Structural Effect on Adjuvanticity of Saponins

Abstract

We have prepared a number of saponin-based vaccine adjuvant candidates. These unnatural saponins have a different terminal-functionalized side chain incorporated into the glucuronic acid unit that is attached to a triterpenoid core at its C3 position. The semisynthetic saponin adjuvants have shown significantly different immunostimulatory activities, suggesting that the structure of the side chain, triterpenoid core, and oligosaccharide domain together orchestrate saponin adjuvant's potentiation of immune responses. Among these new adjuvant candidates, VSA-2 (5b), a derivative of Momordica saponin (MS) II, showed consistent enhancement of immunoglobulin G2a (IgG2a) production when it was in formulation with either ovalbumin or recombinant hemagglutinin B (rHagB) antigen. With rHagB antigen, it induced a significantly higher IgG2a response than the positive control GPI-0100, a well-studied semisynthetic saponin adjuvant mixture derived from Quillaja saponaria Molina saponins, known for its ability to induce a balanced Th1/Th2 immunity. These results confirm that Momordica saponins are a viable natural source to provide potent saponin adjuvants after simple chemical derivatization and identify VSA-2 (5b) as another MS-based promising immunostimulant lead owing to its distinctive ability in potentiating the IgG2a response.

Figure 1.

Natural saponin QS-21.

Figure 2.

MS I (2), MS II (3), and their derivatives.

Figure 3.

QS-21 derivatives with a different side chain.

Figure 4.

Serum IgG (A), IgG1 (B), and IgG2a (C) anti-OVA response in mice immunized by the s.c. route with OVA alone or with GPI-0100 or a MS derivative. Mice were immunized on days 0, 14 and 28. Serum samples were collected prior to each immunization and at 6 weeks after the initial immunization. Values are expressed as mean ± SEM. Statistical significance in antibody responses was evaluated by t tests (with unpaired, nonparametric and Mann-Whiteny test). * P < 0.05 and ** P < 0.01 compared with mice immunized with OVA alone.

Figure 4.

Serum IgG (A), IgG1 (B), and IgG2a (C) anti-OVA response in mice immunized by the s.c. route with OVA alone or with GPI-0100 or a MS derivative. Mice were immunized on days 0, 14 and 28. Serum samples were collected prior to each immunization and at 6 weeks after the initial immunization. Values are expressed as mean ± SEM. Statistical significance in antibody responses was evaluated by t tests (with unpaired, nonparametric and Mann-Whiteny test). * P < 0.05 and ** P < 0.01 compared with mice immunized with OVA alone.

Figure 5.

Serum IgG (A), IgG1 (B), and IgG2a (C) anti-rHagB response in mice immunized by the s.c. route with rHagB alone or with GPI-0100 or a saponin adjuvant. Mice were immunized on days 0, 14 and 28. Serum samples were collected prior to each immunization and at 6 weeks after the initial immunization. Values are expressed as mean ± SEM. Statistical significance in IgG, IgG1, and IgG2a antibody responses was evaluated by t tests (with unpaired, nonparametric and Mann-Whiteny test). * P < 0.05, and ** P < 0.01 compared with mice immunized with rHagB alone, # P < 0.05, compared between the indicated groups.

Figure 6.

Ratio of Serum anti-rHagB IgG2a and IgG1 activity (IgG2a/IgG1).Values are expressed as mean ± SD. Statistical significance compared with rHagB+VSA-2 (5b), Statistical significance was evaluated by t tests (with unpaired, nonparametric and Mann-Whiteny test). *P < 0.05, **P < 0.01.

Scheme 1.

Derivatizing Momordica Saponins^{a a} Reagents and conditions: (a) NMM, HOBt, EDC·HCl, NH₂(CH₂)₁₀COOBn, H₂O/EtOH, r.t., 78% for 4b and 72% for 5b; (b) Pd/C, H₂ (55 psi), 98% for 4c and 96% for 5c.

Scheme 2.

QS Saponin Analogs with Different Side Chains^{a a}Reagents and conditions: (a) Pd/C, H₂ (55 psi); (b) NMM, HOBt, EDC·HCl, NH₂(CH₂)₁₀COOBn, H₂O/EtOH, r.t., 52% over two steps; (c) K₂CO₃, MeOH/H₂O, 57%; (d) Pd/C, H₂ (50 psi), 96%.