Characterization of Inhibitory Anti-Duffy Binding Protein II Immunity: Approach to Plasmodium vivax Vaccine Development in Thailand

Plasmodium vivax Duffy binding protein region II (DBPII) is an important vaccine candidate for antibody-mediated immunity against vivax malaria. A significant challenge for vaccine development of DBPII is its highly polymorphic nature that alters sensitivity to neutralizing antibody responses. Here, we aim to characterize naturally-acquired neutralizing antibodies against DBPII in individual Thai residents to give insight into P. vivax vaccine development in Thailand. Anti-DBPII IgG significantly increased in acute vivax infections compared to uninfected residents and naive controls. Antibody titers and functional anti-DBPII inhibition varied widely and there was no association between titer and inhibition activity. Most high titer plasmas had only a moderate to no functional inhibitory effect on DBP binding to erythrocytes, indicating the protective immunity against DBPII binding is strain specific. Only 5 of 54 samples were highly inhibitory against DBP erythrocyte-binding function. Previously identified target epitopes of inhibitory anti-DBPPII IgG (H1, H2 and H3) were localized to the dimer interface that forms the DARC binding pocket. Amino acid polymorphisms (monomorphic or dimorphic) in H1 and H3 protective epitopes change sensitivity of immune inhibition by alteration of neutralizing antibody recognition. The present study indicates Thai variant H1.T1 (R308S), H3.T1 (D384G) and H3.T3 (K386N) are the most important variants for a DBPII candidate vaccine needed to protect P. vivax in Thai residents

The antibody levels specific to <i>P. vivax</i> antigen.

<p>Graphical display of antibody levels anti-<i>P. vivax</i> shizont protein extract (A) and anti-DBPII (B) in Thai patients (PV), uninfected residents (UR) and naïve controls (NC). Dots represent the mean optical density value in triplicate wells for each sample. Bars represent mean value. Asterisk indicate statistic significant at P<0.05 with non-parametric two independent tests.</p

The synthesized peptides using in antibody purification and testing functional inhibition.

<p>H1, H2 and H3 are target epitopes of naturally acquired inhibitory antibodies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035769#pone.0035769-Chootong1" target="_blank">[25]</a>. H1.T1, H3.T1, H3.T2, H3.T3 and M3.T1 epitopes are the variant strain among Thai vivax isolates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035769#pone.0035769-Gosi1" target="_blank">[19]</a>. NI is the target epitopes of non-inhibitory antibodies.</p

The inhibition activity of anti-DBPII epitope-specific antibody to Sal I and Thai strain epitopes.

<p>(<b>A</b>) Human antibody specific to DBPII epitopes strain Sal I; H1, H2, H3 and NI. (<b>B</b>) Antibodies specific to DBPII epitopes Thai strain; H1.T1, H3.T1, H3.T2, H3.T3, M3.T1 and NI. The purified antibodies were test to determine their inhibitory function against DBPII Sal I binding Duffy positive erythrocyte. The symbol indicates the mean percentage of inhibition of three experiments compared to the result of control experiment with no antibody. For antibody concentration of 8 ug/mL, the <i>P</i> value was <0.001 for comparison of H and NI peptides. Significance of inhibition was measured with the 50% inhibition concentration (1–2 µg/mL) of purified anti- H1, H2, H3 antibodies. (<b>C</b>) Anti-H1 compared with anti-H1.T1; (<b>D</b>) Anti-H3 compared with anti-H3.T3. Antibodies were tested to determine their inhibitory function against DBPII Sal I binding Duffy positive erythrocyte. The symbol indicates the mean percentage of inhibition of three experiments compared to the result of control experiment with no antibody. For antibody concentration of 8 ug/mL, the <i>P</i> value was <0.001 for comparison of H and NI peptides, and comparison H1.T1, H3.T3 and NI peptides.</p

Anti-DBPII response and DBPII-Duffy positive erythrocyte inhibitory.

<p>Scatter plot showing the correlation between anti-DBPII levels and inhibition activity among Thai vivax residents (Spearman's coefficient: 0.042; P = 0.764). Fifty-four samples (1∶200 diluted plasma) were tested for their reactivity to DBPII in standard ELISA procedure and for the inhibition function of DBPII binding to Duffy positive erythrocyte measured by COS7 cell erythrocyte binding assay.</p