Neutralizing and protective epitopes of the 2009 pandemic influenza H1N1 hemagglutinin

Abstract

Aims and Methods To facilitate antigenic characterization of the influenza A 2009 pandemic H1N1 [A(H1N1)pdm09] hemagglutinin (HA), we generated a panel of murine monoclonal antibodies (mAbs) using as the immunogen mammalian‐derived virus‐like particles containing the HA of the A/California/04/2009 virus. The antibodies were specific for the A/California/04/2009 HA, and individual mAbs suitable for use in several practical applications including ELISA, immunofluorescence, and Western blot analysis were identified. Results and Conclusions As the panel of mAbs included antibodies with hemagglutination inhibition (HI) and virus neutralizing activities, this allowed identification and characterization of potentially important antigenic and neutralizing epitopes of the A/California/04/2009 HA and comparison of those epitopes with the HAs of other influenza viruses including seasonal H1N1 viruses as well as the A/South Carolina/1918 and A/New Jersey/1976 H1N1 viruses. Three mAbs with the highest HI and neutralizing titers were able to provide passive protection against virus challenge. Two other mAbs without HI or neutralizing activities were able to provide partial protection against challenge. HA epitopes recognized by the strongest neutralizing mAbs in the panel were identified by isolation and selection of virus escape mutants in the presence of individual mAbs. Cloned viruses resistant to HI and antibody neutralization were sequenced to identify mutations, and two unique mutations (D127E and G155E) were identified, both near the antigenic site Sa. Using human post‐vaccination sera, however, there were no differences in HI titer between A/California/04/2009 and either escape mutant, suggesting that these single mutations were not sufficient to abrogate a protective antibody response to the vaccine.

Virological characterization of influenza H1N1pdm09 in Vietnam, 2010‐2013

“…Beside common amino acid changes of group 6 (D97N, S185T, S203T, E374K, and S541N), study viruses in subgroup 6C also showed substitutions at I216M, V234I, T241I, K283E, and E499K compared to the reference virus A/California/7/09 . Additional significant changes found in Vietnamese isolates in group 6C included D222N (1 isolate) and G155E and N156K (5 isolates); notably, these mutations have previously been associated with increased virulence or severe and fatal disease (D222G/N) or reduced HI titers when using ferret antiserum raised against A/California/7/2009 (G155E and N156K) (Figure , Table ) …”

A monoclonal antibody‐based immunoassay for measuring the potency of 2009 pandemic influenza H1N1 vaccines

“…Additional studies, which compared the binding of the mAbs to A/California HA at neutral and low pH, also indicated that these mAbs bind the globular head of A/California HA (Figure S1). From the previous study, it was known that mAbs 4F8 and 5C12 had hemagglutination inhibition (HI) activity, were strongly neutralizing in multiple types of neutralization assays and were protective in passive antibody transfer experiments . Epitope‐mapping experiments indicated that these two antibodies competed with each other for the antigenic site Sa on HA, but there was some evidence that suggested that the recognition site for these antibodies might not be identical.…”

“…Reference antigens for influenza vaccine candidates X‐179A and X‐181 were produced by the Center for Biologics Evaluation and Research in collaboration with licensed vaccine manufacturers. The preparation and initial characterization of eleven monoclonal antibodies to A/California/4/2009 HA have been previously described …”

Influenza Virus M2 Protein Ion Channel Activity Helps To Maintain Pandemic 2009 H1N1 Virus Hemagglutinin Fusion Competence during Transport to the Cell Surface

“…Mouse anti-(H1N1)pdm09 HA monoclonal antibody 4F8 was described previously (29). Rabbit polyclonal antibodies against PR M2 and actin were purchased from Thermo Scientific (Rockford, IL).…”