Cytokine mRNAs in the nasal-associated lymphoid tissue during in¯uenza virus infection and nasal vaccination Kazutoshi Matsuo a , Takuya Iwasaki a , Hideki Asanuma a , Tomoki Yoshikawa a , Ze Chen a , Hajime Tsujimoto b , Takeshi Kurata a , Shin-ichi Tamura a, * a Department of Pathology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan b Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan Received 26 April 1999; received in revised form 16 August 1999; accepted 3 September 1999 Abstract Intranasal immunization with a current inactivated in¯uenza vaccine together with an adjuvant (cholera toxin B subunit supplemented with a trace amount of whole toxin, CTB Ã ) was con®rmed in BALB/c mice to mimic in¯uenza virus (A/PR/8/34, H1N1) infection with respect to mucosal IgA antibody responses, in which IgA antibody-forming cell responses in the nasal- associated lymphoid tissue (NALT) were involved with a peak around 7 days after infection or vaccination. Next, the expression of various cytokine mRNAs in the NALT was compared in mice either infected with viruses or immunized with CTB Ã -combined vaccine, to examine Th cell and cytokine regulation of mucosal IgA antibody responses. In infected mice, strong IL-2, weak IL- 4, strong IL-6 and strong IFN-g mRNA expressions were induced during early days of infection; especially, IFN-g mRNA was expressed by both CD4 + and CD8 + T cells around 7 days after infection. In mice given CTB Ã -combined vaccine, weak IL-2, strong IL-4, strong IL-6 and weak IFN-g mRNA expressions were induced during early days of vaccination; especially, IL-4 mRNA was expressed by CD4 + T cells. Thus, IL-6 mRNAs were expressed strongly in both infected and vaccinated mice. The IFN-g-rich cytokine mRNA pro®les in the infected mice were re¯ected upon serum IgG2a-rich Ab responses, while the IL-4-rich pro®les in the vaccinated mice were re¯ected upon the IgG1-rich Ab responses. Thus, in¯uenza virus infection and CTB Ã - combined nasal vaccine induced Th1 dominant and Th2 dominant cytokine pro®les, respectively, while the similarity of mucosal IgA antibody responses between infection and vaccination could be explained by the appearance of IL-6 mRNAs. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Cytokine; Nasal-associated lymphoid tissue; In¯uenza 1. Introduction Natural in¯uenza virus infection has been shown to be superior to current inactivated vaccines in inducing cross-protection against variant virus infection within the same subtype [1]. The virus infection induces local IgA antibodies (Abs), serum IgG Abs and cross-reac- tive cytotoxic T cells (CTLs), whereas the inactivated vaccines, given parenterally, induce serum IgG Abs [2,3]. The cross-protection induced by natural infection is correlated with the induction of cross-reacting IgA Abs in the respiratory tract, rather than serum Abs or CTLs [4]. This fact suggests that the development of an immunization procedure to stimulate mucosal IgA production would improve the protective ecacy of the current vaccine [5]. Therefore, we attempted to give the vaccine together with cholera toxin B subunit (CTB) supplemented with a trace amount of CT (CTB Ã ) as an adjuvant [6±8]. Intranasal immunization with the current vaccine, together with CTB Ã , eec- tively provided cross-protection against variants within a subtype of in¯uenza A viruses in the upper respirat- Vaccine 18 (2000) 1344±1350 0264-410X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0264-410X(99)00401-6 www.elsevier.com/locate/vaccine * Corresponding author. Tel.: +81-3-5285-1111; fax: +81-3-5285- 1189. E-mail address: stamura@nih.go.jp (S-I. Tamura).