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 
. 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
in infrequently exposed Thai adults 
and, in a recent report from a rural village in Mali where people are exposed to ~50 infective 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 
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-119
specific 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 accumulation 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 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 circulating 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) 
or as the number of MBCs among all the PBMCs seeded into the plate (MBC/PBMC) 
. 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 
. 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 case.
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 observations, 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 now adults.
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 hypergammaglobulinaemia commonly described in highly malaria endemic areas 
. 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 environmental niches for MBC survival, or by preferential induction of SLPC rather than LLPCs, in frequently infected individuals.