Introduction An unprecedented spread of highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype was observed among wild and domestic birds throughout the last decade. Hundreds of cases of the direct transmission of avian viruses to humans with a case fatality rate exceeding 50% raised great concerns of a possible new pandemic. Numerous clinical studies with vaccines produced from H5N1 viruses have demonstrated that the inactivated vaccines produced from the H5 hemagglutinin (HA) appeared to be poorly immunogenic compared to seasonal influenza strains [1,2]. A broader and longer lasting immunity might be induced by live attenuated influenza vaccines, which are believed to be superior to inactivated vaccines [3,4]. However, H5N1 cold adapted vaccine strains comprising surface antigens derived from A/Vietnam/ 1203/04 (VN1203) or A/Hong Kong/213/03 lacked replication in the human nasal mucosa, correlating with the observed poor immunological outcome [5]. The effectiveness of intranasal live attenuated influenza vaccines is substantially dependent on the efficient virus uptake and subsequent replication in the cells of the upper respiratory tract. Human influenza viruses are known to attach predominantly to the surface of ciliated epithelial cells in the human trachea, bronchi, and bronchioles while avian H5N1 viruses prefer the lower respiratory tract, in turn binding more abundantly to the alveoli [6]. This could be explained by the preferential affinity of H5 HA to sialic acid receptors with an a2,3 galactose (a2,3Gal) linkage dominating on the cells of the lower respiratory tract, but not to the a2,6Gal type, which is abundantly present in the human trachea [7,8]. However, in spite of the difference in the receptor specificity, it was demonstrated that H5N1 viruses are able to infect ex vivo cultures of human nasopharyngeal, adenoid, and tonsillar tissues [9]. Consistently, another live attenuated H5 vaccine candidate comprising the HA of the low pathogenic avian influenza virus (LPAIV) A/duck/Potsdam/86/92 (H5N3) was shown to replicate efficiently in the human upper respiratory tract (for at least 11 days) [10,11]. Therefore, the receptor specificity properties of influenza surface glycoprotein might not be the only responsible reason for the low infectivity of avian viruses in humans. Unlike isolated epithelial cells in vitro, the human airway luminal surface in vivo presents a significant extracellular barrier to influenza infection. It includes the mucociliary clearance system, viscous fluids, and macrophages interfering with the virus access to the cell surface. In addition, the measurement of the pH of the human nasal cavity revealed that the overall pH range at the anterior and posterior sites is 5.2–8.0 [12–15]. Acidic pH, heat, or chemical denaturants are known to promote the conformational change of HA into its fusogenic form, which is responsible for the complete viral inactivation [16]. Therefore, in order to overcome the human mucosal barrier, influenza viruses require a certain level of HA stability towards inactivating factors. It was described that human and LPAIVs are relatively resistant to an acidic pH environment because they perform the pH dependent conformational change of the HA at a pH range of 5.1 to 5.4 [17]. In contrast, the HAs of H5 and H7 HPAIVs are much more sensitive undergoing conformational modification already at a pH of 5.6 to 6.0 [18]. By the mutagenesis of viral HA, Reed et al. succeeded in increasing the environmental stability of an HPAIV H5N1 strain (A/Chicken/Vietnam/C58/ 04) through the reduction of the pH of HA activation [19]. The obtained mutant demonstrated reduced virulence and transmis- sibility in wild ducks. Increased Infectivity of H5N1 Influenza Virus We hypothesized that the poor infectivity of HPAIVs for mammals and, as a consequence, the insufficient efficacy of live attenuated H5 vaccines is related to the low stability of their HA protein. In order to prove our hypothesis, we constructed two H5N1 live attenuated vaccine candidates lacking NS1 based on the VN1203 virus, which differ in a single amino acid substitution 58KRI in the HA2 subunit [20]. Reed et al. described this mutation as decreasing the threshold pH of the activation of the H5 HA protein of approx. 0.5 pH units [21]. The obtained viruses were compared for their stability in vitro and for the infectivity and immunogenicity after intranasal application in vivo in mice. We demonstrated that the stabilization of the HA molecule towards acidic pH and high temperature promotes superior vaccine virus infectivity in the respiratory tract and, consequently, increased immunogenicity in mice. Results HPAIVs but not human seasonal viruses lose their infectious activity at an acidic pH in primary Human Nasal Epithelial cells (HNEpC) The acidification machinery of the human nasal passageway is known to release protons in response to irritation or inflammation [22] acidifying the mucosal surface (down to pH of 5.2). Therefore, viruses must possess a certain level of stability to low pH in order to infect the upper respiratory tract. We analyzed whether human seasonal virus isolates differ from HPAIVs in infectious activity at an acidic pH. Human viruses A/Brisbane/ 59/2007 (H1N1) (BN/59/07), A/Vienna/25/07 (H3N2) (VI/25/ 07), A/St. Petersburg/14/10 (H1N1v) (SP/14/10) and HPAIVs VN1203 (H5N1) and A/Thailand/01/04 (H5N1) (TH/01/04) of an early passage level adjusted to a similar multiplicity of infection (moi) were admixed with acidic (pH 5.4 or/and 5.6) or neutral (pH 7.4) buffer followed by the inoculation of the HNEpCs. After removing the inoculum, the cells were incubated at a neutral pH for 5 hours without trypsin. The effectiveness of the infection was visualized by influenza nucleoprotein (NP) staining (Fig. 1). All human influenza viruses, including the recently appeared pandemic H1N1v virus, were capable of infecting cells at acidic conditions (Fig. 1A), while both highly pathogenic H5N1 viruses infected HNEpCs only at a neutral pH (Fig. 1B). A similar pH dependence of infection was observed in Vero cells (results are not shown). Generation of H5N1 viruses with 58KRI substitution in the HA2 subunit To investigate whether the infectivity and, consequently, the immunogenicity of H5N1 viruses might be improved by decreasing the threshold pH of HA fusion to the range described for human influenza viruses, we modified the HA protein by implementing the 58KRI substitution in the HA2 subunit of the VN1203 virus, according to Reed et al. [21]. This mutation located in the HA2 coiled-coil domain was shown to decrease the pH of the activation of the H5 HA protein. An additional attempt failed to rescue a corresponding virus comprising HA2 23GRC modification, which is described to decrease the pH of the fusion of H7N7 viruses [23]. Increased resistance of influenza HA to an acidic pH is known to be linked to a decreased isoelectric point (pI) of the HA2 protein subunit, the pH value at which the protein charge is neutral [24]. The calculated value pI = 4.59 for the mutated HA2 subunit was lower than that of the natural VN1203 HA2 subunit (pI = 4.67), supporting the assumption of the enhanced resistance of the mutated HA to acidic pH (http://isoelectric.ovh.org/EMBOSS). PLoS ONE | www.plosone.org 2 April 2011 | Volume 6 | Issue 4 | e18577