The Presence, Persistence and Functional Properties of <i>Plasmodium vivax</i> Duffy Binding Protein II Antibodies Are Influenced by HLA Class II Allelic Variants

<div><p>Background</p><p>The human malaria parasite <i>Plasmodium vivax</i> infects red blood cells through a key pathway that requires interaction between Duffy binding protein II (DBPII) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). A high proportion of <i>P</i>. <i>vivax</i>-exposed individuals fail to develop antibodies that inhibit DBPII-DARC interaction, and genetic factors that modulate this humoral immune response are poorly characterized. Here, we investigate if DBPII responsiveness could be HLA class II-linked.</p><p>Methodology/Principal Findings</p><p>A community-based open cohort study was carried out in an agricultural settlement of the Brazilian Amazon, in which 336 unrelated volunteers were genotyped for HLA class II (<i>DRB1</i>, <i>DQA1</i> and <i>DQB1</i> loci), and their DBPII immune responses were monitored over time (baseline, 6 and 12 months) by conventional serology (DBPII IgG ELISA-detected) and functional assays (inhibition of DBPII–erythrocyte binding). The results demonstrated an increased susceptibility of the <i>DRB1*13</i>:<i>01</i> carriers to develop and sustain an anti-DBPII IgG response, while individuals with the haplotype <i>DRB1*14</i>:<i>02-DQA1*05</i>:<i>03-DQB1*03</i>:<i>01</i> were persistent non-responders. HLA class II gene polymorphisms also influenced the functional properties of DBPII antibodies (BIAbs, binding inhibitory antibodies), with three alleles (<i>DRB1*07</i>:<i>01</i>, <i>DQA1*02</i>:<i>01</i> and <i>DQB1*02</i>:<i>02</i>) comprising a single haplotype linked with the presence and persistence of the BIAbs response. Modelling the structural effects of the HLA-DRB1 variants revealed a number of differences in the peptide-binding groove, which is likely to lead to altered antigen binding and presentation profiles, and hence may explain the differences in subject responses.</p><p>Conclusions/Significance</p><p>The current study confirms the heritability of the DBPII antibody response, with genetic variation in HLA class II genes influencing both the development and persistence of IgG antibody responses. Cellular studies to increase knowledge of the binding affinities of DBPII peptides for class II molecules linked with good or poor antibody responses might lead to the development of strategies for controlling the type of helper T cells activated in response to DBPII.</p></div

Map of the state of Amazonas, Northwestern Brazil, showing the study site.

<p>The Rio Pardo settlement is located within the Presidente Figueiredo municipality (grey area in the inset), roughly 160 km from the state capital, Manaus.</p

Association between HLA class II alleles (<i>DRB1</i>, <i>DQA1</i>, and <i>DQB1</i> loci) and <i>Plasmodium vivax</i> Duffy binding protein (DBPII) antibody responses in individuals naturally exposed to malaria.

<p>(<b>A</b>) At enrollment (baseline), adjusted Odds Ratio (aOR) analysis for the association of HLA class II (alleles and haplotypes) and the presence (<i>black circle)</i> or absence (<i>white circle</i>) of DBPII IgG antibody (Ab) response, as detected by ELISA. (<b>B</b>) After 12-month follow-up, adjusted Odds Ratio (aOR) analysis for the association of HLA class II (alleles and haplotypes) and the long-term DBPII antibody response; individuals were classified as persistent non-responder (PNR, <i>white square</i>) or responder (PR, <i>black square</i>), as described in legend of <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005177#pntd.0005177.g002" target="_blank">Fig 2</a>. Adjusted ORs analyses were performed using multivariable logistic regression models adjusted for confounding variables (age, sex, previous malaria episodes, and dwelling location). Variables associated with DBPII antibody at 5% level (p < 0.05) were maintained in the final models.</p

Temporal distribution of <i>P</i>. <i>vivax</i> malaria episodes and immune response targeting region II of the Duffy binding protein (DBPII), Rio Pardo Settlement, Amazonas, Brazil (Nov. 2008 –Nov. 2009).

<p>(<b>A)</b> Monthly malaria notifications in the Rio Pardo settlement (SVS/MS, Surveillance Service database, Brazilian Ministry of Health). Episodes of malaria, as determined by conventional microscopy, varied according to rainfall and season. Arrows indicate the time of the cross-sectional surveys (November, 2008; June 2009; and November, 2009). (<b>B</b>) DBPII IgG ELISA-detected antibody (Anti-DBPII) response remained stable during the follow up period (Baseline, and at 6 or 12 months). Results are expressed as a frequency (%) of responders, and a Reactivity Index (RI) of greater than 1.0 was considered positive. (<b>C</b>) DBPII binding inhibitory antibodies (BIAbs) had a slightly decreased frequency at the 12 months follow-up examination. BIAbs were evaluated by COS-7 cytoadherence assay, using plasma samples diluted to 1:40 (n = 164). Positive results were considered as DBPII–DARC binding inhibition of ≥50%. (<b>D</b>) Based on conventional (ELISA-detected IgG) and functional serology assays (DBPII BIAbs) at 12 months of follow-up, individuals were classified as <i>persistent non-responder</i> (PNR)—no antibody response detected any time-point of the follow-up; <i>transient responder</i> (TR)—antibodies detected in at least one cross-sectional survey; or <i>persistent responder</i> (PR)–antibodies detected in all cross-sectional surveys.</p

Association between HLA class II alleles (<i>DRB1</i>, <i>DQA1</i>, and <i>DQB1</i> loci) and <i>Plasmodium vivax</i> Duffy binding protein (DBPII) binding Inhibitory Antibodies (BIAbs) in individuals naturally exposed to malaria.

<p>(<b>A</b>) At enrollment (baseline), Odds Ratio (OR) analyses for the association of HLA class II (alleles and haplotypes) and DBPII BIAbs response, as detected by functional assays with DBPII- transfected COS cells; <i>black circles</i> represent alleles and haplotypes of HLA class II associated with a positive BIABs response. (<b>B</b>) After 12-month follow-up, Odds Ratio analyses for the association of HLA class II (alleles and haplotypes) and long-term DBPII BIAbs response; individuals were classified as persistent non-responder (PNR) or responder (PR, <i>black square</i>), as described in legend of <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005177#pntd.0005177.g002" target="_blank">Fig 2</a>. Odds Ratio analyses realized using standard contingency tables (p < 0.05; qui-square and Fisher’s exact test, as appropriate).</p