Immunology and Cell Biology (2004) 82, 617–627 doi:10.1111/j.1440-1711.2004.01288.x © 2004 Australasian Society for Immunology Inc. Special Feature Mucosal adjuvants and delivery systems for protein-, DNA- and RNA-based vaccines MICHAEL VAJDY, INDRESH SRIVASTAVA, JOHN POLO, JOHN DONNELLY, DEREK O’HAGAN and MANMOHAN SINGH Chiron Vaccines, Emeryville, California, USA Almost all vaccinations today are delivered through parenteral routes. Mucosal vaccination offers several benefits over parenteral routes of vaccination, including ease of administration, the possibility of self-administration, elimination of the chance of injection with infected needles, and induction of mucosal as well as systemic immunity. However, mucosal vaccines have to overcome several formidable barriers in the form of significant dilution and dispersion; competition with a myriad of various live replicating bacteria, viruses, inert food and dust particles; enzymatic degradation; and low pH before reaching the target immune cells. It has long been known that vaccination through mucosal membranes requires potent adjuvants to enhance immunogenicity, as well as delivery systems to decrease the rate of dilution and degradation and to target the vaccine to the site of immune function. This review is a summary of current approaches to mucosal vaccination, and it primarily focuses on adjuvants as immuno- potentiators and vaccine delivery systems for mucosal vaccines based on protein, DNA or RNA. In this context, we define adjuvants as protein or oligonucleotides with immunopotentiating properties co-administered with pathogen- derived antigens, and vaccine delivery systems as chemical formulations that are more inert and have less immunomodulatory effects than adjuvants, and that protect and deliver the vaccine through the site of administratio n. Although vaccines can be quite diverse in their composition, including inactivated virus, virus-like particles and inactivated bacteria (which are inert), protein-like vaccines, and non-replicating viral vectors such as poxvirus and adenovirus (which can serve as DNA delivery systems), this review will focus primarily on recombinant protein antigens, plasmid DNA, and alphavirus-based replicon RNA vaccines and delivery systems. This review is not an exhaustive list of all available protein, DNA and RNA vaccines, with related adjuvants and delivery systems, but rather is an attempt to highlight many of the currently available approaches in immunopotentiation of mucosal vaccines. Key words: adjuvant, delivery system, immunomodulator, mucosal, vaccine. Recombinant protein vaccines Traditional vaccines have comprised live-attenuated microbes, inactivated microorganisms, purified microbial components, polysaccharide-carrier protein conjugates or recombinant proteins. The first of the latter type of vaccine was derived from the diphtheria and tetanus toxoids, and was developed in the first half of the twentieth century. The two toxins were chemically detoxified to produce the non-toxic toxoids. Conventional approaches to vaccine development have been based on biochemical, immunological and microbiological methods that have been labourious, time-consuming and have allowed identification of the most abundant antigens of any given pathogen. Recent progress in DNA sequencing and subsequently in bioinformatics have resulted in advances in vaccine development. When the whole sequence of a bacterial genome became available, the genomic information was used to discover novel antigens that had been missed by conven- tional methods of vaccine development. This novel approach, now termed reverse vaccinology, involved the in silico analysis of the microbial genome sequence. 1–3 This approach has already resulted in the identification of immunogenic antigens as potential candidates for a vaccine against Neisseria meningitidis. 4 This approach holds great promise for future vaccine development. Immunopotentiating adjuvants for protein vaccines Mutants of heat labile enterotoxin from Escherichia coli and cholera toxin from Vibrio cholera Genetically detoxified mutants of heat labile enterotoxin (LT) have been shown to be potent adjuvants for inducing mucosal and systemic immune responses. To retain the adjuvanticity of these molecules but reduce their toxicity, several mutants have been generated by site directed mutagenesis. Of these, two mutants of the enzymatic A subunit, LTK63 and LTR72, maintain a high degree of adjuvanticity. LTK63 results from the substitution of serine 63 with a lysine in the A subunit, which renders it enzymatically inactive and non-toxic. 5–9 LTR72 is derived from a substitution of alanine 72 with an arginine in the A subunit, and has approximately 0.6% of the enzymatic activity of wild-type LT. LTR72 is shown to be Correspondence: Michael Vajdy, Chiron Vaccines, 4560 Horton Street mail stop, Emeryville, CA 94608, USA. Email: michael_vajdy@chiron.com Received 12 July 2004; accepted 12 July 2004.