Production Of Vesicular Stomatitis Virus Research Articles

Optimization of production of vesicular stomatitis virus (VSV) in suspension serum-free culture medium at high cell density

During the last decade, oncolytic viruses such as vesicular stomatitis virus (VSV) have gained tremendous popularity as efficient vaccines for infectious diseases as well as for the treatment of cancer. Our laboratory has developed two stable cell lines, 293SF-3F6 (derived from HEK293A cells) and SF-BMAdR cells (a variant of A549 that expresses the E1 region of human adenovirus). These two cell lines were adapted to grow efficiently in suspension culture and in serum-free medium. In this report we evaluated the production of a recombinant VSV expressing the green fluorescent protein (VSV-GFP) in these two stable cell lines. At a relatively low cell density of 500,000 cells per ml, 293SF-3F6 produced 4.6 times more infectious particles than SF-BMAdR cells. There was a positive correlation between volumetric virus titer and cell density up to 2.E + 07 cells/ml. A fed-batch process using an in-house medium and feed was developed to support the growth of 293SF-3F6 cells up to a concentration of 1.E + 07 cells/ml for infection at higher cell density and VSV production at high titer. Shifting the temperature from 37 °C to 34 °C at infection time improved VSV titer up to 3.3 fold. After scaling up the optimal condition from small scale (3 ml) to larger volumes (50 & 200 ml), the maximal volumetric titer obtained using the 293SF-3F6 cells was in average 2.9E + 10 extracellular infectious particles/ml. In conclusion, our data demonstrated that 293SF-3F6 cells, for which a cGMP master cell bank is available, is a performant cell line to scale up VSV production in suspension culture using serum-free medium.

Lipid rafts play an important role in the vesicular stomatitis virus life cycle

Lipid rafts are involved in the life cycle of many viruses. In this study, we investigated the role of lipids in the life cycle of vesicular stomatitis virus (VSV). Cholesterol depletion by pretreatment of BHK cells or VSV particles with methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering drug, inhibited the production of VSV dramatically. This effect was reversible, and virus production was restored by the addition of cholesterol, indicating that the reduction was caused by the loss of cholesterol in the cell membrane and virus, respectively. Cholesterol depletion at the adsorption stage also reduced the production of VSV significantly, but in contrast, only had a limited effect on virus production at the post-entry stage. Inhibition of sphingomyelin by myriocin treatment only showed a minor effect on VSV production. However, reduction of cholesterol and sphingomyelin at the same time dramatically reduced VSV production, showed a significant synergistic effect. These results suggest that lipid rafts play an important role in the life cycle of VSV.

Carrier Cell-based Delivery of an Oncolytic Virus Circumvents Antiviral Immunity

Oncolytic viruses capable of tumor-selective replication and cytolysis have shown early promise as cancer therapeutics. However, the host immune system remains a significant obstacle to effective systemic administration of virus in a clinical setting. Here, we demonstrate the severe negative impact of the adaptive immune response on the systemic delivery of oncolytic vesicular stomatitis virus (VSV) in an immune-competent murine tumor model, an effect mediated primarily by the neutralization of injected virions by circulating antibodies. We show that this obstacle can be overcome by administering virus within carrier cells that conceal viral antigen during delivery. Infected cells were delivered to tumor beds and released virus to infect malignant cells while sparing normal tissues. Repeated administration of VSV in carrier cells to animals bearing metastatic tumors greatly improved therapeutic efficacy when compared with naked virion injection. Whole-body molecular imaging revealed that carrier cells derived from solid tumors accumulate primarily in the lungs following intravenous injection, whereas leukemic carriers disseminate extensively throughout the body. Furthermore, xenogeneic cells were equally effective at delivering virus as syngeneic cells. These findings emphasize the importance of establishing cell-based delivery platforms in order to maximize the efficacy of oncolytic therapeutics.

Further characterization of a CD99-related 21-kDa transmembrane protein (VAP21) expressed in Syrian hamster cells and its possible involvement in vesicular stomatitis virus production.

The VAP21, a CD99-related 21-kDa transmembrane protein, was first detected in the enveloped virions that were grown in a Syrian hamster-derived cell line, BHK-21 (Sagara et al., 1997; Yamamoto et al., 1999). We further tried to elucidate the nature and properties of VAP21. The VAP21 was detected in various organs of the Syrian hamster as well as in the Syrian hamster-derived cell lines (BHK-21 and HmLu-1). We could not detect the VAP21 antigen in other cell lines derived from other animal species we examined, including a Chinese hamster (CHO-K1), mouse (neuroblastoma C1300, clone NA), dog (MDCK), monkey (COS-7), and human (HeLa, HepG2). We tried to introduce the VAP21 gene into VAP21-negative cell lines using a tetracycline-regulated gene expression system. All of our trials, however, resulted in failure to establish stably positive inducible cell lines. To the contrary, we could easily establish the VAP21-overexpressing cell lines from the Syrian hamster cell lines, which were successfully grown and maintained without any loss of VAP21 expression even under the induced culture conditions. In such VAP21-overexpressing cells, production of the vesicular stomatitis virus (VSV) was increased several-fold, while suppression of the VAP21 expression resulted in reducing the VSV yields. From these results, we conclude that the VAP21 is a physiologically active cell membrane component of some animal species including the Syrian hamster, and might positively be involved in the VSV replication.

Crystal structure of vesicular stomatitis virus matrix protein.

The vesicular stomatitis virus (VSV) matrix protein (M) interacts with cellular membranes, self-associates and plays a major role in virus assembly and budding. We present the crystallographic structure, determined at 1.96 A resolution, of a soluble thermolysin resistant core of VSV M. The fold is a new fold shared by the other vesiculovirus matrix proteins. The structure accounts for the loss of stability of M temperature-sensitive mutants deficient in budding, and reveals a flexible loop protruding from the globular core that is important for self-assembly. Membrane floatation shows that, together with the M lysine-rich N-terminal peptide, a second domain of the protein is involved in membrane binding. Indeed, the structure reveals a hydrophobic surface located close to the hydrophobic loop and surrounded by conserved basic residues that may constitute this domain. Lastly, comparison of the negative-stranded virus matrix proteins with retrovirus Gag proteins suggests that the flexible link between their major membrane binding domain and the rest of the structure is a common feature shared by these proteins involved in budding and virus assembly.

The role of interleukin-18 in vesicular stomatitis virus infection of the CNS.

Intranasal application of vesicular stomatitis virus (VSV) results in the initial infection of the olfactory receptor neurons and a rapid progression of the virus through the mouse central nervous system (CNS). Interleukin-18 (IL-18) is an 18.3-kd cytokine that induces interferon gamma (IFN-gamma) production in mice. IL-18 is synthesized as an inactive precursor that is cleaved and activated by caspase-1/interleukin-1beta converting enzyme (ICE). IL-18 shares several biological properties with IL-12, including the ability to induce IFN-gamma production in T lymphocytes and natural killer (NK) cells. In the CNS, microglia and astrocytes produce IL-18 and IL-12. We have previously shown that IL-12 promotes recovery from VSV encephalitis. This led us to examine the potential role of IL-18 in the pathogenesis of VSV encephalitis. We show that both IL-18 and caspase-1 mRNA are consistently present in the CNS of mice. The addition of exogenous IL-18 to cell cultures does not affect the production of VSV, and addition of exogenous IL-18 at the time of infection does not alter the morbidity or mortality of BALB/c mice. In vitro studies with neutralizing monoclonal antibody to IL-18 had no effect. From these results we conclude that in this system and under the experimental conditions used, unlike IL-12 and IFN-gamma, IL-18 does not play a significant role in the host response to VSV infection.

Mechanisms of Cytokine-Mediated Inhibition of Viral Replication

In this report, the role of nitric oxide synthase (NOS) and IL-12 administration in inhibition of vesicular stomatitis virus (VSV) from infected neuroblastoma cells was examined. We previously have shown that cytokine treatment of cells results in the induction of NOS-1, and this is associated with a 2 log inhibition of VSV production. We performed these studies to examine the mechanism by which viral replication is suppressed. Neuroblastoma cells (NB41A3) were treated with either IL-12 or medium and subsequently infected with VSV. Viral protein and mRNA were isolated from these cells, and their levels were measured by Western or Northern blots, respectively. mRNA levels were decreased modestly, but viral proteins were decreased substantially in cells pretreated with IL-12, suggesting that the inhibitory effect of NO is working at the translational level. Cytokine treatment of cells was not associated with oxidative stress. The viral proteins also were nitrosylated. These data suggest that the mechanism of NO inhibition of viral replication occurs through translational interference and posttranslational modifications of viral components.

Granzyme B Independently of Perforin Mediates Noncytolytic Intracellular Inactivation of Vesicular Stomatitis Virus

Cytotoxic cells provide a crucial defense against DNA and RNA viral infections. Here we describe anin vitromodel to study the fate of vesicular stomatitis virus (VSV) RNA in cells undergoing apoptosis. Using the [3H]uridine release assay, we show that human LAK cells induce the degradation of RNA in infected U937 cells in addition to inhibiting the production of infectious virions. LAK cell-mediated RNA degradation was blocked by the serine protease inhibitor, 3,4-dichloroisocoumarin. Purified human granzyme B but not inactivated granzyme B, granzyme A, or perforin rapidly induced degradation of RNA in VSV-infected U937 cells in a dose- and time-dependent manner without lysing the cells and suppressed viral production. Northern analysis of RNA extracted from infected cells with a VSV full-length cDNA probe confirmed that levels of viral transcripts were reduced by treatment with granzyme B. Nevertheless, the amount of host β-actin mRNA was also reduced in infected cells, suggesting that treatment with granzyme B induced apoptosis. Consistent with this notion, infected cells exposed to granzyme B rapidly developed DNA strand breakage. Taken together, the data suggest that granzyme B in the absence of perforin reduced VSV production by activating a mechanism that degraded viral transcripts in infected U937 cells.

Effect of combined αIFN and prostaglandin A 1 treatment on vesicular stomatitis virus replication and heat shock protein synthesis in epithelial cells

The antiviral activity of prostaglandin A (PGA) and interferons (IFNs) has been widely described. In the present report, we investigated the effect of combined αIFN and PGA 1 treatment on vesicular stomatitis virus (VSV) replication and on heat shock protein (HSP) induction in monkey epithelial cells. In uninfected cells, PGA 1 caused a dose-dependent induction of HSP70, HSP90 and HSP110, while αIFN did not affect HSP synthesis. Alpha-IFN suppressed VSV replication dose-dependently, even when cells were treated after virus infection. VSV protein synthesis was not affected by αIFN, indicating a block at the level of virus assembly or maturation. PGA 1 caused a dose-dependent inhibition of VSV replication, and suppressed VSV protein synthesis at concentrations which induced the synthesis of high levels of HSP70. The combined treatment with low doses of αIFN or PGA 1, which only moderately inhibited VSV replication when administered separately, was found to suppress VSV production by more than 95%, and resulted in a 3-fold increase of HSP70 synthesis as compared to PGA 1 alone. These results demonstrate a co-operative effect of PGA 1 and αIFN against VSV infection and suggest that αIFN can potentiate the cellular response to HSP induction in virus-infected cells.

Recombinant vesicular stomatitis viruses from DNA.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

Cultivation of mammalian cells on macroporous microcarriers

Adherent cells can be cultivated in a stirred-tank bioreactor by attaching to microcarriers. Macroporous microcarriers, with their intraparticle space and surface area for cell growth, can potentially support a higher cell concentration than conventional microcarriers, which support cell growth only on the external surface. Chinese hamster ovary (CHO) cells and green monkey kidney (Vero) cells were cultivated on macroporous microcarriers, Cultispher-G. Cells attached to the microcarriers at a slow rate and grew to a high density. Thin sections of the microcarriers demonstrate that cells were initially on the exterior of the microcarriers and migrated into the interior as cell concentration increased. Vero cells cultivated on these microcarriers were successfully used for the production of vesicular stomatitis virus (VSV).

Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay

The characteristics of fusion of respiratory syncytial virus (RSV) with HEp-2 cells were studied by the R18 fluorescence dequenching assay of membrane fusion. A gradual increase in fluorescence intensity indicative of virion-cell fusion was observed when R18-labeled RSV was incubated with HEp-2 cells. Approximately 35% dequenching of the probe fluorescence was observed in 1 h at 37 degrees C. Fusion showed a temperature dependence, with significant dequenching occurring above 18 degrees C. The dequenching was also dependent on the relative concentration of target membrane. Thus, increasing the concentration of target membrane resulted in increased levels of dequenching. In addition, viral glycoproteins were shown to be involved in this interaction, since dequenching was significantly reduced by pretreatment of labeled virus at 70 degrees C for 5 min or by trypsinization of R18-labeled virions prior to incubation with HEp-2 cells at 37 degrees C. The fusion of RSV with HEp-2 cells was unaffected over a pH range of 5.5 to 8.5, with some increase seen at lower pH values. Treatment of HEp-2 cells with ammonium chloride (20 and 10 mM), a lysosomotropic agent, during early stages of infection did not inhibit syncytium formation or progeny virion production by RSV. At the same concentrations of ammonium chloride, the production of vesicular stomatitis virus was reduced approximately 4 log10 units. These results suggest that fusion of the virus with the cell surface plasma membrane is the principal route of entry.

Virus production in microsphere-induced aggregate culture of animal cells

Green monkey kidney cells (Vero) were cultivated on microspheres with a median diameter of 23 μm. Aggregates were formed two days after inoculation and eventually reached a few hundred micrometers in diameter. Cells in these aggregates remained viable throughout the cultivation period and were successfully used for the production of vesicular stomatitis virus (VSV).

Effect of monensin on the synthesis, maturation and secretion of vesicular stomatitis virus proteins in a monensin-resistant Chinese hamster ovary cell line.

We compared the effects of the cationic ionophore, monensin, on the synthesis, maturation and release of vesicular stomatitis virus (VSV) in cultures of Chinese hamster ovary (CHO) cells and the monensin-resistant clone, MonR-31. Our results depended on the dose and time of the addition of monensin to the infected cells, from 1 h prior to VSV infection to 1 h after infection. VSV production was more resistant in MonR-31 than in CHO cells when the ionophore was added 1 h prior to VSV infection. Monensin added 1 h after VSV infection showed the opposite phenomenon; release of virus particles into the medium was 10- to 10(5)-fold less in MonR-31 cells than in CHO cells, and the intracellular virus number in the resistant cells was one-third to one-fourth of that in the parental CHO cells. Syntheses of all virus-associated G, N and M proteins were inhibited in both cell lines by monensin, but especially so in the MonR-31 cells. There were no marked qualitative changes in the biochemical properties of viral glycoprotein G in virus-infected CHO and MonR-31 cells treated with monensin after virus infection. An endoglycosidase H-resistant G with a molecular weight smaller than that of normal G and attachments of palmitate or fucose on the truncated G protein appeared. Alteration of the secretion of as well as the synthesis of the enveloped virus is discussed in relation to the monensin susceptibility of the resistant MonR-31 clone.

Prostaglandin A inhibits the replication of vesicular stomatitis virus: effect on virus glycoprotein.

Prostaglandins of the A series were found to strongly suppress the replication of vesicular stomatitis virus (VSV) in mouse L fibroblasts. The highest non-toxic dose of PGA1, 4 micrograms/ml, decreased VSV production by 93.6%. At this dose, PGA1 did not alter DNA, RNA or protein synthesis in uninfected L cells for periods up to 24 h, whereas it further suppressed protein synthesis and slightly increased RNA synthesis in VSV-infected cells. The presence of PGA1 during virus adsorption, with no treatment after infection, reduced VSV yields by 63.6%. However, the presence of PGA1 during an early step of VSV replication was not essential for the antiviral action to occur (PGA1 treatment could be started 1 to 2 h post-infection). Apart from a slight overall inhibition of virus protein synthesis, PGA1 strongly suppressed the synthesis of the VSV glycoprotein G; moreover, it produced an alteration in the mobility of this protein in SDS-polyacrylamide gels. We propose that this slight decrease in molecular weight (about 4000) of the G protein in the presence of PGA1 could be due to an alteration in the glycosylation process.

Search for compounds which have an inhibitory effect on rhabdovirus multiplication In Vitro

Summary Twenty-one substances were investigated in vitro against rhabdovirus infections. The inhibitory effect was evaluated under single-growth cycle conditions. The substance was present in the culture medium during the entire growth cycle at doses varying from 5 to 500 μg/ml. At non-cytotoxic doses, inhibition of rabies virus production was achieved by thirteen substances. Eight of them—7 heteropolyanions and virazole—showed a dose-dependent inhibition, while the others—four acyclic derivatives of adenosine and carbocyclic 3-deazaadenosine—showed a 90 % inhibition whatever the dose. Under the same conditions, none of these thirteen substances inhibited the production of vesicular stomatitis virus. Only one compound, amantadine, inhibited the production of both viruses.

Differential effect of monensin on enveloped viruses that form at distinct plasma membrane domains.

We have observed a striking differential effect of the ionophore, monensin, on replication of influenza virus and vesicular stomatitis virus (VSV) in Madin-Darby canine kidney (MDCK) and baby hamster kidney (BHK21) cells. In MDCK cells, influenza virus is assembled at the apical surfaces, whereas VSV particles bud from the basolateral membranes; no such polarity of maturation is exhibited in BHK21 cells. A 10(-6) M concentration of monensin reduces VSV yields in MDCK cells by greater than 90% as compared with controls, whereas influenza virus yields are unaffected. In BHK21 cells, monensin also inhibits VSV production, but influenza virus is also sensitive to the ionophore. Immunofluorescent staining of fixed and unfixed MDCK monolayers indicates that VSV glycoproteins are synthesized in the presence of monensin, but their appearance on the plasma membrane is blocked. Electron micrographs of VSV-infected MDCK cells treated with monensin show VSV particles aggregated within dilated cytoplasmic vesicles. Monensin-treated influenza virus-infected MDCK cells also contain dilated cytoplasmic vesicles, but virus particles were not found in these structures, and numerous influenza virions were observed budding at the cell surface. These results indicate that influenza virus glycoprotein transport is not blocked by monensin treatment, whereas there is a block in transport of VSV G protein. Thus it appears that at least two distinct pathways of transport of glycoproteins to the plasma membrane exist in MDCK cells, and only one of them is blocked by monensin.

Continuous cell propagation using low‐charge microcarriers

AbstractMicrocarrier cell culture technology has been extended by the finding that two mammalian epithelial cell lines can be continuously subcultured by simple bead‐to‐bead transfer in normal medium in which calcium concentrations have been reduced. Data are reported which show that the hamster ovary line CHO‐Kl and the monkey kidney line LLC‐MK2 can be subcultured simply by adding fresh microcarriers to the stirred suspension culture. Thirteen generations of continuous exponential growth are demonstrated with two such subcultures for the CHO‐Kl cells and with four such subcultures for the LLC‐MK2 cells. Cell generation times were unchanged by this subculturing approach compared to standard subculturing procedure using trypsin to remove cells from surfaces. We have applied this technique to the production of vesicular stomatitis virus (VSV) from CHO‐Kl cells. Viral yields were comparable (less than twofold difference) in microcarrier cultures which were subcultured via bead‐to‐bead transfer or by the standard means of removing cells from microcarriers with trypsin.

Characterization of the non-permissive infection of rabbit cornea cells by vesicular stomatitis virus.

Rabbit cornea cells (RC-60) restrict the reproduction of vesicular stomatitis virus (VSV) (Thacore & Youngner, 1975). In cells infected with VSV alone, an inhibition in the synthesis of VSV genome RNA is observed. A number of parameters which could affect virus RNA synthesis have been examined: virus transcription and post-transcriptional modification, translation, modification of proteins and migration of the G protein to the surface of the cell; they all appear to be normal, although somewhat diminished, in the restricted system. In these cells, therefore, it is the replication of VSV RNA that is directly inhibited, although limited synthesis of both (+) and (-) strand genome length RNA does occur. When the cells are co-infected with rabbit poxvirus (RPV) as a helper virus, however, VSV production is normal. Our studies suggest that RPV plays a role in the maturation as well as in the replication of VSV RNA in RC-60 cells. Certain mutants of RPV have been found to lack the helper function and are unable to convert the RC-60 cells into a permissive host for VSV. These mutants should facilitate the elucidation of the mechanism by which RPV is able to overcome the restriction in these cells.

Production of Vesicular Stomatitis Virus with Low Infectivity by Interferon-treated Cells

In cultures of Ly cells treated with 10 or 30 reference units/ml of mouse interferon there was a 30 to 200 times reduction in the production of infectious vesicular stomatitis virus (VSV); virus particle production, as measured by VSV particle associated virus RNA, virus protein, and virus transcriptase, was inhibited by, at most, six times. These results suggested that interferon-treated cells produce VSV particles with low infectivity and resemble the findings in interferon-treated cells infected with murine leukaemia viruses.