Ectodomain Of Glycoprotein Research Articles

Deep mining reveals the diversity of endogenous viral elements in vertebrate genomes

Integration of viruses into host genomes can give rise to endogenous viral elements (EVEs), which provide insights into viral diversity, host range and evolution. A systematic search for EVEs is becoming computationally challenging given the available genomic data. We used a cloud-computing approach to perform a comprehensive search for EVEs in the kingdoms Shotokuvirae and Orthornavirae across vertebrates. We identified 2,040 EVEs in 295 vertebrate genomes and provide evidence for EVEs belonging to the families Chuviridae, Paramyxoviridae, Nairoviridae and Benyviridae. We also find an EVE from the Hepacivirus genus of flaviviruses with orthology across murine rodents. In addition, our analyses revealed that reptarenaviruses and filoviruses probably acquired their glycoprotein ectodomains three times independently from retroviral elements. Taken together, these findings encourage the addition of 4 virus families and the Hepacivirus genus to the growing virus fossil record of vertebrates, providing key insights into their natural history and evolution.

Enhanced immune response with baculovirus-expressed BoHV-1 glycoprotein D in vaccine development

Bovine herpesvirus 1 (BoHV-1), a significant pathogen in the alpha-herpesvirus subfamily, primarily infects cattle and causes the upper respiratory disease known as infectious bovine rhinotracheitis (IBR). In silico studies evaluated the BoHV-1 D protein to be non-allergenic, non-toxic, and highly antigenic, highlighting its potential as an antigen for vaccine development. Therefore, this study aimed to evaluate the efficacy of a subunit vaccine using the ectodomain of glycoprotein D (gD34–380) as an antigen. The truncated gD was successfully cloned and expressed in both Escherichia coli (E. coli, termed EgD) and baculovirus (termed BgD) systems, with expected molecular weights of 65 kDa and 50 kDa, respectively. For the vaccine formulation, the gD proteins were used either alone or in combination with in-house inactivated BoHV-1. Vaccination of mice and bovines showed that baculovirus-expressed gD34–380 accelerated the antibody response. Moreover, the BgD-vaccinated group also showed significantly higher neutralizing antibody levels against BoHV-1 than the control group (p<0.0001). In conclusion, our study found that BgD from BoHV-1 can increase the immune response and enhance vaccine efficacy.

Genomic Surveillance of Rabies Virus in Georgian Canines.

Rabies is a fatal zoonosis that is considered a re-emerging infectious disease. Although rabies remains endemic in canines throughout much of the world, vaccination programs have essentially eliminated dog rabies in the Americas and much of Europe. However, despite the goal of eliminating dog rabies in the European Union by 2020, sporadic cases of dog rabies still occur in Eastern Europe, including Georgia. To assess the genetic diversity of the strains recently circulating in Georgia, we sequenced seventy-eight RABV-positive samples from the brain tissues of rabid dogs and jackals using Illumina short-read sequencing of total RNA shotgun libraries. Seventy-seven RABV genomes were successfully assembled and annotated, with seventy-four of them reaching the coding-complete status. Phylogenetic analyses of the nucleoprotein (N) and attachment glycoprotein (G) genes placed all the assembled genomes into the Cosmopolitan clade, consistent with the Georgian origin of the samples. An amino acid alignment of the G glycoprotein ectodomain identified twelve different sequences for this domain among the samples. Only one of the ectodomain groups contained a residue change in an antigenic site, an R264H change in the G5 antigenic site. Three isolates were cultured, and these were found to be efficiently neutralized by the human monoclonal antibody A6. Overall, our data show that recently circulating RABV isolates from Georgian canines are predominantly closely related phylogroup I viruses of the Cosmopolitan clade. Current human rabies vaccines should offer protection against infection by Georgian canine RABVs. The genomes have been deposited in GenBank (accessions: OQ603609-OQ603685).

Nipah virus attachment glycoprotein ectodomain delivered by type 5 adenovirus vector elicits broad immune response against NiV and HeV.

Nipah virus (NiV) and Hendra virus (HeV) are newly emerging dangerous zoonotic pathogens of the Henipavirus genus of the Paramyxoviridae family. NiV and HeV (HNVs) which are transmitted by bats cause acute respiratory disease and fatal encephalitis in humans. To date, as there is a lack of antiviral drugs or effective antiviral therapies, the development of vaccines against those two viruses is of primary importance, and the immunogen design is crucial to the success of vaccines. In this study, the full-length protein (G), the ectodomain (Ge) and the head domain (Gs) of NiV attachment glycoprotein were delivered by the replication-defective type 5 adenovirus vector (Ad5) respectively, and the recombinant Ad5-NiV vaccine candidates (Ad5-NiVG, Ad5-NiVGe and Ad5-NiVGs) were constructed and their immunogenicity were evaluated in mice. The results showed that all the vaccine candidates stimulated specific humoral and cellular immune responses efficiently and rapidly against both NiV and HeV, and the Ad5-NiVGe elicited the strongest immune responses after a single-dose immunization. Furthermore, the potent conserved T-cell epitope DTLYFPAVGFL shared by NiV and HeV was identified in the study, which may provide valid information on the mechanism of HNVs-specific cellular immunity. In summary, this study demonstrates that the Ad5-NiVGe could be a potent vaccine candidate against HNVs by inducing robust humoral and cellular immune responses.

Ebola virus shed glycoprotein is toxic to human T, B, and natural killer lymphocytes

Lymphocyte depletion is a distinctive feature of Ebola virus (EBOV) disease. The ectodomain of EBOV glycoprotein (GP) is cleaved off the surface of infected cells into circulation as shed GP. To test the hypothesis that shed GP induces lymphocyte death, we cultured primary human B, NK, or Tcells with shed GP invitro. We found that shed GP dependably decreased B, NK, and Tcell viability across donors. B and NK cells exhibited higher susceptibility than Tcells. Continuous monitoring revealed shed GP began to kill B and NK cells by 4h and Tcells by 5 h. We also demonstrated that shed GP-induced lymphocyte death can be both caspase dependent and caspase independent. Our data are evidence that the cytotoxic effect of shed GP on lymphocytes may contribute to EBOV disease and highlight the need for further research to clarify mechanisms of shed GP-induced death.

Immunogenicity and Antigenicity of the Ectodomain of Rabies Virus Glycoprotein Stably Expressed in HEK293T Cells.

Rabies continues to be a huge threat to public health. The rabies virus envelope glycoprotein (RABV G) is a major rabies virus antigen and contains neutralizing epitopes, which are primary candidates for subunit vaccines and diagnostic antigens. However, the production and purification of rRABV G while retaining its antigenic and immunogenic remains to be a challenge. Here, we aimed to establish a platform for rRABV G production and purification, and determine the immunogenicity and antigenicity of rRABV G. The cDNA fragment encoding the soluble form of RABV G was synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-RABV G-eGFP was packaged, titered, and then transduced into HEK 293T cells. The cell culture supernatant was purified using nickel affinity chromatography and subsequently confirmed through Western Blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). The ELISA utilized human sera obtained from individuals who had been vaccinated with the human commercial Purified Vero Cells Rabies Vaccine (PVRV). Notably, we observed a neutralizing antibody response in immunized pigs rather than in mice. This discrepancy could potentially be attributed to factors such as the instability of the rRABV G protein, variations in host responses, and variances in the adjuvant used. Taking all these findings into account, the rRABV G protein generated in this study exhibits promise as a potential vaccine candidate for the prevention of rabies.

Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner.

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance.

During epidemics, such as the frequent and devastating Ebola virus outbreaks that have historically plagued regions of Africa, serological surveillance efforts are critical for viral containment and the development of effective antiviral therapeutics. Antibody serology can also be used retrospectively for population-level surveillance to provide a more complete estimate of total infections. Ebola surveillance efforts rely on enzyme-linked immunosorbent assays (ELISAs), which restrict testing to laboratories and are not adaptable for use in resource-limited settings. In this manuscript, we describe a paper-based immunoassay capable of detecting anti-Ebola IgG using Ebola virus envelope glycoprotein ectodomain (GP) as the affinity reagent. We evaluated seven monoclonal antibodies (mAbs) against GP—KZ52, 13C6, 4G7, 2G4, c6D8, 13F6, and 4F3—to elucidate the impact of binding affinity and binding epitope on assay performance and, ultimately, result interpretation. We used biolayer interferometry to characterize the binding of each antibody to GP before assessing their performance in our paper-based device. Binding affinity (KD) and on rate (kon) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best KD (3–25 nM) exhibited the lowest limits of detection (ca. μg mL−1), while mAbs with KD > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to kon. These results highlight the importance of ensuring that the quality of recognition reagents is sufficient to support desired assay performance and suggest that the strength of an individual’s immune response can impact the interpretation of assay results.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03317-4.

A single dose investigational subunit vaccine for human use against Nipah virus and Hendra virus

Nipah and Hendra viruses are highly pathogenic bat-borne paramyxoviruses recently included in the WHO Blueprint priority diseases list. A fully registered horse anti-Hendra virus subunit vaccine has been in use in Australia since 2012. Based on the same immunogen, the Hendra virus attachment glycoprotein ectodomain, a subunit vaccine formulation for use in people is now in a Phase I clinical trial. We report that a single dose vaccination regimen of this human vaccine formulation protects against otherwise lethal challenges of either Hendra or Nipah virus in a nonhuman primate model. The protection against the Nipah Bangladesh strain begins as soon as 7 days post immunization with low dose of 0.1 mg protein subunit. Our data suggest this human vaccine could be utilized as efficient emergency vaccine to disrupt potential spreading of Nipah disease in an outbreak setting.

Rational design and expression of a recombinant plant rhabdovirus glycoprotein for production of immunoreactive murine anti-sera

Lettuce necrotic yellows virus (LNYV) is a plant rhabdovirus which has a type-1 transmembrane glycoprotein. Here, we describe the generation of murine anti-sera to the glycoprotein. Rational design, expression, and purification of recombinant glycoprotein, termed rLGe, was undertaken using SignalP4.1 and camSol servers to predict signal peptide cleavage and protein solubility. In order to successfully obtain expression in mammalian cells, LNYV glycoprotein native signal peptide was substituted with that of Rabies virus glycoprotein. In addition, rather than expression of the entire molecule, rLGe consisted of the LNYV glycoprotein ectodomain fused to two affinity tags to minimize the risk of protein aggregation, while facilitating detection and purification. rLGe was transiently expressed in mammalian cell culture, purified using affinity column chromatography, and used to immunize mice. Harvested anti-sera were immunoreactive and specific to the naturally occurring glycoprotein as confirmed by western blotting of plant leaf tissue infected with LNYV.

Optimization of Parameters for Expression and Purification of G Glycoprotein Ectodomain of Respiratory Syncytial Virus

Aim: G glycoprotein ectodomain (Ge) of BA genotype of group B respiratory syncytial virus was expressed and purified to achieve maximum yield of the protein. Materials &amp; methods: We optimized different parameters like strains, temperature, inducer concentration and post induction time period for efficient protein expression in Escherichia coli. The protein was purified using affinity chromatography and confirmed by western blotting. Results: It was concluded that a 5-h induction with 0.75 mM isopropyl β-D-1-thiogalactopyranoside at 37°C in BL21(DE3) cells was the most favorable condition for maximal protein expression. The far-UV circular dichroism spectroscopy suggested that it is an α-helical protein. Conclusion: The purified Ge protein can be characterized by antigenic and biophysical methods in future studies, which will probably assist in vaccine development.

Structure-Based Design of Prefusion-Stabilized Filovirus Glycoprotein Trimers

Ebola virus causes severe hemorrhagic fever, often leading to death in humans. The trimeric fusion glycoprotein (GP) is the sole target for neutralizing antibodies and is the major focus of vaccine development. Soluble GP ectodomains are unstable and mostly monomeric when not fused to a heterologous trimerization domain. Here, we report structure-based designs of Ebola and Marburg GP trimers based on a stabilizing mutation in the hinge loop in refolding region 1 and substitution of a partially buried charge at the interface of the GP1 and GP2 subunits. The combined substitutions (T577P and K588F) substantially increased trimer expression for Ebola GP proteins. We determined the crystal structure of stabilized GP from the Makona Zaire ebolavirus strain without a trimerization domain or complexed ligand. The structure reveals that the stabilized GP adopts the same trimeric prefusion conformation, provides insight into triggering of GP conformational changes, and should inform future filovirus vaccine development.

Expression, Purification, and Crystallization of HSV-1 Glycoproteins for Structure Determination.

Herpes simplex viruses utilize glycoproteins displayed on the viral envelope to perform a variety of functions in the viral infectious cycle. Structural and functional studies of these viral glycoproteinscan benefit frombiochemical, biophysical, and structural analysisof purified proteins. Here, we describe a general protocol for expression and purification ofviral glycoproteins from insect cells based on those developed for the HSV-1 gB and HSV-2 gH/gL ectodomains as well as the protocol for crystallization of these glycoproteins. This protocol can be used for generating milligram amounts of wild-type (WT) or mutant gB and gH/gL ectodomains or can be adapted to produce purified ectodomains of glycoproteins from HSV or other herpesviruses for biochemical and structural studies.

Highly efficient production of rabies virus glycoprotein G ectodomain in Sf9 insect cells.

In the present study, we developed a complete process to produce in insect cells a high amount of the ectodomain of rabies virus glycoprotein G (GE) as suitable antigen for detecting anti-rabies antibodies. Using the baculovirus expression vector system in Sf9 insect cells combined with a novel chimeric promoter (polh-pSeL), the expression level reached a yield of 4.1 ± 0.3mg/L culture, which was significantly higher than that achieved with the standard polh promoter alone. The protein was recovered from the cell lysates and easily purified in only one step by metal ion affinity chromatography, with a yield of 95% and a purity of 87%. Finally, GE was successfully used in an assay to detect specific antibodies in serum samples derived from rabies-vaccinated animals. The efficient strategy developed in this work is an interesting method to produce high amounts of this glycoprotein.

A DNA Vaccine Encoding the Gn Ectodomain of Rift Valley Fever Virus Protects Mice via a Humoral Response Decreased by DEC205 Targeting.

The Rift Valley fever virus (RVFV) is responsible for a serious mosquito-borne viral disease in humans and ruminants. The development of a new and safer vaccine is urgently needed due to the risk of introduction of this arbovirus into RVFV-free continents. We recently showed that a DNA vaccine encoding eGn, the ectodomain of the RVFV Gn glycoprotein, conferred a substantial protection in the sheep natural host and that the anti-eGn IgG levels correlated to protection. Addressing eGn to DEC205 reduced the protective efficacy while decreasing the antibody and increasing the IFNγ T cell responses in sheep. In order to get further insight into the involved mechanisms, we evaluated our eGn-encoding DNA vaccine strategy in the reference mouse species. A DNA vaccine encoding eGn induced full clinical protection in mice and the passive transfer of immune serum was protective. This further supports that antibodies, although non-neutralizing in vitro, are instrumental in the protection against RVFV. Addressing eGn to DEC205 was also detrimental to protection in mice, and in this species, both the antibody and the IFNγ T cell responses were strongly decreased. Conversely when using a plasmid encoding a different antigen, i.e., mCherry, DEC205 targeting promoted the antibody response. Altogether our results show that the outcome of targeting antigens to DEC205 depends on the species and on the fused antigen and is not favorable in the case of eGn. In addition, we bring evidences that eGn in itself is a pertinent antigen to be included in a DNA vaccine and that next developments should aim at promoting the anti-eGn antibody response.

Novel avian paramyxovirus-based vaccine vectors expressing the Ebola virus glycoprotein elicit mucosal and humoral immune responses in guinea pigs

Paramyxovirus vaccine vectors based on human parainfluenza virus type 3 (HPIV-3) and Newcastle disease virus (NDV) have been previously evaluated against Ebola virus (EBOV) challenge. Although both the viral vectored vaccines efficiently induce protective immunity, some concerns remain to be solved. Since HPIV-3 is a common human pathogen, the human population has pre-existing immunity to HPIV-3, which may restrict the replication of the vaccine vector. For NDV, mesogenic (intermediate virulent) strain used in previous studies is currently classified as a Select Agent in the United States, thus making it unsuitable to be used as a vaccine vector. To overcome these concerns, we have developed a modified NDV vector based on a mesogenic NDV strain, in which the ectodomains of envelope glycoproteins were replaced with the corresponding ectodomains from avian paramyxovirus serotype 3 (APMV-3). The modified NDV vector was highly attenuated in chickens and was able to express the EBOV glycoprotein (GP) gene at high level. In addition, the recombinant APMV-3 was also evaluated as a vaccine vector to express the EBOV GP gene. Guinea pigs immunized with these two vector vaccines developed high levels of neutralizing GP-specific IgG and IgA antibodies.

Structure of the zebrafish galectin-1-L2 and model of its interaction with the infectious hematopoietic necrosis virus (IHNV) envelope glycoprotein.

Galectins, highly conserved β-galactoside-binding lectins, have diverse regulatory roles in development and immune homeostasis and can mediate protective functions during microbial infection. In recent years, the role of galectins in viral infection has generated considerable interest. Studies on highly pathogenic viruses have provided invaluable insight into the participation of galectins in various stages of viral infection, including attachment and entry. Detailed mechanistic and structural aspects of these processes remain undetermined. To address some of these gaps in knowledge, we used Zebrafish as a model system to examine the role of galectins in infection by infectious hematopoietic necrosis virus (IHNV), a rhabdovirus that is responsible for significant losses in both farmed and wild salmonid fish. Like other rhabdoviruses, IHNV is characterized by an envelope consisting of trimers of a glycoprotein that display multiple N-linked oligosaccharides and play an integral role in viral infection by mediating the virus attachment and fusion. Zebrafish's proto-typical galectin Drgal1-L2 and the chimeric-type galectin Drgal3-L1 interact directly with the glycosylated envelope of IHNV, and significantly reduce viral attachment. In this study, we report the structure of the complex of Drgal1-L2 with N-acetyl-d-lactosamine at 2.0 Å resolution. To gain structural insight into the inhibitory effect of these galectins on IHNV attachment to the zebrafish epithelial cells, we modeled Drgal3-L1 based on human galectin-3, as well as, the ectodomain of the IHNV glycoprotein. These models suggest mechanisms for which the binding of these galectins to the IHNV glycoprotein hinders with different potencies the viral attachment required for infection.

Structure and flexibility of the extracellular region of the PirB receptor

Murine paired immunoglobulin receptor B (PirB) and its human ortholog leukocyte immunoglobulin-like receptor B2 (LILRB2) are widely expressed inhibitory receptors that interact with a diverse set of extracellular ligands and exert functions ranging from down-regulation of immune responses to inhibition of neuronal growth. However, structural information that could shed light on how PirB interacts with its ligands is lacking. Here, we report crystal structures of the PirB ectodomain; the first full ectodomain structure for a LILR family member, at 3.3-4.5 Å resolution. The structures reveal that PirB's six Ig-like domains are arranged at acute angles, similar to the structures of leukocyte immunoglobulin-like receptor (LILR) and killer-cell immunoglobulin-like receptor (KIR). We observe that this regular arrangement is followed throughout the ectodomain, resulting in an extended zigzag conformation. In two out of the five structures reported here, the repeating zigzag is broken by the first domain that can adopt two alternative orientations. Quantitative binding experiments revealed a 9 μm dissociation constant for PirB-myelin-associated glycoprotein (MAG) ectodomain interactions. Taken together, these structural findings and the observed PirB-MAG interactions are compatible with a model for intercellular signaling in which the PirB extracellular domains, which point away from the cell surface, enable interaction with ligands in trans.

Evaluation of the antiviral potential of the soluble forms of glycoprotein D receptors on ocular herpes caused by HSV-1 and HSV-2 infections in a transgenic mouse model.

Ocular herpes, caused by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections, remains an important corneal disease, which may result in loss of vision. Because the frequency of acyclovir resistance in HSV has increased, novel antiviral agents are needed for therapeutic approaches to ocular herpes. Several studies have demonstrated that fusion proteins containing entire ectodomain of HSV glycoprotein D receptors, including herpesvirus entry mediator A (HVEM), nectin-1 and nectin-2, and the Fc portion of human IgG (HVEMIg, nectin-1Ig, and nectin-2Ig, respectively), can exert antiviral effects in vitro and in vivo. Here, to evaluate the antiviral potential of HVEMIg, nectin-1Ig, and nectin-2Ig against ocular infections with HSV, transgenic mice expressing these fusion proteins were ocularly inoculated with HSV-1 and HSV-2. Transgenic mouse lines expressing HVEMIg and nectin-1Ig showed marked resistance to ocular herpes; on the other hand, mouse lines expressing nectin-2Ig did not. Furthermore, to investigate the therapeutic effects of nectin-1Ig, which can neutralize HSVs in vitro against ocular disease, transgenic mouse serum containing nectin-1Ig was dropped into the eyes of wild-type mice after HSV infection. Reduction of severe symptoms could be observed in mice treated with nectin-1Ig serum. These results warrant further study of soluble HVEM and nectin-1 products as preventive and therapeutic agents against ocular herpes caused by HSV-1 and HSV-2 infections, especially nectin-1Ig as a new eye drop.

Expression of the ectodomain of the rabies virus glycoprotein G in insect cells for diagnostic purposes in vaccinated llamas

This narrative review summarizes the recent literature on deep brain stimulation for treatment resistant obsessive-compulsive disorder highlighting both progress and challenges of this novel treatment. Common targets of psychiatric deep brain stimulation involve both white matter trajectories (anterior limb of the internal capsule, inferior thalamic peduncle, and medial forebrain bundle) and grey matter subcortical nuclei (nucleus accumbens, nucleus subthalamicus, and bed nucleus of the stria terminalis) each of which have been reported with a relevant beneficial effect on obsessive-compulsive symptoms. The mechanisms of action are only partially understood but increasing evidence points towards network effects involving the prefrontal cortex, the striatum and possibly anxiety-related anatomical structures. Deep brain stimulation is a promising therapeutical technique for otherwise treatment refractory patients, but many major issues are unresolved and thorough investigations are needed. Relevant topics for future investigations include treatment predictors and therapeutical augmentation. An international registry of patients treated with deep brain stimulation could improve our understanding of adverse events and target specific effects. In order to step forward, researchers must face inconvenient questions and outperform the status quo of clinical research in this field.