Vesicular Stomatitis Research Articles

Development of a novel minigenome and recombinant VSV expressing Seoul hantavirus glycoprotein-based assays to identify anti-hantavirus therapeutics

Seoul virus (SEOV) is an emerging global health threat that can cause hemorrhagic fever with renal syndrome (HFRS), which results in case fatality rates of ∼2%. There are no approved treatments for SEOV infections. We developed a cell-based assay system to identify potential antiviral compounds for SEOV and generated additional assays to characterize the mode of action of any promising antivirals. To test if candidate antivirals targeted SEOV glycoprotein-mediated entry, we developed a recombinant reporter vesicular stomatitis virus expressing SEOV glycoproteins. To facilitate the identification of candidate antiviral compounds targeting viral transcription/replication, we successfully generated the first reported minigenome system for SEOV. This SEOV minigenome (SEOV-MG) screening assay will also serve as a prototype assay for discovery of small molecules inhibiting replication of other hantaviruses, including Andes and Sin Nombre viruses. Ours is a proof-of-concept study in which we tested several compounds previously reported to have activity against other negative-strand RNA viruses using our newly developed hantavirus antiviral screening systems. These systems can be used under lower biocontainment conditions than those needed for infectious viruses, and identified several compounds with robust anti-SEOV activity. Our findings have important implications for the development of anti-hantavirus therapeutics.

Inflammation-induced development and migration of a novel monocyte-like dendritic cell population that regulates antiviral CD8 T cell immunity.

Abstract We recently discovered a CD11c+MHC-II+CD103+CD11b+ monocyte-like dendritic cell (moDC) population that colocalizes with CD8 T cells in the draining lymph node (dLN) early after respiratory infection. Importantly, depletion of these cells enhances the formation of CD8+ tissue resident memory T cells (T RM) in the lung. Given the importance of T RMafter respiratory challenge, we aimed to understand the origin and migration of these moDCs. To this end, we intranasally (i.n.) infected C57Bl/6 mice with influenza virus A HK-×31 (×31) or with vesicular stomatitis virus (VSV), the latter which allows us to compare with identical systemic infection. moDCs were detected in the bone marrow (BM) ~2 days post infection, one day prior to their peak recovery from the dLN. BM moDCs phenotypically resembled dLN moDCs, including expression of the inflammatory monocyte-associated markers CCR2 and Ly6C. Adoptive transfer of BM Ly6C+ monocytes into congenically mismatched recipients showed a meaningful percentage of moDCs were monocyte-donor derived. Additionally, type I IFNs, which can induce monocyte differentiation and are crucial in early antiviral immunity, were required for moDC development. We showed that moDCs are decreased in the BM, and consequently the dLN of Ifnar1−/− mice compared to WT mice following respiratory infection. Lastly, moDCs were detected in the BM after intravenous (i.v.) VSV but not in the dLN. This could be attributed to the increased expression of CCL2, MIP-1β, and IL-12p40 in the dLN of i.n. VSV or ×31 infected mice compared to naïve or i.v. VSV infected mice, where inflammation is dispersed. Overall, we provide potential targets in the moDC development and migration process that could enhance antiviral CD8 T cell immunity. Supported by grant from NIH (R21 AI131093)

Interferon gamma supresses T follicular helper cell and antibody responses upon viral infection

Abstract Although humoral and cellular immunity upon viral infections usually co-exist, sometimes one of the two responses emerges as dominant and is responsible for most of the antiviral activity. For example, vesicular stomatitis virus (VSV) infection induces early and potent neutralizing antibody (nAb) responses, whereas lymphocytic choriomeningitis virus (LCMV) infection induces strong cellular responses, but weak nAb responses. Recent work from our laboratory has shown that unbalance is observed also at the level of CD4 T cells responses, with subcutaneous VSV infection inducing strong T FHpolarization that supports nAb responses, and subcutaneous LCMV in contrast promoting an extreme T H1 differentiation with no T FH. Single-cell RNA sequencing revealed that LCMV-specific T H1 cells comprise at least to distinct subsets, one of which expresses TCF1, which is usually expressed by T FH. We found that blocking of IFN gamma results in the transition of this atypical T-bet +TCF1 +cells into T FHcells, and in an increased number of germinal center B cells and antibodies. Our results identify IFN gamma as a molecular switch that suppresses T FHand antibody responses while supporting T H1 differentiation upon viral infection. This might be an additional mechanism whereby viruses like LCMV interfere with the generation of efficient antibody responses. Research was supported by the Italian Ministry of Education, University and Research grants SIR-RBSI14BAO5, PRIN-2017ZXT5WR and PRIN-20209Y5YFZ (to M.K.) and by the European Research Council (ERC) Consolidator Grant 725038 (to M.I.)

Comparison of the immunogenicity of nasal-spray rVSV vector, adenovirus vector, and inactivated COVID-19-based vaccines in rodent models.

Intranasal (i.n.) vaccines can induce mucosal and systemic immunity against respiratory pathogens. Previously, we demonstrated that the recombinant vesicular stomatitis virus (rVSV)-based COVID-19 vaccine rVSV-SARS-CoV-2, with poor immunogenicity via the intramuscular route (i.m.), is more suitable for i.n. administration in mice and nonhuman primates. Here, we found that the rVSV-SARS-CoV-2 Beta variant was more immunogenic than the wild-type strain and other variants of concern (VOCs) in golden Syrian hamsters. Furthermore, the immune responses elicited by rVSV-based vaccine candidates via the i.n. route weresignificantly higher than thoseof two licensed vaccines:the inactivated vaccine KCONVAC delivered via the i.m. route and the adenovirus-based Vaxzevria delivered i.n. or i.m. We next assessed the booster efficacy of rVSV following two i.m. doses of KCONVAC. Twenty-eight days after receiving two i.m. doses of KCONVAC, hamsters were boosted with a third dose of KCONVAC (i.m.), Vaxzevria (i.m. or i.n.), or rVSVs (i.n.). Consistent with other heterologous booster studies, Vaxzevria and rVSV elicited significantly higher humoral immunity than the homogenous KCONVAC. In summary, our results confirmed that two i.n. doses of rVSV-Beta elicited significantly higher humoral immune responses than commercial inactivated and adeno-based COVID vaccines in hamsters. As a heterologous booster dose, rVSV-Beta induced potent, persistent, and broad-spectrum humoral and mucosal neutralizing responses against all VOCs, highlighting its potential to be developed into a nasal-spray vaccine.

TGF-β promotes the resolution of acute hepatitis B virus infection

Abstract An estimated 296 million people live with a chronic hepatitis B virus (HBV) infection, resulting in approximately 800,000 annual deaths. Thus, despite the availability of an effective vaccine, HBV remains a global health concern. Most children and a fraction of adults infected with HBV develop a persistent liver infection that yields lifelong complications. It is believed that the immune tolerogenic environment of the liver and the dysfunction of T and B cells contribute to HBV persistence in these individuals. Here, an adeno-associated virus (AAV)-based mouse model of HBV infection was utilized to investigate the underlying mechanisms of immune dysfunction that impede viral clearance. HBV infection was initiated in mice by AAV-HBV transduction, and TGF-β was depleted to determine the role of this cytokine in HBV persistence. As TGF-β is often associated with negative regulation of the immune response, we hypothesized that its inhibition might improve HBV-specific T and B cell responses. However, the lack of TGF-β resulted in impaired HBV surface antigen (HBsAg) clearance and diminished anti-HBsAg antibody levels, revealing a positive role for TGF-β in the antibody response to HBV. This effect of TGF-β on the antibody response to HBsAg was unique to AAV-HBV transduction, as a similar increase in anti-HBsAg was not observed by TGF-β depletion following infection with recombinant vesicular stomatitis virus expressing HBsAg. We also observed differences in B cell activation marker expression during HBsAg clearance in mice transduced with AAV-HBV and depleted of TGF-β. Together, these findings highlight the impact of TGF-β on the B cell response to HBV and have begun to define a unique role for this cytokine in promoting HBV clearance. Supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R01AI148354. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Regulation of respiratory CD8+ T cell immunity by suppressive monocyte-like dendritic cells

Abstract Active immune suppression can mediate the balance between protective cellular immunity and harmful immunopathology. This suppression can occur locally, at an infection site, or in regional draining lymph nodes (dLN). Immune regulation is of particular importance in sites such as the lung where aberrant immunopathology can result in loss of tissue function. We have recently identified a novel population of CD11b+CD103+CCR2+ monocyte-like dendritic cells (moDCs) which directly suppress CD8+ T cell proliferation in vitro. Respiratory infection with RNA viruses recruits these moDCs either exclusively to the dLN (after vesicular stomatitis virus infection) or both the dLN and site of viral replication (after influenza infection). Exclusive depletion of moDCs from the dLN using CCR2-DTR bone marrow chimeras results in enhanced respiratory CD8+ lung tissue resident memory cell formation. By contrast, depletion of moDCs from both the dLN and respiratory tract following influenza infection results in enhanced respiratory CD8+ T cell responses coupled with fatal immunopathology. Together, these data suggest that suppressive moDCs govern key aspects of respiratory immunity by regulating the function and development of long-lived CD8+ T cells, thereby balancing immunity and adverse pathology in the context of viral infection. Supported by grant from NIH (R21 AI131093)

Therapeutic Efficacy of a VSV-GP-based Human Papilloma Virus Vaccine in a Murine Cancer Model

Human papilloma virus (HPV) infections are associated with almost all cervical cancers and to a lower extend also with anogenital or oropharyngeal cancers. HPV proteins expressed in HPV-associated tumors are attractive antigens for cancer vaccination strategies as self-tolerance, which is associated with most endogenous tumor-associated antigens, does not need to be overcome. In this study, we generated a live attenuated cancer vaccine based on the chimeric vesicular stomatitis virus VSV-GP, which has previously proven to be a potent vaccine vector and oncolytic virus. Genes at an earlier position in the genome more to the 3′ end are expressed stronger compared to genes located further downstream. By inserting an HPV16-derived antigen cassette consisting of E2, E6 and E7 into VSV-GP either at first (HPVp1) or fifth (HPVp5) position in VSV-GP’s genome we aimed to analyze the effect of vaccine antigen position and consequently expression level on viral fitness, immunogenicity, and anti-tumoral efficacy in a syngeneic mouse tumor model. HPVp1 expressed higher amounts of HPV antigens compared to HPVp5 in vitro but had a slightly delayed replication kinetic which overall translated into increased HPV-specific T cell responses upon vaccination of mice. Immunization with both vectors protected mice in prophylactic and in therapeutic TC-1 tumor models with HPVp1 being more effective in the prophylactic setting. Taken together, VSV-GP is a promising candidate as therapeutic HPV vaccine and first position of the vaccine antigen in a VSV-derived vector seems to be superior to fifth position.

Syngeneic mouse model of human HER2+ metastatic breast cancer for the evaluation of trastuzumab emtansine combined with oncolytic rhabdovirus.

Established mouse models of HER2+ cancer are based on the over-expression of rodent Neu/Erbb2 homologues, which are incompatible with human HER2 (huHER2) targeted therapeutics. Additionally, the use of immune-deficient xenograft or transgenic models precludes assessment of native anti-tumour immune responses. These hurdles have been a challenge for our understanding of the immune mechanisms behind huHER2-targeting immunotherapies. To assess the immune impacts of our huHER2-targeted combination strategy, we generated a syngeneic mouse model of huHER2+ breast cancer, using a truncated form of huHER2, HER2T. Following validation of this model, we next treated tumour-bearing with our immunotherapy strategy: oncolytic vesicular stomatitis virus (VSVΔ51) with clinically approved antibody-drug conjugate targeting huHER2, trastuzumab emtansine (T-DM1). We assessed efficacy through tumour control, survival, and immune analyses. The generated truncated HER2T construct was non-immunogenic in wildtype BALB/c mice upon expression in murine mammary carcinoma 4T1.2 cells. Treatment of 4T1.2-HER2T tumours with VSVΔ51+T-DM1 yielded robust curative efficacy compared to controls, and broad immunologic memory. Interrogation of anti-tumour immunity revealed tumour infiltration by CD4+ T cells, and activation of B, NK, and dendritic cell responses, as well as tumour-reactive serum IgG. The 4T1.2-HER2T model was used to evaluate the anti-tumour immune responses following our complex pharmacoviral treatment strategy. These data demonstrate utility of the syngeneic HER2T model for assessment of huHER2-targeted therapies in an immune-competent in vivo setting. We further demonstrated that HER2T can be implemented in multiple other syngeneic tumour models, including but not limited to colorectal and ovarian models. These data also suggest that the HER2T platform may be used to assess a range of surface-HER2T targeting approaches, such as CAR-T, T-cell engagers, antibodies, or even retargeted oncolytic viruses.

Engineering Oncolytic Coxsackievirus A21 with Small Transgenes and Enabling Cell-Mediated Virus Delivery by Integrating Viral cDNA into the Genome.

Coxsackievirus A21 (CVA21) is a naturally occurring RNA virus that, in preclinical studies and clinical trials, has demonstrated promising potential in treating a range of malignancies. Other oncolytic viruses, such as adenovirus, vesicular stomatitis virus, herpesvirus, and vaccinia virus, all can be engineered to carry one or more transgenes for various purposes, including immune modulation, virus attenuation, and induction of apoptosis of tumor cells. However, it remained unknown whether CVA21 can express therapeutic or immunomodulatory payloads due to its small size and high mutation rate. Using reverse genetics techniques, we demonstrated that a transgene encoding a truncated green fluorescent protein (GFP) of up to 141 amino acids (aa) can be successfully carried in the 5' end of the coding region. Furthermore, a chimeric virus carrying an eel fluorescent protein, UnaG (139 aa), was also made and shown to be stable, and it maintained efficient tumor cell-killing activity. Similar to other oncolytic viruses, the likelihood of delivering CVA21 by the intravenous route is low due to issues like blood absorption, neutralizing antibodies, and liver clearance. To address this problem, we designed the CVA21 cDNA under the control of a weak RNA polymerase II promoter, and subsequently, a stable cell pool in 293T cells was made by integrating the resulting CVA21 cDNA into the cell genome. We showed that the cells are viable and able to persistently generate rCVA21 de novo. The carrier cell approach described here may pave the way to designing new cell therapy strategies by arming with oncolytic viruses. IMPORTANCE As a naturally occurring virus, coxsackievirus A21 is a promising oncolytic virotherapy modality. In this study, we first used reverse genetics to determine whether A21 can stably carry transgenes and found that it could express up to 141 amino acids of foreign GFP. The chimeric virus carrying another fluorescent eel protein UnaG (139 amino acids) gene also appeared to be stable over at least 7 passages. Our results provided guidance on how to select and engineer therapeutic payloads for future A21 anticancer research. Second, the challenges of delivering oncolytic viruses by the intravenous route hamper the broader use of oncolytic viruses in the clinic. Here, we used A21 to show that cells could be engineered to stably carry and persistently release the virus by harboring the viral cDNA in the genome. The approach we presented here may pave a new way for oncolytic virus administration using cells as carriers.

Low-density lipoprotein receptor-related protein 1 (LRP1) as an auxiliary host factor for RNA viruses.

Viruses with an RNA genome are often the cause of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion-mutagenized mouse embryonic cell library for clones that are resistant to Rift Valley fever virus (RVFV). This screen returned the low-density lipoprotein receptor-related protein 1 (LRP1) as a top hit, a plasma membrane protein involved in a wide variety of cell activities. Inactivation of LRP1 in human cells reduced RVFV RNA levels already at the attachment and entry stages of infection. Moreover, the role of LRP1 in promoting RVFV infection was dependent on physiological levels of cholesterol and on endocytosis. In the human cell line HuH-7, LRP1 also promoted early infection stages of sandfly fever Sicilian virus and La Crosse virus, but had a minor effect on late infection by vesicular stomatitis virus, whereas encephalomyocarditis virus was entirely LRP1-independent. Moreover, siRNA experiments in human Calu-3 cells demonstrated that also SARS-CoV-2 infection benefitted from LRP1. Thus, we identified LRP1 as a host factor that supports infection by a spectrum of RNA viruses.

Abstract CT207: Phase I trial of vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 (VSV-IFNb-TYRP1) in metastatic ocular melanoma

Abstract Oncologic viruses (OV) can induce tumor death through both direct oncolysis and immune-mediated destruction. Our study investigates the use of a novel OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-b) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNb-TYRP1). VSV is an attractive vector since humans are not naturally infected with VSV; thus, pre-existing immunity is minimal. IFN-b leads to increased T-cell responses and tumor cell apoptosis, while TYRP1, a melanocyte differentiation antigen, increases immunogenicity of the therapy. We conducted a Phase 1 clinical trial with a 3+3 design in patients with metastatic uveal melanoma. VSV-IFNb-TYRP1 was injected into a liver metastasis under image guidance, then administered on the same day as a single intravenous (IV) infusion. IV doses started at dose level (DL) 1 1 × 1010 TCID50, then escalated to DL2 3 × 10 10, DL3 1 × 1011, and DL4 3 × 1011. The dose delivered intratumorally (IT) varied based on the IV dose. The primary endpoints were safety and maximum tolerated dose (MTD). Efficacy was a secondary endpoint. Correlative studies focused on understanding viral pharmacokinetics (PK) and immunological responses induced by VSV-IFNb-TYRP1 therapy. Twelve patients with previously treated metastatic uveal melanoma were enrolled. Median follow up was 19.1 months. Four DLs were evaluated with one patient at DL4 experiencing dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). Non-DLTs at DL4 were fatigue, fever, CRS, hematological toxicities (decreased platelets and lymphocytes), and AST elevation. Four patients had stable disease (SD) and 8 had progressive disease (PD) as their best response. ELIspot data show that three of the four patients with response to TYRP1 also had a response to gp100, suggesting possible epitope spreading. These four patients received immune checkpoint inhibitor treatment as the next line of therapy, which led to clinical benefit for two patients. Our study evaluated VSV-IFNb-TYRP1 administered via IT and IV routes in a previously treated population of metastatic uveal melanoma patients and found no major safety events. Although there were no clear objective responses to VSV-IFNb-TYRP1, dose-dependent immunogenicity to melanoma antigens was seen. While OV response alone is not sufficient for clinical benefit, the evidence of epitope spreading is encouraging. Future studies of VSV-IFNb-TYRP1 will evaluate combinations with other therapies. Citation Format: Katherine E. Smith, Adam Weisbrod, Carrie Strand, Jacob Allerd, Jose S. Pulido, Alysha Newsom, Lianwen Zhang, Nandakumar Packiriswamy, Heather Montane, Lisa Kottschade, Anastasios Dimou, Yiyi Yan, Markovic Svetomir, Kah Whye Peng, Richard G. Vile, Matthew S. Block, Roxana S. Dronca. Phase I trial of vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 (VSV-IFNb-TYRP1) in metastatic ocular melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT207.

Development of an Integrated Continuous Manufacturing Process for the rVSV-Vectored SARS-CoV-2 Candidate Vaccine.

The administration of viral vectored vaccines remains one of the most effective ways to respond to the ongoing novel coronavirus disease 2019 (COVID-19) pandemic. However, pre-existing immunity to the viral vector hinders its potency, resulting in a limited choice of viral vectors. Moreover, the basic batch mode of manufacturing vectored vaccines does not allow one to cost-effectively meet the global demand for billions of doses per year. To date, the exposure of humans to VSV infection has been limited. Therefore, a recombinant vesicular stomatitis virus (rVSV), which expresses the spike protein of SARS-CoV-2, was selected as the vector. To determine the operating upstream process conditions for the most effective production of an rVSV-SARS-CoV-2 candidate vaccine, a set of critical process parameters was evaluated in an Ambr 250 modular system, whereas in the downstream process, a streamlined process that included DNase treatment, clarification, and a membrane-based anion exchange chromatography was developed. The design of the experiment was performed with the aim to obtain the optimal conditions for the chromatography step. Additionally, a continuous mode manufacturing process integrating upstream and downstream steps was evaluated. rVSV-SARS-CoV-2 was continuously harvested from the perfusion bioreactor and purified by membrane chromatography in three columns that were operated sequentially under a counter-current mode. Compared with the batch mode, the continuous mode of operation had a 2.55-fold increase in space-time yield and a reduction in the processing time by half. The integrated continuous manufacturing process provides a reference for the efficient production of other viral vectored vaccines.

Analysis of the Differential Expression and Antiviral Activity of Porcine Interferon-α In Vitro.

Porcine interferon α (poIFN-α) is a crucial cytokine that can prevent and treat viral infections. Seventeen functional porcine IFN-α subtypes were found in the porcine genome. In this study, multiple sequence alignment was performed to analyze IFN-α protein structure and function. Phylogenetic tree analysis of the poIFN gene family defined the evolutionary relationship of various subtypes. PoIFN-αs, including poIFN-α1-17, were expressed in an Escherichia coli expression system. The antiviral activities of these IFN-α proteins against vesicular stomatitis virus (VSV) and pseudorabies virus (PRV) were examined in PK-15 cells. We found that the antiviral activity of different poIFN-α molecules greatly differed as follows: the poIFN-α14 and 17 subtypes had the greatest antiviral activities against VSV and PRV in PK-15 cells, poIFN-α1, 2, 3, and 8 exhibited lower biological activities, and poIFN-α4, 5, 6, 7, 9, 10, 11, 12, 13, and 16 had minimal or no effect in the tested target cell‒virus systems. Moreover, our studies demonstrated that the antiviral activity of IFN-α was positively correlated with the induction of IFN-stimulated genes, such as 2'-5'oligoadenylate synthetase 1 (OSA1), interferon-stimulated gene 15 (ISG15), myxoma resistance protein 1 (Mx1), and protein kinase R (PKR). Thus, our experimental results provide important information about the antiviral functions and mechanism of poIFN-α.

FOLR1-induced folate deficiency reduces viral replication via modulating APOBEC3 family expression

Folate receptor alpha (FOLR1) is vital for cells ingesting folate (FA). FA plays an indispensable role in cell proliferation and survival. However, it is not clear whether the axis of FOLR1/FA has a similar function in viral replication. In this study, we used vesicular stomatitis virus (VSV) to investigate the relationship between FOLR1-mediated FA deficiency and viral replication, as well as the underlying mechanisms. We discovered that FOLR1 upregulation led to the deficiency of FA in HeLa cells and mice. Meanwhile, VSV replication was notably suppressed by FOLR1 overexpression, and this antiviral activity was related to FA deficiency. Mechanistically, FA deficiency mainly upregulated apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B) expression, which suppressed VSV replication in vitro and in vivo. In addition, methotrexate (MTX), an FA metabolism inhibitor, effectively inhibited VSV replication by enhancing the expression of APOBEC3B in vitro and in vivo. Overall, our present study provided a new perspective for the role of FA metabolism in viral infections and highlights the potential of MTX as a broad-spectrum antiviral agent against RNA viruses.

Abstract 688: Oncolytic VSV with retargeted glycoprotein and genetic ON switch for precise targeting of liver cancer using a synthetic virology platform

Abstract Introduction: Humane Genomics has developed a platform to make artificial oncolytic viruses with unprecedented selectivity and efficiency. The platform is based on Vesicular Stomatitis Virus (VSV), which has several ideal properties (highly oncolytic, non-pathogenic, small genome that’s well understood and easily modified). Here we present the development of HG001, our first therapy for the treatment of liver cancer. Methods: Genome design of HG001 was completed using our proprietary software. The virus was designed with a GPC3 targeting glycoprotein and a genetic ON-switch (aptazyme) with a liver cancer specific aptamer. DNA fragments were synthesized and then assembled to construct the complete viral genome of HG001. Virus was rescued in BHK-21 using reverse genetics and then verified by nanopore sequencing. We evaluated efficacy and safety in cancer- (Huh-7, SNU-449) and healthy- (NIH/3T3) cells. VSV wildtype was used as a control virus. IncuCyte live cell imaging and analysis software was used to quantify efficacy and safety. Cell counts and cell area per well (µm2/well) were measured to evaluate cell health and proliferation. Apoptosis was determined using Annexin V. Student’s t-test was used to assess statistical significance. Results: Our platform enabled us to complete the process from assembly of the fragments to the start of testing of HG001 in under 2 weeks. To determine selectivity and efficacy, we evaluated serial dilutions in Huh7 (high expression of GPC3) and SNU-449 cells (GPC3 negative). Real time monitoring of HG001 showed cell killing in Huh7 cells at MOI=1 (p<0.001) and cytopathic effects (CPE) in MOI=0.1. However, no impact on cell proliferation and viability was found in SNU-449 (p=ns), indicating specificity for the target receptor. To further validate selectivity and demonstrate safety, we assayed normal NIH/3T3 cells with the same titers. We did not observe any indication of cytotoxicity, and kinetic data of cell growth revealed no significant difference between HG001 and mock infection (p=ns). In stark contrast, VSV-WT induced rapid lysis in NIH/3T3 cells at MOI=0.01 (p<0.01). Taken together, HG001 exhibits at least 2 orders of magnitude improvement in safety. Apoptosis assays conducted in these cell lines corroborated these findings. HG001 was well tolerated and showed no adverse effects during initial in vivo studies. Current in vivo studies are ongoing to confirm efficacy. Conclusion: We have shown that a rationally designed synthetic virus can be made efficiently with high specificity and efficacy. Initial testing is showing high promise for future in vivo testing. With further iterations we hope to develop a strong therapeutic for liver cancer. Going forward, we are actively looking for collaborations with oncology centers on other types of cancer we could target using our platform. Citation Format: Chad Moles, Rupsa Basu, Taylor Flaat, Christopher Collado, Peter Weijmarshausen. Oncolytic VSV with retargeted glycoprotein and genetic ON switch for precise targeting of liver cancer using a synthetic virology platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 688.

Abstract 6417: Deciphering aberrant STING pathway and exploring oncolytic viruses therapy in low grade serous ovarian carcinoma

Abstract There are two serous ovarian cancer histotypes, low and high grade (LGSOC and HGSOC), which are distinct clinical and biological entities. LGSOC is a rare histotype with a relatively stable genome, while HGSC is more common and genomically unstable. Somewhat surprisingly LGSOC expresses high levels of the stimulator of the interferon genes (STING). The STING pathway recognizes cytoplasmic double-stranded DNA and mounts innate cellular immunity through interferon-beta type I production. Our objective is to investigate the aberrant STING signaling in LGSOC and test the effectiveness of oncolytic viruses against LGSOC. We used immunohistochemistry on tissue microarrays (TMAs) to assess STING protein expression in different ovarian cancer histotypes. Whole proteome analysis was applied to identify differentially expressed proteins in LGSOC and HGSOC patient samples (both n=9). Further, a semi-targeted proteomics approach was used to evaluate the expression levels of the STING pathway-related proteins in LGSOC, HGSOC, and LGSOC precursor tumors (each subtype, n=20). To evaluate the key transcription, phosphorylation, and translocation events in STING signaling, we treated LGSOC cell lines with an agonist (dsDNA90) and performed qPCR, immunoblotting, and immunofluorescence experiments, respectively. We tested the viability of the LGSOC cell lines in response to Vaccinia Virus (VV), and Vesicular Stomatitis Virus (VSV) based oncolytic vectors with or without immunostimulatory transgenes. Our results show that STING protein levels were consistently higher in LGSOC TMAs relative to other histotypes. Proteomics analysis showed that the half of the 16 most differentially expressed proteins were the effectors of STING signaling with unexpectedly lower expression in LGSOC, suggesting that despite the robust levels of STING in LGSOC tumors, the pathway is not fully active. Attenuated STING translocation and expression of IFNB1 and other cytokines in LGSOC cell lines confirm the aberrancy in the STING pathway. Semi-targeted proteomics revealed the considerable overexpression of STIM1 in LGSOC patient tumors, which has previously been shown to sequester STING in the endoplasmic reticulum. The treatment with VV and VSV oncolytic viruses significantly reduced the proliferation of LGSOC cell lines. In summary, we find attenuated STING signaling in LGSOC, possibly due to overexpression of STIM1 preventing STING translocation. Although oncolytic viruses show promising results in LGSOC cell lines, more research is needed to determine the optimal treatment strategy, testing oncolytic viruses expressing various transgenes and combination therapies. Citation Format: Almira Zhantuyakova, Dawn Cochrane, Gian Negri, Sandra E. Spencer Miko, Taha Azad, Jutta Huvila, Marta Llaurado Fernandez, Mark Carey, Gregg B. Morin, John Bell, David Huntsman. Deciphering aberrant STING pathway and exploring oncolytic viruses therapy in low grade serous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6417.

Monovalent and trivalent VSV-based COVID-19 vaccines elicit neutralizing antibodies and CD8+ T cells against SARS-CoV-2 variants.

Recombinant vesicular stomatitis virus (rVSV) vaccines expressing spike proteins of Wuhan, Beta, and/or Delta variants of SARS-CoV-2 were generated and tested for induction of antibody and Tcell immune responses following intramuscular delivery to mice. rVSV-Wuhan and rVSV-Delta vaccines and an rVSV-Trivalent (mixed rVSV-Wuhan, -Beta, -Delta) vaccine elicited potent neutralizing antibodies (nAbs) against live SARS-CoV-2 Wuhan (USAWA1), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529) viruses. Prime-boost vaccination with rVSV-Beta was less effective in this capacity. Heterologous boosting of rVSV-Wuhan with rVSV-Delta induced strong nAb responses against Delta and Omicron viruses, with the rVSV-Trivalent vaccine consistently effective in inducing nAbs against all the SARS-CoV-2 variants tested. All vaccines, including rVSV-Beta, elicited a spike-specific immunodominant CD8+ Tcell response. Collectively, rVSV vaccines targeting SARS-CoV-2 variants of concern may be considered in the global fight against COVID-19.

Isoliquiritigenin inhibits virus replication and virus-mediated inflammation via NRF2 signaling

BackgroundThe transcription factor NRF2 is a master redox switch that regulates the cellular antioxidant response. However, recent advances have revealed new roles for NRF2, including the regulation of antiviral responses to various viruses, suggesting that pharmacological NRF2-activating agents may be a promising therapeutic drug for viral diseases. Isoliquiritigenin (ISL), a chalcone isolated from liquorice (Glycyrrhizae Radix) root, is reported to be a natural NRF2 agonist and has has antiviral activities against HCV (hepatitis C virus) and IAV (influenza A virus). However, the spectrum of antiviral activity and associated mechanism of ISL against other viruses are not well defined. PurposeThis study investigated the antiviral activity and underlying mechanism of ISL against vesicular stomatitis virus (VSV), influenza A virus (H1N1), encephalomyocarditis virus (EMCV), herpes simplex virus type 1 (HSV-1). MethodsWe evaluated the antiviral activity of ISL against VSV, H1N1, EMCV, and HSV-1 using flow cytometry and qRT-PCR analysis. RNA sequencing and bioinformatic analysis were performed to investigate the potential antiviral mechanism of ISL. NRF2 knockout cells were used to investigate whether NRF2 is required for the antiviral activity of ISL. The anti-apoptosis and anti-inflammatory activities of ISL were further measured by counting cell death ratio and assessing proinflammatory cytokines expression in virus-infected cells, respectively. In addition, we evaluated the antiviral effect of ISL in vivo by measuring the survival rate, body weights, histological analysis, viral load, and cytokine expression in VSV-infected mouse model. ResultsOur data demonstrated that ISL effectively suppressed VSV, H1N1, HSV-1, and EMCV replication in vitro. The antiviral activity of ISL could be partially impaired in NRF2-deficient cells. Virus-induced cell death and proinflammatory cytokines were repressed by ISL. Finally, we showed that ISL treatment protected mice against VSV infection by reducing viral titers and suppressing the expression of inflammatory cytokines in vivo. ConclusionThese findings suggest that ISL has antiviral and anti-inflammatory effects in virus infections, which are associated with its ability to activate NRF2 signaling, thus indicating that ISL has the potential to serve as an NRF2 agonist in the treatment of viral diseases.

Injectable Scaffolds for In Vivo Programmed Macrophages Manufacture and Postoperative Cancer Immunotherapy

AbstractCancer recurrence and metastasis after surgical resection is a vital reason of treatment failure. The modification of immune cells through implanted biomaterials is a promising postoperative immunotherapy. Herein, an injectable hydrogel scaffold loaded with engineered exosome mimetics that in vivo recruits and programs endogenous macrophages into M1 binding with anti‐CD47 antibody (M1‐aCD47 macrophages) for postoperative cancer immunotherapy is developed. Briefly, M1 macrophages‐derived exosome mimetics co‐modified with vesicular stomatitis virus glycoprotein (VSV‐G) and aCD47 (V‐M1EM‐aCD47) are encapsulated in injectable chitosan hydrogel. Such hydrogel recruits inherent macrophages in situ and releases V‐M1EM‐aCD47 that programs M2 to M1‐aCD47 macrophages. M1‐aCD47 macrophages own dual‐functions of tumor‐homing and enhanced phagocytosis. They can actively target to tumor cells for delivery of aCD47 that blocks the “don't eat me” signal, thereby promoting phagocytosis of macrophages to cancer cells. Furthermore, V‐M1EM‐aCD47 hydrogel implanted into resection site of 4T1 breast tumor inhibits tumor recurrence and metastasis by phagocytosis of M1‐aCD47 macrophages and T cell‐mediated immune responses. The findings demonstrate that biomaterials can be designed in vivo to program inherent macrophages, thereby activating the innate and adaptive immune systems for prevention of postoperative tumor recurrence and metastasis.

P120-catenin promotes innate antiviral immunity through stabilizing TBK1-IRF3 complex

TBK1-IRF3 complex plays vital roles in antiviral immune responses, its regulatory mechanisms are currently incompletely understood. p120-catenin (p120), an armadillo-repeat protein, mainly regulates the stability of classical cadherins and the development of epithelial-to-mesenchymal transitions (EMTs). Here we report that p120 is a positive regulator of type I IFN production. Ectopic expression of p120 enhanced Vesicular stomatitis virus and Sendai-virus-induced type I IFN production, whereas knockdown of p120 expression suppressed type I IFN production. Mechanistically, p120 promoted phosphorylation of IRF3 via stabilizing the TBK1-IRF3 complex. Consistently, p120 knock down mice are more susceptible to VSV infection as indicated by higher tissue viral titers, less IFN-I production and greater infiltration of immune cells. This study reveals p120 as an important positive regulator in innate immunity and identifies that p120 facilitates host antiviral response through stabilizing TBK1-IRF3 complex.