Strain-Transcending Inhibitory Antibodies against Homologous and Heterologous Strains of Duffy Binding Protein region II.

Duffy binding protein region II (DBPII) is a promising vaccine candidate against vivax malaria. However, polymorphisms of DBPII are the major obstacle to designing a successful vaccine. Here, we examined whether anti-DBPII antibodies from individual P. vivax exposures provide strain-transcending immunity and whether their presence is associated with DBPII haplotypes found in patients with acute P. vivax. The ability of antibodies to inhibit DBL-TH-erythrocyte binding was tested by COS7 erythrocyte binding inhibition assay. Seven samples of high responders (HR) were identified from screening anti-DBPII levels. HR no.3 and HR no.6 highly inhibited all DBL-TH binding to erythrocytes, by >80%. Antibodies from these two patients' plasma had the potential to be broadly inhibitory against DBL-TH1, -TH2, -TH6, -TH7, -TH8 and -TH9 haplotypes when plasma was serially diluted from 1:500 to 1:2000. To further examine the association of DBPII haplotypes and the ability of antibodies to broadly inhibit DBL-TH variants, the individual samples underwent sequencing analysis and the inhibitory function of the anti-DBPII antibodies was tested. The patterns of DBPII polymorphisms in acute patients were classified into two groups, DBPII Sal I (55%) and DBL-TH variants (45%). Plasma from Sal I and DBPII-TH patients who had the highest inhibition against Sal I or DBL-TH4 and -TH5 was serially diluted from 1:500 to 1:2000 and their inhibitory capacity was tested against a panel of DBL-TH haplotypes. Results provided evidence of both strain-transcending inhibition as well as strain-specific inhibition by antibodies that blocked erythrocyte binding against some DBL-TH variants and against homologous alleles. This study demonstrated broad inhibition by anti-DBPII antibodies against DBL-TH haplotypes in natural P. vivax exposed individuals. The identification of conserved epitopes among DBL-TH may have implications for vaccine development of a DBPII-based vaccine against diverse P. vivax infections

Functional inhibition of anti-DBPII antibodies in high responder samples against the panel of DBL-TH variants.

<p>Transfected COS7 cells expressing DBL-TH alleles, DBL-TH1, -TH2, -TH3, -TH4, -TH5, -TH6, -TH7, -TH8, -TH9 and reference Sal I were incubated with 1:100 plasma dilution for 1 hr at 37°C followed by incubation with a 10% suspension of human erythrocytes for 2 hrs. The number of rosettes was compared between wells of transfected cells incubated with plasma relative to wells without plasma (30 fields of view, magnification ×200). The symbols represent mean percent inhibition of two experiments tested in duplicate wells.</p

Inhibitory function of anti-DBPII antibodies in <i>P</i>. <i>vivax</i> individuals against heterologous DBL-TH4 and DBL-TH5 or heterologous DBL-TH4, DBL-TH5 and homologous reference Sal I binding to human erythrocytes measured by COS7 cell binding-inhibition assay

<p>Inhibitory function of anti-DBPII antibodies in <i>P</i>. <i>vivax</i> individuals against heterologous DBL-TH4 and DBL-TH5 or heterologous DBL-TH4, DBL-TH5 and homologous reference Sal I binding to human erythrocytes measured by COS7 cell binding-inhibition assay</p

Antibody recognition of recombinant PvDBPII.

<p>The scatter plot graph shows the anti-DBPII antibody levels in Thai patients compare to naive control as measured by ELISA. <b>(A)</b> Anti-DBPII levels were significantly higher in patients with acute <i>P</i>. <i>vivax</i> than in naive controls, <b>(B)</b> ELISA data classified patients into 3 groups: high responder (HR), low responders (LR) and non-responders (NR). Each dot represents the mean of optical density values in double wells for each sample. The line represents the mean value. Significance was determined by non-parametric analysis using the Mann-Whitney U test. The level of significant was set at <i>P</i> < 0.05.</p

Inhibition efficiency of anti-DBPII antibodies in <i>P</i>. <i>vivax</i> exposed individuals.

<p>A and B: Inhibitory function against a panel DBL-TH haplotypes of vivax patients infected with Sal I strain and had the strongest inhibitory immunity against both heterologous DBL-TH4 and DBL-TH5 strain <b>(A)</b> patient no.9, <b>(B)</b> patient no.15. C, D and E:. Inhibitory function against a panel DBL-TH of vivax patients infected with high polymorphism DBL-TH strain and had the strongest inhibitory immunity against heterologous DBL-TH4 or DBL-TH5 or Sal I strain, <b>(C)</b> patient no.25 infected with DBL-TH2 strain, <b>(D)</b> patient no.37 infected with DBL-TH4 strainand <b>(E)</b> infected with new DBL-TH strain, patient no.30. The transfected COS7 cells expressing Thai DBPII alleles were incubated with plasma and with human erythrocytes. The number of rosettes was compared between wells of transfected cells incubated with antibodies relative to wells without antibodies. Each chart represents the mean of two independent experiments with each dilution tested in triplicate. Error bars represent ± standard deviation. Statistical significance was determined using one-way analysis of variance (ANOVA) and multiple comparison analysis by Bonferroni test.</p

Inhibitory function of anti-DBPII antibodies in <i>P</i>. <i>vivax</i> individuals against heterologous DBL-TH4 or DBL-TH5 or homologous reference Sal I binding to human erythrocytes measured by COS7 cell binding-inhibition assay

<p>Inhibitory function of anti-DBPII antibodies in <i>P</i>. <i>vivax</i> individuals against heterologous DBL-TH4 or DBL-TH5 or homologous reference Sal I binding to human erythrocytes measured by COS7 cell binding-inhibition assay</p

Polymorphic residues of DBL-TH haplotypes infected in <i>P</i>. <i>vivax</i> patients at the time of enrollment.

<p>Polymorphic residues of DBL-TH haplotypes infected in <i>P</i>. <i>vivax</i> patients at the time of enrollment.</p

Broad inhibition by high responder samples of erythrocyte binding to DBL-TH haplotypes.

<p>The transfected COS7 cell expressing DBPII reference Sal I or DBL-TH variants were pre-incubated with plasma at dilution 1:500, 1:1000 and 1:2000 for inhibition of DBPII-erythrocyte binding. The charts show the mean inhibition of each DBL-TH variant. Inhibitory function against the panel of DBL-TH variants by (A) HR3 and (B) HR6 samples. Each chart represents the mean of two independent experiments with each dilution tested in triplicate. Error bars represent ± standard deviation. Statistical significance was determined using one-way analysis of variance (ANOVA) and multiple comparison analysis by Bonferroni test.</p