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A Critical HA1 Neutralizing Domain of H5N1 Influenza in an Optimal Conformation Induces Strong Cross-Protection
research-article
Author(s):
Lanying Du
1 ,
Guangyu Zhao
2 ,
Shihui Sun
2 ,
Xiujuan Zhang
1 ,
Xiaojun Zhou
2 ,
Yan Guo
2 ,
Ye Li
1
,
3 ,
Yusen Zhou
2
,
* ,
Shibo Jiang
1
,
4
,
*
Publication date (Electronic):
8 January 2013
Journal:
PLoS ONE
Publisher:
Public Library of Science
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Abstract
The highly pathogenic avian influenza (HPAI) H5N1 viruses, especially the laboratory-generated H5N1 mutants, have demonstrated the potential to cross the species barrier and infect mammals and humans. Consequently, the design of an effective and safe anti-H5N1 vaccine is essential. We previously demonstrated that the full-length hemagglutinin 1 (HA1) could induce significant neutralizing antibody response and protection. Here, we intended to identify the critical neutralizing domain (CND) in an optimal conformation that can elicit strong cross-neutralizing antibodies and protection against divergent H5N1 strains. We thus constructed six recombinant proteins covering different regions of HA1 of A/Anhui/1/2005(H5N1), each of which was fused with foldon (Fd) and Fc of human IgG. We found that the critical fragment fused with Fd/Fc (HA-13–263-Fdc, H5 numbering) that could elicit the strongest neutralizing antibody response is located in the N-terminal region of HA1 (residues 13–263), which covers the receptor-binding domain (RBD, residues 112–263). We then constructed three additional recombinants fused with Fd plus His tag (HA-13–263-Fd-His), Fc only (HA-13–263-Fc), and His tag only (HA-13–263-His), respectively. We found that the HA-13–263-Fdc, which formed an oligomeric conformation, induced the strongest neutralizing antibody response and cross-protection against challenges of two tested H5N1 virus strains covering clade 1: A/VietNam/1194/2004 (VN/1194) or clade 2.3.4: A/Shenzhen/406H/06 (SZ/406H), while HA-13–263-Fc dimer and HA-13–263-Fd-His trimer elicited higher neutralizing antibody response and protection than HA-13–263-His monomer. These results suggest that the oligomeric form of the CND containing the RBD can be further developed as an effective and safe vaccine for cross-protection against divergent strains of H5N1 viruses.
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Most cited references37
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Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus.
James Stevens, Ola Blixt, Terrence Tumpey … (2006)
The hemagglutinin (HA) structure at 2.9 angstrom resolution, from a highly pathogenic Vietnamese H5N1 influenza virus, is more related to the 1918 and other human H1 HAs than to a 1997 duck H5 HA. Glycan microarray analysis of this Viet04 HA reveals an avian alpha2-3 sialic acid receptor binding preference. Introduction of mutations that can convert H1 serotype HAs to human alpha2-6 receptor specificity only enhanced or reduced affinity for avian-type receptors. However, mutations that can convert avian H2 and H3 HAs to human receptor specificity, when inserted onto the Viet04 H5 HA framework, permitted binding to a natural human alpha2-6 glycan, which suggests a path for this H5N1 virus to gain a foothold in the human population.
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Antigenic Fingerprinting of H5N1 Avian Influenza Using Convalescent Sera and Monoclonal Antibodies Reveals Potential Vaccine and Diagnostic Targets
Introduction The recent spread of highly pathogenic (HP) H5N1 avian influenza viruses (AIV) among poultry and transmission of these viruses to humans raised concerns of a potential influenza pandemic. In preparation for such an event, world-wide efforts are under way to test and stockpile preventive vaccines, antiviral drugs, and passive immune therapies [1], [2]. Such efforts could be greatly enhanced by understanding the immune responses of individuals who survived H5N1 virus infection. Recently, human monoclonal antibodies (MAbs) were generated from patients in Vietnam and Turkey who had recovered from H5N1 infections [3], [4]. Some of the human MAbs demonstrated cross-clade neutralization in vitro and protected mice from challenge with lethal H5N1 viruses. The potential benefit of antibody therapy was suggested during the Spanish influenza outbreak when transfusion of convalescent sera reduced mortality by >50% [5], and in a patient infected with an H5N1 (clade 2.3) virus in China [6]. However, there are significant gaps in our knowledge of antibody epitopes against H5N1 viruses, especially in humans, and only a few epitopes have been identified in proteins other than the haemagglutinin (HA) [7]. Characterizing the B cell responses in convalescent individuals could help in the design of future vaccines and therapeutics. Finally, identification of long-lasting antibodies against conserved epitopes could assist in the development of serological assays for surveillance of AIV infections. To address these gaps we have constructed whole-genome–fragment phage display libraries (GFPDL) expressing all the open reading frames of H5N1 A/Vietnam/1203/2004. The H5N1 GFPDL were used to identify recognition sites of antibodies in convalescent sera obtained from five Vietnamese individuals with a history of H5N1 infection and two H5-specific neutralizing MAbs derived from two of these survivors. Materials and Methods Plasma Samples and MAbs Serum samples from five patients who survived H5N1 infection in Vietnam were obtained at one time point within 2–6 mo following H5N1 infection in 2004 and were previously described (Viet-1, 24-year-old male; Viet-2, 8-year-old female; Viet-3 25-year-old female; Viet-4, 26-year-old female; Viet-5, 23-year-old male) [8]. MAbs FLA5.10, and FLD21.140 were derived from two of these donors and were previously described [3]. Plasma samples from 20 Vietnamese adults (all females, aged 20–39 y) and resident in Vietnam but with no history of H5N1 exposure were used as controls in antibody binding experiments. In microneutralization assays using A/Wisconsin/67/05 (H3N2), A/New York/55/04 (H3N2), A/New Caledonia/22/99 (H1N1), and A/Solomon Islands/03/06 (H1N1), 75% of these plasma had neutralizing antibodies against either H3N2 strains, H1N1 strains, or both, with titers ranging between 1∶40 to 1∶1,280. Ten plasma samples from US residents with culture confirmed human seasonal influenza infections who had high HI titers against both H1N1 and H3N2 strains circulation during the 2004–2007 seasons were used as additional controls (no possible H5N1 exposure). All samples were de-identified. All protocols were evaluated by the CBER/NIH Research Involving Human Subjects Committee and were conducted under RIHSC exemption number 03-118B. Construction of H5N1 Gene-Fragmented Phage Display Libraries cDNA corresponding to all eight gene segments of the A/H5N1/Vietnam/1203/2004 were generated from RNA isolated from egg-grown virus strain, and were used for cloning. fSK-9-3 is a gIIIp display-based phage vector where the desired polypeptide can be expressed as gIIIp fusion protein. Phage display libraries were constructed individually for HA and neuraminidase (NA) genes (referred to as HA-NA) and the rest of the six gene segments (PB2, PB1, PA, NP, M, and NS), referred to as FLU-6. Purified DNA containing equimolar ratio of HA and NA (HA-NA) or of the six genes (FLU-6) were digested with DNase shotgun cleavage kit (Novagen) per manufacturer's instructions, to obtain DNA fragments in the size range of 50–200 and 200–1,000 bp for each of the two gene segment pools. Detailed methodology for library construction was described previously [9]. Four libraries were constructed: fSK9-3 H5Viet-HA-NA (50–200 bp), fSK9-3 H5Viet-HA-NA (200–1,000 bp), fSK9-3 H5Viet-FLU-6 (50–200 bp), and fSK9-3 H5Viet-FLU-6 (200–1,000 bp) (Figure S2). Random Peptide Library A random linear dodecapeptide phage display library (Ph.D-12), wherein the displayed peptides (12-mer) are expressed fused to the N terminus of gIII protein was purchased from New England Biolabs. Affinity Selection of H5N1 GFPDL Phages with MAb or Polyclonal Human Sera Prior to panning of GFPDL with plate-bound polyclonal serum antibodies, serum components, which could nonspecifically interact with phage proteins, were removed by incubation with UV-killed M13K07 phage-coated Petri dishes. Subsequent GFPDL affinity selection was carried out on antibody-coated wells as well as in-solution (with Protein A/G). For in-solution panning, 1010 phages (of the Influenza H5 GFPDL) in 500 µl PBST containing 1% BSA were preincubated with 200 µl of 50% Ultralink Protein A/G slurry (Pierce) for 1 h at room temperature (RT) on end-to-end shaker. Following brief centrifugation, 500 µl of supernatant was removed and was added to 5 µg of human anti-H5N1 MAb or 100 µl of VCSM13-preadsorbed human serum (in 1% BSA-PBST), and incubated for 1 h at RT on end-to-end shaker, followed by 200 µl of 50% Ultralink Protein A/G slurry (Pierce) on end-to-end shaker at RT for 1 h. The unbound phages were removed in ten washes with PBST followed by three washes with PBS. The bound phages were eluted by addition of 800 µl of 0.1 N HCl (adjusted to pH 2.2 with glycine and BSA), and incubated for 10 min at RT on end-to-end shaker. The eluates were collected and neutralized by adding 64 µl of 2 M Tris solution. Panning on coated strips has been detailed in [9]. The inserts were PCR amplified and sequenced. Peptide ELISA Biotinylated peptides (1 µg/well) were captured onto wells coated with 500 ng of streptavidin. After blocking with PBST containing 2% milk, serial dilutions of human serum in blocking solution were added to each well, incubated for 1 h at RT, followed by addition of 2,000-fold dilution of HRP-conjugated goat anti-human IgG-Fc specific antibody, and developed by 100 µl of OPD substrate solution. Absorbance was measured at 490–492 nm. As negative controls, peptides derived from HIV and human CCR5 were used. Affinity Measurements by Surface Plasmon Resonance Steady-state equilibrium binding of MAb FLA5.10, and FLD21.140 was monitored at 25°C using a ProteOn surface plasmon resonance biosensor (BioRad Labs). The HA [(-10)-223]-His6 was coupled to a GLC sensor chip using amine coupling with 40 resonance units (RU) in the test flow cells. Samples of 60 µl of freshly prepared antibody at various concentrations were injected at a flow rate of 30 µl/min (120-s contact time). Flow was directed over a mock surface to which no protein was bound, followed by the HA [(-10)-223]-His6 coupled surface. Responses from the peptide surface were corrected for the response from the mock surface and for responses from a separate, buffer only, injection. MAb 2D7 (anti-CCR5) was used as a negative control antibody in various binding experiments. Binding Kinetics for the MAbs and the data analysis was performed using BioRad ProteON manager software (version 2.0.1). Affinity measurements were calculated using the Langmuir with Mass transfer algorithm. Adsorption of Polyclonal Human Sera on H5N1 GFPDL Phages and Residual Reactivity to H5N1-Vietnam HA Prior to panning of GFPDL, 500 µl of 10-fold diluted pooled serum antibodies from H5N1 survivors were adsorbed by incubation with H5N1 (HA+NA) GFPDL phage-coated Petri dishes. To ascertain the residual antibodies specificity, an ELISA was performed with wells coated with 200 ng/100 µl of recombinant H5 HA (A/Vietnam/1203/2004, Protein Sciences). After blocking with PBST containing 2% milk, serial dilutions of human serum (with or without adsorption) in blocking solution were added to each well, incubated for 1 h at RT, followed by addition of 2,000-fold diluted HRP-conjugated goat anti-human IgG-Fc specific antibody and developed by 100 µl of OPD substrate solution. Absorbance was measured at 490–492 nm. Neutralizing Antibodies Adsorption with HA1 peptides Five-fold diluted immune serum (from sheep or ferret) (500 µl) was added to 0.5 mg of purified HA [(-10)-223]-His6 (or shorter HA1-derived peptides) or to control GST-His6 protein, and incubated for 1 h at RT. Ni-NTA Sepharose beads (200 µl; Qiagen) were added for 20 min at RT on end-to-end shaker, to capture the His-tagged peptides and the antibodies bound to them, followed by a brief centrifugation. Supernatants containing the unbound antibodies were collected. The pelleted beads were washed five times with PBST, followed by two washes with PBS. Sepharose-peptide–bound antibodies were eluted by incubating beads with 500 µl of 0.1 N HCl (adjusted to [pH 2.2] with glycine and BSA), for 10 min at RT on end-to-end shaker. The eluates were collected and neutralized by adding 40 µl of 2 M Tris solution. In some cases, the serum adsorption was performed using biotinylated peptides, which were captured using streptavidin-coupled magnetic beads. Neutralization Assay Viral-neutralizing activity was analyzed in a microneutralization assay on the basis of the methods of the pandemic influenza reference laboratories of the US Centers for Disease Control (CDC) [10]–[12]. The sheep anti-H5N1 A/Vietnam/1203/2004 (CBER SRID reagent), and sera from ferrets infected with wild-type H5N1 A/Vietnam/1203/2004, were treated with a Receptor Destroying Enzyme (RDE) overnight followed by heat-inactivation. Low pathogenicity H5N1 viruses, generated by reverse genetics, were obtained from St. Jude, CDC, and NIBSC: A/Vietnam/1203/2004 (SJCRH, clade 1), A/Indo/5/2005 (PR8-IBCDC-RG2; clade 2.1), A/turkey/Turkey/1/05 (NIBRG-23; clade 2.2), A/Anhui/1/05 (IBCDC-RG5, clade 2.3.4). Results Epitope Mapping of H5N1-Neutralizing MAbs Following the outbreak of H5N1 AIV in humans in Vietnam (2004–2005), in which 13/18 patients died [8], [13], memory B cells from peripheral blood mononuclear cells of four surviving patients were immortalized with Epstein-Barr virus to generate human MAbs [3]. Two of the human MAbs exhibited distinct patterns of reactivity with clade 1 and clade 2 H5N1 viruses. FLA5.10 had a narrow (clade 1-specific) neutralization range (Figure 1A) and protected mice from lethal challenge with clade 1 but not clade II viruses [3]. In contrast, FLD21.140 demonstrated broader cross-clade neutralization in vitro with very high neutralization titers against A/Vietnam/1203/2004 (clade 1) and A/Turkey/turkey/1/05 (clade 2.2), and a low neutralization titer against A/Anhui/1/05 (clade 2.3.4) (Figure 1A). Interestingly, although this MAb did not neutralize A/Indonesia/5/05 (clade 2.1) in vitro, it protected BALB/c mice from lethal challenge with A/Indonesia/5/05 as previously reported [3]. We predicted that these MAbs have different binding sites, and the epitope of MAb FLD21.140 could be a potential target for cross-reactive H5N1 vaccines. 10.1371/journal.pmed.1000049.g001 Figure 1 Epitope mapping of broadly neutralizing H5N1 human MAbs. (A) End-point titers (mean of three replicates) using two H5N1 human MAbs (at 1 mg/ml) in a microneutralization assay performed with rgH5N1×PR8 (2∶6) reassorted viruses are shown. *, The information on in vivo protection of mice against wild-type A/Vietnam/1203/2004 and A/Indonesia/5/05 challenge was previously published [3]. (B) HA segment [(-10)-233] was identified by GFPDL panning using MAb FLA5.10 (boxed). Amino acid number +1 corresponds to H3 (A/California/7/2004) amino acid -10. The critical contact residues for FLA5.10, identified using RPL are shown in red circles. (C) Alignment of the critical residues for MAb FLA5.10 binding on the 3-D structure of the HA monomer (Protein Data Base [PDB] identifier 21BX) with amino acid colors corresponding to (B). The predicted glycosylation sites (NXT/NXS) are shown in blue. (D) HA segment (32–320) identified by GFPDL-panning using MAb FLD21.140 (boxed). The putative contact residues identified using RPL, are shown in red circles. (E) Alignment of the critical residues for MAb FLD21.140 binding on the 3-D structure of the HA monomer (PDB Identifier 2IBX) with amino acid colors corresponding to (D). The predicted glycosylation sites (NXT/NXS) are shown in blue. Epitope mapping was addressed following the construction of GFPDL, spanning the genome of A/Vietnam/1203/2004 (H5N1) (Figure S1). The insert sizes in the separately constructed HA+NA and FLU-6 libraries ranged between 50–200 bp and 200–1,000 bp to allow presentation of conformation-dependent epitopes. The four influenza GFPDL consisted of 9.6×106 to 2.6×107 phages (Figure S2A). PCR analysis of 192 transformants per library confirmed that the size and distribution of inserts was random across all eight genes (Figure S2B). Using MAb FLA5.10 for panning of the H5N1 HA+NA libraries, multiple phages expressing an HA segment corresponding to amino acids (aa) [(-10)-223] were selected that included part of the receptor binding site (RBS) preceded by an N-terminal sequence (Figure 1B). This HA segment contains 13 aa differences between A/Vietnam/1203/2004 and A/Indonesia/5/05 viruses, any of which could contribute to the clade-restricted binding of this MAb. To delineate the specific contact residues of FLA5.10, we used a random peptide phage display library (RPL), as previously used to map conformation-dependent MAbs [14]. Most of the selected phages displayed a peptide with the consensus sequence [(H/Q)-I-(T/I)-P-X-X-X-E-(A/V)-T-L, where “X” is any amino acid]. This epitope sequence mimics a nonlinear sequence present in the RBS, which aligns with 115-QIIP-118 and 126-EASL-129 (Figure 1B, circled in red). Comparison of this portion of the HA sequence of A/Vietnam/1203/2004 and A/Indonesia/5/05 shows only one amino acid difference (L129S). Direct binding of FLA5.10 to a chemically synthesized peptide (5.10-101) and a mutated version of the peptide (5.10-101-L/A) confirmed that L129 is a critical contact residue (Figure S3). Importantly, these aa clusters aligned closely on the outer face of the HA globular head on the 3-D structure of HA and are not expected to be masked by glycosylated residues (shown in blue) (Figure 1C). GFPDL panning with MAb FLD21.40 identified a large segment (HA 32–320) including the entire RBS. Subsequent panning with RPL narrowed the putative epitope to two aa clusters, 121-SWS-123 and 164-YNNT-167, which were separated by 40 aa in the linear sequence (Figure 1D, circled in red), but were closely located on the 3-D structure of HA monomer (Figure 1E). Importantly, the sequences identified are highly conserved among clade 1 and clade 2 H5N1 viruses (including clades 2.1, 2.2, 2.3.1, 2.3.2, 2.3.3, 2.3.4, 2.4, and 2.5) as well as clades 5, 6, and 8. This conservation may explain the broader cross-neutralization in vitro and cross-protection in vivo conferred by FLD21.140 (Figure 1A). Binding of Human MAbs to HA Segment To confirm that the HA segments identified using influenza GFPDL can be recognized by neutralizing human MAbs independent of phage presentation, the H5 HA [(-10)-223] peptide was expressed and purified from Escherichia coli. This protein fragment captured on a biosensor chip was used to determine the binding kinetics of FLA5.10 (Figure 2B) and FLD21.140 (Figure 2A). Surprisingly, FLD21.140 bound to this HA segment with 50-fold higher affinity than FLA5.10 (Kd of 0.68 nM versus 34 nM, respectively). Such a difference in binding affinities may predict higher avidity of binding to virus in vivo for FLD21.140, and could contribute to its ability to protect mice against clade 2 viruses. Therefore, both specificity and avidity could be factors in heterologous protection. 10.1371/journal.pmed.1000049.g002 Figure 2 Steady-state binding equilibrium analysis of human MAbs to purified bacterially expressed H5 HA[(-10-223)] fragment. Various concentrations of MAbs FLD21.140 (A) and FLA5.10 (B) were injected simultaneously onto recombinant HA [(-10)-223] (identified in Figure 1B) peptide, immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs). As a control, anti-CCR5 MAb 2D7) was injected at the same concentrations on HA [(-10)-223] coupled chip. RU, resonance units. Antibody Epitopes in the HA and NA Recognized by Sera from Survivors of H5N1 Infection The successful use of the GFPDL/RPL in elucidating the conformation-dependent epitopes of two human MAbs provided proof of concept for this approach. Next, it was important to establish if such antibodies are represented in the polyclonal sera of individuals who had recovered from H5N1 virus infection, and to identify other antibody specificities that may have contributed to virus clearance. Convalescent sera from five H5N1 patients, obtained between 54 and 182 d following hospital admission, were analyzed using the A/Vietnam/1203/2004 GFPDL. We first demonstrated the capacity of the GFPDL to adsorb, at a minimum, 85% of HA-specific antibodies in the pooled convalescent sera, as determined by binding to recombinant H5 HA (A/Vietnam/1203/2004, Protein Sciences) in ELISA (Figure S4), confirming the rationale of using this approach to dissect the antibody repertoires in polyclonal sera. Using the HA+NA GFPDL, the pooled convalescent sera recognized a large number of clones in the HA1, HA2, and NA proteins (Figure 3A). The sequences and frequencies of all the peptides that were bound by the pooled sera are presented in Table S1. The most frequently recognized segments (numbered in Figure 3A) were chemically synthesized or expressed in E. coli and used in ELISA with individual convalescent sera (Viet 1-5, Figure 3B). All five individual sera reacted with the panel of peptides from HA1, HA2, and NA proteins. The strongest reactivity was observed for the large HA1 peptides 1 and 2 encompassing the RBS, which were also recognized by the human MAbs (unpublished data), and against the shorter peptides 7 and 8 in the C terminus of HA1. Phage clones expressing HA2 sequences were isolated at high frequency (Table S1), and individual binding titers for these peptides were confirmed by ELISA (Figure 3B, peptides 9–14). The predominant HA sites recognized by the convalescent sera could be grouped into six antigenic clusters (I–VI) that mapped to the outer face of the HA trimer (Figures 3B and 4A). The HA antigenic clusters were defined on the basis of sequences of the repeatedly selected phage displayed epitopes (Table S1) and the confirmatory ELISA reactivity profiles with the patients' plasma (Figure 3B). Cluster-I, 2,359–2,453; cluster-II: 2,454–2,621; cluster-III: 2,627–2,670; cluster-IV: 2,682–2,703; cluster-V: 2,706–2,814; cluster-VI: 2,823–2,816. Clusters-I and -II encompass the previously described HA1 antigenic sites “a–e” that were defined primarily using mouse monoclonal antibodies against H3 influenza strains [15], [16]. 10.1371/journal.pmed.1000049.g003 Figure 3 Elucidation of the epitope profile in HA and NA proteins recognized by antibodies in individuals that survived H5N1 infections in Vietnam. (A) Alignment of peptides recognized by pooled sera from H5N1-infected individuals identified using H5 (HA+NA) GFPDL. Bars with arrows represent the identified inserts in 5′–3′ orientation. Numbered segments represent inserts that were selected with high frequencies (≥5; Table S1). These peptides were expressed and purified from E .coli or were chemically synthesized and used in ELISA. The sequences of the influenza encoded fragments are numbered according to the intact complete proteome (Figure S1). Peptide ID numbers are the same in (A) and (B). (B) ELISA reactivity of sera from individual H5N1-infected patients (Viet1–5) or pooled sera from 20 healthy Vietnamese adults (75% had neutralizing titers against either H3N2 influenza strains, H1N1 strains, or both) with H5N1 HA1 peptides (1–8), HA2 peptides (9–14), and NA peptides (15–21) (localization indicated in Figure 4A). An initial serum dilution (1∶100) was followed by serial 5-fold dilutions. End-point titers are reported. Days postadmission represent the time of serum collection for each patient. Six antigenic clusters in HA (cluster-I, 2,359–2,453; cluster-II, 2,454–2,621; cluster-III, 2,627–2,670; cluster-IV, 2,682–2,703; cluster-V, 2,706–2,814; cluster-VI, 2,823–2,816) recognized by the convalescent sera are shown. 10.1371/journal.pmed.1000049.g004 Figure 4 Main antigenic clusters in the structures of HA and NA recognized by antibodies from H5N1 virus infected individuals. (A) Antigenic clusters in HA, as identified in Figure 3B, are shown as surface exposed colored patches on one HA monomer within the HA trimer structure (PDB identifier 2IBX). The antigenic clusters (I–III) in HA1 cover the Antigenic Sites a, b, c, d, and e that have been described in the H3 HA1 based on mouse MAbs [15], [22]. (B) The immunodominant conformational epitope in the NA (NA-3676-3854, peptide 15 in Figure 3B) is shown in green on the tetrameric NA structure (PDB Identifier 2HTY) with the predicted site of bound sialic acid shown in red. Side view and bird-eye views are shown. Although the serum samples were obtained at a single time point postinfection in each patient, a general pattern was observed. The ELISA titers of Viet-5 serum (6 mo postinfection) were lower than for earlier convalescence sera (Figure 3B). Interestingly, high antibody titers against peptide 14 in the HA2 C terminus (HA-2838–2866) were detected in all five samples. To control for the potentially confounding influence of cross-reactive antibodies elicited by seasonal influenza, we also evaluated the binding profile of sera from 20 healthy Vietnamese adults with no epidemiological exposure to H5N1 (Figure 3B, right column). While modest binding to peptide 1, encompassing most of the HA1 was observed with these sera (titer 1∶500), binding to all the other peptides was very low or absent (≤1∶100). These results were expanded using 10 US individuals with culture confirmed seasonal influenza infections and high HI titers against both H1N1 and H3N2 (Table S2). Student's t-test analyses (two-tailed distribution with equal variance and a degree of freedom of 13) revealed that the binding to the H5N1 HA peptides were significantly different (p 85% conserved between the human and AIV N1 NAs of A/New Caledonia/20/99 (H1N1) and A/Vietnam/1203/2004 (H5N1), respectively. Surprisingly, Vietnamese control sera bound peptide 15 minimally (≤1∶100) (Figure 3B), and no reactivity was observed with seasonal influenza infected US control sera (Table S2). Antibody Epitopes in the Internal Proteins of Influenza Recognized by Sera from Survivors of H5N1 Infection Panning of GFPDL (FLU-6) expressing inserts from the internal viral proteins (Figure S2A) with the pooled H5N1 convalescent sera resulted in the isolation of many clones across all proteins (Table S1). The majority of the fragments recognized in the internal proteins were shorter than the HA/NA epitopes (Figure 5A). As expected, most of the selected clones were derived from the major structural proteins M1 and NP (531 and 91 clones, respectively, Table S1). Peptides that were recognized at high frequencies were synthesized and tested with individual sera in ELISA (Figure 5B). Interestingly, binding to all four M1 peptides remained at high mean end-point titers (1∶2,500). Control Vietnamese pooled sera reacted weakly with peptides 10 (NP) and 14–15 (M1). The US control sera reacted strongly with M1 peptides 13–16, reflecting the high degree of conservation in these proteins between seasonal human influenza and H5N1 viruses [7]. As indicated before, student's t-test analyses (two-tailed distribution with equal variance and a degree of freedom of 13) revealed that the ELISA reactivities against most of the H5N1 peptides were significantly different (p 0.05 are shown in bold in Table S2, and unpublished data). 10.1371/journal.pmed.1000049.g005 Figure 5 Antibody epitopes in H5N1 internal proteins (FLU-6) recognized by pooled sera from H5N1 infected individuals. (A) Schematic alignment of the peptides identified using GFPDL (H5N1-FLU-6) expressing all internal proteins of influenza A/Vietnam/1203/2004. The predicted influenza encoded proteins are numbered according to the complete proteome (Figure S1). Bars with arrows indicate identified inserts in the 5′–3′ orientation. Numbered segments represent high frequency clones (≥5; Table S1). These peptides were expressed and purified from E. coli or were chemically synthesized and the numbers correspond to the peptide identifiers in the ELISA assay in (B). (B) Reactivities of sera from individual H5N1-infected patients (Viet 1–5) or sera from healthy Vietnamese adults against peptides derived from: PB2 (1); PB1 (2–3); PB1-F2 (4–7); PA (8); NP (9–12); M1 (13–16); M2e (17); M2 (18); NS1 (19–20); and NS2 (21). The M2 ectodomain (M2e) was postulated to contribute to protective immune responses [18], but evidence for anti-M2e responses in humans is lacking. In our study, a strong reactivity against the M2e peptide (17) was found in H5N1 convalescent sera but not with control sera from either Vietnam or the US (Figure 5B; Table S2). PB1-F2 is a 90-aa protein encoded by the PB1 gene. It was identified as a potential virulence factor in the 1918 pandemic H1N1 strain, and in the HP H5N1 (HK/97) viruses [19]. However, evidence for expression of this protein during human infection is lacking. Importantly, the pooled H5N1 convalescent sera selected 35 PB1-F2 phage clones (Table S1), and the synthetic PB1-F2 peptides were recognized by each individual convalescent serum, but not by the Vietnamese or US control sera (Figure 5B, peptides 4–7; Table S2). This is the first report of antibodies against PB1-F2 in H5N1-recovered individuals, and strongly suggests that PB1-F2 is expressed during infection with H5N1 viruses. HA [(-10)-223] Can Adsorb Virus-Neutralizing Activity in Polyclonal Sera To evaluate the functional significance of the main HA epitopes identified using GFPDL, we evaluated the ability of the HA peptide [(-10)-223] (that bound strongly to the H5N1-neutralizing MAbs, FLA5.10 and FLD21.140) to block viral neutralization using hyperimmune sheep sera raised against the reassortant A/Vietnam/1203/04 × PR8 virus that had a titer of 1∶640 in a microneutralization assay using the rgA/Vietnam/1203/2004 virus (Table 1). After incubation with HA [(-10)-223], the unbound antibody fraction lost neutralizing activity (titer 98% loss of FLA5.10 binding. (0.37 MB EPS) Click here for additional data file. Figure S4 Adsorption of anti-HA antibodies in convalescent sera from survivors of H5N1 infection using the H5N1 GFPDL. Pooled sera from five H5N1 survivors were adsorbed with H5 (HA+NA) GFPDL. Binding to recombinant HA protein (A/Vietnam/1203/2004, Protein Sciences Corporation) is shown before (circles) and after (triangles) GFPDL-adsorption. (0.38 MB EPS) Click here for additional data file. Table S1 Frequency of selected phage clones using H5N1 GFPDL after panning with sera from five H5N1-Vietnam infection survivors. Three rounds of affinity selection were performed on pooled sera using each of the four GFPDL under both conditions (antibody coated beads and in-solution). 48 clones were sequenced in each panning round, resulting in sequencing of 2,304 total clones. The peptide sequences displayed on the phage surface and the corresponding frequencies for these phage displayed sequences are shown. Each peptide name indicates the H5N1 protein name and the amino acid numbers corresponding to the complete proteome sequence shown in Figure S1. Sequences in bold letters represent peptides that were used for synthesis and follow up binding assays. (0.14 MB DOC) Click here for additional data file. Table S2 ELISA reactivity of sera from ten individuals with culture-confirmed seasonal influenza infections during the 2006–2007 seasons. End-point antibody titers (based on 5-fold dilutions starting at 1∶100) are reported for US 1–10 against the identical H5N1-Viet peptides used in Figures 4B and 6B). Student t-test was performed for each peptide reactivity using the end-point titers for the control sera (e.g., if end-point titer was 12,500, the value used for statistical analysis was 12,500). p-Values appear in the right column. (0.15 MB DOC) Click here for additional data file.
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Foldon, the natural trimerization domain of T4 fibritin, dissociates into a monomeric A-state form containing a stable beta-hairpin: atomic details of trimer dissociation and local beta-hairpin stability from residual dipolar couplings.
Stephan Grzesiek, Sebastian Meier, Sarah Güthe … (2004)
The C-terminal domain of T4 fibritin (foldon) is obligatory for the formation of the fibritin trimer structure and can be used as an artificial trimerization domain. Its native structure consists of a trimeric beta-hairpin propeller. At low pH, the foldon trimer disintegrates into a monomeric (A-state) form that has similar properties as that of an early intermediate of the trimer folding pathway. The formation of this A-state monomer from the trimer, its structure, thermodynamic stability, equilibrium association and folding dynamics have been characterized to atomic detail by modern high-resolution NMR techniques. The foldon A-state monomer forms a beta-hairpin with intact and stable H-bonds that is similar to the monomer in the foldon trimer, but lacks a defined structure in its N and C-terminal parts. Its thermodynamic stability in pure water is comparable to designed hairpins stabilized in alcohol/water mixtures. Details of the thermal unfolding of the foldon A-state have been characterized by chemical shifts and residual dipolar couplings (RDCs) detected in inert, mechanically stretched polyacrylamide gels. At the onset of the thermal transition, uniform relative changes in RDC values indicate a uniform decrease of local N-HN and Calpha-Halpha order parameters for the hairpin strand residues. In contrast, near-turn residues show particular thermal stability in RDC values and hence in local order parameters. This coincides with increased transition temperatures of the beta-turn residues observed by chemical shifts. At high temperatures, the RDCs converge to non-zero average values consistent with predictions from random chain polymer models. Residue-specific deviations above the unfolding transition reveal the persistence of residual order around proline residues, large hydrophobic residues and at the beta-turn.
Author and article information
Contributors
Jianqing Xu: Role: Editor
Journal
Journal ID (nlm-ta): PLoS One
Journal ID (iso-abbrev): PLoS ONE
Journal ID (publisher-id): plos
Journal ID (pmc): plosone
Title:
PLoS ONE
Publisher:
Public Library of Science
(San Francisco, USA
)
ISSN
(Electronic):
1932-6203
Publication date Collection: 2013
Publication date
(Electronic):
8
January
2013
Volume: 8
Issue: 1
Electronic Location Identifier: e53568
Affiliations
[1
]Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York,
United States of America
[2
]State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology
and Epidemiology, Beijing, China
Author notes
* E-mail:
yszhou@
123456bmi.ac.cn
(Y. Zhou);
sjiang@
123456nybloodcenter.org
(SJ)
Article
Publisher ID: PONE-D-12-28344DOI: 10.1371/journal.pone.0053568
PMC ID: 3539987
PubMed ID: 23320093
SO-VID: 23ee9603-c3ee-468a-98b6-8fdb3b6940f1
Copyright statement:
Copyright @
2013
License:
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
History
Date
received
: 15
September
2012
Date
accepted
: 29
November
2012
Page count
Pages: 13
Funding
This work was supported by grants from National Institutes of Health (NIH)/National
Institute of Allergy and Infectious Diseases (NIAID) of the United States (R03AI088449)
to LD, from the National 973 Basic Research Program of China (2005CB523001) to YZ,
and from the Chinese Ministry of Science & Technology, Hong Kong, Macau, and Taiwan
Collaborative Programs (201200007673) to SJ. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Categories
Subject:
Research Article
Subject:
Biology
Subject:
Microbiology
Subject:
Virology
Subject:
Emerging Viral Diseases
Subject:
Viral Vaccines
Subject:
Medicine
Subject:
Clinical Immunology
Subject:
Immunity
Subject:
Vaccination
Subject:
Vaccines
Subject:
Vaccine Development
Subject:
Infectious Diseases
Subject:
Viral Diseases
Subject:
Influenza
Subject:
Zoonoses
Subject:
Avian influenza A viruses
Subject:
Veterinary Science
Subject:
Veterinary Diseases
Subject:
Zoonotic Diseases
Subject:
Animal Influenza
ScienceOpen disciplines: Uncategorized
Data availability:
ScienceOpen disciplines: Uncategorized