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Anti #Matrix Protein 1 Monoclonal #Antibody Neutralizes #Influenza A Virus Subtypes

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By G.M.

Abstract

Background: 

Research on monoclonal antibodies (mAb) targeting conserved internal proteins of influenza is limited.The matrix protein 1 (M1), the most abundant and conserved internal protein, serves as an endoskeleton bridging cytoplasmic tails of envelope glycoproteins haemagglutinin (HA), neuraminidase (NA) and matrix protein 2 (M2) with viral ribonucleoprotein particles (vRNPs). Clinical studies reveal significant M1 antibody responses post-infection and vaccination, with demonstrated B and T cell recognition. Our study examines 2B-B10-G9, our lab-synthesized mAb targeting conserved linear epitope of M1 at the C-terminal domain (CTD). 

Methods: 

Binding of 2B-B10-G9 to the purified influenza A viruses (IAV) and influenza B viruses (IBV) were assessed using SDS-PAGE and Western blotting with Image J analysis. Purified viruses included IAV (H1N1, Pandemic (H1N1) 2009 (H1N1pdm09), and H3N2 subtypes) and IBV which was first isolated in 1940 (B/Lee/40), and B/Victoria lineage. Cytotoxicity of 2B-B10-G9 on the Madin-Darby canine kidney (MDCK) cells was studied by MTT (3-(4, 5-dimethylthiazol-2)-2, 5-diphenyltetrazolium bromide) cell proliferation assay to establish in vitro and in ovo treatment doses. In ovo studies involved injecting 2B-B10-G9 into the allantoic cavity of specific pathogen free (SPF) 10-day-old embryonated chicken eggs (ECE) immediately post-infection with influenza A/Puerto Rico/8/1934 (H1N1) followed by RT-qPCR analysis of the virulence genes HA, NA, and PB1 expressions in the allantoic fluid (AF) 48 hours post-infection. 

Results: 

mAb 2B-B10-G9 shows significantly stronger binding to the M1 protein of H1N1 subtype of IAV with respect to H3N2 subtype of IAV (p < 0.001), and it has significantly stronger binding to the M1 protein of H1N1 subtype of IAV with respect to H1N1pdm09 subtype, IBV including B/Lee/40, B/Victoria lineage. (p< 0.0001) mAb 2B-B10-G9 at dose of 40 ug/ml which was determined as an optimal dose according to the MTT assay, significantly reduced plaque-forming units (PFU)/ml of IAV H1N1 and H3N2 subtypes in vitro (p< 0.0001). In ovo treatment of IAV H1N1 subtype at the same dose 40 ug/ml, significantly suppressed the virulence genes HA, NA, and PB1 expressions compared to the untreated group (p< 0.0001, p< 0.001, p< 0.0001, respectively) and mouse isotype IgG1 mAb treated groups (p< 0.01). 

Conclusions: 

These findings highlight that anti-M1 mAb 2B-B10-G9, targeting conserved linear epitope at the CTD of M1 protein, might be considered as a possible therapeutic option for IAV H1N1 and H3N2 subtypes related to its significant recognition and binding, viral neutralization and suppression of expression of virulence genes including HA, NA and PB1.

Source: BioRxIV, https://www.biorxiv.org/content/10.1101/2025.06.09.658662v1

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