Recombinant Human Anti-HA Antibody (S9-1-10/5-1) (CAT#: HPAB-N0125-YC)

Figure 1 In vivo protective efficacy of S9-1-10/5-1 in mice.

In vivo protective efficacy of 8 broadly reactive human mAbs in mice. Four mice per group were intraperitoneally injected with PBS or the indicated antibodies at 15 mg/kg. One day later, the mice were challenged with 10 MLD50 of VN1203 virus. Body weight and survival were monitored daily for 14 days.

Yamayoshi, S., Uraki, R., Ito, M., Kiso, M., Nakatsu, S., Yasuhara, A., ... & Kawaoka, Y. (2017). A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine, 17, 182-191.

Figure 2 In vivo protective efficacy of S9-1-10/5-1 in mice.

In vivo protective efficacy of S9-1-10/5-1 against H1, H3, H5, and H7 viruses. Four mice per group were intraperitoneally injected with PBS or S9-1-10/5-1 at 15, 5, 1.7, 0.6, or 0.2 mg/kg.

Yamayoshi, S., Uraki, R., Ito, M., Kiso, M., Nakatsu, S., Yasuhara, A., ... & Kawaoka, Y. (2017). A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine, 17, 182-191.

Figure 3 Inhibition of virus particle release by S9-1-10/5-1.

Plaque reduction assay in MDCK cells infected with CA04 virus pre- or post-treated with S9-1-10/5-1. For the pre-treatment assay, CA04 was incubated with S9-1-10/5-1 (red solid line), CR9114 (blue solid line), or 1429C6/3-3 (black solid line). MDCK cells (3 wells per condition) were incubated with the virusantibody mixture, washed twice to remove the antibody and uninfected virus, and then plaques were counted at 2 days post-infection. For the post-treatment assay, MDCK cells (4 wells per condition) were infected with CA04, washed twice, and then overlaid with agar in the presence of S9-1-10/5-1 (red dashed line), CR9114 (blue dashed line), or 1429C6/3-3 (black dashed line) for 2 days before the plaques were counted. The average plaque number in the mock treatment wells was set to 100%.

Yamayoshi, S., Uraki, R., Ito, M., Kiso, M., Nakatsu, S., Yasuhara, A., ... & Kawaoka, Y. (2017). A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine, 17, 182-191.

Figure 4 Inhibition of virus particle release by S9-1-10/5-1.

Detection of M1 in the cell lysates and supernatants of MDCK cells infected with CA04. MDCK cells were infected with CA04 at an MOI of 1 and then incubated with 10, 2, 0.4, or 0.08 μg/ml of S9- 1-10/5-1, 4-6-19/6 (anti-HA head), or 1429C6/3-3 (anti-B-HA) for 18 h. Cell lysates and supernatants were analyzed by western blotting. ACTB was detected as a loading control.

Yamayoshi, S., Uraki, R., Ito, M., Kiso, M., Nakatsu, S., Yasuhara, A., ... & Kawaoka, Y. (2017). A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine, 17, 182-191.

Figure 5 Inhibition of virus particle release by S9-1-10/5-1.

TEM images of MDCK cells infected with CA04. MDCK cells were infected with CA04 at an MOI of 1 and then incubated with 2 μg/ml of S9-1-10/5-1, 4-6-19/6, or 1429C6/3-3 (anti-B-HA) for 18 h. Ultrathin sections were examined by TEM. Scale bars,

Yamayoshi, S., Uraki, R., Ito, M., Kiso, M., Nakatsu, S., Yasuhara, A., ... & Kawaoka, Y. (2017). A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine, 17, 182-191.

Figure 6 Binding of S9-1-10/5-1 to H1- through H18-HA subtypes.

293T cells were mock transfected or transfected with plasmids encoding H1- to H18-HA subtypes. Expressed HAs were detected by S9-1-10/5-1 or CR9114, followed by an anti-human IgG conjugated with Alexa Fluor 488.

Yamayoshi, S., Uraki, R., Ito, M., Kiso, M., Nakatsu, S., Yasuhara, A., ... & Kawaoka, Y. (2017). A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine, 17, 182-191.

Figure 7 Epitope analysis of S9-1-10/5-1 on H1-HA.

Escape mutations from S9-1-10/5-1. Wild-type HA and mutant HAs expressed in 293T cells were stained with S9-1-10/5-1 or 4-6-19/6 (antiHA head)

Yamayoshi, S., Uraki, R., Ito, M., Kiso, M., Nakatsu, S., Yasuhara, A., ... & Kawaoka, Y. (2017). A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine, 17, 182-191.

Figure 8 Actions of S9-1-10/5-1 on virus entry and release.

Inhibition of viral HA-mediated membrane fusion by S9-1-10/5-1. Hela cells were transfected with a plasmid expressing wildtype CA04-HA. After trypsin treatment, the cells were incubated with the indicated human monoclonal antibody.

Yamayoshi, S., Uraki, R., Ito, M., Kiso, M., Nakatsu, S., Yasuhara, A., ... & Kawaoka, Y. (2017). A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine, 17, 182-191.

Figure 9 Actions of S9-1-10/5-1 on virus entry and release.

Binding of S9-1-10/5-1 to virus particles. Binding of the purified CA04/PB2-KO virus through a sucrose cushion to the immobilized human mAbs, S9-1-10/5-1, CR9114, 4- 6-19/6, and 1429C6/3-3, was measured by bio-layer interferometry.

Yamayoshi, S., Uraki, R., Ito, M., Kiso, M., Nakatsu, S., Yasuhara, A., ... & Kawaoka, Y. (2017). A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine, 17, 182-191.

Figure 10 Actions of S9-1-10/5-1 on virus entry and release.

Relative kobs to virus particles. Relative kobs, which is the observed rate constant, was calculated from the binding kinetics of human mAbs to three concentrations of CA04/PB2-KO virus. The kobs of S9-1-10/5-1 was set to 1.

Yamayoshi, S., Uraki, R., Ito, M., Kiso, M., Nakatsu, S., Yasuhara, A., ... & Kawaoka, Y. (2017). A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine, 17, 182-191.

Figure 11 Actions of S9-1-10/5-1 on virus entry and release.

Viral neuraminidase activity was not inhibited by S9-1-10/5-1. MDCK cells infected with CA04 at an MOI of 1 were mock-incubated or incubated with 100 μM oseltamivir carboxylate (OC), 0.4 or 2 μg/ml S9-1-10/5-1 (S9), or 0.4 or 2 μg/ml 4-6-19/6 (4-6) with or without 20 mU of bacterial sialidase derived from Clostridium perfringens.

Yamayoshi, S., Uraki, R., Ito, M., Kiso, M., Nakatsu, S., Yasuhara, A., ... & Kawaoka, Y. (2017). A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine, 17, 182-191.

Figure 12 Actions of S9-1-10/5-1 on virus entry and release.

Fab fragments of S9-1-10/5-1 failed to inhibit virus particle release. MDCK cells infected with CA04 at an MOI of 1 were incubated with 1, 4, 16, and 64 nM of IgG or Fab fragment of S9-1-10/5-1.

Yamayoshi, S., Uraki, R., Ito, M., Kiso, M., Nakatsu, S., Yasuhara, A., ... & Kawaoka, Y. (2017). A broadly reactive human anti-hemagglutinin stem monoclonal antibody that inhibits influenza A virus particle release. EBioMedicine, 17, 182-191.

Figure 1 Recombinant Human Anti-HA Antibody (HPAB-N0125-YC) in DB

Dot Blot analysis of HPAB-N0125-YC was performed by coating with HA protein (His tag).
HPAB-N0125-YC incubation concentration: 2 μg/mL.
HRP conjugated Anti-human IgG as a secondary antibody (1: 2000)

Figure 2 Recombinant Human Anti-HA Antibody (HPAB-N0125-YC) in ELISA

ELISA analysis of HPAB-N0125-YC was performed by coating with HA protein (His tag). Then blocked with BSA and incubated with HPAB-N0125-YC. The HRP conjugated Anti-human IgG as a secondary antibody (1: 2000). Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.