Novel imiquimod nanovesicles for topical vaccination

Highlights

• High accumulation of imiquimod in human skin.

• Induction of pro-inflammatory cytokines TNF-α and IL-6.

• Topical induction of cell-biased systemic response against seasonal influenza vaccine.

Abstract

Development of needle and pain free noninvasive immunization procedures is a top priority for public health agencies. In this work the topical adjuvant activity of the immunomodulator imiquimod (IMQ) carried by ultradeformable archaeosomes (UDA₂) (nanovesicles containing sn-2,3 ether linked phytanyl saturated archaeolipids) was surveyed and compared with that of ultradeformable liposomes lacking archaeolipids (UDL₂) and free IMQ, using the model antigen ovalbumin and a seasonal influenza vaccine in Balb/c mice. UDA₂ (250 ± 94 nm, -26 ± 4 mV Z potential) induced higher IMQ accumulation in human skin and higher production of TNF-α and IL-6 by macrophages and keratinocytes than free IMQ and UDL₂. Mixed with ovalbumin, UDA₂ was more efficient at generating cellular response, as measured by an increase in serum IgG2a and INF-γ production by splenocytes, compared with free IMQ and UDL₂. Moreover, mixed with a seasonal influenza vaccine UDA₂ produced same IgG titers and IgG2a/IgG1 isotypes ratio (≈1) than the subcutaneously administered influenza vaccine. Topical UDA₂ however, induced highest stimulation index and INF-γ levels by splenocytes. UDA₂ might be a promising adjuvant for topical immunization, since it produced cell-biased systemic response with ≈ 13-fold lower IMQ dose than the delivered as the commercial IMQ cream, Aldara.

Introduction

The development of needle and pain free noninvasive immunization procedures is a top priority for public health agencies [1]. Topical vaccination however, is challenged by the stratum corneum (SC) barrier interposed between antigens-adjuvants and the skin-associated lymphoid tissue (SALT), lying few hundred micrometers depth from skin surface [2,3]. Topical vaccination with soft matter is a new attractive way to induce immune protection without disrupting the SC. In this scenario, we previously showed that topically applied ultradeformable archaeosomes (UDA: made of total polar archaeolipids (TPA) extracted from the archaea Halorubrum tebenquichense plus soybean phosphatidylcholine and sodium cholate (3:3:1 w/w), can penetrate the SC to induce antigen specific IgG serum responses [[4], [5], [6]]. TPA, key components of UDA, are a mixture of sn-2,3 glycerol ether linked fully saturated polyisoprenoid chains phospholipids. TPA extracted from H. tebenquichense are ligands for scavenger receptor class A expressed on phagocytic cells and immature antigen presenting cells (APC) [7]. Vesicles fully or partly made of TPA such as archaeosomes or UDA, are much more pronouncedly captured by APC than TPA-lacking vesicles. On the other hand, TPA confer UDA stability to heat sterilization and storage under cold-free conditions [8]. The immune response elicited by topical UDA against ovalbumin and leishmania antigens, however, is lower than the one obtained after parenteral administration using the universal adjuvant alum or vesicles fully made of TPA [4,5]. Moreover, UDA elicit an IgG2a/IgG1 ratio ≈ 0.5, suggesting an humoral (Th2) biased response [4,8].

Imiquimod (IMQ), on the other hand, is a synthetic ligand of the endosomal Toll Like Receptor 7 (TLR7), that induces the production of cytokines such as IFN-α, TNF-α, IL-1a, IL-6, IL-8 and IL-12 in immune cells like monocytes/macrophages, dendritic cells (DC) (dermal DC and plasmacytoid DC), B cells and granulocytes [9,10]. IMQ induces cellular memory via the activation of effector T cells, Langerhans cells (LC), lymphocytes, and macrophages, which circulate lymphatically and activate the adaptive immune response [11]. Topically administered IMQ induces functional maturation of epidermal LC cells and bone marrow epidermal DC and stimulates migration of these APC to regional lymph nodes where they promote a specific T cell response [9]. A 5% IMQ cream called Aldara is approved to treat dermatologic skin pathologies including external genital and perianal warts, small superficial basal cell carcinoma and actinic keratoses on the face and scalp [12]. Aldara is an oil-in-water emulsion consisting of isostearic acid as the oil phase in which IMQ is solubilized at 5%. In clinical trials, topical application of Aldara to patients with melanoma and prostate cancer vaccinated with peptides has shown to increase cytotoxic responses [13,14]. However, responses induced by Aldara rapidly fade away, resulting in poor memory formation and only partial tumor protection [15,16]. Moreover, isostearic acid cause frequent cutaneous adverse reactions in 75% of the patients and has been recently found that it contributes to inflammatory responses via inflammasome activation [17]. Besides, this emulsion is not stable, Aldara cream is marketed in single-use packaging and unused packets are recommended to be discarded. On the other hand, IMQ shows very poor skin permeability [18]. For topical vaccination, IMQ needs to cross the SC, enter the skin and be taken up by skin-resident LC and DC for activation and migration to draining lymph nodes. At the same time, to avoid any dangerous systemic adverse effect, IMQ should remain at the administration site, not entering the bloodstream. All these data suggest that a new IMQ formulation for topical vaccination is needed.

IMQ has been formulated in different types of particulate carriers such as nanogels [19], solid nanoemulsions [20] and emulsions [21] for topical immunization. All these approaches, however are based on the release of IMQ from the skin surface and not on the target of IMQ loaded nanoparticles to APC. Recently we have showed that aqueous suspension of vesicles fully made by TPA containing IMQ are capable of inducing cell-biased antigen-specific systemic response upon subcutaneous application [22].

The hypothesis of this work is that the immune response elicited by UDA or IMQ alone, may be improved by the inclusion of IMQ in UDA to gain skin penetration and APC and endosomal targeting. IMQ was loaded into and externally associated with nanovesicles (UDA₂ and UDL₂ - ultradeformable nanovesicles lacking TPA-). The in vitro penetration on human skin, cytotoxicity, and the release of proinflammatory cytokines by keratinocytes and macrophages upon incubation with nanovesicles were determined. Then, the ability of topical nanovesicles to stimulate immune responses toward the model antigen ovalbumin and a seasonal influenza vaccine were screened. Finally, storage stability was determined.