We measured IgG responses to 8 P. falciparum antigens at the time of treatment of malaria with AQ+SP to determine whether any of these responses were associated with protection from treatment failure. We found that higher antibody responses to AMA-1, but to none of the other 7 antigens tested, were significantly associated with protection. Furthermore, we found a strong dose response relationship, with higher responses to AMA-1 consistently associated with increased protection against treatment failure.
Does the identified strong association between AMA-1 antibody level and clearance of parasites suggest a causal role in protection? Antibody levels against AMA-1 have been associated with protection from clinical malaria in this 
and other cohorts 
. In addition, data from a recent malaria vaccine trial suggest that vaccination with FMP2.1/AS02A
, a recombinant protein based on AMA-1, may induce strain-specific protection 
. However, an alternative, though not mutually exclusive, explanation of our findings is that antibodies against AMA-1 provide information as a biomarker of immunity beyond any causal effect. Indeed, we found strong associations between epidemiological estimates of P. falciparum
exposure and anti-AMA-1 levels at the time of symptomatic malaria. Furthermore, antibody responses to all 8 antigens were significantly correlated with each other. In addition, despite our prior observation of decreasing efficacy of AQ+SP over time that was likely due to waning immunity 
, antibody responses to AMA-1 at the time of malarial illness did not decrease significantly over time. Furthermore, analysis of anti-AMA-1 levels across multiple episodes of malaria in the same subject suggested that variation within a subject had no association with treatment outcomes. These data argue against a causal effect, but suggest a more robust predictive value of anti-AMA-1 as a biomarker. Thus, antibody responses to AMA-1 may assist in clearance of parasites, but our data more strongly suggest that they offer a useful individual-level biomarker of blood stage immunity.
Associations between antibody responses to various antigens and antimalarial treatment outcome have been examined in prior studies 
. These studies have found associations between positive or high IgG responses to a number of antigens (NANP repeat of CSP 
, K1 and MAD20 block 2 variants of MSP-1 
, and GLURP-R0 and R2 
) and protection from treatment failure. Three of these studies included assessment of AMA-1 antibodies in their analyses, but these did not show strong associations between anti-AMA-1 levels and clearance of parasitemia 
. Differences between our results and prior studies may be attributable to the antimalarial therapy used, due to varying efficacy and pharmacokinetics; differences in the epidemiological setting and patient characteristics; different means of assessing treatment outcome; and the use of different laboratory and analytical methods. It should also be noted that while response to antimalarial therapy may provide information on blood stage protection not directly confounded by prior exposure, such an analysis is inherently limited to subjects who are exposed enough to P. falciparum
to observe a malaria episode and do not have complete immunity against symptomatic malaria. Of note, we obtained highly quantitative values for each response and demonstrated strong associations between anti-AMA-1 levels and protection, whether analyzing responses as reactive/non-reactive, in tertiles, or as a continuous variable.
Interestingly, we found that, for anti-MSP-1 levels assessed 14 days following malaria treatment, lower levels were associated with clearance of parasites. This finding is in contrast to previous studies that found anti-MSP-119
levels at the time of treatment to be protective against treatment failure 
. While our finding may represent a spurious result, given the marginal significance when taking into account multiple testing, there are some biologically plausible mechanisms for this seemingly paradoxical association. For example, persistent parasite replication in the blood after antimalarial therapy may have stimulated responses to MSP-1 on Day 14. Supporting this hypothesis, anti-MSP-1 levels were higher and boosted more from Day 0 to Day 14 in those children with higher parasite densities at diagnosis. Another possibility is the presence of blocking or neutral antibodies that compete for binding sites on the C-terminal epidermal growth factor domain of MSP-119
. Binding of these antibodies (i.e. prevention of beneficial “inhibitory” antibodies that inhibit invasion and secondary processing of MSP-1) lead to MSP-119
processing and resultant invasion of the red blood cell. Indeed, a study in Nigerian children revealed that there was evidence for both blocking and neutral antibodies competing for MSP-119
binding sites as there was absent inhibitory activity observed despite high MSP-119
prevalence and levels by ELISA 
In conclusion, we have shown that the levels of antibodies directed against AMA-1 at the time of presentation with malaria are strongly associated with concurrent and future blood stage immunity, as measured by the ability of the host to clear P. falciparum. A mechanistic role in protection is possible, but would need to be evaluated in the context of broader assessment of immunologic responses. The strong dose-response relationship we observed indicates that quantitative measurement of these antibodies may offer a robust biomarker of immunity and should be validated in other epidemiologic settings.