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