# Highly Conserved Influenza A Nucleoprotein as a Target for Broad-Spectrum Intervention: Characterization of a Monoclonal Antibody with Pan-Influenza Reactivity

## TL;DR

A new monoclonal antibody called 2D8 can detect multiple influenza A subtypes, offering a tool for universal diagnostics and vaccines.

## Contribution

The antibody 2D8 targets a highly conserved NP protein epitope, enabling broad detection of influenza A subtypes.

## Key findings

- 2D8 mAb cross-reacts with H1, H3, H5, H7, and H9 influenza subtypes.
- The antibody binds to a conserved NP motif (38RFYIQMCTEL47) present in 35 global virus samples.
- 2D8 shows no cross-reactivity with unrelated viruses and has an IgG2b/κ subclass with high titer.

## Abstract

The influenza A virus poses a persistent global threat to human health and the poultry industry, as it evolves rapidly and can cross species to spread. To enhance our ability to track these viruses, scientists are seeking “universal” tools that can identify multiple different influenza virus strains at once. In this study, we developed a special monoclonal antibody named 2D8, which targets the NP protein of the influenza virus. This protein is a part of the virus that remains almost unchanged across different influenza subtypes. Our tests showed that this antibody can identify multiple influenza subtypes, including those found in birds, pigs, and humans, such as H1, H3, H5, H7, and H9. We also identified the precise antigenic epitope where this antibody binds to the virus and confirmed that this site is almost identical in 35 different virus samples from around the world. These findings are of great significance to society, as they provide a blueprint for creating faster and more reliable diagnostic tests and help in developing “universal” vaccines that can prevent multiple influenza types with just one injection.

Influenza A viruses remain a persistent global health challenge due to their rapid antigenic evolution, zoonotic potential, and pandemic threat. Universal countermeasures targeting conserved viral components are urgently needed to enhance diagnostic, surveillance, and therapeutic capabilities. Here, we report the generation and characterization of a high-affinity monoclonal antibody (2D8 mAb) against the nucleoprotein (NP) of the H9N2 avian influenza virus, a subtype with increasing relevance to human infections. Importantly, 2D8 mAb exhibited robust cross-reactivity with a broad spectrum of influenza A viruses, including H1, H3, H5, H7, and H9 subtypes, while showing no cross-reactivity with unrelated viral pathogens. Epitope mapping identified its binding target as a highly conserved NP motif 38RFYIQMCTEL47, which is invariant across all major human influenza A lineages. Isotyping revealed 2D8 mAb to be of the IgG2b/κ subclass, with an exceptionally high titer (1:20,480,000) as determined by ELISA. Given the essential role of NP in viral replication and host adaptation, this antibody offers a powerful platform for next-generation diagnostic assays capable of detecting a wide range of human and zoonotic influenza A viruses using a single reagent. Moreover, it holds potential for guiding the design of universal antiviral strategies targeting structurally constrained regions of the influenza virus. Our findings provide a valuable resource for advancing pan-influenza A interventions, with direct implications for improving pandemic preparedness and strengthening global influenza surveillance in both clinical and public health settings.