This study found no evidence to support that the time of first exposure to blood stage P. falciparum infection, as controlled by SP + AS anti-malarial chemoprophylaxis, has a major influence in the acquisition of antibody responses to the MSP-119, AMA-1, EBA-175 and VSA antigens examined. Consistent with the results of the clinical trial , the development of naturally acquired immunity within the parameters analysed (total IgM, IgG and subclasses) was not heavily impacted by a substantial disruption of parasite exposure. Significant decreases were only observed in IgM and IgG responses to some antigens in the early exposure group at month 10.5 following chemoprophylaxis, i.e., lower levels of IgM to EBA-175 and lower levels of positive IgG1 and IgG3 responses to MSP-119. Thus, IgM to blood stage antigens at age 10.5 months appeared to be a surrogate of recent P. falciparum exposure. Maternal IgG levels were high at 2.5 months in all groups before waning over the first year. The conclusion of this first analysis was that, overall, a 3–5 month-long drug intervention had no major impact on P. falciparum-specific IgG levels in these young infants in face of high levels of maternal IgG antibodies still circulating in their plasma. Because of the confounding effect of these passively transferred antibodies, the impact of the study interventions on IgG levels during the first year of life and the impact of IgG responses during the first year of life on subsequent risk of malaria were difficult to ascertain.
Of note, children who received chemoprophylaxis during the first year of life and suffered previous malaria infections were characterized by an increase in antibody levels in the second year, as observed in the stratified cumulative distribution plots (Figures 3, 4 and 5). This pattern appeared to be parallel to the Kaplan Meier survival curves for cumulative proportion of children with at least one episode of malaria during the second year of follow-up, which showed a trend for a higher incidence of malaria in year 2 in the late and early exposure groups when compared to the control group . Thus, these increased IgG antibody levels in children with documented parasite exposure reflected the higher incidence of malaria in the second year in those who received chemoprophylaxis in the first year. Therefore, antibodies to these blood stage antigens appeared to be mostly markers of exposure, and this is consistent with the strong association between previous or concurrent infection and IgG levels and breadth demonstrated here and in previous studies by our group [34, 35]. Indeed, data show that high levels of antibody responses to antigens such as AMA-1 are commonly associated with greater future risk of malaria, presumably because children with increased antibodies levels or breadth are also those who have suffered more previous episodes of malaria and/or prenatal parasite exposure, a known and strong risk factor for subsequent malaria episodes [32, 34, 36]. This is in contrast to other studies showing an association between AMA-1 [3, 16, 37, 38] or MSP-119[4, 16, 39] responses and reduced risk of malaria. Even though, for example, anti-AMA-1 IgG antibodies are described as some times protective  while other non-protective , many studies fail to establish any significant association . Dissimilarities between reports may be partially related to differences in age between participants, under two years old in this study vs older children or adults in other studies. However, a prominent factor that influence immune correlates analysis is heterogeneity in parasite exposure at the individual level. The challenges of establishing protective associations between antibody responses and malaria susceptibility if prior and present infections are not registered and taken into account in the analysis are increasingly being recognized. The only instance in which there was a protective association between higher levels of antibodies and reduced incidence of malaria was for the EBA-175 antigen, and this was mostly shown after analyses were adjusted for previous malaria episodes. Since this result has consistently been reproduced in two other studies in the same area with two different groups of children [35, 42], it is concluded that among the antigen targets evaluated, EBA-175 is the most promising blood stage candidate for vaccine development.
Factors affecting the magnitude of anti-malarial antibodies were analysed in detail. Previous studies report that increasing age (particularly for AMA-1), heterogeneity of exposure, proximity to swamps, and higher previous exposure are significant predictors of higher antibody responses [4, 32, 35]. In this study, age was also the most prominent factor significantly influencing the levels of all antibodies. In addition, factors associated to concurrent or past parasite exposure, including during gestation and birth, generally affected antibody levels in these infants. Season and particular neighbourhoods had increased antibody prevalences, however it was not possible to define the geographic characteristics (e. g., proximity to swamps) explaining the differences. This study was limited in that it could not fully evaluate individual heterogeneity of exposure and differential antibody levels in relation to geographical parameters influencing malaria transmission intensity.
In these very young children, anti-VSA IgG responses were not prominent. Levels were very low and dependent on neighbourhood and age, with an overall pattern showing higher maternally derived IgG waning over time. This was in contrast to studies in a different endemic area of Tanzania where the level of antibodies increased with age from five to 24 months . The results of this study do not support acquisition of VSA IgG by the age of two years in the Manhiça population at the time of the clinical trial. There was a lack of association between these antibodies and current malaria infection, in contrast to what was observed in another study conducted in the same area at a different time , and in Tanzania . This suggests that in these children, anti-VSA IgG were not involved in early acquisition of immunity, presumably due to the high variability of PfEMP-1 antigens and the lower level of exposure during this limited time.
Breadth of antibody response has been suggested to be an important predictor of protection from clinical malaria in Kenyan children , with an inverse association between increasing breadth of IgG antibody specificities and risk of malaria, and none of the children who simultaneously had high antibody levels to five or more antigens experiencing a clinical episode. Studies assessing interaction between breadth and risk of malaria are limited. Here, using only four antigens, a direct association between breadth of IgG response and subsequent incidence of malaria was either not detected or observed at two different follow up periods (5.5-24 and 15–24 months of age), in contrast to the Kenyan study but in the same line as the relationship between magnitude of antibody responses and malaria risk. In Manhiça, the association between IgG antigenic breadth and incidence of malaria was stronger in the unadjusted analysis and weaker when controlling for variables related to parasite exposure, suggesting that it was highly affected by prior infection but that we not were able to remove all the confounders. Thus, in immune correlates analyses in mother-child cohorts it remains a challenge to account for absolute parasite exposures when using a primarily passive surveillance design, particularly to detect those infections that may be asymptomatic and not accounted for in the adjusted analyses.