Single HA2 Mutation Increases the Infectivity and Immunogenicity of a Live Attenuated H5N1 Intranasal Influenza Vaccine Candidate Lacking NS1 Brigitte M. Krenn1., Andrej Egorov1.* , Ekaterina Romanovskaya-Romanko 2 , Markus Wolschek1, Sabine Nakowitsch1, Tanja Ruthsatz1, Bettina Kiefmann 1 , Alexander Morokutti1, Johannes Humer1, Janina Geiler5, Jindrich Cinatl5, Martin Michaelis5, Nina Wressnigg1, Sanda Sturlan1, Boris Ferko1, Oleg V. Batishchev3, Andrey V. Indenbom3, Rong Zhu 4 , Markus Kastner4, Peter Hinterdorfer4, Oleg Kiselev2, Thomas Muster1, Julia Romanova1 1 Avir Green Hills Biotechnology AG, Vienna, Austria, 2 Influenza Research Institute, Russian Academy of Medical Sciences, St. Petersburg, Russia, 3 A.N. Frumkin Institute of Physical Chemistry and Electrochemistry of Russian Academy of Sciences (RAS), Moscow, Russia, 4 Christian Doppler Laboratory of Nanoscopic Methods in Biophysics, Institute for Biophysics, Johannes Kepler University Linz, Linz, Austria, 5 Institute for Medical Virology, Johann Wolfgang Goethe University, Frankfurt, Germany Abstract Background: H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals. Methodology/Principal Findings: We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH#5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58KRI, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose50. Moreover, the reassortants keeping 58KRI mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice. Conclusion/Significance: Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals. Citation: Krenn BM, Egorov A, Romanovskaya-Romanko E, Wolschek M, Nakowitsch S, et al. (2011) Single HA2 Mutation Increases the Infectivity and Immunogenicity of a Live Attenuated H5N1 Intranasal Influenza Vaccine Candidate Lacking NS1. PLoS ONE 6(4): e18577. doi:10.1371/journal.pone.0018577 Editor: Andrew Pekosz, Johns Hopkins University - Bloomberg School of Public Health, United States of America Received November 17, 2010; Accepted March 6, 2011; Published April 7, 2011 Copyright: 2011 Krenn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been partially funded by the European Commission’s 6th Framework Program projects ‘‘Intranasal H5 Vaccine’’ (SP5B-CT-2007-044512) and ‘‘FLUVACC’’ (LSHB-CT-2005-518281). Costs not covered by the European Grants are funded by AVIR Green Hills Biotechnology AG (AGHB) and in part by Russian Foundation for Basic Researches (project #08-03-00971), Program of Presidium of RAS ‘‘Molecular and Cell Biology,’’ Federal Task Program ‘‘Scientific and Scientific-Pedagogic Personnel of Innovative Russia’’ for 2009-2013 (state contract #P337) and Austrian Institute for Biophysics, Christian Doppler Laboratory of Nanoscopic Methods in Biophysics (Johannes Kepler University Linz). No additional external funding was received for this study. AGHB performed the study design, data collection and analysis, decision to publish, and preparation of the manuscript. Competing Interests: AVIR Green Hills Biotechnology (AGHB) is a privately held biopharmaceutical company based in Vienna (Austria) that draws on its own knowledge in virology to develop and commercialize an intranasal live attenuated vaccine targeting pandemic influenza disease among other products in the pipeline. BMK, AE, MW, SN, TR, BK, AM, NW, SS, BF, JH, TM, and RJ were employees of AGHB at the time of study. IPR rights related to this manuscript are covered by the following IP rights: DelNs1 mutants are covered by patents from AGHB and Mount Sinai School of Medicine. Patent applications covering the technology platform of the influenza virus and virus amplification procedure are fully owned by AGHB. In adherence to the PLoS guidelines, AGHB will make freely available any materials and information described in the publication that are reasonably requested by others for the purpose of academic, non-commercial research. * E-mail: a.egorov@greenhillsbiotech.com . These authors contributed equally to this work. PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e18577