A Nanoparticle-Based Trivalent Vaccine Targeting the Glycan Binding VP8* Domains of Rotaviruses

Figure 1. Production, purification, and characterization of the P-VP8* P[8] proteins. (A) A typical elution curve of an anion exchange chromatography of the (NH4)2SO4-precipitated P-VP8* P[8] proteins, showing elution peaks of P5 and P6 containing major P-VP8* P[8] proteins. Y-axis shows A280 absorbances (mAU), while X-axis indicates elusion volume (mL). The red dashed line shows the linear gradient increase of the elution buffer B (0–100%). (B) A typical elution curve of a gel-filtration chromatography of the ion exchange-purified P-VP8* protein from P5 of (A) through a size exclusion column. The major P24-VP8* nanoparticle elution peak and the minimal monomer peak are indicated. The two elution peaks were calibrated using the previously made P24-VP8* P[8] nanoparticles (~1.2 mDa) [15] and P dimers (69 kDa) [43] with their elution positions shown by star symbols on a red dashed line. The Y- and X-axes are the same as in (A). (C) SDS-PAGE of the major elution peaks and fractions showing the purified P-VP8* proteins. M, prestained protein standards with indicated molecular sizes in kDa; inp, the (NH4)2SO4-precipitated P-VP8* P[8] proteins as input proteins. (D) An EM micrograph of the…

Figure 2. Production, purification, and characterization of the P-VP8* P[4] proteins. (A) A typical elution curve of an anion exchange chromatography of the (NH4)2SO4-precipitated P-VP8* P[4] proteins, showing elution peaks of P2, P3, and P[4] containing major P-VP8* P[4] proteins. The Y-axis shows A280 absorbances (mAU), while the X-axis indicates elusion volume (mL). The red dashed line shows the linear gradient increase of the elution buffer B (0–100%). (B) A typical elution curve of a gel-filtration chromatography of the ion exchange-purified P-VP8* protein from P2 of (A) through a size exclusion column. The major P24-VP8* nanoparticle elution peak and the minimal monomer peak are indicated. The two elution peaks were calibrated using the previously made P24-VP8* P[8] nanoparticles (~1.2 mDa) [15] and P dimers (69 kDa) [43] with their elution positions shown by star symbols on a red dashed line. The Y- and X-axes are the same as in (A). (C) SDS-PAGE showing the proteins of the major elution peaks and fractions of the purified P-VP8* P[4] proteins (left) and the (NH4)2SO4-precipitated proteins as input proteins (right, Inp). M, prestained protein standards with indicated molecular sizes…

Figure 3. Production, purification, and characterization of the P-VP8* P[6] proteins. (A) A typical elution curve of an anion exchange chromatography of the (NH4)2SO4-precipitated P-VP8* P[6] proteins, showing elution peaks of P2, P3, and P[4] containing major P-VP8* P[6] proteins. The Y-axis shows A280 absorbances (mAU), while the X-axis indicates elusion volume (mL). The red dashed line shows the linear gradient increase of the elution buffer B (0–100%). (B) A typical elution curve of a gel-filtration chromatography of the ion exchange-purified P-VP8* protein from P2 of (A) through a size exclusion column. The major P24-VP8* elution peak, the minor monomer peak, and a peak representing degraded proteins with small molecular sizes are indicated. The elution peaks were calibrated using the previously made P24-VP8* P[8] nanoparticles (~1.2 mDa) [15] and P dimers (69 kDa) [43] with their elution positions shown by star symbols on a red dashed line. The Y- and X-axes are the same as in (A). (C) SDS-PAGE of the major elution peaks and fractions showing the purified P-VP8* P[6] proteins. M, prestained protein standards with indicated molecular sizes in kDa; inp, the (NH4)2SO4-precipitated…

Figure 4. Interactions of the three P24-VP8* nanoparticles with glycan ligands in the well-characterized saliva samples. (A and B) The P24-VP8* P[8] (A) and the P24-VP8* P[4] (B) nanoparticles bound Leb positive, but not Leb negative saliva samples. (C) The P24-VP8* P[6] nanoparticles bound saliva samples with Lea-b-, but not Leb+ phenotypes. The Y-axis shows binding signals in optical density (OD), while the X-axis indicates various saliva samples with known HBGAs that are coded from OH1 to OH96 from our lab stock.

Figure 5. Antibody responses in mice of the trivalent P24-VP8* nanoparticle vaccine to the three homologous VP8* types compared with those of the three individual P24-VP8* nanoparticles. The Y-axis shows the VP8*-specific IgG titers, while the X-axis indicates different immunogens. Statistical differences between data groups with corresponding P-values are calculated and indicated below the figure. “ns”, non-significant for p-values > 0.05, *, significant for p-values < 0.05, **, highly significant for p-values < 0.01, ***, extremely significant for p-values < 0.001.

Figure 6. Neutralization of the P24-VP8* nanoparticle trivalent vaccine-immunized mouse sera against three homologous rotavirus P types. The 50% neutralization titers (Y-axis) of mouse sera after administration with the trivalent vaccine (trivalent) against a P[8] (Wa strain, G1P8, black columns), a P[4] (DS-1 strain, G2P[4], green columns), and a P[6] (ST-3, G4P6, blue columns) rotavirus in cell culture are shown and compared each other using the mouse sera after immunization with the P24 nanoparticles without the VP8* antigens (P24) as controls (X-axis). Statistical differences between data groups are indicated as “ns” for non-significant for p-values > 0.05, and **** for extremely significant for p-values < 0.0001.