A Nanoparticle Comprising the Receptor-Binding Domains of Norovirus and Plasmodium as a Combination Vaccine Candidate

Figures

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  Figure 1. Production of the GST-tagged P-αTSR protein and self-formation of the P24-αTSR NPs. (A) Schematic representation of the GST-tagged P-αTSR fusion protein construct. The αTSR domain is inserted in loop 2 of the norovirus P domain, with a thrombin cleavage site present between the GST and P-αTSR protein. (B) SDS-PAGE analysis of the GST-P-αTSR fusion protein and its bipartite components after thrombin cleavage. Left lane (M): prestained protein standards, with molecular weights (MWs) indicated in kDa. Middle lane (Fusion): resin-purified GST-P-αTSR fusion protein (~68 kDa) with minor co-purified proteins. Right lane (+Thrombin): two separate components, the P-αTSR protein (~42 kDa) and the GST (~26 kDa), along with minor amounts of uncleaved GST-P-αTSR fusion protein, after thrombin cleavage. (C) Elution curve from gel-filtration chromatography of the thrombin-cleaved GST-P-αTSR protein, showing four distinct elution peaks (P1, P2, P3, and P4). The Y-axis represents relative protein amounts measured by ultraviolet absorbance at 280 nm (mAU) and the X-axis indicates the elution volume (mL). The dashed line at the top, marked with two red stars, indicates the elution positions of the…

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  Figure 2. Generation and characterization of tag-free P-αTSR protein and P24-αTSR NPs. (A) Schematic representation of the tag-free P-αTSR protein construct. (B) SDS-PAGE analysis of the P-αTSR protein precipitated with ammonium sulfate [(NH4)2SO4]. The left lane (M) shows prestained protein standards, with molecular weights (MWs) labeled in kDa. The right lane (PPT) displays the P-αTSR protein (denoted by an arrow) precipitated from bacterial lysate using 1.2 M (NH4)2SO4. (C,D) Representative elution profile from anion exchange chromatography of the (NH4)2SO4-precipitated P-αTSR protein. (D) Is an enlarged view of the framed region in (C). The Y-axis represents relative protein amounts in the eluents, measured by ultraviolet absorbances at 280 nm (A280, mAU), while the X-axis indicates the accumulated elusion volume (mL). The dashed red line shows the linear gradient of buffer B (0–100%), with a red star marking the percentages of buffer B at the elution peak of the P-αTSR protein (33.9%, P6). Six major elution peaks (P1 to P6), analyzed by SDS-PAGE in (E), are indicated. (E) SDS-PAGE analysis of the six major elution peaks from the anion exchange chromatography in (C,D). Lane “Pre”:…

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  Figure 3. The 3D structural models of the P24-αTSR NP. The model was constructed using UCSF ChimeraX software version 1.4, based on the previously elucidated cryo-EM maps of the norovirus P24 NP and the P24-VP8* chimeric NP. In this model, the rotavirus VP8* domain was replaced by the known crystal structure of the αTSR domain (PDB code: 3VDJ). (A–D) Transmission electron microscopy (TEM) images showing the typical morphology of the P24-αTSR NP (indicated by arrows), consisting of a P24 NP core with multiple extended protrusions formed by the αTSR domains. (E–G) Surface representations of the norovirus P24 NP, viewed along the three-fold axis (E), the four-fold axis (F), and at an intermediate angle between these two axes (G). (H–J) Surface representations of the P24-αTSR NP, viewed along the three-fold axis (H), the four-fold axis (I), and at an intermediate angle between these two axes (J). (K–M) Cross-sectional views of the three P24-αTSR NP structures shown in (H–J), cut in half and viewed from the cutting planes along the same three symmetry axes as in (H–J). (N–P) Middle-section cross-sectional views of the three P24-αTSR NP structures shown in (H–J), viewed from the same three…

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  Figure 4. CsCl density gradient ultracentrifugation, specific antibody reactivity, and heparin sulfate glycan binding of the P24-αTSR NP. (A,B) Each of the 23 fractions from the CsCl density gradient was analyzed for the presence of the P24-αTSR NP using EIA assays. Hyperimmune serum from a guinea pig after immunization with norovirus VLP (A) and hyperimmune serum from a mouse after immunization with the Plasmodium αTSR domain (B) were used as detection antibodies, respectively. The Y-axis represents the optical density (OD) as a measure of signal intensity, while the X-axis corresponds to the gradient fractions arranged from the bottom (fraction 1) to the top (fraction 23). (C) EIA-based binding assay showing the interaction of the P24-αTSR NP with heparin sulfate glycans. The GST-αTSR protein served as a positive control, while the P24 NP and GST were used as negative controls. The Y-axis indicates the binding signal intensity of the OD, while the X-axis shows the different proteins at the indicated concentrations. The limit of detection (LOD), indicated by a red dashed line in each panel, was set at OD = 0.1.

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  Figure 5. Immune responses to the P24-αTSR NP toward its bipartite components. (A,B) Plasmodium αTSR-specific IgG (A) and IgA (B) titers in mice immunized with the P24-αTSR NP (blue column) compared to titers induced by free αTSR (red column) and the P24 NP (green column) after three intramuscular immunizations. (C,D) Norovirus P domain-specific IgG (C) and IgA (D) titers in mice immunized with the P24-αTSR NP (blue column) compared to titers induced by free αTSR (red column) and the P24 NP (green column) after three intramuscular immunizations. In (A,C), the Y-axes represent αTSR-specific (A) or P domain-specific (C) IgG titers. In (B,D), the Y-axes represent αTSR-specific (B) or P domain-specific (D) IgA titers. In (A–D), the X-axes indicate various immunogens. (E) The 50% blocking titers (BT50) of sera from mice immunized with the P24-αTSR NP (blue column), P24 NP (green column), and free αTSR (red column) against norovirus VLP-glycan receptor attachment. The Y-axis represents BT50 values, while the X-axis indicates various immunogens. Corresponding statistical p values between the data groups are displayed above the columns. The limit of detection (LOD) is indicated by a red dashed…

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  Figure 6. Specific staining of P. falciparum sporozoites by sera from mice immunized with the P24-αTSR NP, in immunofluorescence assays (IFAs). (A) Representative IFA micrograph showing Plasmodium sporozoites stained by mouse sera (1:8000 dilution) after three immunizations with the P24-αTSR NP. (C) Enlarged view of the marked regions in (A). (E) Sera (1:8000 dilution) from mice immunized with the P24 NP showed no staining of sporozoites (negative control). (B,D,F) Optical views of the same fields correspond to the IFA images in the left panels. Arrows indicate the visualized sporozoites.