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. 2007 Dec;4(12):e337.
doi: 10.1371/journal.pmed.0040337.

Plasmodium vivax invasion of human erythrocytes inhibited by antibodies directed against the Duffy binding protein

Brian T Grimberg  1 , Rachanee Udomsangpetch, Jia Xainli, Amy McHenry, Tasanee Panichakul, Jetsumon Sattabongkot, Liwang Cui, Moses Bockarie, Chetan Chitnis, John Adams, Peter A Zimmerman, Christopher L King
Affiliations

Plasmodium vivax invasion of human erythrocytes inhibited by antibodies directed against the Duffy binding protein

Brian T Grimberg et al. PLoS Med. 2007 Dec.

Abstract

Background: Plasmodium vivax invasion requires interaction between the human Duffy antigen on the surface of erythrocytes and the P. vivax Duffy binding protein (PvDBP) expressed by the parasite. Given that Duffy-negative individuals are resistant and that Duffy-negative heterozygotes show reduced susceptibility to blood-stage infection, we hypothesized that antibodies directed against region two of P. vivax Duffy binding protein (PvDBPII) would inhibit P. vivax invasion of human erythrocytes.

Methods and findings: Using a recombinant region two of the P. vivax Duffy binding protein (rPvDBPII), polyclonal antibodies were generated from immunized rabbits and affinity purified from the pooled sera of 14 P. vivax-exposed Papua New Guineans. It was determined by ELISA and by flow cytometry, respectively, that both rabbit and human antibodies inhibited binding of rPvDBPII to the Duffy antigen N-terminal region and to Duffy-positive human erythrocytes. Additionally, using immunofluorescent microscopy, the antibodies were shown to attach to native PvDBP on the apical end of the P. vivax merozoite. In vitro invasion assays, using blood isolates from individuals in the Mae Sot district of Thailand, showed that addition of rabbit anti-PvDBPII Ab or serum (antibodies against, or serum containing antibodies against, region two of the Plasmodium vivax Duffy binding protein) (1:100) reduced the number of parasite invasions by up to 64%, while pooled PvDBPII antisera from P. vivax-exposed people reduced P. vivax invasion by up to 54%.

Conclusions: These results show, for what we believe to be the first time, that both rabbit and human antibodies directed against PvDBPII reduce invasion efficiency of wild P. vivax isolated from infected patients, and suggest that a PvDBP-based vaccine may reduce human blood-stage P. vivax infection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Inhibition of nDARC-Ig Binding to rPvDBPII by Antibodies
Rabbit (A) and human (B) anti-PvDBPII Ab were tested to observe inhibition of the interaction of the rPvDBPII protein and the N-terminal region of Duffy in the nDARC-Ig chimera. In ELISA-based nDARC-Ig assays, bars indicate the mean binding percentage relative to pre-bleed rabbit serum or nonspecific human IgG (120 μg/ml). Duplicate experiments showed variation of <5%.
Figure 2
Figure 2. Inhibition of rPvDBPII Binding to Human Red Blood Cells by Antibodies
Rabbit (A) and human (B) anti-PvDBPII Ab were tested to observe inhibition of the interaction of the rPvDBPII protein and Duffy-positive human erythrocytes. In erythrocyte-binding assays, the bars indicate the mean binding percentage of four separate experiments (± standard deviation) relative to pre-bleed rabbit serum or nonspecific human IgG (150 μg/ml). In parallel experiments run with Duffy-negative cells, binding was always <5% (unpublished data). Fy6 antibodies (50 μg/ml; recognize N-terminal region of the Duffy receptor) were used to show relative inhibition compared to the affinity-purified human anti-PvDBPII Ab.
Figure 3
Figure 3. Rabbit Anti-PvDBPII Ab Staining of P. vivax Merozoites in Infected Human Erythrocytes
Rabbit anti-PvDBPII serum binding to fixed P. vivax merozoite, trophozoite, and schizont (uninfected cells not shown) was confirmed by immunofluorescence microscopy. Parasites were enriched by Percoll gradient centrifugation from a Thai patient infected with P. vivax. In the left panel, PvDBPII is stained green by a 1:10 dilution of rabbit anti-PvDBPII serum, followed by FITC-conjugated goat anti-rabbit antibody. In the middle panel, DNA is stained blue by Hoechst. The right panel shows the overlay of the left and middle micrographs. Additionally, the antibody concentrated anterior to the nucleus towards the apical end of the merozoite where PvDBPII is known to be expressed in the micronemes [
Figure 4
Figure 4. Inhibition of P. vivax Invasion of Human Red Blood Cells by Anti-PvDBPII Ab
Tests were performed to examine the influence of rabbit (A) and human (B) anti-PvDBPII Ab on P. vivax invasion on a patient sample cultured in PvCM plus 20% AB serum (Experiment 1) or a sample from a second patient cultured in PvCM plus 25% AB serum (Experiment 2). Control cultures (white bars) contained media without antibodies and were the same for both experiments. The concentration of antibodies (pre-bleed rabbit serum or nonspecific human IgG) in the positive control cultures (white bars) was equal to the highest concentration of test serum. Various concentrations of the test antibody (black bars) were added to late-stage P. vivax cultures and grown for 24 h (in duplicate), and the number of newly invaded cells was observed by light microscopy based on examination of 200 high-powered fields of Giemsa-stained thin smears or approximately 20,000 erythrocytes. Bars indicate mean ± standard deviation of the number of invasion events. p-Values (two-sided t-test) are shown for differences of p < 0.1 between test samples and their respective control antibody.
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