Skin immunization with third-generation hepatitis B surface antigen using microneedles

Abstract

L-HBsAg is a third-generation hepatitis vaccine capable of inducing antibodies in non-responders and thus providing potentially therapeutic treatment. In this study, L-HBsAg was administered using microneedles (MN) without an adjuvant to induce intradermal (ID) immunization, and the efficacy of ID immunization was compared with that of intramuscular (IM) immunization that uses a conventional formulation with an adjuvant of aluminum hydroxide (L-HBsAg-AL-IM).

The L-HBsAg was dip-coated onto 800-μm-long microneedles made of polylactic acid (PLA). Delivery efficiency and administration time were determined through in vitro experiments using porcine skin. The denaturation of the formulation against sterilization by gamma rays was observed. A storage test and a freeze-thaw cycle test of the microneedles with trehalose as a stabilizer (L-HBsAg-MN-Tre) were observed. An antibody titer of L-HBsAg-MN-Tre was compared with that of the conventional IM immunization of the L-HBsAg solution with aluminum hydroxide (L-HBsAg-AL-IM).

The formulation containing L-HBsAg was located on the upper third of the microneedle tips. The formulation on the MN was dissolved and delivered within 30 min of insertion into porcine skin in vitro. Trehalose was selected as a stabilizer, and the stabilizing effect increased with the increase of trehalose content in the solidified formulation. L-HBsAg-MN with 15% of trehalose was stable for 7 days at 40 °C and showed increased stability compared to the conventional liquid formulations. L-HBsAg-MN-Tre showed improved stability during the freeze-thaw cycle. The antibody titer of L-HBsAg-MN-Tre at 28 days was higher than that of L-HBsAg-AL-IM.

ID administration of L-HBsAg-MN-Tre showed better efficacy and improved thermal and freeze thaw stability compared to L-HBsAg-AL-IM. Therefore, L-HBsAg-MN-Tre administration showed the possibility of ID delivery of L-HBsAg without the use of an adjuvant for the efficacy, convenience, and safety of pediatric vaccination.

Introduction

Hepatitis B is well known as a major global health problem caused by the hepatitis B virus (HBV), which is 7th leading cause of death worldwide. The most effective prevention currently available is immunization with the hepatitis B vaccine [1].

The first generation of HBV vaccines were used beginning in 1980, when the hepatitis B surface antigen (HBsAg) produced from the plasma of chronic HBV infected individuals [2]. The second generation of HBsAg, yeast-derived HBsAg with a small hepatitis B surface protein (S protein), was introduced to prevent the transmission of blood pathogens from human blood products of those treated with the first generation of HBsAg [3]. The S protein of second-generation HBsAg is introduced into virus-like particles (VLPs) that are 22 nm in size, mimicking the structure of the real virus. This second generation of HBsAg is widely used for vaccination of newborns and adults in over 170 countries [4].

Second-generation HBsAg has not produced effective immunogenicity due to factors such as advanced age, renal dysfunction, liver disease, smoking, and immunosuppression [5], [6]. Potential reasons for non-responsiveness are genetically induced by resistance [7] or an increase in S gene mutations [8]. In attempts to improve the effectiveness of vaccination, large- and middle-sized surface proteins (pre S1 and pre S2) were merged into S proteins to induce improved antibody response against HBV [9], [10] and increase the antibody response of S gene mutants [11]. As the demand for enhanced immunogenicity to overcome the non-responsiveness to the S protein of HBsAg has increased, the third-generation HBsAg (with combination of three envelope proteins: S, pre-S1 [L protein] and pre-S2 [M protein]), has been developed and offers promising results for greatly improved vaccine efficacy [12], [13].

An additional approach to improving immunogenicity against HBV is changing the administration route from intramuscular (IM) to intradermal (ID). The epidermal and dermal layers of the skin are rich in antigen-presenting cells such as Langerhans cells and dendritic cells [14]. These colonized cells have high MHC class II expression and are important mediators of antiviral immunity [15], [16]. Thus an ID administration requires a lower dose of vaccine compared to an IM administration [17]. Furthermore, the safety and efficacy of ID administration of the HBV vaccine have been demonstrated by comparing ID to IM administration. [18], [19], [20], However, ID administration requires application into a skin layer at least 1 mm thick and it is not easy to administer precisely. Thus microneedles (MN) have been introduced as an easy-to-use, painless way to control injection depth and delivery of the right dose of antigen into the skin layer. Various antigens have demonstrated improved efficacy with ID administration using MN compared to IM administration [21], [22], [23], [24], [25], [26]. The second-generation hepatitis B vaccine was delivered into the skin layer using MN administration, and MN encapsulating HBsAg showed effective antibody production and improved storage stability [27].

In this study, immunization through the skin using the third generation of the hepatitis vaccine with pre-S1/pre-S2/S (L-HBsAg) was performed with MN for first time, as shown in Fig. 1. Alum was not included in the MN formulation. Stability of the vaccine was observed during the harsh storage conditions and the freeze-thaw cycle. Animal experiments demonstrated immunological efficacy of the vaccine administered intradermally with MN with a stabilizer compared with IM administration.