Multiple dose vaccination with heterologous H5N2 vaccine: Immune response and protection against variant clade 2.2.1 highly pathogenic avian influenza H5N1 in broiler breeder chickens

Circulation of an antigenically variant lineage of highly pathogenic avian influenza (HPAI) H5N1 virus in chicken breeder flocks in Egypt is a continuing problem. The protective efficacy of multiple repeated vaccinations using the currently available H5N2 vaccines is unclear. Here, broiler breeder chickens were vaccinated at weeks 6, 12 and 18 with an inactivated H5N2 commercial vaccine. HI-titer against an Egyptian H5N1 field isolate of classic clade 2.2.1 (EGYcls/H5N1) were significantly lower after the first immunization but increased after booster vaccinations. In contrast, no HI titers were induced against an antigenically distinct field virus of the variant lineage of clade 2.2.1 (EGYvar/H5N1). Upon challenge at week 50 mild, if any, clinical signs were observed in the group infected with EGYcls/H5N1 although one of eight (12.5%) birds died. Mortality reached 6/8 (75%) in the EGYvar/H5N1 challenge group. Virus excretion in all vaccinated groups was reduced in amplitude, but in vaccinated surviving birds, time of virus excretion was extended to up to ten days. Strikingly, challenged vaccinated birds kept laying eggs almost throughout the observation period. Virus was detected on the outer egg-shell of 17 of 40 eggs. The majority of the infected eggs were derived from the EGYcls/H5N1 challenged animals; here the virus was detected also in the yolk and albumin. Repeated vaccination using a commercial H5N2-based vaccine broadened the antigen profile of induced antibodies but did not provide adequate protection against heterologous virus variant. In addition, the observation of AIV contaminated eggs from infected flocks highlights the risk of silent virus spread by vaccinated animals and point to eggs as a possible vector.