Expression of recombinant Florida clade 2 hemagglutinin in baculovirus expression system: A step for subunit vaccine development against H3N8 equine influenza virus.

Overview

• This study focuses on producing a recombinant hemagglutinin (HA) protein from the Florida clade 2 (FC2) strain of the H3N8 equine influenza virus using a baculovirus expression system.
• The goal is to create a vaccine candidate that better reflects currently circulating EI viruses and supports the development of updated subunit vaccines for equine influenza prevention.

Background on Equine Influenza Virus (EIV)

• Equine influenza (EI) is a contagious respiratory infection affecting horses, caused by influenza A viruses.
• Two subtypes have caused EI historically: H7N7 and H3N8; however, H7N7 has not been detected in many years.
• H3N8 continues to circulate and has evolved into two lineages: Eurasian and American.
• The American lineage further split into sub-lineages, including Kentucky, South America, and Florida types.
• The Florida lineage has two clades currently circulating globally: Florida clade 1 (FC1) and Florida clade 2 (FC2).

Vaccine Context and Need for Update

• Vaccination remains the primary strategy to prevent and control EI infections in horses.
• Several EI vaccines exist, developed using various technologies and platforms.
• The World Organization for Animal Health (WOAH) recommends that all commercial EI vaccines include HA antigens representative of both FC1 and FC2 strains to ensure broad protection.
• Most existing vaccines have not been updated to include FC2 strain antigens, which may reduce their effectiveness against currently circulating viruses.
• This gap motivated the development of a vaccine candidate incorporating the FC2 HA protein.

Research Objectives and Methodology

• The main objective: to express the full-length HA gene from the FC2 strain using a baculovirus expression system.
• The baculovirus system is commonly used for recombinant protein expression, capable of producing properly folded and biologically active proteins.
• The researchers cloned the FC2 HA gene and expressed it recombinantly, aiming to verify that the protein retained its key biological functions.

Validation of Recombinant HA Protein

• Biological activity was assessed through functional assays:
  • Hemadsorption (HAD) assay: tests the ability of HA to bind red blood cells, reflecting proper folding and receptor-binding functionality.
  • Hemagglutination test: measures the capacity of HA to agglutinate red blood cells, confirming functionality.
• Specificity was confirmed using:
  • Immunoperoxidase assay with a reference-specific serum to detect HA antigen expression in cells.
  • Western immunoblotting that identified the HA protein by size and confirmed antigen specificity.
• These experiments demonstrated that the recombinant HA protein is biologically active and antigenically specific to FC2.

Conclusions and Implications

• The study successfully expressed a functional, specific recombinant HA protein from the FC2 strain using baculovirus technology.
• This recombinant HA protein lays the groundwork for developing new EI vaccines, especially subunit vaccines or vaccines based on virus vectors, which incorporate the FC2 strain.
• Inclusion of FC2 antigens aligns with WOAH recommendations and may enhance vaccine efficacy against currently circulating EI viruses.
• The approach could lead to vaccines that better protect horses and reduce EI outbreaks worldwide.