Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes

Respiratory syncytial virus (RSV) invades host cells via a type I fusion (F) glycoprotein that undergoes dramatic structural rearrangements during the fusion process. Neutralizing monoclonal antibodies, such as 101F, palivizumab, and motavizumab, target two major antigenic sites on the RSV F glycoprotein. The structures of these sites as peptide complexes with motavizumab and 101F have been previously determined, but a structure for the trimeric RSV F glycoprotein ectodomain has remained elusive. To address this issue, we undertook structural and biophysical studies on stable ectodomain constructs. Here, we present the 2.8-Å crystal structure of the trimeric RSV F ectodomain in its postfusion conformation. The structure revealed that the 101F and motavizumab epitopes are present in the postfusion state and that their conformations are similar to those observed in the antibody-bound peptide structures. Both antibodies bound the postfusion F glycoprotein with high affinity in surface plasmon resonance experiments. Modeling of the antibodies bound to the F glycoprotein predicts that the 101F epitope is larger than the linear peptide and restricted to a single protomer in the trimer, whereas motavizumab likely contacts residues on two protomers, indicating a quaternary epitope. Mechanistically, these results suggest that 101F and motavizumab can bind to multiple conformations of the fusion glycoprotein and can neutralize late in the entry process. The structural preservation of neutralizing epitopes in the postfusion state suggests that this conformation can elicit neutralizing antibodies and serve as a useful vaccine antigen.     

The major neutralizing antigenic site on herpes simplex virus glycoprotein D overlaps a receptor-binding domain

Herpes simplex virus (HSV) entry is dependent on the interaction of virion glycoprotein D (gD) with one of several cellular receptors. We previously showed that gD binds specifically to two structurally dissimilar receptors, HveA and HveC. We have continued our studies by using (i) a panel of baculovirus-produced gD molecules with various C-terminal truncations and (ii) a series of gD mutants with nonoverlapping 3-amino-acid deletions between residues 222 and 254. Binding of the potent neutralizing monoclonal antibody (MAb) DL11 (group Ib) was unaffected in forms of gD containing residues 1 to 250 but was greatly diminished in molecules truncated at residue 240 or 234. Both receptor binding and blocking of HSV infection were also affected by these C-terminal truncations. gD-1(234t) bound weakly to both HveA and HveC as determined by enzyme-linked immunosorbent assay (ELISA) and failed to block infection. Interestingly, gD-1(240t) bound well to both receptors but blocked infection poorly, indicating that receptor binding as measured by ELISA is not the only gD function required for blocking. Optical biosensor studies showed that while gD-1(240t) bound HveC with an affinity similar to that of gD-1(306t), the rates of complex formation and dissociation were significantly faster than for gD-1(306t). Complementation analysis showed that any 3-amino-acid deletion between residues 222 and 251 of gD resulted in a nonfunctional protein. Among this set of proteins, three had lost DL11 reactivity (those with deletions between residues 222 and 230). One of these proteins (deletion 222-224) was expressed as a soluble form in the baculovirus system. This protein did not react with DL11, bound to both HveA and HveC poorly as shown by ELISA, and failed to block HSV infection. Since this protein was bound by several other MAbs that recognize discontinuous epitopes, we conclude that residues 222 to 224 are critical for gD function. We propose that the potent virus-neutralizing activity of DL11 (and other group Ib MAbs) likely reflects an overlap between its epitope and a receptor-binding domain of gD.     

Location of a highly conserved neutralizing epitope in the F glycoprotein of human respiratory syncytial virus.

Trypsin digestion of the purified F protein from human respiratory syncytial virus (Long strain) generated a set of fragments in the amino-terminal third of the F1 subunit which contained the epitope 47F involved in neutralization. Sequencing of five escape mutant viruses selected with monoclonal antibody 47F allowed us to map precisely two amino acid residues (262 and 268) of the F1 subunit which are essential for the integrity of this important epitope. The results are discussed in terms of the mechanisms involved in virus neutralization and the design of potential synthetic vaccines.     

Purification of human respiratory syncytial virus fusion glycoprotein

Human respiratory syncytial virus (RSV) fusion glycoprotein (F) elicits neutralizing antibodies to RSV and has therefore attracted much attention as a suitable candidate antigen in the development of gene-based vaccines against RSV infections. However, a major obstacle in vaccine development has been the problem of antigen purification. To address this problem, we have developed a new method that combines sucrose gradient ultracentrifugation and a two-step chromatographic process, to purify RSV F from RSV particles propagated in HEp-2 cells. Analysis of the fractions produced using this method showed recovery of a functional homodimer with a molecular weight of 140 kDa, and 54% preservation of the original F.     

Respiratory syncytial virus fusion glycoprotein: further characterization of a major epitope involved in virus neutralization

Competition experiments and biological assays with a panel of 15 monoclonal antibodies confirmed the presence of at least four antigenic sites on the fusion protein of human respiratory syncytial virus, three of which were involved in virus neutralization. One antigenic site, recognized by two strongly neutralizing antibodies, was conserved after reduction and denaturation and shown by immunoblotting to be localized on the F1 fragment of the fusion protein. Cleavage of this protein with staphylococcal protease V8 or papain produced a series of smaller peptides from 11 to 7 kilodaltons that retained this important neutralization determinant. Compared with the other neutralization sites, the epitope defined by monoclonal antibody 7C2 thus appears as the major neutralization epitope. Our peptide mapping results support the hypothesis that this major epitope is composed of a continuous sequence on the viral genome.     

Neutralization epitopes of the F glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function.

Eighteen neutralizing monoclonal antibodies (MAbs) specific for the fusion glycoprotein of the A2 strain of respiratory syncytial virus (RSV) were used to construct a detailed topological and operational map of epitopes involved in neutralization and fusion. Competitive binding assays identified three nonoverlapping antigenic sites (A, B, and C) and one bridge site (AB). Thirteen MAb-resistant mutants (MARMs) were selected, and the neutralization patterns of the MAbs with either MARMs or RSV clinical strains identified a minimum of 16 epitopes. MARMs selected with antibodies to six of the site A and AB epitopes displayed a small-plaque phenotype, which is consistent with an alteration in a biologically active region of the F molecule. Analysis of MARMs also indicated that these neutralization epitopes occupy topographically distinct but conformationally interdependent regions with unique biological and immunological properties. Antigenic variation in F epitopes was examined by using 23 clinical isolates (18 subgroup A and 5 subgroup B) in cross-neutralization assays with the 18 anti-F MAbs. This analysis identified constant, variable, and hypervariable regions on the molecule and indicated that antigenic variation in the neutralization epitopes of the RSV F glycoprotein is the result of a noncumulative genetic heterogeneity. Of the 16 eptiopes, 8 were conserved on all or all but 1 of 23 subgroup A or subgroup B clinical isolates.     

A subgroup-specific antigenic site in the G protein of respiratory syncytial virus forms a disulfide-bonded loop.

An antigenic site (represented by 15 amino acids, residues 174 to 188, designated peptide 12) of the large glycoprotein G of respiratory syncytial virus was demonstrated to be subgroup specific in peptide enzyme-linked immunosorbent assay tests with murine monoclonal antibodies and human postinfection sera. The role of individual amino acids in this subgroup-specific site was determined by use of single-amino-acid-deletion sets of peptides. When monoclonal antibodies were reacted with the deletion sets, a broad amino acid dependence of 11 or 12 residues, Cys-176 (Ile-175 in subgroup B) to Cys-186, was found. Human postinfection sera exhibited a narrower reaction profile (for subgroup A, Cys-182 to Trp-183; for subgroup B, Cys-176 to Lys-183). Reduction of peptides on microtiter plates by treatment with dithiothreitol completely destroyed their antigenic activity in tests with monoclonal antibodies and human postinfection sera of subgroup B. A variant of peptide 12 containing all four cysteines of the G protein (represented by 16 amino acids, residues 172 to 187, designated peptide 12var) also was subgroup specific. We concluded that the activity of the antigenic site in tests with monoclonal antibodies for subgroups A and B appears to depend on intrapeptide disulfide bonds. Reactions with postinfection sera of subgroup B also may depend on a disulfide bond. In contrast, postinfection sera of subgroup A appeared to have the capacity to identify a subgroup-specific site in a linear form of the selected 15-amino-acid-long peptide. Treatment of peptides with dithiothreitol had no effect on their antigenic activity in tests with human postinfection sera of subgroup A. These findings have relevance for molecular engineering of peptide antigens for use in respiratory syncytial virus subgroup-specific site-directed serology.     

Characterization of neutralizing affinity-matured human respiratory syncytial virus F binding antibodies in the sub-picomolar affinity range

In the human adaptation and optimization of a mouse anti-human respiratory syncytial virus neutralizing antibody, affinity assessment was crucial to distinguish among potential candidates and to evaluate whether this correlated with function in vitro and in vivo. This affinity assessment was complicated by the trimeric nature of the antigen target, respiratory syncytial virus F (RSV-F) glycoprotein. In the initial affinity screen, surface plasmon resonance was used to determine the intrinsic binding affinities of anti-RSV-F Fab and immunoglobulin G (IgG) to the extracellular domain of RSV-F. This assessment required minimal biotinylation of the RSV-F protein and design of a capture strategy to minimize avidity effects. Approximately 30 Fabs were selected from three optimization phage display libraries on the basis of an initial ELISA screen. Surface plasmon resonance analysis demonstrated the success of optimization with some candidates from the screened libraries having low picomolar dissociation constants, more than 700-fold tighter than the parental monoclonal antibody (B21M). The affinities of these antibodies were further evaluated by a kinetic exclusion assay, a solution binding technology. One IgG (monoclonal antibody 029) displayed a low picomolar K(D) comparable with that of motavizumab, an RSV antibody in clinical study. Kinetic exclusion assay showed that two other of the matured IgGs (011 and 019) had sub-picomolar dissociation constants that could not be resolved further. We discuss the relevance of these interaction analysis results in the light of recently published data on the mechanism of F-driven viral fusion during paramyxoviral infection and 101F epitope conservation revealed from the recent crystal structure of RSV-F in the post-fusion state.     

Structure of the major antigenic loop of foot-and-mouth disease virus complexed with a neutralizing antibody: direct involvement of the Arg-Gly-Asp motif in the interaction.

The crystal structure of a synthetic peptide representing the major antigenic loop of foot-and-mouth disease virus (FMDV), complexed with the Fab fragment of a neutralizing monoclonal antibody raised against the virus, has been determined at 2.8 A resolution. The peptide shows a high degree of internal structure with a nearly cyclic conformation. The conserved Arg-Gly-Asp motif, involved in the viral attachment of aphtoviruses to cells, participates directly in the interaction with several complementarity determining regions of the antibody molecule. The Arg-Gly-Asp triplet shows the same open turn conformation found in the reduced form of FMDV of another serotype and also in integrin binding proteins. The observed interactions provide a molecular interpretation of the amino acid replacements observed to occur in mutants resistant to neutralization by this antibody. The structure also suggests a number of restrictions to variation within the epitope which are imposed to keep the Arg-Gly-Asp motif in its functional conformation.     

基于融合蛋白的呼吸道合胞病毒疫苗的研究进展

呼吸道合胞病毒(respiratory syncytial virus,RSV)是一种引起严重下呼吸道感染的病原体,易感人群为婴幼儿、老年人及免疫功能低下者。目前尚无有效的抗病毒药物和预防疫苗。RSV融合蛋白(fusion protein,F蛋白)具有高度保守性,其诱导的抗体可同时抑制A型和B型两个亚型的RSV感染。因此,以F蛋白作为靶抗原的RSV亚单位疫苗、颗粒样疫苗和病毒载体疫苗是目前研究的主要策略。现就基于F蛋白的RSV疫苗研究进展作一综述。     

Structure of the major oligosaccharides in the fusion glycoprotein of Newcastle disease virus

The fusion glycoprotein (F0) was isolated from Newcastle disease virus (NDV) particles metabolically labelled with [2-3H]mannose; it was successively digested with protease and with endo-beta-N-acetylglucosaminidase from Streptomyces griseus. In this manner, the majority of the oligosaccharides in NDV F0 could be liberated. After reduction with NaBH4, they were separated by high-performance liquid chromatography, and were subjected to structural analysis. Using micromethylation/capillary gas chromatography/mass fragmentography, alpha-mannosidase digestion, and acetolysis, it was found that the enzymatically released NDV F0 oligosaccharides are common oligomannosidic glycoprotein glycans of size classes (Man)8GlcNAc, Man)7GlcNAc, (Man)6GlcNAc, (Man)9GlcNAc, and (Man)5GlcNAc (in order of prevalence). The major structural isomers present in the NDV F0 (Man)8GlcNAc to (Man)5GlcNAc fractions were shown to lack mannose residues D2, D1D2 or D2D3, D1D2D3, and CD1D2D3, respectively, of (Man)9GlcNAc.     

Marked differences in the antigenic structure of human respiratory syncytial virus F and G glycoproteins.

Monoclonal antibodies directed against the glycoproteins of human respiratory syncytial virus were used in competitive enzyme-linked immunosorbent assays for topological mapping of epitopes. Whereas epitopes of the F glycoprotein could be ascribed to five nonoverlapping antigenic sites, anti-G antibodies recognized unique epitopes, many of whose competition profiles overlapped extensively. Variant viruses selected with a neutralizing (47F) anti-F antibody lost the binding for only 47F and 49F antibodies, which mapped in the same antigenic area. In contrast, viruses selected with an anti-G antibody lost the capacity to bind most of the anti-G antibodies, and their G protein was not recognized by an anti-virus antiserum, indicating major changes in the antigenic structure of the G molecule. Finally, we found great antigenic variation of the G protein among viral isolates. This occurred even within viruses of the same subtype with only limited divergence of amino acid sequence between strains. All of these data indicate marked differences in the antigenic organization of the G and F glycoproteins of respiratory syncytial virus; we discuss these differences in terms of the chemical structure of the glycoproteins.     

Generation and characterization of neutralizing human recombinant antibodies against antigenic site II of rabies virus glycoprotein

The currently recommended treatment for individuals exposed to rabies virus (RV) is post-exposure prophylaxis (PEP) through the combined administration of rabies vaccine and rabies immune globulin (RIG). Human monoclonal antibodies (mAbs) that neutralize RV offer an opportunity to replace RIG for rabies PEP. Here, a combinatorial human Fab library was constructed using antibody genes derived from the blood of RV-vaccinated donors. Selections of this library against purified RV virions resulted in the identification of 11 unique Fab antibodies specific for RV glycoprotein. Of the Fab antibodies, five were converted to full human IgG1 format. The human IgG antibodies revealed high binding affinity and neutralizing activities against RV fixed strains through a rapid fluorescent focus inhibition test in vitro as well as the early stage protective function after exposure to RV infection in vivo. Furthermore, epitope mapping and binding competition analysis showed that all of obtained human neutralizing and protective antibodies were directed to the antigenic site II of RV glycoprotein. Our results provide not only important insight into the protective immune response to RV in humans, but also more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.     

N-glycans of F protein differentially affect fusion activity of human respiratory syncytial virus

The human respiratory syncytial virus (Long strain) fusion protein contains six potential N-glycosylation sites: N27, N70, N116, N120, N126, and N500. Site-directed mutagenesis of these positions revealed that the mature fusion protein contains three N-linked oligosaccharides, attached to N27, N70, and N500. By introducing these mutations into the F gene in different combinations, four more mutants were generated. All mutants, including a triple mutant devoid of any N-linked oligosaccharide, were efficiently transported to the plasma membrane, as determined by flow cytometry and cell surface biotinylation. None of the glycosylation mutations interfered with proteolytic activation of the fusion protein. Despite similar levels of cell surface expression, the glycosylation mutants affected fusion activity in different ways. While the N27Q mutation did not have an effect on syncytium formation, loss of the N70-glycan caused a fusion activity increase of 40%. Elimination of both N-glycans (N27/70Q mutant) reduced the fusion activity by about 50%. A more pronounced reduction of the fusion activity of about 90% was observed with the mutants N500Q, N27/500Q, and N70/500Q. Almost no fusion activity was detected with the triple mutant N27/70/500Q. These data indicate that N-glycosylation of the F2 subunit at N27 and N70 is of minor importance for the fusion activity of the F protein. The single N-glycan of the F1 subunit attached to N500, however, is required for efficient syncytium formation.     

Heptad-repeat regions of respiratory syncytial virus F1 protein form a six-membered coiled-coil complex

The Respiratory Syncytial Virus (RSV) fusogenic glycoprotein F(1) was characterized using biochemical and biophysical techniques. Two heptad-repeat (HR) regions within F(1) were shown to interact. Proteinase-K digestion experiments highlight the HR1 region (located proximal to the fusion peptide sequence) of the F(1) protein to which an HR2-derived (located proximal to the membrane-spanning domain) peptide binds, thus protecting both the protein and peptide from digestion. Solution-phase analysis of HR1-derived peptides shows that these peptides adopt helical secondary structure as measured by circular dichroism. Sedimentation equilibrium studies indicate that these HR1 peptides self-associate in a monomer/trimer equilibrium with an association constant of 5.2 x 10(8) M(-2). In contrast, HR2-derived peptides form random monomers in solution. CD analysis of mixtures containing peptides from the two regions demonstrate their propensity to interact and form a very stable (T(m) = 87 degrees C), helical (86% helicity) complex comprised of three HR1 and three HR2 members.     

A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site.

The structure of a complex of influenza hemagglutinin (HA) with a neutralizing antibody shows that the antibody binds to HA at a distance from the virus receptor binding site. Comparison of the properties of this antibody and its Fab with those of an antibody that recognizes an epitope overlapping the receptor binding site leads to two main conclusions. First, inhibition of receptor binding is an important component of neutralization. Second, the efficiency of neutralization by the antibodies ranks in the same order as their avidities for HA, and their large size makes these antibodies highly efficient at neutralization, regardless of the location of their epitope in relation to the virus receptor binding site. These observations provide rationales for the range of antibody specificities that are detected in immune sera and for the distribution of sequence changes on the membrane-distal surface of influenza HAs that occur during 'antigenic drift.'     

A chimeric respiratory syncytial virus fusion protein functionally replaces the F and HN glycoproteins in recombinant Sendai virus

Entry of most paramyxoviruses is accomplished by separate attachment and fusion proteins that function in a cooperative manner. Because of this close interdependence, it was not possible with most paramyxoviruses to replace either of the two protagonists by envelope glycoproteins from related paramyxoviruses. By using reverse genetics of Sendai virus (SeV), we demonstrate that chimeric respiratory syncytial virus (RSV) fusion proteins containing either the cytoplasmic domain of the SeV fusion protein or in addition the transmembrane domain were efficiently incorporated into SeV particles provided the homotypic SeV-F was deleted. In the presence of SeV-F, the chimeric glycoproteins were incorporated with significantly lower efficiency, indicating that determinants in the SeV-F ectodomain exist that contribute to glycoprotein uptake. Recombinant SeV in which the homotypic fusion protein was replaced with chimeric RSV fusion protein replicated in a trypsin-independent manner and was neutralized by antibodies directed to RSV-F. However, replication of this virus also relied on the hemagglutinin-neuraminidase (HN) as pretreatment of cells with neuraminidase significantly reduced the infection rate. Finally, recombinant SeV was generated with chimeric RSV-F as the only envelope glycoprotein. This virus was not neutralized by antibodies to SeV and did not use sialic acids for attachment. It replicated more slowly than hybrid virus containing HN and produced lower virus titers. Thus, on the one hand RSV-F can mediate infection in an autonomous way while on the other hand it accepts support by a heterologous attachment protein.     

Induction of a neutralizing immune response to human respiratory syncytial virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against monoclonal antibodies that recognized either F glycoprotein 47F or G glycoprotein 63G, 62G, or 74G of the human respiratory syncytial virus Long strain. Anti-Id sera inhibited the virus binding of the immunizing monoclonal antibodies and in some cases the binding of other antibodies reacting with overlapping epitopes. The anti-Id sera also inhibited virus neutralization mediated by the original monoclonal antibodies. Affinity purified anti-Id antibodies were subsequently used to raise a homologous anti-anti-Id response in rabbits. One of the rabbits, inoculated with anti-Id 63G, generated antibodies that reacted with the G protein of respiratory syncytial virus and neutralized the virus to high titers. The antiviral antibodies induced by anti-Id 63G were broadly cross-reactive with strains of the A and B subtypes. However, the specificities of monoclonal antibody 63G and anti-anti-Id 63G were not exactly the same, as indicated by their reaction with escape mutants to antibody 63G. These results demonstrate for the first time the induction of an anti-respiratory syncytial virus response by anti-Id antibodies.     

Screening of phage-displayed peptide libraries with a monoclonal antibody raised against the F protein of the bovine respiratory syncytial virus

Summary Most of the monoclonal antibodies (MAbs) raised against the fusion (F) protein of the bovine respiratory syncytial virus recognize discontinuous epitopes on the protein. In order to find mimotopes of these epitopes, phage-displayed peptide libraries were screened with MAbs. The results obtained with MAb AL11C2 are described here. After four or five pannings, colony immunoscreening with AL11C2 allowed the isolation of positive clones that are specific for this monoclonal antibody. Four different sequences were determined on isolated phages, three of which are cysteine-constrained peptides in fusion with PVIII and one is a hexapeptide in fusion with PIII. In the case of the peptides containing two cysteines, the binding to AL11C2 was shown to be dependent on the presence of a disulfide bridge. The recombinant phages were also shown to inhibit the binding of AL11C2 to its natural antigen in a competitive ELISA assay.     

Immunoprotective activity and safety of a respiratory syncytial virus vaccine: mucosal delivery of fusion glycoprotein with a CpG oligodeoxynucleotide adjuvant

CpG oligodeoxynucleotides (ODN) were identified that stimulated immunoglobulin production and cell proliferation in cotton rat cells in vitro. Three of these ODN were used as a mucosal adjuvant in the noses of cotton rats immunized via this route with respiratory syncytial virus fusion (F) protein. The CpG ODN markedly increased the cotton rat humoral neutralizing-antibody response to respiratory syncytial virus. Such immunized animals had a marked reduction in the production of infectious virus after a live-virus challenge. Animals immunized with the combination of F protein and CpG developed enhanced pulmonary pathology consisting of alveolitis and interstitial pneumonitis after a live-virus challenge. Similar enhanced disease has been seen in cotton rats and children immunized with formalin-inactivated respiratory syncytial virus.