Immunization by avian H5 influenza hemagglutinin mutants with altered receptor binding specificity

Abstract

Influenza virus entry is mediated by the receptor binding domain (RBD) of its spike, the hemagglutinin (HA). Adaptation of avian viruses to humans is associated with HA specificity for alpha2,6- rather than alpha2,3-linked sialic acid (SA) receptors. Here, we define mutations in influenza A subtype H5N1 (avian) HA that alter its specificity for SA either by decreasing alpha2,3- or increasing alpha2,6-SA recognition. RBD mutants were used to develop vaccines and monoclonal antibodies that neutralized new variants. Structure-based modification of HA specificity can guide the development of preemptive vaccines and therapeutic monoclonal antibodies that can be evaluated before the emergence of human-adapted H5N1 strains.

Fig. 1

Structural and genetic basis for hemagglutinin mutations. (A) The RBDs of alternative viral hemagglutinins are shown. (B) Comparison of amino acid sequences in the major 130 and 220 loops and the 190 helix, color-coded in purple, lavender, and green, respectively.

Fig. 2

Altered specificity of the triple-mutant H5 compared with wild-type KAN-1 H5 coexpressed with NA. Glycan microarray analysis of (A) wild-type or (B) triple-mutant HA purified after coexpression with NA was performed by a modification (18) of a previous technique (12) performed by Core H, Consortium for Functional Genomics, Emory University. Glycans with related linkages are grouped by color: selected glycoproteins (orange), predominantly α2,3-sialosides (yellow), α2,6-sialosides (green), α2,8 ligands (blue), or others (purple), as previously shown (table S3).

Fig. 3

Altered neutralization sensitivity of mutant H5N1 pseudovirus. (A) Binding to HA coexpressed with NA in transfected 293T cells was determined by flow cytometry with the indicated mAbs (blue) or isotype control IgG (red). (B) Neutralization sensitivities were assessed with the indicated mAbs. (C) Neutralization sensitivities of the indicated wild-type and mutant HAs to these mAbs (400 ng/ml) are shown (ND, not done). (D) Neutralization sensitivities of wild-type and S137A,T192I mutant to mAb 9E8 and 11H12 are presented.