Intertumoral heterogeneity impacts oncolytic vesicular stomatitis virus efficacy in mouse pancreatic cancer cells.

Oncolytic virus (OV) therapy is a promising virus-based approach against various malignancies, including pancreatic ductal adenocarcinoma (PDAC). Our previous studies demonstrated that human PDAC cell lines are highly variable in their permissiveness to OVs. Mouse PDAC cell lines, which are widely used for in vivo examination of the adaptive immune responses during OV and other cancer therapies, have never been examined systematically for the impact of intertumoral heterogeneity (the differences observed between tumors in different patients) on OV virus efficacy. Here, we examined phenotypically and genotypically three commonly used allograftable mouse PDAC cell lines (C57BL6 genetic background): Panc02 (derived from chemically induced PDAC; also known as Pan02), and two cell lines originated from PDACs developed in two different KPC (KrasG12D, Trp53R172H, and PDX-1-Cre) mouse models. Our study (i) characterized the ability of a widely used attenuated oncolytic vesicular stomatitis virus VSV-ΔM51-GFP to infect, replicate in, and kill mouse PDAC cells; (ii) examined their innate antiviral responses; (iii) compared their permissiveness to a non-attenuated VSV-Mwt-GFP and chemotherapeutic drugs; and (iv) analyzed their karyotype and exome. Mouse PDAC cell lines showed high divergence in their permissiveness to VSV-ΔM51-GFP, which negatively correlated with their abilities to mount innate antiviral responses, while all three cell lines were highly permissive to VSV-Mwt-GFP. No correlation was found between resistance to VSV-ΔM51-GFP and chemotherapy. Also, mouse PDAC cell lines showed high divergence in their karyotype and exome. The exome analysis demonstrated that more VSV-ΔM51-GFP-permissive mouse PDAC cell lines harbor mutations in multiple important antiviral genes, such as TYK2, JAK2, and JAK3. IMPORTANCE Oncolytic virus (OV) therapy is a promising virus-based approach against various malignancies, including pancreatic ductal adenocarcinoma (PDAC). Our previous studies using various human PDAC cell lines demonstrated that they are highly variable in their permissiveness to OVs. In this study, we examined phenotypically and genotypically three commonly used allograftable mouse PDAC cell lines, which are widely used for in vivo examination of the adaptive immune responses during cancer therapies. Mouse PDAC cell lines showed high divergence in their permissiveness to oncolytic vesicular stomatitis virus (VSV), which negatively correlated with their abilities to mount innate antiviral responses. Also, we discovered that more VSV-permissive mouse PDAC cell lines harbor mutations in multiple important antiviral genes, such as TYK2, JAK2, and JAK3. Our study provides essential information about three model mouse PDAC cell lines and proposes a novel platform to study OV-based therapies against different PDACs in immunocompetent mice.

Vesicular stomatitis virus sensitizes immunologically cold tumors to checkpoint blockade by inducing pyroptosis.

BACKGROUND: Traditionally, vesicular stomatitis virus (VSV) and other oncolytic viruses (OVs) are thought to kill tumors by inducing apoptosis. However, cell apoptosis leads to immune quiescence, which is incompatible with the ability of OVs to activate the antitumor immune microenvironment. Thus, studying OVs-mediated oncolytic mechanisms is of great importance for the clinical application of OVs. METHODS: We examined the pyroptosis in tumor cells and tissues by morphological observation, Lactate Dehydrogenase (LDH) assay, frozen section observation, and western-blotting techniques. The critical role of GSDME in VSV-induced pyroptosis was confirmed by CRISPR/Cas9 technique. VSV virotherapy-recruited cytotoxic lymphocytes in the tumors were examined by flow cytometry assay. VSV-activated antitumor immunity was further enhanced by the co-administration with anti-PD-1 antibody. RESULTS: Here, we observed that VSV was able to trigger tumor pyroptosis through Gasdermin E (GSDME) in tumor cells, human tumor samples, and tumor-bearing mouse models. Importantly, the effectiveness of VSV-based virotherapy is highly dependent on GSDME, as depletion of GSDME not only reverses VSV-induced tumor-suppressive effects but also diminishes the ability of VSV to activate antitumor immunity. Notably, VSV treatment makes immunologically 'cold' tumors more sensitive to checkpoint blockade. CONCLUSIONS: Oncolytic VSV induces tumor cell pyroptosis by activating GSDME. GSDME is critical in recruiting cytotoxic T lymphocytes in the context of VSV therapy, which can switch immunologically 'cold' tumors into 'hot' and enhance immune checkpoint therapy efficacy.

Computational prediction of intracellular targets of wild-type or mutant vesicular stomatitis matrix protein.

The matrix (M) protein of vesicular stomatitis virus (VSV) has a complex role in infection and immune evasion, particularly with respect to suppression of Type I interferon (IFN). Viral strains bearing the wild-type (wt) M protein are able to suppress Type I IFN responses. We recently reported that the 22-25 strain of VSV encodes a wt M protein, however its sister plaque isolate, strain 22-20, carries a M[MD52G] mutation that perturbs the ability of the M protein to block NFκB, but not M-mediated inhibition of host transcription. Therefore, although NFκB is activated in 22-20 infected murine L929 cells infected, no IFN mRNA or protein is produced. To investigate the impact of the M[D52G] mutation on immune evasion by VSV, we used transcriptomic data from L929 cells infected with wt, 22-25, or 22-20 to define parameters in a family of executable logical models with the aim of discovering direct targets of viruses encoding a wt or mutant M protein. After several generations of pruning or fixing hypothetical regulatory interactions, we identified specific predicted targets of each strain. We predict that wt and 22-25 VSV both have direct inhibitory actions on key elements of the NFκB signaling pathway, while 22-20 fails to inhibit this pathway.

LDL receptor and pathogen processes: Functions beyond normal lipids.

Although the role of the LDL receptor concerning lipids is well known, its role in various viral and parasitic infections, and in regulating the inflammatory response is poorly understood. Several infectious agents use the LDL receptor as a port of entry, and others depend on it for their cycle of infection. In this review, we focus on the discovery, structure, and normal function of the LDL receptor, as well as its role in a selection of infections. The LDL receptor plays an important role in certain infections and is a potential target for treatment deserving further research.

Characterization of the therapeutic effect of antibodies targeting the Ebola glycoprotein using a novel BSL2-compliant rVSVΔG-EBOV-GP infection model.

Ebola virus (EBOV) infections cause haemorrhagic fever, multi-organ failure and death, and survivors can experience neurological sequelae. Licensing of monoclonal antibodies targeting EBOV glycoprotein (EBOV-GP) improved its prognosis, however, this treatment is primarily effective during early stages of disease and its effectiveness in reducing neurological sequela remains unknown. Currently, the need for BSL4 containment hinders research and therapeutic development; development of an accessible BSL-2 in vivo mouse model would facilitate preclinical studies to screen and select therapeutics. Previously, we have shown that a subcutaneous inoculation with replicating EBOV-GP pseudotyped vesicular stomatitis virus (rVSVΔG-EBOV-GP or VSV-EBOV) in neonatal mice causes transient viremia and infection of the mid and posterior brain resulting in overt neurological symptoms and death. Here, we demonstrate that the model can be used to test therapeutics that target the EBOV-GP, by using an anti-EBOV-GP therapeutic (SAB-139) previously shown to block EBOV infection in mice and primates. We show that SAB-139 treatment decreases the severity of neurological symptoms and improves survival when administered before (1 day prior to infection) or up to 3 dpi, by which time animals have high virus titres in their brains. Improved survival was associated with reduced viral titres, microglia loss, cellular infiltration/activation, and inflammatory responses in the brain. Interestingly, SAB-139 treatment significantly reduced the severe VSV-EBOV-induced long-term neurological sequalae although convalescent mice showed modest evidence of abnormal fear responses. Together, these data suggest that the neonatal VSV-EBOV infection system can be used to facilitate assessment of therapeutics targeting EBOV-GP in the preclinical setting.

A modular self-adjuvanting cancer vaccine combined with an oncolytic vaccine induces potent antitumor immunity.

Functional tumor-specific cytotoxic T cells elicited by therapeutic cancer vaccination in combination with oncolytic viruses offer opportunities to address resistance to checkpoint blockade therapy. Two cancer vaccines, the self-adjuvanting protein vaccine KISIMA, and the recombinant oncolytic vesicular stomatitis virus pseudotyped with LCMV-GP expressing tumor-associated antigens, termed VSV-GP-TAA, both show promise as a single agent. Here we find that, when given in a heterologous prime-boost regimen with an optimized schedule and route of administration, combining KISIMA and VSV-GP-TAA vaccinations induces better cancer immunity than individually. Using several mouse tumor models with varying degrees of susceptibility for viral replication, we find that priming with KISIMA-TAA followed by VSV-GP-TAA boost causes profound changes in the tumor microenvironment, and induces a large pool of poly-functional and persistent antigen-specific cytotoxic T cells in the periphery. Combining this heterologous vaccination with checkpoint blockade further improves therapeutic efficacy with long-term survival in the spectrum. Overall, heterologous vaccination with KISIMA and VSV-GP-TAA could sensitize non-inflamed tumors to checkpoint blockade therapy.

A Methyltransferase-Defective Vesicular Stomatitis Virus-Based SARS-CoV-2 Vaccine Candidate Provides Complete Protection against SARS-CoV-2 Infection in Hamsters.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.

FIP200 restricts RNA virus infection by facilitating RIG-I activation.

Retinoic acid-inducible gene I (RIG-I) senses viral RNA and instigates an innate immune signaling cascade to induce type I interferon expression. Currently, the regulatory mechanisms controlling RIG-I activation remain to be fully elucidated. Here we show that the FAK family kinase-interacting protein of 200 kDa (FIP200) facilitates RIG-I activation. FIP200 deficiency impaired RIG-I signaling and increased host susceptibility to RNA virus infection. In vivo studies further demonstrated FIP200 knockout mice were more susceptible to RNA virus infection due to the reduced innate immune response. Mechanistic studies revealed that FIP200 competed with the helicase domain of RIG-I for interaction with the two tandem caspase activation and recruitment domains (2CARD), thereby facilitating the release of 2CARD from the suppression status. Furthermore, FIP200 formed a dimer and facilitated 2CARD oligomerization, thereby promoting RIG-I activation. Taken together, our study defines FIP200 as an innate immune signaling molecule that positively regulates RIG-I activation.

Defective Interfering Genomes and the Full-Length Viral Genome Trigger RIG-I After Infection With Vesicular Stomatitis Virus in a Replication Dependent Manner.

Replication competent vesicular stomatitis virus (VSV) is the basis of a vaccine against Ebola and VSV strains are developed as oncolytic viruses. Both functions depend on the ability of VSV to induce adequate amounts of interferon-α/ß. It is therefore important to understand how VSV triggers interferon responses. VSV activates innate immunity via retinoic acid-inducible gene I (RIG-I), a sensor for viral RNA. Our results show that VSV needs to replicate for a robust interferon response. Analysis of RIG-I-associated RNA identified a copy-back defective-interfering (DI) genome and full-length viral genomes as main trigger of RIG-I. VSV stocks depleted of DI genomes lost most of their interferon-stimulating activity. The remaining full-length genome and leader-N-read-through sequences, however, still triggered RIG-I. Awareness for DI genomes as trigger of innate immune responses will help to standardize DI genome content and to purposefully deplete or use DI genomes as natural adjuvants in VSV-based therapeutics.

Serine metabolism antagonizes antiviral innate immunity by preventing ATP6V0d2-mediated YAP lysosomal degradation.

Serine metabolism promotes tumor oncogenesis and regulates immune cell functions, but whether it also contributes to antiviral innate immunity is unknown. Here, we demonstrate that virus-infected macrophages display decreased expression of serine synthesis pathway (SSP) enzymes. Suppressing the SSP key enzyme phosphoglycerate dehydrogenase (PHGDH) by genetic approaches or by treatment with the pharmaceutical inhibitor CBR-5884 and by exogenous serine restriction enhanced IFN-ß-mediated antiviral innate immunity in vitro and in vivo. Mechanistic experiments showed that virus infection or serine metabolism deficiency increased the expression of the V-ATPase subunit ATP6V0d2 by inhibiting S-adenosyl methionine-dependent H3K27me3 occupancy at the promoter. ATP6V0d2 promoted YAP lysosomal degradation to relieve YAP-mediated blockade of the TBK1-IRF3 axis and, thus, enhance IFN-ß production. These findings implicate critical functions of PHGDH and the key immunometabolite serine in blunting antiviral innate immunity and also suggest manipulation of serine metabolism as a therapeutic strategy against virus infection.

Oncolytic virotherapy induced CSDE1 neo-antigenesis restricts VSV replication but can be targeted by immunotherapy.

In our clinical trials of oncolytic vesicular stomatitis virus expressing interferon beta (VSV-IFNß), several patients achieved initial responses followed by aggressive relapse. We show here that VSV-IFNß-escape tumors predictably express a point-mutated CSDE1P5S form of the RNA-binding Cold Shock Domain-containing E1 protein, which promotes escape as an inhibitor of VSV replication by disrupting viral transcription. Given time, VSV-IFNß evolves a compensatory mutation in the P/M Inter-Genic Region which rescues replication in CSDE1P5S cells. These data show that CSDE1 is a major cellular co-factor for VSV replication. However, CSDE1P5S also generates a neo-epitope recognized by non-tolerized T cells. We exploit this predictable neo-antigenesis to drive, and trap, tumors into an escape phenotype, which can be ambushed by vaccination against CSDE1P5S, preventing tumor escape. Combining frontline therapy with escape-targeting immunotherapy will be applicable across multiple therapies which drive tumor mutation/evolution and simultaneously generate novel, targetable immunopeptidomes associated with acquired treatment resistance.

In silico trials predict that combination strategies for enhancing vesicular stomatitis oncolytic virus are determined by tumor aggressivity.

BACKGROUND: Immunotherapies, driven by immune-mediated antitumorigenicity, offer the potential for significant improvements to the treatment of multiple cancer types. Identifying therapeutic strategies that bolster antitumor immunity while limiting immune suppression is critical to selecting treatment combinations and schedules that offer durable therapeutic benefits. Combination oncolytic virus (OV) therapy, wherein complementary OVs are administered in succession, offer such promise, yet their translation from preclinical studies to clinical implementation is a major challenge. Overcoming this obstacle requires answering fundamental questions about how to effectively design and tailor schedules to provide the most benefit to patients. METHODS: We developed a computational biology model of combined oncolytic vaccinia (an enhancer virus) and vesicular stomatitis virus (VSV) calibrated to and validated against multiple data sources. We then optimized protocols in a cohort of heterogeneous virtual individuals by leveraging this model and our previously established in silico clinical trial platform. RESULTS: Enhancer multiplicity was shown to have little to no impact on the average response to therapy. However, the duration of the VSV injection lag was found to be determinant for survival outcomes. Importantly, through treatment individualization, we found that optimal combination schedules are closely linked to tumor aggressivity. We predicted that patients with aggressively growing tumors required a single enhancer followed by a VSV injection 1 day later, whereas a small subset of patients with the slowest growing tumors needed multiple enhancers followed by a longer VSV delay of 15 days, suggesting that intrinsic tumor growth rates could inform the segregation of patients into clinical trials and ultimately determine patient survival. These results were validated in entirely new cohorts of virtual individuals with aggressive or non-aggressive subtypes. CONCLUSIONS: Based on our results, improved therapeutic schedules for combinations with enhancer OVs can be studied and implemented. Our results further underline the impact of interdisciplinary approaches to preclinical planning and the importance of computational approaches to drug discovery and development.

Untargeted Lipidomics of Vesicular Stomatitis Virus-Infected Cells and Viral Particles.

The viral lifecycle is critically dependent upon host lipids. Enveloped viral entry requires fusion between viral and cellular membranes. Once an infection has occurred, viruses may rely on host lipids for replication and egress. Upon exit, enveloped viruses derive their lipid bilayer from host membranes during the budding process. Furthermore, host lipid metabolism and signaling are often hijacked to facilitate viral replication. We employed an untargeted HILIC-IM-MS lipidomics approach and identified host lipid species that were significantly altered during vesicular stomatitis virus (VSV) infection. Many glycerophospholipid and sphingolipid species were modified, and ontological enrichment analysis suggested that the alterations to the lipid profile change host membrane properties. Lysophosphatidylcholine (LPC), which can contribute to membrane curvature and serve as a signaling molecule, was depleted during infection, while several ceramide sphingolipids were augmented during infection. Ceramide and sphingomyelin lipids were also enriched in viral particles, indicating that sphingolipid metabolism is important during VSV infection.

Murine GFP-Mx1 forms nuclear condensates and associates with cytoplasmic intermediate filaments: Novel antiviral activity against VSV.

Type I and III interferons induce expression of the "myxovirus resistance proteins" MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles ("biomolecular condensates"). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40-50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20-30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.

Establishment of replication-competent vesicular stomatitis virus-based recombinant viruses suitable for SARS-CoV-2 entry and neutralization assays.

Replication-competent vesicular stomatitis virus (VSV)-based recombinant viruses are useful tools for studying emerging and highly pathogenic enveloped viruses in level 2 biosafety facilities. Here, we used a replication-competent recombinant VSVs (rVSVs) encoding the spike (S) protein of SARS-CoV-2 in place of the original G glycoprotein (rVSV-eGFP-SARS-CoV-2) to develop a high-throughput entry assay for SARS-CoV-2. The S protein was incorporated into the recovered rVSV-eGFP-SARS-CoV-2 particles, which could be neutralized by sera from convalescent COVID-19 patients. The recombinant SARS-CoV-2 also displayed entry characteristics similar to the wild type virus, such as cell tropism and pH-dependence. The neutralizing titers of antibodies and sera measured by rVSV-eGFP-SARS-CoV-2 were highly correlated with those measured by wild-type viruses or pseudoviruses. Therefore, this is a safe and convenient screening tool for SARS-CoV-2, and it may promote the development of COVID-19 vaccines and therapeutics.

A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition.

The VSV matrix protein inhibits NF-κB and the interferon response independently in mouse L929 cells.

The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in immune evasion. While VSV has been thought to suppress the interferon (IFN) response primarily by inhibiting host cell transcription and translation, our recent findings indicate that the M protein also targets NF-κB activation. Therefore, the M protein may utilize two distinct mechanisms to limit expression of antiviral genes, inhibiting both host gene expression and NF-κB activation. Here we characterize a recently reported mutation in the M protein [M(D52G)] of VSV isolate 22-20, which suppressed IFN mRNA and protein production despite activating NF-κB. 22-20 inhibited reporter gene expression from multiple promoters, suggesting that 22-20 suppressed the IFN response via M-mediated inhibition of host cell transcription. We propose that suppression of the IFN response and regulation of NF-κB are independent, genetically separable functions of the VSV M protein.

A tool with many applications: vesicular stomatitis virus in research and medicine.

INTRODUCTION: Vesicular stomatitis virus (VSV) has long been a useful research tool in virology and recently become an essential part of medicinal products. Vesiculovirus research is growing quickly following its adaptation to clinical gene and cell therapy and oncolytic virotherapy. AREAS COVERED: This article reviews the versatility of VSV as a research tool and biological reagent, its use as a viral and vaccine vector delivering therapeutic and immunogenic transgenes and an oncolytic virus aiding cancer treatment. Challenges such as the immune response against such advanced therapeutic medicinal products and manufacturing constraints are also discussed. EXPERT OPINION: The field of in vivo gene and cell therapy is advancing rapidly with VSV used in many ways. Comparison of VSV's use as a versatile therapeutic reagent unveils further prospects and problems for each application. Overcoming immunological challenges to aid repeated administration of viral vectors and minimizing harmful host-vector interactions remains one of the major challenges. In the future, exploitation of reverse genetic tools may assist the creation of recombinant viral variants that have improved onco-selectivity and more efficient vaccine vector activity. This will add to the preferential features of VSV as an excellent advanced therapy medicinal product (ATMP) platform.

Vesicular stomatitis virus nucleocapsids diffuse through cytoplasm by hopping from trap to trap in random directions.

Within 2-6 hours after infection by vesicular stomatitis virus (VSV), newly assembled VSV particles are released from the surface of infected cells. In that time, viral ribonucleoprotein (RNP) particles (nucleocapsids) travel from their initial sites of synthesis near the nucleus to the edge of the cell, a distance of 5-10 µm. The hydrodynamic radius of RNP particles (86 nm) precludes simple diffusion through the mesh of cytoskeletal fibers. To reveal the relative importance of different transport mechanisms, movement of GFP-labeled RNP particles in live A549 cells was recorded within 3 to 4 h postinfection at 100 frames/s by fluorescence video microscopy. Analysis of more than 200 RNP particle tracks by Bayesian pattern recognition software found that 3% of particles showed rapid, directional motion at about 1 µm/s, as previously reported. 97% of the RNP particles jiggled within a small, approximately circular area with Gaussian width σ = 0.06 µm. Motion within such "traps" was not directional. Particles stayed in traps for approximately 1 s, then hopped to adjacent traps whose centers were displaced by approximately 0.17 µm. Because hopping occurred much more frequently than directional motion, overall transport of RNP particles was dominated by hopping over the time interval of these experiments.

Yolkin Isolated from Hen Egg Yolk as a Natural Immunoregulator, Activating Innate Immune Response in BMDM Macrophages.

One of the goals of biomedical sciences is to search and identify natural compounds that are safe, have no side effects, and possess immunostimulatory activity. It has been proven that medicines of natural origin can be effective agents, supporting the therapy of many diseases, not only in the weakened immune system of the body but also in the prevention of many diseases in healthy people. It has been shown that yolkin, a polypeptide complex isolated from hen egg yolk as a fraction accompanying immunoglobulin Y (IgY), possesses potential biological activity. However, the mechanism of its action has not been explained. The objective of this investigation was to examine the molecular mechanisms of innate immune response, activated in response to yolkin, in murine bone marrow-derived macrophages (BMDM). It was shown that yolkin induced phosphorylation of extracellular signal-kinases (ERK1/2) and c-Jun N-terminal kinase (JNK) and upregulated expression and production of type I interferons, TNF-α (tumor necrosis factor α), and nitric oxide (NO), in BMDM cells. Using pharmacological inhibitors of ERK 1/2 and JNK kinases, we revealed that the JNK signaling cascade is required for yolkin-induced inducible NOS expression and upregulation of NO production in mouse macrophages. Using the TLR4-deficient BMDM cell line, we established that yolkin can activate macrophages in a TLR4-dependent manner. It was also shown that NO, TNF-α, and type I IFNs (α/ß) produced by BMDM cells in response to yolkin triggered antiviral activity. These data indicate that yolkin affects the regulation of the immune system and antiviral response; therefore, it can be used as an effective immunostimulator of the innate immunity or as a supplement of the conventional therapy of immunodeficiency.