The Epitope and Neutralization Mechanism of AVFluIgG01, a Broad-Reactive Human Monoclonal Antibody against H5N1 Influenza Virus

The continued spread of highly pathogenic avian influenza (HPAI) H5N1 virus underscores the importance of effective antiviral approaches. AVFluIgG01 is a potent and broad-reactive H5N1-neutralizing human monoclonal antibody (mAb) showing great potential for use either for therapeutic purposes or as a basis of vaccine development, but its antigenic epitope and neutralization mechanism have not been finely characterized. In this study, we first demonstrated that AVFluIgG01 targets a novel conformation-dependent epitope in the globular head region of H5N1 hemagglutinin (HA). By selecting mimotopes from a random peptide library in combination with computational algorithms and site-directed mutagenesis, the epitope was mapped to three conserved discontinuous sites (I-III) that are located closely at the three-dimensional structure of HA. Further, we found that this HA1-specific human mAb can efficiently block both virus-receptor binding and post-attachment steps, while its Fab fragment exerts the post-attachment inhibition only. Consistently, AVFluIgG01 could inhibit HA-mediated cell-cell membrane fusion at a dose-dependent manner and block the acquisition of pH-induced protease sensitivity. These results suggest a neutralization mechanism of AVFluIgG01 by simultaneously blocking viral attachment to the receptors on host cells and interfering with HA conformational rearrangements associated with membrane fusion. The presented data provide critical information for developing novel antiviral therapeutics and vaccines against HPAI H5N1 virus

Development of Co-Based Amorphous Composite Coatings Synthesized by Laser Cladding for Neutron Shielding

Advanced amorphous coatings consisting of Co-based metallic glasses with ultrahigh strength (6 GPa) and high microhardness (up to 17 GPa) can significantly improve the surface properties of matrix materials. However, the intrinsic brittleness of Co-based metallic glasses can lead to the initiation of microcracks caused by the inevitable generation of thermal stress during the laser cladding process, which severely limits the potential application. In this paper, the methods of increasing substrate temperature and fabricating composite coatings with the addition of toughened Fe powders were adopted to inhibit the generation of microcracks in the Co55Ta10B35 amorphous coatings. Moreover, neutron shielding performances of the cladding coatings with high B content were investigated with a wide range of neutron energy (wavelength: 0.15–0.85 nm). The results indicate that the fully amorphous coating and composite ones can be fabricated successfully. The increase in the substrate temperature and the addition of Fe powders can effectively inhibit the initiation and propagation of microcracks. The fully Co-based amorphous coating with high B content (35 at.%) can exhibit excellent neutron shielding performance. With the addition of Fe powders, the neutron shielding performance is reduced gradually due to the dilution effect of B in the composite cladding coatings, but the microcrack will be completely restrained

Reactivity of AVFluIgG01 with different HA antigens.

<p><b>A</b>. Representative sequences of synthesized HA peptides (Viet04). <b>B</b>. Reactivity of AVFluIgG01 with the recombinant HA or HA1 proteins (Viet04) and overlapping peptides in ELISA. <b>C</b>. Reactivity of AVFluIgG01 with the native and reduced HA proteins (HK06) in ELISA. Synthesized peptides at 5 µg/ml or recombinant HA proteins at 1 µg/ml were used to coat 96-well microtiter plates, and AVFluIgG01 or a control mAb was tested at a final concentration of 10 µg/ml.</p

Inhibition of AVFluIgG01 on the pH-induced conformational changes of HA.

<p>The recombinant Viet04 HA proteins were incubated with or without AVFluIgG01 (<b>A</b>) or its Fab (<b>B</b>) at 37°C for 1 h. The mixture was either held at pH 7.5 (lanes 2, 4, 6 and 8) or changed to pH 5.0 (lanes 1, 3, 5 and 7) at 37°C for 1 h. After the pH was restored to 7.5, trypsin (1 µg/ml) was added to detect the acquisition of pH-induced protease susceptibility (lanes 5 to 8). The samples were then analyzed by western blotting. Molecular markers are given on the left.</p

Surface representation of the AVFluIgG01 epitope on the globular head of HA trimer model (PDB ID: 2IBX).

<p>Amino acid positions are designated in H5 numbering. <b>A</b>. Side view of the trimeric HA structure. The epitopic site I residues (I116, I117, P118) is colored in yellow, and site II (W122, S123) and site III (Y164, T167) residues are colored in blue and green, respectively. <b>B</b>. Top view of the globular head. The region encompassing the receptor binding site (RBS) is colored in red. <b>C</b>. Cartoon and sticks illustration of the three-dimensional structure of the AVFluIgG01 epitope. The diagram was generated with PyMOL.</p

Prediction of AVFluIgG01 epitope by minotopes.

<p><b>A</b>. Affinity-selected minotopes from a random peptide phage display library. The selection frequency of a specific minotope is shown in parentheses. <b>B</b>. Predicted amino acid residues by Mapitope. <b>C</b>. Predicted amino acid residues by Pep-3D-Search. The common residues that are predicted by both algorithms are underlined and shown in bold.</p

Inhibition of AVFluIgG01 on HA-mediated cell-cell fusion.

<p>A. AVFluIgG01 inhibits the low-pH induced cell-cell fusion in Vero cells that were transfected with the plasmid encoding Viet04 HA protein (pViet04-HA). The cells were stained with crystal violet. (a) Control cells (no HA transfection); (b) Positive control (without antibodies); (c) Treated with 50 µg/ml AVFluIgG01; (d) Treated with 50 µg/ml control mAb (AVFluIgG03). <b>B</b>. AVFluIgG01 inhibits cell fusion between 293T effector cells and MDCK target cells at a dose-dependent manner. 293T cells were co-transfected with pViet04-HA and pGAL4-VP16 and MDCK cells were transfected with pGal5-luc. After fusion induced by low-pH, the luciferase activity was measured in the cell lysates. The percent inhibition and IC₅₀ were calculated.</p

Mutagenesis analyses of the predicted epitope residues.

<p><b>A</b>. The reactivity of AVFluIgG01 with wild-type (WT) HA or HA containing site-directed mutations analyzed by flow cytometry. <b>B</b>. The reactivity of AVFluIgG01 with the WT (red curve) or HA carrying single mutations (green curve) analyzed by immuofluorescence assay (IFA). In both assays, the WT HA or mutants were transiently expressed on 293T cells by transfection and then immunostanined by AVFluIgG01 or a control mAb at a final concentration of 10 µg/ml.</p