Structure of a major antigenic site on the respiratory syncytial virus fusion glycoprotein in complex with neutralizing antibody 101F

Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling of 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope ～16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.     

Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary,Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein

Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.     

Design and characterization of epitope-scaffold immunogens that present the motavizumab epitope from respiratory syncytial virus

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potent neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 Å resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required.     

A Protective and Safe Intranasal RSV Vaccine Based on a Recombinant Prefusion-Like Form of the F Protein Bound to Bacterium-Like Particles

Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease in infants and the elderly. Currently, no licensed vaccine against RSV is available. Here we describe the development of a safe and effective intranasal subunit vaccine that is based on recombinant fusion (F) protein bound to the surface of immunostimulatory bacterium-like particles (BLPs) derived from the food-grade bacterium Lactococcus lactis. Different variants of F were analyzed with respect to their conformation and reactivity with neutralizing antibodies, assuming that F proteins mimicking the metastable prefusion form of RSV F expose a more extensive and relevant epitope repertoire than F proteins corresponding to the postfusion structure. Our results indicate that the recombinant soluble ectodomain of RSV F readily adopts a postfusion conformation, generation of which cannot be prevented by C-terminal addition of a trimerization motif, but whose formation is prevented by mutation of the two furin cleavage sites in F. While the putative postfusion form of F is recognized well by the monoclonal antibody Palivizumab, this is much less so for the more potently neutralizing, prefusion-specific antibodies D25 and AM22. Both addition of the trimerization motif and mutation of the furin cleavage sites increased the reactivity of F with D25 and AM22, with the highest reactivity being observed for F proteins in which both these features were combined. Intranasal vaccination of mice or cotton rats with BLPs loaded with this latter prefusion-like F protein (BLP-F), resulted in the potent induction of F-specific immunoglobulins and in significantly decreased virus titers in the lungs upon RSV challenge. Moreover, and in contrast to animals vaccinated with formalin-inactivated RSV, animals that received BLP-F exhibited high levels of F-specific secretory IgA in the nose and RSV-neutralizing antibodies in sera, but did not show symptoms of enhanced disease after challenge with RSV.     

Cryo-EM structure of glycoprotein C from Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever virus (CCHFV) is a causative agent of serious hemorrhagic diseases in humans with high mortality rates. CCHFV glycoprotein Gc plays critical roles in mediating virus-host membrane fusion and has been studied extensively as an immunogen. However, the molecular mechanisms involved in membrane fusion and Gc-specific antibody-antigen interactions remain unresolved largely because structural information of this glycoprotein is missing. We designed a trimeric protein including most of the ectodomain region of Gc from the prototype CCHFV strain, IbAr10200, which enabled the cryo-electron microscopy structure to be solved at a resolution of 2.8 ?. The structure confirms that CCHFV Gc is a class II fusion protein. Unexpectedly, structural comparisons with other solved Gc trimers in the postfusion conformation revealed that CCHFV Gc adopted hybrid architectural features of the fusion loops from hantaviruses and domain III from phenuiviruses, suggesting a complex evolutionary pathway among these bunyaviruses. Antigenic sites on CCHFV Gc that protective neutralizing antibodies target were mapped onto the CCHFV Gc structure, providing valuable information that improved our understanding of potential neutralization mechanisms of various antibodies.     

Engineering,Structure and Immunogenicity of the Human Metapneumovirus F Protein in the Postfusion Conformation

Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses.     

Design, expression, and immunogenicity of a soluble HIV trimeric envelope fragment adopting a prefusion gp41 configuration

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) is comprised of non-covalently associated gp120/gp41 subunits that form trimeric spikes on the virion surface. Upon binding to host cells, Env undergoes a series of structural transitions, leading to gp41 rearrangement necessary for fusion of viral and host membranes. Until now, the prefusion state of gp41 ectodomain (e-gp41) has eluded molecular and structural analysis, and thus assessment of the potential of such an e-gp41 conformer to elicit neutralizing antibodies has not been possible. Considering the importance of gp120 amino (C1) and carboxyl (C5) segments in the association with e-gp41, we hypothesize that these regions are sufficient to maintain e-gp41 in a prefusion state. Based on the available gp120 atomic structure, we designed several truncated gp140 variants by including the C1 and C5 regions of gp120 in a gp41 ectodomain fragment. After iterative cycles of protein design, expression and characterization, we obtained a variant truncated at Lys(665) that stably folds as an elongated trimer under physiologic conditions. Several independent biochemical/biophysical analyses strongly suggest that this mini-Env adopts a prefusion e-gp41 configuration that is strikingly distinct from the postfusion trimer-of-hairpin structure. Interestingly, this prefusion mini-Env, lacking the fragment containing the 2F5/4E10 neutralizing monoclonal antibody binding sites, displays no detectable HIV-neutralizing epitopes when employed as an immunogen in rabbits. The result of this immunogenicity study has important implications for HIV-1 vaccine design efforts. Moreover, this engineered mini-Env protein should facilitate three-dimensional structural studies of the prefusion e-gp41 and serve to guide future attempts at pharmacologic and immunologic intervention of HIV-1.     

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 a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation

Enveloped viruses enter cells via a membrane fusion reaction driven by conformational changes of specific viral envelope proteins. We report here the structure of the ectodomain of the tick-borne encephalitis virus envelope glycoprotein, E, a prototypical class II fusion protein, in its trimeric low-pH-induced conformation. We show that, in the conformational transition, the three domains of the neutral-pH form are maintained but their relative orientation is altered. Similar to the postfusion class I proteins, the subunits rearrange such that the fusion peptide loops cluster at one end of an elongated molecule and the C-terminal segments, connecting to the viral transmembrane region, run along the sides of the trimer pointing toward the fusion peptide loops. Comparison with the low-pH-induced form of the alphavirus class II fusion protein reveals striking differences at the end of the molecule bearing the fusion peptides, suggesting an important conformational effect of the missing membrane connecting segment.     

Structural Characterization of HIV gp41 with the Membrane-proximal External Region

Human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein (gp120/gp41) plays a critical role in virus infection and pathogenesis. Three of the six monoclonal antibodies considered to have broadly neutralizing activities (2F5, 4E10, and Z13e1) bind to the membrane-proximal external region (MPER) of gp41. This makes the MPER a desirable template for developing immunogens that can elicit antibodies with properties similar to these monoclonal antibodies, with a long term goal of developing antigens that could serve as novel HIV vaccines. In order to provide a structural basis for rational antigen design, an MPER construct, HR1-54Q, was generated for x-ray crystallographic and x-ray footprinting studies to provide both high resolution atomic coordinates and verification of the solution state of the antigen, respectively. The crystal structure of HR1-54Q reveals a trimeric, coiled-coil six-helical bundle, which probably represents a postfusion form of gp41. The MPER portion extends from HR2 in continuation of a slightly bent long helix and is relatively flexible. The structures observed for the 2F5 and 4E10 epitopes agree well with existing structural data, and enzyme-linked immunosorbent assays indicate that the antigen binds well to antibodies that recognize the above epitopes. Hydroxyl radical-mediated protein footprinting of the antigen in solution reveals specifically protected and accessible regions consistent with the predictions based on the trimeric structure from the crystallographic data. Overall, the HR1-54Q antigen, as characterized by crystallography and footprinting, represents a postfusion, trimeric form of HIV gp41, and its structure provides a rational basis for gp41 antigen design suitable for HIV vaccine development.     

Crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes

West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.     

Crystal Structure of the Human Cytomegalovirus Glycoprotein B

Human cytomegalovirus (HCMV), a dsDNA, enveloped virus, is a ubiquitous pathogen that establishes lifelong latent infections and caused disease in persons with compromised immune systems, e.g., organ transplant recipients or AIDS patients. HCMV is also a leading cause of congenital viral infections in newborns. Entry of HCMV into cells requires the conserved glycoprotein B (gB), thought to function as a fusogen and reported to bind signaling receptors. gB also elicits a strong immune response in humans and induces the production of neutralizing antibodies although most anti-gB Abs are non-neutralizing. Here, we report the crystal structure of the HCMV gB ectodomain determined to 3.6-Å resolution, which is the first atomic-level structure of any betaherpesvirus glycoprotein. The structure of HCMV gB resembles the postfusion structures of HSV-1 and EBV homologs, establishing it as a new member of the class III viral fusogens. Despite structural similarities, each gB has a unique domain arrangement, demonstrating structural plasticity of gB that may accommodate virus-specific functional requirements. The structure illustrates how extensive glycosylation of the gB ectodomain influences antibody recognition. Antigenic sites that elicit neutralizing antibodies are more heavily glycosylated than those that elicit non-neutralizing antibodies, which suggest that HCMV gB uses glycans to shield neutralizing epitopes while exposing non-neutralizing epitopes. This glycosylation pattern may have evolved to direct the immune response towards generation of non-neutralizing antibodies thus helping HCMV to avoid clearance. HCMV gB structure provides a starting point for elucidation of its antigenic and immunogenic properties and aid in the design of recombinant vaccines and monoclonal antibody therapies.     

Analysis of Human RSV Immunity at the Molecular Level: Learning from the Past and Present

Human RSV is one of the most prevalent viral pathogens of early childhood for which no vaccine is available. Herein we provide an analysis of RSV epitope data to examine its application to vaccine design and development. Our objective was to provide an overview of antigenic coverage, identify critical antibody and T cell determinants, and then analyze the cumulative RSV epitope data from the standpoint of functional responses using a combinational approach to characterize antigenic structure and epitope location. A review of the cumulative data revealed, not surprisingly, that the vast majority of epitopes have been defined for the two major surface antigens, F and G. Antibody and T cell determinants have been reported from multiple hosts, including those from human subjects following natural infection, however human data represent a minority of the data. A structural analysis of the major surface antigen, F, showed that the majority of epitopes defined for functional antibodies (neutralizing and/or protective) were either shown to bind pre-F or to be accessible in both pre- and post-F forms. This finding may have has implications for on-going vaccine design and development. These interpretations are in agreement with previous work and can be applied in the larger context of functional epitopes on the F protein. It is our hope that this work will provide the basis for further RSV-specific epitope discovery and investigation into the nature of antigen conformation in immunogenicity.     

Residues within the C-terminal arm of the herpes simplex virus 1 glycoprotein B ectodomain contribute to its refolding during the fusion step of virus entry

Herpesvirus entry into cells requires coordinated interactions among several viral glycoproteins. The final membrane fusion step of entry is executed by glycoprotein B (gB), a class III viral fusion protein that is conserved across all herpesviruses. Fusion proteins are metastable proteins that mediate fusion by inserting into a target membrane and refolding from a prefusion to postfusion conformation to bring the viral and cell membranes together. Although the structure of gB has been solved in a conformation that likely represents its postfusion form, its prefusion structure and the details of how it refolds to execute fusion are unknown. The postfusion gB structure contains a trimeric coiled-coil at its core and a long C-terminal arm within the ectodomain packs against this coil in an antiparallel manner. This coil-arm complex is reminiscent of the six-helix bundle that provides the energy for fusion in class I fusogens. To determine the role of the coil-arm complex, we individually mutated residues in the herpes simplex virus 1 gB coil-arm complex to alanine and assessed the contribution of each residue to cell-cell and virus-cell fusion. Several coil mutations resulted in a loss of cell surface expression, indicating that the coil residues are important for proper processing of gB. Three mutations in the arm region (I671A, H681A, and F683A) reduced fusion without affecting expression. Combining these three arm mutations drastically reduced the ability of gB to execute fusion; however, fusion function could be restored by adding known hyperfusogenic mutations to the arm mutant. We propose that the formation of the coil-arm complex drives the gB transition to a postfusion conformation and the coil-arm complex performs a function similar to that of the six-helix bundle in class I fusion. Furthermore, we suggest that these specific mutations in the arm may energetically favor the prefusion state of gB.     

In Silico Vaccine Design Based on Molecular Simulations of Rhinovirus Chimeras Presenting HIV-1 gp41 Epitopes

A cluster of promising epitopes for the development of human immunodeficiency virus (HIV) vaccines is located in the membrane-proximal external region (MPER) of the gp41 subunit of the HIV envelope spike structure. The crystal structure of the peptide corresponding to the so-called ELDKWA epitope (HIV-1 HxB2 gp41 residues 662-668), in complex with the corresponding broadly neutralizing human monoclonal antibody 2F5, provides a target for structure-based vaccine design strategies aimed at finding macromolecular carriers that are able to present this MPER-derived epitope with optimal antigenic activity. To this end, a series of replica exchange molecular dynamics computer simulations was conducted to characterize the distributions of conformations of ELDKWA-based epitopes inserted into a rhinovirus carrier and to identify those with the highest fraction of conformations that are able to bind 2F5. The length, hydrophobic character, and precise site of insertion were found to be critical for achieving structural similarity to the target crystal structure. A construct with a high degree of complementarity to the corresponding determinant region of 2F5 was obtained. This construct was employed to build a high-resolution structural model of the complex between the 2F5 antibody and the chimeric human rhinovirus type 14:HIV-1 ELDKWA virus particle. Additional simulations, which were conducted to study the conformational propensities of the ELDKWA region in solution, confirm the hypothesis that the ELDKWA region of gp41 is highly flexible and capable of assuming helical conformations (as in the postfusion helical bundle structure) and β-turn conformations (as in the complex with the 2F5 antibody). These results also suggest that the ELDKWA epitope can be involved in intramolecular—and likely intermolecular—hydrophobic interactions. This tendency offers an explanation for the observation that mutations decreasing the hydrophobic character of the MPER in many cases result in conformational changes that increase the affinity of this region for the 2F5 antibody.     

HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.     

Structural insights into the neutralization mechanism of a higher primate antibody against dengue virus

The four serotypes of dengue virus (DENV-1 to -4) cause the most important emerging viral disease. Protein E, the principal viral envelope glycoprotein, mediates fusion of the viral and endosomal membranes during virus entry and is the target of neutralizing antibodies. However, the epitopes of strongly neutralizing human antibodies have not been described despite their importance to vaccine development. The chimpanzee Mab 5H2 potently neutralizes DENV-4 by binding to domain I of E. The crystal structure of Fab 5H2 bound to E from DENV-4 shows that antibody binding prevents formation of the fusogenic hairpin conformation of E, which together with in-vitro assays, demonstrates that 5H2 neutralizes by blocking membrane fusion in the endosome. Furthermore, we show that human sera from patients recovering from DENV-4 infection contain antibodies that bind to the 5H2 epitope region on domain I. This study, thus, provides new information and tools for effective vaccine design to prevent dengue disease.     

Glycoprotein B of herpes simplex virus 2 has more than one intracellular conformation and is altered by low pH

The crystal structure of herpes simplex virus (HSV) gB identifies it as a class III fusion protein, and comparison with other such proteins suggests this is the postfusion rather than prefusion conformation, although this is not proven. Other class III proteins undergo a pH-dependent switch between pre- and postfusion conformations, and a low pH requirement for HSV entry into some cell types suggests that this may also be true for gB. Both gB and gH undergo structural changes at low pH, but there is debate about the extent and significance of the changes in gB, possibly due to the use of different soluble forms of the protein and different assays for antigenic changes. In this study, a complementary approach was taken, examining the conformations of full-length intracellular gB by quantitative confocal microscopy with a panel of 26 antibodies. Three conformations were distinguished, and low pH was found to be a major influence. Comparison with previous studies indicates that the intracellular conformation in low-pH environments may be the same as that of the soluble form known as s-gB at low pH. Interestingly, the antibodies whose binding was most affected by low pH both have neutralizing activity and consequently must block either the function of a neutral pH conformation or its switch from an inactive form to an activated form. If one of the intracellular conformations is the fusion-active form, another factor required for fusion is presumably absent from wherever that conformation is present in infected cells so that inappropriate fusion is avoided.     

A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus

Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by “DS-Cav1” mutations and by an appended C-terminal trimerization motif or “foldon” from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide “rings”, with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen.     

The broadly neutralizing anti-human immunodeficiency virus type 1 4E10 monoclonal antibody is better adapted to membrane-bound epitope recognition and blocking than 2F5

The broadly neutralizing 2F5 and 4E10 monoclonal antibodies (MAbs) recognize epitopes within the membrane-proximal external region (MPER) that connects the human immunodeficiency virus type 1 (HIV-1) envelope gp41 ectodomain with the transmembrane anchor. By adopting different conformations that stably insert into the virion external membrane interface, such as helical structures, a conserved aromatic-rich sequence within the MPER is thought to participate in HIV-1-cell fusion. Recent experimental evidence suggests that the neutralizing activity of 2F5 and 4E10 might correlate with the MAbs' capacity to recognize epitopes inserted into the viral membrane, thereby impairing MPER fusogenic activity. To gain new insights into the molecular mechanism underlying viral neutralization by these antibodies, we have compared the capacities of 2F5 and 4E10 to block the membrane-disorganizing activity of MPER peptides inserted into the surface bilayer of solution-diffusing unilamellar vesicles. Both MAbs inhibited leakage of vesicular aqueous contents (membrane permeabilization) and intervesicular lipid mixing (membrane fusion) promoted by MPER-derived peptides. Thus, our data support the idea that antibody binding to a membrane-inserted epitope may interfere with the function of the MPER during gp41-induced fusion. Antibody insertion into a cholesterol-containing, uncharged virion-like membrane is mediated by specific epitope recognition, and moreover, partitioning-coupled folding into a helix reduces the efficiency of 2F5 MAb binding to its epitope in the membrane. We conclude that the capacity to interfere with the membrane activity of conserved MPER sequences is best correlated with the broad neutralization of the 4E10 MAb.