A small-molecule IRF3 agonist functions as an influenza vaccine adjuvant by modulating the antiviral immune response

Abstract

Vaccine adjuvants are essential to drive a protective immune response in cases where vaccine antigens are weakly immunogenic, where vaccine antigen is limited, or where an increase in potency is needed for a specific population, such as the elderly. To discover novel vaccine adjuvants, we used a high-throughput screen (HTS) designed to identify small-molecule agonists of the RIG-I-like receptor (RLR) pathway leading to interferon regulatory factor 3 (IRF3) activation. RLRs are a group of cytosolic pattern-recognition receptors that are essential for the recognition of viral nucleic acids during infection. Upon binding of viral nucleic acid ligands, the RLRs become activated and signal to transcription factors, including IRF3, to initiate an innate immune transcriptional program to control virus infection. Among our HTS hits were a series of benzothiazole compounds from which we designed the lead analog, KIN1148. KIN1148 induced dose-dependent IRF3 nuclear translocation and specific activation of IRF3-responsive promoters. Prime-boost immunization of mice with a suboptimal dose of a monovalent pandemic influenza split virus H1N1 A/California/07/2009 vaccine plus KIN1148 protected against a lethal challenge with mouse-adapted influenza virus (A/California/04/2009) and induced an influenza virus-specific IL-10 and Th2 response by T cells derived from lung and lung-draining lymph nodes. Prime-boost immunization with vaccine plus KIN1148, but not prime immunization alone, induced antibodies capable of inhibiting influenza virus hemagglutinin and neutralizing viral infectivity. Nevertheless, a single immunization with vaccine plus KIN1148 provided increased protection over vaccine alone and reduced viral load in the lungs after challenge. These findings suggest that protection was at least partially mediated by a cellular immune component and that the induction of Th2 and immunoregulatory cytokines by a KIN1148-adjuvanted vaccine may be particularly beneficial for ameliorating the immunopathogenesis that is associated with influenza viruses.

Keywords: Adjuvant; IRF3; Influenza; Vaccine.

Fig. 1.

HTS hits and derivatives induce the nuclear translocation of IRF3 and IRF3-dependent gene expression. (A) Structure of KIN1000 and its derivative KIN1148. (B) KIN1000 and KIN1148 induce dose-dependent nuclear translocation of IRF3 in PH5CH8 cells. (C) KIN1148 is more potent than KIN1000 as an activator of IRF3-dependent ISG54 and OASL expression by PH5CH8. (D) KIN1148 induces the production of IP-10 by PMA activated THP-1 cells. Experiments in were performed in duplicates for (B, C) and triplicates (C). Data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Prime-boost immunization with vaccine plus KIN1148 protects against lethal H1N1 challenge. Groups of 10 C57BL/6N mice were immunized twice, 21 days apart, with vaccine plus PBS, vaccine plus vehicle, or vaccine plus KIN1148. The mice were then challenged with 10× the LD₅₀ of mouse-adapted influenza virus A/California/04/2009 21 days after boost. Mice were observed for 14 days after challenge (A) Survival. (B) Weight loss. Data are representative of two independent experiments. ***P < 0.001, ****P < 0.0001.

Fig. 3.

Prime-boost immunization with vaccine plus KIN1148 enhances influenza virus-specific Th2 and immunoregulatory responses in response to viral challenge. Groups of C57BL/6N mice were immunized twice, 21 days apart, with vaccine plus vehicle (n = 4), or vaccine plus KIN1148 (n = 8). Mice were then challenged with 10× the LD₅₀ of mouse-adapted influenza virus A/California/04/2009 21 days after boost. T cells from lung and lung-draining lymph nodes (collected on day 7 after challenge) were stimulated for 18 h with split vaccine (SV), CD4 epitope peptide MA-CA/04 NP_260–283 (CD4 Pep), CD8 epitope peptide MA-CA/04 NP_366–374 (CD8 Pep), or concanavalin A (Con A). Cytokines in culture supernatants were then measured using a multiplex ELISA. (A) T cells derived from lungs were evaluated from individual mice. Bars represent the mean and SEM of KIN1148/vaccine (n = 8 mice) and vehicle/vaccine (n = 4 mice) groups. (B) Cells derived from lung-draining lymph nodes were evaluated as separate pools derived from mice receiving KIN1148/vaccine (n = 8 mice) or vehicle/vaccine (n = 4 mice). Stimulations were performed in triplicate with bars representing the mean and STD. Student’s two-tailed t-test was performed to compare the respective antigen-specific T cell responses from SV/vehicle control and SV/KIN1148 groups. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4.

Prime-boost immunization with vaccine plus KIN1148 induces a strong humoral response. Groups of C57BL/6N mice were immunized twice, 21 days apart, with vaccine plus PBS, vaccine plus vehicle, or vaccine plus KIN1148. Mice were bled 18 days after prime and 14 days after boost and sera was used for determination of IgG, neutralizing antibody, or HAI titer. (A) IgG titers at day 18 (post prime). (B) IgG titers at day 35 (14 days post boost). (C) Neutralizing antibody titer at day 35. (D) HAI titer at day 35. Data shown as individual mice with median (horizontal line) and interquartile range (error bar) of the respective groups (n = 15, A/B; n = 8 C/D). One-way ANOVA (Kruskal-Wallis test) followed by Dunn’s multiple comparison test was performed for statistical analysis. **P < 0.01, ***P < 0.001. Data are representative of three individual experiments.

Fig. 5.

Passive transfer of serum from mice immunized with vaccine plus KIN1148 protects mice against lethal H1N1 challenge. Immune passive transfer sera was generated from mice immunized either once (prime), or twice (prime/boost), 21 days apart, with vaccine plus vehicle, or vaccine plus KIN1148. Groups of 6 C57BL/6N mice received 200 μl of pooled immune sera by intraperitoneal injection on day −2 and day −1 prior to challenge. Mice were then challenged with 10× the LD₅₀ dose of mouse-adapted influenza virus A/California/04/2009 and observed for 14 days after challenge. (A) Passive transfer of prime sera. (B) Passive transfer of prime-boost sera. **P < 0.01, ****P < 0.0001.

Fig. 6.

Prime immunization alone with vaccine plus KIN1148 protects mice against lethal H1N1 challenge. Groups of C57BL/6N mice were immunized twice, 21 days apart, with vaccine plus PBS, vaccine plus vehicle, or vaccine plus KIN1148. (A) Survival (10 mice per group). (B) Viral load in lungs 3 days post challenge (5 mice per group). (C/D) Cytokine response of T cells from (C) lung and (D) lung-draining lymph nodes (5 mice per group). T cells from lung and lung-draining lymph nodes (collected on day 7 after challenge) were stimulated for 18 h as described in the legend to Fig. 3. Cytokines in culture supernatants were then measured using a multiplex ELISA. T cells derived from lungs were evaluated from individual mice. Bars represent the mean and SEM of KIN1148/vaccine and vehicle/vaccine groups. Cells derived from lung-draining lymph nodes were pooled by group. Stimulations were performed in triplicate with bars representing the mean and STD. Student’s two-tailed t-test was performed to compare the respective antigen-specific T cell responses from SV/vehicle control and SV/KIN1148 groups. *P < 0.05 **P < 0.01, ***P < 0.001, ****P < 0.0001 Data represent the results from at least two independent experiments.