Human pregnancy‐associated malaria‐specific B cells target polymorphic, conformational epitopes in VAR2CSA

Abstract

Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes (IEs) that bind to chondroitin sulphate A (CSA) in the placenta by PAM-associated clonally variant surface antigens (VSA). Pregnancy-specific VSA (VSAPAM), which include the PfEMP1 variant VAR2CSA, are targets of IgG-mediated protective immunity to PAM. Here, we report an investigation of the specificity of naturally acquired immunity to PAM, using eight human monoclonal IgG1 antibodies that react exclusively with intact CSA-adhering IEs expressing VSAPAM. Four reacted in Western blotting with high-molecular-weight (> 200 kDa) proteins, while seven reacted with either the DBL3-X or the DBL5-ε domains of VAR2CSA expressed either as Baculovirus constructs or on the surface of transfected Jurkat cells. We used a panel of recombinant antigens representing DBL3-X domains from P. falciparum field isolates to evaluate B-cell epitope diversity among parasite isolates, and identified the binding site of one monoclonal antibody using a chimeric DBL3-X construct. Our findings show that there is a high-frequency memory response to VSAPAM, indicating that VAR2CSA is a primary target of naturally acquired PAM-specific protective immunity, and demonstrate the value of human monoclonal antibodies and conformationally intact recombinant antigens in VSA characterization.

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

“…S2) and their inability to inhibit IE adhesion to CSA (24) indicate that DBL3X is positioned near DBL5ε in the correctly folded VAR2CSA molecule and that neither domain is directly involved in VAR2CSA-mediated adhesion to CSA. It is noteworthy in this context that VAR2CSA-specific immunity acquired in response to placental P. falciparum infection seems highly focused on these two domains (23). Taken together, the present evidence suggests that nonspecific binding of IgM to VAR2CSA has evolved to shield epitopes that are immunogenic but not critical to molecular function (adhesion to CSA) from recognition by specific IgG, whereas functionally important epitopes are kept clear of this masking.…”

“…2D. ‡ 3D7-VAR2CSA does not contain the epitope recognized by PAM8.1 (23), and no IE labeling was seen in the experiments conducted here.…”

“…S2). All of the IgG antibodies that were inhibited by IgM preincubation are specific for either the DBL3X or DBL5ε domain of VAR2CSA (23). The unaffected antibody (PAM1.4) recognizes neither of these domains and instead, seems to recognize a conformational and possibly discontinuous epitope in VAR2CSA (23,24).…”

“…The isolation and characterization of the seven VAR2CSA-specific human monoclonal IgG1 antibodies used here (PAM1.4, PAM2.8, PAM3.10, PAM5.2, PAM6.1, PAM7.5, and PAM8.1) have been described in detail elsewhere (23,24). PAM2.8, PAM6.1, and PAM8.1 recognize epitopes in the DBL3X domain, whereas PAM3.10, PAM5.2, and PAM7.5 recognize epitopes in DBL5ε (23). PAM1.4 recognizes a conformational, possibly discontinuous, epitope in VAR2CSA (24).…”

Binding affinity of Plasmodium falciparum-infected erythrocytes from infected placentas and laboratory selected strains to chondroitin 4-sulfate

“…However, it should be noted that the level of var2 PfEMP1 expression on the IRBC surface could vary in a strain dependent manner and that its expression could decrease over successive generations. Furthermore, although recent studies have raised monoclonal and polyclonal antibodies against var2 PfEMP1 [30,31], so far the ability of antibody to block IRBC binding to C4S has not been demonstrated, suggesting that var2 PfEMP1 might not be directly binding to C4S. Further studies are required to determine whether var2 PfEMP1 mediates IRBC binding.…”