Utilizing nanobody technology to target non-immunodominant domains of VAR2CSA

Abstract

Placental malaria is a major health problem for both pregnant women and their fetuses in malaria endemic regions. It is triggered by the accumulation of Plasmodium falciparum-infected erythrocytes (IE) in the intervillous spaces of the placenta and is associated with foetal growth restriction and maternal anemia. IE accumulation is supported by the binding of the parasite-expressed protein VAR2CSA to placental chondroitin sulfate A (CSA). Defining specific CSA-binding epitopes of VAR2CSA, against which to target the immune response, is essential for the development of a vaccine aimed at blocking IE adhesion. However, the development of a VAR2CSA adhesion-blocking vaccine remains challenging due to (i) the large size of VAR2CSA and (ii) the extensive immune selection for polymorphisms and thereby non-neutralizing B-cell epitopes. Camelid heavy-chain-only antibodies (HcAbs) are known to target epitopes that are less immunogenic to classical IgG and, due to their small size and protruding antigen-binding loop, able to reach and recognize cryptic, conformational epitopes which are inaccessible to conventional antibodies. The variable heavy chain (VHH) domain is the antigen-binding site of camelid HcAbs, the so called Nanobody, which represents the smallest known (15 kDa) intact, native antigen-binding fragment. In this study, we have used the Nanobody technology, an approach new to malaria research, to generate small and functional antibody fragments recognizing unique epitopes broadly distributed on VAR2CSA.

Figure 1. Sequence alignment of the 17 VAR2CSA-specific Nbs.

The clones used for Nanobody production were sequenced, converted to amino acid sequences and aligned. The complementarity determining regions (CDRs) 1–3 make up the binding paratope and the framework regions (FRs) 1–4 are indicated.

Figure 2. Recognition of immobilized full-length VAR2CSA by each of the 17 Nbs.

Microtiter plates were coated with VAR2CSA protein (50 nM) and incubated with individual Nbs (50 nM). Binding was detected with rabbit anti-camel antibody and HRP-conjugated goat anti-rabbit antibody. Optical density was measured at 490 nm after 20 min. A non-VAR2CSA-PfEMP1 (50 nM) was used as negative control. Data are represented as mean and standard deviations of three independent experiments.

Figure 3. Recognition of immobilized VAR2CSA domains by Nbs (Nb01–Nb17).

Baculovirus-produced domains of VAR2CSA (50 nM) were coated on microtiter plates and incubated with each of the Nbs (50 nM). The binding was detected with rabbit anti-camel and goat anti-rabbit HRP-conjugated antibodies and optical density measured at 490 nm after 20 min. Non-VAR2CSA PfEMP1 (50 nM) was used as negative control. The assay was performed several times with similar result.

Figure 4. Recognition of the minimal CSA-binding region of VAR2CSA.

(A) Reactivity of Nbs to specific minimal CSA-binding regions produced in different expression systems. ID1–ID2a proteins produced either in a baculo–virus expression system (BV), or in E. coli (coli) or in Schneider 2 (S2) cells were coated (50 nM) on microtiter plates and incubated with 50 nM Nb01, Nb07, Nb09, Nb10 or Nb12 (Nbs specific for VAR2CSA minimal CSA-binding domain). Nb02 (DBL5-specific) was used as a negative control. (B) Cross-reacitivity of Nbs specific for the minimal CSA-binding region of FCR3 to recombinant proteins covering the minimal CSA-binding region (DBL1-ID2a and ID1–ID2a) of the heterologous 3D7 parasite line produced in the baculovirus expression system. In both assays, the binding was detected with rabbit-anti-camel antibody and HRP-labeled goat anti-rabbit antibody and the optical density was measured at 490 nm after 20 min. A non-VAR2CSA protein was used as a negative control.

Figure 5. Nbs recognize both continuous and discontinuous epitopes.

Reduced and non-reduced baculovirus-produced VAR2CSA domains were transferred onto membranes by Western blotting and probed with the corresponding domain-specific Nbs; (A) DBL4ε protein probed with Nb03, Nb13, Nb16 and Nb17. (B) DBL5ε protein probed with Nb02, Nb05, Nb11, Nb14. (C) DBL6ε probed with Nb15, Nb08, Nb06 and Nb04. (D) DBL1-ID2a protein (containing minimal CSA-binding region) probed with Nb12, Nb10, Nb09, Nb07 and Nb01. Binding was detected with rabbit-anti-camel and HRP-labeled goat-anti-rabbit antibodies. Bands larger than monomer size correspond to multimers formed by intermolecular disulfide bonds (monomers are marked with arrows).

Figure 6. Nanobody recognition of native VAR2CSA expressed on the surface of P. falciparum-infected erythrocytes (IE).

Binding of VAR2CSA-specific Nbs to VAR2CSA-expressing IE was measured by flow cytometry. Three different P. falciparum strains (FCR3, 7201 and NF54) were tested. Each bar represents reactivity of 25 µl nanobody (0.1 mg/ml) to 50 µl IE (2×10⁵ parasites/ml). Values are normalized to the mean fluorescence intensity (MFI) of the negative control (neg ctr; IE incubated only with the secondary and tertiary antibodies). Specific recognition was defined by an MFI ratio >1.2. Error bars in A) represents standard deviation of three independent experiments.

Figure 7. Adhesion-inhibitory capacity of VAR2CSA-specific nanobodies.

(A) Ability of the individual Nbs to inhibit the adhesion of VAR2CSA-expressing IE (FCR3 line) to Decorin. Nbs specific for the minimal CSA-binding-region of VAR2CSA (Nb01, Nb07, Nb09, Nb10 and Nb12) were tested three times on the homologous parasite FCR3-CSA. (B) Adhesion inhibition capacity of the minimal CSA-binding-specific Nbs to VAR2CSA-expressing heterologous parasite lines NF54 and (C) 7201. Parasite binding to CSA ligand without Nbs was set to 100.