Development and Characterization of Novel Swine Influenza Virus-Like Particles (VLPs)

Swine influenza (SI) is an acute infectious disease of pigs caused by Swine influenza virus (SIV). To develop virus-like particles (VLPs) of SIV H1 and H3 subtype and determine their morphological structure and biological characteristics, this study synthesized a gene encoding lumazine synthase (LS) derived from a hyperthermophilic bacterium and introduced an immunoglobulin-binding domain sequence from streptococcal protein G (pG) to construct the pG-LS recombinant plasmid. The plasmid was then expressed in both prokaryotic and eukaryotic systems.After the conjugation of the 2 proteins, the morphology and the formation of VLP could be observed by transmission electron microscopy. Average particle diameters of the conjugated proteins were measured with dynamic light scattering method while the biological properties of the complex protein were analyzed by SDSPAGE, Western blot and hemagglutination assay. Both expression methods successfully yielded pG-LS nanoparticles, with eukaryotic expression demonstrating higher purity. The complexes formed between the two HA-Fc proteins and eukaryotic-expressed pG-LS nanoparticles exhibited a spherical morphology with an average diameter of approximately 80 nm.Further immunological analysis and particle diameter measurement both showed the successful construction of VLPs. Hemagglutination assays indicated HA-Fc alone could not agglutinate erythrocytes, while the virus-like particles prepared from 2 conjugated proteins showed exponential affinity, which could efficiently agglutinate red blood cells of different species. This study successfully constructed pG-LS nanoparticles and 2 pG-LS-HA VLPs, where HA proteins successfully displayed at the VLP surface. Both developed pG-LS-HA had stable property and biological function, with a potential of large-scale production. In the meantime, this study found that both H1 and H3 HA could be effectively displayed to form VLP, which shed light on the future use of multi-subtype influenza virus HA protein co-presentation strategy to develop novel, multivalent VLP vaccine candidates.