A Pseudovirus Nanoparticle-Based Trivalent Rotavirus Vaccine Candidate Elicits High and Cross P Type Immune Response

Figure 1. Expression and selective precipitation of the S-VP8* fusion proteins. (A) Schematic diagram of the S-VP8* fusion proteins. S, modified norovirus shell (S) domain; VP8*, rotavirus VP8* domain. (B) SDS-PAGE of the ammonium sulfate [(NH4)2SO4] precipitated S-VP8* fusion proteins at about 43 kDa, which contain the VP8* domains of P[6], P[4], P[8], and P[11], respectively. Lane M is a pre-stained protein standards with indicated molecular weights in kDa.

Figure 2. Purification of tag-free S-VP8* P[4] protein and its self-assembly into pseudovirus nanoparticles (PVNPs). (A) An anion exchange elution curve of the ammonium sulfate [(NH4)2SO4] precipitated S-VP8* P[4] protein. The X-axis indicates elution volume (mL), whereas the Y-axis shows UV (A280) absorbances (mAU). The red dashed line indicates the linear increase of elution buffer B (0–100%), with a red star symbol indicating the percentage of buffer B at the elution peak of the S-VP8* P[4] protein (34.7%). Two major peaks that were analyzed by SDS-PAGE are indicated as P1 and P2. (B) SDS-PAGE of the pre-loaded protein (Pre), as well as proteins from peak 1 (P1) and peak 2 (P2) from the anion exchange chromatography. Pre is the (NH4)2SO4 precipitated protein samples before loading to the column; M is the pre-stained protein standards with indicated molecular weights in kDa. The S-VP8* P[4] protein was eluted in P2. (C) A micrograph of negative-staining transmission electron microscopy (TEM) of the protein from P2 shows spheric-shaped PVNPs.

Figure 3. Analyses of the four S60-VP8* PVNPs using cesium chloride (CsCl) density gradient centrifugation and transmission electron microscopy (TEM). (A,C,E,G) Following centrifugation, the CsCl density gradients containing the S60-VP8* PVNPs of P[4] (A), P[6] (C), P[8] (E), and P[11] (G) rotaviruses, respectively, were fractionated into 22 portions. The relative S60-VP8* protein amounts in the fractions were measured by EIA assays using a hyperimmune antibody against norovirus VLP [52]. Y-axes show signal intensities in optical density (OD), with red dashed lines showing the cut-off signal at OD = 0.1, while X-axes indicate the fraction numbers. (B,D,F,H) Negative stain TEM micrographs of the S60-VP8* PVNPs from fractions 17 (B,F,H), or 18 (D), showing uniform, ring-shaped PVNPs containing VP8* antigens of the P[4] (B), P[6] (D), P[8] (F) rotaviruses, and P[11] (H) rotaviruses, respectively.

Figure 4. The trivalent S60-VP8* PVNP vaccine (trivalent) induced higher and broader IgG responses in mice toward the three homologous VP8* antigens (black columns) compared with those elicited by the three single-valent S60-VP8* PVNPs (brawn/cyan/green columns) after two (A) and three (B) immunizations. The Y-axis shows the VP8*-specific IgG titers, while the X-axis shows different vaccines or immunogens, as indicated. Statistical differences between data groups with corresponding p-values are calculated and shown in the Supplementary Materials. “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; “****”, extremely significant for p-values < 0.0001.

Figure 5. The trivalent S60-VP8* PVNP vaccine (trivalent) induced higher and broader serum IgA responses in mice toward the three homologous VP8* antigens (black columns) compared with those elicited by the three single-valent S60-VP8* PVNPs (brawn/cyan/green columns) after three immunizations. The Y-axis shows the VP8*-specific serum IgA titers, while the X-axis shows different vaccines or immunogens, as indicated. Statistical differences between data groups with corresponding p-values are calculated and shown in the Supplementary Materials. “NS”, non-significant for p-values > 0.05; “*”, significant for p-values < 0.05; “**”, highly significant for p-values < 0.01.

Figure 6. Blocking (A) and neutralization (B) titers of the mouse sera after immunization with the trivalent PVNP vaccine and controls against predominant rotavirus P types. (A) 50% blocking titers (BT50, Y-axis) of the mouse sera after immunization with the trivalent PVNP vaccine (trivalent) and the three single-valent PVNPs (X-axis) against attachment of the P24 nanoparticle displaying P[8] VP8* to glycan receptors in a Leb positive saliva sample. (B) 50% neutralization titers (Y-axis) of mouse sera after administration with the trivalent vaccine (trivalent) against replication of a P[8] (Wa strain, G1P8, black columns), a P[4] (DS-1 strain, G2P4, brown columns), and a P[6] (ST-3, G4P6, green columns) rotavirus, respectively, in cell culture, using the mouse sera after immunization with the three single-valent PVNPs and the S60 nanoparticle without VP8* antigens (S60) as controls (indicated on the top). Statistical differences between data groups with corresponding p-values are calculated and shown in the Supplementary Materials. “NS”, non-significant for p-values > 0.05; “*”, significant for p-values < 0.05; “**”, highly significant for p-values < 0.01; “***”, extremely significant for…