An approach involving the preparation of biodegradable microparticles with a cationic surface was developed to improve the delivery of adsorbed DNA into antigen-presenting cells after i.m. injection. The microparticles released intact and functional DNA over 2 weeks in vitro. In addition, the microparticles induced higher levels of marker gene expression in vivo. After i.m. immunization, the microparticles induced significantly enhanced serum antibody responses in comparison to naked DNA. Moreover, the level of antibodies induced by the microparticles was significantly enhanced by the addition of a vaccine adjuvant, aluminum phosphate. In addition, in contrast to naked DNA, the cationic microparticles induced potent cytotoxic T lymphocyte responses at a low dose.DNA delivery ͉ poly(lactide-co-glycolide) microparticles F ollowing up on reports that direct injection of plasmid DNA resulted in gene expression (1), several groups pursued the possibility that direct injection of plasmid DNA could be exploited as a vaccine strategy. The first peer-reviewed report of protective immunity and cytotoxic T lymphocyte (CTL) induction in mice after i.m. injection of a DNA plasmid appeared in 1993 (2). Subsequently, the use of DNA vaccines in preclinical studies has become well established, with reports of protective immunity in many different independent studies (3). In recent studies, both antibody and CTL responses were induced in nonhuman primates, although 1-2 mg of DNA was immunized on multiple occasions in these studies (4). Antibody and CTL responses also have been induced in human volunteers, but again, high doses of DNA were used (5-7). For example, in one study in naïve subjects, optimal CTL responses were induced with 2.5 mg of DNA from Plasmodium falciparum (6). Nevertheless, DNA vaccines have proven very effective in small animal models and are also effective in larger animals, including cattle, horses, and swine (8, 9). However, although the use of DNA vaccines at milligram doses is feasible, it would impose serious limitations on the number of constructs that could be included in a vaccine. In addition, the use of very high doses of DNA is less favorable from a process economics standpoint. Therefore, there is a clear need to induce effective immunity in humans with lower and fewer doses of DNA, as well as to increase the magnitude of the immune responses obtained.There are a number of strategies available that have the potential to improve the potency of DNA vaccines. These strategies include: (i) vector modification to enhance antigen expression, which may involve targeting of the expressed protein to a particular cellular location, the inclusion of immunostimulatory sequences, or the elimination of inhibitory sequences in the plasmid; (ii) improvements in DNA delivery; or (iii) the inclusion of adjuvants, either as a gene or as a coadministered agent. Our group has focused predominantly on the use of DNA delivery systems to enhance the response to DNA vaccines. To achieve this, we have developed cationic mi...

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SUMMARYIt is estimated that Helicobacter pylori infects the stomachs of over 50% of the world's population and if not treated may cause chronic gastritis, peptic ulcer disease, gastric adenocarcinoma and gastric B-cell lymphoma. The aim of this study was to enhance the mucosal and systemic immune responses against the H. pylori antigens cytotoxin-associated gene A (CagA) and neutrophil-activating protein (NAP), through combinations of mucosal and systemic immunizations in female BALB/c mice. We found that oral or intranasal (i.n.) followed by i.m. immunizations induced signi®cantly higher serum titres against NAP and CagA compared to i.n. alone, oral alone, i.m. alone, i.m. followed by i.n. or i.m. followed by oral immunizations. However, only oral followed by i.m. immunizations induced anti-NAP antibody-secreting cells in the stomach. Moreover, mucosal immunizations alone or in combination with i.m., but not i.m. immunizations alone, induced mucosal immunoglobulin A (IgA) responses in faeces. Any single route or combination of immunization routes with NAP and CagA preferentially induced antigen-speci®c splenic interleukin-4-secreting cells and far fewer interferon-g-secreting cells in the spleen. Moreover, i.n. immunizations alone or in combination with i.m. immunizations induced predominantly serum IgG1 and far less serum IgG2a. Importantly, we found that while both i.n. and i.m. recall immunizations induced similar levels of serum antibody responses, mucosal IgA responses in faeces were only achieved through i.n. recall immunization. Collectively, our data show that mucosal followed by systemic immunization signi®cantly enhanced local and systemic immune responses and that i.n. recall immunization is required to induce both mucosal and systemic memory type responses.

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