Inhibition of receptor binding stabilizes Newcastle disease virus HN and F protein-containing complexes

Receptor binding of paramyxovirus attachment proteins and the interactions between attachment and fusion (F) proteins are thought to be central to activation of the F protein activity; however, mechanisms involved are unclear. To explore the relationships between Newcastle disease virus (NDV) HN and F protein interactions and HN protein attachment to sialic acid receptors, HN and F protein-containing complexes were detected and quantified by reciprocal coimmunoprecipitation from extracts of transfected avian cells. To inhibit HN protein receptor binding, cells transfected with HN and F protein cDNAs were incubated with neuraminidase from the start of transfection. Under these conditions, no fusion was observed, but amounts of HN and F protein complexes increased twofold over amounts detected in extracts of untreated cells. Stimulation of attachment by incubation of untransfected target cells with neuraminidase-treated HN and F protein-expressing cells resulted in a twofold decrease in amounts of HN and F protein complexes. In contrast, high levels of complexes containing HN protein and an uncleaved F protein (F-K115Q) were detected, and those levels were unaffected by neuraminidase treatment of cell monolayers or by incubation with target cells. These results suggest that HN and F proteins reside in a complex in the absence of receptor binding. Furthermore, the results show that not only receptor binding but also F protein cleavage are necessary for disassociation of the HN and F protein-containing complexes

Cotton rat immune responses to virus-like particles containing the pre-fusion form of respiratory syncytial virus fusion protein

BACKGROUND: Virus-like particles (VLPs) based on Newcastle disease virus (NDV) core proteins, M and NP, and containing two chimera proteins, F/F and H/G, composed of the respiratory syncytial virus (RSV) fusion protein (F) and glycoprotein (G) ectodomains fused to the transmembrane and cytoplasmic domains of the NDV F and HN proteins, respectively, stimulate durable, protective anti-RSV neutralizing antibodies in mice. Furthermore, immunization of mice with a VLP containing a F/F chimera protein with modifications previously reported to stabilize the pre-fusion form of the RSV F protein resulted in significantly improved neutralizing antibody titers over VLPs containing the wild type F protein. The goal of this study was to determine if VLPs containing the pre-fusion form of the RSV F protein stimulated protective immune responses in cotton rats, a more RSV permissive animal model than mice. METHODS: Cotton rats were immunized intramuscularly with VLPs containing stabilized pre-fusion F/F chimera protein as well as the H/G chimera protein. The anti-RSV F and RSV G antibody responses were determined by ELISA. Neutralizing antibody titers in sera of immunized animals were determined in plaque reduction assays. Protection of the animals from RSV challenge was assessed. The safety of the VLP vaccine was determined by monitoring lung pathology upon RSV challenge of immunized animals. RESULTS: The Pre-F/F VLP induced neutralizing titers that were well above minimum levels previously proposed to be required for a successful vaccine and titers significantly higher than those stimulated by RSV infection. In addition, Pre-F/F VLP immunization stimulated higher IgG titers to the soluble pre-fusion F protein than RSV infection. Cotton rats immunized with Pre-F/F VLPs were protected from RSV challenge, and, importantly, the VLP immunization did not result in enhanced respiratory disease upon RSV challenge. CONCLUSIONS: VLPs containing the pre-fusion RSV F protein have characteristics required for a safe, effective RSV vaccine

Comparisons of Antibody Populations in Different Pre-Fusion F VLP-Immunized Cotton Rat Dams and Their Offspring

Respiratory syncytial virus (RSV) infection poses a significant risk for infants. Since the direct vaccination of infants is problematic, maternal vaccination may provide a safer, more effective approach to their protection. In the cotton rat (CR) model, we have compared the immunization of pregnant CR dams with virus-like particles assembled with the prototype mutation stabilized pre-fusion F protein, DS-Cav1, as well two alternative mutation stabilized pre-fusion proteins (UC-2 F, UC-3 F) and showed that the alternative pre-fusion F VLPs protected the offspring of immunized dams significantly better than DS-Cav1 F VLPs (Blanco, et al. J. Virol. 93: e00914). Here, we have addressed the reasons for this increased protection by characterizing the specificities of antibodies in the sera of both immunized dams and their offspring. The approach was to measure the levels of total anti-pre-F IgG serum antibodies that would block the binding of representative pre-fusion specific monoclonal antibodies to soluble pre-fusion F protein targets. Strikingly, we found that the sera in most offspring of DS-Cav1 F VLP-immunized dams had no mAb D25-blocking antibodies, although their dams had robust levels. In contrast, all offspring of UC-3 F VLP-immunized dams had robust levels of these D25-blocking antibodies. Both sets of pup sera had significant levels of mAb AM14-blocking antibodies, indicating that all pups received maternal antibodies. A lack of mAb D25-blocking antibodies in the offspring of DS-Cav1 F VLP-immunized dams may account for the lower protection of their pups from challenge compared to the offspring of UC-3 F VLP-immunized dams

Murine immune responses to virus-like particle-associated pre- and postfusion forms of the respiratory syncytial virus F protein

Virus-like particles (VLPs) built on the Newcastle disease virus (NDV) core proteins, NP and M, and containing two chimeric proteins, F/F and H/G, composed of respiratory syncytial virus (RSV) fusion protein (F) and glycoprotein (G) ectodomains fused to the transmembrane and cytoplasmic domains of the NDV F and HN proteins, respectively, stimulate durable, protective RSV neutralizing antibodies in mice. Here, we report the properties of VLPs constructed to contain mutant RSV F protein ectodomains stabilized in prefusion (pre-F/F) or postfusion (post-F/F) configurations. The structures of the chimeric proteins assembled into VLPs were verified immunologically by their reactivities with a conformationally restricted anti-F protein monoclonal antibody. Following immunization of mice, without adjuvant, pre-F/F-containing VLPs induced significantly higher neutralizing antibody titers than the post-F/F-containing VLPs or the wild-type F/F-containing VLPs after a single immunization but not after prime and boost immunization. The specificities of anti-F IgG induced by the two mutant VLPs were assessed by enzyme-linked immunosorbent assay (ELISA) using soluble forms of the prefusion and postfusion forms of the F protein as targets. While both types of VLPs stimulated similar levels of IgG specific for the soluble postfusion F protein, titers of IgG specific for prefusion F induced by the pre-F/F-containing VLPs were higher than those induced by post-F/F-containing VLPs. Thus, VLPs containing a stabilized prefusion form of the RSV F protein represent a promising RSV vaccine candidate. IMPORTANCE: The development of vaccines for respiratory syncytial virus has been hampered by a lack of understanding of the requirements for eliciting high titers of neutralizing antibodies. The results of this study suggest that particle-associated RSV F protein containing mutations that stabilize the structure in a prefusion conformation may stimulate higher titers of protective antibodies than particles containing F protein in a wild-type or postfusion conformation. These findings indicate that the prefusion F protein assembled into VLPs has the potential to produce a successful RSV vaccine candidate

A chimeric EBV gp350/220-based VLP replicates the virion B-cell attachment mechanism and elicits long-lasting neutralizing antibodies in mice

Epstein-Barr virus (EBV), an oncogenic gammaherpesvirus, causes acute infectious mononucleosis (AIM) and is linked to the development of several human malignancies. There is an urgent need for a vaccine that is safe, prevents infection and/or limits disease. Unique among human herpesviruses, glycoprotein (gp)350/220, which initiates EBV attachment to susceptible host cells, is the major ligand on the EBV envelope and is highly conserved. Interaction between gp350/220 and complement receptor type 2 (CR2)/CD21 and/or (CR1)/CD35 on B-cells is required for infection. Potent antibody responses to gp350/220 occur in animal models and humans. Thus, gp350/220 provides an attractive candidate for prophylactic subunit vaccine development. However, in a recent Phase II clinical trial immunization with soluble recombinant gp350 reduced the incidence of AIM, but did not prevent infection. Despite various attempts to produce an EBV vaccine, no vaccine is licensed. Herein we describe a sub-unit vaccine against EBV based on a novel Newcastle disease virus (NDV)-virus-like particle (VLP) platform consisting of EBVgp350/220 ectodomain fused to NDV-fusion (F) protein. The chimeric protein EBVgp350/220-F is incorporated into the membrane of a VLP composed of the NDV matrix and nucleoprotein. The particles resemble native EBV in diameter and shape and bind CD21 and CD35. Immunization of BALB/c mice with EBVgp350/220-F VLPs elicited strong, long-lasting neutralizing antibody responses when assessed in vitro. This chimeric VLP is predicted to provide a superior safety profile as it is efficiently produced in Chinese hamster ovary (CHO) cells using a platform devoid of human nucleic acid and EBV-transforming genes

Role of Cotranslational Disulfide Bond Formation in the Folding of the Hemagglutinin-Neuraminidase Protein of Newcastle Disease Virus

AbstractThe role of cotranslational disulfide bond formation in the folding pathway of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was explored. Electrophoresis of pulse-labeled HN protein in the presence or absence of reducing agent showed that, characteristic of many glycoproteins, the nascent HN protein contains intramolecular disulfide bonds. As reported by Braakmanet al.(EMBO J.11, 1717–1722, 1992), incubation of cells in dithiothreitol (DTT) blocked the formation of these bonds. Removal of DTT after a pulse-label allowed for the subsequent formation of intramolecular disulfide bonds and folding of the molecule as assayed by the appearance of conformationally sensitive antigenic sites and by the formation of disulfide-linked dimers. However, thet1/2for the formation of a conformationally sensitive antigenic site after synthesis in the presence of DTT was over twice that of the control. Furthermore, the order of appearance of the antigenic sites was different from the control, suggesting that inhibition of cotranslational disulfide bond formation altered the folding pathway of the protein. Similar results were obtained in a cell-free system containing membranes. The HN protein forced to form intramolecular disulfide bonds posttranslationally had no detectable neuraminidase or cell attachment activity, suggesting that the protein had an abnormal conformation

Thiol/Disulfide Exchange Is Required for Membrane Fusion Directed by the Newcastle Disease Virus Fusion Protein

Newcastle disease virus (NDV), an avian paramyxovirus, initiates infection with attachment of the viral hemagglutinin-neuraminidase (HN) protein to sialic acid-containing receptors, followed by fusion of viral and cell membranes, which is mediated by the fusion (F) protein. Like all class 1 viral fusion proteins, the paramyxovirus F protein is thought to undergo dramatic conformational changes upon activation. How the F protein accomplishes extensive conformational rearrangements is unclear. Since several viral fusion proteins undergo disulfide bond rearrangement during entry, we asked if similar rearrangements occur in NDV proteins during entry. We found that inhibitors of cell surface thiol/disulfide isomerase activity—5′5-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin, and anti-protein disulfide isomerase antibody—inhibited cell-cell fusion and virus entry but had no effect on cell viability, glycoprotein surface expression, or HN protein attachment or neuraminidase activities. These inhibitors altered the conformation of surface-expressed F protein, as detected by conformation-sensitive antibodies. Using biotin maleimide (MPB), a reagent that binds to free thiols, free thiols were detected on surface-expressed F protein, but not HN protein. The inhibitors DTNB and bacitracin blocked the detection of these free thiols. Furthermore, MPB binding inhibited cell-cell fusion. Taken together, our results suggest that one or several disulfide bonds in cell surface F protein are reduced by the protein disulfide isomerase family of isomerases and that F protein exists as a mixture of oxidized and reduced forms. In the presence of HN protein, only the reduced form may proceed to refold into additional intermediates, leading to the fusion of membranes