Formulation and preclinical studies with a trivalent rotavirus P2-VP8 subunit vaccine

Figures

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  Figure 1.. Percent adsorption of P2-VP8* P[8] protein to aluminum adjuvants in 0.9% (w/v) saline. Adsorption was measured using the bicinchoninic acid assay (n = 3 at each time point).

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  Figure 2.. Influence of phosphate (PO4) concentration in buffer on P2-VP8* monovalent and trivalent adsorption to Alhydrogel, using the bicinchoninic acid assay. (a) The percent adsorption of monovalent P2-VP8* P[8] (120 µg/mL) to Alhydrogel (1.125 mg Al/mL) at different concentrations of PO4 buffer with 0.9% saline, pH ~7.2, was determined at time of formulation (circles) and after 24-hour incubation at 2°C to 8°C (squares) (n = 3 measurements). (b) The percent adsorption of P2-VP8* monovalent and trivalent antigens in 0.5 mM and 1 mM PO4 buffer for antigen concentrations ranging from 30 to 360 µg/mL. Values are the means ± standard deviations (n = 3 measurements).

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  Figure 3.. Ion exchange high-performance liquid chromatography data comparing monovalent standard elution peaks P[4], P[6], and P[8] with that of the trivalent vaccine antigen.

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  Figure 4.. SDS-PAGE images of the trivalent formulation at t = 0 and after storage at different temperatures for one, four, and 12 weeks. The lanes are as follows: (1) ladder; (2) trivalent standard; (3) whole vaccine at 2°C to 8°C; (4) whole vaccine at 25°C; and (5) whole vaccine at 37°C. Double bands representing the three antigens (upper: P[6] and lower: P[4] and P[8]) are at approximately 20 kDa. Reduction in band intensity over time is due to incomplete desorption of antigen from adjuvant particles related to increased strength of antigen binding to adjuvant.

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  Figure 5.. SDS-PAGE images of the trivalent formulation during storage at 2°C to 8°C. The lanes are as follows: (1) blank; (2) ladder; (3) trivalent standard; (4) whole vaccine; (5) vaccine supernatant; and (6) vaccine pellet. Double bands representing the three antigens are at approximately 20 kDa.

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  Figure 6.. Serum antibody responses after intramuscular injections with P2-VP8* P[8] antigen with and without Alhydrogel adjuvant. Six animals for each antigen group with and without adjuvant were immunized on days 0, 14, and 28, and blood samples were analyzed on day 42 (two weeks after the final immunization). Three naïve animals for each antigen group were included as negative controls, and blood samples were analyzed on day 42. (a) Neutralizing antibodies and total binding immunoglobulin G (IgG) detected against homologous antigen. (b) Neutralizing antibody responses generated against heterologous antigens. Geometric mean titers with 95% confidence intervals are shown.

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  Figure 7.. Serum neutralizing antibody responses (y-axis) after intramuscular injections with P2-fused and non-fused monovalent antigens with Alhydrogel adjuvant (x-axis). Animals were immunized on days 0, 14, and 28, and blood samples were analyzed on day 42 (two weeks after final immunization). Neutralizing antibody results against DS-1 (P[4]), ST3 (P[6]), and Wa (P[8]) rotavirus strains. Geometric mean titers with 95% confidence intervals are shown.

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  Figure 8.. Serum immunoglobulin G (IgG) antibody responses after intramuscular injections with P2-fused and non-fused monovalent antigens adsorbed to Alhydrogel adjuvant (x-axis). Animals were immunized on days 0, 14, and 28, and blood samples were analyzed on day 42 (two weeks after final immunization). Binding immunoglobulin G results against VP8* and P2-VP8* antigens are shown as geometric mean titers with 95% confidence intervals.