Unraveling of a neutralization mechanism by two human antibodies against conserved epitopes in the globular head of H5 hemagglutinin

Abstract

The rapid spread of highly pathogenic avian influenza (HPAI) H5N1 virus underscores the importance of effective antiviral treatment. Previously, we developed human monoclonal antibodies 65C6 and 100F4 that neutralize almost all (sub)clades of HPAI H5N1. The conserved 65C6 epitope was mapped to the globular head of HA. However, neither the 100F4 epitope nor the neutralization mechanism by these antibodies was known. In this study, we determined the 100F4 epitope and unraveled a neutralization mechanism by antibodies 65C6 and 100F4.

Fig 1

Amino acid residues involved in the neutralization epitope of 100F4. (A) Broad neutralization activity (95% inhibitory concentration [IC₉₅]) of antibodies 65C6 and 100F4 against a panel of H5N1 pseudotypes reproduced from reference . Green, >1 μg/ml required to reach IC₉₅; yellow, between 0.5 and 1 μg/ml required to reach IC₉₅; red, <0.5 μg/ml required to reach IC₉₅. (B) List of 15 single amino acid mutants that antibody 100F4 can no longer bind to, obtained by using yeast Saccharomyces cerevisiae that displays a random mutagenesis library of HA fragment comprising amino acid residues 51 to 260 in fine epitope mapping. Among them, 8 amino acid mutants are underneath the HA surface and the other 7 are on the surface of HA. (C) Titration of antibody 100F4 against H5N1 pseudotypes expressing 7 single surface amino acid mutants compared to the results against H5N1 pseudotype expressing the parental HA. (D) Amino acid residues D68 and E112 (D72 and E116 in H3 numbering) involved in 100F4 epitope are highlighted by blue shading. (E) Amino acid residues D68 and E112 are highlighted in red and blue, respectively, in space-filling model of HA. Yellow, pink, and grey each indicate one of three monomers that make up an HA trimer. (F) 100F4 and 65C6 epitopes in the context of known neutralization epitopes in the H1 HA structure (Protein Data Bank [PDB] structure accession number IRU7), as follows: Ca1, cyan; Ca2, light green; Cb, magenta; Sa, forest green; and Sb, orange. Yellow, overlap amino acid residues between the 65C6 epitope and the Sa site. (G) 100F4 and 65C6 epitopes in the context of known neutralization epitopes in H3 HA structure (PBD structure accession number 2VIU), as follows: site A, forest green; site B, orange; site C, yellow-green; site D, cyan; and site E, magenta. Yellow, overlap amino acid residues between the 65C6 epitope and site A. (H) 100F4 and 65C6 epitopes in the context of known neutralization epitopes detected by human MAb in H5 HA structure (PBD structure accession number 2ibx). Neutralization epitopes detected by human MAb are highlighted in forest green, and amino acid residues that overlap amino acid residues of the 65C6 epitope are highlighted in yellow. (I) 100F4 and 65C6 epitopes in the context of known neutralization epitopes detected by mouse MAb in H5 HA structure (PBD structure accession number 2ibx). Neutralization epitopes detected by mouse MAb are highlighted in forest green, and amino acid residues that overlap amino acid residues of 65C6 epitope are highlighted in yellow.

Fig 2

Determination of antibody-driven escape mutants. (A) Antibody-driven escape mutants were generated through a series of passages. (B) Point mutations found in two 100F4 antibody-driven escape clones compared to the sequence of parental H5 HA. (C) Sequence alignment of amino acid residues 65 to 75 and 110 to 119 among 21 HA covering all H5 clades and subclades. Residues 68 and 112 are highlighted in boxes.

Fig 3

Determination of effects of antibodies 65C6 and 100F4 on virus neutralization, attachment, postattachment, and fusion. (A) Neutralization activity against H5N1 pseudotype A/Shenzhen/406H/06 by indicated concentrations of antibodies 65C6, 100F4, AVIg03, F10, and PG16. The neutralization activity was calculated as follows: relative luciferase activity (RLA) of pseudotype with a given concentration of an antibody/RLA of pseudotype alone without antibodies. (B) Effects of indicated concentrations of antibodies 65C6, 100F4, AVIg03, F10, and PG16 on binding of H5N1 pseudotype A/Shenzhen/406H/06 to MDCK cells compared to binding of pseudotype alone (without antibodies). (C) Effects of indicated concentrations of antibodies 65C6, 100F4, AVIg03, F10, and PG16 on postattachment entry of H5N1 pseudotype A/Shenzhen/406H/06 into MDCK cells compared to entry of pseudotype alone (without antibodies). (D) Effects of antibodies 65C6, 100F4, AVIg03, F10, and PG16 on pH 5.0-triggered, HA-mediated cell-cell fusion among influenza H5 HA-transduced HeLa cells. (E) Mean and standard deviation of fusion percentages in each image in panel D. The fusion percentages were calculated as described in the text.

Fig 4

Bound antibodies 65C6 and 100F4 are internalized and block low-pH-induced fusion. (A) Confocal microscopy of virus internalization into CHO-K1 cells with or without antibody 100F4. Cell samples were collected at 2, 40, and 360 min postinfection as indicated. Green, antibody 100F4 staining; red, viral NP staining; blue, DAPI staining for nucleus. (B) Colocalization of antibody 100F4 and viral NP, as determined by Pearson's correlation coefficients, was quantified at 2, 40, and 360 min postinfection. ***, P < 0.001. (C) Blockage of low-pH-triggered, wild-type H5 HA-mediated fusion by antibodies 65C6 and 100F4 but not PG16 control. Mean and standard deviation of fusion percentage in each image is shown below. (D) Blockage of low-pH-triggered, escape A of H5 HA-mediated fusion by antibody 65C6 but not by antibody 100F4 and PG16 control. Mean and standard deviation of fusion percentage in each image is shown below.