The role of toll-like receptor 7 agonist imiquimod in enhancing influenza vaccine immunogenicity

Abstract

Vaccination is the most effective approach for the prevention and control of influenza virus infection. Conventional influenza vaccines induce suboptimal immune responses in elderlies and immunocompromized pateints. Therefore it is important to investigate new strategies to improve vaccine efficacy. Recently, studies on novel adjuvants targeting on TLRs showed promisingresults.TLR7 agonist, imiquimod, was shown to enhance vaccine efficacy against many pathogens. However, its potential as adjuvant for influenza vaccine remains unknown.

In this study, we firstly demonstrated that imiquimod expedited and augmented the immune responses when administrated in combination with 2009 pandemic H1N1 influenza vaccine. This immunization approach conferred very early protection against lethal virus challenge only 3 days after vaccination in mice. Improved survival rates, reduced lung viral load and attenuated lung inflammatory responses were observed in immunized mice. The protective effect was attributed mainly to the rapid and strong antibody production. Mice immunized with vaccine and imiquimod combination (VCI) 3 days before virus challenge produced significantly higher level of virus-specific IgA, IgM, IgG as early as at day 2-3 p.i. (post infection). High titer of viral neutralizing antibody was detected in the sera as early as day 4 p.i..

To elucidate how VCI conferred immediate protection in mice, we first examined the direct immuno-modulatory effect of imiquimod. We found that imiquimod induced remarkable cytokine gene up-regulation, but could not directly inhibit influenza virus replication in vitro. However, intranasal administration of imiquimod to infected mice provided partial protection against mouse adapted A(H1N1)pdm09mutantvirus challenge, but without reduction in lung viral titer. Study of the infected mouse lung showed attenuated pro-inflammatory cytokine expression and neutrophil infiltration, but enriched T and B cells recruitment in mice treated by intranasal imiquimod. This finding suggested that imiquimod may confer beneficial and balanced immunological changes when immunized mice are challenged by influenza virus. We further investigated how VCI induced B and T cell responses, which may lead to accelerated and augmented antibody production in VCI immunized mice. Firstly, compared to vaccine only, VCI stimulation of ex vivo cultured mouse peritoneal cells induced higher expression of B cell activation and migration markers. After intraperitoneal administration of VCI, peritoneal B cell number reduced dramatically while B cell number and activation markers significantly increased in the lymph nodes. Following virus challenge 3 days after VCI immunization, increased number of B cells, CD4+ and CD8+ T cells, but decreased number of neutrophil were observed in the lung tissues and bronchoalevolar lavage fluid (BALF). In the spleen and mediastinal lymph node (MLN)of VCI immunized mice, the number of B cells expressing activation and differentiation marker increased at day 3 p.i.. These B cells were functionally active and participating in antibody responses, as indicated by the large number of plasma cells and antibody-secreting cells in spleen and MLN.

Our findings on the role of imiquimod in influenza virus vaccination and infection may provide information in new strategies for influenza vaccination by targeting TLR7 for the induction of rapid and augmented immune responses for prophylactic and therapeutic purpose.