Whereas the EBAs and PfRh1 are important for SA-dependent invasion. Expression of PfRh4 varies among isolates, but knowledge on the extent of variation and the frequency of expression of PfRh4 by isolates is limited. There are data on expression of the Pfrh4 gene by isolates from infected individuals in Africa, and data on PfRh4 expression by a small number of laboratoryadapted isolates; however, there are presently no data on expression of PfRh4 protein by clinical isolates, or data from Lomitapide Mesylate populations outside Africa. Protective immunity to malaria eventually develops after repeated exposure, and is thought to prevent disease by controlling blood-stage parasitemia. Despite an expanding knowledge of the genomics and proteomics of P. falciparum, few antigens have been studied as immune targets in humans and there is a paucity of data on functional immune responses to specific antigens. These gaps have restricted our knowledge of immunity and impeded progress towards developing effective vaccines. Antibodies to erythrocyte invasion ligands may act by directly inhibiting parasite replication, possibly also through antibody dependent cellular inhibition, and opsonization of merozoites for phagocytosis. Very little is known about immune responses to PfRh proteins. An initial study in Kenya reported that antibodies to PfRh2 and PfRh4 were acquired in an age-dependent manner, reflecting the acquisition of immunity in the population, and antibodies to PfRh2 were associated with protective immunity in a prospective study of children. In light of the important role of PfRh4 in invasion, we have evaluated PfRh4 as a target of human immunity. To determine the association between antibody levels and subsequent episodes of P. falciparum infection and symptomatic malaria, children were stratified into three equal groups reflecting low, medium and high responders according to OD values for each antigen; the risk of malaria and Tulathromycin B parasitemia was compared between responder groups, as used previously. Although some children had multiple episodes of parasitemia or malaria, analysis included time to first re-infection or first symptomatic episode only. All children were treated with 7 days artesunate, orally, at enrolment to clear existing parasitemia, and treatment efficacy was 91.4%. Treatment failures were differentiated from re-infection by genotyping of msp2, and were excluded from the analysis. The Cox proportional hazards model was used to calculate hazard ratios for risk of malaria between antibody responder groups. For antibody variables that showed non-proportional hazards, an interaction term between the antibody response and time was included in the analysis. A range of demographic, clinical and biological variables were assessed as potential confounders of associations between antibodies and malaria outcomes. Only host age and location of residence were identified as being significantly associated with antibodies and malaria outcomes. Of note, parasitemia status at enrolment and red blood cell polymorphisms were not associated with malaria outcomes. Therefore, multivariate analysis was used to calculate adjusted hazard ratios using covariates of age and location. Age was used as binary variable, as previous studies indicated that this stratification was the most informative approach for assessing the effect of age. Additional age groupings with narrower age ranges were explored in the multivariate analysis; however, results using these groupings in the analysis were not different to using age as a dichotomous variable. Adjusting for host gender did not influence protective associations. Some previous studies have stratified analyses based on the presence or absence of parasitemia at baseline because it was found to influence antibody associations.