Isolation and Characterization of Human Monoclonal Antibodies That Recognize the Influenza A(H1N1)pdm09 Virus Hemagglutinin Receptor-Binding Site and Rarely Yield Escape Mutant Viruses.
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ABSTRACT:
SUBMITTER: Yasuhara A
PROVIDER: S-EPMC6222141 | biostudies-literature | 2018
REPOSITORIES: biostudies-literature
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Isolation and Characterization of Human Monoclonal Antibodies That Recognize the Influenza A(H1N1)pdm09 Virus Hemagglutinin Receptor-Binding Site and Rarely Yield Escape Mutant Viruses.
Yasuhara Atsuhiro A Yamayoshi Seiya S Ito Mutsumi M Kiso Maki M Yamada Shinya S Kawaoka Yoshihiro Y
Frontiers in microbiology 20181101
The influenza A virus rapidly mutates to escape from antibodies. Here, we isolated and characterized three human monoclonal antibodies (mAbs) that neutralize A(H1N1)pdm09 viruses. Generation of escape mutant viruses suggested that these antibodies recognized conserved residues of the receptor-binding site (RBS) of hemagglutinin (HA) and that mutant viruses that escaped from these mAbs rarely appeared. Moreover, the escape mutant viruses grew significantly slower than wild-type virus, indicating ...[more]
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Project description:UNLABELLED:We determined the antigenic structure of pandemic influenza A(H1N1)pdm09 virus hemagglutinin (HA) using 599 escape mutants that were selected using 16 anti-HA monoclonal antibodies (MAbs) against A/Narita/1/2009. The sequencing of mutant HA genes revealed 43 amino acid substitutions at 24 positions in three antigenic sites, Sa, Sb, and Ca2, which were previously mapped onto A/Puerto Rico/8/34 (A/PR/8/34) HA (A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417-427, 1982), and an undesignated site, i.e., amino acid residues 141, 142, 143, 171, 172, 174, 177, and 180 in the Sa site, residues 170, 173, 202, 206, 210, 211, and 212 in the Sb site, residues 151, 154, 156, 157, 158, 159, 200, and 238 in the Ca2 site, and residue 147 in the undesignated site (numbering begins at the first methionine). Sixteen MAbs were classified into four groups based on their cross-reactivity with the panel of escape mutants in the hemagglutination inhibition test. Among them, six MAbs targeting the Sa and Sb sites recognized both residues at positions 172 and 173. MAb n2 lost reactivity when mutations were introduced at positions 147, 159 (site Ca2), 170 (site Sb), and 172 (site Sa). We designated the site consisting of these residues as site Pa. From 2009 to 2013, no antigenic drift was detected for the A(H1N1)pdm09 viruses. However, if a novel variant carrying a mutation at a position involved in the epitopes of several MAbs, such as 172, appeared, such a virus would have the advantage of becoming a drift strain. IMPORTANCE:The first influenza pandemic of the 21st century occurred in 2009 with the emergence of a novel virus originating with swine influenza, A(H1N1)pdm09. Although HA of A(H1N1)pdm09 has a common origin (1918 H1N1) with seasonal H1N1, the antigenic divergence of HA between the seasonal H1N1 and A(H1N1)pdm09 viruses gave rise to the influenza pandemic in 2009. To take precautions against the antigenic drift of the A(H1N1)pdm09 virus in the near future, it is important to identify its precise antigenic structure. To obtain various mutants that are not neutralized by MAbs, it is important to neutralize several plaque-cloned parent viruses rather than only a single parent virus. We characterized 599 escape mutants that were obtained by neutralizing four parent viruses of A(H1N1)pdm09 in the presence of 16 MAbs. Consequently, we were able to determine the details of the antigenic structure of HA, including a novel epitope.
| S-EPMC4248900 | biostudies-literature
Project description:Immune imprinting is a driver known to shape the anti-hemagglutinin (HA) antibody landscape of individuals born within the same birth cohort. With the HA and neuraminidase (NA) proteins evolving at different rates under immune selection pressures, anti-HA and anti-NA antibody responses since childhood influenza virus infections have not been evaluated in parallel at the individual level. This is partly due to the limited knowledge of changes in NA antigenicity, as seasonal influenza vaccines have focused on generating neutralizing anti-HA antibodies against HA antigenic variants. Here, we systematically characterized the NA antigenic variants of seasonal A(H1N1) viruses from 1977 to 1991 and completed the antigenic profile of N1 NAs from 1977 to 2015. We identified that NA proteins of A/USSR/90/77, A/Singapore/06/86, and A/Texas/36/91 were antigenically distinct and mapped N386K as a key determinant of the NA antigenic change from A/USSR/90/77 to A/Singapore/06/86. With comprehensive panels of HA and NA antigenic variants of A(H1N1) and A(H1N1)pdm09 viruses, we determined hemagglutinin inhibition (HI) and neuraminidase inhibition (NI) antibodies from 130 subjects born between 1950 and 2015. Age-dependent imprinting was observed for both anti-HA and anti-NA antibodies, with the peak HI and NI titers predominantly detected from subjects at 4 to 12 years old during the year of initial virus isolation, except the age-independent anti-HA antibody response against A(H1N1)pdm09 viruses. More participants possessed antibodies that reacted to multiple antigenically distinct NA proteins than those with antibodies that reacted to multiple antigenically distinct HA proteins. Our results support the need to include NA proteins in seasonal influenza vaccine preparations. IMPORTANCE Seasonal influenza vaccines have aimed to generate neutralizing anti-HA antibodies for protection since licensure. More recently, anti-NA antibodies have been established as an additional correlate of protection. While HA and NA antigenic changes occurred discordantly, the anti-HA and anti-NA antibody profiles have rarely been analyzed in parallel at the individual level, due to the limited knowledge on NA antigenic changes. By characterizing NA antigenic changes of A(H1N1) viruses, we determined the anti-HA and anti-NA antibody landscape against antigenically distinct A(H1N1) and A(H1N1)pdm09 viruses using sera of 130 subjects born between 1950 and 2015. We observed age-dependent imprinting of both anti-HA and anti-NA antibodies against strains circulated during the first decade of life. A total of 67.7% (88/130) and 90% (117/130) of participants developed cross-reactive antibodies to multiple HA and NA antigens at titers ≥1:40. With slower NA antigenic changes and cross-reactive anti-NA antibody responses, including NA protein in influenza vaccine preparation may enhance vaccine efficacy.
| S-EPMC10294682 | biostudies-literature
Project description:The hemagglutinin (HA) is a major influenza virus antigen, which, once recognized by antibodies and substitutions in HA genes, helps virus in escaping the human immune response. It is therefore critical to perform genetic and phylogenetic analysis of HA in circulating influenza viruses. We performed phylogenetic and genetic analysis of isolates from Ukraine, the vaccine strain and reference strains were used to phylogenetically identify trends in mutation locations and substitutions. Ukrainian isolates were collected between 2009-2017 and clustered in the influenza genetic groups 2, 6, 7, and 8. Genetic changes were observed in each of the antigenic sites: Sa - S162T, K163Q, K163I; Sb - S185T, A186T, S190G, S190R; Ca1 - S203T, R205K, E235V, E235D, S236P; Ca2 - P137H, H138R, A141T, D222G, D222N; Cb - A73S, S74R, S74N. In spite of detected mutations in antigenic sites, Ukrainian isolates retained similarity to the vaccine strain A/California/07/09 circulated during 2009-2017. However, WHO recommended a new vaccine strain A/Michigan/45/2015 for the Southern Hemisphere after the emergence of the new genetic groups 6B.1 and 6B.2. Our study demonstrated genetic variability of HA protein of A(H1N1)pdm09 viruses isolated in 2009-2017 in Ukraine. Influenza surveillance is very important for understanding epidemiological situations.
| S-EPMC6963832 | biostudies-literature
Project description:Although it has been suggested that antigenic drift does not occur in a single epidemic season in temperate countries, there is not enough evidence on the circulation period of influenza virus with identical nucleotide sequences. Therefore, strains of influenza virus were isolated sequentially during five consecutive epidemic seasons in Japan and their nucleotide sequences were determined.Nasal swabs or aspirated nasal discharges were collected from influenza A virus antigen-positive individuals living in Tottori Prefecture, Japan for five consecutive winters starting in 2009-2010, and subjected to viral isolation, determination of hemagglutinin nucleotide sequence and phylogenic analyses. The nucleotide sequences were compared with each other and also with those of foreign strains in the International Nucleotide Sequence Database.Totally 288 A(H1N1)pdm09 strains were tested and those composed 38 clusters with identical ones displaying 100% nucleotide homology. One strain showed sequential infections more than three months without any detectable mutation, and a maximum interval of two detection timings of strains was 94 days. This implies that influenza viruses mutate rarely in an epidemic season in Japan if they can be hypothesized, mutation frequency of influenza viruses being mostly the same among strains. Among these identical strains, two strains were not only identical to other Japanese isolates, but also to those isolated in Mongolia and Thailand in the same epidemic season.These results suggest that genetic drift has occurred infrequently in Japan as shown in some other countries. The drifted strains may have generated somewhere else and entered into Japan. These results support the proposed 'sink-source' model of viral ecology in which new lineages are seeded from a persistent influenza reservoir in tropical countries to 'sink' populations in temperate regions including Japan.
| S-EPMC4701988 | biostudies-literature
Project description:The last influenza A pandemic provided an excellent opportunity to study the adaptation of the influenza A(H1N1)pdm09 virus to the human host. Particularly, due to the availability of sequences taken from isolates since the beginning of the pandemic until date, we could monitor amino acid changes that occurred in the hemagglutinin (HA) as the virus spread worldwide and became the dominant H1N1 strain. HA is crucial to viral infection because it binds to sialidated cell-receptors and mediates fusion of cell and viral membranes; because antibodies that bind to HA may block virus entry to the cell, this protein is subjected to high selective pressure. Multiple alignment analysis of sequences of the HA from isolates taken since 2009 to date allowed us to find amino acid changes that were positively selected as the pandemic progressed. We found nine changes that became prevalent: HA1 subunits D104N, K166Q, S188T, S206T, A259T, and K285E; and HA2 subunits E47K, S124N, and E172K. Most of these changes were located in areas involved in inter- and intrachain interactions, while only two (K166Q and S188T) were located in known antigenic sites. We conclude that selective pressure on HA was aimed to improve its functionality and hence virus fitness, rather than at avoidance of immune recognition.
| S-EPMC4198066 | biostudies-literature
Project description:We report characteristics of oseltamivir-resistant influenza A(H1N1)pdm09 viruses and patients infected with these viruses in the United States. During 2013-14, fifty-nine (1.2%) of 4,968 analyzed US influenza A(H1N1)pdm09 viruses had the H275Y oseltamivir resistance-conferring neuraminidase substitution. Our results emphasize the need for local surveillance for neuraminidase inhibitor susceptibility among circulating influenza viruses.
| S-EPMC4285251 | biostudies-literature
Project description:Hemagglutinin is the major surface glycoprotein of influenza viruses. It participates in the initial steps of viral infection through receptor binding and membrane fusion events. The influenza pandemic of 2009 provided a unique scenario to study virus evolution. We performed molecular dynamics simulations with four hemagglutinin variants that appeared throughout the 2009 influenza A (H1N1) pandemic. We found that variant 1 (S143G, S185T) likely arose to avoid immune recognition. Variant 2 (A134T), and variant 3 (D222E, P297S) had an increased binding affinity for the receptor. Finally, variant 4 (E374K) altered hemagglutinin stability in the vicinity of the fusion peptide. Variants 1 and 4 have become increasingly predominant, while variants 2 and 3 declined as the pandemic progressed. Our results show some of the different strategies that the influenza virus uses to adapt to the human host and provide an example of how selective pressure drives antigenic drift in viral proteins.
| S-EPMC3720954 | biostudies-literature
Project description:The continuous antigenic variation of influenza A viruses remains a major hurdle for vaccine selection; however, the molecular determinants and mechanisms of antigenic change remain largely unknown. In this study, two escape mutants were generated by serial passages of the Eurasian avian-like H1N1 swine influenza virus (EA H1N1 SIV) A/swine/Henan/11/2005 (HeN11) in the presence of two neutralizing monoclonal antibodies (mAbs) against the hemagglutinin (HA) protein, which were designated HeN11-2B6-P5 and HeN11-4C7-P8, respectively. The HeN11-2B6-P5 mutant simultaneously harbored the N190D and I230M substitutions in HA, whereas HeN11-4C7-P8 harbored the M269R substitution in HA (H3 numbering). The effects of each of these substitutions on viral antigenicity were determined by measuring the neutralization and hemagglutination inhibition (HI) titers with mAbs and polyclonal sera raised against the representative viruses. The results indicate that residues 190 and 269 are key determinants of viral antigenic variation. In particular, the N190D mutation had the greatest antigenic impact, as determined by the HI assay. Further studies showed that both HeN11-2B6-P5 and HeN11-4C7-P8 maintained the receptor-binding specificity of the parent virus, although the single mutation N190D decreased the binding affinity for the human-type receptor. The replicative ability in vitro of HeN11-2B6-P5 was increased, whereas that of HeN11-4C7-P8 was decreased. These findings extend our understanding of the antigenic evolution of influenza viruses under immune pressure and provide insights into the functional effects of amino acid substitutions near the receptor-binding site and the interplay among receptor binding, viral replication, and antigenic drift. IMPORTANCE The antigenic changes that occur continually in the evolution of influenza A viruses remain a great challenge for the effective control of disease outbreaks. Here, we identified three amino acid substitutions (at positions 190, 230, and 269) in the HA of EA H1N1 SIVs that determine viral antigenicity and result in escape from neutralizing monoclonal antibodies. All three of these substitutions have emerged in nature. Of note, residues 190 and 230 have synergistic effects on receptor binding and antigenicity. Our findings provide a better understanding of the functional effects of amino acid substitutions in HA and their consequences for the antigenic drift of influenza viruses.
| S-EPMC9400474 | biostudies-literature
Project description:BACKGROUND:In patients with A(H1N1)pdm09 infection, severe lung involvement requiring admission to intensive care units (ICU) has been reported. Mutations at the hemagglutinin (HA) receptor binding site (RBS) have been associated with increased virulence and disease severity, representing a potential marker of critical illness. OBJECTIVES:To assess the contribution of HA-RBS variability in critically ill patients, A(H1N1)pdm09 virus from adult patients with severe infection admitted to ICU for extracorporeal membrane oxygenation support (ECMO) during influenza season 2009-2011 in Piemonte (4·2 million inhabitants), northwestern Italy, was studied. PATIENTS AND METHODS:We retrospectively analyzed HA-RBS polymorphisms in ICU patients and compared with those from randomly selected inpatients with mild A(H1N1)pdm09 disease and outpatients with influenza from the local surveillance program. RESULTS:By HA-RBS direct sequencing of respiratory specimens, D222G and D222N viral variants were identified in a higher proportion in ICU patients (n=8/24, 33·3%) than in patients with mild disease (n=2/34, 6%) or in outpatients (n=0/44) (Fisher's exact test P<0·0001; OR 38·5; CI 95% 4·494-329·9). Eleven ICU patients died (42%), three of them carrying the D222G variant, which was associated with RBS mutation S183P in two. D222G and D222N mutants were identified in upper and lower respiratory samples. CONCLUSIONS:A(H1N1)pdm09 HA substitutions D222G and D222N were harbored in a significantly higher proportion by patients with acute respiratory distress for A(H1N1)pdm09 severe infection requiring ICU admission and ECMO. These data emphasize the importance of monitoring viral evolution for understanding virus-host adaptation aimed at the surveillance of strain circulation and the study of viral correlates of disease severity.
| S-EPMC4634302 | biostudies-literature
Project description:D222G/N substitutions in A(H1N1)pdm09 hemagglutinin may be associated with increased binding of viruses causing low respiratory tract infections and human pathogenesis. We assessed the impact of such substitutions on the balance between hemagglutinin binding and neuraminidase cleavage, viral growth and in vivo virulence.Seven viruses with differing polymorphisms at codon 222 (2 with D, 3 G, 1 N and 1 E) were isolated from patients and characterized with regards hemagglutinin binding affinity (Kd) to α-2,6 sialic acid (SAα-2,6) and SAα-2,3 and neuraminidase enzymatic properties (Km, Ki and Vmax). The hemagglutination assay was used to quantitatively assess the balance between hemagglutinin binding and neuraminidase cleavage. Viral growth properties were compared in vitro in MDCK-SIAT1 cells and in vivo in BALB/c mice. Compared with D222 variants, the binding affinity of G222 variants was greater for SAα-2,3 and lower for SAα-2,6, whereas that of both E222 and N222 variants was greater for both SAα-2,3 and SAα-2,6. Mean neuraminidase activity of D222 variants (16.0 nmol/h/10(6)) was higher than that of G222 (1.7 nmol/h/10(6) viruses) and E/N222 variants (4.4 nmol/h/10(6) viruses). The hemagglutination assay demonstrated a deviation from functional balance by E222 and N222 variants that displayed strong hemagglutinin binding but weak neuraminidase activity. This deviation impaired viral growth in MDCK-SIAT1 cells but not infectivity in mice. All strains but one exhibited low infectious dose in mice (MID50) and replicated to high titers in the lung; this D222 strain exhibited a ten-fold higher MID50 and replicated to low titers. Hemagglutinin-neuraminidase balance status had a greater impact on viral replication than hemagglutinin affinity strength, at least in vitro, thus emphasizing the importance of an optimal balance for influenza virus fitness. The mouse model is effective in assessing binding to SAα-2,3 but cannot differentiate SAα-2,3- from SAα-2,6- preference, nor estimate the hemagglutinin-neuraminidase balance in A(H1N1)pdm09 strains.
| S-EPMC4131921 | biostudies-literature