Gnarled-trunk evolutionary model of influenza A virus hemagglutinin

Human influenza A viruses undergo antigenic changes with gradual accumulation of amino acid substitutions on the hemagglutinin (HA) molecule. A strong antigenic mismatch between vaccine and epidemic strains often requires the replacement of influenza vaccines worldwide. To establish a practical model enabling us to predict the future direction of the influenza virus evolution, relative distances of amino acid sequences among past epidemic strains were analyzed by multidimensional scaling (MDS). We found that human influenza viruses have evolved along a gnarled evolutionary pathway with an approximately constant curvature in the MDS-constructed 3D space. The gnarled pathway indicated that evolution on the trunk favored multiple substitutions at the same amino acid positions on HA. The constant curvature was reasonably explained by assuming that the rate of amino acid substitutions varied from one position to another according to a gamma distribution. Furthermore, we utilized the estimated parameters of the gamma distribution to predict the amino acid substitutions on HA in subsequent years. Retrospective prediction tests for 12 years from 1997 to 2009 showed that 70% of actual amino acid substitutions were correctly predicted, and that 45% of predicted amino acid substitutions have been actually observed. Although it remains unsolved how to predict the exact timing of antigenic changes, the present results suggest that our model may have the potential to recognize emerging epidemic strains.     

Antigenic and genetic evolution of swine influenza A (H3N2) viruses in Europe

In the early 1970s, a human influenza A/Port Chalmers/1/73 (H3N2)-like virus colonized the European swine population. Analyses of swine influenza A (H3N2) viruses isolated in The Netherlands and Belgium revealed that in the early 1990s, antigenic drift had occurred, away from A/Port Chalmers/1/73, the strain commonly used in influenza vaccines for pigs. Here we show that Italian swine influenza A (H3N2) viruses displayed antigenic and genetic changes similar to those observed in Northern European viruses in the same period. We used antigenic cartography methods for quantitative analyses of the antigenic evolution of European swine H3N2 viruses and observed a clustered virus evolution as seen for human viruses. Although the antigenic drift of swine and human H3N2 viruses has followed distinct evolutionary paths, potential cluster-differentiating amino acid substitutions in the influenza virus surface protein hemagglutinin (HA) were in part the same. The antigenic evolution of swine viruses occurred at a rate approximately six times slower than the rate in human viruses, even though the rates of genetic evolution of the HA at the nucleotide and amino acid level were similar for human and swine H3N2 viruses. Continuous monitoring of antigenic changes is recommended to give a first indication as to whether vaccine strains may need updating. Our data suggest that humoral immunity in the population plays a smaller role in the evolutionary selection processes of swine H3N2 viruses than in human H3N2 viruses.     

Antigenic structure of the influenza B virus hemagglutinin: nucleotide sequence analysis of antigenic variants selected with monoclonal antibodies.

We report here the complete nucleotide sequence of the hemagglutinin (HA) gene of influenza B virus B/Oregon/5/80 and, through comparative sequence analysis, identify amino acid substitutions in the HA1 polypeptide responsible for the antigenic alterations in laboratory-selected antigenic variants of this virus. The complete nucleotide sequence of the B/Oregon/5/80 HA gene was established by a combination of chemical sequencing of a full-length cDNA clone and dideoxy sequencing of the virion RNA. The nucleotide sequence is very similar to previously reported influenza B virus HA gene sequences and differs at only nine nucleotide positions from the B/Singapore/222/79 HA gene (Verhoeyen et al., Nucleic Acids Res. 11:4703-4712, 1983). The nucleotide sequences of the HA1 portions of the HA genes of 18 laboratory-selected antigenic variants were determined by the dideoxy method. Comparison of the deduced amino acid sequences of the parental and variant HA1 polypeptides revealed 16 different amino acid substitutions at nine positions. All amino acid substitutions resulted from single-point mutations, and no double mutants were detected, demonstrating that as in the influenza A viruses, single amino acid substitutions are sufficient to alter the antigenicity of the HA molecule. Many of the amino acid substitutions in the variants occurred at positions also observed to change in natural drift strains. The substitutions appear to identify at least two immunodominant regions which correspond to proposed antigenic sites A and B on the influenza A virus H3 HA.     

Molecular characterization and phylogenetic analysis of H3N2 human influenza A viruses in Cheongju, South Korea

To investigate the genetic characteristics of human influenza viruses circulating in Chungbuk province, we tested 510 clinical samples of nasopharyngeal suction from pediatric patients diagnosed with respiratory illness between June 2007 and June 2008. Genetic characterization of the HA genes of H3N2 isolates indicated the relative higher similarity to A/Virginia/04/07 (99.6%) rather than that of A/Wisconsin/67/2005 (98.4%), a Northern Hemisphere 2007∼2008 vaccine strain, based on amino acid sequences. We found several altered amino acids at the H3 HA1 antigenic sites compared with the vaccine strain; K140I at site A, K158R at site B, and K173N (H471) or K173Q, and S262N at site E, but there was no antigenic shift among the H3N2 viruses. Interestingly, A/Cheongju/H383/08 and A/Cheongju/H407/08 isolates had single amino acid substitution at D151G on the catalytic site of the N2 NA while A/Cheongju/H412/08 and A/Cheongju/ H398/07 isolates had one amino acid deletion at residue 146. Furthermore, we found that 25% (3 out of 12 isolates) of the H3N2 subtype viruses had the amino acid substitution at position 31 on the M2 protein (Aspartic acid to Asparagine) and confirmed their drug-resistance by biological assays. Taken together, the results of this study demonstrated continuous evolutions of human H3N2 viruses by antigenic drift and also highlighted the need to closely monitor antigenic drug resistance in influenza A viruses to aid in the early detection of potentially pandemic strains, as well as underscore the need for new therapeutics.     

Distinct epitopes recognized by I-Ad-restricted T-cell clones within antigenic site E on influenza virus hemagglutinin.

A total of 14 I-Ad-restricted helper T-cell clones specific for the hemagglutinin (HA) molecule of influenza virus were isolated from spleens of BALB/c or (BALB/c X C57BL/10)F1 mice immunized with the H3 subtype influenza virus A/Memphis/71 (Mem 71) and from lymph nodes of BALB/c mice primed with purified HA. The specificity of these T-cell clones was assessed in proliferation assays by reactivity with naturally occurring strains of viruses that arose by antigenic drift and contain known amino acid sequence changes in HA and with a panel of monoclonal antibody (MAb)-selected mutants of Mem 71 with single amino acid substitutions in HA. The HA genes of those mutant viruses that failed to stimulate one or more of the T-cell clones were sequenced. The clones could be allocated to at least four groups, each group having a distinct pattern of reactivity with the panel of natural field strains. The epitopes recognized by the four groups of clones were found, by reactivity with MAb-selected mutants, to be in very close proximity to one another and probably overlapping. All of the distinct epitopes recognized by the T-cell clones were adversely affected by a single amino acid substitution, either at residue 60 or at residue 63 in the HA1 polypeptide chain, within the region known from antibody-binding studies as site E. Some, but not all, of the epitopes may be influenced by the addition of a carbohydrate side chain to the HA of a particular MAb-selected mutant and certain field strains containing an Asp----Asn substitution at residue 63. Site E is therefore a major site of H-2d helper T-cell recognition on the H3 HA.     

Minor changes in the hemagglutinin of influenza A(H1N1)2009 virus alter its antigenic properties

Background

The influenza A(H1N1)2009 virus has been the dominant type of influenza A virus in Finland during the 2009–2010 and 2010–2011 epidemic seasons. We analyzed the antigenic characteristics of several influenza A(H1N1)2009 viruses isolated during the two influenza seasons by analyzing the amino acid sequences of the hemagglutinin (HA), modeling the amino acid changes in the HA structure and measuring antibody responses induced by natural infection or influenza vaccination.

Methods/Results

Based on the HA sequences of influenza A(H1N1)2009 viruses we selected 13 different strains for antigenic characterization. The analysis included the vaccine virus, A/California/07/2009 and multiple California-like isolates from 2009–2010 and 2010–2011 epidemic seasons. These viruses had two to five amino acid changes in their HA1 molecule. The mutation(s) were located in antigenic sites Sa, Ca1, Ca2 and Cb region. Analysis of the antibody levels by hemagglutination inhibition test (HI) indicated that vaccinated individuals and people who had experienced a natural influenza A(H1N1)2009 virus infection showed good immune responses against the vaccine virus and most of the wild-type viruses. However, one to two amino acid changes in the antigenic site Sa dramatically affected the ability of antibodies to recognize these viruses. In contrast, the tested viruses were indistinguishable in regard to antibody recognition by the sera from elderly individuals who had been exposed to the Spanish influenza or its descendant viruses during the early 20^th century.

Conclusions

According to our results, one to two amino acid changes (N125D and/or N156K) in the major antigenic sites of the hemagglutinin of influenza A(H1N1)2009 virus may lead to significant reduction in the ability of patient and vaccine sera to recognize A(H1N1)2009 viruses.     

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.     

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.     

Molecular evolution of hemagglutinin genes of H1N1 swine and human influenza A viruses

Summary The hemagglutinin (HA) genes of influenza type A (H1N1) viruses isolated from swine were cloned into plasmid vectors and their nucleotide sequences were determined. A phylogenetic tree for the HA genes of swine and human influenza viruses was constructed by the neighbor-joining method. It showed that the divergence between swine and human HA genes might have occurred around 1905. The estimated rates of synonymous (silent) substitutions for swine and human influenza viruses were almost the same. For both viruses, the rate of synonymous substitution was much higher than that of nonsynonymous (amino acid altering) substitution. It is the case even for only the antigenic sites of the HA. This feature is consistent with the neutral theory of molecular evolution. The rate of nonsynonymous substitution for human influenza viruses was three times the rate for swine influenza viruses. In particular, nonsynonymous substitutions at antigenic sites occurred less frequently in swine than in humans. The difference in the rate of nonsynonymous substitution between swine and human influenza viruses can be explained by the different degrees of functional constraint operating on the amino acid sequence of the HA in both hosts.     

Accumulation of amino acid substitutions promotes irreversible structural changes in the hemagglutinin of human influenza AH3 virus during evolution

In order to clarify the effect of an accumulation of amino acid substitutions on the hemadsorption character of the influenza AH3 virus hemagglutinin (HA) protein, we introduced single-point amino acid changes into the HA1 domain of the HA proteins of influenza viruses isolated in 1968 (A/Aichi/2/68) and 1997 (A/Sydney/5/97) by using PCR-based random mutation or site-directed mutagenesis. These substitutions were classified as positive or negative according to their effects on the hemadsorption activity. The rate of positive substitutions was about 50% for both strains. Of 44 amino acid changes that were identical in the two strains with regard to both the substituted amino acids and their positions in the HA1 domain, 22% of the changes that were positive in A/Aichi/2/68 were negative in A/Sydney/5/97 and 27% of the changes that were negative in A/Aichi/2/68 were positive in A/Sydney/5/97. A similar discordance rate was also seen for the antigenic sites. These results suggest that the accumulation of amino acid substitutions in the HA protein during evolution promoted irreversible structural changes and therefore that antigenic changes in the H3HA protein may not be limited.     

Glycosylation and an amino acid insertion in the head of hemagglutinin independently affect the antigenic properties of H5N1 avian influenza viruses

Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies(MAbs) targeted to the hemagglutinin(HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131 N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.     

Distinct glycoprotein inhibitors of influenza A virus in different animal sera.

Normal horse and guinea pig sera contain the glycoprotein inhibitor alpha 2-macroglobulin, which inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H2 and H3 subtypes. In the current study, the presence of inhibitors of influenza A virus in pig and rabbit sera was investigated. Variants of influenza virus type A/Los Angeles/2/87(H3N2) that were resistant to horse, pig, or rabbit serum were isolated. Analysis of the variant viruses with anti-hemagglutinin (HA) monoclonal antibodies revealed that antigenic changes occurred with the development of serum inhibitor resistance. Characterization of the inhibitors in pig and rabbit sera by using periodate and receptor-destroying enzyme demonstrated that carbohydrate is an important constituent of the active portion of both inhibitor molecules and that sialic acid is involved in the interaction of the inhibitors with influenza virus HA. Nucleotide sequence analysis of the HA molecule revealed that the serum-resistant variants each acquired a different set of amino acid alterations. The multiply resistant variants maintained the original amino acid changes and acquired additional changes. Sequence modifications in the HA involved the conserved amino acids within the receptor binding site (RBS) at position 137 and the second-shell RBS residues at positions 155 and 186. Amino acid changes also occurred within antigenic site A (position 145) and directly behind the receptor binding pocket (position 220). Amino acid alterations resulted in the acquisition of a potential glycosylation site at position 128 and the loss of potential glycosylation sites at positions 246 and 248. The localization of the amino acid changes in HA1 to the region of the RBS supports the concept of serum inhibitors as receptor analogs. The unique set of mutations acquired by the serum inhibitor-resistant variants strongly suggests that horse, pig, and rabbit sera each contain distinct glycoprotein inhibitors of influenza A virus.     

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.     

Comparison of epitope structures of H3HAs through protein modeling of influenza A virus hemagglutinin: mechanism for selection of antigenic variants in the presence of a monoclonal antibody

Starting with nine plaques of influenza A/Kamata/14/91(H3N2) virus, we selected mutants in the presence of monoclonal antibody 203 (mAb203). In total, amino acid substitutions were found at nine positions (77, 80, 131, 135, 141, 142, 143, 144 and 146), which localized in the antigenic site A of the hemagglutinin (HA). The escape mutants differed in the extent to which they had lost binding to mAb203. HA protein with substitutions of some amino acid residues created by site-directed mutagenesis in the escape mutants retained the ability to bind to mAb203. Changes in the amino acid character affecting charge or hydrophobicity accounted for the binding capacity to the antibody of the HA with most of the substitutions in the escape mutants and binding-positive mutants. However, the effect of some amino acid substitutions remained unexplained. A three-dimensional model of the 1991 HA was constructed and used to analyze substituted amino acids in these mutants for the accessible surface hydrophobic and hydrophilic characters. One amino acid substitution in an escape mutant and another amino acid substitution in a binding-positive mutant seemed to be explained by the changes noted on this model.     

Subtype- and antigenic site-specific differences in biophysical influences on evolution of influenza virus hemagglutinin

ABSTRACT: BACKGROUND: Influenza virus undergoes rapid evolution by both antigenic shift and antigenic drift. Antibodies, particularly those binding near the receptor-binding site of hemagglutinin (HA) or the neuraminidase (NA) active site, are thought to be the primary defense against influenza infection, and mutations in antibody binding sites can reduce or eliminate antibody binding. The binding of antibodies to their cognate antigens is governed by such biophysical properties of the interacting surfaces as shape, non-polar and polar surface area, and charge. Methods: To understand forces shaping evolution of influenza virus, we have examined HA sequences of human influenza A and B viruses, assigning each amino acid values reflecting total accessible surface area, non-polar and polar surface area, and net charge due to the side chain. Changes in each of these values between neighboring sequences were calculated for each residue and mapped onto the crystal structures. Results: Areas of HA showing the highest frequency of changes agreed well with previously identified antigenic sites in H3 and H1 HAs, and allowed us to propose more detailed antigenic maps and novel antigenic sites for H1 and influenza B HA. Changes in biophysical properties differed between HAs of different subtypes, and between different antigenic sites of the same HA. For H1, statistically significant differences in several biophysical quantities compared to residues lying outside antigenic sites were seen for some antigenic sites but not others. Influenza B antigenic sites all show statistically significant differences in biophysical quantities for all antigenic sites, whereas no statistically significant differences in biophysical quantities were seen for any antigenic site is seen for H3. In many cases, residues previously shown to be under positive selection at the genetic level also undergo rapid change in biophysical properties. Conclusions: The biophysical consequences of amino acid changes introduced by antigenic drift vary from subtype to subtype, and between different antigenic sites. This suggests that the significance of antibody binding in selecting new variants may also be variable for different antigenic sites and influenza subtypes.     

Mechanism of antigenic drift in influenza virus. Amino acid sequence changes in an antigenically active region of Hong Kong (H3N2) influenza virus hemagglutinin

During antigenic drift in influenza viruses, changes in antigenicity are associated with changes in amino acid sequence of the large hemagglutinin polypeptide, HA1. In ten variants of Hong Kong (H3N2) influenza virus selected with monoclonal antibodies, the proline residue at position 143 in HA1 changed to serine, threonine, leucine or histidine. In other variants, asparagine 133 changed to lysine, glycine 144 to aspartic acid and serine 145 to lysine. All these changes are possible by single base changes in the RNA except the last, which requires a double base change. Residues 142 to 146 also changed in field strains of Hong Kong influenza isolated between 1968 and 1977 (Laver et al., 1980). The single amino acid sequence changes in HA1 of the monoclonal variants were detected by comparing the compositions of the soluble tryptic peptides from the variants with the known sequences of these peptides from wild-type virus. Two insoluble tryptic peptides, comprising residues 110 to 140 and 230 to 255 in the HA1 molecule, were not examined and we do not know if additional changes occurred in these regions.In order to determine whether sequential changes at the same position occurred during antigenic drift, antibody prepared against the new antigenic site on the variants in which proline 143 changed to histidine or threonine was used to select second generation variants of these variants. In the first case, the glycine residue (144) next to the histidine changed to aspartic acid, and in the second, the threonine residue at position 143 reverted to proline and the virus regained the antigenicity of wild-type.Although monoclonal antibodies revealed dramatic antigenic differences between the variants and wild-type virus, only those variants with changes at position 144 of glycine to aspartic acid or at position 145 of serine to lysine could be distinguished from wild-type virus using heterogeneous rabbit or ferret antisera. The other variants, including those which showed sequence changes in widely separated positions of HA1, could not be distinguished from wild-type with heterogeneous antisera.These findings suggest that sequence changes in the region comprising residues 142 to 146 of HA1 affect an important antigenic site on the hemagglutinin molecule, but how these changes affect the antigenic properties, or whether this region actually forms part of the antigenic site is not known.     

Computational Identification of Antigenicity-Associated Sites in the Hemagglutinin Protein of A/H1N1 Seasonal Influenza Virus

The antigenic variability of influenza viruses has always made influenza vaccine development challenging. The punctuated nature of antigenic drift of influenza virus suggests that a relatively small number of genetic changes or combinations of genetic changes may drive changes in antigenic phenotype. The present study aimed to identify antigenicity-associated sites in the hemagglutinin protein of A/H1N1 seasonal influenza virus using computational approaches. Random Forest Regression (RFR) and Support Vector Regression based on Recursive Feature Elimination (SVR-RFE) were applied to H1N1 seasonal influenza viruses and used to analyze the associations between amino acid changes in the HA1 polypeptide and antigenic variation based on hemagglutination-inhibition (HI) assay data. Twenty-three and twenty antigenicity-associated sites were identified by RFR and SVR-RFE, respectively, by considering the joint effects of amino acid residues on antigenic drift. Our proposed approaches were further validated with the H3N2 dataset. The prediction models developed in this study can quantitatively predict antigenic differences with high prediction accuracy based only on HA1 sequences. Application of the study results can increase understanding of H1N1 seasonal influenza virus antigenic evolution and accelerate the selection of vaccine strains.     

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.     

Antigenic and genetic evolution of equine influenza A (H3N8) virus from 1968 to 2007

Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.     

Defects in antigen presentation of mutant influenza haemagglutinins are reversed by mutations in the MHC class II molecule

A functional analysis was undertaken of the effects of mutating single amino acid residues in the alpha chain of the I-Ak molecule (to alanine; residues 50-79) on the ability of I-Ak transfectants to process and present influenza haemagglutinin to CD4+ T cell clones specific for two major antigenic sites of the HA1 subunit. In each instance, T cells were insensitive to a majority of substitutions in Ak with the exception of a few critical residues that differed for individual T cell clones. But more significantly, the failure of T cell clones to respond to mutant influenza viruses, containing drift substitutions within a T cell recognition site, in association with wild type I-Ak, could be reversed by single substitutions in Ak alpha. A T cell clone specific for HA1 120-139 failed to respond to a laboratory mutant virus (HA1 135 Gly----Arg) whereas optimal responses were observed with a mutant Ak transfectant (Ak alpha 56 Arg----Ala). Similarly, mutant transfectant 62 (Ak alpha 62 Gly----Ala) was able to present a natural variant virus A/TEX/77 to a T cell clone specific for HA1 48-67. We propose that Ak alpha 56 and Ak alpha 62 increase the affinity of association of mutant HA1 peptides for class II and therefore confer T cell recognition of variant viruses.