Elsevier

Vaccine

Volume 27, Issue 42, 25 September 2009, Pages 5791-5799
Vaccine

Topical resiquimod promotes priming of CTL to parenteral antigens

https://doi.org/10.1016/j.vaccine.2009.07.062Get rights and content

Abstract

We explored the topical use of resiquimod (R-848), a Toll-like receptor (TLR) 7/8 agonist, in gel formulation, to enhance cross-priming to subcutaneously administered protein antigen in a murine model. Resiquimod application at the time of subcutaneous administration of ovalbumin generated robust antigen-specific CTL as detected by tetramers, IFN-γ ELISPOT assays and standard cytotoxicity assays. Induced CTL were capable of mediating antigen-specific killing in vivo as measured by in vivo cytotoxicity assays and an ability to protect against B16-OVA tumor challenge. Multiple serial applications of topical resiquimod increased the frequency of antigen-specific CTL when compared to single application. This enhanced frequency was noted despite a marked inhibition of adjuvant mediated pro-inflammatory cytokine release following repeated administration. Topical resiquimod is a potent adjuvant for locally administered subcutaneous vaccines, inducing clinically relevant CTL responses following single application at the time of subcutaneous vaccination.

Introduction

Cytotoxic T Lymphocytes (CTL) assist the immune system in the control of tumors and viruses. Current vaccination strategies using inactivated vaccines are unfortunately limited by inefficient CTL priming [1]. The abundance and diversity of antigen presenting cells, called dendritic cells (DC), in the skin suggest the potential to elicit potent CTL responses to antigens, making the skin an ideal target for vaccine strategies to overcome this limitation. Toll-like receptor (TLR)-agonists activate DC resulting in cytokine production and co-stimulatory molecule up-regulation and are thus useful agents to improve vaccine efficacy [2]. Resiquimod is a synthetic TLR7 and 8 agonist that has been shown to have potential as an adjuvant when given parenterally (reviewed in [3]). Resiquimod produces anti-viral effects through the induction of pro-inflammatory cytokines [4] mediated at least in part through the activation of plasmacytoid DC [5]. Imiquimod (Aldara™), a related imidazoquinoline and TLR7 agonist, has been studied as a topical vaccine adjuvant to induce CTL responses to co-injected protein [6], topically co-applied peptides [7], [8], and subcutaneously injected dendritic cell [9] and DNA vaccines [10]. Topical resiquimod has been shown to induce pro-inflammatory cytokine release [11], [12] and to improve antibody responses to subcutaneously administered antigen [3] and may thus also be an effective topical adjuvant for promoting CTL responses to vaccination.

CTL priming to foreign antigens is mediated through cross-presentation in which antigen presenting cells acquire and process exogenous antigen and target it to a pathway that culminates in the loading of antigen-derived peptides onto MHC class I molecules. TLR 3, 7 -9 agonists effectively promote cross-presentation of soluble protein antigen through the activation of DC [13]. In this study we investigate the ability of resiquimod as a topical vaccine adjuvant to promote CTL priming. Using a murine model, we show that topical resiquimod induces the generation of functional cell-mediated immune responses to locally co-administered parenteral protein antigen. We demonstrate that repeated topical administration of resiquimod following parenteral vaccination has discrepant effects on local cytokine production (with induction of local inflammatory cytokine induction upon single administration but inhibition of local inflammatory cytokine production upon repeated administration albeit with the persistent induction of type 1 interferon-related myxovirus resistance protein A – MxA expression) and enhances the frequency of antigen-specific CTL at an early time-point in the immune response.

Section snippets

Mice and cell lines

C57Bl/6 mice were obtained from Charles River Laboratories (Wilmington, MA) and maintained in a specific pathogen-free environment. Animal experiments were approved by the Animal Care Committee of the University of British Columbia. The OVA-expressing melanoma cell line B16-OVA (B16 transfected to express OVA) [14] was obtained from Dr. Hung-Sia Teh (University of British Columbia, BC, Canada). B16-OVA cells were cultured in complete medium (CM) consisting of RPMI 1640 (Invitrogen, Burlington,

Topical resiquimod application increases the antigen-specific CTL response to subcutaneous protein antigen with or without tape stripping

Topical TLR-9 agonists such as CpG-oligodeoxynucleotide (CpG-ODN) induce effective cross-priming of soluble protein antigen [16]. Benefits of topical adjuvant application over parenteral adjuvant administration include diminished toxicity and improved cross-priming due to the activation of local DC [16]. Topical application of the TLR7 agonist imiquimod promotes immune responses to locally injected protein antigen [6]. Resiquimod, a TLR7/8 agonist, has been reported to be anywhere from 10 to

Discussion

The need for better adjuvants, which effectively elicit strong CTL responses, is clearly important for vaccine development. Topical adjuvants have shown potential for generating CTL in a safe and effective manner [6], [16]. Here, we show that resiquimod applied to the skin has strong potential as an epicutaneous adjuvant through its ability to elicit potent CTL responses to parenteral antigen. Robust antigen-specific CTL populations were detected through a variety of CTL assays. Levels of CTL

Acknowledgements

This work was funded by Canadian Institutes of Health Research Grant MOP-69004 and the Canadian Dermatology Foundation. We thank Drs. Mark Tomai and TC Meng from 3M for the supply of resiquimod, careful reading of the manuscript and helpful discussions. We also thank Dr. Mehran Ghoreishi for assistance with immunohistochemistry.

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