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The H5 highly pathogenic avian influenza viruses (HPAIVs) in China pose a serious challenge to public health and the poultry industry. In this study, we constructed a replication-competent recombinant influenza A virus of clade 2.3.4.4 Н5N1 expressing the clade 2.3.2.1 H5 HA1 protein from a tricistronic NS segment. We used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. H5 HA1 and nuclear export information were fused in frame with a truncated NS1 open reading frame, separated by 2A self-processing sites. The resulting PR8-H5-NS1(73)H5 stably expressed clade 2.3.4.4 H5 HA and clade 2.3.2.1 H5 HA1 proteins and exhibited similar  in vitro  growth kinetics as the parental PR8-2344H5 virus. PR8-H5-NS1(73)H5 induced specific hemagglutination-inhibition (HI) antibody against clade 2.3.4.4 H5 that was comparable to that of the combination vaccine of PR8-2344H5 and PR8-2321H5. HI antibody titers were significantly lower against clade 2.3.2.1 H5 virus than with the combination vaccine. PR8-H5-NS1(73)H5 completely protected chickens from both clade 2.3.4.4 and clade 2.3.2.1 H5 HPAIVs challenge. Our results suggested that PR8-H5-NS1(73)H5 was highly immunogenic and efficacious against both clade 2.3.4.4 and clade 2.3.2.1 H5 HPAIVs in chickens.

Influenza viral vectors expressing two kinds of HA proteins for bivalent vaccines against clade 2.3.4.4 and clade 2.3.2.1 H5 HPAIVs