Systemic and mucosal immune response induced by transcutaneous immunization using Hepatitis B surface antigen-loaded modified liposomes

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Abstract

We have evaluated the efficiency of novel modified liposomes (ethosomes) for transcutaneous immunization (TCI) against Hepatitis B. Antigen-loaded ethosomes were prepared and characterized for shape, lamellarity, fluidity, size distribution, and entrapment efficiency. Spectral bio-imaging and flow cytometric studies showed efficient uptake of Hepatitis B surface antigen (HBsAg)-loaded ethosomes by murine dendritic cells (DCs) in vitro, reaching a peak by 180 min. Transcutaneous delivery potential of the antigen-loaded system using human cadaver skin demonstrated a much higher skin permeation of the antigen in comparison to conventional liposomes and soluble antigen preparation. Topically applied HBsAg-loaded ethosomes in experimental mice showed a robust systemic and mucosal humoral immune response compared to intramuscularly administered alum-adsorbed HBsAg suspension, topically applied plain HBsAg solution and hydroethanolic (25%) HBsAg solution. The ability of the antigen-pulsed DCs to stimulate autologous peripheral blood lymphocytes was demonstrated by BrdU assay and a predominantly TH1 type of immune response was observed by multiplex cytometric bead array analysis. HBsAg-loaded ethosomes are able to generate a protective immune response and their ability to traverse and target the immunological milieu of the skin may find a potential application in the development of a transcutaneous vaccine against Hepatitis B virus (HBV).

Introduction

Vaccination is one of the most powerful tools available in the ongoing battle against infectious agents. Virtually all recommended immunizations require parenteral administration, and many require a series of injections, therefore, new vaccine delivery methods, specifically alternatives to injections, are being sought. Topically applied (transcutaneous) vaccines, transgenic edible plants that contain genes for human vaccine antigens, and controlled delivery depot systems with vaccine antigens encapsulated in biodegradable polymers are possibilities currently under study (Tacket et al., 1998, Gupta et al., 1998) Such new delivery methods could decrease reliance on repeated injections, the need for trained healthcare workers, and perhaps the need for a stringent cold chain for vaccine storage (Poland et al., 2002).

Transcutaneous immunization (TCI) offers a new method for the delivery of vaccines, that relies on the application of antigen with adjuvant onto the outer layer of the skin and subsequent delivery to underlying Langerhans cells that serve as antigen-presenting cells (Glenn et al., 1999). It has many merits compared to injectable routes of administration. This needle-free method of vaccine delivery could decrease the risk of needle-borne diseases, reduce the complications related to physical skin penetration, and improve access to vaccination by eliminating the need for trained personnel and sterile equipment. As an initial step towards the development of a carrier system for TCI against Hepatitis B virus (HBV), we recently reported elastic liposome-based HBsAg vaccine (Mishra et al., 2006, Mishra et al., 2007).

Ethosomes are interesting and innovative vesicular carriers that have appeared in the fields of pharmaceutical technology and drug delivery in recent years (Touitou et al., 2000). These ethanol-containing liposomes present an ample opportunity to transport active substances more efficaciously through the stratum corneum into the deeper layers of the skin than conventional liposomes. In fact, ethosomes are soft, malleable vesicles tailored for enhanced delivery of active agents (Dubey et al., 2007a). This aspect is of great importance for the design of carriers to be applied topically both for local and systemic administration. Furthermore, the ethosomal carrier is also able to provide an effective intracellular delivery of both hydrophilic and lipophilic molecules (Dayan and Touitou, 2000).

Hepatitis B is a serious global public health problem with about 2 billion people infected with the virus. 360 million people have chronic infection and 600,000 deaths occur each year from HBV related disease or hepatocellular carcinoma worldwide (Shepard et al., 2006). Vaccination is the most effective measure to prevent HBV infection. Although available anti-Hepatitis B vaccines produce good and sustained immune response, the effectiveness falls with age and they are less effective in special groups like patients with chronic renal failure, liver transplant recipients and HIV-affected individuals (Akbar et al., 2006). Also, the elimination of the disease would require different vaccination efforts with improved delivery modules in the population living with chronic HBV infection (Inchauspé and Michel, 2007).

The aim of present work was to incorporate HBsAg in ethosomes further exploring its immunoadjuvant property for TCI, to induce both systemic and mucosal immune responses. Ethosomes were prepared and characterized for vesicular size, shape, lamellarity, entrapment efficiency and fluidity. In vitro cellular uptake and internalization studies of ethosomal carriers by dendritic cells (DCs) and fibroblasts were performed using flow cytometric analysis and spectral bio-imaging, respectively.

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Materials

Purified HBsAg (source-genetically modified yeast cells) was obtained as gift sample from the National Institute of Immunology, New Delhi, India. Bovine albumin (fraction V), soya phosphatidylcholine (SPC), sephadex G-150, fluorescein isothiocyanate (FITC) and Rhodamine were obtained from Sigma–Aldrich, St. Louis, MO, USA. All reagents used in ELISA were purchased from Genei, Bangalore, India and Imx HBsAg (V2) kit was obtained from Abbott, Wiesbaden, Germany. RPMI-1640, alamar blue, foetal

Preparation and characterization

The ethosomes prepared with 2% SPC and 25% ethanol using classical mechanical dispersion method appeared to be unilamellar and spherical in shape in a TEM study (Fig. 1). Surface morphology and three-dimensional nature of ethosomes were further confirmed by SEM, suggesting the vesicular characteristics possessed by these novel carriers (Fig. 2). The developed formulation indicated a narrow size distribution having nanometric range (153 ± 8.5 nm) with a very low polydispersity. This nanometric size

Discussion

This is the first report of testing a novel preparation for eliciting an immune response against Hepatitis B which has used HBsAg-loaded ethosomes as antigens and DCs as antigen-presenting cells. The immune response was studied in a murine system. The antigen-loaded ethanolic liposomes were efficiently internalized by the DCs as evidenced by flow cytometry and spectral bio-imaging analysis. The antigen-pulsed DCs were able to initiate an immune response predominantly of TH1 type. However, a

Acknowledgements

The authors are indebted to Dr. A.K. Panda, National Institute of Immunology, New Delhi, India for providing pure HBsAg and the All India Institute of Medical Sciences, New Delhi, India for providing facility for Electron Microscopy. Mr. Dinesh Mishra is a recipient of Senior Research Fellowship of the University Grants Commission (UGC), New Delhi, India.

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