Influenza Neuraminidase Characteristics and Potential as a Vaccine Target

Abstract

Neuraminidase of influenza A and B viruses plays a critical role in the virus life cycle and is an important target of the host immune system. Here, we highlight the current understanding of influenza neuraminidase structure, function, antigenicity, immunogenicity, and immune protective potential. Neuraminidase inhibiting antibodies have been recognized as correlates of protection against disease caused by natural or experimental influenza A virus infection in humans. In the past years, we have witnessed an increasing interest in the use of influenza neuraminidase to improve the protective potential of currently used influenza vaccines. A number of well-characterized influenza neuraminidase-specific monoclonal antibodies have been described recently, most of which can protect in experimental challenge models by inhibiting the neuraminidase activity or by Fc receptor-dependent mechanisms. The relative instability of the neuraminidase poses a challenge for protein-based antigen design. We critically review the different solutions that have been proposed to solve this problem, ranging from the inclusion of stabilizing heterologous tetramerizing zippers to the introduction of inter-protomer stabilizing mutations. Computationally engineered neuraminidase antigens have been generated that offer broad, within subtype protection in animal challenge models. We also provide an overview of modern vaccine technology platforms that are compatible with the induction of robust neuraminidase-specific immune responses. In the near future, we will likely see the implementation of influenza vaccines that confront the influenza virus with a double punch: targeting both the hemagglutinin and the neuraminidase.

Keywords: antigenic drift; correlate of protection; influenza; monoclonal antibodies; neuraminidase; vaccines.

Figure 1

Structure of neuraminidase and its catalytic site. (A) Side view and (B) top view of N1 NA (PDB 6Q23). NA is a homotetrameric type II membrane protein consisting of a head domain, stalk domain, and a transmembrane domain (TMD) and cytoplasmic tail that together form the signal anchor sequence. In general, NA stalk domains contain a cysteine residue (Cys) involved in intermolecular disulphide bond formation. The inset in panel (B) shows the catalytic site with the residues that interact with the sialic acid-containing substrate depicted in red, and the residues that stabilize the catalytic site labelled in orange. Ca²⁺ ions are shown as green spheres.

Figure 2

Important role for NA in the virus life cycle. NA contributes to virus motility, allowing the virus to move through the mucus layer and to reach functional receptors at the cell surface. NA also plays an essential role at the end of the virus life cycle by removing sialic acids from the cell surface thereby allowing efficient release of virions and preventing virion aggregation.

Figure 3

NA structural conservation. Structural alignments of all seasonal influenza vaccine included influenza strains in the period 1970-2021.The sequence conservation of Influenza A N1, Influenza A N2 and Influenza B NA amino acid residues was visualized using MUSCLE and shown on a crystal structure of the respective NA subtype using the render by conservation function in CHIMERA. Residues conserved in all sequences of a specific subtype NA are shown in red. Further distinction of conservation is indicated from dark blue (99% conservation) till yellow (1% conservation). Structure representations based on N1: PDB 4B7Q, N2: PDB 4GZX, and IBV NA: PDB 4CPL. Arrows point towards the catalytic site.

Figure 4

Antigenic regions of N1 NA-specific mAbs. The A/California/04/2009 (H1N1) tetramer is depicted with one protomer in ribbon representation. The catalytic residues are depicted in red. For each region, a representative antibody footprint is shown: 1G01 (105) in pink (PDB 6Q23) with antibody contact residues in NA that overlap with the catalytic site in dark red; CD6 (103) in gold (PDB 4QNP) and N1-7D3 (113) in light blue.

Figure 5

Design of next-generation NA-based vaccines. (A) The NA antigen can be presented in the native membrane-bound form, as a soluble protein that lacks the transmembrane domain with or without modifications to retain or stabilize the tetrameric structure, or as peptides containing an epitope of interest. (B) Depending on the antigen design, various methods for vaccine delivery are possible. Soluble NA can be administered as a subunit vaccine or coupled to a nanoparticle carrier. Membrane-bound NA can be presented on a virus-like particle or on the cell surface when encoded as RNA or DNA delivered directly or by a viral vector. (C) Strategies to increase the breadth of the immune response include mixing of NAs from different strains, computational design of consensus NAs, or hetero-multivalent mosaic presentation of NAs from different strains on a single particle.