A broadly neutralizing human monoclonal antibody against the hemagglutinin of avian influenza virus H7N9

Abstract

Background: The new emerging avian influenza A H7N9 virus, causing severe human infection with a mortality rate of around 41%. This study aims to provide a novel treatment option for the prevention and control of H7N9.

Methods: H7 hemagglutinin (HA)-specific B cells were isolated from peripheral blood plasma cells of the patients previously infected by H7N9 in Jiangsu Province, China. The human monoclonal antibodies (mAbs) were generated by amplification and cloning of these HA-specific B cells. First, all human mAbs were screened for binding activity by enzyme-linked immunosorbent assay. Then, those mAbs, exhibiting potent affinity to recognize H7 HAs were further evaluated by hemagglutination-inhibiting (HAI) and microneutralization in vitro assays. Finally, the lead mAb candidate was selected and tested against the lethal challenge of the H7N9 virus using murine models.

Results: The mAb 6-137 was able to recognize a panel of H7 HAs with high affinity but not HA of other subtypes, including H1N1 and H3N2. The mAb 6-137 can efficiently inhibit the HA activity in the inactivated H7N9 virus and neutralize 100 tissue culture infectious dose 50 (TCID50) of H7N9 virus (influenza A/Nanjing/1/2013) in vitro, with neutralizing activity as low as 78 ng/mL. In addition, the mAb 6-137 protected the mice against the lethal challenge of H7N9 prophylactically and therapeutically.

Conclusion: The mAb 6-137 could be an effective antibody as a prophylactic or therapeutic biological treatment for the H7N9 exposure or infection.

Figure 1

In vitro binding and inhibition of HA for the human mAb. (A) The binding of mAb 6-137 with a panel of HAs was determined by ELISA. (B) The highest dilution Inhibited four HA unit of the inactivated H7N9 A/Nanjing/1/2013 was measured by the modified horse red blood cell haemagglutination-inhibition assay. The mAb 3-2 is an H7N9-irrelevant human mAb and is used as a human mAb control. The initial human mAb concentration (6-137 and 3-2) is 1.0 mg/mL. The initial dilution of the donor's serum is 1:10. ELISA: Enzyme-linked immunosorbent assay; HA: Hemagglutinin; HAI: Hemagglutinin-inhibiting; mAb: Monoclonal antibody.

Figure 2

Prophylactic and therapeutic efficacy of human mAb 6-137 protects mice against the challenge of H7N9. (A) BALB/c mice were dosed with two different concentrations of the human neutralizing mAbs and subsequently challenged for five MLD₅₀ of H7N9 influenza A/Anhui/1-YK_RG25/2013; (B) BALB/c mice were infected intranasally with 5 MLD₅₀ of H7N9 influenza A/Anhui/1-YK_RG25/2013 2 h before passively immunizing with the human neutralizing mAbs. Mice were followed for survival (left) and body weight change (right). mAb: Monoclonal antibody; MLD: Median lethal doses.

Figure 3

Therapeutic efficacy of human mAb 6-137 in mice after the challenge of H7N9 at 2, 6, and 24 h post-infection. Mice received mAb 6-137 at 20 mg/kg at 2, 6, and 24 h postinfection with Ave MLD₅₀ of H7N9 influenza A/Anhui/1/2013. Mice were followed for survival curves (left) and body weight loss (right). Control antibody (mAb 3-2) was administered at the highest concentration at 2 h post-challenge. mAb: Monoclonal antibody; MLD: Median lethal doses; p.i.: post infection.

Figure 4

The binding affinity of human neutralizing mAb 6-137 germline version. The binding affinity of the germline antibodies 6-137 (H-GL or L-GLor H-GL+ L-GL) with (A) H1, (B) H7 HA trimers, and (C) H7 HA1 were compared with the matured mAb 6-137 and determined by ELISA. ELISA: Enzyme-linked immunosorbent assay; HA: Hemagglutinin; H-GL: High-chain germline; L-GL: Light-chain germline; mAb: Monoclonal antibody.