Fig. 1: Engineering, expression, and antibody recognition of the PfCSP-spirulina VLP vaccine. | npj Vaccines

Fig. 1: Engineering, expression, and antibody recognition of the PfCSP-spirulina VLP vaccine.

From: Needle-free, spirulina-produced Plasmodium falciparum circumsporozoite vaccination provides sterile protection against pre-erythrocytic malaria in mice

Fig. 1

a Light micrograph of spirulina filaments. Magnification ×400. b, c Spirulina were engineered to carry Woodchuck hepadnavirus core proteins (WHcAg) by cloning a single copy (b) or two copies linked in tandem by five Gly-Gly-Ser amino acid repeats (5xGGS) (c) of the WHcAg sequence into a plasmid with arms for homologous recombination into the spirulina chromosome. b Insertion of the PfCSP antigen at serine 78 in the Major Insertion Region (MIR) of the single copy construct forms dimers containing two copies of each antigen within the spike. c In cases where the WHcAg sequence was linked in tandem, only the second MIR site was used for placement of the PfCSP antigen, forcing the formation of dimers displaying only one antigen within the spike. d Spirulina strains were tested by sucrose density ultracentrifugation and fractionation to demonstrate their ability to form particles; results for sp82 shown. Pre- (left) versus post-centrifugation (right) images show the sedimentation fractions of different pigments present in spirulina, such as phycocyanin (blue). e 1-mL fractions were collected by bottom puncture of the tube with every other fraction resolved by SDS-PAGE and Western blotted using anti-Myc antibody (middle panel). A separate sucrose gradient was carried out on lysate following pre-treatment with SDS to show particle disruption (bottom panel). Molecular weight markers are shown in kDa. f PfCSP-VLPs were also screened by ELISA for their ability to be recognized by serum from BALB/cJ mice previously immunized intravenously three times with 2 × 104 PfCSP-expressing P. yoelii spz. Bars show ELISA absorbance at 450 nm (A450) for the displayed three-fold dilutions of hyperimmune sera ranging from 1:300 to 1:218,700. Sera from naïve mice served as a control. sp646 and sp648 were PfCSP-carrying particles and sp79 was the control no-antigen particle. Images of VLP dimers were designed using PyMol software with PDB 6ECS73 as template.

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