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DNA vaccines represent a new approach to the control of infectious disease. Both cellular and humoral immune responses are induced without the attendant concerns associated with live, attenuated vaccines. The vast majority of DNA vaccines are delivered by parental routes, which rarely elicit immune responses at the mucosal epithelia, the primary sites of pathogen transmission. In view of the importance of mucosal and regional lymph node immunity in the control of pathogens transmitted across the mucosal epithelia, a number of groups, including our own, have developed immunization strategies that target plasmid DNA to mucosal inductive sites associated with the lymphoid tissues of the respiratory, gastrointestinal, and genital tracts. Here, we describe the procedures for the formulation and delivery of plasmid DNA to mucosal inductive tissues and address the theoretical basis to selection of particular mucosal locations for the induction of effective immune responses.

The development of DNA vaccination to mucosal surfaces has continued apace over the last 2 years, with the investigation of several novel delivery vehicles. There have been advances in the understanding of the basic immunological mechanisms behind the induction of immune responses by plasmid DNA. The mechanistic insights are paving the way for the design of a second generation of mucosally delivered DNA vaccines. This article reviews the recent progress in the field of microparticle, cationic lipid and bacterial delivery systems. All these mechanisms afford some protection from environmental degradation and facilitate DNA uptake. These methods have been compared with respect to transfection efficiency, ability to elicit a full range of immune responses and their relative safety for in vivo applications.

The nasal mucosa provides a simple, non-invasive route to deliver DNA encoding the gene of interest to stimulate mucosal and systemic immune responses. However, unlike the intradermal or intramuscular routes for plasmid DNA (pDNA) delivery, immune responsiveness to antigen exposure at the respiratory mucosa is tightly regulated, consistent with the balance between active immunity and non-responsiveness to pathogenic or inert environmental antigens. We have characterised the antigen presenting cell types, their distribution and activation status following nasal vaccination with pDNA-cytofectin complexes encoding model antigens. We demonstrate that nasal immunisation is associated with expression of the encoded protein in a small population of dendritic cells and macrophages at the site of pDNA delivery, in the draining lymph nodes (LN) and in the spleen. Antigen expression by nasal dendritic cells was associated with up-regulation of surface MHC class II and CD86 expression and functional activation of T-lymphocytes. The results highlight the potential of intranasal vaccination with pDNA, provided the activation / costimulatory requirements for an active immune response are achieved.

The development of vaccines against pathogens transmitted across the genito-rectal mucosa that effectively stimulate both secretory IgA Abs and cytotoxic T lymphocytes in the genital tract and CTL in the draining lymph nodes (LN) has proven a major challenge. Here we report a novel, noninvasive approach of genetic vaccination via the intranasal route. Such vaccination elicits immune responses in the genital and rectal mucosa, draining LNs, and central lymphoid system. Intranasal immunization with plasmid DNA-lipid complexes encoding the model Ag firefly luciferase resulted in dissemination of the DNA and the encoded transcript throughout the respiratory and gastrointestinal tracts, draining LNs, and spleen. Complexing the plasmid DNA with the lipid DMRIE/DOPE enhanced expression of the encoded protein in the respiratory tract, increased specific secretory IgA Ab in the vaginal and rectal tracts, and increased the circulating levels of specific IgA and IgG. In addition, intranasal DNA immunization resulted in generation of Ag-specific CTL that were localized in the genital and cervical LNs and spleen. These results suggest that intranasal immunization with plasmid DNA-lipid complexes may represent a generic immunization strategy against pathogens transmitted across the genito-rectal and other mucosal surfaces.

The mucosal surfaces represent the primary site for transmission of several viruses including HIV. To prevent mucosal transmission and dissemination to the regional lymph nodes, an effective HIV vaccine may need to stimulate immune responses at the genital and rectal mucosa. Optimal induction of mucosal immunity in general requires targeting antigens to the specialized antigen presenting cells of mucosal associated lymphoid tissues. The nasal mucosa may provide a simple, non-invasive route to deliver DNA encoding the introduced gene to stimulate mucosal immunity. As a first step to evaluate the feasibility of this approach, we have investigated as a model system, systemic and mucosal immune responses elicited to firefly luciferase generated by DNA immunization. Incorporating DNA into liposomes with cationic lipids enhanced luciferase expression in nasal tissue, and was associated with induction of a humoral response in serum and vaginal fluids and also a proliferative and cytotoxic T lymphocyte response in the spleen and iliac lymph nodes draining the genital and rectal mucosa.