Antibody titer has positive predictive value for vaccine protection against challenge with natural antigenic-drift variants of H5N1 high-pathogenicity avian influenza viruses from Indonesia

Abstract

Vaccines are used in integrated control strategies to protect poultry against H5N1 high-pathogenicity avian influenza (HPAI). H5N1 HPAI was first reported in Indonesia in 2003, and vaccination was initiated in 2004, but reports of vaccine failures began to emerge in mid-2005. This study investigated the role of Indonesian licensed vaccines, specific vaccine seed strains, and emerging variant field viruses as causes of vaccine failures. Eleven of 14 licensed vaccines contained the manufacturer's listed vaccine seed strains, but 3 vaccines contained a seed strain different from that listed on the label. Vaccines containing A/turkey/Wisconsin/1968 (WI/68), A/chicken/Mexico/28159-232/1994 (Mex/94), and A/turkey/England/N28/1973 seed strains had high serological potency in chickens (geometric mean hemagglutination inhibition [HI] titers, ≥ 1:169), but vaccines containing strain A/chicken/Guangdong/1/1996 generated by reverse genetics (rg; rgGD/96), A/chicken/Legok/2003 (Legok/03), A/chicken/Vietnam/C57/2004 generated by rg (rgVN/04), or A/chicken/Legok/2003 generated by rg (rgLegok/03) had lower serological potency (geometric mean HI titers, ≤ 1:95). In challenge studies, chickens immunized with any of the H5 avian influenza vaccines were protected against A/chicken/West Java/SMI-HAMD/2006 (SMI-HAMD/06) and were partially protected against A/chicken/Papua/TA5/2006 (Papua/06) but were not protected against A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Experimental inactivated vaccines made with PWT/06 HPAI virus or rg-generated PWT/06 low-pathogenicity avian influenza (LPAI) virus seed strains protected chickens from lethal challenge, as did a combination of a commercially available live fowl poxvirus vaccine expressing the H5 influenza virus gene and inactivated Legok/03 vaccine. These studies indicate that antigenic variants did emerge in Indonesia following widespread H5 avian influenza vaccine usage, and efficacious inactivated vaccines can be developed using antigenic variant wild-type viruses or rg-generated LPAI virus seed strains containing the hemagglutinin and neuraminidase genes of wild-type viruses.

Importance: H5N1 high-pathogenicity avian influenza (HPAI) virus has become endemic in Indonesian poultry, and such poultry are the source of virus for birds and mammals, including humans. Vaccination has become a part of the poultry control strategy, but vaccine failures have occurred in the field. This study identified possible causes of vaccine failure, which included the use of an unlicensed virus seed strain and induction of low levels of protective antibody because of an insufficient quantity of vaccine antigen. However, the most important cause of vaccine failure was the appearance of drift variant field viruses that partially or completely overcame commercial vaccine-induced immunity. Furthermore, experimental vaccines using inactivated wild-type virus or reverse genetics-generated vaccines containing the hemagglutinin and neuraminidase genes of wild-type drift variant field viruses were protective. These studies indicate the need for surveillance to identify drift variant viruses in the field and update licensed vaccines when such variants appear.

FIG 1

Indirect potency test based on the mean serological titers obtained following a single immunization of 3-week-old chickens with 15 different vaccines and by use of the homologous vaccine antigen in the HI test. Threshold lines for the minimum titers that protect from mortality (dotted line; titer, 1:32) and challenge virus shedding from the oropharynx (dashed line; titer, 1:128) are included.

FIG 2

Phylogenetic tree of representative H5N1 isolates from Indonesia and other countries. DK and Dk, duck; TK, turkey; CK and Ck, chicken; GS, goose. The numbers on nodes are bootstrap values.

FIG 3

Cartographic map of H5 avian influenza viruses produced with chicken antisera. The colored shapes (viruses) and open shapes (antisera) are relative positions adjusted such that the distances between viruses and antisera in the map represent the corresponding HI measurements with the least error. The blue fill represents the antigenic root from classic H5 influenza viruses, including four viruses of wild bird origin (A/mallard/Netherlands/3/1999 [MA/NETHERLANDS/3/1999], A/mallard/Sweden/49/2002 [MA/SWEDEN/49/2002], A/mallard/Sweden/7/2002 [MA/SWEDEN/7/2002], and A/mallard/Sweden/21/2002 [MA/SWEDEN/21/2002]) and four viruses of poultry origin (A/turkey/Wisconsin/1968 [TU/WISCONSIN/1968], A/turkey/England/N28/1973 (TU/ENGLAND/N28/1973], A/turkey/Ireland/1983 [TU/IRELAND/1983], and A/chicken/Mexico/23294/1994 [CH/Mexico/23294/1994]). The other solid colors represent six H5N1 Guangdong lineage viruses (A/chicken/Hong Kong/220/1997 [CH/HK/220/1997], A/chicken/Legok/2003 [CH/LEGOK/2003], A/Vietnam/1203/2004 [VN/1203/04], A/chicken/West Java/SMI-HAMD/06 [SMI-HAMD/06], A/chicken/Papua/TA5/06 [Papua/06], and A/chicken/West Java/PWT-WIJ/06 [PWT/06]). Since the relative positions of antigens and antisera are determined and both the vertical and horizontal axes represent antigenic distance, the orientation of the map within these axes is free. The spacing between grid lines is 1 unit of antigenic distance, corresponding to a 2-fold dilution of antiserum in the HI assay.

FIG 4

Alignment of the HA1 amino acid sequences of the vaccine and challenge viruses used in these studies. Orange shading, amino acids identified to be important for antigenic recognition by previous studies (41, 42, 44, 45, 75); blue shading, amino acids identified to be part of the receptor binding site; asterisks, potential N-linked glycosylation sites; red arrow, the proteolytic cleavage site; boxes, the loop at position 130, the helix at position 190, and the loop at position 220. Note that amino acids at positions higher than position 138 appear to have numbering 1 greater than their typical numbering due to an insertion in the A/turkey/England/N28/1973 isolate.

FIG 5

Survival of 3-week-old chickens vaccinated with each vaccine seed strain and challenged 3 weeks later with H5N1 HPAI viruses SMI-HAMD/06 (A), Papua/06 (B), and PWT/06 (C).

FIG 6

Survival of 3-week-old chickens immunized with vaccines containing seed strains Eng/73, rgLegok/03 or Legok/03, rgGD/96, and Mex/94 and challenged 3 weeks later with antigenic variant H5N1 HPAI virus PWT/06.

FIG 7

Survival of 3-week-old chickens immunized with experimental vaccines containing inactivated wild-type PWT/06 or rgPWT/06 or a combination of rFPV-AI-H5 (1 day of age) and Legok/03 at 3 weeks of age and challenged 3 weeks later with antigenic variant H5N1 HPAI virus PWT/06.