Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5N1 influenza virus vaccines

Abstract

If H5N1 influenza viruses become transmissible among humans, vaccination will offer the most effective option to limit their spread. Two human vaccine candidates recently generated by reverse genetics are based on antigenically different hemagglutinin (HA) glycoproteins derived from the A/HK/213/03 (H5N1) and A/Vietnam/1203/04 (H5N1) viruses. Their HA1 amino acid sequences differ at 10 positions, one of which (N154) introduces a potential glycosylation site in A/Vietnam/1203/04 (H5N1). To assess the impact of five amino acids in the putative antigenic sites on immunogenicity and immune protection, we generated a series of whole-virus vaccines that differed only in one or two HA amino acids. Sera from ferrets vaccinated with these inactivated preparations had high virus neutralization titers, but their hemagglutination inhibition (HI) titers were usually low. Interestingly, a recombinant virus in which the HA amino acid S223 (characteristic of 2004 viruses) was converted to N223 (as in A/HK/213/03) resulted in higher HI titers. This observation indicates that specific HA residues, such as N223, increase the sensitivity of the HI assay by altering receptor specificity and/or antibody-antigen binding. Ferrets vaccinated with mutant vaccine viruses were protected against lethal challenge with wild-type A/Vietnam/1203/04 virus. Our results suggest that inclusion of the N223 residue in the HA glycoproteins of diagnostic reference viruses may facilitate the evaluation of vaccine efficacy in humans.

Fig. 1.

HI antibody titers in ferrets inoculated with H5N1 influenza viruses isolated in 2003 and 2004 (A) and HI and virus neutralizing titers in ferrets immunized with ΔH5N1/03 and ΔH5N1/04 viruses (B). (A) Sera were collected on day 28 after inoculation with 10⁶ eID₅₀ of H5N1 viruses and titrated against 4 HAUs of homologous virus. Data are representative values from two or four sera. (B) Sera were collected from ferrets vaccinated twice with 7 μg of HA (see Materials and Methods) of ΔH5N1/03 and ΔH5N1/04 viruses and titrated against 4 HAUs and 100 tissue culture 50% infective dose of homologous virus, respectively.

Fig. 2.

Virus titers in nasal washes of vaccinated and control ferrets after challenge with A/Vietnam/1203/04 (H5N1). Ferrets vaccinated with ΔH5N1/04 or ΔH5/04 recombinant viruses were inoculated intranasally with 10⁶ eID₅₀ of A/Vietnam/1203/04 virus. Titers are the mean values (log₁₀ eID₅₀/0.1 ml) ± SD determined in the nasal washes of three ferrets.

Fig. 3.

Location of amino acid at positions 154 and 223 in the 3D structure of H5 HA. (A) The amino acids in the 3D structure of the HA of A/duck/Singapore/3/97 (H5N3) virus (18). The glycosylation site at position 154 is located at the top of the HA molecule. (B) The circle represents the interface between the monomer shown and two other monomers (data not shown) in the trimeric HA. Amino acid at position 223 is located at the surface of the trimer.