An Anti-Influenza Virus Antibody Inhibits Viral Infection by Reducing Nucleus Entry of Influenza Nucleoprotein

<div><p>To date, four main mechanisms mediating inhibition of influenza infection by anti-hemagglutinin antibodies have been reported. Anti-globular-head-domain antibodies block either influenza virus receptor binding to the host cell or progeny virion release from the host cell. Anti-stem region antibodies hinder the membrane fusion process or induce antibody-dependent cytotoxicity to infected cells. In this study we identified a human monoclonal IgG₁ antibody (CT302), which does not inhibit both the receptor binding and the membrane fusion process but efficiently reduced the nucleus entry of viral nucleoprotein suggesting a novel inhibition mechanism of viral infection by antibody. This antibody binds to the subtype-H3 hemagglutinin globular head domain of group-2 influenza viruses circulating throughout the population between 1997 and 2007.</p></div

Effect of CT302 IgG₁ on the membrane fusion and the traffic of viral nucleoprotein to nucleus.

<p>Entry fusion (EF) assay: Virus particles were labeled with DiOC18 and R18, and were allowed to enter CT302 IgG1-, Bafilomycin A1- or control IgG1-treated cells. Fusion of viral and vacuolar membranes of cells triggered dequenching of DiOC18 (green) signal co-localized with the R18 (Red) signal. Nuclear import (EI assay): In the CT302 IgG₁-, BafilomycinA1- or control IgG₁-treated cells, virus particles were allowed to enter nucleus. Incoming NP proteins (green) were detected within the nucleus (blue) by the anti-NP antibody. Magnification: 20×.</p

Microneutralization activity of CT302 IgG₁ against influenza virus strains.

<p>^aMN (microneutralization) titers are expressed as the lowest concentration of purified CT302 IgG₁ that completely neutralized the influenza virus.</p><p>^bNo detectable neutralization activity at the concentration of 20 μg/ml.</p><p>Microneutralization activity of CT302 IgG₁ against influenza virus strains.</p

Inhibition of hemagglutination and cell fusion by CT302 IgG₁.

<p>(A) Two-fold dilutions of CT302 IgG₁ (over the range of 20 to 0.039 μg/ml in PBS) were added to wells harboring 8 hemagglutination units of influenza virus, and chicken red blood cells (RBCs) were added to a final concentration of 0.5%. Control A wells contained only RBCs and influenza virus, without CT302 IgG₁. Control B wells contained only RBCs, without influenza virus or CT302 IgG₁. (B) CHO cells expressing HA protein of A/Brisbane/10/2007 strain were incubated with CT302 IgG₁ and then exposed to low-pH medium (pH 5.0) to induce membrane fusion. As controls, cells not treated with CT302 IgG₁ but exposed to low pH, or cells not treated with CT302 IgG₁ and not exposed to low pH, are shown. Magnification: 40×, 100×. TCID₅₀, tissue-culture infective dose required to cause cytopathic effects on 50% of inoculated cells.</p

Affinity constants (<i>K</i>_<i>a</i>, <i>K</i>_<i>d</i>) of CT302 IgG₁ determined by real-time interaction analysis.

<p><i>K</i>_<i>a</i>, association rate constant; <i>K</i>_<i>d</i>, dissociation rate constant; <i>K</i>_<i>D</i>, dissociation equilibrium constant calculated based on <i>K</i>_<i>d</i>/<i>K</i>_<i>a</i>; M, molarity.</p><p>Affinity constants (<i>K</i>_<i>a</i>, <i>K</i>_<i>d</i>) of CT302 IgG₁ determined by real-time interaction analysis.</p

Reactivity of CT302 IgG₁ to H3N2 trimeric HA.

<p>(A) After recombinant trimeric HA protein was coated onto 96-well plates, CT302 IgG₁ (■) or negative-control anti-respiratory syncytial virus IgG₁ (□) was incubated in wells as primary detection antibodies. Antibody bound to the recombinant His-tagged trimeric HA protein was detected with an HRP-conjugated anti-human IgG by addition of HRP substrate. Results represent the mean±S.D. obtained from duplicate wells per each condition. (B) Recombinant His-tagged trimeric HA proteins from H1, H3, and H5 strains were resolved by SDS-PAGE and transferred to nitrocellulose membranes, which were probed with CT302 IgG₁ or anti-His antibody. (C) Amino acid sequences in the V_H and V_L regions of CT302 IgG₁. HA, hemagglutinin; HRP, horseradish peroxidase.</p

Protective and therapeutic effects of CT302 IgG₁ in influenza virus-infected mice and ferrets.

<p>(A) CT302 IgG₁ was administered intraperitoneally to mice at -24, +24, or +48 hr from infection with A/NYMC X-171 strain (mouse-adapted A/Brisbane/10/2007 strain) (a, c) or A/Hong kong/1/1968 (b, d), and survival rate (a, b) and body weight change (c, d) were monitored twice daily up to 14 days. (B) Ferrets were challenged with the influenza virus and treated intravenously either with 30 mg/kg CT302 IgG₁ or control IgG₁ after 1 day. Subsequently, viral titers in the nasal fluid and lung tissue were determined. ^#p<0.05 (vs. PBS) determined by log-rank (Mantel-Cox) test. *p<0.05 (vs. control) on day 5, determined by Student’s t-test. TCID₅₀, tissue-culture infective dose required to cause cytopathic effects on 50% of inoculated cells.</p

Binding activity of CT302 IgG₁ to HA mutants.

<p>(A) Globular head domain of HA protein is composed of residues 52 to 277. The amino-acid sequences for HA protein were downloaded from the Influenza Virus Resource at the National Center for Biotechnology Information (NCBI). The NCBI accession numbers are indicated next to each strain name. (B) Locations of the seven residues (62, 121, 124, 133, 158, 196, and 276) that vary among the influenza strains that are either neutralized or not neutralized by CT302 IgG₁ are indicated in the three-dimensional structure of HA. The illustrations were constructed on H3N2 HA (Protein Data Bank accession code 4FNK) using molecular-visualization system software PyMOL 1.3. (C) HEK293T cells expressing wild-type or mutant HA were analyzed by flow cytometry using CT302 IgG₁, CT149 IgG₁ (an anti-stem-region IgG₁), or an anti-respiratory syncytial virus IgG₁ (negative control). (D) In real-time interaction analysis, sensor chips were first conjugated with anti-His antibody. Next, His-tagged recombinant HA protein of A/Brisbane/10/2007 stain was loaded, and the interaction of HA protein with CT302 IgG₁ or CT149 IgG₁ was examined by sequentially applying these antibodies. Either antibody bound after presaturation with the other antibody, indicating that these two antibodies bind noncompetitively to different epitopes. RU, resonance units.</p

SDS-PAGE of A/bat/Peru/10 HA0 (lanes 1 to 4) and its mature HA (lanes 5 to 8) in trypsin susceptibility assay.

<p>A/bat/Peru/10 HA with a monobasic cleavage site was expressed in its HA0 form in a baculovirus expression system. Lanes 1 and 2 show A/bat/Peru/10 native HA0 at pH 8.0 and pH 4.9, respectively, while lanes 3 and 4 show the equivalent reducing gel of HA0 treated with trypsin at pH 8.0 and pH 4.9, respectively. Similarly, lanes 5 and 6 show A/bat/Peru/10 mature HA at pH 8.0 and pH 4.9, respectively, while lanes 7 and 8 show the equivalent reducing gel of mature HA treated with trypsin at pH 8.0 and pH 4.9, respectively.</p

Important functional motifs in non-bat influenza A internal proteins are largely conserved in bat influenza proteins.

†<p>Letters written as subscripts denote amino acid substitutions in bat viruses compared to the consensus non-bat amino acid sequence.</p