Utilizing Nanobody Technology to Target Non-Immunodominant Domains of VAR2CSA

<div><p>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 <i>Plasmodium falciparum</i>-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>i</i>) the large size of VAR2CSA and (<i>ii</i>) 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.</p></div

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

<p>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.</p

Sequence alignment of the 17 VAR2CSA-specific Nbs.

<p>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.</p

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

<p>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.</p

Summary of the reactivity of the individual Nbs against VAR2CSA (full length and domains).

<p>A cross in the box indicates specific binding.</p

Adhesion-inhibitory capacity of VAR2CSA-specific nanobodies.

<p>(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.</p

Recognition of the minimal CSA-binding region of VAR2CSA.

<p>(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 <i>E. coli</i> (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.</p

Other PV VLPs with AviTag^TM inserted in DE-loop.

<p>The HPV16 L1 VLP was used as VLP platform for proof of concept in this study. However, the AviTag^TM can also be inserted into the DE loop of the major capsid protein from other papilloma viruses while retaining the ability to self-assemble into VLPs, as demonstrated in this figure. <b>a</b> Multiple sequence alignment of the HPV16 L1, HPV118 L1 and major capsid protein from European Elk papilloma virus (PAPVE). <b>b</b> Purification of HPV118 Avi-L1 and PAPVE Avi-L1 VLPs were performed by UC over an Optiprep^TM density gradient (27%/33%/39). Subsequent reduced SDS-PAGE analysis of high-density UC fractions (3–5) show the presence of a protein band of 56 kDa corresponding to the full-length Avi-L1 protein. These fractions also contain an intense protein band of approximately 43kDa, which may represent a truncated Avi-L1 product.</p

A Novel Virus-Like Particle Based Vaccine Platform Displaying the Placental Malaria Antigen VAR2CSA

<div><p>Placental malaria caused by <i>Plasmodium falciparum</i> is a major cause of mortality and severe morbidity. Clinical testing of a soluble protein-based vaccine containing the parasite ligand, VAR2CSA, has been initiated. VAR2CSA binds to the human receptor chondroitin sulphate A (CSA) and is responsible for sequestration of <i>Plasmodium falciparum</i> infected erythrocytes in the placenta. It is imperative that a vaccine against malaria in pregnancy, if administered to women before they become pregnant, can induce a strong and long lasting immune response. While most soluble protein-based vaccines have failed during clinical testing, virus-like particle (VLP) based vaccines (<i>e</i>.<i>g</i>., the licensed human papillomavirus vaccines) have demonstrated high efficacy, suggesting that the spatial assembly of the vaccine antigen is a critical parameter for inducing an optimal long-lasting protective immune response. We have developed a VLP vaccine display platform by identifying regions of the HPV16 L1 coat protein where a biotin acceptor site (AviTag^TM) can be inserted without compromising VLP-assembly. Subsequent biotinylation of Avi-L1 VLPs allow us to anchor monovalent streptavidin (mSA)-fused proteins to the biotin, thereby obtaining a dense and repetitive VLP-display of the vaccine antigen. The mSA-VAR2CSA antigen was delivered on the Avi-L1 VLP platform and tested in C57BL/6 mice in comparison to two soluble protein-based vaccines consisting of naked VAR2CSA and mSA-VAR2CSA. The mSA-VAR2CSA Avi-L1 VLP and soluble mSA-VAR2CSA vaccines induced higher antibody titers than the soluble naked VAR2CSA vaccine after three immunizations. The VAR2CSA Avi-L1 VLP vaccine induced statistically significantly higher endpoint titres compared to the soluble mSA-VAR2CSA vaccine, after 1^st and 2^nd immunization; however, this difference was not statistically significant after 3^rd immunization. Importantly, the VLP-VAR2CSA induced antibodies were functional in inhibiting the binding of parasites to CSA. This study demonstrates that the described Avi-L1 VLP-platform may serve as a versatile system for facilitating optimal VLP-display of large and complex vaccine antigens.</p></div

Display of VAR2CSA on HPV16 L1-AviTag VLPs assessed by parasite inhibition assay.

<p>The functional antibody response was assessed by measuring the capacity of mouse anti-sera to inhibit binding between native VAR2CSA expressed on parasitized erythrocytes and CSA in a static binding-assay. <i>P</i>. <i>falciparum</i> (FCR3 genotype)-infected red blood cells, expressing the native VAR2CSA, were first incubated with mouse anti-serum (4 fold dilution series, starting from 1:50) and then allowed to incubate on decorin coated plates for 90 min. Unbound IE were washed away and the remaining IEs were quantified. Normalized parasite binding after incubation with pooled anti-sera from mice (n = 5) vaccinated with mSA-VAR2CSA-coupled HPV16 Avi-L1 VLPs (blue) or soluble mSA-VAR2CSA (red) are shown after first (<b>a</b>), second (<b>b</b>) and third (<b>c</b>) immunization. The green piles in Fig 6<b>c</b> represent anti-sera from mice vaccinated with soluble naked VAR2CSA and is a pool of sera from 2^nd and 3^rd bleed.</p