Henju Marjuki, Vasiliy P. Mishin, Ning Chai, Man-Wah Tan, Elizabeth M. Newton, John Tegeris, Karl Erlandson, Melissa Willis, Joyce Jones, Todd Davis, James Stevens, Larisa V. Gubareva

The pandemic threat posed by emerging zoonotic influenza A viruses necessitate development of antiviral agents effective against various antigenic subtypes. Human monoclonal antibody (hmAb) targeting the hemagglutinin (HA) stalk offers a promising approach to control influenza virus infections. Here we investigated the ability of hmAb 81.39a to inhibit in vitro replication of human and zoonotic viruses, representing 16 HA subtypes. The majority of viruses were effectively neutralized by 81.39a, EC50 <0.01-4.9μg/ml. Among group 2 HA viruses tested, a single A(H7N9) virus was not neutralized at 50μg/ml; it contained HA2-Asp19Gly, an amino acid position previously associated with resistance to neutralization by the group 2 HA-neutralizing mAb CR8020. Notably, among group 1 HA viruses, H11-H13, and H16 subtypes were not neutralized at 50μg/ml; they shared a substitution HA2-Asp19Asn/Ala. Conversely, H9 viruses harboring HA2-Asp19Ala were fully susceptible to neutralization. Therefore, amino acid variance at HA2-Asp19 has subtype-specific adverse effects on in vitro neutralization. Mice given a single injection (15 or 45 mg/kg) at 24 or 48 hours after infection with recently emerged A(H5N2), A(H5N8), A(H6N1) or A(H7N9) viruses were protected from mortality and showed drastically reduced lung viral titers. Furthermore, 81.39a protected mice infected with A(H7N9) harboring HA2-Asp19Gly, although the antiviral effect was lessened. A(H1N1)pdm09-infected ferrets receiving a single dose (25 mg/kg) had reduced viral titers and showed less lung tissue injury, despite 24-72 hours delayed treatment. Taken together, this study provides experimental evidence for the therapeutic potential of 81.39a against diverse influenza A viruses. Zoonotic influenza viruses, such as A(H5N1) and A(H7N9) subtypes, have caused severe disease and deaths in humans raising public health concerns. Development of novel anti-influenza therapeutics with a broad spectrum of activity against various subtypes is necessary to mitigate disease severity. Here we demonstrate that the hemagglutinin (HA) stalk-targeting human monoclonal antibody 81.39a effectively neutralized the majority of influenza A viruses tested, representing 16 HA subtypes. Furthermore, 81.39a delayed treatment significantly suppressed virus replication in the lungs, prevented dramatic body weight loss and increased survival rates of mice infected with A(H5Nx), A(H6N1) or A(H7N9) viruses. When tested in ferrets, 81.39a delayed treatment reduced viral titers, particularly in the lower respiratory tract, and substantially alleviated disease symptoms associated with severe A(H1N1)pdm09 influenza. Collectively, our data demonstrated the effectiveness of 81.39a against both seasonal and emerging influenza A viruses.