Differential recognition of P. falciparum VAR2CSA domains by naturally acquired antibodies in pregnant women from a malaria endemic area

Abstract

Background: Plasmodium falciparum infected red blood cells (iRBC) express variant surface antigens (VSA) of which VAR2CSA is involved in placental sequestration and causes pregnancy-associated malaria (PAM). Primigravidae are most susceptible to PAM whereas antibodies associated with protection are often present at higher levels in multigravid women. However, HIV co-infection with malaria has been shown to alter this parity-dependent acquisition of immunity, with more severe symptoms as well as more malaria episodes in HIV positive women versus HIV negative women of a similar parity.

Methods: Using VAR2CSA DBL-domains expressed on the surface of CHO-745 cells we quantified levels of DBL-domain specific IgG in sera from pregnant Malawian women by flow cytometry. Dissociations constants of DBL5epsilon specific antibodies were determined using a surface plasmon resonance technique, as an indication of antibody affinities.

Results: VAR2CSA DBL5epsilon was recognized in a gender and parity-dependent manner with anti-DBL5epsilon IgG correlating significantly with IgG levels to VSA-PAM on the iRBC surface. HIV positive women had lower levels of anti-DBL5epsilon IgG than HIV negative women of similar parity. In primigravidae, antibodies in HIV positive women also showed significantly lower affinity to VAR2CSA DBL5epsilon.

Conclusions: Pregnant women from a malaria-endemic area had increased levels of anti-DBL5epsilon IgG by parity, indicating this domain of VAR2CSA to be a promising vaccine candidate against PAM. However, it is important to consider co-infection with HIV, as this seems to change the properties of antibody response against malaria. Understanding the characteristics of antibody response against VAR2CSA is undoubtedly imperative in order to design a functional and efficient vaccine against PAM.

Figure 1. VSA-PAM and VAR2CSA DBL-domain recognition of naturally acquired antibodies in pregnant women.

A: IgG levels against VSA-PAM expressed on the surface of CS2 parasites. Groups are divided into primigravidae (PG), secundigravidae (SG) and multigravidae (MG) (x-axis) and antibody levels are expressed as relative median fluorescence intensity (rMFI, y-axis). MG women have significantly higher levels of antibodies against VSA-PAM than PG women (Kruskal-Wallis ANOVA, p = 0.0001; Dunn's Multiple Comparison test, p<0.001). B: IgG levels against VAR2CSA domains DBL3x (n = 93 pregnancy and 10 male sera), DBL5ε (n = 125 pregnancy and 13 male sera), and DBL6ε (n = 122 pregnancy and 9 male sera), Groups are divided into pregnant women (all parities shown together, labeled P) and males (labeled M) from the same endemic areas. Pregnant women had significantly higher levels of antibodies against each domain than their male counterparts, except for DBL3x, probably due to the lower sample size for this domain (Kruskal-Wallis ANOVA, p<0.0001; Dunn's Multiple Comparison test, p>0.05 (ns) (DBL3x), p<0.01 (DBL5ε) and p = 0.05 (DBL6ε)). C: IgG levels against DBL3x showing no significant difference in antibody levels in PG and MG women (Kruskal-Wallis ANOVA, p = 0.0589). D: Correlation of IgG levels against DBL3x and total VSA-PAM, expressed as rMFI. No correlation was found (Spearman r = −0.08980, p = 0.3972). E: IgG levels against DBL5ε showing significantly higher levels of antibodies in MG women than PG women (Kruskal-Wallis ANOVA, p = 0.0141, Dunn's Multiple Comparison test, p<0.05). F: Correlation of IgG levels against DBL5ε and total VSA-PAM, expressed as rMFI. A significant positive moderate correlation was found (Spearman r = 0.5632, p<0.0001). G: IgG levels against DBL6ε showing no significant difference in antibody levels in PG and MG women (Kruskal-Wallis ANOVA, p = 0.5118). H: Correlation of IgG levels against DBL6ε and total VSA-PAM, expressed as rMFI. A significant positive weak correlation was found (Spearman r = 0.2905, p = 0.0013).

Figure 2. The effect of HIV infection on antibody levels to DBL5ε.

IgG levels against DBL5ε showing HIV negative women to have higher antibody levels than HIV positive women in all parity groups, however only significantly so in multigravidae (Kruskal-Wallis ANOVA, p = 0.023). Groups are divided into primigravidae (PG), secundigravidae (SG) and multigravidae (MG) and HIV positive (+) or negative (−) (x-axis) and antibody levels are expressed as relative median fluorescence intensity (rMFI, y-axis).

Figure 3. Affinity of naturally acquired antibodies in pregnant women to VAR2CSA DBL5ε.

A: Affinity displayed as the dissociation rate constant (k_d×10∧-4), comparing PG and MG (both HIV positive (+) and HIV negative (−) women). There is no significant difference between antibody affinities to DBL5ε comparing PG and MG women (t-test, p = 0.7630). B: Affinity displayed as the dissociation rate constant (k_d×10∧-4), comparing primigravid women divided into HIV status. Antibodies from PG HIV- women have a significantly higher affinity to DBL5ε than antibodies from PG HIV+ women (t-test, p = 0.0230). C: Affinity displayed as the dissociation rate constant (k_d×10∧-4), comparing multigravid women divided into HIV status. No significant difference in antibody affinity is seen comparing groups MG- women and MG+ women (t-test, p = 0.1152).

Figure 4. Correlation of VSA-PAM IgG, DBL5ε IgG and antibody affinity.

A: Correlation of IgG levels against total VSA-PAM and antibody affinity against DBL5ε, expressed as rMFI and dissociation rate constant respectively. These show a negative moderate significant correlation (Spearman r = −0.3571, p = 0.0002). B: Correlation of IgG levels against DBL5ε and antibody affinity against DBL5ε, expressed as rMFI and dissociation rate constant respectively. These show a negative moderate significant correlation (Spearman r = −0.3414, p = 0.0024).