Naturally acquired Duffy-binding protein-specific binding inhibitory antibodies confer protection from blood-stage Plasmodium vivax infection

Abstract

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.

Fig. 1.

Association of anti-PvDBPII BIAbs with reinfection by P. vivax and P. falciparum. Children with high PvDBPII BIAbs (n = 18, dashed line) vs. those with low BIAbs (n = 148, solid line) show a delay in time to first blood-stage P. vivax infection detected by LM of Giemsa-stained blood smears (A, P = 0.02, Log-rank test) after parasite clearance with artesunate. The presence of anti-PvDBPII high BIAbs did not affect the time to reinfection with P. falciparum as measured by LM of Giemsa-stained blood smears (B). Binding BIAbs did not affect the time to reacquisition of blood-stage infection for either P. vivax or P. falciparum as measured by PCR-based LDR-FM assay (C and D).

Fig. 2.

The association of anti-PvDBPII BIAbs antibodies with lower P. vivax density. A shows that the presence of high anti-PvDBPII BIAbs before treatment (n = 18) correlates with lower P. vivax density compared with individuals with intermediate (n = 40) or low (n = 148) BIAb levels as estimated by LM (black bars) or RTQ-PCR (stippled bars). There was no association of high anti-PvDBPII BIAbs levels with lower P. falciparum parasite densities. Parasite densities were determined from an average of 12 observations per individuals as described in Table 1 over the 6-month followup period after treatment. B shows that the presence of high overall PvDBPII reactivity measured by ELISA was also associated with a reduction in parasitemia for P. vivax and not P. falciparum. Measurement of parasitemia and the statistical methods for testing their differences between groups are described in Materials and Methods.

Fig. 3.

Anti-PvDBPII-specific binding BIAb levels to different variants of PvDBPII. A shows inhibition of receptor binding by six variants of PvDBPII at a 1:5 plasma dilution for the 18 individuals with high BIAbs. The other images show variant-specific binding inhibitory activities at different plasma dilutions for an individual with high (B) and intermediate BIAb levels (C and D).

Fig. 4.

Repeated measurements on inhibition of PvDBPII-receptor binding by endemic sera during the study. (A) Serial measurements of PvDBPII-specific BIAbs for 15 children for whom three or more plasma samples were collected over 1 year. Time 0 represents a sample collected before artesunate treatment and time 11–12 months are collected on asymptomatic individuals 1 year after treatment. All intervening samples were collected at the time of an acute malaria episode (>90% due to P. falciparum). (B) BIAb levels from one subject (no. 5) at different plasma dilutions collected at four time intervals over the course of the study. The solid, shaded, and hatched bars represent binding inhibition at plasma dilutions of 1:5, 1:10, and 1:20, respectively.