Intranasal delivery of Norwalk virus-like particles formulated in an in situ gelling, dry powder vaccine.

The development of a vaccine to prevent norovirus infections has been focused on immunization at a mucosal surface, but has been limited by the low immunogenicity of self-assembling Norwalk virus-like particles (NV VLPs) delivered enterically or at nasal surfaces. Nasal immunization, which offers the advantage of ease of immunization, faces obstacles imposed by the normal process of mucociliary clearance, which limits residence time of applied antigens. Herein, we describe the use of a dry powder formulation (GelVac) of an inert in situ gelling polysaccharide (GelSite) extracted from Aloe vera for nasal delivery of NV VLP antigen. Powder formulations, with or without NV VLP antigen, were similar in structure in dry form or when rehydrated in simulated nasal fluids. Immunogenicity of the dry powder VLP formulation was compared to equivalent antigen/adjuvant liquid formulations in animals. For the GelVac powder, we observed superior NV-specific serum and mucosal (aerodigestive and reproductive tracts) antibody responses relative to liquid formulations. Incorporation of the TLR7 agonist gardiquimod in dry powder formulations did not enhance antibody responses, although its inclusion in liquid formulations did enhance VLP immunogenicity irrespective of the presence or absence of GelSite. We interpret these data as showing that GelSite-based dry powder formulations (1) stabilize the immunogenic structural properties of VLPs and (2) induce systemic and mucosal antibody titers which are equal or greater than those achieved by VLPs plus adjuvant in a liquid formulation. We conclude that in situ gelation of the GelVac dry powder formulation at nasal mucosal surfaces delays mucociliary clearance and thereby prolongs VLP antigen exposure to immune effector sites.

Delivery of DNA into skin via electroporation.

Delivery of DNA into skin is an attractive method, because skin is the most accessible somatic tissue for gene transfer and can be monitored conveniently. Skin is especially suitable for immunization using plasmid-DNA-based vaccines; however, a low level of transfection is the major limitation to the use of DNA-based therapeutics. Several chemical and physical methods are being investigated to improve the transfection of target cells with plasmid DNA. Electroporation is a physical method of gene transfer by applying electric pulses to the target cells. Most of the electroporation studies involve insertion of electrode needles into the tissues. In this chapter, we discuss that the DNA delivery into skin can be greatly enhanced by topical electroporation of the DNA injection site in rabbits using a tweezer electrode. Furthermore, the immune responses following a DNA vaccine delivery by using electroporation have been explored. Electroporation shows great potential for enhancing the DNA delivery into the skin.

Prospects for vaccines for allergic and other immunologic skin disorders.

The human skin hosts a variety of immune response-associated components that together form the skin immune system. Any abnormality in the functioning of the skin immune system leads to a variety of dermatologic complications, including dermatitis, psoriasis, and eczema. Exposure to antigens/allergens can lead to allergic skin disorders such as atopic dermatitis, urticaria, and allergic contact dermatitis. Recent investigations have provided new insights into the immunologic processes leading to the development of skin diseases. T cells play a central role in the activation and regulation of immune responses by recognizing antigen and inducing cytokine production. Despite advances in the understanding of the immunologic events leading to the development of skin diseases, no effective prevention measure exists. Current therapeutic treatments are based on either alleviating the symptoms or suppressing the immune system with immunosuppressive drugs. Allergen-specific immunotherapy is expected to induce specific T cells that abolish allergen-induced proliferation of T helper cells, as well as their cytokine production. Recent approaches using recombinant protein, polycytosine guanine oligonucleotides, and plasmid DNA for vaccination suggest the possibility of protection against these skin disorders. The involvement of T cells in psoriasis indicates that the development of a T-cell receptor peptide vaccine may be beneficial. Dendritic cell-based vaccines using tolerogenic dendritic cells that can induce T-cell tolerance have been shown to be useful in dealing with autoimmune disorders and allergic conditions. In the light of these developments, this article presents the current status and prospects of developing vaccines for allergic and other immunologic skin disorders.

Skin targeted DNA vaccine delivery using electroporation in rabbits II. Safety.

The Achilles heel of gene-based therapy is gene delivery into the target cells efficiently with minimal toxic effects. Viral vectors for gene/DNA vaccine delivery are limited by the safety and immunological problems. Recently, nonviral gene delivery mediated by electroporation has been shown to be efficient in different tissues including skin. There are no detailed reports about the effects of electroporation on skin tissue, when used for gene/DNA vaccine delivery. In a previous study we demonstrated the efficacy of skin targeted DNA vaccine delivery using electroporation in rabbits [Medi, B.M., Hoselton, S., Marepalli, B.R., Singh, J., 2005. Skin targeted DNA vaccine delivery using electroporation in rabbits. I. Efficacy. Int. J. Pharm. 294, 53-63]. In the present study, we investigated the safety aspects of the electroporation technique in vivo in rabbits. Different electroporation parameters (100-300 V) were tested for their effects on skin viability, macroscopic barrier property, irritation and microscopic changes in the skin. Skin viability was not affected by the electroporation protocols tested. The electroporation pulses induced skin barrier perturbation and irritation as indicated by elevated transepidermal water loss (TEWL) and erythema/edema, respectively. Microscopic studies revealed inflammatory responses in the epidermis following electroporation using 200 and 300 V pulses. However, these changes due to electroporation were reversible within a week. The results suggest that the electroporation does not induce any irreversible changes in the skin and can be a useful technique for skin targeted DNA vaccine delivery.

Skin targeted DNA vaccine delivery using electroporation in rabbits. I: efficacy.

Genetic immunization through skin is highly desirable as skin has plenty of antigen presenting cells (APCs) and is easily accessible. The purpose of this study was to investigate the effects of electroporation pulse amplitude, pulse length and number of pulses on cutaneous plasmid DNA vaccine delivery and immune responses, following intradermal injection in vivo in rabbits. Expression of the delivered plasmid was studied using a reporter plasmid, coding for beta-galactosidase. The efficiency of DNA vaccine delivery was investigated using a DNA vaccine against Hepatitis B, coding for Hepatitis B surface antigen (HBsAg). Serum samples and peripheral blood mononuclear cells (PBMC) were analyzed for humoral and cellular immunity, respectively, following immunization. The expression of transgene in the skin was transient and reached its peak in 2 days post-delivery with 200 and 300 V pulses. The expression levels with 200 and 300 V pulses were 48- and 129-fold higher, respectively, compared with the passive on day 2. In situ histochemical staining of skin with X-gal demonstrated the localized expression of beta-galactosidase with electroporation pulses of 200 and 300 V. Electroporation mediated cutaneous DNA vaccine delivery significantly enhanced both humoral and cellular immune responses (p<0.05) to Hepatitis B compared to passive delivery. The present study demonstrates the enhanced DNA vaccine delivery to skin and immune responses by topical electroporation. Hence, electroporation mediated cutaneous DNA vaccine delivery could be developed as a potential alternative for DNA vaccine delivery.

Electronically facilitated transdermal delivery of human parathyroid hormone (1-34).

Electronically facilitated transdermal delivery of human parathyroid hormone (1-34), hPTH (1-34), was investigated in vitro, using dermatomed porcine skin. The effect of iontophoretic current density, electroporative pulse voltages and also electroporation followed by iontophoresis was investigated on the in vitro percutaneous absorption of hPTH (1-34). Iontophoresis at 0.5 mA/cm2 current density significantly enhanced (P<0.05) the flux of hPTH (1-34) in comparison to passive flux. Electroporation pulses of 100, 200 and 300 V significantly increased (P<0.05) the flux of hPTH (1-34) in comparison with the passive as well as iontophoretic flux at 0.5 mA/cm2. The electroporative flux of hPTH (1-34) was found to vary linearly (R2 = 0.97) with the pulse amplitude. The principal barrier of the skin, stratum corneum, was found perturbed following the pulses as evident by light microscopy studies. The application of electroporation pulses followed by iontophoresis further increased the flux by several fold. The flux of hPTH (1-34) with the electroporation pulses of 100 and 300 V followed by iontophoresis at 0.2 mA/cm2 was 10- and 5-fold higher, respectively, in comparison to the flux with corresponding pulses alone. This shows the synergistic effect of iontophoresis in combination with electroporation on skin permeability of hPTH (1-34). The results indicate the possibility of designing controlled transdermal delivery systems for hPTH (1-34) using electroporation followed by iontophoresis.