The molecular basis of influenza virus antigenic change

Influenza viruses escape antibody-mediated neutralization by accumulating amino acid substitutions in the hemagglutinin surface protein, necessitating frequent updates of the influenza vaccine. Previous studies identified more than 130 potentially important amino acid positions that could cause antibody escape upon mutation. The 20 amino acids that could theoretically occupy each of these positions imply a seemingly endless number of possibilities to escape from antibody recognition. For this thesis the exact amino acid changes responsible for antigenic change of influenza viruses in the past were identified, with the ultimate goal to provide new insights in the evolution of influenza viruses that will help to improve selection of vaccine strains. All major antigenic differences were due to one or very few substitutions on a small number of key positions near the domain ofthe hemagglutinin responsible for binding to the host cell, and generally involved substantial alterations of the biophysical properties of the amino acids. It was shown that the ability of key substitutions to cause antigenic change is largely independent of the amino acid context, and that substitutions similar to those that caused antibody escape in the past are potentially important for future antigenic change of the influenza viruses responsible for the 2009 influenza pandemic. These results indicate that the possibilities for influenza viruses to escape antibody-mediated neutralization are limited and that genetically vastly different influenza viruses have analogous ways to change antigenically. The small number of critical positions and restricted amino acid usage decimates the number of possible escape variants from thousands to dozens and thus substantially increases the possibility to predict the antigenic component of influenza virus evolution

Antigenic variation of clade 2.1 H5N1 virus is determined by a few amino acid substitutions immediately adjacent to the receptor binding site

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are genetically highly variable and have diversified into multiple phylogenetic clades over the past decade. Antigenic drift is a well-studied phenomenon for seasonal human influenza viruses, but much less is known about the antigenic evolution of HPAI H5N1 viruses that circulate in poultry. In this study, we focused on HPAI H5N1 viruses that are enzootic to Indonesia. We selected representative viruses from genetically distinct lineages that are currently circulating and determined their antigenic properties by hemagglutination inhibition assays. At least six antigenic variants have circulated between 2003, when H5N1 clade 2.1 viruses were first detected in Indonesia, and 2011. During this period, multiple antigenic variants cocirculated in the same geographic regions. Mutant viruses were constructed by site-directed mutagenesis to represent each of the circulating antigenic variants, revealing that antigenic differences between clade 2.1 viruses were due to only one or very few amino acid substitutions immediately adjacent to the receptor binding site. Antigenic variants of H5N1 virus evaded recognition by both ferret and chicken antibodies. The molecular basis for antigenic change in clade 2.1 viruses closely resembled that of seasonal human influenza viruses, indicating that the hemagglutinin of influenza viruses from different hosts and subtypes may be similarly restricted to evade antibody recognition