Human vaccines are typically injected into muscle despite its inconsequential function in the induction of subsequent immune responses. In contrast, skin, a potent immunological induction site, is rarely used for vaccination because of its poor accessibility by needle and its poor permeability to topically applied vaccines. When special delivery systems are used, administering vaccines to the skin is very efficacious. Vaccination with a live attenuated vaccine by skin scarification led to global eradication of the deadly smallpox disease 1,2 . Particle-mediated DNA immunization to the skin requires 0.4-4% the DNA required for intramuscular injection 3,4 .Human skin consists of an epidermis of columnar epithelium and a dermis of fibrous connective tissue. The keratinized epidermal cells with their tight conjunction form the stratum corneum that prevents molecules of 500 Da or greater to penetrate 5,6 . The underlying viable epidermis, with its dense network of antigen-presenting cells (Langerhans cells) and relative lack of sensory nerve endings, has long been recognized as a safe and effective target tissue for vaccination 7 . However, the epidermis is too thin for needle injection. Topical application of protein antigens can result in an immune response, although high antigen dose and toxic adjuvant may be required 8-10 . Here we describe a new technology, epidermal powder immunization (EI), that effectively delivers powdered vaccines to the viable epidermis by using a heliumpowered, needle-free PowderJect system. EI induced serum antibody response to the influenza vaccines and provided protection against homologous and heterologous challenge in a mouse challenge model. Powder delivery systemThe helium-powered PowderJect device for delivering powdered vaccines has been described 11,12 . The device used here was a reusable research model 15 cm in length with an 'actuation' button, a helium gas chamber, a vaccine cassette, a nozzle and a 'silencer' (Fig. 1). The stainless steel gas chamber was filled with approximately 5 ml medical-grade helium gas to a pressure of 50 bar. When the device was activated, the released helium gas ruptured the membranes of the trilaminate cassette and accelerated the entrapped vaccine powders to a high speed so that the particles perforated the stratum corneum and landed in the epidermis. The helium gas was reflected off the skin and was 'exhausted' through the vented silencer. Comparison of EI and needle injectionAlthough the intramuscular route is commonly used for administering human vaccines, subcutaneous, intraperitoneal and intramuscular routes are often used interchangeably for immunizing experimental animals such as mice. To compare EI with needle injection through these conventional routes, we used a formalin-inactivated Aichi/68 influenza virus to immunize BALB/c mice (n = 8 per group). For EI, the vaccine was formulated with trehalose into a powder with microscopic particles 20-53 µm in diameter. We administered 1 mg powder containing 5 µg vaccine (total viral protein) to t...

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Cytotoxic T lymphocytes (CTL) play a vital role in host defense against viral and intracellular bacterial infections. However, nonreplicating vaccines administered by intramuscular injection using a syringe and needle elicit predominantly humoral responses and not CTL responses. Here we report that epidermal powder immunization (EPI), a technology that delivers antigens on 1.5-to 2.5-m gold particles to the epidermis using a needle-free powder delivery system, elicits CTL responses to nonreplicating antigens. Following EPI, a majority of the antigen-coated gold particles were found in the viable epidermis in the histological sections of the target skin. Further studies using transmission electron microscopy revealed the intracellular localization of the gold particles. Many Langerhans cells (LCs) at the vaccination site contained antigen-coated particles, as revealed by two-color immunofluorescence microscopy, and these cells were found in the draining lymph nodes 20 h later. Immune responses to several viral protein antigens after EPI were studied in mice.

Both circulating and mucosal antibodies are considered important for protection against infection by influenza virus in humans and animals. However, current inactivated vaccines administered by intramuscular injection using a syringe and needle elicit primarily circulating antibodies. In this study, we report that epidermal powder immunization (EPI) via a unique powder delivery system elicits both serum and mucosal antibodies to an inactivated influenza virus vaccine. Serum antibody responses to influenza vaccine following EPI were enhanced by codelivery of cholera toxin (CT), a synthetic oligodeoxynucleotide containing immunostimulatory CpG motifs (CpG DNA), or the combination of these two adjuvants. In addition, secretory immunoglobulin A (sIgA) antibodies were detected in the saliva and mucosal lavages of the small intestine, trachea, and vaginal tract, although the titers were much lower than the IgG titers. The local origin of the sIgA antibodies was further shown by measuring antibodies released from cultured tracheal and small intestinal fragments and by detecting antigen-specific IgA-secreting cells in the lamina propria using ELISPOT assays. EPI with a single dose of influenza vaccine containing CT or CT and CpG DNA conferred complete protection against lethal challenges with an influenza virus isolated 30 years ago, whereas a prime and boost immunizations were required for protection in the absence of an adjuvant. The ability to elicit augmented circulating antibody and mucosal antibody responses makes EPI a promising alternative to needle injection for administering vaccines against influenza and other diseases.