Vaccine 21 (2003) 3972–3981 Mucosal delivery of the human immunodeficiency virus-1 Tat protein in mice elicits systemic neutralizing antibodies, cytotoxic T lymphocytes and mucosal IgA Mariarosaria Marinaro a , Antonella Riccomi a , Rino Rappuoli b , Mariagrazia Pizza b , Valeria Fiorelli c , Antonella Tripiciano c , Aurelio Cafaro c , Barbara Ensoli c , Maria Teresa De Magistris a,∗ a Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy b Chiron SpA, Via Fiorentina 1, 53100 Siena, Italy c Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy Received 8 July 2002; accepted 28 March 2003 Abstract Human immunodeficiency virus (HIV)-1 Tat protein induces protection in non-human primates upon systemic vaccination. In view of the design of mucosal vaccines against HIV-1 we studied the immune response to native Tat (aa 1–86) in mice following intranasal delivery of the protein with two mucosal adjuvants, Escherichia coli heat-labile enterotoxin (LT) and LT-R72, a non-toxic mutant of LT. Immunization with Tat and the two adjuvants induced in BALB/c but not in C57BL/6 mice high and persistent levels of serum IgG and secretory IgA in vaginal and intestinal fluids. Mice sera neutralized Tat and recognized two epitopes mapping in the regions 1–20 and 46–60. Furthermore, their splenocytes proliferated and secreted IFN- and IL-6 in response to Tat. Finally, CTLs were also elicited and they recognized an epitope localized within aa 11–40 of Tat. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Mucosal vaccines; Adjuvants; HIV-1 Tat protein 1. Introduction An ideal vaccine against human immunodeficiency virus (HIV) should elicit immune responses able to prevent tis- sue invasion at the mucosal level and to neutralize the virus and its pathogenic factors at the systemic level. While par- enteral vaccines generally fail to induce mucosal responses, mucosal vaccines are very effective at eliciting high levels of systemic IgG as well as secretory IgA and cell-mediated responses, including CTLs [1]. In this respect, the correlates of protection against HIV infection or disease are still elu- sive [2]. Studies in non-human primates and in infected or exposed individuals have shown that systemic neutralizing Abs and mucosal Ab responses against HIV are all impor- tant [3–8], although they may play a role in different phases of HIV infection. In addition, cell-mediated responses, and particularly CTLs, are presently considered critical for con- ∗ Corresponding author. Tel.: +39-06-4990-2734; fax: +39-06-4990-2934. E-mail address: mtdemagi@iss.it (M.T. De Magistris). ferring an effective protection against HIV and simian im- munodeficiency virus (SIV) infection and disease progres- sion [9–14]. Thus, a mucosal vaccine against HIV has the potential to stimulate the full range of immune responses relevant for protection. Among the candidate Ags for an anti-HIV vaccine, the regulatory protein Tat has received much attention in the late years [15–17]. Tat is produced very early after infec- tion, prior to expression of the structural genes env, gag, and pol and is essential for viral replication [18]. Further- more, Tat is released by the infected T lymphocytes in the extracellular milieu [19–21] and enters both infected cells, where it promotes HIV replication, and uninfected cells in which it causes activation or repression of cytokine and cel- lular genes controlling the cell cycle [18,21–23]. Tat also in- duces the expression of the chemokine receptors (and HIV-1 co-receptors) CCR5 and CXCR4 [24,25], responsible for the transmission of macrophage- and T-cell-tropic HIV-1 strains, respectively. Thus, extracellular Tat plays a role in the spreading of infection by recruiting new cell targets. The Tat protein also affects the function of different cell types 0264-410X/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0264-410X(03)00295-0