Abstract
The highly pathogenic avian influenza virus H5N1 is a major threat to global public health and therefore a high-priority target of current vaccine development. The receptor-binding site (RBS) on the globular head of hemagglutinin (HA) in the viral envelope is one of the major target sites for antibody recognition against H5N1 and other influenza viruses. Here, we report the identification and characterization of a pair of human RBS–specific antibodies, designated FLD21.140 and AVFluIgG03, that are mutually complementary in their neutralizing activities against a diverse panel of H5N1 viruses. Crystallographic analysis and site-directed mutagenesis revealed that the two antibodies share a similar RBS-binding mode, and their individual specificities are governed by residues at positions 133a, 144, and 145. Specifically, FLD21.140 preferred Leu-133a/Lys-144/Ser-145, whereas AVFluIgG03 favored Ser-133a/Thr-144/Pro-145 residue triplets, both of which perfectly matched the most prevalent residues in viruses from epidemic-originating regions. Of note, according to an analysis of 3758 H5 HA sequences available in the Influenza Virus Database at the National Center for Biotechnology Information, the residues Leu-133a/Ser-133a and Ser-145/Pro-145 constituted more than 87.6 and 99.3% of all residues at these two positions, respectively. Taken together, our results provide a structural understanding for the neutralizing complementarity of these two antibodies and improve our understanding of the RBS-specific antibody response against H5N1 infection in humans.
Highlights
5, FLD21.140 and AVFluIgG03 demonstrated strong neutralizing activities against these mutants and a high degree of tolerance toward residue variations within and surrounding the RBS. Both antibodies maintained equivalent or even stronger neutralizing potency against mutant H5N1 strains that acquired ability in respiratory droplets transmission among ferrets (Table 5) [8, 9, 48]. These results suggest that FLD21.140 and AVFluIgG03 are capable of neutralizing almost all mutant H5N1 strains either induced in the mouse model or artificially generated in vitro
RBS-specific antibodies play critical roles in protecting the host from influenza virus infection. They are by far the most predominant among the isolated antibodies and perhaps the best characterized despite their structural and functional diversity [34, 42,43,44,45,46,47,48,49]. Identifying their unique as well as common features will greatly improve our understanding of their protective role in vivo and guide the future design of more effective vaccines against influenza virus infection
Over the last few years, while studying a panel of human monoclonal antibodies isolated from H5N1-infected and convalescent patients, we identified the two antibodies FLD21.140 and AVFluIgG03 that are mutually complementary in their binding and neutralizing activities against a diverse panel of H5N1 strains
Summary
FLD21.140 and AVFluIgG03 neutralize distinct but complementary subsets of H5N1 influenza viruses. To identify critical residues that differentiate the specificity and complementarity of FLD21.140 and AVFluIgG03, we generated a total of 52 single, 6 double, and 2 triple mutant HA envelope sequences on the backbone of clade 1 A/Vietnam/ 1203/2004 and clade 2.3.4 A/Anhui/1/2005 (Table 3). For mutation were the contacting residues within and surrounding the RBS identified through the structural analysis (Table S3) and their prevalence among 18 diverse H5N1 strains initially used for characterization of the two antibodies (Fig. 3). Pseudoviruses bearing these mutant HA envelope sequences were generated and subjected to sensitivity analysis by FLD21.140 and AVFluIgG03 neutralization. 10.5 b together, these results indicate that the residues at positions 133a, 144, and 145 play a critical role in determining the specificity of FLD21.140 and AVFluIgG03, whereas those at the remaining positions of the epitope were recognized by both antibodies with similar activity
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