In a treatment re-infection study of 206 Papua New Guinean school children, we examined risk of reinfection and symptomatic malaria caused by different Plasmodium species. Although children acquired a similar number of polymerase chain reaction-detectable Plasmodium falciparum and P. vivax infections in six months of active follow-up (P. falciparum = 5.00, P. vivax = 5.28), they were 21 times more likely to develop symptomatic P. falciparum malaria (1.17/year) than P. vivax malaria (0.06/year). Children greater than nine years of age had a reduced risk of acquiring P. vivax infections of low-to-moderate (>150/microL) density (adjusted hazard rate [AHR] = 0.65 and 0.42), whereas similar reductions in risk with age of P. falciparum infection was only seen for parasitemias > 5,000/microL (AHR = 0.49) and symptomatic episodes (AHR = 0.51). Infection and symptomatic episodes with P. malariae and P. ovale were rare. By nine years of age, children have thus acquired almost complete clinical immunity to P. vivax characterized by a very tight control of parasite density, whereas the acquisition of immunity to symptomatic P. falciparum malaria remained incomplete. These observations suggest that different mechanisms of immunity may be important for protection from these malaria species.

Individuals residing in malaria-endemic regions acquire protective immunity after repeated infection with malaria parasites; however, mechanisms of protective immunity and their immune correlates are poorly understood. Blood-stage infection with Plasmodium vivax depends completely on interaction of P. vivax Duffy-binding protein (PvDBP) with the Duffy antigen on host erythrocytes. Here, we performed a prospective cohort treatment/reinfection study of children (5-14 years) residing in a P. vivax-endemic region of Papua New Guinea (PNG) in which children were cleared of blood-stage infection and then examined biweekly for reinfection for 25 weeks. To test the hypothesis that naturally acquired binding inhibitory antibodies (BIAbs) targeting PvDBP region II (PvDBPII) provide protection against P. vivax infection, we used a quantitative receptor-binding assay to distinguish between antibodies that merely recognize PvDBP and those that inhibit binding to Duffy. The presence of high-level BIAbs (>90% inhibition of PvDBPII-Duffy binding, n ‫؍‬ 18) before treatment was associated with delayed time to P. vivax reinfection diagnosed by light microscopy (P ‫؍‬ 0.02), 55% reduced risk of P. vivax reinfection (Hazard's ratio ‫؍‬ 0.45, P ‫؍‬ 0.04), and 48% reduction in geometric mean P. vivax parasitemia (P < 0.001) when compared with children with low-level BIAbs (n ‫؍‬ 148). Further, we found that stable, high-level BIAbs displayed strain-transcending inhibition by reducing reinfection with similar efficiency of PNG P. vivax strains characterized by six diverse PvDBPII haplotypes. These observations demonstrate a functional correlate of protective immunity in vivo and provide support for developing a vaccine against P. vivax malaria based on PvDBPII.Duffy antigen ͉ immunity ͉ protective antibodies P rotective immunity against malaria is acquired by residents of endemic regions over a period of years after repeated exposure to malaria parasites (1). Naturally acquired immunity to Plasmodium falciparum and Plasmodium vivax malaria does not prevent infection by these parasites completely, but limits parasite densities, reduces the frequency of clinical malaria episodes, and prevents severe disease (2). Humoral immune responses against blood-stage antigens are an important component of naturally acquired immunity to malaria (2, 3). Therefore, parasite proteins engaged in erythrocyte invasion are potential targets of protective antibody responses.Interaction between P. vivax Duffy-binding protein (PvDBP) and the Duffy antigen receptor (DA) on host erythrocytes is central to blood-stage infection by P. vivax (4, 5). Adhesion to DA is mediated by the 140-kDa PvDBP (6-8). The receptor-binding domain of PvDBP maps to the conserved, N-terminal cysteine-rich region II (PvDBPII) (9, 10). Given the complete dependence of P. vivax on the PvDBPII-Duffy interaction for blood-stage infection and recent observations that PvDBPII-specific antibodies inhibit P. vivax invasion of human erythrocytes in vitro (11), we examined the hypothesis that...

Improving strategies for diagnosing infection by the four human Plasmodium species parasites is important as field-based epidemiologic and clinical studies focused on malaria become more ambitious. Expectations for malaria diagnostic assays include rapid processing with minimal expertise, very high specificity and sensitivity, and quantitative evaluation of parasitemia to be delivered at a very low cost. Toward fulfilling many of these expectations, we have developed a post-polymerase chain reaction (PCR)/ligase detection reaction-fluorescent microsphere assay (LDR-FMA). This assay, which uses Luminex FlexMAP microspheres, provides simultaneous, semi-quantitative detection of infection by all four human malaria parasite species at a sensitivity and specificity equal to other PCR-based assays. In blinded studies using P. falciparum-infected blood from in vitro cultures, we identified infected and uninfected samples with 100% concordance. Additionally, in analyses of P. falciparum in vitro cultures and P. vivax-infected monkeys, comparisons between parasitemia and LDR-FMA signal intensity showed very strong positive correlations (r > 0.95). Application of this multiplex Plasmodium species LDR-FMA diagnostic assay will increase the speed, accuracy, and reliability of diagnosing human Plasmodium species infections in epidemiologic studies of complex malaria-endemic settings.

Plasmodium vivax Duffy binding protein (DBP) is a merozoite microneme ligand vital for blood-stage infection, which makes it an important candidate vaccine for antibody-mediated immunity against vivax malaria. A differential screen with a linear peptide array compared the reactivities of noninhibitory and inhibitory high-titer human immune sera to identify target epitopes associated with protective immunity. Naturally acquired anti-DBP-specific serologic responses observed in the residents of a region of Papua New Guinea where P. vivax is highly endemic exhibited significant changes in DBP-specific titers over time. The anti-DBP functional inhibition for each serum ranged from complete inhibition to no inhibition even for high-titer responders to the DBP, indicating that epitope specificity is important. Inhibitory immune human antibodies identified specific B-cell linear epitopes on the DBP (SalI) ligand domain that showed significant correlations with inhibitory responses. Affinity-purified naturally acquired antibodies on these epitopes inhibited the DBP erythrocyte binding function greatly, confirming the protective value of specific epitopes. These results represent an important advance in our understanding of part of blood-stage immunity to P. vivax and some of the specific targets for vaccine-elicited antibody protection.