Elsevier

Vaccine

Volume 22, Issues 13–14, 16 April 2004, Pages 1799-1809
Vaccine

Imiquimod and resiquimod in a mouse model: adjuvants for DNA vaccination by particle-mediated immunotherapeutic delivery

https://doi.org/10.1016/j.vaccine.2003.09.052Get rights and content

Abstract

Imiquimod, an immune response modifier and inducer of cytokines in vitro and in vivo, has been shown to have potent antiviral and antitumour activity and to act as an adjuvant for protein vaccination. We have undertaken studies in mice to investigate the potential of imiquimod and resiquimod to adjuvant DNA vaccination. These imidazoquinolines were administered by subcutaneous injection at the vaccination site immediately after particle-mediated immunotherapeutic delivery of plasmid DNA using a gene gun. Imiquimod was found to increase the number and maturation status of dendritic cells in draining lymph nodes, and to enhance antigen-specific CD4+ and CD8+ T cell responses, as assessed by analyses of clonal expansion, and the quantity and kinetics of cytokine production from these cells in lymph nodes and spleens collected after vaccination. A more substantial increase in IFN-γ-producing, compared with IL-4-producing CD4+ T cells suggested that imiquimod biased the immune response towards a predominance of Th1 cells. The analogue resiquimod was found to be to produce a similar Th1 biased immune response with a 10-fold reduced dose compared with imiquimod. Collectively, these studies suggest that both imiquimod and resiquimod may be suitable adjuvants for therapeutic DNA vaccines requiring induction of potent cytotoxic T cell responses.

Introduction

Immunisation with DNA is a promising new vaccination strategy [1], [2], [3]. Unlike traditional vaccine approaches in which proteins or heat killed organisms provide an extracellular source of immunogens for uptake by antigen presenting cells and subsequent processing and presentation in MHC class II molecules, DNA vaccination provides the immunogens intracellularly after transfection of cells in vivo in a similar way to virus infection. This endogenous production of immunogens, providing an efficient mechanism for processing and presentation of antigens in MHC class I molecules, offers the potential of a broader immune response and is thus a distinguishing feature of DNA vaccination.

It is becoming apparent that adjuvants will be required to fully maximise the efficacy of DNA vaccines, probably because plasmid DNA encoding only antigenic components, or parts thereof, lack the danger signals required to mount a full and optimal immune response [3]. Many adjuvant strategies are currently under investigation including methods for invoking inflammation and cytokine release at the vaccine site. For DNA vaccination this has involved administering combinations of cytokine-encoding DNA, together with the antigen-encoding DNA plasmid [4]. Alternatively, cytokine proteins themselves or substances which induce immune response modification or the recruitment of inflammatory cells to the vaccination site have been employed [4].

The small molecular weight imidazoquinolines, imiquimod and resiquimod, are compounds which have been shown to have immune response modifying properties both in vitro and in vivo, and to provide antiviral and antitumour activity via the stimulation of endogenous cytokine production [5], [6]. Results from in vitro studies using non-human or human monocytes treated with imiquimod or resiquimod have shown increased mRNA levels in cell lysates and/or cytokine levels in supernatants including interferon-α, interleukin-1β, interleukin-6 and tumour necrosis factor-α [7], [8]. In vivo studies have confirmed the induction of interferon-α and tumour necrosis factor-α, as well as other cytokines such as IL-12p40, in humans and animals treated with topical imiquimod cream or resiquimod gel [9], [10].

Such cytokine-inducing properties have provided the basis for investigations of the adjuvant activities of these imidazoquinolines. Imiquimod has been reported to decrease the severity of the acute disease, viral replication and disease recurrence, when given as an adjuvant with HSV-2 glycoprotein immunisation before intravaginal HSV-2 inoculation [11]. Imiquimod has also been shown to enhance efficacy of protein vaccination for the rejection of tumours. In E7 immunised mice, imiquimod had no effect on EL4 tumour growth but reduced the weight of E7 expressing tumours [12].

Investigations of the physiologic target of these small molecular weight compounds has recently yielded evidence that they act through stimulation of members of the toll-like receptor (TLR) family [13]. Such promising initial reports on the biology and mechanism of action of these imidazoquinolines suggest that the scope of these compounds to act as adjuvants across a broader range of vaccination strategies warrants investigation.

Accordingly, we have investigated the effects of imiquimod and resiquimod on immune responses to immunisation using DNA rather than protein as the vaccine agent. Here, DNA has been administered by particle-mediated immunotherapeutic delivery (PMID) using a device colloquially known as a gene gun. We report that both CD4+ and CD8+ T cell responses are augmented, with a bias towards a Th1 immune profile evident, when these imidazoquinolines are used in combination with PMID.

Section snippets

Mice

C57Bl/6 and Balb/c mice were purchases from Charles River United Kingdom Ltd. (Margate, UK) and were used between 6 and 10 weeks of age. D0.11.10 mice (Balb/c background) carrying ovalbumin-specific transgenic T cells, originally obtained from Dr. Ken Murphy (Washington University School of Medicine, Howard Hughes Medical Institute, St. Louis, MO), were maintained at the specific pathogen-free breeding facilities at Bury Green Farm, GlaxoSmithKline, UK, and were used at 6–12 weeks of age. The

Imiquimod increases cell traffic and upregulates co-stimulatory molecules and activation markers on CD11c+ cells in draining lymph nodes

Application of FITC at the skin surface was used to label cells in the skin and thereby enable analyses of migration of dendritic cells following subsequent treatments at the skin site. The dendritic cell-specific marker, CD11c, was used to identify dendritic cells in lymph nodes. Fig. 1A shows that the percentage of FITC-labelled cells in inguinal lymph nodes draining the site of FITC and drug administration was increased two-fold following administration of imiquimod compared with control

Discussion

In this study, we have demonstrated that the two imidazoquinoline compounds, imiquimod and resiquimod, are adjuvants for DNA vaccination. Utilising murine models to analyse immune responses, we have shown that both these compounds induce significant increases in cytokine production from antigen-specific T cells when administered in combination with DNA vaccination by PMID, compared with vaccination without these adjuvants. Although the adjuvant effects of imiquimod and resiquimod have

Acknowledgements

The authors are grateful to Barrie Kirk, High Throughput Chemistry Department, GlaxoSmithKline, Stevenage, UK, for synthesis of imiquimod and resiquimod, and to Andrew Lloyd, Biostatistics and Data Sciences Department, GlaxoSmithKline, Harlow, UK, for advise and assistance with the statistical analyses.

References (29)

  • J.P. Vasilakos et al.

    Adjuvant activities of immune response modifier R-848: comparison with CpG ODN

    Cell. Immunol.

    (2000)
  • T.L. Wagner et al.

    Induction of cytokines in cynomolgus monkeys by the immune response modifiers, imiquimod, S-27609 and S-28463

    Cytokine

    (1997)
  • K.V. Anderson

    Toll signaling pathways in the innate immune response

    Curr. Opin. Immunol.

    (2000)
  • A. Reyes-Sandoval et al.

    DNA vaccines

    Curr. Mol. Med.

    (2001)
  • Cited by (93)

    • Polyphosphazene immunoadjuvants: Historical perspective and recent advances

      2021, Journal of Controlled Release
      Citation Excerpt :

      In vivo studies demonstrated that the PCPP-R adjuvanted HCV formulation induced higher serum neutralization titers in BALB/c mice and shifted the response towards desirable cellular immunity, as evaluated by antibody isotype ratio (IgG2a/IgG1) and ex vivo analysis of cytokine secreting splenocytes (higher levels of interferon gamma (IFN-γ) single and tumor necrosis factor alpha (TNF-α)/IFN-γ double producing cells). PCPP-enabled non-covalent multimerization of R848 stands in contrast to previously suggested delivery methods for this compound, which involve covalent conjugation or encapsulation [69–81]. The ion-pairing concept can be extended to different members of the polyphosphazene homologous series, such as PCMP and PCEP, as well as other representatives of the imidazoquinoline family, such as gardiquimod and newer derivatives [82], or TLR 7/8 agonists of the oxoadenine series [83] among others.

    View all citing articles on Scopus
    View full text