Figure 4 - Glycan binding specificity of SC18 and Mos99 HAs, as well as H5N1 viruses.

(a) Both SC18 and Mos99 HAs show substantial and preferential binding to the apical side of the tracheal tissue (green against as against propidium iodide staining in red) compared with the alveolar tissue sections, although binding of SC18 is more restricted than that of Mos99. The sialic acid–specific binding of HA is demonstrated by a substantial reduction in binding after pretreatment of tissues with Sialidase A (Supplementary Fig. 4 online). Moreover, competition experiments involving SC18 HA and SNA-I showed dramatic reductions in SC18 HA binding to tracheal sections (data not shown). Further AV18 HA does not bind to tracheal sections (data not shown). The binding specificity of SC18 and Mos99 HAs was demonstrated with dose-dependent direct binding to defined 2-3 and 2-6 oligosaccharides (shown on the right). The characteristic binding pattern of SC18 and Mos99 HAs is their binding at saturating levels to the long 2-6 (6'SLN-LN) over a range of HA dilution from 40 to 5 g/ml. The narrow specificity of SC18 HA correlates with its restrictive tracheal tissue binding. On the other hand, the ability of Mos99 HA to bind to diverse sialylated glycans is consistent with its more extensive binding to tracheal tissue sections (as compared with SC18). The high affinity binding of SC18 and Mos99 HAs to long 2-6 was confirmed using another human-adapted H3N2 (A/Wyoming/3/03) HA (Supplementary Fig. 5 and Supplementary Methods online). (b) In contrast to the dose-dependent binding profile of the human-adapted SC18 and Mos99 HAs, Viet0304 and HK486 H5N1 viruses bind with high affinity to the 2-3 and minimal affinity to the long 2-6 oligosaccharide. The dose-dependent direct binding of the whole virus corroborates with the binding of soluble HA protein and the deep lung tissue tropism of the H5N1 HA is consistent with its 2-3 binding specificity (Supplementary Fig. 6 and Supplementary Methods online). HAU, Virus titer in Hemagglutinating Unit.