Llama immunization with full-length VAR2CSA generates cross-reactive and inhibitory single-domain antibodies against the DBL1X domain

Abstract

VAR2CSA stands today as the leading vaccine candidate aiming to protect future pregnant women living in malaria endemic areas against the severe clinical outcomes of pregnancy associated malaria (PAM). The rational design of an efficient VAR2CSA-based vaccine relies on a profound understanding of the molecular interactions associated with P. falciparum infected erythrocyte sequestration in the placenta. Following immunization of a llama with the full-length VAR2CSA recombinant protein, we have expressed and characterized a panel of 19 nanobodies able to recognize the recombinant VAR2CSA as well as the surface of erythrocytes infected with parasites originating from different parts of the world. Domain mapping revealed that a large majority of nanobodies targeted DBL1X whereas a few of them were directed towards DBL4ε, DBL5ε and DBL6ε. One nanobody targeting the DBL1X was able to recognize the native VAR2CSA protein of the three parasite lines tested. Furthermore, four nanobodies targeting DBL1X reproducibly inhibited CSA adhesion of erythrocytes infected with the homologous NF54-CSA parasite strain, providing evidences that DBL1X domain is part or close to the CSA binding site. These nanobodies could serve as useful tools to identify conserved epitopes shared between different variants and to characterize the interactions between VAR2CSA and CSA.

Structure of the DBL3X-DBL4ε region of the VAR2CSA placental malaria vaccine candidate: insight into DBL domain interactions

“…Several studies analysing the immune response to VAR2CSA have demonstrated that DBL4ε-specific antibodies are able to label and inhibit the adhesion of P. falciparum infected erythrocytes in a strain-independent manner, showing that this domain is surface-exposed in the full-length VAR2CSA 29 35 36 37 . Nanobodies raised against the full-length VAR2CSA, but with specificity to DBL4ε domains, were also found to be surface-reactive although not inhibitory 38 39 . These data and the fact that DBL4ε does not participate in the minimal binding region of VAR2CSA 21 22 , suggest that inhibition could involve steric hindrance and/or abolition of the native higher-order domain organisation.…”

A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells

“…Since nanobodies have only three hypervariable regions (CDRs), compared to antibody formats derived from conventional IgGs, these binders preferably recognize and bind conformational epitopes e.g. formed by enzymatic pockets of regulatory domains [ 57 , 58 ]. Consequently, binding of nanobodies often interferes with the function of the targeted antigen [ 59 – 61 ].…”

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…Likewise, Ditlev et al . 70 attributed the binding of a panel of alpaca V H Hs to multiple domains of the malarial VAR2CSA protein to an inherent ability of V H Hs to recognize subdominant epitopes, although limited understanding of the human conventional antibody response against VAR2CSA as well as irreproducibility of these reactivity patterns by llama V H Hs 71 complicated this assessment.…”