Investigation of Avian Influenza H5N6 Virus-like Particles as a Broad-Spectrum Vaccine Candidate against H5Nx Viruses

Abstract

Highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses have been reported to be the source of infections in several outbreaks in the past decades. In a previous study, we screened out a broad-spectrum virus strain, H5N6-Sichuan subtype, by using a lentiviral pseudovirus system. In this project, we aimed to investigate the potential of H5N6 virus-like particles (VLPs) serving as a broad-spectrum vaccine candidate against H5Nx viruses. We cloned the full-length M1 gene and H5, N6 genes derived from the H5N6-Sichuan into pFASTBac vector and generated the VLPs using the baculovirus-insect cell system. H5N6 VLPs were purified by sucrose gradient centrifugation, and the presence of H5, N6 and M1 proteins was verified by Western blot and SDS-PAGE. The hemagglutination titer of H5N6 VLPs after purification reached 5120 and the particle structure remained as viewed by electron microscopy. The H5N6 VLPs and 293T mammalian cell-expressed H5+N6 proteins were sent for mice immunization. Antisera against the H5+N6 protein showed 80 to 320 neutralizing antibody titers to various H5Nx pseudoviruses. In contrast, H5N6 VLPs not only elicited higher neutralizing antibody titers, ranging from 640 to 1280, but also induced higher IL-2, IL-4, IL-5, IFN-γ and TNF production, thus indicating that H5N6 VLPs may be a potential vaccine candidate for broad-spectrum H5Nx avian influenza vaccines.

Enhance immune response to H9 AIV DNA vaccine based on polygene expression and DGL nanoparticle encapsulation

“…To enhance the expression efficacy of the DNA vaccine gene, the AIV antigen gene isolated from the chicken was optimized and cloned into the pCAGGS vector, which is widely known to be highly expressed in eukaryotic cells because of its sequence containing the chicken β-actin promoter and CMV enhancer ( Jiang et al, 2007 ; Zhao et al, 2020 ).Using the optimized genes, we constructed 4 pCAGGS-optiDNAs expressing multiple HA or NA protein-coding genes, and found that the group containing 3 AIV fragments (pβH9N1SH5, pβH5N1SH9) had the fastest conversion rate and produced more significant HI, confirming that compared to the other vector combinations we investigated (monovalent or bivalent vaccines), trivalent antigenic vaccines were more able to induce H9N2 AIV HI antibodies. Subsequently, the virus-like particles ( VLPs ) formed by pβH9N1SH5 showed particle diameters of approximately 100 to 200 nm, which are consistent with the diameters of influenza viruses and influenza VLPs produced in different expression systems, such as mammalian cell expression systems and baculovirus-insect cell systems ( Wu et al, 2017 ; Yang et al, 2022 ). These results suggest that the pβH9N1SH5 plasmid expression efficiently assembles and releases VLPs, which in turn induce AIV HI antibodies.…”

Genetic and Cross Neutralization Analyses of Coxsackievirus A16 Circulating in Taiwan from 1998 to 2021 Suggest Dominant Genotype B1 can Serve as Vaccine Candidate

“…One milligram protein of rg-viruses was used to immunize BALB/c mice with the addition of Freund’s complete adjuvant (CFA, Sigma, St. Louis, MO, USA) for priming and Freund’s incomplete adjuvant (IFA, Sigma, St. Louis, MO, USA). At the end of the immunization period, mice were sacrificed and the cardiac chambers serum sample were collected [ 47 , 48 ].…”

Respiratory Viruses and Virus-like Particle Vaccine Development: How Far Have We Advanced?

“…The neutralizing potential of these sera was tested against pseudoviruses expressing H5 and a wide array of NA antigens. In almost all cases, irrespective of H5N2, H5N6, or H5N8 subtypes, neutralizing activity was greater in the sera acquired from the H5N6 VLP-immunized mice [ 29 ]. Insect cell-derived VLPs expressing the HA antigens of multiple clades were demonstrated to be more efficacious than single antigens.…”