Immunoinformatics based designing a multi-epitope vaccine against pathogenic Chandipura vesiculovirus.

Chandipura vesiculovirus (CHPV) is a rapidly emerging pathogen responsible for causing acute encephalitis. Due to its widespread occurrence in Asian and African countries, this has become a global threat, and there is an urgent need to design an effective and nonallergenic vaccine against this pathogen. The present study aimed to develop a multi-epitope vaccine using an immunoinformatics approach. The conventional method of vaccine design involves large proteins or whole organism which leads to unnecessary antigenic load with increased chances of allergenic reactions. In addition, the process is also very time-consuming and labor-intensive. These limitations can be overcome by peptide-based vaccines comprising short immunogenic peptide fragments that can elicit highly targeted immune responses, avoiding the chances of allergenic reactions, in a relatively shorter time span. The multi-epitope vaccine constructed using CTL, HTL, and IFN-γ epitopes was able to elicit specific immune responses when exposed to the pathogen, in silico. Not only that, molecular docking and molecular dynamics simulation studies confirmed a stable interaction of the vaccine with the immune receptors. Several physicochemical analyses of the designed vaccine candidate confirmed it to be highly immunogenic and nonallergic. The computer-aided analysis performed in this study suggests that the designed multi-epitope vaccine can elicit specific immune responses and can be a potential candidate against CHPV.

Effects of Gamma Irradiation of Human Serum Samples from rVSVΔG-ZEBOV-GP (V920) Ebola Virus Vaccine Recipients on Plaque-Reduction Neutralization Assays.

Gamma irradiation (GI) is included in the CDC guidance on inactivation procedures to render a group of select agents and toxins nonviable. The Ebola virus falls within this group because it potentially poses a severe threat to public health and safety. To evaluate the impact of GI at a target dose of 50 kGy on neutralizing antibody titers induced by the rVSVΔG-ZEBOV-GP vaccine (V920), we constructed a panel of 48 paired human serum samples (GI-treated versus non-GI-treated) from healthy participants selected from a phase 3 study of V920 (study V920-012; NCT02503202). Neutralizing antibody titers were determined using a validated plaque-reduction neutralization test. GI of sera from V920 recipients was associated with approximately 20% reduction in postvaccination neutralizing antibody titers. GI was not associated with any change in pre-vaccination neutralizing antibody titers.

Niosomal virosome derived by vesicular stomatitis virus glycoprotein as a new gene carrier.

Virosomes as membranous vesicles with viral fusion protein in their membrane are versatile vehicles for cargo delivery. The vesicular stomatitis virus glycoprotein (VSV-G) is a common fusogenic protein used in virosome preparation. This glycoprotein has been used in liposomal systems so far, but in this study, we have tried to use the niosomal form instead of liposome for. Niosomes are vesicular systems composed of non-ionic surfactants. Niosomes were constructed by the thin-film hydration method. VSV-G gene in pMD2.G plasmid was expressed in the HEK293T cell line and then was reconstituted in the niosome bilayer. The formation of niosomal virosomes was confirmed with different methods such as SDS-PAGE gel, western blotting, and transmission electron microscopy (TEM). The efficiency of niosomal virosome was investigated with the pmCherry reporter gene. SDS-PAGE and western blotting proved the expression and successful insertion of protein into the bilayer. The TEM images showed the spike projection of VSV-G on the surface of niosomes. The transfection results showed high efficiency of niosomal virosomes as a novel carrier. This report has verified that niosome could be used as an efficient bilayer instead of liposome to construct virosomes.

Spray dried VSV-vectored vaccine is thermally stable and immunologically active in vivo.

Effective vaccine delivery and coverage to rural and resource-poor countries is hindered by the dependence on cold chain storage. As such, developments of cold chain-free technologies are highly sought. Although spray dried adenoviral vectors have shown long term stability at ambient temperatures and relatively low humidity, it remains to be determined whether similar excipient formulations are applicable to other viral vectors. To address this, we have spray dried vesicular stomatitis virus (VSV)-vectors with a panel of well-characterized sugar excipients to determine the optimal formulation for vector stabilization. Upon reconstitution, we show that trehalose conferred superior stability of VSV both in vitro and in vivo. Importantly, following cold chain-free storage at elevated temperatures at 37 °C for 15 days, we show that a VSV-vectored vaccine retains its in vivo immunogenicity, whereas a liquid control completely lost its immune-stimulating ability. Our results provide foundational evidence that spray drying with properly tested excipients can stabilize viral vectors such as VSV, allowing them to be stored long-term at elevated temperatures without dependency on cold chain conditions.

The Connector Domain of Vesicular Stomatitis Virus Large Protein Interacts with the Viral Phosphoprotein.

Vesicular stomatitis virus (VSV) is an archetypical member of Mononegavirales, viruses with a genome of negative-sense single-stranded RNA (-ssRNA). Like other viruses of this order, VSV encodes a unique polymerase, a complex of viral L (large, the enzymatic component) protein and P (phosphoprotein, a cofactor component). The L protein has a modular layout consisting of a ring-shaped core trailed by three accessory domains and requires an N-terminal segment of P (P N-terminal disordered [PNTD]) to perform polymerase activity. To date, a binding site for P on L had not been described. In this report, we show that the connector domain of the L protein, which previously had no assigned function, binds a component of PNTD We further show that this interaction is a positive regulator of viral RNA synthesis, and that the interfaces mediating it are conserved in other members of Mononegavirales Finally, we show that the connector-P interaction fits well into the existing structural information of VSV L.IMPORTANCE This study represents the first functional assignment of the connector domain of a Mononegavirales L protein. Furthermore, this study localizes P polymerase cofactor activity to specific amino acids. The functional necessity of this interaction, combined with the uniqueness of L and P proteins to the order Mononegavirales, makes disruption of the P-connector site a potential target for developing antivirals against other negative-strand RNA viruses. Furthermore, the connector domain as an acceptor site for the P protein represents a new understanding of Mononegavirales L protein biology.

The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion.

The L-protein of vesicular stomatitis virus (VSV) is a single-chain multi-domain RNA-dependent RNA polymerase. Previously reported attempts of intramolecular insertions of fluorescent proteins into the L-protein resulted in temperature-sensitive and highly attenuated polymerase activity. Here, we describe a novel insertion site that was selected based on in silico prediction. Of five preselected locations, insertion of the fluorescent protein mCherry in the VSV polymerase between amino acids 1620 and 1621 preserved polymerase function even after extended passaging and showed only mild attenuation compared to wildtype VSV polymerase. High magnification fluorescence imaging revealed a corpuscular cytosolic pattern for the L-protein. To confirm that the insertion site tolerates inclusion of proteins others than mCherry, we cloned mWasabi into the same position in L, generating a VSV-LmWasabi, which was also functional. We also generated a functional dual-color-dual-insertion VSV construct with intramolecularly labeled P and L-proteins. Together, our data present an approach to tag VSV polymerase intramolecularly without perturbing enzymatic activity. This L fusion protein might enable future tracing studies to monitor intracellular location of the VSV transcription and replication machinery in real-time life-imaging studies.

The stress granule protein G3BP1 binds viral dsRNA and RIG-I to enhance interferon-ß response.

RIG-I senses viral RNA in the cytosol and initiates host innate immune response by triggering the production of type 1 interferon. A recent RNAi knockdown screen yielded close to hundred host genes whose products affected viral RNA-induced IFN-ß production and highlighted the complexity of the antiviral response. The stress granule protein G3BP1, known to arrest mRNA translation, was identified as a regulator of RIG-I-induced IFN-ß production. How G3BP1 functions in RIG-I signaling is not known, however. Here, we overexpress G3BP1 with RIG-I in HEK293T cells and found that G3BP1 significantly enhances RIG-I-induced ifn-b mRNA synthesis. More importantly, we demonstrate that G3BP1 binds RIG-I and that this interaction involves the C-terminal RGG domain of G3BP1. Confocal microscopy studies also show G3BP1 co-localization with RIG-I and with infecting vesicular stomatitis virus in Cos-7 cells. Interestingly, immunoprecipitation studies using biotin-labeled viral dsRNA or poly(I·C) and cell lysate-derived or in vitro translated G3BP1 indicated that G3BP1 could directly bind these substrates and again via its RGG domain. Computational modeling further revealed a juxtaposed interaction between G3BP1 RGG and RIG-I RNA-binding domains. Together, our data reveal G3BP1 as a critical component of RIG-I signaling and possibly acting as a co-sensor to promote RIG-I recognition of pathogenic RNA.

Identification of a conserved neutralizing epitope in the G-protein of Chandipura virus.

Chandipura virus (CHPV), associated with an encephalitic illness in humans, has caused multiple outbreaks with high mortality in central and western India in recent years. The present study compares surface glycoprotein (G-protein) from prototype and recent outbreak strains using in silico tools and in vitro experiments. In silico epitope predictions (B-cell and T-helper cell) for the sequences, 3D structure prediction and comparison of the G-proteins of the strains: I653514 (Year 1965), CIN0327 (Year 2003) and 148974 (Year 2014) revealed that the CHPV G-protein is stable and antigenic determinants are conserved. A monoclonal antibody developed against strain CIN0327 (named NAbC) was found to neutralize prototype I653514 as well as the currently circulating strain 148974. In silico antigen-antibody interaction studies using molecular docking of predicted structures of NAbC and G-proteins of various CHPV strains led to the identification of a conserved neutralizing epitope in the fusion domain of G-protein, which also contained a putative T-helper peptide. The identification of a conserved neutralizing epitope in domain IV (fusion domain amino acids 53 to 172) of CHPV G-protein is an important finding that may have the scope towards the development of protective targets against CHPV infection.

Constitutively Active MDA5 Proteins Are Inhibited by Paramyxovirus V Proteins.

Excessive interferon (IFN) production and signaling can lead to immunological and developmental defects giving rise to autoimmune diseases referred to collectively as "type I interferonopathies." A subset of these diseases is caused by monogenic mutations affecting proteins involved in nucleic acid sensing, homeostasis, and metabolism. Interferonopathic mutations in the cytosolic antiviral sensor MDA5 render it constitutively hyperactive, resulting in chronic IFN production and IFN-stimulated gene expression. Few therapeutic options are available for patients with interferonopathic diseases, but a large number of IFN evasion and antagonism strategies have evolved in viral pathogens that can counteract IFN production and signaling to enhance virus replication. To test the hypothesis that these natural IFN suppressors could be used to subdue the activity of interferonopathic signaling proteins, hyperactive MDA5 variants were assessed for susceptibility to a family of viral MDA5 inhibitors. In this study, Paramyxovirus V proteins were tested for their ability to counteract constitutively active MDA5 proteins. Results indicate that the V proteins are able to bind to and disrupt the signaling activity of these MDA5 proteins, irrespective of their specific mutations, reducing IFN production and IFN-stimulated gene expression to effectively suppress the hyperactive antiviral response.

Reversible Fusion Proteins as a Tool to Enhance Uptake of Virus-Functionalized LbL Microcarriers.

For the efficient treatment of an increasing number of diseases the development of new therapeutics as well as novel drug delivery systems is essential. Such drug delivery systems (DDS) must not only consider biodegradability and protective packaging but must also target and control the release of active substances, which is one of the most important points in DDS application. We highlight the improvement of these key aspects, the increased interaction rate of Layer-by-Layer (LbL) designed microcarriers as a promising DDS after functionalization with vesicular stomatitis virus (VSV). We make use of the unique conformational reversibility of the fusion protein of VSV as a surface functionalization of LbL microcarriers. This reversibility allows for VSV to be used both as a tool for assembly onto the DDS and as an initiator for an efficient cellular uptake. We could show that the evolutionary optimized viral fusion machinery can be successfully combined with a biophysical DDS for optimization of its cellular interaction.

Pseudotyping of lentiviral vector with novel vesiculovirus envelope glycoproteins derived from Chandipura and Piry viruses.

While the envelope glycoprotein of vesicular stomatitis virus (VSV-G) is widely used for pseudotyping of lentiviral vectors, sub-optimal gene transfer into certain cell types and its sensitivity to inactivation by human complement hinders its broader applications. To find alternative candidates, here we evaluated two serologically distinct novel viral envelopes derived from Chandipura (CNV-G) and Piry (PRV-G) vesiculoviruses. Both permitted generation of high titer psuedotyped lentiviral vectors with a capacity for high efficiency gene transfer into various cell types from different species. In human lymphoid and hematopoietic stem cells, their transduction efficiency was significantly lower than that of VSV-G. However, both novel envelopes were found to be more resistant to inactivation by human serum complement compared to VSV-G. Thus CNV-G and PRV-G envelopes can be harnessed for multiple uses in the future based on the cell type that needs to be gene transduced and possibly for in vivo gene transfer.

Structure and Function of the N-Terminal Domain of the Vesicular Stomatitis Virus RNA Polymerase.

UNLABELLED: Viruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of the Rhabdoviridae, Paramyxoviridae, and Filoviridae share sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses. IMPORTANCE: Negative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the design of new therapeutics against negative-strand RNA viruses.

Structure of the low pH conformation of Chandipura virus G reveals important features in the evolution of the vesiculovirus glycoprotein.

Chandipura virus (CHAV), a member of the vesiculovirus genus, is an emerging human pathogen. As for other rhabdoviruses, CHAV entry into susceptible cells is mediated by its single envelope glycoprotein G which is both involved in receptor recognition and fusion of viral and cellular membranes. Here, we have characterized the fusion properties of CHAV-G. As for vesicular stomatitis virus (VSV, the prototype of the genus) G, fusion is triggered at low pH below 6.5. We have also analyzed the biochemical properties of a soluble form of CHAV-G ectodomain (CHAV-Gth, generated by thermolysin limited-proteolysis of recombinant VSV particles in which the G gene was replaced by that of CHAV). The overall behavior of CHAV-Gth is similar to that previously reported for VSV-Gth. Particularly, CHAV-Gth pre-fusion trimer is not stable in solution and low-pH-induced membrane association of CHAV-Gth is reversible. Furthermore, CHAV-Gth was crystallized in its low pH post-fusion conformation and its structure was determined at 3.6Å resolution. An overall comparison of this structure with the previously reported VSV-Gth post-fusion conformation, shows a high structural similarity as expected from the comparison of primary structure. Among the three domains of G, the pleckstrin homology domain (PHD) appears to be the most divergent and the largest differences are confined to the secondary structure of the major antigenic site of rhabdoviruses. Finally, local differences indicate that CHAV has evolved alternate structural solutions in hinge regions between PH and fusion domains but also distinct pH sensitive switches. Globally the comparison between the post fusion conformation of CHAV and VSV-G highlights several features essential for the protein's function. It also reveals the remarkable plasticity of G in terms of local structures.

Vesiculoviral matrix (M) protein occupies nucleic acid binding site at nucleoporin pair (Rae1 • Nup98).

mRNA export factor 1 (Rae1) and nucleoporin 98 (Nup98) are host cell targets for the matrix (M) protein of vesicular stomatitis virus (VSV). How Rae1 functions in mRNA export and how M protein targets both Rae1 and Nup98 are not understood at the molecular level. To obtain structural insights, we assembled a 1:1:1 complex of M•Rae1•Nup98 and established a crystal structure at 3.15-Å resolution. We found that the M protein contacts the Rae1•Nup98 heterodimer principally by two protrusions projecting from the globular domain of M like a finger and thumb. Both projections clamp to the side of the ß-propeller of Rae1, with the finger also contacting Nup98. The most prominent feature of the finger is highly conserved Methionine 51 (Met51) with upstream and downstream acidic residues. The complementary surface on Rae1 displays a deep hydrophobic pocket, into which Met51 fastens like a bolt, and a groove of basic residues on either side, which bond to the acidic residues of the finger. Notably, the M protein competed for in vitro binding of various oligonucleotides to Rae1•Nup98. We localized this competing activity of M to its finger using a synthetic peptide. Collectively, our data suggest that Rae1 serves as a binding protein for the phosphate backbone of any nucleic acid and that the finger of M mimics this ligand. In the context of mRNA export, we propose that a given mRNA segment, after having been deproteinated by helicase, is transiently reproteinated by Nup98-tethered Rae1. We suggest that such repetitive cycles provide cytoplasmic stopover sites required for ratcheting mRNA across the nuclear pore.

Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

Characterization of the chandipura virus leader RNA-phosphoprotein interaction using single tryptophan mutants and its detection in viral infected cells.

The phosphoprotein (P protein) of the chandipura virus (CHPV), a negative strand RNA virus, is involved in both transcription and replication of the viral life cycle. Interaction between the P protein and the viral leader (le) RNA under in vitro conditions has been previously reported for CHPV and other negative strand RNA viruses such as the rinderpest virus (RPV). However, till date, the region of the P protein involved in le RNA binding remains undefined. Moreover, the in vivo occurrence of this interaction has not been studied before. Here, we have characterised the P protein-le RNA interaction, using single tryptophan mutants of the P protein. The CHPV P protein contains two tryptophan residues located at amino acid position 105 and 135 respectively. Our previous study showed that Trp 135 is located in a buried region within a less polar environment whereas Trp 105 is more solvent-exposed. In this study we have used steady state and time resolved fluorescence spectroscopy at 298 K to show that the buried tryptophan (Trp 135) is involved in the interaction with the le RNA and the more solvent exposed Trp 105 is only slightly perturbed during this interaction. We also show that Trp 135 is responsible for the dimerization of the CHPV P protein. In addition, we have been able to demonstrate for the first time that the P protein-le RNA interaction is detectable in CHPV-infected Vero-76 cells and this interaction is augmented during the replication phase of the viral cycle.

Modulation of lentiviral vector tropism in cerebellar Purkinje cells in vivo by a lysosomal cysteine protease cathepsin K.

We previously reported that vesicular stomatitis virus-derived glycoprotein (VSV-G)-pseudotyped lentiviral vectors harvested 2 days post-transfection preferred to infect Purkinje cells (PCs), whereas those harvested after a longer cultivation period exhibited Bergmann glia-preferential transduction. However, the mechanisms by which lentiviral tropism was altered remained unsolved. Here, we investigated whether proteases released from the cells during viral production affect lentiviral tropism. Enhanced green fluorescence protein-expressing lentiviral vectors were produced using human embryonic kidney (HEK) 293FT or 293 T cells and injected into the mouse cerebellum to examine tropism in PCs. We found that the addition of a protease inhibitor-in particular, the cathepsin K (CatK) inhibitor-into the culture medium significantly increased lentiviral tropism in PCs. Moreover, the concentration of CatK in the culture medium drastically increased upon prolonged cultivation, concomitant with the expression levels of CatK in HEK 293 T cells. An increase in CatK activity by the addition of recombinant CatK enzyme to PC-preferential viral solution, which was obtained 2 days post-transfection, shifted the viral tropism toward Bergmann glia. In contrast, a decrease in CatK activity in the Bergmann glia-preferential viral solution, which was obtained 6 days post-transfection by the addition of CatK inhibitor or by the removal of a CatK-containing fraction, restored the PC preference of viruses. These results suggest that the CatK released from deteriorated HEK 293 T cells plays a key role in reducing lentiviral tropism in PCs, presumably by affecting a receptor molecule for lentiviral VSV-G, resulting in the preferential transduction of Bergmann glia.

Crystallization and preliminary X-ray analysis of Chandipura virus glycoprotein G.

Fusion in members of the Rhabdoviridae virus family is mediated by the G glycoprotein. At low pH, the G glycoprotein catalyzes fusion between viral and endosomal membranes by undergoing a major conformational change from a pre-fusion trimer to a post-fusion trimer. The structure of the G glycoprotein from vesicular stomatitis virus (VSV G), the prototype of Vesiculovirus, has recently been solved in its trimeric pre-fusion and post-fusion conformations; however, little is known about the structural details of the transition. In this work, a soluble form of the ectodomain of Chandipura virus G glycoprotein (CHAV G(th)) was purified using limited proteolysis of purified virus; this soluble ectodomain was also crystallized. This protein shares 41% amino-acid identity with VSV G and thus its structure could provide further clues about the structural transition of rhabdoviral glycoproteins induced by low pH. Crystals of CHAV G(th) obtained at pH 7.5 diffracted X-rays to 3.1 Å resolution. These crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 150.3, b = 228.2, c = 78.8 Å. Preliminary analysis of the data based on the space group and the self-rotation function indicated that there was no trimeric association of the protomers. This unusual oligomeric status could result from the presence of fusion intermediates in the crystal.

Anti-Fab aptamers for shielding virus from neutralizing antibodies.

Oncolytic viruses are promising therapeutics that can selectively replicate in and kill tumor cells. However, repetitive administration of viruses provokes the generation of neutralizing antibodies (nAbs) that can diminish their anticancer effect. In this work, we selected DNA aptamers against the antigen binding fragment (Fab) of antivesicular stomatitis virus polyclonal antibodies to shield the virus from nAbs and enhance its in vivo survival. For the first time, we used flow cytometry and electrochemical immunosensing to identify aptamers targeting the Fab region of antibodies. We evaluated the aptamers in a cell-based infection assay and found that several aptamer clones provide more than 50% shielding of VSV from nAbs and thus have the potential to enhance the delivery of VSV without compromising the patient's immune system. In addition, we developed a bifunctional label-free electrochemical immunosensor for the quantitation of aptamer-mediated degree of shielding and the amount of vesicular stomatitis virus (VSV) particles. Electrochemical impedance spectroscopy was employed to interrogate the level of VSV in a linear range from 5 × 10(4) to 5 × 10(6) PFU mL(-1) with a detection limit of 10(4) PFU mL(-1).