Wild-type Vesicular Stomatitis Virus Research Articles

Calpain-2 mediates SARS-CoV-2 entry via regulating ACE2 levels.

Many efforts in small-molecule screens have been made to counter SARS-CoV-2 infection by targeting the viral main protease, the major element that processes viral proteins after translation. Here, we discovered that calpain inhibitors further block SARS-CoV-2 infection in a main protease-independent manner. We identified the host cysteine protease calpain-2 as an important positive regulator of the cell surface levels of SARS-CoV-2 cellular receptor ACE2 and, thus, a facilitator of viral infection. By either pharmacological inhibition or genetic knockout of calpain-2, the SARS-CoV-2 binding to host cells is blocked and viral infection is decreased. Our findings highlight a novel mechanism of ACE2 regulation, which presents a potential new therapeutic target. Since calpain inhibitors also potently interfere with the viral main protease, our data also provide a mechanistic understanding of the potential use of calpain inhibitors as dual inhibitors (entry and replication) in the clinical setting of COVID-19 diseases. Our findings bring mechanistic insights into the cellular process of SARS-CoV-2 entry and offer a novel explanation to the mechanism of activities of calpain inhibitors.

Recombinant vesicular stomatitis vaccine against Nipah virus has a favorable safety profile: Model for assessment of live vaccines with neurotropic potential.

Nipah virus (NiV) disease is a bat-borne zoonosis responsible for outbreaks with high lethality and is a priority for vaccine development. With funding from the Coalition of Epidemic Preparedness Innovations (CEPI), we are developing a chimeric vaccine (PHV02) composed of recombinant vesicular stomatitis virus (VSV) expressing the envelope glycoproteins of both Ebola virus (EBOV) and NiV. The EBOV glycoprotein (GP) mediates fusion and viral entry and the NiV attachment glycoprotein (G) is a ligand for cell receptors, and stimulates neutralizing antibody, the putative mediator of protection against NiV. PHV02 is identical in construction to the registered Ebola vaccine (Ervebo) with the addition of the NiV G gene. NiV ephrin B2 and B3 receptors are expressed on neural cells and the wild-type NiV is neurotropic and causes encephalitis in affected patients. It was therefore important to assess whether the NiV G alters tropism of the rVSV vector and serves as a virulence factor. PHV02 was fully attenuated in adult hamsters inoculated by the intramuscular (IM) route, whereas parental wild-type VSV was 100% lethal. Two rodent models (mice, hamsters) were infected by the intracerebral (IC) route with graded doses of PHV02. Comparator active controls in various experiments included rVSV-EBOV (representative of Ebola vaccine) and yellow fever (YF) 17DD commercial vaccine. These studies showed PHV02 to be more neurovirulent than both rVSV-EBOV and YF 17DD in infant animals. PHV02 was lethal for adult hamsters inoculated IC but not for adult mice. In contrast YF 17DD retained virulence for adult mice inoculated IC but was not virulent for adult hamsters. Because of the inconsistency of neurovirulence patterns in the rodent models, a monkey neurovirulence test (MNVT) was performed, using YF 17DD as the active comparator because it has a well-established profile of quantifiable microscopic changes in brain centers and a known reporting rate of neurotropic adverse events in humans. In the MNVT PHV02 was significantly less neurovirulent than the YF 17DD vaccine reference control, indicating that the vaccine will have an acceptable safety profile for humans. The findings are important because they illustrate the complexities of phenotypic assessment of novel viral vectors with tissue tropisms determined by transgenic proteins, and because it is unprecedented to use a heterologous comparator virus (YF vaccine) in a regulatory-enabling study. This approach may have value in future studies of other novel viral vectors.

Differential infection of murine and human dendritic cell subsets by oncolytic vesicular stomatitis virus variants

ABSTRACT Oncolytic viruses (OVs) can eradicate tumor cells and elicit antitumor immunity. VSV-GP, a chimeric vesicular stomatitis virus (VSV) with the glycoprotein (GP) of the lymphocytic choriomeningitis virus, is a promising new OV candidate. However, the interaction of VSV-GP with host immune cells is not fully understood. Dendritic cells (DCs) are essential for inducing efficient antitumor immunity. Thus, we aimed to investigate the interaction of VSV-GP with different murine and human DCs subsets in direct comparison to the less cytopathic variant VSV-dM51-GP and wild type VSV. Immature murine bone marrow-derived DCs (BMDCs) were equally infected and killed by VSV and VSV-GP. Human monocyte-derived DCs (moDCs) were more permissive to VSV. Interestingly, VSV-dM51-GP induced maturation instead of killing in both BMDCs and moDCs as well as a pronounced release of pro-inflammatory cytokines. Importantly, matured BMDCs and moDCs were no longer susceptible to VSV-GP infection. Mouse splenic conventional DC type 1 (cDC1) could be infected ex vivo by VSV and VSV-GP to a higher extent than cDC2. Systemic infection of mice with VSV-GP and VSV-dM51-GP resulted in strong activation of cDCs despite low infection rates in spleen and tumor tissue. Human blood cDC1 were equally infected by VSV and VSV-GP, whereas cDC2 showed preferential infection with VSV. Our study demonstrated differential DC infection, activation, and cytokine production after the treatment with VSV and VSV-GP variants among species and subsets, which should be taken into account when investigating immunological mechanisms of oncolytic virotherapy in mouse models and human clinical trials.

High dose of vesicular stomatitis virus-vectored Ebola virus vaccine causes vesicular disease in swine without horizontal transmission

ABSTRACT The recent impact of Ebola virus disease (EVD) on public health in Africa clearly demonstrates the need for a safe and efficacious vaccine to control outbreaks and mitigate its threat to global health. ERVEBO® is an effective recombinant Vesicular Stomatitis Virus (VSV)-vectored Ebola virus vaccine (VSV-EBOV) that was approved by the FDA and EMA in late 2019 for use in prevention of EVD. Since the parental virus VSV, which was used to construct VSV-EBOV, is pathogenic for livestock and the vaccine virus may be shed at low levels by vaccinated humans, widespread deployment of the vaccine requires investigation into its infectivity and transmissibility in VSV-susceptible livestock species. We therefore performed a comprehensive clinical analysis of the VSV-EBOV vaccine virus in swine to determine its infectivity and potential for transmission. A high dose of VSV-EBOV resulted in VSV-like clinical signs in swine, with a proportion of pigs developing ulcerative vesicular lesions at the nasal injection site and feet. Uninoculated contact control pigs co-mingled with VSV-EBOV-inoculated pigs did not become infected or display any clinical signs of disease, indicating the vaccine is not readily transmissible to naïve pigs during prolonged close contact. In contrast, virulent wild-type VSV Indiana had a shorter incubation period and was transmitted to contact control pigs. These results indicate that the VSV-EBOV vaccine causes vesicular illness in swine when administered at a high dose. Moreover, the study demonstrates the VSV-EBOV vaccine is not readily transmitted to uninfected pigs, encouraging its safe use as an effective human vaccine.

A mutant vesicular stomatitis virus with reduced cytotoxicity and enhanced anterograde trans-synaptic efficiency

Understanding the connecting structure of brain network is the basis to reveal the principle of the brain function and elucidate the mechanism of brain diseases. Trans-synaptic tracing with neurotropic viruses has become one of the most effective technologies to dissect the neural circuits. Although the retrograde trans-synaptic tracing for analyzing the input neural networks with recombinant rabies and pseudorabies virus has been broadly applied in neuroscience, viral tools for analyzing the output neural networks are still lacking. The recombinant vesicular stomatitis virus (VSV) has been used for the mapping of synaptic outputs. However, several drawbacks, including high neurotoxicity and rapid lethality in experimental animals, hinder its application in long-term studies of the structure and function of neural networks. To overcome these limitations, we generated a recombinant VSV with replication-related N gene mutation, VSV-NR7A, and examined its cytotoxicity and efficiency of trans-synaptic spreading. We found that by comparison with the wild-type tracer of VSV, the NR7A mutation endowed the virus lower rate of propagation and cytotoxicity in vitro, as well as significantly reduced neural inflammatory responses in vivo and much longer animal survival when it was injected into the nucleus of the mice brain. Besides, the spreading of the attenuated VSV was delayed when injected into the VTA. Importantly, with the reduced toxicity and extended animal survival, the number of brain regions that was trans-synaptically labeled by the mutant VSV was more than that of the wild-type VSV. These results indicated that the VSV-NR7A, could be a promising anterograde tracer that enables researchers to explore more downstream connections of a given brain region, and observe the anatomical structure and the function of the downstream circuits over a longer time window. Our work could provide an improved tool for structural and functional studies of neurocircuit.

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.

M51R and Delta-M51 matrix protein of the vesicular stomatitis virus induce apoptosis in colorectal cancer cells.

Colorectal cancer (CRC) is the third most common cancer in both men and women. Oncolytic viral-based therapy methods seem to be promising for CRC treatment. Vesicular stomatitis virus (VSV) is considered as a potent candidate in viral therapy for several tumors. VSV particles with mutated matrix (M) protein are capable of initiating cell death cascades while not being harmful to the immune system. In the current study, the effects of the VSV M-protein was investigated on the apoptosis of the colorectal cancer SW480 cell. Wild-type, M51R, and ΔM51 mutants VSV M-protein genes were cloned into the PCDNA3.1 vector and transfected into the SW480 cells. The results of the MTT assay, Western blotting, and Caspase 3, 8, and 9 measurement, illustrated that both wild and M51R mutant M-proteins can destroy the SW480 colorectal cancer cells. DAPI/TUNEL double-staining reconfirmed the apoptotic effects of the M-protein expression. The ΔM51 mutant M-protein is effective likewise M51R, somehow it can be considered as a safer substitution.

A Highly Attenuated Vesicular Stomatitis Virus-Based Vaccine Platform Controls Hepatitis B Virus Replication in Mouse Models of Hepatitis B.

Therapeutic vaccines may be an important component of a treatment regimen for curing chronic hepatitis B virus (HBV) infection. We previously demonstrated that recombinant wild-type vesicular stomatitis virus (VSV) expressing the HBV middle surface glycoprotein (MHBs) elicits functional immune responses in mouse models of HBV replication. However, VSV has some undesirable pathogenic properties, and the use of this platform in humans requires further viral attenuation. We therefore generated a highly attenuated VSV that expresses MHBs and contains two attenuating mutations. This vector was evaluated for immunogenicity, pathogenesis, and anti-HBV function in mice. Compared to wild-type VSV, the highly attenuated virus displayed markedly reduced pathogenesis but induced similar MHBs-specific CD8+ T cell and antibody responses. The CD8+ T cell responses elicited by this vector in naive mice prevented HBV replication in animals that were later challenged by hydrodynamic injection or transduction with adeno-associated virus encoding the HBV genome (AAV-HBV). In mice in which persistent HBV replication was first established by AAV-HBV transduction, subsequent immunization with the attenuated VSV induced MHBs-specific CD8+ T cell responses that corresponded with reductions in serum and liver HBV antigens and nucleic acids. HBV control was associated with an increase in the frequency of intrahepatic HBV-specific CD8+ T cells and a transient elevation in serum alanine aminotransferase activity. The ability of VSV to induce a robust multispecific T cell response that controls HBV replication combined with the improved safety profile of the highly attenuated vector suggests that this platform offers a new approach for HBV therapeutic vaccination.IMPORTANCE A curative treatment for chronic hepatitis B must eliminate the virus from the liver, but current antiviral therapies typically fail to do so. Immune-mediated resolution of infection occurs in a small fraction of chronic HBV patients, which suggests the potential efficacy of therapeutic strategies that boost the patient's own immune response to the virus. We modified a safe form of VSV to express an immunogenic HBV protein and evaluated the efficacy of this vector in the prevention and treatment of HBV infection in mouse models. Our results show that this vector elicits HBV-specific immune responses that prevent the establishment of HBV infection and reduce viral proteins in the serum and viral DNA/RNA in the liver of mice with persistent HBV replication. These findings suggest that highly attenuated and safe virus-based vaccine platforms have the potential to be utilized for the development of an effective therapeutic vaccine against chronic HBV infection.

Cell Cycle Arrest in G2/M Phase Enhances Replication of Interferon-Sensitive Cytoplasmic RNA Viruses via Inhibition of Antiviral Gene Expression.

Vesicular stomatitis virus (VSV) (a rhabdovirus) and its variant VSV-ΔM51 are widely used model systems to study mechanisms of virus-host interactions. Here, we investigated how the cell cycle affects replication of these viruses using an array of cell lines with different levels of impairment of antiviral signaling and a panel of chemical compounds arresting the cell cycle at different phases. We observed that all compounds inducing cell cycle arrest in G2/M phase strongly enhanced the replication of VSV-ΔM51 in cells with functional antiviral signaling. G2/M arrest strongly inhibited type I and type III interferon (IFN) production as well as expression of IFN-stimulated genes in response to exogenously added IFN. Moreover, G2/M arrest enhanced the replication of Sendai virus (a paramyxovirus), which is also highly sensitive to the type I IFN response but did not stimulate the replication of a wild-type VSV that is more effective at evading antiviral responses. In contrast, the positive effect of G2/M arrest on virus replication was not observed in cells defective in IFN signaling. Altogether, our data show that replication of IFN-sensitive cytoplasmic viruses can be strongly stimulated during G2/M phase as a result of inhibition of antiviral gene expression, likely due to mitotic inhibition of transcription, a global repression of cellular transcription during G2/M phase. The G2/M phase thus could represent an "Achilles' heel" of the infected cell, a phase when the cell is inadequately protected. This model could explain at least one of the reasons why many viruses have been shown to induce G2/M arrest.IMPORTANCE Vesicular stomatitis virus (VSV) (a rhabdovirus) and its variant VSV-ΔM51 are widely used model systems to study mechanisms of virus-host interactions. Here, we investigated how the cell cycle affects replication of VSV and VSV-ΔM51. We show that G2/M cell cycle arrest strongly enhances the replication of VSV-ΔM51 (but not of wild-type VSV) and Sendai virus (a paramyxovirus) via inhibition of antiviral gene expression, likely due to mitotic inhibition of transcription, a global repression of cellular transcription during G2/M phase. Our data suggest that the G2/M phase could represent an "Achilles' heel" of the infected cell, a phase when the cell is inadequately protected. This model could explain at least one of the reasons why many viruses have been shown to induce G2/M arrest, and it has important implications for oncolytic virotherapy, suggesting that frequent cell cycle progression in cancer cells could make them more permissive to viruses.

Chikungunya-vesicular stomatitis chimeric virus targets and eliminates brain tumors

Vesicular stomatitis virus (VSV) shows potential for targeting and killing cancer cells, but can be dangerous in the brain due to its neurotropic glycoprotein. Here we test a chimeric virus in which the VSV glycoprotein is replaced with the Chikungunya polyprotein E3-E2-6K-E1 (VSVΔG-CHIKV). Control mice with brain tumors survived a mean of 40 days after tumor implant. VSVΔG-CHIKV selectively infected and eliminated the tumor, and extended survival substantially in all tumor-bearing mice to over 100 days. VSVΔG-CHIKV also targeted intracranial primary patient derived melanoma xenografts. Virus injected into one melanoma spread to other melanomas within the same brain with little detectable infection of normal cells. Intravenous VSVΔG-CHIKV infected tumor cells but not normal tissue. In immunocompetent mice, VSVΔG-CHIKV selectively infected mouse melanoma cells within the brain. These data suggest VSVΔG-CHIKV can target and destroy brain tumors in multiple animal models without the neurotropism associated with the wild type VSV glycoprotein.

Porcine monocyte-derived dendritic cells can be differentially activated by vesicular stomatitis virus and its matrix protein mutants

Vesicular stomatitis virus (VSV) can cause serious vesicular lesions in pigs, and the matrix (M) protein is its predominant virulence factor. Dendritic cells (DCs) act as the bridge between innate and adaptive immune responses. However, the susceptibility of porcine DCs to VSV infection and the role of M protein in modulating the function of infected DCs are still poorly defined. Thus, this study aimed to determine the ability of virulent wild-type VSV(wtVSV) and two attenuated M protein variants (VSVΔM51 and VSVMT) to induce maturation of porcine monocyte-derived DCs (MoDCs) in vitro. It was found that both wtVSV and the M protein mutant VSVs could productively replicate in porcine MoDCs. Infection with wtVSV resulted in weak proinflammatory cytokine responses and interfered with DC maturation via downregulation of the costimulatory molecule complex CD80/86. Whilst VSVΔM51 could activate porcine MoDCs, VSVMT, a highly attenuated recombinant VSV with triple mutations in the M protein, induced a potent maturation of MoDCs, as evidenced by efficient cytokine induction, and upregulation of CD80/86 and MHC class II. Overall, our findings reveal that porcine MoDCs are differentially activated by VSV, dependent on the presence of a functional M protein. M protein plays a crucial role in modulating porcine DC-VSV interactions. The data further support the potential use of VSVMT as a vaccine vector for pigs.

Chikungunya, Influenza, Nipah, and Semliki Forest Chimeric Viruses with Vesicular Stomatitis Virus: Actions in the Brain.

Recombinant vesicular stomatitis virus (VSV)-based chimeric viruses that include genes from other viruses show promise as vaccines and oncolytic viruses. However, the critical safety concern is the neurotropic nature conveyed by the VSV glycoprotein. VSVs that include the VSV glycoprotein (G) gene, even in most recombinant attenuated strains, can still show substantial adverse or lethal actions in the brain. Here, we test 4 chimeric viruses in the brain, including those in which glycoprotein genes from Nipah, chikungunya (CHIKV), and influenza H5N1 viruses were substituted for the VSV glycoprotein gene. We also test a virus-like vesicle (VLV) in which the VSV glycoprotein gene is expressed from a replicon encoding the nonstructural proteins of Semliki Forest virus. VSVΔG-CHIKV, VSVΔG-H5N1, and VLV were all safe in the adult mouse brain, as were VSVΔG viruses expressing either the Nipah F or G glycoprotein. In contrast, a complementing pair of VSVΔG viruses expressing Nipah G and F glycoproteins were lethal within the brain within a surprisingly short time frame of 2 days. Intranasal inoculation in postnatal day 14 mice with VSVΔG-CHIKV or VLV evoked no adverse response, whereas VSVΔG-H5N1 by this route was lethal in most mice. A key immune mechanism underlying the safety of VSVΔG-CHIKV, VSVΔG-H5N1, and VLV in the adult brain was the type I interferon response; all three viruses were lethal in the brains of adult mice lacking the interferon receptor, suggesting that the viruses can infect and replicate and spread in brain cells if not blocked by interferon-stimulated genes within the brain.IMPORTANCE Vesicular stomatitis virus (VSV) shows considerable promise both as a vaccine vector and as an oncolytic virus. The greatest limitation of VSV is that it is highly neurotropic and can be lethal within the brain. The neurotropism can be mostly attributed to the VSV G glycoprotein. Here, we test 4 chimeric viruses of VSV with glycoprotein genes from Nipah, chikungunya, and influenza viruses and nonstructural genes from Semliki Forest virus. Two of the four, VSVΔG-CHIKV and VLV, show substantially attenuated neurotropism and were safe in the healthy adult mouse brain. VSVΔG-H5N1 was safe in the adult brain but lethal in the younger brain. VSVΔG Nipah F+G was even more neurotropic than wild-type VSV, evoking a rapid lethal response in the adult brain. These results suggest that while chimeric VSVs show promise, each must be tested with both intranasal and intracranial administration to ensure the absence of lethal neurotropism.

Live virus vaccines based on a vesicular stomatitis virus (VSV) backbone: Standardized template with key considerations for a risk/benefit assessment

The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety of live, recombinant viral vaccines incorporating genes from heterologous viral and other microbial pathogens in their genome (so-called “chimeric virus vaccines”). Many such viral vector vaccines are now at various stages of clinical evaluation. Here, we introduce an attenuated form of recombinant vesicular stomatitis virus (rVSV) as a potential chimeric virus vaccine for HIV-1, with implications for use as a vaccine vector for other pathogens. The rVSV/HIV-1 vaccine vector was attenuated by combining two major genome modifications. These modifications acted synergistically to greatly enhance vector attenuation and the resulting rVSV vector demonstrated safety in sensitive mouse and non-human primate neurovirulence models. This vector expressing HIV-1 gag protein has completed evaluation in two Phase I clinical trials. In one trial the rVSV/HIV-1 vector was administered in a homologous two-dose regimen, and in a second trial with pDNA in a heterologous prime boost regimen. No serious adverse events were reported nor was vector detected in blood, urine or saliva post vaccination in either trial. Gag specific immune responses were induced in both trials with highest frequency T cell responses detected in the prime boost regimen. The rVSV/HIV-1 vector also demonstrated safety in an ongoing Phase I trial in HIV-1 positive participants. Additionally, clinical trial material has been produced with the rVSV vector expressing HIV-1 env, and Phase I clinical evaluation will initiate in the beginning of 2016. In this paper, we use a standardized template describing key characteristics of the novel rVSV vaccine vectors, in comparison to wild type VSV. The template facilitates scientific discourse among key stakeholders by increasing transparency and comparability of information. The Brighton Collaboration V3SWG template may also be useful as a guide to the evaluation of other recombinant viral vector vaccines.

Protective efficacy of a recombinant Newcastle disease virus expressing glycoprotein of vesicular stomatitis virus in mice

BackgroundVesicular stomatitis virus (VSV) causes severe losses to the animal husbandry industry. In this study, a recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of VSV (rL-VSV-G) was constructed and its pathogenicity and immune protective efficacy in mouse were evaluated.ResultsIn pathogenicity evaluation test, the analysis of the viral distribution in mouse organs and body weight change showed that rL-VSV-G was safe in mice. In immune protection assay, the recombinant rL-VSV-G triggered a high titer of neutralizing antibodies against VSV. After challenge, the wild-type (wt) VSV viral load in mouse organs was lower in rL-VSV-G group than that in rLaSota groups. wt VSV was not detected in the blood, liver, or kidneys of mice, whereas it was found in these tissues in control groups. The mice body weight had no significant change after challenge in the rL-VSV-G group. Additionally, suckling mice produced from female mice immunized with rL-VSV-G were partially protected from wt VSV challenge.ConclusionsThese results demonstrated that rL-VSV-G may be a suitable candidate vaccine against vesicular stomatitis (VS).

Abstract A191: Augmenting the therapeutic efficacy of oncolytic LCMV-GP pseudotyped vesicular stomatitis virus via modulation of the innate immune system

Abstract Introduction: Vesicular stomatitis virus (VSV) is a promising oncolytic virus candidate due to its fast mode of action, high titer production, absence of pre-existing anti-VSV immunity and broad tumor tropism. However, potential neurotoxicity of wild-type VSV and a rapid induction of neutralizing antibodies have been a hindrance for further clinical advancement. Our group previously reported that pseudotyping VSV with the LCMV glycoprotein (VSV-GP) results in complete abrogation of neurotoxicity and the absence of neutralizing antibody induction [1]. Here we addressed to what extent the innate immune response to VSV-GP limits its oncolytic efficacy and analyze means to counter these inhibiting factors. Methods: MTT cell viability and TCID50 viral replication assays were used to quantify oncolytic activity in vitro. VSV-GP oncolytic efficiency was also tested in vivo using both xenograft and syngeneic mouse models. Results: VSV-GP exhibited high oncolytic efficiency in vitro, achieving 80-100% killing at 48-72 h post infection in most ovarian, melanoma, and lung cancer cell lines. Further analysis of resistant cell lines showed that these cells were able to mount an interferon (IFN-I) induced antiviral response. VSV-GP efficacy was also tested for ovarian cancer in vivo using s.c xenograft models and in an orthotopic model. Intratumoral injection of VSV-GP into ovarian s.c. xenografts led to tumor remission in all animals. Relapse was observed and relapsing tumors remained sensitive for successive VSV-GP treatment. Significantly prolonged survival was also observed in an ovarian cancer orthotopic model using IVIS in vivo imaging. Oncolytic potency of VSV-GP was enhanced with combination treatment with Jak1/2 inhibitors. Conclusion: These results confirm that LCMV GP-pseudotyped VSV exhibits a highly beneficial toxicity and efficacy profile. However, tumors escaping complete oncolyse via innate immune system activation require coapplication of targeted immune modulators. Reference: [1] - Muik, A. et al. Re-engineering Vesicular Stomatitis Virus to Abrogate Neurotoxicity, Circumvent Humoral Immunity, and Enhance Oncolytic Potency. Cancer Res 74, 3567-3578, (2014). Citation Format: Carles Rodriguez Urbiola, Catherine Dold, Janine Kimpel, Christian Marth, Frederic Santer, Zoran Culig, Alexander Muik, Tanja Knapp, Ira Winkler, Guido Wollmann, Dorothee Von Laer, Patrik Erlmann. Augmenting the therapeutic efficacy of oncolytic LCMV-GP pseudotyped vesicular stomatitis virus via modulation of the innate immune system. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A191.

Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly.

Vesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) with the large polymerase protein (L). To study the assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of viruses with insertions in the M gene a replication-competent virus containing a fluorescent M and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintained the same bullet shape appearance as wild-type VSV but had a modest increase in particle length, reflecting the increased genome size. Imaging of the released particles revealed a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the late domains in M, which serve to recruit the endosomal sorting complex required for transport (ESCRT) machinery during budding. Mutations in late domains resulted in the accumulation of virions that failed to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M tagged with enhanced green fluorescent protein and M tagged with mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late-domain mutant into the released virions. In contrast, the intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV. Assembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein, to the plasma membrane. The matrix protein contains a motif termed a "late domain" that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate the release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. In the study described here, we developed new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We used those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P.

Lassa-vesicular stomatitis chimeric virus safely destroys brain tumors.

High-grade tumors in the brain are among the deadliest of cancers. Here, we took a promising oncolytic virus, vesicular stomatitis virus (VSV), and tested the hypothesis that the neurotoxicity associated with the virus could be eliminated without blocking its oncolytic potential in the brain by replacing the neurotropic VSV glycoprotein with the glycoprotein from one of five different viruses, including Ebola virus, Marburg virus, lymphocytic choriomeningitis virus (LCMV), rabies virus, and Lassa virus. Based on in vitro infections of normal and tumor cells, we selected two viruses to test in vivo. Wild-type VSV was lethal when injected directly into the brain. In contrast, a novel chimeric virus (VSV-LASV-GPC) containing genes from both the Lassa virus glycoprotein precursor (GPC) and VSV showed no adverse actions within or outside the brain and targeted and completely destroyed brain cancer, including high-grade glioblastoma and melanoma, even in metastatic cancer models. When mice had two brain tumors, intratumoral VSV-LASV-GPC injection in one tumor (glioma or melanoma) led to complete tumor destruction; importantly, the virus moved contralaterally within the brain to selectively infect the second noninjected tumor. A chimeric virus combining VSV genes with the gene coding for the Ebola virus glycoprotein was safe in the brain and also selectively targeted brain tumors but was substantially less effective in destroying brain tumors and prolonging survival of tumor-bearing mice. A tropism for multiple cancer types combined with an exquisite tumor specificity opens a new door to widespread application of VSV-LASV-GPC as a safe and efficacious oncolytic chimeric virus within the brain. Many viruses have been tested for their ability to target and kill cancer cells. Vesicular stomatitis virus (VSV) has shown substantial promise, but a key problem is that if it enters the brain, it can generate adverse neurologic consequences, including death. We tested a series of chimeric viruses containing genes coding for VSV, together with a gene coding for the glycoprotein from other viruses, including Ebola virus, Lassa virus, LCMV, rabies virus, and Marburg virus, which was substituted for the VSV glycoprotein gene. Ebola and Lassa chimeric viruses were safe in the brain and targeted brain tumors. Lassa-VSV was particularly effective, showed no adverse side effects even when injected directly into the brain, and targeted and destroyed two different types of deadly brain cancer, including glioblastoma and melanoma.

Independent of plasmacytoid dendritic cell (pDC) infection, pDC triggered by virus-infected cells mount enhanced type I IFN responses of different composition as opposed to pDC stimulated with free virus.

Upon treatment with vesicular stomatitis virus (VSV) particles, plasmacytoid dendritic cells (pDC) are triggered to mount substantial type I IFN responses, whereas myeloid DC (mDC) are only minor producers. Interestingly, bone marrow-derived (BM-)mDC were more vulnerable to infection with enhanced GFP (eGFP)-expressing VSV (VSVeGFP) than BM-pDC. BM-pDC stimulated with wild-type VSV mounted TLR-dependent IFN responses that were independent of RIG-I-like helicase (RLH) signaling. In contrast, in BM-pDC the VSV variant M2 induced particularly high IFN responses triggered in a TLR- and RLH-dependent manner, whereas BM-mDC stimulation was solely RLH-dependent. Importantly, VSVeGFP treatment of BM-pDC derived from IFN-β yellow fluorescent protein (YFP) reporter mice (messenger of IFN-β) resulted in YFP(+) and eGFP(+) single-positive cells, whereas among messenger of IFN-β-BM-mDC most YFP(+) cells were also eGFP(+). This observation indicated that unlike mDC, direct virus infection was not required to trigger IFN responses of pDC. VSV-infected BM-mDC triggered BM-pDC to mount significantly higher IFN responses than free virus particles. Stimulation with infected cells enhanced the percentages of pDC subsets expressing either IFN-β(+) or IFN-α6(+) plus IFN-β(+). Irrespective of whether stimulated with free virus or infected cells, IFN induction was dependent on autophagy of pDC, whereas autophagy of the infected mDC was dispensable. Collectively, these results indicated that productive VSV infection was needed to trigger IFN responses of mDC, but not of pDC, and that IFN responses were primarily induced by virus-infected cells that stimulated pDC in a TLR-dependent manner.

Digital sensing and sizing of vesicular stomatitis virus pseudotypes in complex media: a model for Ebola and Marburg detection.

Rapid, sensitive, and direct label-free capture and characterization of nanoparticles from complex media such as blood or serum will broadly impact medicine and the life sciences. We demonstrate identification of virus particles in complex samples for replication-competent wild-type vesicular stomatitis virus (VSV), defective VSV, and Ebola- and Marburg-pseudotyped VSV with high sensitivity and specificity. Size discrimination of the imaged nanoparticles (virions) allows differentiation between modified viruses having different genome lengths and facilitates a reduction in the counting of nonspecifically bound particles to achieve a limit-of-detection (LOD) of 5 × 10(3) pfu/mL for the Ebola and Marburg VSV pseudotypes. We demonstrate the simultaneous detection of multiple viruses in a single sample (composed of serum or whole blood) for screening applications and uncompromised detection capabilities in samples contaminated with high levels of bacteria. By employing affinity-based capture, size discrimination, and a "digital" detection scheme to count single virus particles, we show that a robust and sensitive virus/nanoparticle sensing assay can be established for targets in complex samples. The nanoparticle microscopy system is termed the Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and is capable of high-throughput and rapid sizing of large numbers of biological nanoparticles on an antibody microarray for research and diagnostic applications.

Oncolytic vesicular stomatitis virus as a treatment for neuroendocrine tumors

Therapeutic goals for neuroendocrine tumors (NETs) not amenable to operative cure are limited to relieving symptoms and slowing progression. Many malignancies acquire defective antiviral responses as they undergo unregulated proliferation. Therefore, we explored the abilities of recombinant wild-type vesicular stomatitis virus and an attenuated matrix protein mutant (M51R-VSV) to exploit defective antiviral pathways in NETs. Viral infectivity and lethality were evaluated in a panel of human NET cell lines H727, UMC-11, and CNDT2.5. We evaluated β-interferon pathways in these cells to define the acquired defect. Murine xenografts were treated with a single intratumoral injection of M51R-VSV to study viral efficacy in vivo. VSV infected >99% of cells within 24 hours and killed >95% within 72 hours. NET cells did not produce relevant amounts of β-interferon after infection, but exogenous β-interferon protected cells from oncolysis. Treatment with M51R-VSV resulted in suppressed tumor growth (mean value ± standard error of the mean) compared with mock-infected xenografts for H727 (87 ± 72% vs. 2,197 ± 335%; P < .001), UMC-11 (13 ± 59% vs. 1,471 ± 324%; P < .001), and CNDT2.5 (81 ± 121% vs. 1,576 ± 349%; P = .001). VSV infects and kills human NETs by exploiting their inability to produce a type I antiviral response. Therefore, M51R-VSV is an excellent candidate for the treatment of advanced NETs.