Changes in the antigenicity of the hemagglutinin molecule of H3 influenza virus at acidic pH

In order to determine the location and biological significance of the acid-induced conformational change in influenza virus hemagglutinin (HA) reported by Skehel et al., monoclonal antibodies were prepared to the molecule before and after treatment at pH 5.0. These antibodies together with monoclonal antibodies to the different antigenic regions of the H3 HA were used in immunoprecipitation and ELISA binding studies to show that antigenic changes accompanied the conformational change in the HA. Treatment at pH 5.2 or less exposed new determinants on the HA while two antigenic regions, located at the tip and interface of the molecule at neutral pH, were lost or modified. Antigenic sites in the loop and hinge regions defined by the available monoclonal antibodies were not affected by the conformational change. Monoclonal antibodies specific for the acid-induced conformation efficiently inhibited hemagglutination of the virus at low pH but were extremely poor inhibitors of virus-induced red blood cell hemolysis at its pH optimum of 5.1. These antibodies were unable to neutralize viral infectivity under neutral or acidic conditions. Antibodies specific for the non-acid-treated HA conformation failed to inhibit hemagglutination at low pH values but were able to both inhibit hemolysis of red blood cells and neutralize virus infectivity. Residual unmodified HA after pH 5.0 treatment could explain the inhibition of hemolysis and infectivity by monoclonal antibodies in each of the different antigenic areas.     

Inhibition of virus-induced hemolysis with monoclonal antibodies to different antigenic areas on the hemagglutinin molecule of A/seal/Massachusetts/1/80 (H7N7) influenza virus

A/seal/Massachusetts/1/80 (H7N7) influenza virus caused maximal hemolysis at pH 5.9 Monoclonal antibodies to each of the four nonoverlapping antigenic areas on the hemagglutinin molecule of the virus inhibited the hemolysis whereas those belonging to two of the groups did not inhibit hemagglutination of the virus. Hemolysis also occurred when the virus was incubated at pH 5.9 prior to addition of erythrocytes. Such hemolysis caused by acid-treated virus was inhibited with the antibodies as well. At pH 5.9, hemagglutination of neither intact virus nor hemagglutinin rosettes was inhibited with any of the monoclonal antibodies, indicating conformational change in the hemagglutinin molecule, at this pH. On the other hand, hemagglutination-inhibition was observed when the antigens were incubated with the monoclonal antibodies at pH 7.0 and then the pH was later shifted to 5.9, suggesting that antibody-binding interferes with the conformational change in the hemagglutinin molecule at pH 5.9. The present findings indicate that antibodies to the hemagglutinin of influenza virus can inhibit virus-induced hemolysis by blocking conformational change in the hemagglutinin molecule and blocking later step of fusion than the conformational change, in addition to blocking attachment of virus to the receptor of erythrocytes.     

Characterization of H2 influenza virus hemagglutinin with monoclonal antibodies: influence of receptor specificity

Antigenic analysis of human and avian H2 influenza viruses were done with monoclonal antibodies to the HA molecules in hemagglutination inhibition (HI) assays. These studies revealed that the receptor-binding specificity of the hemagglutinin can markedly influence the antigenic analysis obtained with monoclonal antibodies in HI tests. Influenza viruses that are sensitive or resistant to inhibition by horse serum inhibitors showed marked differences in their reactivity with monoclonal antibodies to the hemagglutinin. This was apparent with the A/RI/5+/57 and A/RI/5-/57 strains of H2N2 viruses isolated by Choppin and Tamm (1960a), half of the panel of different monoclonal antibodies failed to inhibit hemagglutination of the RI/5- variant, whereas all of the 18 monoclonal antibodies inhibited RI/5+. These findings have important implications in the antigenic analysis of influenza viruses where HI assays are conventionally used to determine the extent of antigenic drift in nature. Antigenic differences were detectable between different human H2 influenza virus isolates from 1957 that were sensitive to inhibition by horse serum, indicating that minor antigenic variation occurs within the first year of appearance of the new subtype. Minor antigenic variation continued in the H2 viruses until 1961, but by 1962 antigenically distinguishable variants that could be discriminated with both monoclonal antibodies and postinfection ferret antisera predominated. Analysis of avian H2 influenza viruses with a panel of monoclonal antibodies indicated that antigenic variation occurs and that multiple different variants cocirculate in the population. There was no progressive antigenic change in the avian H2 influenza viruses with time, as was found with the human H2N2 strains. Topographical mapping of the H2 hemagglutinin by selection of antigenic variants with monoclonal antibodies and analysis of their reactivity patterns by HI showed overlap between the epitopes examined. These results may reflect restriction in the antibody repertoire of the mice used in preparation of the monoclonal antibodies or that the H2 hemagglutinin does not have such discrete nonoverlapping antigenic regions found in the early H3 influenza virus.     

Analysis of antigenic drift in recently isolated influenza A (H1N1) viruses using monoclonal antibody preparations.

Monoclonal antibodies to the hemagglutinin (HA) and neuraminidase (NA) of A/USSR/ 90/77(HlN1) were prepared and used to study antigenic drift in the H1N1 subtype of influenza viruses. The results obtained with five different monoclones to each molecule were compared with the results obtained with postinfection ferret sera. Monoclonal antibodies and postinfection ferret sera detected antigenic drift in the hemagglutinin and neuraminidase molecules of H1N1 viruses and monoclonal antibodies showed that the A/USSR/90/77, A/ Roma/1/49, and A/Fort Warren/1/50 viruses were identical at five sites on both the hemagglutinin and neuraminidase molecules. To further evaluate monoclonal antibodies, they were used in hemagglutination inhibition tests with recently isolated influenza A(HlNl) viruses, including some shown in tests with postinfection ferret sera to have undergone antigenic drift. Three patterns of reactivity with the monoclonal antibodies were detected with the variants: two variants differed at two of the five sites identified by the monoclonal antibodies and a third variant differed at only one of the five sites. Two of these variant groupings had not been distinguished from each other in HI tests with ferret sera, whereas ferret sera were capable of distinguishing between two groups of variants that had similar reaction patterns with the five monoclonal antibody preparations. Monoclonal antibodies failed to detect antigenic drift in the NA molecules of the recent isolates of H1N1 influenza viruses, suggesting that antigenic changes occur less frequently in this molecule. The observation that antigenic differences could be detected between the recent variants with such a small panel of monoclonal antibodies (less than 10% of the number obtained to the HA of A/PR/8/34) suggests that monoclonal antibodies may provide an exquisitely sensitive method for antigenic mapping of influenza viruses.     

Is bivalent binding of monoclonal antibodies to different antigenic areas on the hemagglutinin of influenza virus required for neutralization of viral infectivity?

Summary Biological activities of Fab fragments of monoclonal IgG antibodies to each of four nonoverlapping antigenic areas on the hemagglutinin molecule of A/seal/Massachusetts/1/80 (H7N7) influenza virus were examined. Fab fragments of the antibodies belonging to groups I and II neutralized viral infectivity. These Fab fragments inhibited hemagglutination of the virus and virus-induced hemolysis at pH 5.9. On the other hand, Fab fragments of groups III and IV antibodies showed neither neutralization nor hemolysis-inhibition activities, while intact IgG molecules of groups III and IV effectively neutralized viral infectivity and inhibited virus-induced hemolysis, as previously found. These IgG molecules scarcely or did not inhibit hemagglutination of the virus. Neutralization of viral infectivity, however, was observed when the virus was coated with Fab fragments of groups III and IV antibodies and then incubated with anti-Fab fragment antibodies. These findings suggest that bivalent binding of the IgG antibodies of groups III and IV is required for neutralization of viral infectivity through a proposed mechanism by which these antibodies interfere with a low pH-induced conformational change resulting in inhibition of the fusion step of the viral replication process (6).With 2 Figures     

Spin-labeling of influenza virus hemagglutinin permits analysis of the conformational change at low pH and its inhibition by antibody

To study the conformational changes in the hemagglutinin (HA) molecule of A/seal/Mass/1/80 (H7N7) (Seal) influenza virus at low pH, a spin-labeling method was used. This method also permits study of antibody interaction with the HA. A synthetic nitroxide compound was used for spin-labeling of tyrosine residues of the isolated HA molecule. Electron spin resonance (ESR) spectra of the spin-labeled HA at various pH values indicated that a conformational transition occurred under acidic conditions, and around pH 5.8 the HA molecule has maximal flexibility. Since virus-induced hemolysis occurs optimally at pH 5.8-5.9, the HA molecule in the maximally flexible conformation is considered to mediate membrane fusion. The ESR spectra of the antibody-bound HA at various pH values revealed that monoclonal antibodies to different regions on the molecule may inhibit the conformational change by different modes. One antibody inhibited the changes in the HA that resulted in flexibility, while the other did not. These results support the assumption that monoclonal antibodies, which failed to inhibit hemagglutination of the virus yet neutralized viral infectivity, inhibited the fusion step in the viral replication process by interfering with a low pH-induced conformational change in the HA molecule (Kida, H., Webster, R.G. and Yanagawa, R. (1983) Arch. Virol. 76, 91-99).     

Characterization of monoclonal antibodies against human rotavirus hemagglutinin

Three monoclonal antibodies capable of specifically inhibiting hemagglutination of human rotavirus were produced. Their hemagglutination inhibition (HI) activity was specific to the homologous strain (KUN) used for immunization. The monoclonal antibodies with HI activity were highly effective in neutralizing the infectivity of the KUN strain. These antibodies reacted with Vp80, and 80,000 molecular weight (MW) protein present in the viral outer shell. It was confirmed by immunoblotting assay with the monoclonal antibodies that the antigenic site of human rotavirus hemagglutinin (HA) resides on Vp80 and on its smaller trypsin cleavage products Vp30 (MW 30,000) and Vp24 (MW 24,000). Immunofluorescence studies using the antibodies revealed that the HA antigen of the KUN strain developed at the final stage of virus maturation.     

The antigenic structure of the influenza B virus hemagglutinin: operational and topological mapping with monoclonal antibodies

We have probed the antigenic structure of the influenza B virus hemagglutinin (HA) with monoclonal antibodies specific for the HA of influenza B virus, B/Oregon/5/80. Seventeen laboratory-selected antigenic variants of this virus were analyzed by hemagglutination-inhibition (HI) assays or ELISA and an operational antigenic map was constructed. In addition, the monoclonal antibodies were tested in a competitive binding assay to construct a topological map of the antigenic sites. In contrast to the influenza A virus HA, only a single immunodominant antigenic site composed of several overlapping clusters of epitopes was defined by the HI-positive antibodies. Three variants could be distinguished from the parental virus with polyclonal antisera by HI and infectivity reduction assays suggesting that changes in this antigenic site may be sufficient to provide an epidemiological advantage to influenza B viruses in nature. In addition, two nonoverlapping epitopes of unknown biological significance were identified in the competitive binding analysis by two monoclonal antibodies with no HI activity and little or no neutralizing activity. We previously identified single amino acid substitutions in the HAs of the antigenic variants used in this study (M. T. Berton, C. W. Naeve, and R. G. Webster (1984), J. Virol. 52, 919-927). These changes occurred in regions of the molecule which, by amino acid sequence alignment, appeared to correspond to proposed antigenic sites A and B on the H3 HA of influenza A virus. Correlation with the antigenic map established in this report, however, demonstrates that the amino acid residues actually contribute to a single antigenic site on the influenza B virus HA and suggests significant differences in the antigenic structures of the influenza A and B virus HAs.     

Evaluation of the Single Radial Hemolysis Test for Measuring Hemagglutinin- and Neuraminidase-Specific Antibodies to H3N2 Influenza Strains and Antibodies to Influenza B

Antibodies to the H3 hemagglutinin of influenza A virus could be specifically measured by single radial hemolysis (SRH) when test antigens were recombinant viruses containing the relevant H3 hemagglutinin antigen and irrelevant Neq1 neuraminidase of A/equine/Prague/1/56 virus. Antibodies to influenza B virus could also be measured by the SRH technique. Antibody rises to influenza A or B virus measured by SRH agreed with results of hemagglutination inhibition (HI) tests for about 80% of the sera tested, including sera from volunteers receiving killed influenza vaccine and sera from patients naturally infected with influenza. Correlation between antibody titers measured by SRH and HI was also good. Antibodies to the N2 neuraminidase of influenza A virus could be specifically measured by SRH when test antigens were recombinant viruses containing the relevant N2 neuraminidase antigen and irrelevant Heq1 hemagglutinin of A/equine/Prague/1/56 virus. The SRH test for neuraminidase antibodies was more strain specific than was the SRH test for hemagglutinin antibodies. Probably for this reason, agreement between neuraminidase antibody determinations in human sera by the SRH test and by the neuraminidase inhibition test was poorer than agreement between the SRH test for hemagglutinin antibodies and the HI test.     

An analysis of the antigenic structure of the hemagglutinins from different strains of the influenza B virus by using monoclonal antibodies

A comparative immunological analysis of the antigenic composition of hemagglutinin (HA) of influenza B virus drift-variants isolated during 46 years was carried out using monoclonal antibodies (MAb) to HA of B/Oregon 5/80 virus in HI test and solid-phase enzyme immunoassay. The presence of type- and group-specific antigenic determinants was demonstrated which agreed with our previous data obtained in studies with polyvalent sera. However, using Mab three more group-specific determinants were detected which characterize HA of influenza B viruses isolated in 1970-1979, 1970-1984, and 1970-1986.     

Antigenic structure of the hemagglutinin of influenza virus B/Hong Kong/8/73 as determined from gene sequence analysis of variants selected with monoclonal antibodies

Antigenic variation among influenza B viruses is different from that of influenza A in several ways. Antigenic shift has not been observed, distinct antigenic variants of influenza B cocirculate, and antigenically similar viruses have been isolated many years apart. To study the mechanism of antigenic drift in influenza B viruses, monoclonal antibodies were used to select antigenic variants of B/Hong Kong/8/73 virus hemagglutinin (HA). Analyses of the nucleotide sequences of the HA gene of B/Hong Kong/8/73 and the eight variants identified specific regions of the influenza B HA molecule involved in antigenicity, and enabled antigenic mapping data to be correlated with the structure of the protein. The altered amino acids in the variants, when compared to the HA of A/Aichi/2/68, were found in two of the four antigenic regions previously identified for type A viruses. In addition, four of the eight variants showed multiple nucleotide changes some of which gave rise to double amino acid changes. In addition, in the present study monoclonal antibodies which belong to the same antigenic group recognize amino acid changes in regions corresponding to antigenic sites A and B of the H3 HA. These results are in contrast to those obtained with HA variants of A/Memphis/1/71 virus. In the influenza A studies only single amino acid changes were found and these correlated well with the three-dimensional structure as determined by D. C. Wiley, I. A. Wilson, and J. J. Skehel, (1981, Nature (London) 289, 366-373); monoclonal antibodies which recognized one region did not recognize any of the other antigenic sites. Our results suggest that although the basic three-dimensional structure of the influenza B HA may be similar to that of A viruses, the B HA molecule may be folded in a more compact manner so that antigenic sites A and B are in closer proximity to each other than in the H3 structure.     

Antigenic properties of variants of the influenza virus H7N7 and its recombinants

Passages of the A/seal/Massachusetts/1/80 strain in different biological systems (chick embryos, mice, chickens, organ cultures of human embryo trachea) yielded variants differing from the original virus both in their biological and antigenic properties. The M20 variant selected in passages of the seal virus in mouse lungs differed from the latter only in biological but not antigenic properties. The OC20 and C20 variants produced by passages of the seal virus in organ cultures and chicken lungs differed from the original virus only antigenically. The antigenic properties were examined by the HI test using monoclonal antibodies to the A/seal/Massachusetts/1/80 strain. In addition, the HI test with the selected variants and individual clones of the seal virus as well as recombinants produced with this virus revealed variability of the epitope of hemagglutinin reacting with monoclone 71/4. Because mice, chickens and chick embryos had no specific antibodies to the A/seal/Massachusetts/1/80 strain, a conclusion is drawn on the possibility of antigenic variability of influenza A virus in nonimmune hosts.     

Migration of the Swine Influenza Virus δ-Cluster Hemagglutinin N-Linked Glycosylation Site from N142 to N144 Results in Loss of Antibody Cross-Reactivity

Routine antigenic characterization of swine influenza virus isolates in a high-throughput serum neutralization (HTSN) assay found that approximately 20% of isolates were not neutralized by a panel of reference antisera. Genetic analysis revealed that nearly all of the neutralization-resistant isolates possessed a seasonal human-lineage hemagglutinin (HA; δ cluster). Subsequent sequencing analysis of full-length HA identified a conserved N144 residue present only in neutralization-resistant strains. N144 lies in a predicted N-linked glycosylation consensus sequence, i.e., N-X-S/T (where X is any amino acid except for proline). Interestingly, neutralization-sensitive viruses all had predicted N-linked glycosylation sites at N137 or N142, with threonine (T) occupying position 144 of HA. Consistent with the HTSN assay, hemagglutination inhibition (HI) and serum neutralization (SN) assays demonstrated that migration of the potential N-linked glycosylation site from N137 or N142 to N144 resulted in a >8-fold decrease in titers. These results were further confirmed in a reverse genetics system where syngeneic viruses varying only by predicted N-glycosylation sites at either N142 or N144 exhibited distinct antigenic characteristics like those observed in field isolates. Molecular modeling of the hemagglutinin protein containing N142 or N144 in complex with a neutralizing antibody suggested that N144-induced potential glycosylation may sterically hinder access of antibodies to the hemagglutinin head domain, allowing viruses to escape neutralization. Since N-linked glycosylation at these sites has been implicated in genetic and antigenic evolution of human influenza A viruses, we conclude that the relocation of the hemagglutinin N-linked glycosylation site from N142 to N144 renders swine influenza virus δ-cluster viruses resistant to antibody-mediated neutralization.     

Characterization of host cell binding variants of influenza virus by monoclonal antibodies

We have previously shown that a plaque-type mutant of influenza virus A/WSN has a growth advantage in MDBK cells because its hemagglutinin (HA) has a greater affinity for host cell receptors than does the HA of the parent virus. We show here that the mutant is also less sensitive than the parent to neutralization by antibodies to epitopes in at least two regions on the HA. WSN-specific monoclonal antibodies which had higher radioimmunoassay (RIA) titers against the parent than the mutant virus also had higher plaque inhibition (PI) and hemagglutination inhibition (HI) titers. In contrast, cross-reacting antibodies bound equally well to the parent and mutant viruses as judged by RIA but those which bound to the Cb region of the HA exhibited higher PI and HI titers against the parent virus. The results suggest that preferential neutralization of the parental virus by antibodies can contribute to the selective advantage of mutants which have increased affinity for cellular receptors.     

2006年中国流感病毒抗原性及基因特性研究

目的分析2006年中国季节性流感的流行状况，以及病毒的抗原性和基因变异情况。方法对来自流感监测网络的毒株进行单向血凝抑制试验，在此基础上选择不同时间、地点分离的毒株进行血凝素基因的序列测定，然后分析其基因特性。结果2006年我国同时流行A型（H1N1亚型、H3N2亚型）和B型流感病毒。H1N1亚型毒株和B型Victoria系流感病毒为优势毒株。对H1N1亚型毒株的HA1区序列比较发现，2006年分离的毒株与A，湖北洪山／53／2005（H1N1）比较，在192、193、196、198位发生氨基酸替换的毒株．这些位点位于抗原决定簇的B区。H3N2亚型毒株与A，云南，1145／2005（H3N2）比较，在142、144位发生氨基酸替换。我国流行的B型流感毒株无论是Victoria系和Yamagata系毒株的抗原性均没有发生变异，与2005--2006年我国的流行株B／shenzhen／155／2005、B／tianjin／144／2005类似。结论2006年中国流行的H1N1亚型和H3N2亚型流感病毒的抗原性及基因特性已经发生改变；B型流感病毒的抗原性和基因特性没有改变。     

Characterization of reference strains of Newcastle disease virus (NDV) and NDV-like isolates by monoclonal antibodies to HN subunits

Summary The hemagglutinin-neuraminidase (HN) subunits of NDV and NDV-like isolates were analyzed antigenically by monoclonal antibodies to the HN of Miyadera and Taka viruses. In immuno-double-diffusion (IDD) tests, all NDVs examined gave clear lines of precipitation with some of the potent monoclonal antibodies, but it was difficult to determine with certainty the immunological properties of HN subunits due to a rare disagreement with the results obtained in other immunological tests. Monoclonal antibodies used in the tests were found to show different immunological reactivities with the viruses.Monoclonal antibodies belonging to the 1st group (1/29) inhibited the hemagglutinating (HA) activity of all strains but not the neuraminidase (NA) activity. The second monoclonal antibody (5/205) inhibited both the HA and NA activities of the restrictive NDV strains, indicating antigenic changes in HN molecules. However, the inhibitory activity of this monoclone to neuraminidase appeared to be greatly diminished when neuraminyl lactose was used as substrate. Although the 3rd type of monoclonal antibody (5/220) showed HI activity against several strains, this antibody did not inhibit NA activity of any viruses.The remaining monoclone to the HN of Taka virus inhibited the HA activity of all reference strains of NDV and many NDV-like isolates but did not affect NA activity. Two inhibitory activities of four monoclonal antibodies against different viruses, HI and hemolysis-inhibition, were not always consistent with inhibition of virus growth. HI and NI tests with the above four monoclonal antibodies showed that the strains tested fell into five antigenic groups according to their reaction patterns with mouse hybridoma antibodies.With 3 Figures     

Antigenic variants of influenza viruses: marked differences in the frequencies of variants selected with different monoclonal antibodies

Monoclonal antibodies against the hemagglutinin of two influenza A viruses and one influenza B virus were used to analyze the frequencies of antigenic variant subpopulations in cloned virus seeds. Antigenic variants selected with monoclonal antibodies specific for different antigenic determinants were obtained over a wide range of frequencies. With one monoclonal antibody directed to the hemagglutinin of A/PR/8/34 virus antigenic variants were isolated at a frequency of 10-^4.4, whereas another monoclonal antibody, specific for the hemagglutinin of X-31 virus, completely neutralized all infectious virus in the X-31 virus seeds examined, indicating a frequency of antigenic variation, for one seed at least, of less than 10^?8.1.     

Antigenic analysis of H4 influenza virus isolates using monoclonal antibodies to defined antigenic sites on the hemagglutinin of A/budgerigar/Hokkaido/1/77 strain

Summary Three non-overlapping antigenic sites were defined on the hemagglutinin of avian influenza virus A/budgerigar/Hokkaido/1/77 (H4N6) by competitive binding assay of monoclonal antibodies to the virus and comparative antigenic analysis of variants selected with monoclonal antibodies. Antigenic relationship among 25 H4 influenza viruses of different bird origin was examined by ELISA with the monoclonal antibodies to each of defined antigenic sites. Two of the three antigenic sites contained epitopes specific to the H4 influenza viruses of budgerigar and mynah origin, and the remaining site contained an epitope which was cross-reactive with almost all of the H4 influenza viruses.     

Broadly neutralizing antibodies recognizing different antigenic epitopes act synergistically against the influenza B virus

The discovery of broadly neutralizing monoclonal antibodies against influenza viruses has raised hope for the successful development of new antiviral drugs. However, due to the speed and variety of mutations in influenza viruses, single-component antibodies that recognize specific epitopes are susceptible to viral escape and have limited efficacy when administration is delayed. Hence, it is necessary to develop alternative strategies with better antiviral activity. Influenza B virus infection can cause severe illness in children and the elderly. Commonly used anti-influenza drugs have low clinical efficacy against influenza B virus. In this study, we investigated the antiviral efficacy of combinations of representative monoclonal antibodies targeting different antigenic epitopes against the influenza B virus. We found that combinations of antibodies recognizing the hemagglutinin (HA) head and stem regions showed a stronger neutralizing activity than single antibodies and other antibody combinations in vitro. In addition, we found that pair-wise combinations of antibodies recognizing the HA head region, HA stem region, and neuraminidase enzyme-activated region showed superior antiviral activity than single antibodies in both mouse and ferret in vivo protection assays. Notably, these antibody combinations still displayed good antiviral efficacy when treatment was delayed. Mechanistic studies further revealed that combining antibodies recognizing different epitope regions resulted in extremely strong antibody-dependent cell-mediated cytotoxicity, which may partly explain their superior antiviral effects. Together, the findings of this study provide new avenues for the development of better antiviral drugs and vaccines against influenza viruses.     

Enumeration of antigenic sites of influenza virus hemagglutinin.

The antigenic sites on the hemagglutinin of X-31 (H3) influenza virus have been defined by using a competitive radioimmunoassay with a panel of monoclonal antibodies which includes those known to select variants with substitutions of particular amino acids. The capacity of each monoclonal antibody to block the binding of other radioiodinated monoclones to purified hemagglutinin permitted classification of the panel into four separate groups, each of which defined a particular antigenic site on the hemagglutinin molecule. Three of these are located on the polypeptide backbone and correspond to the "hinge," the "loop," and the "tip/interface" of the X-ray crystallographic model of Wiley et al. (Nature [London] 289:373-378, 1981). Nonreciprocal blocking of certain anti-interface antibodies by anti-loop antibody suggests that much of the exposed surface of the head of the hemagglutinin molecule extending from the loop to the interface may be a continuum of epitopes. A fourth antigenic site is carbohydrate in nature, presumably situated on the antigenic oligosaccharide side chains. These four domains are in addition to two antigenic sites defined by monoclonal antibodies that inhibit neither hemagglutination nor infectivity (Breschkin et al., Virology 113:130-140, 1981;' Yewdell et al., Nature [London] 279:246-248, 1979).