Fig 1.
Schematic representation of the VAR2CSA VLP-vaccine components and the assembly system.
a Structure of HPV16 L1 major capsid protein. The biotin acceptor sequence (AviTagTM) was successfully inserted into the protruding DE and HI loops and the H4-βJ coil (blue boxes) of the HPV16 L1. Gray boxes represent regions (loops and coils) of the L1 protein where the AviTagTM could not be inserted (FG loop) or has not been investigated. The VAR2CSA antigen encompasses the extracellular ID1-ID2a (ID1 –Interdomain 1, DBL2X –Duffy binding like 2X and ID2a –Interdomain 2a) domains of the full-length VAR2CSA fused at the N-terminus to a previously described monovalent streptavidin (mSA) [29]. b Production process of HPV16 Avi-L1 VLPs displaying consistently oriented mSA-VAR2 antigens in a high-density repetitive manner. The HPV16 Avi-L1 and the mSA-vaccine antigen are separately expressed. The HPV16 Avi-VLPs are subsequently in vitro biotinylated (the triangle represents BirA ligase) and finally mixed with the soluble mSA-vaccine antigen. The HPV16 Avi-L1 VLP is theoretically expected to bind one mSA- VAR2CSA antigen per Avi-L1.
Fig 2.
Verification of self-assembly and subsequent in vitro biotinylation of HPV16 Avi-L1 VLPs a Purification of HPV16 Avi-L1 VLPs (HI) VLPs was performed by ultracentrifugation (UC) on an iodixanol (OptiprepTM) density-gradient (27%/33%/39%).
Subsequent reduced SDS-PAGE analyses showed the presence of a 56kDa protein band (theoretical size of Avi-L1) in the high-density UC fractions (4–6) containing particulate material. b Transmission-electron microscopy (TEM) analysis of material representing UC fraction 4 post UC purification. To verify the integrity of the chimeric HPV16 Avi-L1 (HI) VLPs, an aliquot of diluted particles was placed on carbon-coated grids, negatively stained with 2% phosphotungstic acid (pH = 7.0) and examined by transmission electron microscopy (TEM) using a CM 100 BioTWIN at magnification x 36,000 (Å), scale bar 80 nm. c Western blot analysis of fraction 4 post UC purification. The blot demonstrates the presence of HPV16 Avi-L1 (56kDa) detected by Camvir-1 (lane one) and successful biotinylation of HPV16 Avi-L1 using Strep-HRP to detect biotin (lane two).
Fig 3.
HPV16 Avi-L1 VLP coupled to mSA-ID1-ID2a analyzed by ultracentrifugation followed by SDS-PAGE, Western blot and TEM analysis.
a After coupling of the mSA-ID1-ID2a antigen to the HPV16 Avi-L1 VLPs, excess antigen was removed by UC over an OptiprepTM gradient (27%/33%/39%). Reducing SDS-PAGE analysis showed the presence of two protein bands corresponding to the size of HPV16 Avi-L1 (56kDa) and mSA-VAR2CSA (85kDa), respectively, in the high-density fractions (4–6) post UC purification. Excess unbound mSA-VAR2CSA was present in the higher UC fractions (12–14) containing soluble proteins. b Transmission electron microscopy (TEM) analysis of material from UC fraction 4 containing HPV16 Avi-L1 VLPs coupled with mSA-VAR2CSA. An aliquot of diluted particles was placed on carbon-coated grids, negatively stained with 2% phosphotungstic acid (pH = 7.0) and examined by transmission electron microscopy (TEM) using a CM 100 BioTWIN at magnification x 36,000 (Å). Black scale bar 200 nm, enhanced section white scale bar 40 nm. c Western blot analysis of fraction 4 post UC purification of mixed HPV16 Avi-L1 and mSA-VAR2CSA. The blot confirms the presence of HPV16 Avi-L1 and mSA-VAR2CSA detected by Camvir-1 and α-PENTA HIS-tag, respectively.
Fig 4.
Other PV VLPs with AviTagTM inserted in DE-loop.
The HPV16 L1 VLP was used as VLP platform for proof of concept in this study. However, the AviTagTM 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. a Multiple sequence alignment of the HPV16 L1, HPV118 L1 and major capsid protein from European Elk papilloma virus (PAPVE). b Purification of HPV118 Avi-L1 and PAPVE Avi-L1 VLPs were performed by UC over an OptiprepTM 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.
Fig 5.
Reactivity of sera from vaccinated mice by ELISA.
C57BL/6 mice were immunized three times with three-week intervals. Serum samples were collected two weeks after each immunization. Total VAR2CSA-specific immunoglobulin was measured in a serial dilution of mouse anti-sera by ELISA using recombinant VAR2CSA as the solid phase capturing antigen. HRP conjugated anti-mouse Ig antibodies were used for detection by measuring absorbance at OD 490 nm. Serum reactivity from individual mice vaccinated with mSA-VAR2CSA coupled HPV16 Avi-L1 (HI) VLPs (blue) or uncoupled mSA-VAR2CSA (red) are shown after first (a), second (b) and third (c) immunization where each line shows the reactivity of one animal. Green curves represent sera from mice vaccinated with soluble naked VAR2CSA and is a pool of sera obtained after 2nd and 3rd bleed.
Table 1.
Serum endpoint titers (median {25 and 75 percentiles}) obtained with the different immunogens.
Fig 6.
Display of VAR2CSA on HPV16 L1-AviTag VLPs assessed by parasite inhibition assay.
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. P. falciparum (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 (a), second (b) and third (c) immunization. The green piles in Fig 6c represent anti-sera from mice vaccinated with soluble naked VAR2CSA and is a pool of sera from 2nd and 3rd bleed.