Summary Newcastle disease virus (NDV) vectors expressing avian influenza virus (AIV) hemagglutinin (HA) of subtype H5 protect specific pathogen free (SPF) chickens from Newcastle disease (ND) and avian influenza (AI). Protection from ND of chickens with maternally derived NDV immunity was achieved after the replacement of the surface proteins F and HN of NDV with those of avian paramyxovirus serotype 8. However, maternal anti-AIV antibodies (AIV-MDA+) still interfered with active immunization, resulting in inefficient protection. To overcome this disadvantage, we inserted a transgene encoding a truncated soluble hemagglutinin (HA) in addition to the gene encoding membrane-bound HA from highly pathogenic avian influenza virus (HPAIV) H5N1 into lentogenic NDV Clone 30 genome (rNDVsolH5_H5). The biallelic expression increased the total amount of HA, expressed by the recombinant virus 5.25-fold compared to a single membrane-bound expressing NDV. Vaccination of three-weeks-old AIV-MDA+ chickens with rNDVsolH5_H5 and subsequent challenge infection with HPAIV H5N1 three weeks later resulted in 100% protection. Vaccination of younger chickens with higher AIV-MDA levels one week and two weeks after hatch, resulted in protection rates of 40 and 85%, respectively. However, all vaccinated chickens showed strongly reduced shedding of challenge virus, compared to age-matched, non-vaccinated control chickens. All control chickens succumbed to the HPAIV infection with a grading in disease progression between the three groups, indicating the influence of AIV-MDA even at a low level. Furthermore, the shedding and serological data gathered after immunization indicate sufficient replication of the vaccine virus. This leads to the assumption that lower protection rates in younger AIV-MDA+ chickens are caused by an H5 antigen-specific block and not by the interference of the AIV-MDA and the vaccine virus itself. In summary, solid protective efficacy and reduced virus transmission were achieved in three-weeks-old AIV-MDA+ chickens, which is relevant especially in regions endemically infected with HPAIV H5N1.

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