Journal of General Virology (2011), 92, 326–335 DOI 10.1099/vir.0.026419-0 Correspondence Yoshikazu Kurosawa kurosawa@fujita-hu.ac.jp Received 13 August 2010 Accepted 9 November 2010 Localization of epitopes recognized by monoclonal antibodies that neutralized the H3N2 influenza viruses in man Jun Okada,1,2 Nobuko Ohshima,3 Ritsuko Kubota-Koketsu,4 Yoshitaka Iba,1 Sayuri Ota,3 Wakana Takase,3 Tetsushi Yoshikawa,5 Toyokazu Ishikawa,6 Yoshizo Asano,4 Yoshinobu Okuno6 and Yoshikazu Kurosawa1 1Division of Antibody Project, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan 2Institute for Antibody Ltd, Toyoake, Aichi 470-1192, Japan 321st Century COE Research Center, Fujita Health University, Toyoake, Aichi 470-1192, Japan 4Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan 5Department of Pediatrics, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan 6The Research Foundation for Microbial Diseases, Osaka University, Kannonji, Kagawa 768-0061, Japan Through extensive isolation of neutralizing mAbs against H3N2 influenza viruses representing the in vivo repertoire in a human donor, we examined the relationships between antigenic drift of influenza virus and protective antibodies generated in an infected individual. The majority of mAbs isolated from a donor born in 1960 were divided into three major groups with distinct strain specificity: 1968–1973, 1977–1993 and 1997–2003. In the present study, we developed a new method that allowed us to comprehensively determine the location of epitopes recognized by many mAbs. Original haemagglutinins (HAs) of several strains and chimaeric variants, in which one of the seven sites (A, B1, B2, C1, C2, D or E) was replaced by some other strain-derived sequence, were artificially expressed on the cell surface. The binding activity of mAbs to the HAs was examined by flow cytometry. By using this method, we determined the location of epitopes recognized by 98 different mAbs. Clones that neutralize the 1968–1973 strains bind to site B2/D, A or A/B1. While sites C, E and B were recognized by clones that neutralized the 1977–1993 strains, the majority of these clones bind to site C. Clones that neutralize the 1997–2003 strains bind to site B, A/B1, A/B2 or E/C2. INTRODUCTION Antibodies (Abs) play important roles in protection against and recovery from influenza virus infection, and haemag- glutinin (HA) is the main target for virus-neutralizing Abs (Gerhard et al., 1997). Viruses with mutations in critical residues of HA could preferentially survive under the pressure of neutralizing Abs and cause future influenza epidemics (Hay et al., 2001). Mutations mainly accumulate in the five sites (A, B, C, D and E) on HA1 that should include neutralizing epitopes (Underwood, 1982; Wiley Supplementary materials describing the construction of plasmid DNAs for cell-surface expression of HA and chimaeric HAs are available with the online version of this paper. 326 et al., 1981). To the best of our knowledge, however, very little is known about the nature, range and specificity of protective mAbs that are generated in infected individuals. Since the outbreak of ‘Hong Kong’ influenza in 1968, the H3N2 influenza viruses remain the most common cause of flu. Therefore, in the present study, we examined the neutralizing Abs against the H3 strains isolated between 1968 and 2004. We attempted to reveal the relationships between the location of epitopes recognized by neutralizing mAbs against influenza viruses in a human donor and that of mutations introduced in the epidemic strains. Previously (Okada et al., 2010), we collected a large number of B lymphocytes by apheresis from a donor born in 1960. Since 026419 G 2011 SGM Printed in Great Britain