Glycosylation at 158N of the hemagglutinin protein and receptor binding specificity synergistically affect the antigenicity and immunogenicity of a live attenuated H5N1 A/Vietnam/1203/2004 vaccine virus in ferrets

Abstract

A live attenuated influenza A/Vietnam/1203/2004 (H5N1) vaccine virus (VN04 ca) has receptor binding specificity to alpha2,3-linked sialosides (alpha2,3SAL), and a single dose induces a minimal serum antibody response in mice and ferrets. In contrast, A/Hong Kong/213/2003 (H5N1) vaccine virus (HK03 ca) binds to both alpha2,6SAL and alpha2,3SAL and generates a stronger serum antibody response in animals. Among the 9 amino acids that differed between the two H5 HA1 proteins, several HK03-specific residues enabled the VN04 ca virus to bind to both alpha2,3SAL and alpha2,6SAL receptors, but only the removal of the 158N glycosylation, together with an S227N change, resulted in more-efficient viral replication in the upper respiratory tract of ferrets and an increased serum antibody response. However, the antibody response was HK03 strain specific and did not significantly cross-neutralize VN04 virus. A second approach was taken to adapt the H5N1 VN04 ca virus in MDCK cells to select HA variants with larger plaque morphology. Although a number of large-plaque-size HA variants with amino acid changes in the HA receptor binding region were identified, none of these mutations affected virus receptor binding preference and immunogenicity. In addition, the known receptor binding site changes, Q226L and G228S, were introduced into the HA protein of the VN04 ca virus. Only in conjunction with the removal of the 158N glycosylation did the virus replicate efficiently in the upper respiratory tract of ferrets and became more immunogenic, yet the response was also HK03 specific. Thus, the mask of the antigenic epitopes by 158N glycosylation at the HA globular head and its alpha2,3SAL binding preference of VN04 ca virus affect virus antigenicity and replication in the host, resulting in a lower antibody response.

FIG. 1.

Plaque morphology of the H5N1 VN04 ca virus passaged in MDCK cells. MDCK cells were infected with the VN04 ca virus at a MOI of 0.01, and virus supernatants collected at passages 1, 3, and 6 were examined by plaque assay in MDCK cells. The viruses in the later passages exhibited large-plaque morphology compared to the viruses in the earlier passage.

FIG. 2.

Examination of glycosylation of the H5N1 HA proteins by Western blotting. Viruses were amplified in eggs, concentrated by centrifugation, and electrophoresed on 10% polyacrylamide gel containing 0.1% SDS. The HA1 proteins were detected by using polyclonal anti-VN04 antibody. The 158N glycosylated (CHO-HA1) with 160T and nonglycosylated HA1 (HA1) with 160A proteins are indicated.