Development of Virus-like Particle Plant-Based Vaccines against Avian H5 and H9 Influenza A Viruses

Abstract

Avian influenza A virus (AIV) is a significant cause of mortality in poultry, causing substantial economic loss, particularly in developing countries, and has zoonotic potential. For example, highly pathogenic avian influenza (HPAI) viruses of the H5 subtype have been circulating in Egypt for around two decades. In the last decade, H5N1 viruses of clade 2.2.1 have been succeeded by the antigenically distinct H5N8 clade 2.3.4.4b viruses. Furthermore, H9N2 viruses co-circulate with the H5N8 viruses in Egyptian poultry. It is widely recognised that effective vaccination against IAV requires a close antigenic match between the vaccine and viruses circulating in the field. Therefore, approaches to develop cost-effective vaccines that can be rapidly adapted to local virus strains are required for developing countries such as Egypt. In this project, the haemagglutinin (HA) proteins of Egyptian H5 and H9 viruses were expressed by transient transfection of plants (Nicotiana benthamiana). The formation of virus-like particles (VLPs) was confirmed by transmission electron microscopy. Mice were immunised with four doses of either H5 or H9 VLPs with adjuvant. Antibody and cellular immune responses were measured against the corresponding recombinant protein using ELISA and enzyme-linked immunosorbent assay (ELISpot), respectively. Chickens were immunised with one dose of H5 VLPs, eliciting HA-specific antibodies measured by ELISA and a pseudotyped virus neutralisation test using a heterologous H5 HA. In conclusion, plant-based VLP vaccines have potential for producing an effective vaccine candidate within a short time at a relatively low cost.

Keywords: ELISpot; Nicotiana benthamiana; influenza; plant expression; pseudotyped virus neutralisation test; virus-like particles.

Figure 1

Purification of H5 and H9 VLPs using sucrose cushion ultracentrifugation. Panels (A) and (B) illustrate the stained SDS-PAGE gel and western blot of the H9 VLP purification, respectively, while panels (C,D) illustrate the stained SDS-PAGE gel and western blot of the H5 VLP purification, respectively. Panels (A,B), lane 1: H9 pellet, lane 2: H9 plant lysate, lane 3: syringe-filtered H9 plant lysate, lane 4: concentrated H9 VLPs and lane 5: collected supernatant after ultracentrifugation. Panels (C,D), lane 1: H5 plant lysate, lane 2: syringe-filtered H5 plant lysate, lane 3: concentrated H5 VLPs, lane 4: collected supernatant after ultracentrifugation and lane 5: negative control plant lysate infiltrated with an empty pEAQ-HT vector. Bands (6–9) represent the H9 VLP, trimer, dimer and monomer patterns, respectively. A smaller band (10) is suggestive of some protein degradation.

Figure 2

Negatively stained TEM of the H5 and H9 VLPs. Figures (A,B) represent the H9 and H5 VLP, respectively. The VLPs measure around 100 nm.

Figure 3

ELISA using H5 and H9 virus-like particles (VLPs) and pooled mice sera. Pre-immunisation sera sampled at day 0 and post-immunisation sera sampled at day 36 and day 63 were pooled from each pair of mice immunised with either H5 or H9 VLPs and diluted 1:1000. Pre-immunisation samples were added to wells of an ELISA plate coated with either H5 or H9 VLPs and post-immunisation sera samples were added to wells coated with either H5 or H9 VLPs or negative control plant lysate (NC). Mean OD at 450 nm for duplicate wells (±SEM) are shown.

Figure 4

Interferon-γ ELISpot results for spleen cells restimulated with either H5 or H9 VLPs or negative control plant lysate (NC) at 0.9 µg/mL. Cell-only control wells had a maximum of 6 spots. The results were calculated as mean (±SEM) of duplicate wells.

Figure 5

ELISA using H5 VLPs and sera from six chickens immunised with the H5 VLP vaccine. The negative control plant lysate derived from plants infiltrated with Agrobacteria containing an empty pEAQ-HT vector showed negligible reaction with an OD at 450nm of around 0.5. The results were calculated as mean (±SEM) of duplicate wells.

Figure 6

Percentage of neutralisation obtained with A/Vietnam/1194/2004 (H5N1) pseudotyped virus VNT with sera from six chickens immunised with the A/chicken/Egypt/FL6/2018 (H5N8) H5 VLP vaccine. The mean percentage neutralisation of negative chicken sera was −0.3%. The percentage of neutralisation from duplicate wells was calculated using the Excel sheet supplied by Nie et al. [35].