Universal flu vaccines aim to produce broadly neutralising antibodies (bnAbs) that target the conserved but immuno-subdominant HA2 stalk region of influenza viral haemagglutinin (HA). This study aims to determine the likelihood of bnAbs inducing viral escape mutations by evaluating the fitness landscape of helix A, a key conserved region of HA2. Human bnAbs T1-3B, T2-6C, T3-5D, and MEDI8852 were obtained to test the virus mutants generated later in the thesis.
Structural bioinformatics guided the design of cDNA libraries with targeted mutations in helix A of the HA2 stalk from the A/England/195/2009 H1N1 strain. Through reverse genetics and site saturation mutagenesis, recombinant viruses with single or double mutations were generated. Sequencing identified over 22 viable variants with changes at 10 different residues. Ten of these recombinant viruses were selected for detailed phenotypic analysis to assess their fitness and potential to escape antibody recognition.
The study examined viral replication, plaque morphology, and pH stability. Some mutants exhibited altered pH stability, and differences in replication were observed between MDCK cells and primary human airway epithelial tissue. Notably, the V52IHA2 mutation significantly increased viral fitness.
However, none of the viable mutants managed to escape bnAb recognition. A double mutation that did evade antibody detection was poorly expressed and did not yield a viable virus. Despite bnAbs generally inhibiting the virus within endosomes, two out of four tested bnAbs failed to retain full HA binding at low pH.
In summary, this work demonstrates that the escape potential of influenza virus from bnAbs is constrained by the local fitness landscape of the HA stalk epitope. The study underscores the importance of evaluating bnAb efficacy under low pH conditions and with recent human-adapted strains to ensure robust control of H1N1pdm09-lineage flu outbreaks.