Vaccine 22 (2004) 4365–4373 Immunisation against plague by transcutaneous and intradermal application of subunit antigens J.E. Eyles a,∗ , S.J. Elvin a , A. Westwood a , C.S. LeButt a , H.O. Alpar b , S. Somavarapu b , E.D. Williamson a a Biomedical Sciences, Dstl, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK b The School of Pharmacy, University of London, London WC1N 1AX, UK Received 8 September 2003; accepted 2 February 2004 Abstract We have investigated immunological responses in BALB/c mice following transcutaneous (TC) delivery of fraction 1 (F1) and V subunits from Yersinia pestis in conjunction with an enterotoxin-derived adjuvant (cholera toxin, CT). It was found that two or more TC applications of F1 and V subunits (admixed with cholera toxin) served to elicit significant levels of anti-F1 and V antibodies in the serum of immunised mice. IL-6 secretion from cultured splenocytes derived from immunised mice indicated that a single TC application of F1 and V subunits (admixed with cholera toxin) conferred a cell-mediated response. As compared with intranasal or direct intradermal injection of F1 and V, the numbers of F1/V-specific antibody-forming cells in the spleens of animals immunised by TC application of F1 and V (admixed with CT) was relatively low. It was noted that TC application of F1 and V admixed with CT was very effective for priming responses that were boosted by intranasal or intradermal routes. Similarly, it was found that TC application of F1 and V admixed with CT could be used to efficiently boost pre-existing responses engendered by intradermal injection or intranasal instillation of F1 and V. In order to assess if TC application of F1 and V admixed with CT could protect experimental animals from plague, immunised mice were injected with a virulent strain of Y. pestis. It was found that two TC applications of F1 and V admixed with CT conferred only limited protection against 10 2 MLDs. However, three TC applications of F1 and V admixed with CT conferred solid protection against 10 2 MLDs. Hence we have shown, for the first time, that TC application of F1 and V admixed with CT can protect animals against challenge with a virulent strain of plague causing bacteria. These data suggest that transcutaneous immunisation may be a simple and non-invasive method for immunising individuals against plague. © 2004 Elsevier Ltd. All rights reserved. Keywords: Immunisation; Non-invasive; Skin; Mucosal; Enterotoxin; Subunit vaccines 1. Introduction Plague is a zoonotic infection caused by the Gram- negative bacterium Yersinia pestis [1]. To date, immunisation procedures for plague have involved parenteral administra- tion of killed Y. pestis bacilli [2]. Despite their widespread use, inactivated Y. pestis vaccines have an unsatisfactory in- cidence of transient local and systemic side effects [3]. More- over, it is apparent that killed whole cell vaccines are unable ∗ Corresponding author. Tel.: +44 1980 613528; fax: +44 1980 614307. E-mail address: jeeyles@dstl.gov.uk (J.E. Eyles). to induce appropriate immunity to counteract the form of plague that can be acquired by inhalation of aerosolised Y. pestis [4,5]. In recent years, a possible alternative to the use of killed whole cell plague vaccines has emerged. Fraction 1 (F1) and V are recombinant proteins from Y. pestis that are produced as virulence factors. When injected into experimental animals in conjunction with aluminum salt based adjuvants, recombi- nant (r) F1 and V elicit robust protection against high levels of injected or aerosolised plague challenge [6]. Injected formu- lations containing rF1 and rV are in the process of evaluation as components of an improved human plague vaccine [7]. 0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.02.049