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Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies
Published on: August 29, 2017
Interaction between a 1998 human influenza virus N2 neuraminidase and monoclonal antibody Mem5
1Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, 73104, USA.
Abstract:
Influenza virus constantly escapes antibody inhibition by introducing mutations that disrupt protein-protein interactions. Based on the structure of the complex between neuraminidase (NA) of influenza A/Memphis/31/98 (H3N2) and the Fab of a monoclonal antibody (Mem5) that binds and inhibits the Memphis/98 NA, we investigated the contribution made by individual amino acids of NA to the stability of the complex. We made mutations D147A, D147N, H150A, H197A, D198A, D198N, E199A, E199Q, K221R, A246K, D251N, and D251A. Binding of each mutant to NA was quantitated by NA inhibition assays and ELISA. Most of the mutant NAs were inhibited by Mem5 to the same extent as wild-type, but with lower affinity. The exceptions were E199A, E199Q, and K221R, in which binding was abrogated. The ELISA results confirmed a correlation between NA inhibition and binding. The Mem5 epitope is dominated by a few high-energy interactions as was found in the epitope on an avian subtype N9 NA that binds antibody NC41 and different to the more diffuse energy distribution in the NC10 epitope on N9 NA. Energetic dominance of a particular interaction, which is associated with potential for antibody escape mutations, may be associated with the absence of water molecules in the vicinity. Critical contacts in a dominant antigenic site are likely to mutate, allowing some predictions of antigenic drift.
Insights
Influenza virus neuraminidase (NA) mutations can evade antibody binding. Key amino acid interactions in the NA-Mem5 antibody complex were identified, revealing sites prone to mutations that drive viral escape and antigenic drift.
Area of Science:
- Virology
- Immunology
- Structural Biology
Background:
- Influenza viruses continuously evolve to escape host immunity.
- Neuraminidase (NA) is a key surface protein and a target for neutralizing antibodies.
- Mutations in NA can alter antibody binding, leading to viral escape.
Purpose of the Study:
- To investigate the contribution of individual amino acids in influenza A H3N2 neuraminidase (NA) to the stability of its complex with the monoclonal antibody Mem5.
- To identify critical interactions within the NA epitope that are susceptible to mutations driving antibody escape.
Main Methods:
- Site-directed mutagenesis of specific amino acids in the H3N2 neuraminidase (NA).
- Quantification of antibody binding affinity using NA inhibition assays and ELISA.
- Structural analysis of antibody-epitope interactions.
Main Results:
- Mutations at E199A, E199Q, and K221R abrogated binding of the Mem5 antibody to NA.
- Most other mutations resulted in reduced binding affinity without complete loss of inhibition.
- The Mem5 epitope is characterized by a few high-energy interactions, unlike broader epitopes.
Conclusions:
- Specific amino acid residues in the NA epitope are critical for high-affinity antibody binding.
- Energetically dominant interactions in antigenic sites are prone to mutations, facilitating influenza virus immune escape.
- Understanding these interactions aids in predicting antigenic drift and developing more effective influenza vaccines.
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