Vaccine 20 (2002) 2836–2839 Effect of nasal immunization with protective antigen of Bacillus anthracis on protective immune response against anthrax toxin Reetika Gaur a , Pradeep K. Gupta a , Akhil C. Banerjea b , Yogendra Singh a,∗ a Centre for Biochemical Technology Mall Road, Near Jubilee Hall, Delhi-110 007, India b National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India Received 23 October 2001; received in revised form 30 January 2002; accepted 26 February 2002 Abstract Anthrax toxin consists of three proteins: protective antigen (PA), lethal factor (LF) and edema factor (EF). PA in combination with LF (lethal toxin) is lethal to mammalian cells and is the major component of human anthrax vaccine. Immunization with PA elicits the production of neutralizing antibodies that form a major component of the protective immunity against anthrax. Recent reports have shown that neutralizing antibody titres can serve as a reliable surrogate marker for protection against anthrax. In the present study, the use of non-invasive routes such as bare skin and nose for immunization with PA on its protective immune response was investigated. Mice were inoculated intranasally (i.n.), subcutaneously (s.c.) or through the skin on days 0, 15 and 28 with purified PA. Intranasal and subcutaneous immunization with PA resulted in high IgG ELISA titers. The predominant subclass in each group was IgG1. High titres of IgA were observed only in i.n. immunized mice. In a cytotoxicity assay these sera protected J774A.1 cells from lethal toxin challenge. The results suggest that non-invasive nasal immunization may be useful in improving vaccination strategies against anthrax. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Anthrax toxin; Protective antigen; Nasal immunization 1. Introduction Anthrax is a bacterial disease caused by Bacillus an- thracis. The disease is normally confined to herbivores such as sheep, goat and cattle, but can also infect humans [1]. The major virulence factor associated with B. anthracis is a tri- partite protein exotoxin called anthrax toxin. The three com- ponents of anthrax toxin are protective antigen (PA), lethal factor (LF), and edema factor (EF). Individually, all the three components are non-toxic to mammalian cells. However, in binary combination, PA plus LF (lethal toxin) causes lysis of mouse macrophages [2] and PA plus EF (edema toxin) raises the intracellular cAMP levels causing cellular edema [3]. PA is the most immunogenic component of anthrax toxin and is a necessary component of anthrax vaccine [4,5]. We have previously shown that a non-toxic mutant PA protein completely protected guinea pigs from B. anthracis spore challenge [6]. The ability of this organism to form resistant spores and infect via the aerosol route has led it to be a po- tential agent of bioterrorism. There has been a continuous demand for a vaccine against anthrax in various countries [7]. The currently available human vaccines are far from ∗ Corresponding author. Tel.: +91-11-7666156; fax: +91-11-7667471. E-mail address: ysingh@cbt.res.in (Y. Singh). ideal; they are expensive to produce, require repeated doses and may invoke transient side effects in some individuals. The current US human anthrax vaccine, anthrax vaccine adsorbed (AVA), consists of aluminium hydroxide-adsorbed supernatant material from fermentor cultures of a toxi- genic non-encapsulated strain of B. anthracis, V770-NPI-R. Currently, the human vaccination strategy against anthrax utilizes a course of three subcutaneous injections 2 weeks apart (0–2–4), followed by three injections 6 months apart (6–12–18) and annual booster doses as long as the indi- vidual remains at risk [8]. These vaccines cause local pain, edema, erythema and require several boosters [9]. Recent reports have also shown that there has been a shortage in the supply of anthrax vaccine [7]. Thus, there is an urgent need to improve the efficacy of the anthrax vaccine to overcome these limitations. In recent years, it has been shown that non-invasive routes for vaccine delivery such as the bare skin or the nose improve the efficacy of vaccination against various diseases due to the presence of a large amount of associated lym- phoid tissue and antigen presenting cells [10–12]. However, no such studies have been carried out in the case of an- thrax. The present study was thus, undertaken to investigate the protective efficacy of intranasal and skin immunization against anthrax toxin. 0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII:S0264-410X(02)00207-4