Monoclonal antibodies targeting the influenza virus N6 neuraminidase

Abstract

Influenza A viruses are a diverse species that include 16 true hemagglutinin (HA) subtypes and 9 true neuraminidase (NA) subtypes. While the antigenicity of many HA subtypes is reasonably well studied, less is known about NA antigenicity, especially when it comes to non-human subtypes that only circulate in animal reservoirs. The N6 subtype NAs are mostly found in viruses infecting birds. However, they have also been identified in viruses that infect mammals, such as swine and seals. More recently, highly pathogenic H5N6 subtype viruses have caused rare infections and mortality in humans. Here, we generated murine mAbs to the N6 NA, characterized their breadth and antiviral properties in vitro and in vivo and mapped their epitopes by generating escape mutant viruses. We found that the antibodies had broad reactivity across the American and Eurasian N6 lineages, but relatively little binding to the H5N6 NA. Several of the antibodies exhibited strong NA inhibition activity and some also showed activity in the antibody dependent cellular cytotoxicity reporter assay and neutralization assay. In addition, we generated escape mutant viruses for six monoclonal antibodies and found mutations on the lateral ridge of the NA. Lastly, we observed variable protection in H4N6 mouse challenge models when the antibodies were given prophylactically.

Development and characterization of an antibody that recognizes influenza virus N1 neuraminidases

“…DA03E17 [ 28 ] and HCA-2 [ 36 ] are analogous to 1G04, 1E01, and 1G01, with consistent binding breadth. N1-C4 [ 37 ], reported by Job E R et al, and multiple antibodies [ 17 ], reported by Strohmeier et al, are some of the subtype-specific antibodies that target N1 and N6 NAs, respectively. These antibodies can also mediate NI and reduce virus replication in vitro.…”

“…As a sialidase, NA cleaves terminal sialic acids on glycans expressed on the host cell surface, enabling the release and spread of newly synthesized viruses [ 17 , 18 ]. Additionally, NA can break down mucins in the respiratory tract at the early stage of infection, allowing the virus to more efficiently penetrate the mucus layers, thereby playing an important role during infection [ 19 , 20 ].…”

Epitopes in the HA and NA of H5 and H7 avian influenza viruses that are important for antigenic drift

“…Amino acids 220 and 221 flank variable segment VI and are likely part of the same epitope, and the Mem5 epitope includes these amino acids (Venkatramani et al 2006 ). Several immune escape mutants at these positions have been reported, including N6 escape mutant with G220E that was associated with a strong reduction in NI activity (Strohmeier et al 2022 ) and an N9 escape mutant with R220Q (Webster et al 1987 ). A N2 immune escape mutant with D221H substitution has been described (Laver et al 1982 ).…”

“…Specifically, N6 escape mutants contained P245Q, N248S, or R249G/K substitutions in this region. N248S resulted in a significant reduction in neuraminidase inhibition (NI) activity and P246Q and R250G/K resulted in a complete loss of NI activity (Strohmeier et al 2022 ). The Mem5 Fab footprint maps to amino acids 249 and 251 in this region (Venkatramani et al 2006 ).…”

“…The first antigenic evolution study to experimentally characterize antigenic drift was first described in 1950 (Archetti and Horsfall 1950 ). To examine residues crucial to the antigenicity of H5 and H7 AIVs, experimentally derived HA and NA antigenic variants have been generated by in vitro or in ovo selection of escape mutants in the presence of monoclonal antibodies (mAbs) (Lentz et al 1984 , Air et al 1985 , 1990 , Webster et al 1987 , Philpott et al 1989 , Saito et al 1994 , Kaverin et al 2002 , 2007 , Chen et al 2009 , Krylov et al 2009 , Ferreira et al 2010 , Prabakaran et al 2010 , Rudneva et al 2010 , He and Kwang 2013 , Zhu et al 2013 , Itoh et al 2014 , Kobayashi-Ishihara et al 2014 , Henry Dunand et al 2015 , Kaverin et al 2015 , Tan et al 2015 , Claes et al 2016 , Henry Dunand et al 2016 , Thornburg et al 2016 , Wan et al 2016 , Gronsang et al 2017 , Ohkawara et al 2017 , Ito et al 2019 , Li et al 2019a , Okuda et al 2019 , Timofeeva et al 2020a , Xiong et al 2020 , Strohmeier et al 2022 , Chang et al 2023 , Lyashko et al 2024 ), polyclonal antiserum (Lambkin et al 1994 , Cleveland et al 1997 , Höper et al 2012 , Kalthoff et al 2013 , Sitaras et al 2014 , Chang et al 2019 , Sitaras et al 2020 ), or following infection of vaccinated birds (Hinshaw et al 1990 , Beato et al 2014 , Nguyen et al 2017 ). Antigenic variants that lead to evasion of the neutralizing host immune response may also be associated with changes in functional attributes, such as receptor binding dynamics (Hensley et al 2009 ), pH of fusion, or thermostability.…”