These data suggest that multicomponent malaria vaccines mimicking naturally acquired immunity should induce antibody responses that can be boosted by natural infections. Supplementary Data Supplementary materials are available at online (http://cid.oxfordjournals.org/). been repeatedly infected and have gradually developed some degree of immunity against malaria. The number of clones, ML-098 as characterized by polymorphisms within merozoite surface antigen genes, vary with age and malaria transmission intensity [4, 5]. Cohort studies in areas of high malaria transmission have shown that the number of clones at baseline in cross-sectional surveys is associated with reduced subsequent risk of malaria [4, 6C8]. Multiclonal infections have been proposed to confer protection against malaria by preventing superinfection or by mediating tolerance to infection [3]. However, the precise immunological mechanisms underlying this protective association are unknown. Antibodies are well recognized as important components of malaria immunity [9]. In malaria-endemic areas, anti-merozoite antibody titers are higher in children with asymptomatic infections compared to aparasitemic children [10C15]. Moreover, increasing breadth of antibody responses to merozoite antigens (ie, the number of antigens to which an individual has high antibody titers) was associated with increasing protection against malaria [11, 16]. Whereas it has been postulated that the tolerance of multiclonal infections is associated with a broad repertoire of immune responses that control parasitemia and prevent malaria [3], this hypothesis has not been empirically tested. Here, we test the hypothesis that asymptomatic multiclonal infections are associated with increasing breadth of anti-merozoite antibody responses, and that, in combination, these factors would be more strongly associated with protection against malaria than either factor individually. In a cohort study in Tanzania that included children aged 16 years, we determined the number of clones in asymptomatic infections at baseline by genotyping the merozoite surface protein 2 gene (genotypes in asymptomatic infections and antibody responses to these merozoite antigens in relation to risk of malaria. MATERIALS AND METHODS Study Population The study was conducted within a longitudinally monitored population in Nyamisati village in the Rufiji River delta, coastal Tanzania [18]. A cross-sectional survey including 890 individuals (aged 1C84 years) was conducted in ML-098 MarchCApril 1999 in which venous blood was collected in ethylenediaminetetraacetic acid and stored frozen as plasma and packed cells. Parasite prevalence was 46% by PCR, with the highest prevalence in children aged 3C5 years (74% by PCR) [7]. All participants were monitored for 40 weeks and malaria was recorded by passive case detection by researchers who operated the only PCDH9 health facility in the village [18]. All individuals who reported to the research clinic with fever and parasites detected by microscopy were administered free antimalarial treatment. In this study, malaria was defined as fever (axillary temperature >37.5C or history of hot body within 24 hours) and 5000 parasites/L in peripheral blood [19]. The present analysis was restricted to children aged 16 years who were healthy at the time of the survey (n = 320). Children with history of fever in the 4 weeks preceding or within 1 week of the survey were excluded. The project was scientifically and ethically approved by the National Institute for Medical Research in Tanzania and the Stockholm Ethical Review Board (Dnr00-084 and 2012/1151-32). Informed consent was obtained from the guardians of all participants. Genotyping of Infections DNA was extracted from packed erythrocytes using ABI6100 PrepStation (Applied Biosystems). Genotyping of the gene was ML-098 performed as described previously [17]. In brief, the PCR included an initial amplification of the outer domain, followed by nested reactions with fluorescent primers targeting the FC27 and IC-1/3D7allelic types of Allelic fragments were separated by capillary electrophoresis and analyzed using GeneMapper software (Applied Biosystems). The number of genotypes identified by this method determines the number of clones within an individual infection. Considering that PCR can amplify genomic DNA encoding the gene from both asexual [20] and sexual [21] parasite stages, genotypes detected could be from either stage. The gene of a subset of infections with only 1 1 genotype were sequenced using BigDye terminator technology (Applied Biosystems). Recombinant Merozoite Antigens Seven recombinant antigens representing 4 malaria vaccine candidate antigens were expressed in and purified by high-performance liquid chromatography. Two allelic forms of MSP-2, MSP-2_Dd2 and MSP-2_CH150/9 (representing the FC27 and IC-1 allelic families of respectively), and the 19-kilodalton fragment.