Recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates

The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses an individual filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV) GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV) GP; three animals received rVSV-wild type (wt) vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use.     

Homology between the glycoproteins of vesicular stomatitis virus and rabies virus

We compared the predicted amino acid sequences of the vesicular stomatitis virus and rabies virus glycoproteins by using a computer program which provides an optimal alignment and a statistical significance for the match. Highly significant homology between these two proteins was detected, including identical positioning of one glycosylation site. A significant homology between the predicted amino acid sequences of vesicular stomatitis virus and influenza virus matrix proteins was also found.     

Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and Sindbis virus.

Tunicamycin, an antibiotic which prevents the glycosylation of newly synthesized proteins, inhibits the replication of both vesicular stomatitis virus and Sindbis virus. In tunicamycin-treated infected cells, all of the viral proteins are synthesized but the glycoproteins are devoid of carbohydrate. The nonglycosylated glycoproteins could not be detected on the outside of the plasma membrane by lactoperoxidase labeling, indirect immunofluorescence staining, or chymotrypsin treatment of intact cells, whereas the glycosylated glycoproteins were readily detected by all three methods. These results indicate that the bulk of the nonglycosylated glycoproteins are unable to undergo the normal migration to the cell surface. In contrast to the normal glycosylated viral glycoproteins, the nonglycosylated glycoproteins were insoluble in nonionic detergents such as Triton X-100. The nonglycosylated glycoprotein of vesicular stomatitis virus could be solubilized using a combination of 6 M guanidine hydrochloride and 0.2% Triton X-100, but precipitated when the 6 M guanidine was removed by dialysis. These results suggest that the lack of carbohydrate alters the properties of the glycoproteins, which may explain their impaired mobility through the intracellular membranous system.     

Attenuated vesicular stomatitis viruses as vaccine vectors

We showed previously that a single intranasal vaccination of mice with a recombinant vesicular stomatitis virus (VSV) expressing an influenza virus hemagglutinin (HA) protein provided complete protection from lethal challenge with influenza virus (A. Roberts, E. Kretzschmar, A. S. Perkins, J. Forman, R. Price, L. Buonocore, Y. Kawaoka, and J. K. Rose, J. Virol. 72:4704-4711, 1998). Because some pathogenesis was associated with the vector itself, in the present study we generated new VSV vectors expressing HA which are completely attenuated for pathogenesis in the mouse model. The first vector has a truncation of the cytoplasmic domain of the VSV G protein and expresses influenza virus HA (CT1-HA). This nonpathogenic vector provides complete protection from lethal influenza virus challenge after intranasal administration. A second vector with VSV G deleted and expressing HA (DeltaG-HA) is also protective and nonpathogenic and has the advantage of not inducing neutralizing antibodies to the vector itself.     

Pseudotypes of vesicular stomatitis virus with the mixed coat of reticuloendotheliosis virus and vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) forms pseudotypes with envelope components of reticuloendotheliosis virus (REV). The VSV pseudotype possesses the limited host range and antigenic properties of REV. Approximately 70% of the VSV, Indiana serotype, and 45% of VSV, New Jersey serotype, produced from the REV strain T-transformed chicken bone marrow cells contain mixed envelope components of both VSV and REV. VSV pseudotypes with mixed envelope antigens can be neutralized with excess amounts of either anti-VSV antiserum or anti-REV antiserum.     

Role of heterologous and homologous glycoproteins in phenotypic mixing between Sendai virus and vesicular stomatitis virus.

Phenotypic mixing between Sendai virus and vesicular stomatitis virus (VSV) or the mutant VSV ts045 was studied. Conditions were optimized for double infection, as shown by immunofluorescence microscopy. Virions from double-infected cells were separated by sequential velocity and isopycnic gradient centrifugations. Two types of particles with mixed protein compositions were found. One type was VSV particles with Sendai virus spikes, i.e., phenotypically mixed particles. A second type was Sendai virus-VSV associations, which in plaque assays also behaved as phenotypically mixed particles. The ratio of VSV G protein to Sendai virus glycoproteins on the cell surface was varied, using the VSV mutant ts045 in double infections. Thus, different amounts of the VSV G protein were allowed to reach the cell surface at 32, 38, and 39 degrees C in Sendai virus-infected cells. However, a fixed number of Sendai virus spikes was always found in the ts045 virions. This represented 12 to 16% of the number of G proteins present in normal VSV. Furthermore, the yield of ts045 virions was radically reduced during double infection when the temperature was raised to block G-protein transport to the cell surface, suggesting that the Sendai virus glycoproteins were not able to compensate for G protein in budding. These results emphasize the role of the G protein in VSV assembly.     

Generation and characterization of a recombinant vesicular stomatitis virus expressing the glycoprotein of Borna disease virus

Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of mononegavirales. However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. BDV cell entry occurs via receptor-mediated endocytosis, a process initiated by the recognition of an as yet unidentified receptor at the cell surface by the BDV surface glycoprotein (G). The paucity of cell-free virus associated with BDV infection has hindered studies aimed at the elucidation of cellular receptors and detailed mechanisms involved in BDV cell entry. To overcome this problem, we generated and characterized a replication-competent recombinant vesicular stomatitis virus expressing BDV G (rVSVDeltaG*/BDVG). Cells infected with rVSVDeltaG*/BDVG produced high titers (10(7) PFU/ml) of cell-free virus progeny, but this virus exhibited a highly attenuated phenotype both in cell culture and in vivo. Attenuation of rVSVDeltaG*/BDVG was associated with a delayed kinetics of viral RNA replication and altered genome/N mRNA ratios compared to results for rVSVDeltaG*/VSVG. Likewise, incorporation of BDV G into virions appeared to be restricted despite its high levels of expression and efficient processing in rVSVDeltaG*/BDVG-infected cells. Notably, rVSVDeltaG*/BDVG recreated the cell tropism and entry pathway of bona fide BDV. Our results indicate that rVSVDeltaG*/BDVG represents a unique tool for the investigation of BDV G-mediated cell entry, as well as the roles of BDV G in host immune responses and pathogenesis associated with BDV infection.     

Characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis C virus glycoproteins

We generated recombinant vesicular stomatitis viruses (VSV) expressing genes encoding hybrid proteins consisting of the extracellular domains of hepatitis C virus (HCV) glycoproteins fused at different positions to the transmembrane and cytoplasmic domains of the VSV G glycoprotein (E1G and E2G). We show that these chimeric proteins are transported to the cell surface and incorporated into VSV virions efficiently. We also generated VSV recombinants in which the gene encoding the VSV G protein was deleted and replaced by one or both of the E1G and E2G genes, together with a green fluorescent protein gene. These DeltaG viruses incorporated E1G and E2G proteins at levels approximately equivalent to the normal level of VSV G itself, or about 1,200 molecules of each protein per virion. Given the potency of VSV recombinants as vaccines in other studies, this high-level expression and incorporation of HCV proteins into virions could be very important for development of an HCV vaccine. Despite the presence of E1G and E2G proteins at high levels in the virions, these virions did not infect cell lines that have been reported to support at least a low level of HCV infection and replication.     

Pseudotyping vesicular stomatitis virus with lymphocytic choriomeningitis virus glycoproteins enhances infectivity for glioma cells and minimizes neurotropism

Vesicular stomatitis virus (VSV)-based oncolytic virotherapy has the potential to significantly improve the prognosis of aggressive malignancies such as brain cancer. However, VSV's inherent neurotoxicity has hindered clinical development so far. Given that this neurotropism is attributed to the glycoprotein VSV-G, VSV was pseudotyped with the nonneurotropic envelope glycoprotein of the lymphocytic choriomeningitis virus (LCMV-GP→VSV-GP). Compared to VSV, VSV-GP showed enhanced infectivity for brain cancer cells in vitro while sparing primary human and rat neurons in vitro and in vivo, respectively. In conclusion, VSV-GP has a much wider therapeutic window than VSV and is thus more suitable for clinical applications, especially in the brain.     

Expression of the M gene of vesicular stomatitis virus cloned in various vaccinia virus vectors.

Initial attempts to clone the matrix (M) gene of vesicular stomatitis virus (VSV) in a vaccinia virus expression vector failed, apparently because the expressed M protein, and particularly a carboxy-terminus-distal two-thirds fragment, was lethal for the virus recombinant. Therefore, a transient eucaryotic expression system was used in which a cDNA clone of the VSV M protein mRNA was inserted into a region of plasmid pTF7 flanked by the promoter and terminator sequences for the T7 bacteriophage RNA polymerase. When CV-1 cells infected with recombinant vaccinia virus vTF1-6,2 expressing the T7 RNA polymerase were transfected with pTF7-M3, the cells produced considerable amounts of M protein reactive by Western blot (immunoblot) analysis with monoclonal antibodies directed to VSV M protein. Evidence for biological activity of the plasmid-expressed wild-type M protein was provided by marker rescue of the M gene temperature-sensitive mutant tsO23(III) at the restrictive temperature. Somewhat higher levels of M protein expression were obtained in CV-1 cells coinfected with a vaccinia virus-M gene recombinant under control of the T7 polymerase promoter along with T7 polymerase-expressing vaccinia virus vTF1-6,2.     

Biophysical studies of vesicular stomatitis virus

McCombs, Robert M. (Baylor University College of Medicine, Houston, Tex.), Matilda Benyesh-Melnick, and Jean P. Brunschwig. Biophysical studies of vesicular stomatitis virus: J. Bacteriol. 91:803-812. 1966.-The infectivity and morphology of vesicular stomatitis virus (VSV) were studied after density gradient centrifugation in cesium chloride (CsCI), potassium tartrate (KT), and sucrose. Centrifugation in CsCl revealed two equally infectious bands corresponding to densities of 1.19 and 1.22 g/ml, and a third (density, 1.26 g/ml) band of low infectivity. Two bands (densities of 1.16 and 1.18 g/ml) were observed in the KT gradient, in which the lighter band contained most of the infectivity. Centrifugation in sucrose resulted in a single broad infectious band (density, 1.16 g/ml). The typical rod-shaped VSV particles were found mainly in the lighter bands obtained in CsCl (1.19 g/ml) and KT (1.16 g/ml) and in the single sucrose gradient band (1.16 g/ml). Bent particles equally as infectious as the rod-shaped particles were a constant finding in the CsCl preparations, and were observed mainly in the second band (density, 1.19 g). Numerous strands 15mmu wide were found in the third CsCl (density, 1.26 g/ml) and the second KT (1.18 g/ml) bands. Similar strands could be liberated from VSV particles after treatment with deoxycholate. Internal transverse striations were found to be a regular feature of VSV particles examined with the pseudoreplication negative-staining technique. For crude virus stocks, the physical particle-to-infectivity ratio ranged from 73 to 194. Several morphological similarities between VSV and myxoviruses were observed, including 10 mmu surface projections, pleomorphic morphological forms, and 15 mmu seemingly nucleoprotein strands.     

Cerulenin blocks fatty acid acylation of glycoproteins and inhibits vesicular stomatitis and Sindbis virus particle formation

Cerulenin, an antibiotic that inhibits de novo fatty acid and cholesterol biosynthesis, effectively inhibited the formation and release of virus particles from chicken embryo fibroblasts infected with Sindbis or vesicular stomatitis virus (VSV). When added for 1 h at 3 h postinfection, the antibiotic blocked VSV particle production by 80 to 90% and inhibited incorporation of [3H]palmitic acid into the VSV glycoprotein by an equivalent amount. The effect of this antibiotic on virus protein and RNA biosynthesis was significantly less than that on fatty acid acylation. Nonacylated virus glycoproteins accumulated inside and on the surface of cerulenin-treated cells. These data indicate that fatty acid acylation is not essential for intracellular transport of these membrane proteins, but it may have an important role in the interaction of glycoproteins with membranes during virus assembly and budding.     

Density-dependent selection in vesicular stomatitis virus

We used vesicular stomatitis virus to test the effect of complementation on the relative fitness of a deleterious mutant, monoclonal antibody-resistant mutant (MARM) N, in competition with its wild-type ancestor. We carried out competitions of MARM N and wild-type populations at different multiplicities of infection (MOIs) and initial ratios of the wild type to the mutant and found that the fitness of MARM N relative to that of the wild type is very sensitive to changes in the MOI (i.e., the degree of complementation) but depends little, if at all, on the initial frequencies of MARM N and the wild type. Further, we developed a mathematical model under the assumption that during coinfection both viruses contribute to a common pool of protein products in the infected cell and that they both exploit this common pool equally. Under such conditions, the fitness of all virions that coinfect a cell is the average fitness in the absence of coinfection of that group of virions. In the absence of coinfection, complementation cannot take place and the relative fitness of each competitor is only determined by the selective value of its own products. We found good agreement between our experimental results and the model predictions, which suggests that the wild type and MARM N freely share all of their gene products under coinfection.     

Monensin blocks endocytosis of vesicular stomatitis virus

Monensin inhibits the infection of mouse cells by Vesicular Stomatitis Virus (VSV). At low drug concentrations (0.5 μM), endocytosis of VSV is inhibited whereas viral binding is unaffected. Monensin may be useful for analyzing the internalization of other viruses as well as soluble ligands.