Hemagglutinin Residues Research Articles

Broadly neutralizing influenza hemagglutinin stem-specific antibody CR8020 targets residues that are prone to escape due to host selection pressure.

Broadly neutralizing antibodies (bNAb) that target a conserved region of a viral antigen hold significant therapeutic promise. CR8020 is a bNAb that targets the stem region of influenza A virus (IAV) hemagglutinin (HA). CR8020 is currently being evaluated forprophylactic use against group 2 IAVs in phase II studies. Structural and computational analyses reported here indicate that CR8020 targets HA residues that are prone to antigenic drift and host selection pressure. Critically, CR8020 escape mutation is seen in certain H7N9 viruses from recent outbreaks. Furthermore, the ability of the bNAb Fc region to effectively engage activating Fcγ receptors (FCγR) is essential for antibody efficacy. In this regard, our data indicate that the membrane could sterically hinder the formation of HA-CR8020-FcγRIIa/HA-IgG-FcγRIIIa ternary complexes. Altogether, our analyses suggest that epitope mutability and accessibility to immune complex assembly are important attributes to consider when evaluating bNAb candidates for clinical development.

A Broadly Neutralizing Human Monoclonal Antibody Directed against a Novel Conserved Epitope on the Influenza Virus H3 Hemagglutinin Globular Head

Most neutralizing antibodies elicited during influenza virus infection or vaccination target immunodominant, variable epitopes on the globular head region of hemagglutinin (HA), which leads to narrow strain protection. In this report, we describe the properties of a unique anti-HA monoclonal antibody (MAb), D1-8, that was derived from human B cells and exhibits potent, broad neutralizing activity across antigenically diverse influenza H3 subtype viruses. Based on selection of escape variants, we show that D1-8 targets a novel epitope on the globular head region of the influenza virus HA protein. The HA residues implicated in D1-8 binding are highly conserved among H3N2 viruses and are located proximal to antigenic site D. We demonstrate that the potent in vitro antiviral activity of D1-8 translates into protective activity in mouse models of influenza virus infection. Furthermore, D1-8 exhibits superior therapeutic survival benefit in influenza virus-infected mice compared to the neuraminidase inhibitor oseltamivir when treatment is started late in infection. The present study suggests the potential application of this monoclonal antibody for the therapeutic treatment of H3N2 influenza virus infection. Recently, a few globular head-targeting MAbs have been discovered that exhibit activity against different subtypes of influenza subtypes, such as H1; however, none of the previously described MAbs showed broadly neutralizing activity against diverse H3 viruses. In this report, we describe a human MAb, D1-8, that exhibits potent, broadly neutralizing activity against antigenically diverse H3 subtype viruses. The genotypic analysis of escape mutants revealed a unique putative epitope region in the globular head of H3 HA that is comprised of highly conserved residues and is distinct from the receptor binding site. Furthermore, we demonstrate that D1-8 exhibits superior therapeutic efficacy in influenza virus-infected mice compared to the neuraminidase inhibitor oseltamivir when treatment is started late in infection. In addition to describing a novel anti-globular head of H3 HA MAb with potent broadly neutralizing activity, our report suggests the potential of D1-8 for therapeutic treatment of seasonal influenza virus H3 infection.

Polymorphisms at Residue 222 of the Hemagglutinin of Pandemic Influenza A(H1N1)pdm09: Association of Quasi-Species to Morbidity and Mortality in Different Risk Categories

The D222G substitution in the hemagglutinin (HA) gene of the pandemic influenza A(H1N1)pdm09 virus has been identified as a potential virulence marker, because this change allows for virus invasion deeper into the respiratory tract. In this study, we analyzed D, G and N polymorphisms at residue 222 by pyrosequencing (PSQ). We initially analyzed 401 samples from Brazilian patients. These were categorized with respect to clinical conditions due to influenza infection (mild, serious or fatal) and sub-stratified by risky factors. The frequency of mixed population of virus, with more than one polymorphism at residue 222, was significantly higher in serious (10.6%) and fatal (46.7%) influenza cases, whereas those who showed mild influenza infections were all infected by D222 wild-type. Mixtures of quasi-species showed a significant association of mortality, especially for those with risk factors, in special pregnant women. These results not only reinforce the association between D222G substitution and influenza A(H1N1)pdm09-associated morbidity and mortality, but also add the perspective that a worse clinical prognosis is most likely correlated with mixtures of quasi-species at this HA residue. Therefore, quasi-species may have a critical and underestimated role in influenza-related clinical outcomes.

Molecular Mechanisms of Inhibition of Influenza by Surfactant Protein D Revealed by Large-Scale Molecular Dynamics Simulation

Surfactant protein D (SP-D), a mammalian C-type lectin, is the primary innate inhibitor of influenza A virus (IAV) in the lung. Interactions of SP-D with highly branched viral N-linked glycans on hemagglutinin (HA), an abundant IAV envelope protein and critical virulence factor, promote viral aggregation and neutralization through as yet unknown molecular mechanisms. Two truncated human SP-D forms, wild-type (WT) and double mutant D325A+R343V, representing neck and carbohydrate recognition domains are compared in this study. Whereas both WT and D325A+R343V bind to isolated glycosylated HA, WT does not inhibit IAV in neutralization assays; in contrast, D325A+R343V neutralization compares well with that of full-length native SP-D. To elucidate the mechanism for these biochemical observations, we have determined crystal structures of D325A+R343V in the presence and absence of a viral nonamannoside (Man9). On the basis of the D325A+R343V-Man9 structure and other crystallographic data, models of complexes between HA and WT or D325A+R343V were produced and subjected to molecular dynamics. Simulations reveal that whereas WT and D325A+R343V both block the sialic acid receptor site of HA, the D325A+R343V complex is more stable, with stronger binding caused by additional hydrogen bonds and hydrophobic interactions with HA residues. Furthermore, the blocking mechanism of HA differs for WT and D325A+R343V because of alternate glycan binding modes. The combined results suggest a mechanism through which the mode of SP-D-HA interaction could significantly influence viral aggregation and neutralization. These studies provide the first atomic-level molecular view of an innate host defense lectin inhibiting its viral glycoprotein target.

Genetic Requirement for Hemagglutinin Glycosylation and Its Implications for Influenza A H1N1 Virus Evolution

Influenza A virus has evolved and thrived in human populations. Since the 1918 influenza A pandemic, human H1N1 viruses had acquired additional N-linked glycosylation (NLG) sites within the globular head region of hemagglutinin (HA) until the NLG-free HA head pattern of the 1918 H1N1 virus was renewed with the swine-derived 2009 pandemic H1N1 virus. Moreover, the HA of the 2009 H1N1 virus appeared to be antigenically related to that of the 1918 H1N1 virus. Hence, it is possible that descendants of the 2009 H1N1 virus might recapitulate the acquisition of HA head glycosylation sites through their evolutionary drift as a means to evade preexisting immunity. We evaluate here the evolution signature of glycosylations found in the globular head region of H1 HA in order to determine their impact in the virulence and transmission of H1N1 viruses. We identified a polymorphism at HA residue 147 associated with the acquisition of glycosylation at residues 144 and 172. By in vitro and in vivo analyses using mutant viruses, we also found that the polymorphism at HA residue 147 compensated for the loss of replication, virulence, and transmissibility associated with the presence of the N-linked glycans. Our findings suggest that the polymorphism in H1 HA at position 147 modulates viral fitness by buffering the constraints caused by N-linked glycans and provide insights into the evolution dynamics of influenza viruses with implications in vaccine immunogenicity.

Identification of Critical Residues in the Hemagglutinin and Neuraminidase of Influenza Virus H1N1pdm for Vaccine Virus Replication in Embryonated Chicken Eggs

In 2009, we successfully produced a high-yield live attenuated H1N1pdm A/California/7/2009 vaccine (CA/09 LAIV) by substitution of three residues (K119E, A186D, and D222G) in the hemagglutinin (HA) protein. Since then, we have generated and evaluated additional H1N1pdm vaccine candidates from viruses isolated in 2010 and 2011. The 2010 strains with the new HA substitutions near the HA receptor binding site (N125D and D127E or D127E and K209E) grew well in eggs and formed large plaques in Madin-Darby canine kidney (MDCK) cells. Introduction of these acidic amino acids into the HA of CA/09 also improved vaccine virus growth in eggs to a titer comparable to that of CA/09 LAIV. However, the high growth of A/Gilroy/231/2011 (Gil/11) vaccine virus required modification in both the HA and the NA segments. The residue at position 369 of the NA was found to be critical for virus replication in MDCK cells and eggs. These HA and NA residues had minimal impact on viral entry but greatly improved viral release from infected cells. Our data implied that the HA receptor binding and NA receptor cleaving function of the poor-growth H1N1pdm virus was not well balanced for virus replication in host cells. The high-growth vaccine candidates described in this study maintained vaccine virus antigenicity and induced high levels of neutralizing antibodies in immunized ferrets, making them suitable for vaccine production. The identification of the amino acids and their roles in viral replication should greatly help vaccine manufacturers to produce high-yield reassortant vaccine viruses against the future drifted H1N1pdm viruses.

A recombinant protein containing highly conserved hemagglutinin residues 81-122 of influenza H5N1 induces strong humoral and mucosal immune responses

Influenza has long been considered a serious global health threat. The highly pathogenic avian influenza A virus (IAV) H5N1, particularly the currently identified IAV/H7N9 in humans in China, illustrates that influenza is still a significant public health problem. Due to the high mortality of H5N1, development of safe and effective vaccines against divergent strains of H5N1 influenza virus, especially the one capable of inducing both strong systemic and local immune responses in the vaccinated targets, is a challenge of immediate importance. In the present study, we designed two recombinant proteins containing highly conserved hemagglutinin (HA) residues 81-122 of H5N1 fused with Fc of human IgG (HA-81-122-Fc) and/or foldon (Fd) trimeric motif (HA-81-122-Fdc), and identified their immunogenicity in vaccinated mice. We found that HA-81-122-Fc and HA-81-122-Fdc proteins formed high molecular weight dimer and oligomer, respectively, and induced potent IgG antibodies in vaccinated mouse sera and lung wash. Stronger IgG1 (Th2-associated) and IgG2 (Th1-associated) antibody responses could be raised in the sera of mice following last vaccination of HA-81-122-Fdc than those raised by HA-81-122-Fc vaccination. Importantly, HA-81-122-Fdc is able to elicit high titers of IgA antibodies in vaccinated mouse lung wash and sera through the parenteral immunization pathway. Our data demonstrated that the recombinant protein containing highly conserved HA residues 81-122 of H5N1 fused with Fd and Fc could induce strong local mucosal and systemic humoral immune responses in the vaccinated animals, revealing the possibility of developing an effective Fc-mediated mucosal influenza vaccine.

Nectin-4-Dependent Measles Virus Spread to the Cynomolgus Monkey Tracheal Epithelium: Role of Infected Immune Cells Infiltrating the Lamina Propria

After the contagion measles virus (MV) crosses the respiratory epithelium within myeloid cells that express the primary receptor signaling lymphocytic activation molecule (SLAM), it replicates briskly in SLAM-expressing cells in lymphatic organs. Later, the infection spreads to epithelia expressing nectin-4, an adherens junction protein expressed preferentially in the trachea, but how it gets there is not understood. To characterize the mechanisms of spread, we infected groups of 5 or 6 cynomolgus monkeys (Macaca fascicularis) with either a wild-type MV or its "N4-blind" derivative, which is unable to enter nectin-4-expressing cells because of the targeted mutation of two hemagglutinin residues. As expected, both viruses caused similar levels of immunosuppression, as monitored by reductions in white blood cell counts and lymphocyte proliferation activity. However, monkeys infected with the N4-blind MV cleared infection more rapidly. Wild-type virus-infected monkeys secreted virus, while marginal virus titers were detected in tracheal lavage fluid cells of N4-blind MV-infected hosts. Analyses of tracheal rings obtained at necropsy (day 12) documented widespread infection of individual cells or small cell clusters in the subepithelial lamina propria of monkeys infected with either virus. However, only wild-type MV spread to the epithelium, forming numerous infectious centers comprised of many contiguous columnar cells. Infected CD11c(+) myeloid (macrophage or dendritic) cells were frequently observed in the lamina propria below epithelial infectious centers. Thus, MV may use myeloid cells as vehicles not only immediately after contagion but also to infect epithelia of tissues expressing nectin-4, including the trachea.

Molecular evolution of HA1 in pandemic H1N1 influenza viruses isolated in Guangdong during 2009 to 2011

Objective To understand the evolutionary characterization of hemagglutinin (HA)gene of pandemic H1N1 influenza virus in Guangdong during 2009-2011. Methods We selected 83 pandemic H1N1 strains isolated in Guangdong during 2009-2011. The HA1 genes were sequenced and analyzed comparatively by Bioedit 7.0 and MEGA 4.0. Results The evolutionary rate of Hal gene of pandemic H1N1 and seasonal H1N1 viruses was 5.2×10-3 substitutions/site/year, higher than that of seasonal H1N1 viruses. Most amino acid changes in HA1 molecules accumulated on the surface of the molecule and were partly located in antigenic sites. Two fatal infections were detected with a mutation at HA residue 222, in one virus with a change D222G, and in one virus D222N. Conclusion The phylogenetic analysis demonstrates that the influenza epidemic in Guangdong at the beginning of 2011 are due to occurrence of genetic changes of pandemic H1N1 virus. The amino acid change at residue 222 of the HA1 are likely to be associated with severe or even fatal illness. Key words: Pandemic H1N1 influenza viruses; Hemagglutinin gene; Sequencing; Evolution

Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt.

Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.

Genetic diversity of the 2009 pandemic influenza A(H1N1) viruses in Finland.

BackgroundIn Finland, the first infections caused by the 2009 pandemic influenza A(H1N1) virus were identified on May 10. During the next three months almost all infections were found from patients who had recently traveled abroad. In September 2009 the pandemic virus started to spread in the general population, leading to localized outbreaks and peak epidemic activity was reached during weeks 43–48.Methods/ResultsThe nucleotide sequences of the hemagglutinin (HA) and neuraminidase (NA) genes from viruses collected from 138 patients were determined. The analyzed viruses represented mild and severe infections and different geographic regions and time periods. Based on HA and NA gene sequences, the Finnish pandemic viruses clustered in four groups. Finnish epidemic viruses and A/California/07/2009 vaccine virus strain varied from 2–8 and 0–5 amino acids in HA and NA molecules, respectively, giving a respective maximal evolution speed of 1.4% and 1.1%. Most amino acid changes in HA and NA molecules accumulated on the surface of the molecule and were partly located in antigenic sites. Three severe infections were detected with a mutation at HA residue 222, in two viruses with a change D222G, and in one virus D222Y. Also viruses with change D222E were identified. All Finnish pandemic viruses were sensitive to oseltamivir having the amino acid histidine at residue 275 of the neuraminidase molecule.ConclusionsThe Finnish pandemic viruses were quite closely related to A/California/07/2009 vaccine virus. Neither in the HA nor in the NA were changes identified that may lead to the selection of a virus with increased epidemic potential or exceptionally high virulence. Continued laboratory-based surveillance of the 2009 pandemic influenza A(H1N1) is important in order to rapidly identify drug resistant viruses and/or virus variants with potential ability to cause severe forms of infection and an ability to circumvent vaccine-induced immunity.

Evolution of Human Receptor Binding Affinity of H1N1 Hemagglutinins from 1918 to 2009 Pandemic Influenza A Virus

The recent outbreak of the novel 2009 H1N1 influenza in humans has focused global attention on this virus, which could potentially have introduced a more dangerous pandemic of influenza flu. In the initial step of the viral attachment, hemagglutinin (HA), a viral glycoprotein surface, is responsible for the binding to the human SIA alpha2,6-linked sialopentasaccharide host cell receptor (hHAR). Dynamical and structural properties, based on molecular dynamics simulations of the four different HAs of Spanish 1918 (H1-1918), swine 1930 (H1-1930), seasonal 2005 (H1-2005), and a novel 2009 (H1-2009) H1N1 bound to the hHAR were compared. In all four HA-hHAR complexes, major interactions with the receptor binding were gained from HA residue Y95 and the conserved HA residues of the 130-loop, 190-helix, and 220-loop. However, introduction of the charged HA residues K145 and E227 in the 2009 HA binding pocket was found to increase the HA-hHAR binding efficiency in comparison to the three previously recognized H1N1 strains. Changing of the noncharged HA G225 residue to a negatively charged D225 provides a larger number of hydrogen-bonding interactions. The increase in hydrophilicity of the receptor binding region is apparently an evolution of the current pandemic flu from the 1918 Spanish, 1930 swine, and 2005 seasonal strains. Detailed analysis could help the understanding of how different HAs effectively attach and bind with the hHAR.

In Vitro Generation of Neuraminidase Inhibitor Resistance in A(H5N1) Influenza Viruses

To identify mutations that can arise in highly pathogenic A(H5N1) viruses under neuraminidase inhibitor selective pressure, two antigenically different strains were serially passaged with increasing levels of either oseltamivir or zanamivir. Under oseltamivir pressure, both A(H5N1) viruses developed a H274Y neuraminidase mutation, although in one strain the mutation occurred in combination with an I222M neuraminidase mutation. The H274Y neuraminidase mutation reduced oseltamivir susceptibility significantly (900- to 2,500-fold compared to the wild type). However the dual H274Y/I222M neuraminidase mutation had an even greater impact on resistance, with oseltamivir susceptibility reduced significantly further (8,000-fold compared to the wild type). A similar affect on oseltamivir susceptibility was observed when the dual H274Y/I222M mutations were introduced, by reverse genetics, into a recombinant seasonal human A(H1N1) virus and also when an alternative I222 substitution (I222V) was generated in combination with H274Y in A(H5N1) and A(H1N1) viruses. These viruses remained fully susceptible to zanamivir but demonstrated reduced susceptibility to peramivir. Following passage of the A(H5N1) viruses in the presence of zanamivir, the strains developed a D198G neuraminidase mutation, which reduced susceptibility to both zanamivir and oseltamivir, and also an E119G neuraminidase mutation, which demonstrated significantly reduced zanamivir susceptibility (1,400-fold compared to the wild type). Mutations in hemagglutinin residues implicated in receptor binding were also detected in many of the resistant strains. This study identified the mutations that can arise in A(H5N1) under either oseltamivir or zanamivir selective pressure and the potential for dual neuraminidase mutations to result in dramatically reduced drug susceptibility.

Possibility of Mutation Prediction of Influenza Hemagglutinin by Combination of Hemadsorption Experiment and Quantum Chemical Calculation for Antibody Binding

We have performed a quantum-chemical MP2/6-31G* calculation for the hemagglutinin (HA) antigen-antibody system of the H3N2 influenza virus with the fragment molecular orbital method, which provides one of the world's largest ab initio electron-correlated calculations for biomolecular systems. On the basis of the calculated interfragment interaction energies (IFIEs) representing the molecular interactions between the amino acid residues in the antigen-antibody complex, we have identified those residues in the antigenic region E of HA protein that are significantly recognized by the Fab fragment of antibody with strongly attractive interactions. Combining these IFIE results with those of hemadsorption experiments by which the mutation-prohibited sites are specified has enabled us to explain most of the historical mutation data (five of six residues), which would thus provide a promising method for predicting the HA residues that have a high probability of forthcoming mutation.

808. The Two Phases of Measles Virus Cell Entry through SLAM: Hemagglutinin Residues Necessary for Primary Binding or Membrane Fusion

808. The Two Phases of Measles Virus Cell Entry through SLAM: Hemagglutinin Residues Necessary for Primary Binding or Membrane Fusion

Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5N1 influenza virus vaccines

If H5N1 influenza viruses become transmissible among humans, vaccination will offer the most effective option to limit their spread. Two human vaccine candidates recently generated by reverse genetics are based on antigenically different hemagglutinin (HA) glycoproteins derived from the A/HK/213/03 (H5N1) and A/Vietnam/1203/04 (H5N1) viruses. Their HA1 amino acid sequences differ at 10 positions, one of which (N154) introduces a potential glycosylation site in A/Vietnam/1203/04 (H5N1). To assess the impact of five amino acids in the putative antigenic sites on immunogenicity and immune protection, we generated a series of whole-virus vaccines that differed only in one or two HA amino acids. Sera from ferrets vaccinated with these inactivated preparations had high virus neutralization titers, but their hemagglutination inhibition (HI) titers were usually low. Interestingly, a recombinant virus in which the HA amino acid S223 (characteristic of 2004 viruses) was converted to N223 (as in A/HK/213/03) resulted in higher HI titers. This observation indicates that specific HA residues, such as N223, increase the sensitivity of the HI assay by altering receptor specificity and/or antibody-antigen binding. Ferrets vaccinated with mutant vaccine viruses were protected against lethal challenge with wild-type A/Vietnam/1203/04 virus. Our results suggest that inclusion of the N223 residue in the HA glycoproteins of diagnostic reference viruses may facilitate the evaluation of vaccine efficacy in humans.

Improvement of Influenza A/Fujian/411/02 (H3N2) Virus Growth in Embryonated Chicken Eggs by Balancing the Hemagglutinin and Neuraminidase Activities, Using Reverse Genetics

The H3N2 influenza A/Fujian/411/02-like virus strains that circulated during the 2003-2004 influenza season caused influenza epidemics. Most of the A/Fujian/411/02 virus lineages did not replicate well in embryonated chicken eggs and had to be isolated originally by cell culture. The molecular basis for the poor replication of A/Fujian/411/02 virus was examined in this study by the reverse genetics technology. Two antigenically related strains that replicated well in embryonated chicken eggs, A/Sendai-H/F4962/02 and A/Wyoming/03/03, were compared with the prototype A/Fujian/411/02 virus. A/Sendai differed from A/Fujian by three amino acids in the neuraminidase (NA), whereas A/Wyoming differed from A/Fujian by five amino acids in the hemagglutinin (HA). The HA and NA segments of these three viruses were reassorted with cold-adapted A/Ann Arbor/6/60, the master donor virus for the live attenuated type A influenza vaccines (FluMist). The HA and NA residues differed between these three H3N2 viruses evaluated for their impact on virus replication in MDCK cells and in embryonated chicken eggs. It was determined that replication of A/Fujian/411/02 in eggs could be improved by either changing minimum of two HA residues (G186V and V226I) to increase the HA receptor-binding ability or by changing a minimum of two NA residues (E119Q and Q136K) to lower the NA enzymatic activity. Alternatively, recombinant A/Fujian/411/02 virus could be adapted to grow in eggs by two amino acid substitutions in the HA molecule (H183L and V226A), which also resulted in the increased HA receptor-binding activity. Thus, the balance between the HA and NA activities is critical for influenza virus replication in a different host system. The HA or NA changes that increased A/Fujian/411/02 virus replication in embryonated chicken eggs were found to have no significant impact on antigenicity of these recombinant viruses. This study demonstrated that the reverse genetics technology could be used to improve the manufacture of the influenza vaccines.

Hemagglutinin residues of recent human A(H3N2) viruses that affect agglutination of chicken erythrocytes

Background: The hemagglutination remains crucial in diagnosis of influenza virus and for the antigenic characterization of the hemagglutinin (HA) and neuraminidase. However, the human influenza viruses A(H3N2) isolated recently appear to have lost the ability to agglutinate chicken erythrocytes (ER) (RBC). The molecular determinants of this phenomenon are not known. Methods: Two viruses isolated in Paris were studied, since their ability to agglutinate chicken RBC was observed after serial passages either in Madin Darby canine kidney (MDCK) cells or embryonated hen's eggs. Sequencing analyses and hemadsorption assays were performed to demonstrate the role of amino acid substitutions in the HA gene. Results: Sequencing of the HA gene revealed the presence of either the Leu194Ile or the Val226Ile mutation following the phenotypic change. Hemadsorption assays performed following transfection of COS-1 cells, with plasmids expressing wild-type or mutated HA molecules, showed that the Leu194Ile and Val226Ile mutations were responsible for the ability of the HA to bind chicken RBCs. Conclusion: These findings suggest that a valine at position 226 in the HA molecule, found in recent clinical isolates of human A(H3N2) viruses, could be responsible for their inability to agglutinate chicken erythrocytes.

Hemagglutinin residues of recent human A(H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes.

To identify the molecular determinants contributing to the inability of recent human influenza A(H3N2) viruses to agglutinate chicken erythrocytes, phenotypic revertants were selected upon passage in eggs or MDCK cells. The Leu194Ile or Val226Ile substitutions were detected in their hemagglutinin (HA) sequence concomitantly with the phenotypic reversion. Remarkably, as little as 3.5% of variants bearing a Val226Ile substitution was found to confer the ability to agglutinate chicken erythrocytes to the virus population. Hemadsorption assays following transient expression of mutated HA proteins showed that the successive Gln226 --> Leu --> Ile --> Val changes observed on natural isolates resulted in a progressive loss of the ability of the HA to bind chicken erythrocytes. The Val226Ile change maintained the preference of the HA for SAalpha2,6Gal over SAalpha2,3Gal and enhanced binding of the HA to alpha2,6Gal receptors present on chicken erythrocytes. In contrast, simultaneous Ser193Arg and Leu194Ile substitutions that were found to confer the ability to agglutinate sheep erythrocytes increased the affinity of the HA for SAalpha2,3Gal.

T cell selection and differential activation on structurally related HLA-DR4 ligands.

Plasticity of TCR interactions during CD4(+) T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4(+) T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307-319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307-319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.