Elsevier

Vaccine

Volume 35, Issue 14, 27 March 2017, Pages 1802-1809
Vaccine

Skin vaccination via fractional infrared laser ablation - Optimization of laser-parameters and adjuvantation

https://doi.org/10.1016/j.vaccine.2016.11.105Get rights and content

Abstract

Background

Methods to deliver an antigen into the skin in a painless, defined, and reproducible manner are essential for transcutaneous immunization (TCI). Here, we employed an ablative fractional infrared laser (P.L.E.A.S.E. Professional) to introduce clinically relevant vaccines into the skin. To elicit the highest possible antibody titers with this system, we optimized different laser parameters, such as fluence and pore number per area, and tested various adjuvants.

Methods

BALB/c mice were immunized with Hepatitis B surface antigen (HBsAg) by laser-microporation. Adjuvants used were alum, CRM197, monophosphoryl lipid A, heat-labile enterotoxin subunit B of E. coli (LT-B), and CpG ODN1826. The influence of different fluences (2.1 to 16.8 J/cm2) and pore densities (5–15%) was investigated. Furthermore, immunogenicity of HBsAg and the commercially available conjugate vaccines ActHIB® and Menveo® applied via TCI was compared to standard i.m. injection. Antigen-specific antibody titers were assessed by luminometric ELISA.

Results

Antibody titers against HBsAg were dependent on pore depth and peaked at a fluence of 8.4 J/cm2. Immunogenicity was independent of pore density. Adjuvantation with alum significantly reduced antibody titers after TCI, whereas other adjuvants only induced marginal changes in total IgG titers. LT-B and CpG shifted the polarization of the immune response as indicated by decreased IgG1/IgG2a ratios. HBsAg/LT-B applied via TCI induced similar antibody titers compared to i.m. injection of HBsAg/alum. In contrast to i.m. injection, we observed a dose response from 5 to 20 μg after TCI. Both, ActHIB® and Menveo® induced high antibody titers after TCI, which were comparable to i.m. injection.

Conclusions

Alum, the most commonly used adjuvant, is contraindicated for transcutaneous vaccination via laser-generated micropores. TCI with optimized laser parameters induces high antibody titers, which cannot be significantly increased by the tested adjuvants. Commercially available vaccines formulated without alum have the potential for successful TCI via laser-generated micropores, without the need for reformulation.

Introduction

Though the successful worldwide eradication of smallpox was achieved by administering vaccinia virus to the skin [1], nowadays the intradermal route is only used for delivery of Bacille Calmette-Guérin and in some Asian countries for post-exposure prophylaxis against rabies [2]. The only recent example of approval for intradermal immunization in Europe and the US is a new type of influenza vaccine, which benefits from lower antigen dose compared to the standard subcutaneous or intramuscular routes [3]. From an immunological point of view it is surprising that the skin has been largely ignored as a target for vaccination over the past decades. In contrast to subcutaneous fat tissue and muscle, the skin is rich in cell types with innate and adaptive immune functions, such as dendritic cells [4], keratinocytes [5], mast cells [6], and T lymphocytes [7]. Moreover, with its ease in accessibility and efficient drainage to regional lymph nodes, the skin represents an attractive target for vaccination, provided that some remaining challenges can be addressed. These include development of suitable devices for painless delivery, establishment of optimal doses, and issues of reformulation, i.e. addition of potent and safe adjuvants.

Several non- or minimally invasive technologies for circumvention of the uppermost skin layer, the stratum corneum, have become available. Pulsed jet injectors [8], ballistic powder injection [9], electroporation [10], sonoporation [11], and various types of microneedle arrays, which enable contact of cutaneous antigen presenting cells with the vaccine [12], have been used for cutaneous immunization. Successful vaccine delivery has also been reported following disruption of the skin barrier by tape stripping [13] or mild abrasion [14], and even by application of antigen to intact skin using occlusive patches [15].

Recently, Er:YAG and CO2 infrared laser devices, so far mainly employed for dermatologic treatments or delivery of low molecular weight drugs via the skin, have entered the vaccination field [16], [17], [18]. In contrast to CO2 lasers, the wavelength of the Er:YAG lasers of 2940 nm corresponds to the main absorption peak of water, enabling “cold ablation”, i.e., production of little or no microthermal zones around the site of application. By fractional ablation, an array of skin micropores with intact tissue in between can be produced, leading to fast wound healing. Whereas some devices use a grid to split the laser beam into microbeams [19], the Precise Laser Epidermal System (P.L.E.A.S.E., Pantec Biosolutions) relies on a scanner, which adjusts the laser beam in a pre-defined pattern to generate individual micropores. We and others have used this device for prophylactic and therapeutic approaches against type I allergies [20], [21] and tumors [22].

In the current study, we optimized the laser parameters and compared different adjuvants for induction of high antibody titers via transcutaneous immunization (TCI) using laser-microporation against hepatitis B surface antigen. Based on these findings, we assessed whether commercially available conjugate vaccines (ActHIB® and Menveo®) could by employed for vaccination via micropores without reformulation.

Section snippets

Laser microporation

Laser-microporation was performed using the P.L.E.A.S.E. Professional fractional infrared laser system (Pantec Biosolutions, Ruggell, Liechtenstein). Laserporation was performed at 500 Hz, 50 μs pulse length, and 0.5 or 0.7 W, corresponding to a fluence of 2.1 or 2.8 J/cm2 per pulse, respectively. Area size was 1 cm2. All experiments were performed at a pore density of 5%, unless otherwise indicated.

Antigens & adjuvants

Hepatitis B Surface antigen subtype adw (HBsAg) was purchased from Aldevron (Freiburg, Germany) or

Pore depth influences the magnitude of humoral immune responses

To determine, which pore depth would be optimal for induction of specific antibodies, BALB/c mice were immunized two times with 5 μg HBsAg delivered via laser-microporated skin areas. Micropores were produced by application of 1, 2, 4, 6, or 8 pulses (500 Hz, 50 μs pulse length, 0.5 W), corresponding to 2.1, 4.2, 8.4, 12.6, and 16.8 J/cm2, respectively. Pore density was 5%. As shown in Fig. 1, HBsAg -specific total IgG peaked at a pore depth produced by 4 laser pulses. IgG levels were significantly

Discussion

Laser-microporation is a novel method to bypass the stratum corneum in a highly controlled and adjustable way. We and others have previously shown that laser-assisted microporation can be an efficient tool for transfer of high molecular weight drugs [33] and antigens into the skin [34]. Moreover, controlled tissue damage induced by lasers has been shown to elicit an adjuvant effect [35], making this an interesting technology for pain-free topical application of vaccines. Here we tested

Author contribution

SS, AK, VH, and TT performed experiments and did data acquisition. RW and SS designed the study, performed data analysis and interpretation, and drafted the manuscript. MS took part in study conception and critically discussed the data. JT performed study conception, data interpretation and manuscript drafting. All authors contributed to revising the manuscript and approved the final version.

Conflict of interest

AK, VH, and SS received funding from Pantec Biosolutions. RW has received research support from Pantec Biosolutions and is a member of their scientific advisory board. MS works as a consultant for Pantec Biosolutions.

Funding

This work was funded by Pantec Biosolutions. The funders had no role in collection, analysis, and interpretation of the data.

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