Antigen reversal finds the targets of opsonizing IgGs against pregnancy-associated malaria

More than 100 million pregnant women in malaria-endemic areas are at risk of developing pregnancy-associated malaria (PAM), a potentially severe consequence of Plasmodium falciparum infection. Afflicted women suffer from the extensive accumulation of infected erythrocytes (IE) and leukocytes in the intervillous spaces of the placenta, which increases their risk of anemia, hypertension, premature delivery, and the potential death of low-birth-weight infants. Clinical immunity to pregnancy associated-malaria (PAM) in multigravid women has been attributed to antibodies that recognize VAR2CSA on the IE surface. VAR2CSA is a large multidomain variant of P. falciparum erythrocyte membrane protein 1 (PfEMP1). It enables IEs to bind to the chondroitin sulfate A (CSA) on the surface of placental cells called syncytiotrophoblasts. The size and complexity of VAR2CSA has focused efforts on selecting one or more of its six Duffy binding-like (DBL) domains for vaccine development. Presently, however, there is no consensus as to which of the DBL domain(s) would be most effective in eliciting immunity. This is because antibodies to a number of the DBL domains have been found to block the adhesion of VAR2CSA-expressing erythrocytes to CSA — a major criterion for evaluating vaccine candidacy. The accumulation of circulating monocytes and macrophages within the placenta during PAM underscores the importance of opsonization, an often-overlooked aspect of the acquired immune response. Opsonization of IEs via antibodies that recognize VAR2CSA represents an important yet understudied effector mechanism in acquired immunity to PAM. The dearth of such studies is partly a consequence of the labor-intensive and time-consuming nature of the experiments. To date, no studies have sought to determine the targets of those antibodies. In this study we found that IgGs from multigravid Malian women showed (1) higher reactivity to recombinant DBL domains by ELISA, (2) more binding to VAR2CSA-expressing IEs, and (3) greater opsonization of these IEs by human monocytic cells, than IgGs from malaria-exposed Malian men and malaria-naïve American adults. Preincubation of multigravid IgGs with recombinant DBL2χ, DBL3χ, or DBL5ϵ domains significantly diminished opsonization of VAR2CSA-expressing IEs by human monocytes. These data identify the DBL2χ, DBL3χ, and DBL5ϵ domains as the primary targets of opsonizing IgGs for the first time. Our study introduces a new approach to determining the antigenic targets of opsonizing IgGs in phagocytosis assays.