Establishment of replication-competent vesicular stomatitis virus recapitulating SADS-CoV entry.

Zoonotic coronaviruses pose a continuous threat to human health, with newly identified bat-borne viruses like swine acute diarrhea syndrome coronavirus (SADS-CoV) causing high mortality in piglets. In vitro studies indicate that SADS-CoV can infect cell lines from diverse species, including humans, highlighting its potential risk to human health. However, the lack of tools to study viral entry, along with the absence of vaccines or antiviral therapies, perpetuates this threat. To address this, we engineered an infectious molecular clone of Vesicular Stomatitis Virus (VSV), replacing its native glycoprotein (G) with SADS-CoV spike (S) and inserting a Venus reporter at the 3' leader region to generate a replication-competent rVSV-Venus-SADS S virus. Serial passages of rVSV-Venus-SADS S led to the identification of an 11-amino-acid truncation in the cytoplasmic tail of the S protein, which allowed more efficient viral propagation due to increased cell membrane anchoring of the S protein. The S protein was integrated into rVSV-Venus-SADS SΔ11 particles, susceptible to neutralization by sera from SADS-CoV S1 protein-immunized rabbits. Additionally, we found that TMPRSS2 promotes SADS-CoV spike-mediated cell entry. Furthermore, we assessed the serum-neutralizing ability of mice vaccinated with rVSV-Venus-SADS SΔ11 using a prime-boost immunization strategy, revealing effective neutralizing antibodies against SADS-CoV infection. In conclusion, we have developed a safe and practical tool for studying SADS-CoV entry and exploring the potential of a recombinant VSV-vectored SADS-CoV vaccine.IMPORTANCEZoonotic coronaviruses, like swine acute diarrhea syndrome coronavirus (SADS-CoV), pose a continual threat to human and animal health. To combat this, we engineered a safe and efficient tool by modifying the Vesicular Stomatitis Virus (VSV), creating a replication-competent rVSV-Venus-SADS S virus. Through serial passages, we optimized the virus for enhanced membrane anchoring, a key factor in viral propagation. This modified virus, rVSV-Venus-SADS SΔ11, proved susceptible to neutralization, opening avenues for potential vaccines. Additionally, our study revealed the role of TMPRSS2 in SADS-CoV entry. Mice vaccinated with rVSV-Venus-SADS SΔ11 developed potent neutralizing antibodies against SADS-CoV. In conclusion, our work presents a secure and practical tool for studying SADS-CoV entry and explores the promise of a recombinant VSV-vectored SADS-CoV vaccine.

Translational success of fundamental virology: a VSV-vectored Ebola vaccine.

Ebola virus disease (EVD) caused by Ebola virus (EBOV) is a severe, often fatal, hemorrhagic disease. A critical component of the public health response to curb EVD epidemics is the use of a replication-competent, recombinant vesicular stomatitis virus (rVSV)-vectored Ebola vaccine, rVSVΔG-ZEBOV-GP (ERVEBO). In this Gem, we will discuss the past and ongoing development of rVSVΔG-ZEBOV-GP, highlighting the importance of basic science and the strength of public-private partnerships to translate fundamental virology into a licensed VSV-vectored Ebola vaccine.

Chandipura viral glycoprotein (CNV-G) promotes Gectosome generation and enables delivery of intracellular therapeutics.

Overexpression of vesicular stomatitis virus G protein (VSV-G) elevates the secretion of EVs known as gectosomes, which contain VSV-G. Such vesicles can be engineered to deliver therapeutic macromolecules. We investigated viral glycoproteins from several viruses for their potential in gectosome production and intracellular cargo delivery. Expression of the viral glycoprotein (viral glycoprotein from the Chandipura virus [CNV-G]) from the human neurotropic pathogen Chandipura virus in 293T cells significantly augments the production of CNV-G-containing gectosomes. In comparison with VSV-G gectosomes, CNV-G gectosomes exhibit heightened selectivity toward specific cell types, including primary cells and tumor cell lines. Consistent with the differential tropism between CNV-G and VSV-G gectosomes, cellular entry of CNV-G gectosome is independent of the Low-density lipoprotein receptor, which is essential for VSV-G entry, and shows varying sensitivity to pharmacological modulators. CNV-G gectosomes efficiently deliver diverse intracellular cargos for genomic modification or responses to stimuli in vitro and in the brain of mice in vivo utilizing a split GFP and chemical-induced dimerization system. Pharmacokinetics and biodistribution analyses support CNV-G gectosomes as a versatile platform for delivering macromolecular therapeutics intracellularly.

Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development.

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

Shear-reversible clusters of HIV-1 in solution: stabilized by antibodies, dispersed by mucin.

IMPORTANCE: The phenomenon of reversible clustering is expected to further nuance HIV immune stealth because virus surfaces can escape interaction with antibodies (Abs) by hiding temporarily within clusters. It is well known that mucin reduces HIV virulence, and the current perspective is that mucin aggregates HIV-1 to reduce infections. Our findings, however, suggest that mucin is dispersing HIV clusters. The study proposes a new paradigm for how HIV-1 may broadly evade Ab recognition with reversible clustering and why mucin effectively neutralizes HIV-1.

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration.

Cell culture-based production of vector-based vaccines and virotherapeutics is of increasing interest. The vectors used not only retain their ability to infect cells but also induce robust immune responses. Using two recombinant vesicular stomatitis virus (rVSV)-based constructs, we performed a proof-of-concept study regarding an integrated closed single-use perfusion system that allows continuous virus harvesting and clarification. Using suspension BHK-21 cells and a fusogenic oncolytic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV), a modified alternating tangential flow device (mATF) or tangential flow depth filtration (TFDF) systems were used for cell retention. As the hollow fibers of the former are characterized by a large internal lumen (0.75 mm; pore size 0.65 µm), membrane blocking by the multi-nucleated syncytia formed during infection could be prevented. However, virus particles were completely retained. In contrast, the TFDF filter unit (lumen 3.15 mm, pore size 2-5 µm) allowed not only to achieve high viable cell concentrations (VCC, 16.4-20.6×106 cells/mL) but also continuous vector harvesting and clarification. Compared to an optimized batch process, 11-fold higher infectious virus titers were obtained in the clarified permeate (maximum 7.5×109 TCID50/mL). Using HEK293-SF cells and a rVSV vector expressing a green fluorescent protein, perfusion cultivations resulted in a maximum VCC of 11.3×106 cells/mL and infectious virus titers up to 7.1×1010 TCID50/mL in the permeate. Not only continuous harvesting but also clarification was possible. Although the cell-specific virus yield decreased relative to a batch process established as a control, an increased space-time yield was obtained. KEY POINTS: • Viral vector production using a TFDF perfusion system resulted in a 460% increase in space-time yield • Use of a TFDF system allowed continuous virus harvesting and clarification • TFDF perfusion system has great potential towards the establishment of an intensified vector production.

Optimal delivery of RNA interference by viral vectors for cancer therapy.

In recent years, there has been a surge in the innovative modification and application of the viral vector-based gene therapy field. Significant and consistent improvements in the engineering, delivery, and safety of viral vectors have set the stage for their application as RNA interference (RNAi) delivery tools. Viral vector-based delivery of RNAi has made remarkable breakthroughs in the treatment of several debilitating diseases and disorders (e.g., neurological diseases); however, their novelty has yet to be fully applied and utilized for the treatment of cancer. This review highlights the most promising and emerging viral vector delivery tools for RNAi therapeutics while discussing the variables limiting their success and suitability for cancer therapy. Specifically, we outline different integrating and non-integrating viral platforms used for gene delivery, currently employed RNAi targets for anti-cancer effect, and various strategies used to optimize the safety and efficacy of these RNAi therapeutics. Most importantly, we provide great insight into what challenges exist in their application as cancer therapeutics and how these challenges can be effectively navigated to advance the field.

Preexisting Immunity Does Not Prevent Efficacy of Vesicular Stomatitis Virus-Based Filovirus Vaccines in Nonhuman Primates.

Ebola virus (EBOV) and Marburg virus (MARV) made headlines in the past decade, causing outbreaks of human disease in previously nonendemic yet overlapping areas. While EBOV outbreaks can be mitigated with licensed vaccines and treatments, there is not yet a licensed countermeasure for MARV. Here, we used nonhuman primates (NHPs) previously vaccinated with vesicular stomatitis virus (VSV)-MARV and protected against lethal MARV challenge. After a resting period of 9 months, these NHPs were revaccinated with VSV-EBOV and challenged with EBOV, resulting in 75% survival. Surviving NHPs developed EBOV glycoprotein (GP)-specific antibody titers and no viremia or clinical signs of disease. The single vaccinated NHP succumbing to challenge showed the lowest EBOV GP-specific antibody response after challenge, supporting previous findings with VSV-EBOV that antigen-specific antibodies are critical in mediating protection. This study again demonstrates that VSVΔG-based filovirus vaccine can be successfully used in individuals with preexisting VSV vector immunity, highlighting the platform's applicability for consecutive outbreak response.

Limited Benefit of Postexposure Prophylaxis With VSV-EBOV in Ebola Virus-Infected Rhesus Macaques.

Vesicular stomatitis virus-Ebola virus (VSV-EBOV) vaccine has been successfully used in ring vaccination approaches during EBOV disease outbreaks demonstrating its general benefit in short-term prophylactic vaccination, but actual proof of its benefit in true postexposure prophylaxis (PEP) for humans is missing. Animal studies have indicated PEP efficacy when VSV-EBOV was used within hours of lethal EBOV challenge. Here, we used a lower EBOV challenge dose and a combined intravenous and intramuscular VSV-EBOV administration to improve PEP efficacy in the rhesus macaque model. VSV-EBOV treatment 1 hour after EBOV challenge resulted in delayed disease progression but little benefit in outcome. Thus, we could not confirm previous results indicating questionable benefit of VSV-EBOV for EBOV PEP in a nonhuman primate model.

A high-throughput screening system for SARS-CoV-2 entry inhibition, syncytia formation and cell toxicity.

BACKGROUND: The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell is mediated through the binding of the SARS-CoV-2 Spike protein via the receptor binding domain (RBD) to human angiotensin-converting enzyme 2 (hACE2). Identifying compounds that inhibit Spike-ACE2 binding would be a promising and safe antiviral approach against COVID-19. METHODS: In this study, we used a BSL-2 compatible replication-competent vesicular stomatitis virus (VSV) expressing Spike protein of SARS-CoV-2 with eGFP reporter system (VSV-eGFP-SARS-CoV-2) in a recombinant permissive cell system for high-throughput screening of viral entry blockers. The SARS-CoV-2 permissive reporter system encompasses cells that stably express hACE2-tagged cerulean and H2B tagged with mCherry, as a marker of nuclear condensation, which also enables imaging of fused cells among infected EGFP positive cells and could provide real-time information on syncytia formation. RESULTS: A limited high-throughput screening identified six natural products that markedly inhibited VSV-eGFP-SARS-CoV-2 with minimum toxicity. Further studies of Spike-S1 binding using the permissive cells showed Scillaren A and 17-Aminodemethoxygeldanamycin could inhibit S1 binding to ACE2 among the six leads. A real-time imaging revealed delayed inhibition of syncytia by Scillaren A, Proscillaridin, Acetoxycycloheximide and complete inhibition by Didemnin B indicating that the assay is a reliable platform for any image-based drug screening. CONCLUSION: A BSL-2 compatible assay system that is equivalent to the infectious SARS-CoV-2 is a promising tool for high-throughput screening of large compound libraries for viral entry inhibitors against SARS-CoV-2 along with toxicity and effects on syncytia. Studies using clinical isolates of SARS-CoV-2 are warranted to confirm the antiviral potency of the leads and the utility of the screening system.

Vesicular Stomatitis Virus-Based Epstein-Barr Virus Vaccines Elicit Strong Protective Immune Responses.

Epstein-Barr virus (EBV), the first identified human tumor virus, is etiologically associated with various kinds of malignant and benign diseases, accounting for 265,000 cancer incident cases and 164,000 cancer deaths in 2017. EBV prophylactic vaccine development has been gp350 centered for several decades. However, clinical studies show that gp350-centered vaccines fail to prevent EBV infection. Advances in the EBV infection mechanisms shed light on gB and gHgL, the two key components of the infection apparatus. In this study, for the first time, we utilized recombinant vesicular stomatitis virus (VSV) to display EBV gB (VSV-ΔG-gB/gB-G) or gHgL (VSV-ΔG-gHgL). In vitro studies confirmed successful virion production and glycoprotein presentation on the virion surface. In mouse models, VSV-ΔG-gB/gB-G or VSV-ΔG-gHgL elicited potent humoral responses. Neutralizing antibodies elicited by VSV-ΔG-gB/gB-G were prone to prevent B cell infection, while those elicited by VSV-ΔG-gHgL were prone to prevent epithelial cell infection. Combinatorial vaccination yields an additive effect. The ratio of endpoint neutralizing antibody titers to the endpoint total IgG titers immunized with VSV-ΔG-gHgL was approximately 1. The ratio of IgG1/IgG2a after VSV-ΔG-gB/gB-G immunization was approximately 1 in a dose-dependent, adjuvant-independent manner. Taken together, VSV-based EBV vaccines can elicit a high ratio of epithelial and B lymphocyte neutralizing antibodies, implying their unique potential as EBV prophylactic vaccine candidates. IMPORTANCE Epstein-Barr virus (EBV), one of the most common human viruses and the first identified human oncogenic virus, accounted for 265,000 cancer incident cases and 164,000 cancer deaths in 2017 as well as millions of nonmalignant disease cases. So far, no prophylactic vaccine is available to prevent EBV infection. In this study, for the first time, we reported the VSV-based EBV vaccines presenting two key components of the EBV infection apparatus, gB and gHgL. We confirmed potent antigen-specific antibody generation; these antibodies prevented EBV from infecting epithelial cells and B cells, and the IgG1/IgG2a ratio indicated balanced humoral-cellular responses. Taken together, we suggest VSV-based EBV vaccines are potent prophylactic candidates for clinical studies and help eradicate numerous EBV-associated malignant and benign diseases.

A Recombinant VSV-Based Bivalent Vaccine Effectively Protects against Both SARS-CoV-2 and Influenza A Virus Infection.

COVID-19 and influenza are both highly contagious respiratory diseases that have been serious threats to global public health. It is necessary to develop a bivalent vaccine to control these two infectious diseases simultaneously. In this study, we generated three attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates against both SARS-CoV-2 and influenza viruses. These rVSV-based vaccines coexpress SARS-CoV-2 Delta spike protein (SP) bearing the C-terminal 17 amino acid (aa) deletion (SPΔC) and I742A point mutation, or the SPΔC with a deletion of S2 domain, or the RBD domain, and a tandem repeat harboring four copies of the highly conserved influenza M2 ectodomain (M2e) that fused with the Ebola glycoprotein DC-targeting/activation domain. Animal immunization studies have shown that these rVSV bivalent vaccines induced efficient humoral and cellular immune responses against both SARS-CoV-2 SP and influenza M2 protein, including high levels of neutralizing antibodies against SARS-CoV-2 Delta and other variant SP-pseudovirus infections. Importantly, immunization of the rVSV bivalent vaccines effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads. Overall, this study provides convincing evidence for the high efficacy of this bivalent vaccine platform to be used and/or easily adapted to produce new vaccines against new or reemerging SARS-CoV-2 variants and influenza A virus infections. IMPORTANCE Given that both COVID-19 and influenza are preferably transmitted through respiratory droplets during the same seasons, it is highly advantageous to develop a bivalent vaccine that could simultaneously protect against both COVID-19 and influenza. In this study, we generated the attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates that target both spike protein of SARS-Cov-2 Delta variant and the conserved influenza M2 domain. Importantly, these vaccine candidates effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads.

Whole genome CRISPR screening strategy to identify genes contributing to SARS-CoV-2 spike and VSV-G mediated entry.

Understanding the cellular host factors that promote and inhibit viral entry is important for identifying viral countermeasures. CRISPR whole-genome screens can be used to rapidly discover host factors that contribute to or impair viral entry. However, when using live viruses and cellular lethality for selection, these screens can identify an overwhelming number of genes without specificity for the stage of the viral infection cycle. New screening methods are needed to identify host machinery contributing to specific steps of viral infection. Here, we developed a CRISPR whole-genome screen and counter-screen strategy based on a pseudoviral platform that allowed identification of genes specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike and vesicular stomatitis virus glycoprotein (VSV-G) mediated entry. Screening of SARS-CoV-2 spike and VSV-G on the same lentiviral pseudovirus allowed the identification of entry-specific genes relative to genes associated with retro-transcription, integration, and reporter expression from the lentiviral pseudovirus. Second, a Cre-Gag fusion protein packaged into the pseudovirus was used to bypass retro-transcription and integration by directly activating a floxed fluorescent protein reporter upon entry  reduced the number of gene hits and increase specificity for viral entry. Our approach correctly identified SARS-CoV-2 and VSV-G receptors ACE2 and low-density lipoprotein receptors, respectively, and distinguished genes associated with retroviral reporter expression from envelope-mediated entry. Moreover, the CRE-Gag fusion/flox reporter increased the screen specificity for viral entry-associated genes. Validation of a few hits demonstrates that this approach distinguishes envelope-specific host factors from genes affecting reporter expression. Overall, this approach provides a new strategy for identifying host genes influencing viral entry without the confounding complexity of live-viral screens which produce long gene lists associated with all aspects of viral pathogenesis and replication.

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 were significantly higher than those of 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.

The roles of BST-2 in murine B cell development and on virus propagation.

The bone marrow (BM) stromal cell antigen-2 (BST-2), also known as tetherin, CD317, PDCA-1, or HM1.24, is a membrane protein overexpressed in several types of tumors and may act as a promising target for cancer treatment via antibody-dependent cellular cytotoxicity. BST-2 is also expressed in human BM stromal cells (BMSC), which support B cell development. While the activity of BST-2 as an antiviral factor has been demonstrated, the expression patterns and the role of BST-2 on B-cell development and activation have not been investigated, especially in vivo. In this study, Bst2 knockout (Bst2-/- ) mice were generated to assess the role of BST-2 on B cell development and activation. It was observed that BST-2 was not expressed in BMSC or all B cell progenitors even in wild-type mice and does not play a significant role in B cell development. In addition, the loss of BST-2 had no effect on B cell activation. Furthermore and in contrast to the well-known antiviral role of BST-2, infection of vesicular stomatitis Indiana virus to the BM cells collected from the Bst2-/- mice produced less infectious virus compared with that from the WT mice. These results suggest that murine BST-2 is different from human BST-2 in the expression pattern, physiological function, in vivo, and might possess positive role on VSV replication.

Pseudotyped Viruses for Marburgvirus and Ebolavirus.

Marburg virus (MARV) and Ebola virus (EBOV) of the Filoviridae family are the most lethal viruses in terms of mortality rate. However, the development of antiviral treatment is hampered by the requirement for biosafety level-4 (BSL-4) containment. The establishment of BSL-2 pseudotyped viruses can provide important tools for the study of filoviruses. This chapter summarizes general information on the filoviruses and then focuses on the construction of replication-deficient pseudotyped MARV and EBOV (e.g., lentivirus system and vesicular stomatitis virus system). It also details the potential applications of the pseudotyped viruses, including neutralization antibody detection, the study of infection mechanisms, the evaluation of antibody-dependent enhancement, virus entry inhibitor screening, and glycoprotein mutation analysis.

Pseudotyped Viruses for Mammarenavirus.

Mammarenaviruses are classified into New World arenaviruses (NW) and Old World arenaviruses (OW). The OW arenaviruses include the first discovered mammarenavirus-lymphocytic choriomeningitis virus (LCMV) and the highly lethal Lassa virus (LASV). Mammarenaviruses are transmitted to human by rodents, resulting in severe acute infections and hemorrhagic fever. Pseudotyped viruses have been widely used as a tool in the study of mammarenaviruses. HIV-1, SIV, FIV-based lentiviral vectors, VSV-based vectors, MLV-based vectors, and reverse genetic approaches have been applied in the construction of pseudotyped mammarenaviruses. Pseudotyped mammarenaviruses are commonly used in receptor research, neutralizing antibody detection, inhibitor screening, viral virulence studies, functional analysis of N-linked glycans, and studies of viral infection, endocytosis, and fusion mechanisms.

Development of Recombinant Oncolytic rVSV-mIL12-mGMCSF for Cancer Immunotherapy.

Anti-cancer therapy based on oncolytic viruses (OVs) is a targeted approach that takes advantage of OVs' ability to selectively infect and replicate in tumor cells, activate the host immune response, and destroy malignant cells over healthy ones. Vesicular stomatitis virus (VSV) is known for its wide range of advantages: a lack of pre-existing immunity, a genome that is easily amenable to manipulation, and rapid growth to high titers in a broad range of cell lines, to name a few. VSV-induced tumor immunity can be enhanced by the delivery of immunostimulatory cytokines. The targeted cytokine delivery to tumors avoids the significant toxicity associated with systemic delivery while also boosting the immune response. To demonstrate this enhanced effect on both tumor growth and survival, a novel recombinant VSV (rVSV)-mIL12-mGMCSF, co-expressing mouse IL-12 (interleukin-12) and GM-CSF (granulocyte-macrophage colony-stimulating factor), was tested alongside rVSV-dM51-GFP (rVSV-GFP) that was injected intratumorally in a syngeneic in vivo C57BL/6 mouse model infused subcutaneously with B16-F10 melanoma cells. The pilot study tested the effect of two viral injections 4 days apart and demonstrated that treatment with the two rVSVs resulted in partial inhibition of tumor growth (TGII of around 40%) and an increased survival rate in animals from the treatment groups. The effect of the two VSVs on immune cell populations will be investigated in future in vivo studies with an optimized experimental design with multiple higher viral doses, as a lack of this information presents a limitation of this study.

Direct trafficking pathways from the Golgi apparatus to the plasma membrane.

In eukaryotic cells, protein sorting is a highly regulated mechanism important for many physiological events. After synthesis in the endoplasmic reticulum and trafficking to the Golgi apparatus, proteins sort to many different cellular destinations including the endolysosomal system and the extracellular space. Secreted proteins need to be delivered directly to the cell surface. Sorting of secreted proteins from the Golgi apparatus has been a topic of interest for over thirty years, yet there is still no clear understanding of the machinery that forms the post-Golgi carriers. Most evidence points to these post-Golgi carriers being tubular pleomorphic structures that bud from the trans-face of the Golgi. In this review, we present the background studies and highlight the key components of this pathway, we then discuss the machinery implicated in the formation of these carriers, their translocation across the cytosol, and their fusion at the plasma membrane.

A method for the generation of pseudovirus particles bearing SARS coronavirus spike protein in high yields.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 spike protein in high yield. We found that a large proportion of pseudovirions produced with the conventional transient expression system lacked coronavirus spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus spike protein allowed the efficient production of progeny pseudoviruses decorated with spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.Key words: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, pseudovirus, vesicular stomatitis virus (VSV), spike protein.