Molecular evolution of influenza A (H3N2) viruses circulated in Fujian Province, China during the 1996–2004 period

We studied the genetic and epidemic characteristics of influenza A (H3N2) viruses circulated in human in Fujian Province, south of China from 1996 to 2004. Phylogenetic analysis was carried out for genes encoding hemagglutinin1 (HA1) of influenza A virus (14 new and 11 previously reported reference se-quences). Our studies revealed that in the 8 flu seasons, the mutations of HA1 genes occurred from time to time, which were responsible for about four times of antigenic drift of influenza H3N2 viruses in Fujian, China. The data demonstrated that amino acid changes were limited to some key codons at or near antibody binding sites A through E on the HA1 molecule. The changes at the antibody binding site B or A or sialic acid receptor binding site 226 were critical for antigenic drift. But the antigenic sites might change and the key codons for antigenic drift might change as influenza viruses evolve. It seems important to monitor new H3 isolates for mutations in the positively selected codons of HA1 gene in south of Asia.     

Patterns of predicted T-cell epitopes associated with antigenic drift in influenza H3N2 hemagglutinin

Antigenic drift allowing escape from neutralizing antibodies is an important feature of transmission and survival of influenza viruses in host populations. Antigenic drift has been studied in particular detail for influenza A H3N2 and well defined antigenic clusters of this virus documented. We examine how host immunogenetics contributes to determination of the antibody spectrum, and hence the immune pressure bringing about antigenic drift. Using uTOPE™ bioinformatics analysis of predicted MHC binding, based on amino acid physical property principal components, we examined the binding affinity of all 9-mer and 15-mer peptides within the hemagglutinin 1 (HA1) of 447 H3N2 virus isolates to 35 MHC-I and 14 MHC-II alleles. We provide a comprehensive map of predicted MHC-I and MHC-II binding affinity for a broad array of HLA alleles for the H3N2 influenza HA1 protein. Each HLA allele exhibited a characteristic predicted binding pattern. Cluster analysis for each HLA allele shows that patterns based on predicted MHC binding mirror those described based on antibody binding. A single amino acid mutation or position displacement can result in a marked difference in MHC binding and hence potential T-helper function. We assessed the impact of individual amino acid changes in HA1 sequences between 10 virus isolates from 1968–2002, representative of antigenic clusters, to understand the changes in MHC binding over time. Gain and loss of predicted high affinity MHC-II binding sites with cluster transitions were documented. Predicted high affinity MHC-II binding sites were adjacent to antibody binding sites. We conclude that host MHC diversity may have a major determinant role in the antigenic drift of influenza A H3N2.     

Reactivity of human convalescent sera with influenza virus hemagglutinin protein mutants at antigenic site A

How the antibodies of individual convalescent human sera bind to each amino acid residue at the antigenic sites of hemagglutinin (HA) of influenza viruses, and how the antigenic drift strains of influenza viruses are selected by human sera, is not well understood. In our previous study, it was found by a binding assay with a chimeric HA between A/Kamata/14/91 (Ka/91) and A/Aichi/2/68 that convalescent human sera, following Ka/91 like (H3N2) virus infection, bind to antigenic site A of Ka/91 HA. Here using chimeric HAs possessing single amino acid substitutions at site A, it was determined how those human sera recognize each amino acid residue at antigenic site A. It was found that the capacity of human sera to recognize amino acid substitutions at site A differs from one person to another and that some amino acid substitutions result in all convalescent human sera losing their binding capacity. Among these amino acid substitutions, certain ones might be selected by chance, thus creating successive antigenic drift. Phylogenetic analysis of the drift strains of Ka/91 showed amino acid substitutions at positions 133, 135 and 145 were on the main stream of the phylogenetic tree. Indeed, all of the investigated convalescent sera failed to recognize one of them.     

Influenza Human Monoclonal Antibody 1F1 Interacts with Three Major Antigenic Sites and Residues Mediating Human Receptor Specificity in H1N1 Viruses

Most monoclonal antibodies (mAbs) to the influenza A virus hemagglutinin (HA) head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates). The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca₂. The 1F1 heavy chain also reaches into the receptor binding site (RBS) and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute to its ability to inhibit divergent HAs.     

Immunodominance of Antigenic Site B over Site A of Hemagglutinin of Recent H3N2 Influenza Viruses

H3N2 influenza viruses have now circulated in the human population for 43 years since the pandemic of 1968, accumulating sequence changes in the hemagglutinin (HA) and neuraminidase (NA) that are believed to be predominantly due to selection for escape from antibodies. Examination of mutations that persist and accumulate led to identification of antigenically significant mutations that are contained in five antigenic sites (A-E) mapped on to the H3 HA. In early H3N2 isolates, antigenic site A appeared to be dominant while in the 1990s site B seemed more important. To obtain experimental evidence for dominance of antigenic sites on modern H3 HAs, we have measured antibodies in plasma of human subjects who received the 2006-07 trivalent subunit influenza vaccine (H3 component A/Wisconsin/67/05) or the 2008-09 formulation (H3 component A/Uruguay/716/07). Plasmas were tested against expressed HA of Wisconsin-like influenza A/Oklahoma/309/06 and site-directed mutants in antigenic site A (NNES121-124ITEG, N126T, N133D, TSSS135-138GSNA, K140I, RSNNS142-146PGSG), and antigenic site B (HL156-157KS, KFK158-160GST, NDQI189-192QEQT, A196V). "Native ELISA" analysis and escape mutant selection with two human monoclonal antibodies demonstrated that antibody E05 binds to antigenic site A and 1_C02 binds to site B. We find that most individuals, after vaccination in seasons 2006-07 and/or 2008-09, showed dominance of antigenic site B recognition over antigenic site A. A minority showed dominance of site A in 2006 but these were reduced in 2008 when the vaccine virus had a site A mutation. A better understanding of immunodominance may allow prediction of future antigenic drift and assist in vaccine strain selection.     

Compensatory Hemagglutinin Mutations Alter Antigenic Properties of Influenza Viruses

Influenza viruses routinely acquire mutations in antigenic sites on the globular head of the hemagglutinin (HA) protein. Since these antigenic sites are near the receptor binding pocket of HA, many antigenic mutations simultaneously alter the receptor binding properties of HA. We previously reported that a K165E mutation in the Sa antigenic site of A/Puerto Rico/8/34 (PR8) HA is associated with secondary neuraminidase (NA) mutations that decrease NA activity. Here, using reverse genetics, we show that the K165E HA mutation dramatically decreases HA binding to sialic acid receptors on cell surfaces. We sequentially passaged reverse-genetics-derived PR8 viruses with the K165E antigenic HA mutation in fertilized chicken eggs, and to our surprise, viruses with secondary NA mutations did not emerge. Instead, viruses with secondary HA mutations emerged in 3 independent passaging experiments, and each of these mutations increased HA binding to sialic acid receptors. Importantly, these compensatory HA mutations were located in the Ca antigenic site and prevented binding of Ca-specific monoclonal antibodies. Taken together, these data indicate that HA antigenic mutations that alter receptor binding avidity can be compensated for by secondary HA or NA mutations. Antigenic diversification of influenza viruses can therefore occur irrespective of direct antibody pressure, since compensatory HA mutations can be located in distinct antibody binding sites.     

Fatty acids on the A/USSR/77 influenza virus hemagglutinin facilitate the transition from hemifusion to fusion pore formation

Influenza virus hemagglutinin (HA) has three highly conserved acylation sites close to the carboxyl terminus of the HA2 subunit, one in the transmembrane domain and two in the cytoplasmic domain. Each site is modified by palmitic acid through a thioester linkage to cysteine. To elucidate the biological significance of HA acylation, the acylation sites of HA of influenza virus strain A/USSR/77 (H1N1) were changed by site-directed mutagenesis, and the membrane fusion activity of mutant HAs lacking the acylation site(s) was examined quantitatively using transfer assays of lipid (R18) and aqueous (calcein) dyes. Lipid mixing, so-called hemifusion, activity was not affected by deacylation, whereas transfer of aqueous dye, so-called fusion pore formation, was dramatically restricted. When the fusion reaction was induced by a lower pH than the optimal one, calcein transfer with the mutant HAs was improved, but simultaneously a considerable calcein leakage into the medium was observed. From these results, we conclude that the palmitic acids on the H1 subtype HA facilitate the transition from hemifusion to fusion pore formation.     

Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls

In wild aquatic birds and poultry around the world, influenza A viruses carrying 15 antigenic subtypes of hemagglutinin (HA) and 9 antigenic subtypes of neuraminidase (NA) have been described. Here we describe a previously unidentified antigenic subtype of HA (H16), detected in viruses circulating in black-headed gulls in Sweden. In agreement with established criteria for the definition of antigenic subtypes, hemagglutination inhibition assays and immunodiffusion assays failed to detect specific reactivity between H16 and the previously described subtypes H1 to H15. Genetically, H16 HA was found to be distantly related to H13 HA, a subtype also detected exclusively in shorebirds, and the amino acid composition of the putative receptor-binding site of H13 and H16 HAs was found to be distinct from that in HA subtypes circulating in ducks and geese. The H16 viruses contained NA genes that were similar to those of other Eurasian shorebirds but genetically distinct from N3 genes detected in other birds and geographical locations. The European gull viruses were further distinguishable from other influenza A viruses based on their PB2, NP, and NS genes. Gaining information on the full spectrum of avian influenza A viruses and creating reagents for their detection and identification will remain an important task for influenza surveillance, outbreak control, and animal and public health. We propose that sequence analyses of HA and NA genes of influenza A viruses be used for the rapid identification of existing and novel HA and NA subtypes.     

Glycosylation focuses sequence variation in the influenza A virus H1 hemagglutinin globular domain

Antigenic drift in the influenza A virus hemagglutinin (HA) is responsible for seasonal reformulation of influenza vaccines. Here, we address an important and largely overlooked issue in antigenic drift: how does the number and location of glycosylation sites affect HA evolution in man? We analyzed the glycosylation status of all full-length H1 subtype HA sequences available in the NCBI influenza database. We devised the “flow index” (FI), a simple algorithm that calculates the tendency for viruses to gain or lose consensus glycosylation sites. The FI predicts the predominance of glycosylation states among existing strains. Our analyses show that while the number of glycosylation sites in the HA globular domain does not influence the overall magnitude of variation in defined antigenic regions, variation focuses on those regions unshielded by glycosylation. This supports the conclusion that glycosylation generally shields HA from antibody-mediated neutralization, and implies that fitness costs in accommodating oligosaccharides limit virus escape via HA hyperglycosylation.     

A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site.

The structure of a complex of influenza hemagglutinin (HA) with a neutralizing antibody shows that the antibody binds to HA at a distance from the virus receptor binding site. Comparison of the properties of this antibody and its Fab with those of an antibody that recognizes an epitope overlapping the receptor binding site leads to two main conclusions. First, inhibition of receptor binding is an important component of neutralization. Second, the efficiency of neutralization by the antibodies ranks in the same order as their avidities for HA, and their large size makes these antibodies highly efficient at neutralization, regardless of the location of their epitope in relation to the virus receptor binding site. These observations provide rationales for the range of antibody specificities that are detected in immune sera and for the distribution of sequence changes on the membrane-distal surface of influenza HAs that occur during 'antigenic drift.'     

HA2 subunit of influenza A H1 and H2 subtype viruses induces a protective cross-reactive cytotoxic T lymphocyte response

Influenza H1 subtype-specific CTL can be induced by secondary stimulation of a hybrid protein of the first 81 amino acids of the viral NS1 non-structural protein and the HA2 subunit of A/Puerto Rico/8/34(H1N1) hemagglutinin. In addition, a derivative of this protein with 65 amino acids deleted from the N-terminal end of HA2 can also generate H1 subtype-specific CTL in bulk cultures. CTL clones established by stimulation with the derivative protein demonstrated cross-reactive lysis of target cells infected with virus strains of the H1 and H2 subtypes. Cold target competition experiments with CTL clones as effectors demonstrated that the Ag specificity between these two hybrid proteins is identical. Adoptive transfer of the CTL clone significantly reduced virus titers in the lungs of mice infected with the virus strains of the H1 or H2 subtype but not those infected with the H3 subtype virus in vivo, which reflects the in vitro CTL clone activity. These experiments demonstrate that an epitope on the hemagglutinin that is conserved on virus strains of the H1 and H2 subtypes induces a protective CTL response. These results suggest an alternative approach for developing influenza vaccines by using conserved antigenic sites on the hemagglutinin HA2 subunit to avoid the problem of frequent antigenic mutations of the HA1 subunit antibody binding sites.     

I-Ad restricted T cell recognition of influenza hemagglutinin. Synthetic peptides identify multiple epitopes corresponding to antibody-binding regions of the HA1 subunit

In a recent study, we reported extensive diversity in the Iak-restricted T cell repertoire for the hemagglutinin molecule (HA) of influenza A viruses (H3 subtype). Synthetic peptides identified six nonoverlapping epitopes on the HA1 subunit, and CD4+ T cell clones, specific for these regions, discriminated between natural variant viruses that had accumulated amino acid substitutions during antigenic drift. Here, we demonstrate similar specificity and diversity for the Iad haplotype and have identified multiple T cell epitopes within the sequences HA1 56-76, 71-91, 81-97, 177-199, 186-205, and 206-227. These also include recognition sites for neutralizing antibodies and correlations can be made between antigenic drift substitutions in H3 subtype viruses and the specificity of individual CD4+ clones for mutant HA. Moreover, these peptides appear not to exhibit structural homology and fail to compete for Ag presentation, indicating heterogeneity in peptide-Ia interaction. To explain the observation that CD4+ T cells, from two major haplotypes, recognize antibody binding regions of the HA molecule, we propose that surface Ig receptors of the Ag-specific B memory cell exert a direct effect on the processing of HA peptides and subsequent selection of the class II-restricted T cell memory repertoire after natural infection.     

Antigenic drift in influenza virus H3 hemagglutinin from 1968 to 1980: multiple evolutionary pathways and sequential amino acid changes at key antigenic sites

Surveys of the antigenic properties of a wide range of variants of the H3N2 (Hong Kong) influenza virus subtype have revealed complex patterns of variants cocirculating during each of the main epidemic eras of the subtype. We determined hemagglutinin (HA) gene sequences for 14 isolates chosen to give the wildest possible spread of variant types. The addition of these data to existing HA gene sequence information for other variants provides a comprehensive picture of HA gene evolution during antigenic drift among H3N2 subtype viruses. The data reveal the existence of multiple evolutionary pathways during at least one period of development of the subtype and strikingly demonstrate that amino acid changes are limited to a small number of locations on the HA molecule during antigenic drift. The occurrence of sequential amino acid changes at key positions within these variable regions suggests that the HA structure has remained constant during subtype evolution so that only limited possibilities remain for further antigenic drift among H3N2 viruses.     

A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains.

When mice were immunized with the A/Okuda/57 (H2N2) strain of influenza virus, a unique monoclonal antibody designated C179 was obtained. Although C179 was confirmed to recognize the hemagglutinin (HA) glycoprotein by immunoprecipitation assays, it did not show hemagglutination inhibition activity to any of the strains of the three subtypes of influenza A virus. However, it neutralized all of the H1 and H2 strains but not the H3 strains. Moreover, it inhibited polykaryon formation induced by the H1 and H2 strains but not by the H3 strains. Two antigenic variants against C179 were obtained, and nucleotide sequence analysis revealed that amino acid sequences, from 318 to 322 of HA1 and from 47 to 58 of HA2, conserved among H1 and H2 strains were responsible for the recognition of C179. Since the two sites were located close to each other at the middle of the stem region of the HA molecule, C179 seemed to recognize these sites conformationally. These data indicated that binding of C179 to the stem region of HA inhibits the fusion activity of HA and thus results in virus neutralization and inhibition of cell-cell fusion. This is the first report which describes the presence of conserved antigenic sites on HA not only in a specific subtype but also in two subtypes of influenza A virus.     

Effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza A/H3N2 virus hemagglutinin

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.     

The predicted antigenicity of the haemagglutinin of the 1918 Spanish influenza pandemic suggests an avian origin.

In 1982 we characterized the antigenic sites of the haemagglutinin of influenza A/PR/8/34, which is an influenza strain of the H1 subtype that was isolated from humans in 1934, by studying mutants which escaped neutralization by antibody. Four antigenic sites, namely Cb, Sa, Sb and Ca, were found to be located near the tip of the trimeric haemagglutinin spike. Based on the sequence of the haemagglutinin of the 1918 Spanish influenza, we can now specify the extent of divergence of antigenic sites of the haemagglutinin during the antigenic drift of the virus between 1918 and 1934. This divergence was much more extensive (40%) than the divergence (20%) in predicted antigenic sites between the 1918 Spanish influenza and an avian H1 subtype consensus sequence. These results support the hypothesis that the human 1918 pandemic originated from an avian virus of the H1 subtype that crossed the species barrier from birds to humans and adapted to humans, presumably by mutation and/or reassortment, shortly before 1918.     

Selection pressure on Haemagglutinin genes of H9N2 influenza viruses from different hosts

Positive selection and differential selective pressure analyses were carried out to study Haemagglutinin (HA) genes of H9N2 influenza viruses from different hosts in this paper. Results showed that, although most positions in HAs were under neutral or purifying evolution, a few positions located in the antigenic regions and receptor binding sites were subject to positive selection and some of them were even positively selected at the population level. In addition, there were always some positions differentially selected for viruses from different hosts. Both selection pressure working on HA codons and positions differentially selected might account for the extension of the host range and adaptations to different hosts of H9N2 influenza viruses.     

Identifying antigenicity-associated sites in highly pathogenic H5N1 influenza virus hemagglutinin by using sparse learning

Since the isolation of A/goose/Guangdong/1/1996 (H5N1) in farmed geese in southern China, highly pathogenic H5N1 avian influenza viruses have posed a continuous threat to both public and animal health. The non-synonymous mutation of the H5 hemagglutinin (HA) gene has resulted in antigenic drift, leading to difficulties in both clinical diagnosis and vaccine strain selection. Characterizing H5N1's antigenic profiles would help resolve these problems. In this study, a novel sparse learning method was developed to identify antigenicity-associated sites in influenza A viruses on the basis of immunologic data sets (i.e., from hemagglutination inhibition and microneutralization assays) and HA protein sequences. Twenty-one potential antigenicity-associated sites were identified. A total of 17 H5N1 mutants were used to validate the effects of 11 of these predicted sites on H5N1's antigenicity, including 7 newly identified sites not located in reported antibody binding sites. The experimental data confirmed that mutations of these tested sites lead to changes in viral antigenicity, validating our method.     

Antigenic drift in H5N1 avian influenza virus in poultry is driven by mutations in major antigenic sites of the hemagglutinin molecule analogous to those for human influenza virus

H5N1 highly pathogenic avian influenza virus has been endemic in poultry in Egypt since 2008, notwithstanding the implementation of mass vaccination and culling of infected birds. Extensive circulation of the virus has resulted in a progressive genetic evolution and an antigenic drift. In poultry, the occurrence of antigenic drift in avian influenza viruses is less well documented and the mechanisms remain to be clarified. To test the hypothesis that H5N1 antigenic drift is driven by mechanisms similar to type A influenza viruses in humans, we generated reassortant viruses, by reverse genetics, that harbored molecular changes identified in genetically divergent viruses circulating in the vaccinated population. Parental and reassortant phenotype viruses were antigenically analyzed by hemagglutination inhibition (HI) test and microneutralization (MN) assay. The results of the study indicate that the antigenic drift of H5N1 in poultry is driven by multiple mutations primarily occurring in major antigenic sites at the receptor binding subdomain, similarly to what has been described for human influenza H1 and H3 subtype viruses.     

Genetic control and fine specificity of the immune response to a synthetic peptide of influenza virus hemagglutinin.

The immune response to a synthetic peptide, H3 HA1(305-328), representing the C'-terminal 24 amino acid residues of the HA1 chain of the hemagglutinin of the H3 subtype of influenza virus is controlled by genes in the I region of the major histocompatibility complex. Mice of the H-2d haplotype are high responders and produce antibody for several months after a single injection of peptide without carrier. Mice of the H-2b, H-2k, and H-2q haplotypes are low antibody responders. Investigation of recombinant and congenic mouse strains revealed that high responsiveness requires the genes that encode the I-Ed molecule. Immunoassays, involving direct binding to analogs of this peptide and inhibition by both these analogs and synthetic epitopes, were used to analyze the specificity of the polyclonal response. In BALB/c mice, the primary antibody response is directed principally against the antigenic site 314-LKLAT-318, whereas the secondary response after a boost is predominantly directed to a distinct site, 320-MRNVPEKQT-328. The T-cell response to the peptide H3 HA1(305-328), as measured by antigen-induced proliferation of primed T cells in vitro, is also I-Ed restricted in high-responder H-2d mice and is directed against an antigenic site that does not require the four C-terminal residues unique to the H3 influenza subtype. A different epitope appears to be recognized by T cells from CBA (H-2k) mice, which proliferate to a moderate extent on exposure to the peptide but, nevertheless, do not provide help for an antibody response.