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

Abstract

Rotavirus causes severe gastroenteritis in children. Although vaccines are implemented, rotavirus-related diarrhea still claims ~200,000 lives annually worldwide, mainly in low-income settings, pointing to a need for improved vaccine tactics. To meet such a public health need, a P₂₄-VP8* nanoparticle displaying the glycan-binding VP8* domains, the major neutralizing antigens of rotavirus, was generated as a new type of rotavirus vaccine. We reported here our development of a P₂₄-VP8* nanoparticle-based trivalent vaccine. First, we established a method to produce tag-free P₂₄-VP8* nanoparticles presenting the VP8*s of P[8], P[4], and P[6] rotaviruses, respectively, which are the three predominantly circulating rotavirus P types globally. This approach consists of a chemical-based protein precipitation and an ion exchange purification, which may be scaled up for large vaccine production. All three P₂₄-VP8* nanoparticle types self-assembled efficiently with authentic VP8*-glycan receptor binding function. After they were mixed as a trivalent vaccine, we showed that intramuscular immunization of the vaccine elicited high IgG titers specific to the three homologous VP8* types in mice. The resulted mouse sera strongly neutralized replication of all three rotavirus P types in cell culture. Thus, the trivalent P₂₄-VP8* nanoparticles are a promising vaccine candidate for parenteral use against multiple P types of predominant rotaviruses.

Keywords: P24-VP8* nanoparticle; non-replicating rotavirus vaccine; norovirus P domain; rotavirus; rotavirus VP8*; rotavirus vaccine.

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 P-VP8* (8) protein from the major peak of (B) showing typical P₂₄-VP8 nanoparticles (arrows). (E) A Western blot analysis of the three ion-exchange-purified P-VP8* proteins using antibody against norovirus VLPs. The positions of both monomers and dimers are shown. Control, the previously made P-VP8* P[8] protein using the GST tag approach.

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 in kDa. (D) An EM micrograph of the P-VP8* (4) proteins from the major peak of (B) showing typical P₂₄-VP8 nanoparticles (arrows).

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 proteins as input proteins. (D) An EM micrograph of the P-VP8*(6) proteins from the major peak of (B) showing typical P₂₄-VP8 nanoparticles (arrows).

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 Le^b positive, but not Le^b negative saliva samples. (C) The P₂₄-VP8* P[6] nanoparticles bound saliva samples with Le^a-b-, but not Le^b+ 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.