2'-Fluoro-c-di-GMP as an oral vaccine adjuvant

Abstract

Bis-(3'-5')-cyclic dimeric 2'-deoxy-2'-fluoroguanosine monophosphate (2'-F-c-di-GMP) was synthesized through the modified H-phosphonate chemistry. Oral immunization of C57BL/6 mice with Helicobacter pylori cell-free sonicate extract adjuvanted with 2'-F-c-di-GMP led to the production of antigen-specific antibodies in feces and sera, and lowered bacterial counts in the stomach upon post-vaccination infections in immunized mice. Similarly, oral vaccination of BALB/c mice with flagillin proteins from Clostridium difficile and Listeria monocytogenes adjuvanted with 2'-F-c-di-GMP led to production of antigen-specific antibodies both systemically and mucosally. The adjuvanticity of 2'-F-c-di-GMP is associated with the enhanced induction of interferon γ. These results demonstrated the excellent oral adjuvanticity of 2'-F-c-di-GMP.

Scheme 1. Reagents and conditions: (i) (a) (CH₃)₃SiCl, (CH₃)₂CHCOCl, C₅H₅N; (b) aq. NH₃; (ii) DMTrCl, C₅H₅N; (iii) (a) 10, (CH₃)₃CCOCl, C₅H₅N; (b) H₂O; (iv) (a) 12, C₅H₅N; (b) Cl₂CHCOOH, pyrrole, CH₂Cl₂; (v) (a) (CH₃)₃CCOCl, C₅H₅N; (b) 11, C₅H₅N; (vi) NH₂NH₂·H₂O, CH₃COOH, C₅H₅N, H₂O; (vii) (a) 10, (CH₃)₃CCOCl, C₅H₅N; (b) H₂O; (c) Cl₂CHCOOH, pyrrole, CH₂Cl₂; (viii) (a) (PhO)₂P(O)Cl, C₅H₅N; (b) 11, C₅H₅N; (ix) (a) 13, TMG; (b) aq. NH₃, 55 °C.

Fig. 1. Induction of antigen-specific mucosal IgA responses by intranasal administration of 2′-F-c-di-GMP. Groups of 5 BALB/c mice were intranasally immunized with 2 μg PsaA admixed with 2.5 μg 2′-F-c-di-GMP, 2.5 μg c-di-GMP or 10 μg c-di-GMP at day 0, 14 and 21. Additional group of mice were immunized with phosphate-buffered saline (PBS) and served as sham-immunized group. The feces, vaginal washing and blood samples were collected at day 28 and assayed by ELISA for PsaA-specific IgA and IgG isotypes (IgG1 and IgG2a) responses. *P < 0.05 vs. PsaA + c-di-GMP groups.

Fig. 2. Reduced nasopharyngeal colonization by S. pneumoniae in mice intranasally immunized with 2′-F-c-di-GMP-adjuvanted vaccine. Groups of 5 BALB/c mice were intranasally immunized with 2 μg PsaA admixed with 2.5 μg 2′-F-c-di-GMP or sham-immunized with PBS at day 0, 14 and 21. The mice were intranasally challenged at day 35 with 5 × 10⁶ CFU type 14 S. pneumoniae and the bacterial numbers in the nasal cavity of challenged mice were determined 3 days later. *P < 0.05 vs. PBS group.

Fig. 3. Induction of antigen-specific mucosal IgA responses by oral administration of 2′-F-c-di-GMP. Groups of 5 C56BL/6 mice were orally immunized with varying amount of H. pylori cell free sonicate extract (HPCE) (A) or flagillin proteins from C. difficile and L. monocytogenes (B) admixed with either 100 μg 2′-F-c-di-GMP or 10 μg cholera toxin (CT, positive control) at day 0, 14 and 21. Additional group of mice were immunized with PBS and served as sham-immunized group. Feces and blood samples were collected at day 28 and assayed by ELISA for antigen-specific IgA and IgG isotypes (IgG1 and IgG2a) responses.

Fig. 4. Reduced gastric colonization by H. pylori in mice orally immunized with 2′-F-c-di-GMP-adjuvanted vaccine. Groups of 5 C56BL/6 mice were orally immunized with varying amount of H. pylori cell free sonicate extract (HPCE) admixed with either 100 μg 2′-F-c-di-GMP or 10 μg cholera toxin (CT, positive control) at day 0, 14 and 21. Additional group of mice were immunized with PBS and served as sham-immunized group. The mice were orally challenged 3× between day 35 and 42 with 10⁸ CFU H. pylori SS1 and the bacterial numbers in the gastric mucosa of challenged mice were determine 4 weeks later. ***P < 0.001 vs. immunized groups.

Fig. 5. Induction of antigen-specific Th1/Th17 cytokine responses by oral administration of 2′-F-c-di-GMP. Groups of 5 C56BL/6 mice were orally immunized with varying amount of H. pylori cell free sonicate extract (HPCE) admixed with either 100 μg 2′-F-c-di-GMP or 10 μg cholera toxin (CT, positive control) at day 0, 14 and 21. Additional group of mice were immunized with PBS and served as sham-immunized group. The spleens were collected at day 28 and single cell suspension was prepared for the determination of antigen-specific cytokine responses. The cells were stimulated by 10 mg ml⁻¹ HPCE and cultured at 37 °C in a 5% CO₂ atmosphere for 72 hours. At the end of the culture, the supernatant was collected and assayed for the levels of IFN-γ, IL-2 and IL-17 by Luminex. *P < 0.05 and ***P < 0.001 vs. PBS group.