Recombinant expression systems for production of stabilised virus-like particles as next-generation polio vaccines

Figures

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  Fig. 1. Schematic of PV genome and VLP expression strategy. The capsid region P1 and the viral protease, 3CD, were introduced into each of mammalian, yeast, insect and plant expression systems for production of PV VLPs.

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  Fig. 2. Sequence and structural arrangement of stabilising mutations designed in PV rsVLPs. a Schematic display of stabilising mutations designed for each PV serotype with a description of their location relative to capsid features. Those mutations present in each serotype are denoted in bold. The four-digit sequence numbering denotes the mature capsid subunit in the first digit (e.g. R4018G refers to VP4 R18G). b Cartoon of an icosahedral PV capsid, with a single pentamer shaded in pale yellow. Five-fold, three-fold and two-fold symmetry axes are labelled with symbols. A single protomeric subunit within the pentamer is highlighted and subunits coloured blue (VP1), green (VP0) and red (VP3). The lipid bound in the VP1 hydrophobic pocket is depicted in black. Key capsid features from (a) are labelled and the canyon around the five-fold axis is shown as a semi-transparent grey ring. The expanded view shows the capsid protomer as a molecular cartoon with the positions of all stabilising mutations from (a) mapped onto the structure and colour-coded based on their insertion into the PV1-SC6b (blue), PV2-SC6b (green) and PV3-SC8 (red) rsVLPs. Mutations present in more than one rsVLP are…

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  Fig. 3. CryoEM reconstructions of PV1-SC6b and PV2-SC6b rsVLPs and the PV1-SC6bGPP3+GSH complex. a Top panel, PV1-SC6b rsVLPs from yeast and mammalian (MVA) cells depicted as isosurface representations of the electron potential maps at a threshold of 4σ (σ is the standard deviation of the map) and radially coloured by distance (Å) from the particle centre according to the colour key. Representative five-fold, three-fold and two-fold symmetry axes, and an icosahedral AU are shown as in Fig. 2c. Bottom panel, zoomed-in views of the two-fold interface of each rsVLP. b Left panel, cartoon depiction of the VP1 hydrophobic pocket of the yeast PV1-SC6b D Ag particle, with palmitic acid shown as an orange stick model fitted into the cryoEM map (1.0 σ). Amino acid residues interacting with palmitic acid are labelled. Right panel, VP1 pockets of the C Ag particles from yeast (cyan) and mammalian cells (magenta) are superposed on the D Ag particle VP1 pocket (grey). CryoEM maps at 4σ of PV2-SC6b rsVLP expressed in mammalian (c) and insect (d) cells, respectively (radial colouring same as a), shown alongside their respective VP1 pockets, with sphingosine (SPH) modelled as orange sticks. Electron…

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  Fig. 4. Immunogenicity of rsVLPs in TgPVR mice. Neutralising antibody titres following immunisation with VLPs from each of the 3 PV serotypes produced in different expression systems. Groups of 8 mice received 2 injections (on days 0 and 14) of 0.5 human doses of either PV1-SC6b (16 D Ag), PV2-SC6b (4 D Ag), PV3-SC8 (14 D Ag), IPV or PBS as the negative control. Sera were collected 35 dpi and neutralisation assays performed as described91. Each IPV bar is representative of a different experiment positive control. The position of each bar and the colour of the stripes in the IPV bars indicate for which expression system(s) IPV was the positive control (Insect; purple, Mammalian; red, Plant; brown, and Yeast; orange). N.D indicates the experiment was not done due to insufficient D Ag. Error bars represent the Geomean Standard Deviation of the data points. Source data are provided as a Source Data file.

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  Fig. 5. Protection against live challenge in TgPVR mice immunised with PV1-SC6b VLPs. a Groups of eight mice received either a single or two doses of rsVLPs (orange) produced in yeast (day 0 and 14) prior to challenge on day 35 with 25 × PD50 dose of live PV1 Mahoney and compared to IPV (blue) or a PBS negative control (black). Neutralisation titres prior to boost and on the day of challenge were determined. b Animals were monitored for survival for 14 days following challenge. Error bars represent the Geomean Standard Deviation of the data points. Source data are provided as a Source Data file.

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  Fig. 6. Immunogenicity of rsVLP vaccines in female rats. Dose response in neutralising antibodies following a single immunisation of female Wistar rats in the absence (a) or the presence (b) of adjuvant. Groups of 10 rats received rsVLPs at various multiples of human doses and compared with IPV (blue). Sera were collected 21 dpi and neutralisation titres against the Sabin strains of PV1, PV2 or PV3 determined. VLPs produced from each expression system are depicted as follows: Insect; purple, Mammalian; red, Plant; brown, and Yeast; orange. Error bars represent the Geomean Standard Deviation of the data points. Source data are provided as a Source Data file.