Evasion of immunity to Plasmodium falciparum malaria by IgM masking of protective IgG epitopes in infected erythrocyte surface-exposed PfEMP1

Abstract

Plasmodium falciparum malaria is a major cause of mortality and severe morbidity. Its virulence is related to the parasite's ability to evade host immunity through clonal antigenic variation and tissue-specific adhesion of infected erythrocytes (IEs). The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family is central to both. Here, we present evidence of a P. falciparum evasion mechanism not previously documented: the masking of PfEMP1-specific IgG epitopes by nonspecific IgM. Nonspecific IgM binding to erythrocytes infected by parasites expressing the PfEMP1 protein VAR2CSA (involved in placental malaria pathogenesis and protective immunity) blocked subsequent specific binding of human monoclonal IgG to the Duffy binding-like (DBL) domains DBL3X and DBL5ε of this PfEMP1 variant. Strikingly, a VAR2CSA-specific monoclonal antibody that binds outside these domains and can inhibit IE adhesion to the specific VAR2CSA receptor chondroitin sulfate A was unaffected. Nonspecific IgM binding protected the parasites from FcγR-dependent phagocytosis of VAR2CSA(+) IEs, but it did not affect IE adhesion to chondroitin sulfate A or lead to C1q deposition on IEs. Taken together, our results indicate that the VAR2CSA affinity for nonspecific IgM has evolved to allow placenta-sequestering P. falciparum to evade acquired protective immunity without compromising VAR2CSA function or increasing IE susceptibility to complement-mediated lysis. Furthermore, functionally important PfEMP1 epitopes not prone to IgM masking are likely to be particularly important targets of acquired protective immunity to P. falciparum malaria.

Fig. 1.

Nonspecific IgM binding to the surface of P. falciparum-infected erythrocytes. (A) Relationship between concentration of IgM and nonspecific IgM binding to field isolate A1149 (△), 3D7-VAR2CSA⁺ (○), FCR3-VAR2CSA⁺ (□), and 3D7-VAR4⁺ (●) late-stage IEs (identified by ethidium bromide nuclear labeling). Representative flow cytometry histograms showing nonspecific IgM binding (at 10 nM concentration) to the surface of VAR2CSA⁺ (B) and VAR4⁺ late-stage IEs (C). Background labeling is shown in gray. Data typical of at least three experiments are shown.

Fig. 2.

Relationships between nonspecific IgM binding and VAR2CSA-specific IgG binding to VAR2CSA⁺-infected erythrocytes. (A) The effect of preincubation of FCR3-VAR2CSA⁺ IEs with nonspecific IgM (10 nM) on subsequent labeling by VAR2CSA-specific monoclonal IgG antibodies (150 nM) that either are (white bars) or are not (black bar) specific for the DBL3X or DBL5ε domains of VAR2CSA. IgG labeling is expressed as the percentage (mean and SD) of IgG labeling without IgM preincubation (control). A pool of all of the IgG antibodies (all mAbs) at a final concentration of 150 nM is also shown (gray bar). Inset shows representative flow cytometry histograms of binding of PAM1.4 and PAM7.5 to VAR2CSA⁺ IEs without (heavy line) or after (thin line) preincubation with nonspecific IgM (background labeling is shown in gray). Labeling of 3D7-VAR2CSA⁺ IEs by (B) PAM3.10 (150 nM) or (C) PAM1.4 (150 nM) in the presence of 10 nM IgM (●; IgM and IgG added simultaneously) or in the absence of IgM (○). IgG labeling is expressed as the percentage of IgG labeling at 150 nM in the absence of IgM (control). Drop lines corresponding to the IgG concentration required to obtain half-maximal IgG labeling in the absence and presence of IgM are indicated (Table 1). (D) Nonspecific IgM labeling of FCR3-VAR2CSA⁺ IEs in the presence of various concentrations of VAR2CSA-specific monoclonal IgG antibodies (IgM and IgG added simultaneously). IgM labeling is expressed as the percentage of IgM labeling in the absence of IgG (control). Drop lines corresponding to the PAM2.8 and PAM3.10 concentrations required to obtain half-maximal IgM labeling are indicated (Table 1). Figure data (means and SD) are typical (A) or complete (B–D) from at least three experiments.

Fig. 3.

IgM domain specificity of nonspecific IgM binding to VAR2CSA. (A) Binding of nonspecific IgM (10 nM) to FCR3-VAR2CSA⁺ IEs after preincubation of the IgM with various mouse monoclonal antibodies (100 nM) with specificity for the Cμ2 (HB57), Cμ3 (196.6b, 4–3, and 5D7), or Cμ4 (1G6) domain of IgM. Inhibition of IgM binding by preincubation of IEs with a pool of all seven human monoclonal VAR2CSA-specific IgG antibodies (PAM1-8; final concentration is 150 nM) is shown for comparison. (B) Binding to VAR2CSA⁺ IEs of recombinant chimeric 3-iodo-4-hydroxy-5-nitrophenacetyl (NIP)-specific human IgM, IgA, domain-swapped IgA in which the Cα3 domain has been replaced by Cμ4 from human IgM (AAM), and IgG2. Binding of the recombinant human antibodies to IEs was detected with a phycoerythrin (PE)-conjugated antibody specific for the mouse λ-light chain shared by all these antibodies [described previously by Ghumra et al. (17)]. Only IgM or the domain-swapped IgA containing Cμ4 bound to the IEs. (C) Dose-dependent competition for binding to VAR2CSA⁺ IEs between the AAM antibody (10 nM) on the one hand and varying concentrations of nonspecific IgM and monoclonal VAR2CSA-specific IgG antibodies PAM1.4 and PAM3.10 on the other hand. Data (means and SD) typical of three independent experiments are shown.

Fig. 4.

Phagocytosis of antibody-opsonized VAR2CSA⁺ IEs. FcγR-dependent THP-1 phagocytosis of monoclonal VAR2CSA-specific IgG- or plasma-opsonized FCR3-VAR2CSA⁺ IEs without (white bars) or with (black bars) preincubation of IEs with nonspecific IgM (10 nM). Plasma pools were prepared from adult donors without P. falciparum exposure (nonexp. pool), P. falciparum-exposed men (male pool), and previously pregnant P. falciparum-exposed women (female pool). Summary (means and SDs) of three independent experiments is shown.

Fig. 5.

CSA-specific adhesion of VAR2CSA⁺ IEs. (A) Adhesion of VAR2CSA⁺ IEs to CSA under flow after preincubation of IEs in PBS, nonspecific IgM (10 nM), or nonimmune human serum (10%) in the absence (open boxes) or presence (shaded boxes) of a pool of VAR2CSA-specific monoclonal IgG antibodies (final concentration of 150 nM). (B) Adhesion of VAR2CSA⁺ IEs (shaded boxes) or VAR4⁺ IEs (open box) to placental cryosections after preincubation of IEs in PBS, nonspecific IgM (10 nM), soluble CSA (7.5 μg/mL), or nonimmune human serum (10%). The box plots show median (central line), central 50% (box), central 80% (whiskers), and outlying data points. The statistical significance levels of intergroup differences are indicated. All data from a minimum of three independent experiments, normalized to assay mean adhesion without IgM (control), are included.