title: Single HA2 mutation increases the infectivity and immunogenicity of a live attenuated H5N1 intranasal influenza vaccine candidate lacking NS1.
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.
  
  
  RESULTS
  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 58K→I, 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 58K→I 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Avir Green Hills Biotechnology AG, Vienna, Austria.
authors.forename: Brigitte M
authors.initials: BM
authors.lastname: Krenn
authors.forename: Andrej
authors.initials: A
authors.lastname: Egorov
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Romanovskaya-Romanko
authors.forename: Markus
authors.initials: M
authors.lastname: Wolschek
authors.forename: Sabine
authors.initials: S
authors.lastname: Nakowitsch
authors.forename: Tanja
authors.initials: T
authors.lastname: Ruthsatz
authors.forename: Bettina
authors.initials: B
authors.lastname: Kiefmann
authors.forename: Alexander
authors.initials: A
authors.lastname: Morokutti
authors.forename: Johannes
authors.initials: J
authors.lastname: Humer
authors.forename: Janina
authors.initials: J
authors.lastname: Geiler
authors.forename: Jindrich
authors.initials: J
authors.lastname: Cinatl
authors.forename: Martin
authors.initials: M
authors.lastname: Michaelis
authors.forename: Nina
authors.initials: N
authors.lastname: Wressnigg
authors.forename: Sanda
authors.initials: S
authors.lastname: Sturlan
authors.forename: Boris
authors.initials: B
authors.lastname: Ferko
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Batishchev
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Indenbom
authors.forename: Rong
authors.initials: R
authors.lastname: Zhu
authors.forename: Markus
authors.initials: M
authors.lastname: Kastner
authors.forename: Peter
authors.initials: P
authors.lastname: Hinterdorfer
authors.forename: Oleg
authors.initials: O
authors.lastname: Kiselev
authors.forename: Thomas
authors.initials: T
authors.lastname: Muster
authors.forename: Julia
authors.initials: J
authors.lastname: Romanova
mesh_terms: D000281:Administration, Intranasal; D000818:Animals; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics* / Q000276:immunology* / Q000472:pathogenicity; D007252:Influenza Vaccines / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D009154:Mutation; D009976:Orthomyxoviridae Infections / Q000235:genetics* / Q000276:immunology*; D014613:Vaccines, Attenuated / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D014709:Vero Cells
chemical_list: D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007252:Influenza Vaccines; D014613:Vaccines, Attenuated