A commonly accepted tenet in malaria research is that clinical immunity to symptomatic P. falciparum
infection is achievable but requires several years of constant parasite exposure. Although humoral responses are thought to be the mechanism driving immunity to malaria infection, most prior epidemiologic studies performed in high-transmission areas demonstrate short-lived antimalaria antibody responses and irregularities in cellular memory responses (3
). These findings help to explain the delayed acquisition of protective immunity in high-transmission areas, which is likely due to multiple factors that impair effective immune response development, including high infection rate, diverse parasite genetic characteristics, host genetics, and/or concomitant infections.
However, as most prior studies evaluating the humoral response to malaria infection were performed in high-transmission areas, they were limited in their ability to consider postinfection antibody dynamics by the rapidity of reinfection that defines these regions (1
). Interestingly, several studies performed at low-malaria-transmission sites suggest more rapid development of clinical immunity (4
) and stable anti-MSP119
), although these studies were complicated by widely spaced sampling time points. Other groups have reported robust antimalarial antibody responses to low levels of parasite exposure (26
). Thus, we hypothesized that antibody responses to malaria infection could develop more effectively in low-transmission areas, even after only one or two documented P. falciparum
The communities around Iquitos, Peru, are an ideal setting for testing this hypothesis. The malaria transmission rate in this region is less than 0.5 infection/person/year (7
), leaving little possibility of overlapping infections. We used a unique longitudinal sampling strategy that enabled us to capture pre- and postinfection antibody dynamics to evaluate our Peruvian cohort. In addition, by comparing personal histories to health post records, we were able to estimate each individual's number of prior P. falciparum
In this study, we first evaluated the seroprevalence of IgG to four different malaria vaccine candidate antigens (AMA-1, CSP, EBA-175, and MSP-119
) at time points before, during, and after infection. Individuals in our cohort had a more robust response to MSP119
than to any of the other antigens tested (), and thus we chose to focus on the MSP119
antigen for the remainder of the study. We sampled our study subjects for at least 180 days after infection, during which time, we used blood smear microscopy to establish that there was no continuing P. falciparum
infection after treatment. The majority of individuals (>60% of adults and 40% of children) maintained a positive anti-MSP119
response for more than 5 months postinfection (A and B), indicating that most adults as well as a large percentage of children are able to maintain an appropriate antibody response to infection. This finding supports the results of prior studies in which adults were shown to maintain a stronger antibody response than children; however, those studies did not explore the idea that this phenomenon is due to prior P. falciparum
exposure rather than the physiologic changes of aging (1
). Due to the low malaria transmission rate and highly regulated regional malaria drug treatment policies, we were able to consider previous parasite exposure separate from age.
Our study region contains both adults and children with zero or very few prior symptomatic P. falciparum infections in their lifetime. Even among groups with various levels of exposure (0, 1, 2, or >2 prior infections), adult MSP119 antibody responses had a median seropositivity time of >180 days (C). With respect to the adults reporting 0 prior infections, the fact that their anti-MSP119 responses last for >180 days indicates that either they mount a robust response to their first infection or they had previously been exposed to Plasmodium but were not diagnosed at the health post. In addition, more than 60% of children reporting either 1 or 2 prior infections had median antibody seropositivity times of at least 180 days postinfection, suggesting that only moderate P. falciparum exposure (1 or 2 prior infections) is necessary to maintain a robust anti-MSP119 IgG response. Individuals who had antibody responses that lasted <180 days were frequently children with no history of infection (this being their first infection) or children reporting >2 prior infections (B). Initial antibody level did not explain the shorter-duration response, as individuals included in the Kaplan-Meier analysis did not have significantly different levels of anti-MSP119 antibody at the time of infection. (The analysis only included individuals who had an IgG OD of ≥0.500 at day 0.) These individuals may have a diminished capacity to develop a lasting response due to experiencing more than the average number of prior infections early in life.
The presence of serum antibody in the absence of infection can be considered secondary evidence of plasma cell maintenance (34
). Thus, we examined cellular immunologic memory in this Peruvian cohort by evaluating the dynamics of B cell subsets during and just after infection. Upon antigenic stimulation, both naïve and memory B cells proliferate and differentiate into antibody-secreting plasmablasts and eventually become nondividing, highly productive plasma cells (38
). In this study, we used whole blood samples taken shortly after P. falciparum
diagnosis, as well as follow-up samples from these patients, to evaluate changes in the dynamics of certain B cell subsets, namely, CD19+
plasmablasts and CD19+
memory B cells over time. We observed a larger population of CD19+
plasmablasts at day 0 compared to the recovery phase 11 or more days later in both adults and children (), indicating that these individuals are successfully generating pathogen-initiated, antibody-producing plasma cells at the time of infection. While this plasmablast increase following infection could reflect polyclonal B cell activation with accompanying hypergammaglobulinemia, that is more characteristic of chronic malaria infections occurring in high-transmission regions. We also observed a larger population of CD19+
memory B cells in children at day 0, but not in adults, perhaps because adults already have an established malaria-specific memory B cell pool. We did not observe the disruption in B cell memory and plasmablast responses that Asito et al. described in high-transmission settings (3
), which could be due to the lower transmission setting of our study. Because of the small number of individuals with >2 prior infections in our study, we could not directly test the association between B cell population dynamics and prior P. falciparum
exposure. Such a test may provide further insight into the discrepancy between our findings and those of Asito et al.
To determine whether the memory B cells we detected via flow cytometry were Plasmodium specific, we conducted malaria antigen-specific memory B cell ELISPOT assays. Although memory B cells are a rare population in the peripheral blood, especially as time increases after infection, we found that a subset of individuals demonstrated MSP1-specific memory B cells at day 0 as well as at various postinfection time points (). This indicates that individuals in a low-transmission setting generate Plasmodium-specific memory B cells, even after a single or few P. falciparum infections. The combined observations of robust MSP119 antigen-specific antibody responses, infection-specific plasmablast population expansion, and MSP1-specific memory B cells suggest that immunologic memory is achievable in individuals living in this low-transmission region.
Few studies have identified malaria-specific memory B cells (19
), perhaps due to the relatively recent availability of assays that make it possible to test antigen-specific memory B cell populations. However, Weiss et al. recently reported that an FCRL4(+) hyporesponsive subset of memory B cells, coined “atypical” memory B cells, are increased in individuals exposed to high transmission rates in Mali (42
). These authors proposed that this newly defined memory population could represent a clue into the delayed acquisition of immunity to malaria. Recently, we directly compared the memory B cell responses of our Peruvian cohort to those of a high-transmission population in Mali (41
). We found that, compared to P. falciparum
-naive controls, “atypical” memory B cells were increased in Peruvian adults exposed to low P. falciparum
transmission, but to a lesser degree than that observed in Malian adults exposed to intense P. falciparum
), suggesting that our low-transmission setting provides the appropriate stimulus for developing B cell memory to malaria. The data from our current study further support the hypothesis that antimalarial immune responses are more stable in regions of low transmission because they are not disrupted by extreme parasite exposure.
The exact mechanism(s) that occur in high-transmission areas to produce a dysfunctional humoral response remain unclear. In areas of high transmission, increased parasite genetic diversity results in infections being mixtures of many different genetic types. In a study in western Kenya, we found that individuals having mixed-genotype infections (antigenically complex infections) were more rapidly reinfected. We suggested that complex infections could result in mediocre immune response development to conserved antigens, such as MSP119
). In addition, Crompton et al. hypothesized that this poor response could be related to the host immune system being overwhelmed by the large number of parasite antigens presented to it during infection (14
). Thus, infection spacing, parasite antigenic diversity/quantity, and geographic differences might together explain why many prior studies have not found evidence for functional immunologic memory to malaria.
There are several limitations to our study. We were unable to demonstrate an independent measure of prior P. falciparum exposure. Although this variable may always remain inextricably tied to age, perhaps with a larger sample size, more definitive differences in exposure groups could be demonstrated. In addition, our study involved a very long sample collection period, during which sample degradation could have occurred. During this sample collection period, we did not begin to collect cellular data until 2009, and thus our cellular data cannot be directly compared to our antibody data.
In this study, we present a model of effective immunity against P. falciparum malaria that may be the result of human genetic or environmental factors specific to this low-transmission region or perhaps to the presence of fewer and less antigenically complex infections. These results may signify that, as vaccine strategies and other malaria-blocking interventions work to decrease the malaria burden, the resulting reduced transmission intensity would pave the way for the rapid development of naturally acquired immunity, further reducing malaria pathology and transmission.