Characterization Of Antibody Recognition In Convalescents From Highly Pathogenic Avian Influenza H5N1 Infection

Highly pathogenic avian influenza virus H5N1(HPAIV H5N1), a subtype of influenza virus A, is a persistent threat to public health worldwide because of its high morbidity and mortality and broad host spectrum. To prepare effective therapies and vaccines, it is critical to understand the host protective antibody response against the viral surface glycoprotein hemagglutinin(HA). Here, we performed a comprehensive analysis of the antigenic sites recognized by five monoclonal antibodies(mAbs) and convalescent sera from two individuals who had recovered from HPAIV H5N1 infection.We first developed a novel epitope-mapping method, which involves construction and expression of a random fragment library of target antigen on the surface of yeast,screening the library via fluorescence-activated cell sorting(FACS) and sequence analysis of sorted yeast clones. We constructed a fragment library of H5N1 HA and screened the library with a mAb recognizing a conformational epitope and three serum samples from mice immunized with HA peptides. Our results demonstrated this method can be used to analyze both linear and conformational epitopes. We also studied the sera from mice immunized with recombinant HA protein and identified the major linear epitopes recognized by the polyclonal antibodies, which further proved the method can be applied to characterize polyclonal antibody recognition.Using this method, we first analyzed the epitopes of the five mAbs and found that they all targeted the globular head of H5 HA. Then we further pinpointed the epitopes with a random mutagenesis library and identified their key neutralizing residues based on pseudovirus neutralization assay, which suggested that the five mAbs bound to three distinct sites on the globular head. To get a more precise view of their epitopes, we selected three representative mAbs and determined their crystal structures in complex with HA globular head. From structural analysis of these epitopes and others reported elsewhere, we further clustered the epitopes on the globular head into four major antigenic sites. Finally, we analyzed the polyclonal antibody recognition in the convalescent sera and found that these four antigenic sites are the major targets for the neutralizing antibodies in the sera, although the sera contained antibodies targeting the globular head and stem region.In sum, we characterized the epitopes of monoclonal and polyclonal antibodies against HA in convalescents from HPAIV H5N1 infection. These results could not only explain the neutralization mechanisms of antibodies against HPAIV H5N1, but also facilitate the combination and optimization of these five mAbs and rational design of novel influenza vaccine.