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Many broadly neutralizing antibodies (bnAbs) bind to conserved areas of the hemagglutinin (HA) stalk region and can inhibit the low pH induced HA conformational changes necessary for viral membrane fusion activity. We developed and evaluated a high-throughput virus-free and cell-free ELISA based low pH induced HA Conformational Change Inhibition Antibody Detection Assay (HCCIA) and a complementary proteinase susceptibility assay. Human serum samples (n = 150) were tested by HCCIA using H3 recombinant HA. Optical density (OD) ratios of mAb HC31 at pH 4.8 to pH 7.0 ranged from 0.87 to 0.09. Our results demonstrated that low pH induced HA conformational change inhibition antibodies (CCI) neutralized multiple H3 strains after removal of head-binding antibodies. The results suggest that HCCIA can be utilized to detect and characterize CCI in sera, that are potentially broadly neutralizing, and serves as a useful tool for evaluating universal vaccine candidates targeting the HA stalk., Competing Interests: The authors have declared that no competing interests exist.

Unlabelled: We determined the antigenic structure of pandemic influenza A(H1N1)pdm09 virus hemagglutinin (HA) using 599 escape mutants that were selected using 16 anti-HA monoclonal antibodies (MAbs) against A/Narita/1/2009. The sequencing of mutant HA genes revealed 43 amino acid substitutions at 24 positions in three antigenic sites, Sa, Sb, and Ca2, which were previously mapped onto A/Puerto Rico/8/34 (A/PR/8/34) HA (A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417-427, 1982), and an undesignated site, i.e., amino acid residues 141, 142, 143, 171, 172, 174, 177, and 180 in the Sa site, residues 170, 173, 202, 206, 210, 211, and 212 in the Sb site, residues 151, 154, 156, 157, 158, 159, 200, and 238 in the Ca2 site, and residue 147 in the undesignated site (numbering begins at the first methionine). Sixteen MAbs were classified into four groups based on their cross-reactivity with the panel of escape mutants in the hemagglutination inhibition test. Among them, six MAbs targeting the Sa and Sb sites recognized both residues at positions 172 and 173. MAb n2 lost reactivity when mutations were introduced at positions 147, 159 (site Ca2), 170 (site Sb), and 172 (site Sa). We designated the site consisting of these residues as site Pa. From 2009 to 2013, no antigenic drift was detected for the A(H1N1)pdm09 viruses. However, if a novel variant carrying a mutation at a position involved in the epitopes of several MAbs, such as 172, appeared, such a virus would have the advantage of becoming a drift strain., Importance: The first influenza pandemic of the 21st century occurred in 2009 with the emergence of a novel virus originating with swine influenza, A(H1N1)pdm09. Although HA of A(H1N1)pdm09 has a common origin (1918 H1N1) with seasonal H1N1, the antigenic divergence of HA between the seasonal H1N1 and A(H1N1)pdm09 viruses gave rise to the influenza pandemic in 2009. To take precautions against the antigenic drift of the A(H1N1)pdm09 virus in the near future, it is important to identify its precise antigenic structure. To obtain various mutants that are not neutralized by MAbs, it is important to neutralize several plaque-cloned parent viruses rather than only a single parent virus. We characterized 599 escape mutants that were obtained by neutralizing four parent viruses of A(H1N1)pdm09 in the presence of 16 MAbs. Consequently, we were able to determine the details of the antigenic structure of HA, including a novel epitope., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Influenza A/H3N2 viruses have developed an increased number of glycosylation sites on the globular head of the hemagglutinin (HA) protein since their appearance in 1968. Here, the effect of addition of oligosaccharide chains to the HA of A/H3N2 viruses on its biological activities was investigated. We constructed seven mutant HAs of A/Aichi/2/68 virus with one to six glycosylation sites on the globular head, as found in natural isolates, by site-directed mutagenesis and analyzed their intracellular transport, receptor binding, and cell fusion activities. The glycosylation sites of mutant HAs correspond to representative A/H3N2 isolates (A/Victoria/3/75, A/Memphis/6/86, or A/Sydney/5/97). The results showed that all the mutant HAs were transported to the cell surface as efficiently as wild-type HA. Although mutant HAs containing three to six glycosylation sites decreased receptor binding activity, their cell fusion activity was not affected. The reactivity of mutant HAs having four to six glycosylation sites with human sera collected in 1976 was much lower than that of wild-type HA. Thus, the addition of new oligosaccharides to the globular head of the HA of A/H3N2 viruses may have provided the virus with an ability to evade antibody pressures by changing antigenicity without an unacceptable defect in biological activity., (Copyright 2004 American Society for Microbiology)

The haemagglutinin (HA) protein of influenza A/H2N2 virus possesses five oligosaccharide attachment sites, two of which have overlapping glycosylation sequons at positions 20-23 (NNST) and 169-172 (NNTS). Here, the role of these two oligosaccharide attachment sites is investigated with regard to antigenic property, intracellular transport and biological activity of the HA protein. Glycosylation-site HA mutants with mutation(s) in their overlapping glycosylated sequons, each of which had one or two oligosaccharide attachment sites removed, were constructed. Comparison of electrophoretic mobility between the wt and mutant HA proteins showed that both Asn residues 20 and 21 and Asn residues 169 and 170 could be used for glycosylation. Analysis of reactivity of the mutants with anti-HA monoclonal antibodies suggested that amino acid changes at these two positions result in a conformational change of the HA molecule. Even if oligosaccharide chains linked to Asn 20 or 21 and Asn 169 or 170 are eliminated, the antigenic properties, intracellular transport and biological activities are not influenced strongly. Thus it is reasonable to conclude that the two overlapping glycosylation sequons at positions 20-23 and 169-172 are conserved among all of the HAs of influenza A/H2N2 viruses because conservation of the amino acid sequence itself rather than that of N-glycosylation is essential for the formation of the proper conformation, intracellular transport and biological activities of the H2 subtype HA.

The haemagglutinin (HA) of influenza A/H2N2 virus possesses six antigenic sites (I-A to I-D, II-A and II-B), and sites I-A, I-B and I-C are located in the regions corresponding to sites A, B and D on the H3 HA. We demonstrated previously that most escape mutants selected by mAbs to site I-A, I-B or I-C had acquired a new oligosaccharide at position 160, 187 or 131, respectively, but this has never occurred during circulation of A/H2N2 virus in humans. Here, to examine whether the H2 HA has the potential to gain two new oligosaccharides on its tip, 31 double escape mutants were isolated by using a single escape mutant with an oligosaccharide at position 160, 187 or 131 as a parental virus and a mAb to an antigenic site different from that to which the mAb used for selection of the parental virus was directed as a selecting antibody, but there were no mutants with two new oligosaccharides. Glycosylation-site HA mutants containing one to three oligosaccharides at positions 160, 187 and 131 were also constructed and their intracellular transport and biological activities were analysed. The results showed that all of the mutant HAs were transported to the cell surface but exhibited a decrease in both receptor-binding and cell-fusing activities. Thus, influenza A/H2N2 virus may have failed to increase the number of oligosaccharides on the HA because, if this happens, the biological activities of the HA are reduced, decreasing the ability of the virus to replicate in humans.

The antigenic structure of influenza A/H2N2 virus haemagglutinin (HA) was analysed using 19 monoclonal antibodies (MAbs) against the HA of A/Kayano/57. The antibodies were classified into three groups: group I had both haemagglutination inhibition and neutralization activities, group II had neutralization activity but no haemagglutination inhibition activity and group III had neither activity. Analysis of escape mutants selected by each of the group I and II antibodies identified six distinct antigenic sites: four (I-A to I-D) were recognized by group I MAbs and two (II-A and II-B) were recognized by group II MAbs. Sequence analysis of the HA genes of the escape mutants demonstrated that sites I-A, I-B and I-C form a contiguous antigenic area that contains the regions corresponding to antigenic sites A, B and D on the H3 molecule and that sites I-D and II-B are the equivalents of sites E and C, respectively, suggesting that the antigenic structure of the H2 molecule is largely similar to that of the H3 molecule. However, the H2 molecule differed from the H3 molecule in having a highly conserved antigenic site (II-A) in the stem domain. It was also found that most of the escape mutants selected by antibodies to sites I-A, I-B and I-C acquired a new glycosylation site at position 160, 187 or 131, respectively, which indicates that A/H2N2 viruses have the potential to gain at least one additional oligosaccharide on the tip of the HA, although this has never occurred during 11 years of its circulation in humans.