PMC

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.

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.

(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.

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.

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).

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.

(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.