Evaluating replication-defective vesicular stomatitis virus as a vaccine vehicle

We have generated replication-competent (VSV-C/E1/E2) and nonpropagating (VSVDeltaG-C/E1/E2) vesicular stomatitis virus (VSV) contiguously expressing the structural proteins of hepatitis C virus (HCV; core [C] and glycoproteins E1 and E2) and report on their immunogenicity in murine models. VSV-C/E1/E2 and VSVDeltaG-C/E1/E2 expressed high levels of HCV C, E1, and E2, which were authentically posttranslationally processed. Both VSV-expressed HCV E1-E2 glycoproteins were found to form noncovalently linked heterodimers and appeared to be correctly folded, as confirmed by coimmunoprecipitation analysis using conformationally sensitive anti-HCV-E2 monoclonal antibodies (MAbs). Intravenous or intraperitoneal immunization of BALB/c mice with VSV-C/E1/E2 or VSVDeltaG-C/E1/E2 resulted in significant and surprisingly comparable HCV core or E2 antibody responses compared to those of control mice. In addition, both virus types generated HCV C-, E1-, or E2-specific gamma interferon (IFN-gamma)-producing CD8(+) T cells, as determined by enzyme-linked immunospot (ELISPOT) analysis. Mice immunized with VSVDeltaG-C/E1/E2 were also protected against the formation of tumors expressing HCV E2 (CT26-hghE2t) and exhibited CT26-hghE2t-specific IFN-gamma-producing and E2-specific CD8(+) T-cell activity. Finally, recombinant vaccinia virus (vvHCV.S) expressing the HCV structural proteins replicated at significantly lower levels when inoculated into mice immunized with VSV-C/E1/E2 or VSVDeltaG-C/E1/E2, but not with control viruses. Our data therefore illustrate that potentially safer replication-defective VSV can be successfully engineered to express high levels of antigenically authentic HCV glycoproteins. In addition, this strategy may therefore serve in effective vaccine and immunotherapy-based approaches to the treatment of HCV-related disease.     

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.     

Cell fusion by Semliki Forest, influenza, and vesicular stomatitis viruses

Representatives of three families of enveloped viruses were shown to fuse tissue culture cells together. These were: Semliki Forest virus (SFV, a togavirus), vesicular stomatitis virus (a rhabdovirus), and two myxoviruses, fowl plaque virus and Japan influenza virus (Japan)/A/305/57). Unlike paramyxoviruses, whose fusion activity is known to occur over a broad pH range, fusion by these viruses was restricted to mildly acidic pH. The pH thresholds for the four viruses were 6.0, 6.1, 5.5, and 5.1, respectively. The precursor form of Japan influenza, which is not infectious and which contains the uncleaved hemagglutinin, had no fusion activity. This result suggested a role for the influenza hemagglutinin in the low-pH-dependent membrane fusion activity. Taken together, our results show that low-pH-induced fusion is a widespread property of enveloped animal viruses and that it may play a role in the infective process. The fusion reactions with all four viruses were fast, efficient, and easy to induce. With UV-inactivated SFV, the fusion was shown to be nonlytic and the polykaryons were viable for at least 12 h. 30 ng of SFV/1 x 10(6) BHK-21 cells were required for 50% fusion, and 250 ng sufficed to fuse the entire culture into a single polykaryon.     

Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses.

Tunicamycin (TM), an antibiotic that inhibits the formation of N-acetylglucosamine-lipid intermediates, thereby preventing the glycosylation of newly synthesized glycoproteins, inhibits the growth of Sindbis virus and vesicular stomatitis virus in BHK cells. At 0.5 mug of TM per ml, the replication of both viruses is inhibited 99.9%. Noninfectious particles were not detected. All the viral proteins were synthesized in the presence of TM, but the glycoproteins were selectively altered in that they migrated faster than normal viral glycoproteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting defective glycosylation. Within 1 h after TM addition, [14C]glucosamine incorporation into glycoproteins was inhibited 20%, whereas [35S]methionine incorporation was unaffected. By 2 to 3 h after TM addition, glucosamine incorporation had fallen to 15% of control value, with methionine incorporation being 60% of normal. TM did not affect the growth of the nomenveloped encephalomyocarditis virus in BHK cells, demonstrating that TM is not a general inhibitor of protein synthesis. These data demonstrate that TM specifically inhibits the glycosylation of viral glycoproteins and that glycosylation may be essential for the normal assembly of enveloped viral particles.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.     

An effective AIDS vaccine based on live attenuated vesicular stomatitis virus recombinants

We developed an AIDS vaccine based on attenuated VSV vectors expressing env and gag genes and tested it in rhesus monkeys. Boosting was accomplished using vectors with glycoproteins from different VSV serotypes. Animals were challenged with a pathogenic AIDS virus (SHIV89.6P). Control monkeys showed a severe loss of CD4+ T cells and high viral loads, and 7/8 progressed to AIDS with an average time of 148 days. All seven vaccinees were initially infected with SHIV89.6P but have remained healthy for up to 14 months after challenge with low or undetectable viral loads. Protection from AIDS was highly significant (p = 0.001). VSV vectors are promising candidates for human AIDS vaccine trials because they propagate to high titers and can be delivered without injection.     

Proposal for a uniform nomenclature for defective interfering viruses of vesicular stomatitis virus.

Defective interfering particles of vesicular stomatitis virus have been named according to their parental derivation and to their genomic length and physical properties. This suggested uniform nomenclature can be adapted for other virus systems.     

Production of neutralizing antibody to rabies and vesicular stomatitis viruses by hybrid cell lines

Hybrid cell lines were obtained following fusion of P 3 × 63 Ag-8 myeloma cells with spleen cells derived from BALB/c mice immunized either with rabies virus or with vesicular stomatitis virus. Hybrid cell lines were selected which continued to secrete rabies virus or vesicular stomatitis virus neutralizing antibody specifically directed against coat glycoprotein of respective viruses.     

Fluorescence photobleaching recovery measurements reveal differences in envelopment of sindbis and vesicular stomatitis viruses

Fluorescence photobleaching recovery (FPR) measurements of virus glycoproteins on the surfaces of cells infected with vesicular stomatitis virus (VSV) and Sindbis virus showed that the VSV glycoprotein (G) remained mobile throughout the infectious cycle, whereas Sindbis virus glycoproteins (E1, E2) were partially mobile early after infection and immobile at later times when greater amounts of these proteins were on the cell surface. A highly mobile fraction of Sindbis virus glycoproteins was detected throughout the replication cycle of a temperature-sensitive mutant unable to form virus particles. Thus immobilization of E1 and E2 was the result of increasing surface glycoprotein concentrations and virus budding. Together with other data, which included the detection of E1 and E2 in particles as soon as these proteins were transported to the cell surface, the FPR results suggest that Sindbis virus assembly initiates on intracellular vesicles, where glycoproteins aggregate and bind nucleocapsids. In contrast, our FPR data on VSV support a model previously suggested by others, in which a small fraction of cell-surface G is immobilized into budding sites formed by interactions with virus matrix and nucleoproteins. FPR measurements also provide direct evidence for strong interactions between E1 and E2, as well as between E1 and PE2, the precursor form of E2.     

Ribosome topography in baby hamster kidney cells infected with Sindbis and vesicular stomatitis viruses

The topography of polysomal ribosomes in mock-infected and in Sindbis virus- and vesicular stomatitis virus-infected BHK cells was investigated using a double, radioactive labelling technique. Ribosomal proteins in intact polysomes were surface labelled by reductive methylation using [14C]formaldehyde. Following removal of ribosomal RNA, proteins were denatured in 6 M guanidine and labelled with [3H]borohydride. Labelled ribosomal proteins were separated by electrophoresis in two-dimensional gels and the 3H/14C ratio for each ribosomal protein was taken as an index of its relative surface exposure in intact ribosomes. Comparison of the ratios for individual ribosomal proteins in Sindbis virus-infected vs. control polysomes indicated that proteins L7, L8, L17, L26 and S19 became more 'buried' and others such as L4, L29, L36, S2 and S26 became more 'exposed' in infected cells. Most of the topographical alterations occurred in the large ribosomal subunit. In contrast, infection of BHK cells with vesicular stomatitis virus induced little or no topographical alteration.     

Parvovirus-like particles as vaccine vectors.

A wide array of systems have been developed to improve "classic" vaccines. The use of small polypeptides able to elicit potent antibody and cytotoxic responses seems to have enormous potential in the design of safer vaccines. While peptide coupling to large soluble proteins such as keyhole limpet hemocyanin is the current method of choice for eliciting antibody responses and insertion in live viruses for cytotoxic T-lymphocyte responses, alternative cheaper and/or safer methods will clearly be required in the future. Virus-like particles constitute very immunogenic molecules that allow for covalent coupling of the epitopes of interest in a simple way. In this article, we detail the methodology employed for the preparation of efficient virus vectors as delivery systems. We used parvovirus as the model for the design of new vaccine vectors. Recently parvovirus-like particles have been engineered to express foreign polypeptides in certain positions, resulting in the production of large quantities of highly immunogenic peptides, and to induce strong antibody, helper-T-cell, and cytotoxic T-lymphocyte responses. We discuss the different alternatives and the necessary steps to carry out this process, placing special emphasis on the flow of decisions that need to be made during the project.     

High-performance liquid chromatographic total particles quantification of retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein

A novel and rapid method for the total particles quantification of murine leukemia virus derived retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein was developed using high performance liquid chromatography. Virus particles were detected by absorbance at 260 nm and quantified using a calibration curve generated from highly purified and concentrated viral stock characterized by negative stain electron microscopy. The method requires Benzonase digestion and concentration of the supernatant prior to analysis. The virus eluted in 12.55 min at a flow rate of 1 mL/min in 20 mM Tris-Cl, pH 7.4 + 1.1 M NaCl. The limits of detection and quantification of this assay were 4.71 x 10(8) and 1.57 x 10(9) viral particles/mL, respectively. Linearity was between 3.0 x 10(9) and 1.0 x 10(11) viral particles/mL with a correlation coefficient of 0.9923 and a slope of 6 x 10(-6). The assay precision was <5% and <10% for intra- and inter-day analysis, respectively. This assay was used for the total particles quantification of a 7-day, large-scale perfusion culture production of a retroviral vector grown in 293 cells expressing the beta-galactosidase gene.     

Recombinant viruses as transformation vectors of marine macroalgae

The large dsDNA viruses that are known to infect eukaryotic algae show promise as genetic vectors for algal biotechnology. The large size (150–330 kbp) of these viral genomes may permit insertion of large sequences of foreign DNA. The viruses infecting filamentous marine brown algae appear to be integrated into the genomes of their hosts, and may provide integration mechanisms that can be used for directing insertion of foreign genes into algal chromosomes.     

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.