Receptor Specificity Of Hemagglutinin Research Articles

Re-Emergence of Circulation of Seasonal Influenza during COVID-19 Pandemic in Russia and Receptor Specificity of New and Dominant Clade 3C.2a1b.2a.2 A(H3N2) Viruses in 2021-2022.

The circulation of seasonal influenza in 2020-2021 around the world was drastically reduced after the start of the COVID-19 pandemic and the implementation of mitigation strategies. The influenza virus circulation reemerged in 2021-2022 with the global spread of the new genetic clade 3C.2a1b.2a.2 of A(H3N2) viruses. The purpose of this study was to characterize influenza viruses in the 2021-2022 season in Russia and to analyze the receptor specificity properties of the 3C.2a1b.2a.2 A(H3N2) viruses. Clinical influenza samples were collected at the local Sanitary-and-Epidemiological Centers of Rospotrebnadzor. Whole genome sequencing was performed using NGS. The receptor specificity of hemagglutinin was evaluated using molecular modeling and bio-layer interferometry. Clinical samples from 854 cases of influenza A and B were studied; A(H3N2) viruses were in the majority of the samples. All genetically studied A(H3N2) viruses belonged to the new genetic clade 3C.2a1b.2a.2. Molecular modeling analysis suggested a higher affinity of hemagglutinin of 3C.2a1b.2a.2. A(H3N2) viruses to the α2,6 human receptor. In vitro analysis using a trisaccharide 6'-Sialyl-N-acetyllactosamine receptor analog did not resolve the differences in the receptor specificity of 3C.2a1b.2a.2 clade viruses from viruses belonging to the 3C.2a1b.2a.1 clade. Further investigation of the A(H3N2) viruses is required for the evaluation of their possible adaptive advantages. Constant monitoring and characterization of influenza are critical for epidemiological analysis.

COMPARISON OF INFLUENZA A(H1N1)pdm09 GENOMES OBTAINED FROM DIFFERENT TYPES OF VIRUS-CONTAINING MATERIAL

This paper describes results of comparison of genetic sequences of inf luenza viruses obtained from clinical samples (nasopharyngeal swabs) and viruses isolated on MDCK cells and in developing chick embryos. The obtained data shows that 1–2x fold passaging of inf luenza viruses in MDCK cells does not lead to additional mutations in the genome of the inf luenza virus, and the genetic material of this virus is identical to the genetic material from the clinical sample. When passaging viruses in chick embryos within 1–2 passages, the genetic material of the virus differs from the genetic material from the clinical sample, because of mutations in the receptor region of hemagglutinin, leading to aminoacid substitution Q223R. The mutation Q223R changes the receptor specificity of hemagglutinin from human-type receptors (α-2.6-sialic acids) to avian receptors (α-2.3-sialic acids). The data obtained coincide with the data of Japanese researchers who showed that a similar substitution in the hemagglutinin gene was detected in Japanese strains of inf luenza virus subtype A (H1N1) pdm09, passaged in chick embryos for 1–2 passages. With their further passaging in chick embryos, the number of acquired mutations in the surface proteins of these viruses increases. Thus, we have shown that for the genetic analysis of epidemic-relevant strains of inf luenza viruses for routine epidemiological surveillance tasks, the use of direct sequencing of clinical samples containing the genetic material of viruses makes it possible to obtain the most reliable data, to increase their efficiency, since it saves time normally spent on virus isolation procedure. If preliminary isolation of viruses is necessary, for example, if it is not possible to carry out full genome amplification from a clinical sample (low content of the material, presence of polymerase chain reaction inhibitors), virus isolation on MDCK cells with a minimal amount of passage is optimal, which is used in practice of epidemiological surveillance.

In vitro evolution of an influenza broadly neutralizing antibody is modulated by hemagglutinin receptor specificity

The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. However, the factors that influence the evolution of high-affinity bnAbs remain elusive. We therefore explore the functional sequence space of bnAb C05, which targets the receptor-binding site (RBS) of influenza haemagglutinin (HA) via a long CDR H3. We combine saturation mutagenesis with yeast display to enrich for C05 variants of CDR H3 that bind to H1 and H3 HAs. The C05 variants evolve up to 20-fold higher affinity but increase specificity to each HA subtype used in the selection. Structural analysis reveals that the fine specificity is strongly influenced by a highly conserved substitution that regulates receptor binding in different subtypes. Overall, this study suggests that subtle natural variations in the HA RBS between subtypes and species may differentially influence the evolution of high-affinity bnAbs.

Attenuation of an influenza A virus due to alteration of its hemagglutinin-neuraminidase functional balance in mice

Influenza A viruses possess two surface glycoproteins, hemagglutinin (HA), which binds to sialic-acid-containing receptors, and neuraminidase (NA), which removes sialic acid from host cells. It is well established that the HA-NA functional balance regulates the efficiency of virus replication. Here, we selected a plaque variant of the WSN (H1N1) strain that grew better than the wild-type virus in NA-expressing MDCK cell culture. A reverse genetics study revealed that the single mutation HA E190K, which occurs infrequently in naturally isolated H1N1 viruses, was responsible for the phenotype of this variant. Receptor assays indicated that this mutation did not affect the receptor specificity of HA but enhanced its receptor-binding affinity, resulting in altered HA-NA functional balance relative to that of the wild-type virus. We also found that this variant replicated in nasal turbinates at an equivalent level but in lungs at a lower level compared with wild-type virus, demonstrating its attenuation in mice. Together, our data demonstrated the importance of the HA-NA functional balance for influenza virus replication in an in vivo biological setting.

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.

Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic

Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only for α2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For α2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with α2-6- and α2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for α2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.

Glycans as receptors for influenza pathogenesis

Influenza A viruses, members of the Orthomyxoviridae family, are responsible for annual seasonal influenza epidemics and occasional global pandemics. The binding of viral coat glycoprotein hemagglutinin (HA) to sialylated glycan receptors on host epithelial cells is the critical initial step in the infection and transmission of these viruses. Scientists believe that a switch in the binding specificity of HA from Neu5Acα2-3Gal linked (α2-3) to Neu5Acα2-6Gal linked (α2-6) glycans is essential for the crossover of the viruses from avian to human hosts. However, studies have shown that the classification of glycan binding preference of HA based on sialic acid linkage alone is insufficient to establish a correlation between receptor specificity of HA and the efficient transmission of influenza A viruses. A recent study reported extensive diversity in the structure and composition of α2-6 glycans (which goes beyond the sialic acid linkage) in human upper respiratory epithelia and identified different glycan structural topologies. Biochemical examination of the multivalent HA binding to these diverse sialylated glycan structures also demonstrated that high affinity binding of HA to α2-6 glycans with a characteristic umbrella-like structural topology is critical for efficient human adaptation and human-human transmission of influenza A viruses. This review summarizes studies which suggest a new paradigm for understanding the role of the structure of sialylated glycan receptors in influenza virus pathogenesis.

Influenza Virus Receptor Specificity: Disease and Transmission

Influenza Virus Receptor Specificity: Disease and Transmission

A Two-Amino Acid Change in the Hemagglutinin of the 1918 Influenza Virus Abolishes Transmission

The 1918 influenza pandemic was a catastrophic series of virus outbreaks that spread across the globe. Here, we show that only a modest change in the 1918 influenza hemagglutinin receptor binding site alters the transmissibility of this pandemic virus. Two amino acid mutations that cause a switch in receptor binding preference from the human alpha-2,6 to the avian alpha-2,3 sialic acid resulted in a virus incapable of respiratory droplet transmission between ferrets but that maintained its lethality and replication efficiency in the upper respiratory tract. Furthermore, poor transmission of a 1918 virus with dual alpha-2,6 and alpha-2,3 specificity suggests that a predominant human alpha-2,6 sialic acid binding preference is essential for optimal transmission of this pandemic virus. These findings confirm an essential role of hemagglutinin receptor specificity for the transmission of influenza viruses among mammals.

Changes in H3 influenza A virus receptor specificity during replication in humans

Influenza A viruses of the H3 subtype caused the 1968 Hong Kong pandemic, the hemagglutinin (HA) gene being introduced into humans following a reassortment event with an avian virus. Receptor specificity and serum inhibitor sensitivity of the HA of influenza A viruses are linked to the host species. Human H3 viruses preferentially recognize N-acetyl sialic acid linked to galactose by α2,6 linkages (Neu5Ac α2,6Gal) and are sensitive to serum inhibitors, whereas avian and equine viruses preferentially recognize Neu5Ac α2,3Gal linkages and are resistant to serum inhibitors. We have examined the receptor specificity and serum inhibitor sensitivity of H3 human influenza A viruses from the time they were introduced into the human population to gain insight into the mechanism of viral molecular evolution and host tropism. All of the viruses were sensitive to neutralization and hemagglutination inhibition by horse serum. Early H3 viruses were resistant to pig and rabbit serum inhibitors. Viruses isolated after 1977 were uniformly sensitive to inhibition by pig and rabbit sera. The recognition of Neu5Ac α2,3Gal or Neu5Ac α2,6Gal linkages was not correlated with the serum sensitivity. These data showed that the receptor specificity of HA, measured as inhibitor sensitivity, has changed during replication in humans since its introduction from an avian virus.

Characterization of the hemagglutinin receptor specificity and neuraminidase substrate specificity of clinical isolates of human influenza A viruses.

Six clinical isolates of influenza A viruses were examined for hemagglutinin receptor specificity and neuraminidase substrate specificity. All of the viral isolates minimally passaged in mammalian cells demonstrated preferential agglutination of human erythrocytes enzymatically modified to contain NeuAc alpha 2,6Gal sequences, with no agglutination of cells bearing NeuAc alpha 2,3Gal sequences. This finding is consistent with the hemagglutination receptor specificity previously demonstrated for laboratory strains of influenza A viruses. The neuraminidase substrate specificities of the clinical isolates examined were also identical to that described for the N2 neuraminidase of recent laboratory strains of human influenza viruses. The H3N2 viruses all displayed the ability to release sialic acid from both alpha 2, 3 and alpha 2, 6 linkages. In addition, two clinical isolates of H1N1 viruses also demonstrated this dual neuraminidase substrate specificity, a characteristic which has not been previously described for the N1 neuraminidase. These results demonstrate that complementary hemagglutinin and neuraminidase specificities are found in recent isolates of both H1N1 and H3N2 influenza viruses.

Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity

The complement of sialyloligosaccharides present on the surface of human tracheal epithelium has been implicated as an important factor in the selection of hemagglutinin receptor specificity of human influenza A virus. Human strains of influenza A virus preferentially recognize host cell receptors bearing SAα2,6Gal sequences, a sequence which is found on the surface of ciliated tracheal epithelium. A fluorescently-labelled H3 human virus strain bound avidly to the apical surface of human tracheal epithelium, while a fluorescently-labelled receptor variant strain, which preferentially binds SAα2,3Gal sequences, showed little binding to the epithelial surface and localized primarily to intracellular mucin droplets. Extracts of human bronchial mucin, which is known to contain sialic acid primarily in the SAα2,3Gal linkage, was a potent inhibitor of the binding of the receptor variant strain to trachea sections, while the binding of the parent strain was unaffected by the presence of mucin. Human bronchial mucin also inhibited the binding of the receptor variant strains, but not the parent virus strains, to human erythrocytes derivatized to contain SAα2,6Gal sequences. These results suggest that a combination of selection pressures present in the respiratory tract environment have resulted in the evolution of a hemagglutinin receptor specificity in human influenza A virus strains which optimizes recognition of, binding to and infection of host cells.

Effects of body temperature on respiratory rate in young children.

<h3>Abstract</h3> Influenza A viruses (IAV) initiate infection <i>via</i> binding of the viral hemagglutinin (HA) to sialylated glycan receptors on host cells. HAs receptor specificity towards sialic acid (SA) is well studied and clearly critical for virus infection, but the contribution of the highly complex cellular plasma membrane to the cellular specificity remains elusive. In addition, some studies indicated that other host cell factors such as the epidermal growth factor receptor might contribute to the initial virus-cell contact and further downstream signaling¹. Here we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS) to compare influenza virus receptor specificity with actual host cell binding. Unexpectedly, our study reveals that HAs receptor binding preference does not necessarily reflect virus-cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAV thereby including the complex environment of sialylated receptors within the plasma membrane of living cells.

The N2 neuraminidase of human influenza virus has acquired a substrate specificity complementary to the hemagglutinin receptor specificity

A survey of 10 human influenza A viruses of the N2 serotype, isolated between 1957 and 1987, has revealed a drift in neuraminidase linkage specificity. While the earliest N2 strains examined exhibit strict specificity for cleavage of the NeuAcα2,3Gal sequence, N2 isolates from 1967 to 1968 also show limited activity towards the NeuAcα2,6Gal linkage. In strains isolated in 1972 and later, the N2 neuraminidase has approximately equal activity towards both types of linkages. The NeuAcα2,6Gal linkage cleaved by the later N2 neuraminidases is the preferred receptor determinant of human H2 and H3 hemagglutinins. Thus, the acquired neuraminidase specificity of the later isolates allows elution of bound virus from erythrocytes derivatized to contain the NeuAcα2,6Gal linkage, while earlier isolates, which cleave only the NeuAcα2,3Gal sequence, fail to elute from these erythrocytes. These results suggest that the observed drift in N2 neuraminidase specificity in the direction of the preferred H2 and H3 receptor determinant may facilitate release of progeny virus from host cells.