Development of recombinant vesicular stomatitis viruses that exploit defects in host defense to augment specific oncolytic activity

Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus normally sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta). Recently, we reported that VSV replicates to high levels in many transformed cells due, in part, to susceptible cells harboring defects in the IFN system. These observations were exploited to demonstrate that VSV can be used as a viral oncolytic agent to eradicate malignant cells in vivo while leaving normal tissue relatively unaffected. To attempt to improve the specificity and efficacy of this system as a potential tool in gene therapy and against malignant disease, we have genetically engineered VSV that expresses the murine IFN-beta gene. The resultant virus (VSV-IFNbeta) was successfully propagated in cells not receptive to murine IFN-alpha/beta and expressed high levels of functional heterologous IFN-beta. In normal murine embryonic fibroblasts (MEFs), the growth of VSV-IFNbeta was greatly reduced and diminished cytopathic effect was observed due to the production of recombinant IFN-beta, which by functioning in a manner involving autocrine and paracrine mechanisms induced an antiviral effect, preventing virus growth. However, VSV-IFNbeta grew to high levels and induced the rapid apoptosis of transformed cells due to defective IFN pathways being prevalent and thus unable to initiate proficient IFN-mediated host defense. Importantly, VSV expressing the human IFN-beta gene (VSV-hIFNbeta) behaved comparably and, while nonlytic to normal human cells, readily killed their malignant counterparts. Similar to our in vitro observations, following intravenous and intranasal inoculation in mice, recombinant VSV (rVSV)-IFNbeta was also significantly attenuated compared to wild-type VSV or rVSV expressing green fluorescent protein. However, VSV-IFNbeta retained propitious oncolytic activity against metastatic lung disease in immunocompetent animals and was able to generate robust antitumor T-cell responses. Our data indicate that rVSV designed to exploit defects in mechanisms of host defense can provide the basis for new generations of effective, specific, and safer viral vectors for the treatment of malignant and other disease.     

Arrested spread of vesicular stomatitis virus infections in vitro depends on interferon-mediated antiviral activity

A quantitative understanding of the innate immune response will enable its recruitment against emerging, poorly characterized, or weaponized viral pathogens. To gain insights into how the innate responses can limit viral spread, we used quantitative focal infections to study how the spread of recombinant vesicular stomatitis viruses (VSV) on baby hamster kidney (BHK) and delayed brain tumor (DBT) cell monolayers is affected by innate cellular antiviral responses. We observed that rates of infection spread correlated with one-step growth rankings for four ectopic VSV strains: N1, N2, N3, and N4. However, this correlation was lost for M51R, a recombinant VSV mutant that lacks the ability to shut-off host gene expression. In BHK cells, M51R spread at two-thirds the rate of the recombinant control virus, XK3.1, even though their one-step growth was comparable. In DBT cells, M51R infections failed to spread beyond the site of inoculation. Addition of anti-interferon antibody restored M51R spread and one-step growth to wild-type levels. Interestingly, the antibody enhanced the spread of wild-type virus but not its growth. These results suggest that while the rate of viral spread generally correlates with the rate of viral growth, the induction of cellular antiviral activities can be in some cases, the overriding factor in both spread and growth. In summary, focal infections enabled us to visualize and quantify how viral spread was inhibited by cellular antiviral activities. This study demonstrates a mechanism for quantifying how innate cellular responses can mitigate infection spread in vitro.     

The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avian-human influenza A/Pintail/79 reassortant viruses for monkeys.

Reassortant viruses which possessed the hemagglutinin and neuraminidase genes of wild-type human influenza A viruses and the remaining six RNA segments (internal genes) of the avian A/Pintail/Alberta/119/79 (H4N6) virus were previously found to be attenuated in humans. To study the genetic basis of this attenuation, we isolated influenza A/Pintail/79 X A/Washington/897/80 reassortant viruses which contained human influenza virus H3N2 surface glycoprotein genes and various combinations of avian or human influenza virus internal genes. Twenty-four reassortant viruses were isolated and first evaluated for infectivity in avian (primary chick kidney [PCK]) and mammalian (Madin-Darby canine kidney [MDCK]) tissue culture lines. Reassortant viruses with two specific constellations of viral polymerase genes exhibited a significant host range restriction of replication in mammalian (MDCK) tissue culture compared with that in avian (PCK) tissue culture. The viral polymerase genotype PB2-avian (A) virus, PB1-A virus, and PA-human (H) virus was associated with a 900-fold restriction, while the viral polymerase genotype PB2-H, PB1-A, and PA-H was associated with an 80,000-fold restriction of replication in MDCK compared with that in PCK. Fifteen reassortant viruses were subsequently evaluated for their level of replication in the respiratory tract of squirrel monkeys, and two genetic determinants of attenuation were identified. First, reassortant viruses which possessed the avian influenza virus nucleoprotein gene were as restricted in replication as a virus which possessed all six internal genes of the avian influenza A virus parent, indicating that the nucleoprotein gene is the major determinant of attenuation of avian-human A/Pintail/79 reassortant viruses for monkeys. Second, reassortant viruses which possessed the viral polymerase gene constellation of PB2-H, PB1-A, and PA-H, which was associated with the greater degree of host range restriction in vitro, were highly restricted in replication in monkeys. Since the avian-human influenza reassortant viruses which expressed either mode of attenuation in monkeys replicated to high titer in eggs and in PCK tissue culture, their failure to replicate efficiently in the respiratory epithelium of primates must be due to the failure of viral factors to interact with primate host cell factors. The implications of these findings for the development of live-virus vaccines and for the evolution of influenza A viruses in nature are discussed.     

Delayed appearance of pseudotypes between vesicular stomatitis virus influenza virus during mixed infection of MDCK cells.

In intact Madin-Darby canine kidney (MDCK) cell monolayers, vesicular stomatitis virus (VSV) matures only at basolateral membranes beneath tight junctions, whereas influenza virus buds from apical cell surfaces. Early in the growth cycle, the viral glycoproteins are restricted to the membrane domain from which each virus buds. We report here that phenotypic mixing and formation of VSV pseudotypes occurred when influenza virus-infected MDCK cells were superinfected with VSV. Up to 75% of the infectious VSV particles from such experiments were neutralized by antiserum specific for influenza virus, and a smaller proportion (up to 3%) were resistant to neutralization with antiserum specific for VSV. The latter particles, which were neutralized by antiserum to influenza A/WSN virus, are designated as VSV(WSN) pseudotypes. During mixed infections, both wild-type viruses were detected 1 to 2 h before either phenotypically mixed VSV or VSV(WSN) pseudotypes. Coincident with the appearance of cytopathic effects in the monolayer, the yield of pseudotypes rose dramatically. In contrast, in doubly infected BHK-21 cells, which do not show polarity in virus maturation sites and are not connected by tight junctions, VSV(WSN) pseudotypes were detected as soon as VSV titers rose to the minimum levels which allowed detection of pseudotypes, and the proportion observed remained relatively constant at later times. Examination of thin sections of doubly infected MDCK monolayers revealed that polarity in maturation sites was preserved for both viruses until approximately 12 h after inoculation with influenza virus, when disruption of junctional complexes was evident. Even at later periods, the majority of each virus type was associated with its normal membrane domain, suggesting that the sorting mechanisms responsible for directing the glycoproteins of VSV and influenza virus to separate surface domains continue to operate in doubly infected MDCK cells. The time course of VSV(WSN) pseudotype formation and changes in virus maturation sites are compatible with progressive mixing of viral glycoproteins at either intracellular or plasma membranes of doubly infected cells.     

Characterization of nonpathogenic, live, viral vaccine vectors inducing potent cellular immune responses

Experimental vaccines based on recombinant vesicular stomatitis viruses (VSV) expressing foreign viral proteins are protective in several animal disease models. Although these attenuated viruses are nonpathogenic in nonhuman primates when given by nasal, oral, or intramuscular routes, they are pathogenic in mice when given intranasally, and further vector attenuation may be required before human trials with VSV-based vectors can begin. Mutations truncating the VSV glycoprotein (G) cytoplasmic domain from 29 to 9 or 1 amino acid (designated CT9 or CT1, respectively) were shown previously to attenuate VSV growth in cell culture and pathogenesis in mice. Here we show that VSV recombinants carrying either the CT1 or CT9 deletion and expressing the human immunodeficiency virus (HIV) Env protein are nonpathogenic in mice, even when given by the intranasal route. We then carried out a detailed analysis of the CD8+ T-cell responses, including in vivo cytotoxic T-cell activity, induced by these vectors. When given by either the intranasal or intraperitoneal route, the VSV-CT9 vector expressing HIV Env elicited primary and memory CD8+ T-cell responses to Env equivalent to those elicited by recombinant wild-type VSV expressing Env. The VSV-CT1 vector also induced potent CD8+ T-cell responses after intraperitoneal vaccination, but was less effective when given by the intranasal route. The VSV-CT1 vector was also substantially less effective than the VSV-CT9 or wild-type vector at inducing antibody to Env. The VSV-CT9 vector appears ideal because of its lack of pathogenesis, propagation to high titers in vitro, and stimulation of strong cellular and humoral immune responses.     

Morphological and biochemical characterization of viral particles produced by the tsO45 mutant of vesicular stomatitis virus at restrictive temperature.

The growth at restrictive temperature of tsO45, a group V (glycoprotein) conditional lethal mutant of vesicular stomatitis virus (VSV), was demonstrated to result in the production of large numbers of noninfectious viral particles. The infectivity of these tsO45 particles could be enhanced by procedures known to promote membrane fusion. Morphologically and biochemically these particles differed from wild-type VSV by their lack of viral glycoprotein. The other structural proteins of VSV were present and indistinguishable by size and relative proportion from those of virus grown at the permissive temperature. Examination of glycoprotein maturation at the restrictive temperature (39.5 degrees C) in tsO45-infected cells demonstrated the synthesis of normal viral glycoprotein but failed to demonstrate the presence of this glycoprotein in either the cell membrane or the envelope of free virions. The further absence of soluble viral glycoprotein from the supernatants of such cells strongly suggests that viral glycoprotein may not be necessary for the successful budding of VSV.     

A Chimeric Vesiculo/Alphavirus Is an Effective Alphavirus Vaccine

While a large number of mosquito-transmitted alphaviruses are known to cause serious human diseases, there are no licensed vaccines that protect against alphavirus infections. The alphavirus chikungunya virus (CHIKV) has caused multiple recent outbreaks of chikungunya fever. This virus has the potential to cause a worldwide epidemic and has generated strong interest in development of a prophylactic CHIKV vaccine. We report here on the development of a potent experimental vaccine for CHIKV based on a chimeric vesicular stomatitis virus (VSV) expressing the entire CHIKV envelope polyprotein (E3-E2-6K-E1) in place of the VSV glycoprotein (G). These VSVΔG-CHIKV chimeras incorporated functional CHIKV glycoproteins into the viral envelope in place of VSV G. The chimeric viruses were attenuated for growth in tissue culture but could be propagated to high titers without VSV G complementation. They also generated robust neutralizing antibody and cellular immune responses to CHIKV in mice after a single dose and protected mice against CHIKV infection. VSVΔG-alphavirus chimeras could have general applicability as alphavirus vaccines.     

Wide occurrence of measles virus subgenomic RNAs in attenuated live-virus vaccines

Nine measles vaccine preparations, including four different viral strains, provided by eight different manufacturers were analysed by Northern blot for the nature of their nucleocapsid RNAs. Out of nine preparations, six were shown to contain subgenomic RNAs, along with the full length genomic RNA. Presence or absence of the subgenomic RNAs correlated strictly with the viral strains used. The role of the defective interfering particles in measles virus vaccine attenuation, and in its seroconversion efficacy upon vaccination, as well as the potential hazard of the presence of defective interfering particles in live-virus vaccine preparations, is discussed.     

Cellular Inflammatory Response to Flaviviruses in the Central Nervous System of a Primate Host

Flaviviruses such as tick-borne encephalitis virus, Japanese encephalitis virus, West Nile virus, and St. Louis encephalitis virus are important neurotropic human pathogens, typically causing a devastating and often fatal neuroinfection. Flaviviruses induce neuroinflammation with typical features of viral encephalitides, including inflammatory cell infiltration, activation of microglia, and neuronal degeneration. Development of safe and effective live-virus vaccines against neurotropic flavivirus infections demands a detailed knowledge of their neuropathogenesis in a primate host that is evolutionarily close to humans. Here, we used computerized morphometric analysis to quantitatively assess the cellular inflammatory responses in the central nervous system (CNS) of rhesus monkeys infected with three antigenically divergent attenuated flaviviruses. The kinetics, spatial pattern, and magnitude of microglial activation, trafficking of T and B cells, and changes in T cell subsets within the CNS define unique phenotypic signatures for each of the three viruses. Our results provide a benchmark for investigation of cellular inflammatory responses induced by attenuated flaviviruses in the CNS of primate hosts and provide insight into the neuropathogenesis of flavivirus encephalitis that might guide the development of safe and effective live-virus vaccines. (J Histochem Cytochem 57:973–989, 2009)     

The Double-Edged Sword: How Evolution Can Make or Break a Live-Attenuated Virus Vaccine

Even students who reject evolution are often willing to consider cases in which evolutionary biology contributes to, or undermines, biomedical interventions. Moreover, the intersection of evolutionary biology and biomedicine is fascinating in its own right. This review offers an overview of the ways in which evolution has impacted the design and deployment of live-attenuated virus vaccines, with subsections that may be useful as lecture material or as the basis for case studies in classes at a variety of levels. Live-attenuated virus vaccines have been modified in ways that restrain their replication in a host so that infection (vaccination) produces immunity but not disease. Applied evolution, in the form of serial passage in novel host cells, is a “classical” method to generate live-attenuated viruses. However, many live-attenuated vaccines exhibit reversion to virulence through back-mutation of attenuating mutations, compensatory mutations elsewhere in the genome, recombination or reassortment, or changes in quasispecies diversity. Additionally, the combination of multiple live-attenuated strains may result in competition or facilitation between individual vaccine viruses, resulting in undesirable increases in virulence or decreases in immunogenicity. Genetic engineering informed by evolutionary thinking has led to a number of novel approaches to generate live-attenuated virus vaccines that contain substantial safeguards against reversion to virulence and that ameliorate interference among multiple vaccine strains. Finally, vaccines have the potential to shape the evolution of their wild-type counterparts in counter-productive ways; at the extreme, vaccine-driven eradication of a virus may create an empty niche that promotes the emergence of new viral pathogens.     

Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture

Cultured hippocampal neurons were infected with a temperature-sensitive mutant of vesicular stomatitis virus (VSV) and a wild-type strain of the avian influenza fowl plague virus (FPV). The intracellular distribution of viral glycoproteins was monitored by immunofluorescence microscopy. In mature, fully polarized neurons the VSV glycoprotein (a basolateral protein in epithelial MDCK cells) moved from the Golgi complex to the dendritic domain, whereas the hemagglutinin protein of FPV (an apically sorted protein in MDCK cells) was targeted preferentially, but not exclusively, to the axon. The VSV glycoprotein appeared in clusters on the dendritic surface, while the hemagglutinin was distributed uniformly along the axonal membrane. Based on the finding that the same viral glycoproteins are sorted in a polarized fashion in both neuronal and epithelial cells, we propose that the molecular mechanisms of surface protein sorting share common features in the two cell types.