Filoviruses, like the Marburg (MARV) and Ebola (EBOV) viruses, have caused outbreaks associated with significant hemorrhagic morbidity and high fatality rates. Vaccines offer one of the best countermeasures for fatal infection, but to date only the EBOV vaccine has received FDA licensure. Given the limited cross protection between the EBOV vaccine and Marburg hemorrhagic fever (MHF), we analyzed the protective efficacy of a similar vaccine, rVSV-MARV, in the lethal cynomolgus macaque model. NHPs vaccinated with a single dose (as little as 1.6 × 107 pfu) of rVSV-MARV seroconverted to MARV G-protein prior to challenge on day 42. Vaccinemia was measured in all vaccinated primates, self-resolved by day 14 post vaccination. Importantly, all vaccinated NHPs survived lethal MARV challenge, and showed no significant alterations in key markers of morbid disease, including clinical signs, and certain hematological and clinical chemistry parameters. Further, apart from one primate (from which tissues were not collected and no causal link was established), no pathology associated with Marburg disease was observed in vaccinated animals. Taken together, rVSV-MARV is a safe and efficacious vaccine against MHF in cynomolgus macaques.

There are currently no prophylactic vaccines licensed to protect against Lassa fever caused by Lassa virus (LASV) infection. The Emergent BioSolutions (EBS) vaccine candidate, EBS-LASV, is being developed for the prevention of Lassa fever. EBS-LASV is a live-attenuated recombinant Vesicular Stomatitis Virus (rVSV)-vectored vaccine encoding the surface glycoprotein complex (GPC) from LASV and has two attenuating vector modifications: a gene shuffle of the VSV N gene and a deletion of the VSV G gene. Preclinical studies were performed to evaluate EBS-LASV\'s neurovirulence potential following intracranial (IC) injection and to determine the biodistribution and vector replication following intramuscular (IM) inoculation in mice. In addition, the potential EBS-LASV toxicity was assessed using repeated-dose IM EBS-LASV administration to rabbits. All mice receiving the IC injection of EBS-LASV survived, while mice administered the unattenuated control vector did not. The vaccine was only detected in the muscle at the injection site, draining lymph nodes, and the spleen over the first week following IM EBS-LASV injection in mice, with no detectable plasma viremia. No toxicity was observed in rabbits receiving a three-dose regimen of EBS-LASV. These studies demonstrate that EBS-LASV is safe when administered to animals and supported a first-in-human dose-escalation, safety, and immunogenicity clinical study.

Marburg virus (MARV), a filovirus, was first identified in 1967 in Marburg, Germany, and Belgrade, former Yugoslavia. Since then, MARV has caused sporadic outbreaks of human disease with high case fatality rates in parts of Africa, with the largest outbreak occurring in 2004/05 in Angola. From 2021 to 2023, MARV outbreaks occurred in Guinea, Ghana, New Guinea, and Tanzania, emphasizing the expansion of its endemic area into new geographical regions. There are currently no approved vaccines or therapeutics targeting MARV, but several vaccine candidates have shown promise in preclinical studies. We compared three vaccine platforms simultaneously by vaccinating hamsters with either a single dose of an adenovirus-based (ChAdOx-1 MARV) vaccine, an alphavirus replicon-based RNA (LION-MARV) vaccine, or a recombinant vesicular stomatitis virus-based (VSV-MARV) vaccine, all expressing the MARV glycoprotein as the antigen. Lethal challenge with hamster-adapted MARV 4 weeks after vaccination resulted in uniform protection of the VSV-MARV and LION-MARV groups and 83% of the ChAdOx-1 MARV group. Assessment of the antigen-specific humoral response and its functionality revealed vaccine-platform-dependent differences, particularly in the Fc effector functions.

Vesicular stomatitis virus (VSV) outbreaks periodically occur in livestock in the western US and are thought to originate from outside this country. Feral swine (Sus scrofa) have been identified as an amplifying host for vesicular stomatitis New Jersey virus (VSNJV) and have been used to better understand the epidemiology of this virus through serosurveillance. This study aimed to determine if antibodies to vesicular stomatitis Indiana virus (VSIV) and VSNJV were present in feral swine in the western US and to determine if seropositive animals were associated with areas of previously detected VSV in livestock. A total of 4,541 feral swine samples was tested using virus neutralization (VN); samples exhibiting neutralizing activity against one or more of the viruses were confirmed using competitive ELISA (cELISA). Eight sera exhibited neutralizing activity by VN assay and a single serum sample from an animal from Kinney County, Texas sampled in December 2019 tested positive for antibodies to VSIV by cELISA. This finding is supported by a local outbreak of VSIV in horses in the same county in June 2019. The low prevalence of antibodies against VSNJV and VSIV was unexpected but indicates that feral swine in the western US do not represent an endemic reservoir for either of these viruses.

The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3-6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2-8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.

Vectored RNA vaccines offer a variety of possibilities to engineer targeted vaccines. They are cost-effective and safe, but replication competent, activating the humoral as well as the cellular immune system.This chapter focuses on RNA vaccines derived from negative-strand RNA viruses from the order Mononegavirales with special attention to Newcastle disease virus-based vaccines and their generation. It shall provide an overview on the advantages and disadvantages of certain vector platforms as well as their scopes of application, including an additional section on experimental COVID-19 vaccines.

Virus particle (VP) quantification plays a pivotal role in the development of production processes of VPs for virus-based therapies. The yield based on total VP count serves as a process performance indicator for evaluating process efficiency and consistency. Here, a label-free particle quantification method for enveloped VPs was developed, with potential applications in oncolytic virotherapy, vaccine development, and gene therapy. The method comprises size-exclusion chromatography (SEC) separation using high-performance liquid chromatography (HPLC) instruments. Ultraviolet (UV) was used for particle quantification and multi-angle light scattering (MALS) for particle characterization. Consistent recoveries of over 97% in the SEC were achieved upon mobile phase screenings and addition of bovine serum albumin (BSA) as sample stabilizer. A calibration curve was generated, and the method\'s performance and applicability to in-process samples were characterized. The assay\'s repeatability variation was <1% and its intermediate precision variation was <3%. The linear range of the method spans from 7.08 × 108 to 1.72 × 1011 VP/mL, with a limit of detection (LOD) of 7.72 × 107 VP/mL and a lower limit of quantification (LLOQ) of 4.20 × 108 VP/mL. The method, characterized by its high precision, requires minimal hands-on time and provides same-day results, making it efficient for process development.

METHODS: Active ingredients in functional foods exhibit broad-spectrum antiviral activity. The objective of this study is to investigate the protective effect of quercetin derived from bee propolis, a natural product with antiviral activity and modulating effects on the gut microbiota, against vesicular stomatitis virus (VSV) infection.
RESULTS: Through a cellular-based study, this study demonstrates that quercetin can modulate the activity of interferon-regulating factor 3 (IRF3). In vivo, it shows that quercetin protects mice from VSV infection by enhancing interferon production and inhibiting the production of proinflammatory cytokines. The study conducts 16S rRNA-based gut microbiota and nontargets metabolomics analyses to elucidate the mechanisms underlying quercetin-mediated bidirectional communication between the gut microbiome and host metabolome during viral infection. Quercetin not only ameliorates VSV-induced dysbiosis of the intestinal flora but also alters serum metabolites related to lipid metabolism. Cross-correlations between the gut bacteriome and the serum metabolome indicate that quercetin can modulate phosphatidylcholine (16:0/0:0) and 5-acetylamino-6-formylamino-3-methyluracil to prevent VSV infection.
CONCLUSIONS: This study systematically elucidates the anti-VSV mechanism of quercetin through gut bacteriome and host metabolome assays, offering new insights into VSV treatment and revealing the mechanisms behind a novel disease management strategy using dietary flavonoid supplements.

BACKGROUND: Metastatic breast cancer is a leading cause of cancer death in woman. Current treatment options are often associated with adverse side effects and poor outcomes, demonstrating the need for effective new treatments. Immunotherapies can provide durable outcomes in many cancers; however, limited success has been achieved in metastatic triple negative breast cancer. We tested whether combining different immunotherapies can target metastatic triple negative breast cancer in pre-clinical models.
METHODS: Using primary and metastatic 4T1 triple negative mammary carcinoma models, we examined the therapeutic effects of oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express reovirus-derived fusion associated small transmembrane proteins p14 (VSV-p14) or p15 (VSV-p15). These viruses were delivered alone or in combination with natural killer T (NKT) cell activation therapy mediated by adoptive transfer of α-galactosylceramide-loaded dendritic cells.
RESULTS: Treatment of primary 4T1 tumors with VSV-p14 or VSV-p15 alone increased immunogenic tumor cell death, attenuated tumor growth, and enhanced immune cell infiltration and activation compared to control oncolytic virus (VSV-GFP) treatments and untreated mice. When combined with NKT cell activation therapy, oncolytic VSV-p14 and VSV-p15 reduced metastatic lung burden to undetectable levels in all mice and generated immune memory as evidenced by enhanced in vitro recall responses (tumor killing and cytokine production) and impaired tumor growth upon rechallenge.
CONCLUSIONS: Combining NKT cell immunotherapy with enhanced oncolytic virotherapy increased anti-tumor immune targeting of lung metastasis and presents a promising treatment strategy for metastatic breast cancer.

The design of new effective cancer treatment methods is a promising and important research field in translational medicine. Oncolytic viruses can induce immunogenic cell death by activating the body\'s immune system to recognize tumor cells. This work presents the results for optimizing the production of recombinant vesicular stomatitis viruses (rVSVs). To ensure the assembly of viral particles, we developed the HEK293TN-T7 cell line, which stably expresses DNA-dependent RNA polymerase 7 for viral genome transcription, and obtained helper plasmids encoding viral genes under the control of the CAG promoter. The oncolytic activity of the purified virus preparation was assessed in a murine model of B16F10Red melanoma cells expressing a red fluorescent protein. The presented method makes it possible to obtain purified viral preparations with a high titer and oncolytic activity. The amplification of viral particles in a HEK293 suspension culture allows for rapid scalability. Therefore, the developed approach can be used to obtain other recombinant VSV-based oncolytic viruses for tumor immunotherapy.