Production of an immunogenic trivalent poliovirus virus-like particle vaccine candidate in yeast using controlled fermentation

Figures

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  Fig. 1. VLP purification schematic. To compare yields of yeast-derived PV VLPs produced by either flask or bioreactor mediated expression, yeast pellets are processed in the same way. Pellets derived from equivalent culture volumes were mechanically disrupted at 40 kpsi ( ~ 275 MPa) and the cell lysates collected for further processing. Following removal of insoluble material, samples were treated with nuclease prior to a chemical precipitation step using PEG. The pelleted material was then resuspended and pelleted through 30% sucrose. The resulting pellet was resuspended in a small volume prior to sucrose density gradient ultracentrifugation. The resultant fractions were analysed for the presence of PV VLPs by ELISA.

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  Fig. 2. Production of PV VLPs under fermentation conditions. A Antigenicity of PV VLPs purified from 10 L bioreactor material. Reactivity of gradient fractions using PV serotype-specific monoclonal antibodies, for D antigen (MAb 234, 1050, and 520 for PV1, PV2 and PV3, respectively) or C antigen (MAb 1588 and 517.3 for PV1 and PV3, respectively) in ELISA. The pink dashed line represents the positive control, BRP, for the D antigen ELISA. OD at λ = 492 nm is represented in arbitrary units. The figure is a representative example of three separate experiments for each construct. B Representative micrographs of PV VLPs purified from 10 L bioreactor material (Scale bar shows 200 nm). C Comparative analysis of VLP production in flask and bioreactor. Amount of purified D Ag produced per 100 mL culture as determined by ELISA using MAb 234, 1050, and 520 for PV1, PV2 and PV3, respectively. OD at λ = 492 nm is represented in arbitrary units (n = 3). Means +/− standard deviation. Statistical analysis determined by two-tailed t test (**, p value > 0.01, ***, p value > 0.001).

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  Fig. 3. Comparison of PV2-SC6b and PV2-SC5a VLPs. A Antigenicity of thermally stabilised PV2 VLPs purified from flask-based expression. Reactivity of gradient fractions using a PV2 specific monoclonal antibody for D antigen (MAb 1050) in ELISA. The pink dashed line represents the positive control, BRP. OD at λ = 492 nm is represented in arbitrary units. The figure is a representative example of three separate experiments for each variant. B Representative micrographs of PV2 VLPs purified from 10 L bioreactor material (Scale bar shows 200 nm). C Comparative analysis of PV2 VLP production in flask and bioreactor. Amount of purified D Ag produced per 100 mL culture as determined by ELISA using MAb 1050. OD at λ = 492 nm is represented in arbitrary units (n = 3) Means +/− standard deviation. Statistical analysis determined by two-tailed t test (**, p value > 0.01, ***, p value > 0.001). D Reactivity of purified PV2-SC6b and PV2-SC5a VLPs and BRP aliquots to D-antigen specific MAb 1050 in ELISA after incubation at different temperatures for 10 min, normalised to corresponding aliquot incubated at 4 °C. This figure is a representative example of two separate experiments for each variant.

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  Fig. 4. Cryo-EM analysis of bioreactor produced PV2-SC6b and PV2-SC5a VLPs. Gold standard Fourier shell correlation (GSFSC) curves calculated between two independent half sets of data as a function of resolution are shown for PV2-SC6b (A) and PV2-SC5a (B), generated after local resolution analysis in CryoSPARC. Isosurface representations of the electron potential maps for PV2-SC6b (C) and PV2-SC5a (D) are shown at a threshold of 4σ (σ is the standard deviation of the map). Representative fivefold, threefold and twofold symmetry axes are indicated with symbols, and the white triangle delimits an icosahedral asymmetric unit (AU). A central slice through half of each VLP is viewed along the icosahedral twofold axis. Maps are coloured by local resolution (in Å) according to the colour key. Molecular cartoon representations of a single capsid protomer for PV2-SC6b (E) and PV2-SC5a (F) is shown within a triangle depicting an icosahedral AU, with symmetry axes labelled with symbols as in (C) and (D). Individual subunits of the capsid protomer are coloured blue (VP1), green (VP0) and red (VP3). Stabilising mutations engineered into each VLP are shown as spheres coloured magenta and labelled. For…

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  Fig. 5. Immunogenicity of PV2-SC5a VLPs. Dose response in neutralizing antibodies following a single immunization of Wistar rats with PV2-SC5a VLPs in either the absence or presence of adjuvant. Groups of 10 rats received PV2-SC5a VLPs at various multiples of human doses and compared to a group of 10 that were immunized with IPV as positive control. Sera were collected 21 dpi and neutralisation titres against the S19 S2 Sabin strain of PV2 was determined. Error bars represent the Geomean Standard Deviation of the data points. Statistical analysis determined by two-tailed t test in comparison to IPV (**, p value > 0.01, ***, p value > 0.001). * colour denotes the comparison group.

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  Fig. 6. Trivalent immunogenicity of PV rsVLPs. Dose response in neutralizing antibodies following a single immunization of Wistar rats with trivalent PV VLPs containing PV1-SC6b, PV2-SC5a and PV3-SC8 VLPs, in either the absence or presence of adjuvant. Groups of 10 rats received VLPs at various multiples of human doses and were compared to a group that had received IPV as a positive control. Sera were collected 21 dpi and neutralisation titres against the Sabin strains of PV1, PV2 or PV3 were determined. Error bars represent the Geomean Standard Deviation of the data points. Statistical analysis determined by two-tailed t test in comparison to IPV (*, p value > 0.05, ***, p value > 0.001). * colour denotes the comparison group.