tetanus and diphtheria responses respectively), it is far from clear
that this is due to the activity of a parasite-derived mitogen as
opposed to generalised cytokine-mediated B cell activation.
The functional significance of the relatively high frequencies of
cells with the phenotype of so-called ‘‘atypical MBCs’’ in malaria-
exposed individuals is not known. One potential explanation is
that these cells - which have a substantially shorter life span than
classical MBC  – have been displaced from bone marrow
niches as a result of intermittent polyclonal bystander activation of
B cells (after a transient malaria infection, for example) and are
destined to die. The observation that such atypical cells are present
at much higher frequencies in the high transmission area in Mali
 than in our low transmission setting or in another area of low
transmission in Peru  further supports this notion. The fact
that the age-related expansion of classical MBCs that we observed
is not accompanied by a similar expansion of atypical MBCs in a
low endemicity setting tends to support the hypothesis that the
frequency of atypical MBCs reflects cumulative malaria exposure
and is not just a function of increasing age but, currently, it is not
possible to rule out other chronic or recurrent infections (such as
helminths, for example), nutritional status or other environmental
exposures as contributory causes of this atypical B cell phenotype.
In summary, although the prevalence of individuals with
malaria-specific MBCs increases with increasing age (and/or
malaria exposure) the magnitude of an individual’s malaria specific
MBC response is similar in children with minimal prior malaria
exposure to adults with a considerably higher cumulative malaria
exposure. However, the antigenic repertoire of the B cell response
is more limited in children than in adults and only increases with
increasing exposure. Since the magnitude of the malaria-specific B
cell response is similar to that induced by diphtheria vaccination,
we find no evidence that mounting a B cell response to malarial
antigens is more difficult than to other antigens. Nevertheless, the
possibility remains that the development and/or retention of
malaria specific memory B cells is more efficient in areas of low
malaria transmission, such as The Gambia or Thailand, than in
very highly endemic areas such as Mali. If so, this might be
explained either by competition for limited numbers of environ-
mental niches for MBC survival, or by preferential induction of
SLPC rather than LLPCs, in frequently infected individuals.
Materials and Methods
The studies were approved by both the Gambia Government/
Medical Research Council (MRC) Joint Ethics Committee and the
Ethics Committee at the London School of Hygiene and Tropical
Medicine. Participants were enrolled after individual written
informed consent was obtained from the participant or their
Study site and sample collection
Blood samples from 118 healthy individuals were collected in
May–June 2009 (prior to the transmission season) from Brefet,
Foni District, The Gambia. Brefet is a rural village situated 55 km
inland from the Atlantic coast and 1 km from the river Gambia. In
The Gambia, malaria transmission is seasonal, starting in July and
ending in December with the peak of transmission being in
November . The entomological inoculation rate in the area of
Brefet was estimated as 3.24 infective mosquito bites per person
per year in 1991 , as 0.92 in 2001 , and as 0.62 in 2006
. At the time of this survey (in 2009), 82% of beds in Brefet
were covered with an insecticide treated bed net.
Villagers were grouped according to age into the following six
age categories (1–4, 5–9, 10–14, 15–24, 25–39 and .40 years,
respectively) and 20 individuals from each group were randomly
selected. To avoid household clustering, all volunteers in each
group received consecutive numbers, the total number of villagers
per group was noted and out of this figure a random number
generator drew 20 numbers to identify 20 individuals per age
group. For individuals absent on the day of sampling, a new
number was drawn. There was an equal distribution of males and
females in each group.
A venous blood sample of 5 to 20 ml was obtained from each
participant in accordance with the age-specific guidance provided
by the Gambian Government / MRC Joint Ethics Committee. A
thick blood film was prepared to test for presence of parasitaemia
by slide microscopy, 0.5 ml of blood was collected into EDTA
tubes for parasite detection by PCR and the remainder of the
sample was collected into heparinised tubes and used for B cell
ELISpot and Ab measurement by ELISA. HIV testing was not
done on any of the samples, and HIV status was not reported.
Adult HIV prevalence in rural areas of The Gambia is estimated
to be approx 2% in 2009 ; thus undiagnosed HIV infection is
unlikely to have markedly affected the immune responses reported
here. Stool samples from 38% of randomly selected study
participants were also collected to assess worm carriage (Table S1).
A village health worker was present in Brefet throughout the
transmission season to diagnose malaria infections by use of RDT
(OptiMALH) for malaria. Study participants had been asked to
report to the village health centre if they suspected they had
malaria. Anti-malarial treatment was only given if the rapid
diagnostic test was positive.
At the end of the transmission season (December 2009), a finger
prick sample from each study participant was obtained to assess i)
anti-malarial Ab levels, and ii) parasitaemia levels by PCR.
Samples from 20 individuals were missing (18 had travelled away
from the village and 2 had withdrawn their consent). A week later,
a further venous sample for B cell ELISpot was requested from
those individuals who showed a$1.5 fold increase in Ab
concentration for at least 1 Ag in comparison to the value
recorded at the start of the malaria season in May and/or for
whom a clinical episode of malaria during the last transmission
season was confirmed by a positive RDT. Individuals with
increased Ab concentrations and/or a clinical episode of malaria
are collectively referred to as cases. Age-matched (plus/minus 1
year) controls (study participants without evidence of malaria
exposure during the transmission season) were selected from the
study cohort for comparison.
For the measurement of AMB, blood collected from children
enrolled in an ongoing malaria case-control study based in the
coastal area of The Gambia [33,34] was used.
The MSP-119 and MSP-2 Ags used were glutathione s-
transferase (GST) fusion proteins representing amino acids
1631–1726 of the Wellcome allele of MSP-119 and amino
acids 22–247 of the Dd2 allele of MSP-2 , respectively. MSP-
119is highly conserved among P. falciparum isolates with minor
sequence variations having a minimal effect on antibody
recognition . Parasites belonging to the MSP2-Dd2 family
are widely represented amongst parasite isolates in The Gambia
Malaria-Specific MBC in Low Transmission Areas
PLoS ONE | www.plosone.org11 October 2011 | Volume 6 | Issue 10 | e25582
. The MSP-3 Ag used is a maltose binding protein (MBP)
fusion protein representing amino acids 2–354 of the 3D7 allele of
MSP-3 . The AMA-1 Ag is a 66Histidine tagged protein
representing amino acids 22–545 of the 3D7 allele sequence
[48,49]. Although there is almost an infinite array of AMA-1
alleles, differing slightly from each other in sequence and
antigenicity, polyclonal antibody responses of individuals living
in endemic areas appear to cross-react extensively with heterol-
ogous AMA-1 sequences . All the Ags were further diluted in
16PBS at the appropriate working concentrations (0.002 mg/ml).
Purified diphtheria toxoid (DT) (NIBSC, UK), derived by
formaldehyde deactivation of diphtheria toxin, a 535 amino acid
long polypeptide, was used at 0.002 mg/ml to establish diphtheria
vaccine induced responses. KLH (from Calbiochem) was used as a
control Ag at 0.002 mg/ml.
PBMC were isolated from the participant’s whole blood as
described elsewhere . Following a protocol adapted from
Crotty et al. [1,19], 16106PBMCs in RPMI containing 10%
fetal calf serum, 100 U/ml penicillin, 100 ug/ml streptomycin
and 2 mM L-glutamine (all Sigma) were added to each well of a
24-well culture plate, and supplemented with 50 mM b-Mercap-
toethanol, 0.5 mg/ml Phytolacca Americana pokeweed mitogen
(PWE, Sigma), Staphylococcus aureus Cowan (SAC, Sigma),
3 mg/ml CpG-2006 (Eurofins MWG-Operon- 59TCG TCG
TTT TGT CGT TTTGTC
recombinant human IL-10 (R&D Systems) and placed in the
incubator (37uC, 5% CO2) for six days. On day 5, quadruplicate
wells of multiscreen-HA plates (Millipore, MAHAS4510) were
coated overnight with either unbiotinylated AffiniPure F(ab9)2
fragment donkey anti-human IgG (Jackson ImmunoResearch
Laboratories), or with one of the four malarial Ags, DT or KLH,
respectively. On day six, 400,000 cultured cells were added to
each antigen coated well (2000 cells for IgG coated wells), and
incubated for 6 hours (37uC, 5% CO2). Cells were then
incubated overnight (at 4uC) with biotin-SP-conjugated Affini-
Pure fragment donkey anti-human IgG (Jackson ImmunoRe-
search). The next day, strepavidin-AKP (BD Biosciences)
followed by detection solution (AP conjugate substrate kit
(BioRad)) was added and plates were read using the AID
ELISpot reader. All samples in the IgG coated wells yielded more
than 1000 spots/million PBMC, a threshold previously defined
for a valid assay . The median of the coefficients of variation
for the replicates from all subjects and all antigens was 8.03%.
Malaria-specific ASC are presented either as the percentage of
the average number of spots counted in the IgG coated wells (%
MBC), or as spots per million PBMC (MBC/PBMC) seeded onto
the ELISpot plate after the 6 day culture.
Only values above the upper limit of the 99% confidence
interval of the median obtained for the negative control KLH
(0.004% or more than 1.88 spots/million PBMC) were considered
as positive responses.
GTT39) and 25 ng/ml
ELISA for serum antibodies to malaria antigens
Concentrations of IgG binding to MSP-119, MSP-2, MSP-3 and
AMA-1 were measured by ELISA, using the method described
elsewhere [12,51]. Ab levels to the different malaria antigens tested
were compared to a pool of sera samples collected in Brefet in
2008, and expressed as arbitrary units. Results obtained with a 50
fold dilution of the pooled sample were defined as 20 arbitrary
A positive response was defined by a value above the upper limit
of the 99% confidence interval obtained for this Ag with a pool of
plasma samples obtained from 20 malaria naı ¨ve tourists from
Europe. Only individuals which were above the cut-off value were
Diphtheria toxoid IgG ELISA
Ab levels to DT were quantified using a commercial kit (MP
Biomedicals Diphtheria Toxoid IgG antibody ELISA kit catalogue
number 071-524002) following the manufacturer’s instructions.
OD values are converted to International Units (IU/ml) by
comparison with the standard curve. The manufacturer considers
antibody titres .0.1 IU/ml sufficient for protection.
DNA was extracted from 150 ml of EDTA blood using the X-
tractor GeneTM robot, according to the manufacturer’s instruc-
tions (Corbett Robotics) . PCR amplification of Plasmodium
falciparum ribosomal DNA was performed as described previously
. PCR products were resolved by 2% agarose gel electropho-
resis (30 mins, 100 v); gels were stained with ethidium bromide
and read by a trans-illuminator (BioRad).
Flow cytometry analysis
Surface staining of AMB was performed on freshly isolated
PBMC. The cells were stained with the following fluorochrome-
labelled mouse anti-human antibodies: ECD-anti-CD19 (Beckman
Coulter), and APC-anti-CD21, APC-Cy7-anti-CD27, Pe-Cy7-
anti-CD10 (all from eBioscience). AMB were identified as follows:
CD19+CD212CD272CD102. All samples were acquired on a 9-
colour CyAn ADP flow cytometer and were analysed by FlowJo
software (TreeStar Inc., Ashland, OR, USA).
Stool samples were collected into fixative (10% formalin) and
processed using the ParasiTrap fecal diagnostic system (Biosepar)
and read by the routine microbiology laboratories at the MRC in
Fajara, The Gambia.
Data were analysed using GraphPad Prism 5 and STATA 10.1.
Chi-squared tests for trend were used to assess whether the
percentage of individuals having Ab and MBC changed
significantly with age for each Ag as well as for measuring the
breadth of the immune response. Kruskal-Wallis tests were used to
assess whether Ab concentration or MBC numbers differed
significantly between age groups, and to test for differences in
MBC numbers for the different Ags. As appropriate, Wilcoxon
matched paired tests or Mann-Whitney tests were performed to
compare two groups. Where multiple tests were performed on the
same individuals for multiple responses to different malarial Ags, p
values were adjusted for multiple comparisons using the Bonferoni
correction. To assess the agreement between the two read outs
used for the ELISpot results (% MBC or MBC/PBMC) in
identifying ‘‘responders’’ Kappa statistics were calculated, and the
results graded using the classification by Landis & Koch .
Correlation of two quantitative variables was assessed by
calculating the Spearman’s correlation coefficient. A logistic
regression model was used to analyse the changes in the frequency
of MBC with age.
Baseline characteristics of the study cohort.
Malaria-Specific MBC in Low Transmission Areas
PLoS ONE | www.plosone.org12 October 2011 | Volume 6 | Issue 10 | e25582
We express our deepest appreciation for the village community of Brefet, in
The Gambia for their participation in our study. A special thank you goes
to our study nurse Kebba Jobe, our field worker Lamin Manneh and to
Ousman Sanyang our village coordinator for ensuring the smooth running
of the study. We are grateful to David Cavanagh and Jana McBride for
provision of the plasmid for MSP-2 protein expression. Finally, we would
like to thank Simon Correa, Madi Njie and Idrissa Sambou for their
Conceived and designed the experiments: MW EMR. Performed the
experiments: SN. Analyzed the data: SN MW EMR BW. Contributed
reagents/materials/analysis tools: JCH EJR KT DJC. Wrote the paper:
SN MW EMR.
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