Structure-Activity Relationship Study of Momordica Saponin II Derivatives as Vaccine Adjuvants

Abstract

VSA-2 is a recently developed semisynthetic saponin immunostimulant. It is prepared by incorporating a terminal-functionalized side chain to the branched trisaccharide domain at the C3 position of Momordica saponin II (MS II) isolated from the seeds of perennial Momordica cochinchinensis Spreng. Direct comparison of VSA-2 and the clinically proven saponin adjuvant QS-21 shows that VSA-2 is comparable to QS-21 in enhancing humoral and cellular immune responses. Structure-activity relationship studies show that structural changes in the side chain have a significant impact on saponins' adjuvant activity. However, with the VSA-2 molecular framework intact, the new VSA-2 analogues with various substitution(s) at the terminal benzyl group of the side chain retain the ability of potentiating antigen-specific humoral and cellular responses.

Figure 1.

Serum IgG (A), IgG1 (B), and IgG2a (C) anti-OVA responses in mice immunized by the s.c. route with OVA (20.0 μg) alone (none) or with QS-21 (20.0 μg) or an MS derivative (50.0 μg). 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 unpaired t tests (nonparametric and Mann–Whiteny test). *P < 0.05 and **P < 0.01 compared with mice immunized with OVA alone. #P < 0.05 and ##P < 0.01.

Figure 2.

Serum IgG1 (A) and IgG2a (B) anti-OVA responses in mice immunized by the s.c. route with OVA (20 μg) alone (none) or with QS-21 (20 μg), VSA-2 (100 μg), or an MS derivative (50 μg). 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 unpaired t tests (nonparametric and Mann–Whiteny test). *P < 0.05 and **P < 0.01 compared with mice immunized with OVA alone. #P < 0.05 and ##P < 0.01.

Figure 3.

CD8⁺ T-cell surface expression of PD-1 (A), intracellular levels of IFNγ in CD8⁺ T-cells (B), expression of granzyme B in CD8⁺ T-cells (C), CD4⁺ T-cell surface expression of PD-1 (D), intracellular levels of IFNγ in CD4⁺ T-cells (E), expression of IL-4 in CD4⁺ T-cells (F), and expression of IL-21 CD4⁺ T-cells (G) in mice immunized by the s.c. route with OVA (20 μg) alone (none) or with QS-21 (20 μg), VSA-2 (100 μg), or an MS derivative (50 μg). Mice were immunized on days 0, 14, and 28. Spleens were collected at 6 weeks after the initial immunization. Mean Fluorescence Intensity (MPI) values are expressed as mean ± SEM. Statistical significance in each comparison evaluated by unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 4.

Analysis of splenic cell types affected by saponin adjuvants: follicular helper T cells (A), follicular regulatory T cells (B), ratio of T_FH to T_RF (C), CD19-expressing B-cells (D), and dark zone B-cells (E) in mice immunized by the s.c. route with OVA (20 μg) alone (none) or with QS-21 (20 μg), VSA-2 (100 μg), or an MS derivative (50 μg). Mice were immunized on days 0, 14, and 28. Spleens were collected at 6 weeks after the initial immunization. Values are expressed as mean ± SEM. Statistical significance in each comparison is evaluated by unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Scheme 1.. Preparation of VSA-2 Adjuvant from Natural Momordica Saponin II

Scheme 2.. Synthesis of MS II derivatives 1D–1H^a

^aReagents and conditions: (a) Boc₂O, NaHCO₃, DMF, 0 °C—r.t.; (b) BzCl, Et₃N, DCM; (c) BnBr, NaH, DMF, 0 °C—r.t.; (d) TFA, DCM; (e) NMM, HOBt, EDC^.HCl, R′NH₂, H₂O/EtOH, r.t.; (f) Pd/C, H₂ (55 psi), MeOH/H₂O; (g) benzoyl NHS ester, methanol.

Scheme 3.. Synthesis of MS II derivatives 2D–2H^a

^aReagents and conditions: (a) SOCl₂, ArCH₂OH, r.t.—70 °C; (b) NMM, HOBt, EDC.HCl, RNH₂, H₂O/EtOH, r.t.