Dose immunogenicity study of a plant-produced influenza virus-like particle vaccine in layer hens.

Avian influenza poses one of the largest known threats to global poultry production and human health, but effective poultry vaccines can reduce infections rates, production losses and prevent mortalities, and reduce viral shed to limit further disease spread. The antigenic match between a vaccine and the circulating field influenza A viruses (IAV) is a critical determinant of vaccine efficacy. Here, an Agrobacterium tumefaciens-mediated transient tobacco plant (Nicotiana benthamiana) system was used to rapidly update an H6 influenza subtype virus-like particle (VLP) vaccine expressing the hemagglutininn (HA) protein of South African H6N2 IAVs circulating in 2020. Specific pathogen free White Leghorn layer hens vaccinated twice with ≥125 hemagglutinating unit (HAU) doses elicited protective antibody responses associated with prevention of viral shedding, i.e. hemaglutination inhibition (HI) mean geometric titres (GMTs) of ≥7 log2, for at least four months before dropping to approximately 5-6 log2 for at least another two months. A single vaccination with a 250 HAU dose induced significantly higher HI GMTs compared lower or higher doses, and was thus the optimal dose for chickens. Use of an adjuvant was essential, as the plant-produced H6 HA VLP alone did not induce protective antibody responses. Plant-produced IAV VLPs enable differentiation between vaccinated and infected animals (DIVA principle), and with sucrose density gradient-purified yields of 20,000 doses per kg of plant material, this highly efficacious, safe and economical technology holds enormous potential for improving poultry health in lower and middle-income countries.

Emergence of novel reassortant H6N2 avian influenza viruses in ducks in India.

The recent reports of human infection due to H6 subtype avian influenza viruses (AIV), which are prevalent in terrestrial poultry, indicate evolution of the virus to a possible pandemic strain. Here, we report antigenic and genetic characterization of two H6N2 viruses isolated from apparently healthy domestic ducks in Kerala and Assam, India during 2014 and 2015, respectively. Hemagglutination inhibition assay revealed antigenic divergence between the two isolates, which was corroborated by amino acid differences at 55 positions (15.98%) between their hemagglutinin (HA) 1.The sequence analyses indicated that both the viruses are avian origin with avian receptor specificity, low pathogenic to poultry and sensitive to oseltamivir. However, Kerala14 had V27I mutation marker for amantadine resistance in M2. The Assam15 virus had an additional N-linked glycosylation on HA2 (position 557) compared to Kerala14 virus. Phylogenetic analysis of the HA gene revealed that both the viruses belonged to distinct lineages (Eurasian and Asia II). Phylogeny of neuraminidase and internal gene segments revealed that both the viruses are novel reassortants and are genetically distinct with different gene constellations. The results suggest independent introductions of the two H6N2 viruses into India and migratory wild birds in the Central Asian flyway might be the source of H6N2 viruses in ducks in India. Therefore, continued AIV surveillance in poultry and wild birds is essential for early detection of emergence of novel strains with pandemic potential and control of their spread.