The Fine Antigenic Variation Within The Antigenic Sites Of Haemagglutinin Of H5N1 Influenza Virus

In 1997, an H5N1 influenza virus crossed the species barrier to directly transmit from birds to humans in Hong Kong and caused 18 cases of illness including six deaths. New genotypes of the H5N1 virus continued to circulate and occasionally caused severe human disease in Asia. A lack of protective immunity in the human population against the H5 influenza virus subtype presents a significant risk of an influenza pandemic.The rapid spread of influenza viruses provide an opportunity to study the host-virus evolutionary system. Mutations within the virus protein can allow virus to escape from antibody recognition. Antigenic drift is a continuous process in influenza viruses and is the basis for the evolution of influenza viruses.Antigenic drift of the influenza virus is mainly caused by the accumulation of mutations in the antigenic regions of the haemagglutinin (HA). The HA protein on all influenza viruses consists of two chains, HA1 and HA2. The HA1 mutates more frequently than HA2 and undergoes strong Darwinian selection for novel variants. Codon usage at the H3 HA epitopic residues are biased toward diversification relative to the nonepitopic residues. In fact, one or two amino acids changes in the HA1 of H3 subtype viruses could influence their antigenicity. Previous studies have indicated the existence of immunodominant positions in the H3N2 viruses. It would be also worthwhile to identify the immunodominant positions of H5 HA to improve the immunogenicity of vaccine strains.Epitope mapping of HA is essential to understanding the mechanism of antigenic drift in this protein. We demonstrated that all of the 15 mAbs-specific epitopes are located in the HA1 region of HA in the A/HongKong/482/97 H5N1 isolate. 10 mAbs (H5M2, 5, 9, 11, 12, 24-26 and 28-29) recognized non-overlapping or partially overlapping antigenic sites in amino acid region 1-302 of HA. The smaller H5 HA fragments within this region disrupt this disulfide bond, resulting in the structural alterations to which antibody binding is sensitive. The mAb H5M20 recognized a linear epitope located in the region of amino acids 69-133.We analyzed the evolution of H5 HA by using publicly available H5N1 sequence data. We have found surprising differences in residue variation of H5 HA and H3 HA. We have found that many residues which show a signature of diversifying selection in the H3 HA exhibit little codon or amino acid diversifying in the H5 HA. We have also found some residues outside the defined 131 residues in the antigenic region that do show diversifying selection. It is possible that these residues may be new antigenic sites of H5N1 viruses.The epitopes recognized by mAbs H5M6, H5M7, H5M8 and H5M10 is located in the region of amino acids 69-302. Seven point mutations that we generated in this region provided additional information about the contribution of single amino acid substitution on the antigen-antibody reactivity. The results demonstrated that all the substitutions except on position 217 altered the reactivity pattern of HA13F with mAbs to different extent. The substitution on position 124 dramatically altered the reactivity pattern with mAbs, indicating the existence of immunodominant positions on H5 HA protein.Homology modeling of the HA1 subunit structure before and after point mutation indicated that structural changes caused by point mutation might in some cases explain the altered reactivity patterns. The substitution at position 124 changed the partial electric charge distribution in the neighboring area. It seems that position 124, although not located in the antigenic site B in the H3 HA, might provide the structural foundation of the epitopes recognized by these 4 mAbs as changes in this position lead to the structural alterations to which antibody binding is sensitive.One of the major questions we face in making a human vaccine for avian influenza virus is which candidate strain should be used. The antigenic drift observed since 1997 within the H5N1 subtype influenza virus demonstrates an urgent need for vaccine updating to make sure it continually represents the latest strains. Our study revealed differences in the evolution of H5 HA and H3 HA. We have discussed how the structural changes caused by a point mutation might in some cases explain the altered antigenicity of the HA protein. The results also demonstrate the existence of immunodominant positions in the H5 HA protein. Therefore, it may be worthwhile to explore the vaccine potential of H5 HA with alteration of immunodominant amino acids in future study.