Search Results (243)

Schmallenberg virus (SBV) is a negative-sense RNA virus transmitted by insect vectors, causing arthrogryposis-hydranencephaly syndrome in newborn ruminants. Since its discovery in Germany and the Netherlands in 2011, SBV has rapidly spread across multiple European countries, resulting in significant economic losses in the livestock industry. With the increasing global animal trade and the expanded range of insect transmission, the risk of SBV introduction into non-endemic regions is also rising. As the gold standard for serological testing, the virus neutralization test (VNT) is crucial for tracking the spread of SBV and evaluating the efficacy of vaccines. However, in non-endemic regions, the lack of local viral strains and the biosafety risks associated with introducing foreign strains pose challenges to the implementation of VNT. In this study, we employed reverse genetics techniques using vesicular stomatitis virus (VSV) to substitute the VSV G protein with the envelope glycoproteins of SBV, thereby successfully generating and rescuing the recombinant virus rVSVΔG-eGFP-SBVGPC. The recombinant virus was then thoroughly characterized in terms of SBV Gc protein expression, viral morphology, and growth kinetics. Importantly, rVSVΔG-eGFP-SBVGPC exhibited SBV-specific cell tropism and was capable of reacting with SBV-positive serum, enabling the measurement of neutralizing antibody titers. The results suggest that this recombinant virus can serve as a feasible alternative for SBV neutralization tests, with promising potential for application in serological screening and vaccine evaluation. Full article

Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic bunyavirus with a high case-fatality ratio for which there is no approved vaccine. Studies have assessed different vaccine technologies. However, few studies have yet assessed the immunogenicity of heterologous prime-boost regimens. Methods: Here, we compare a lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA-based vaccine encoding the SFTSV glycoproteins, Gn and Gc, to our recently described recombinant VSV SFTSV (rVSV-SFTSV) vaccine in single dose, homologous, and heterologous prime-boost regimens in mice. Results: We show that all regimens protect from pathogenic SFTSV challenge and elicit strong long-lasting antibody responses. Furthermore, strong cellular immunity is elicited by mRNA-LNP immunizations and by heterologous immunization with an rVSV-SFTSV prime and mRNA-LNP boost. Cellular responses robustly polarized towards a type 1 response, characterized by high levels of IFNγ, TNFα, and IL-2. Immunization with mRNA led to a mixed type 1/type 2 immune response, as determined by antibody isotypes IgG1 and IgG2c. We found that homologous immunization leads to stronger antibody responses while heterologous immunization drives a slightly stronger cellular response. Conclusions: Taken together, the vaccine platforms described here represent strong vaccine candidates for further development. Full article

Background/Objectives: Leukemia is associated with high recurrence rates and cancer vaccines are emerging as a promising immunotherapy against the disease. Here, we investigate the mechanism of action by which a personalized vaccine made from leukemia cells infected with an oncolytic virus (ICV) induces anti-tumor immunity. Methods: Using the L1210 murine model, leukemia cells were infected and irradiated to create the ICV. The CRISPR-Cas9 system was used to engineer knockout cells to test in treatment efficacy studies. Results: We found that pro-inflammatory interferons (IFNs) that are produced by infected vaccine cells induce the immunoproteasome (ImP), a specialized proteasome subtype that is found in immune cells. Interestingly, we show that while a vaccine using the oncolytic vesicular stomatitis virus (oVSV) completely protects against tumor challenge, the wild-type (wt) virus, which does not induce the ImP, is not as effective. To delineate the contribution of the ImP for vaccine efficacy, we generated ImP-knockout cell lines and found no differences in treatment efficacy compared to wild-type cells. Furthermore, an ICV using another murine leukemia model that expresses the ImP only when infected by an IFN gamma-encoding variant of the virus demonstrated similar efficacy as the parental virus. Conclusions: Taken together, our data show that ImP expression by vaccine cells was not required for the efficacy of leukemia ICVs. Full article

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the etiologic agent that causes the coronavirus disease (COVID-19) identified in Wuhan, in 2019. Men are more prone to developing severe manifestations than women, suggesting a possible crucial role of sex hormones. 17,β-Estradiol (E2) and 1,25 α dihydroxyvitamin D₃ (calcitriol) act upon gene pathways as immunomodulators in several infectious respiratory diseases. In this study, we aimed to evaluate the influence of E2 and calcitriol on the VSV-based pseudovirus SARS-CoV-2 and SARS-CoV-2 infection in vitro. We infected Vero E6 cells with the recombinant VSV-based pseudovirus SARS-CoV-2 and the SARS-CoV-2 viruses according to the pre-treatment and pre–post-treatment models. The Angiotensin-Converting Enzyme 2 (ACE2) and Vitamin D Receptor (VDR) gene expression did not change under different treatments. The VSV-based pseudovirus SARS-CoV-2 infection showed a significant decrease in the focus-forming unit count in the presence of E2 and calcitriol (either alone or in combination) in the pre-treatment model, while in the pre–post-treatment model, the infection was inhibited only in the presence of E2. Th SARS-CoV-2 infection highlighted a decrease in viral titres in the presence of E2 and calcitriol only in the pre–post-treatment model. 17,β-Estradiol and calcitriol can exert an inhibitory effect on SARS-CoV-2 infections, demonstrating their protective role against viral infections. Full article

Nipah virus (NiV) is a highly pathogenic bat-borne zoonotic pathogen that poses a significant threat to human and animal health, with fatality rates exceeding 70% in some outbreaks. Despite its significant public health impact, there are currently no licensed vaccines or specific therapeutics available. Various virological tools—such as reverse genetics systems, replicon particles, VSV-based pseudoviruses, and recombinant Cedar virus chimeras—have been widely used to study the molecular mechanisms of NiV and to support vaccine development. Building upon these platforms, we developed a replication-competent recombinant vesicular stomatitis virus (rVSVΔG-eGFP-NiV_BD F/G) expressing NiV attachment (G) and fusion (F) glycoproteins. This recombinant virus serves as a valuable tool for investigating NiV entry mechanisms, cellular tropism, and immunogenicity. The virus was generated by replacing the VSV G protein with NiV F/G through reverse genetics, and protein incorporation was confirmed via immunofluorescence and electron microscopy. In vitro, the virus exhibited robust replication, characteristic cell tropism, and high viral titers in multiple cell lines. Neutralization assays showed that monoclonal antibodies HENV-26 and HENV-32 effectively neutralized the recombinant virus. Furthermore, immunization of golden hamsters with inactivated rVSVΔG-eGFP-NiV_BD F/G induced potent neutralizing antibody responses, demonstrating its robust immunogenicity. These findings highlight rVSVΔG-eGFP-NiV_BD F/G as an effective platform for NiV research and vaccine development. Full article

In December 2023, infections of western equine encephalitis virus (WEEV) within Argentina were reported to the World Health Organization (WHO). By April 2024, more than 250 human infections, 12 of which were fatal, and 2500 equine infections were identified in South America. Laboratory diagnosis and surveillance in affected countries were hindered by a lack of facilities equipped with BSL-3 laboratories, as confirmatory serodiagnosis for WEEV requires live virus in the plaque reduction neutralization test (PRNT). To expand serodiagnosis for WEEV in the Americas, we developed a virus chimera composed of vesicular stomatitis virus (VSV) engineered to display the E2-E1 glycoproteins of WEEV (VSV/WEEV) in place of the VSV glycoprotein (G). PRNT₉₀ and IC₉₀ values of parental WEEV and VSV/WEEV were analogous using sera collected from mice, horses, and chickens. VSV/WEEV rapidly formed plaques with clear borders and reduced the assay readout time by approximately 8 h compared to the parental virus. Overall, we demonstrate that chimeric VSV/WEEV is a suitable surrogate for WEEV in a diagnostic PRNT. Use of chimeric VSV/WEEV in place of authentic WEEV will dramatically expand testing capacity by enabling PRNTs to be performed at BSL-2 containment, while simultaneously decreasing the health risk to testing personnel. Full article

Recombinant vesicular stomatitis virus (rVSV) is a promising viral vaccine vector for addressing the COVID-19 pandemic. Inducing mucosal immunity via the intranasal route is an ideal strategy for rVSV-based vaccines, but it requires extremely stringent safety standards. In this study, we constructed two rVSV variants with amino acid mutations in their M protein: rVSV-M2 with M33A/M51R mutations and rVSV-M4 with M33A/M51R/V221F/S226R mutations, and developed COVID-19 vaccines based on these attenuated vectors. By comparing viral replication capacity, intranasal immunization, intracranial injection, and blood cell counts, we demonstrated that the M protein mutation variants exhibit significant attenuation effects both in vitro and in vivo. Moreover, preliminary investigations into the mechanisms of virus attenuation revealed that these attenuated viruses can induce a stronger type I interferon response while reducing inflammation compared to the wild-type rVSV. We developed three candidate vaccines against SARS-CoV-2 using the wildtype VSV backbone with either wild-type M (rVSV-JN.1) and two M mutant variants (rVSV-M2-JN.1 and rVSV-M4-JN.1). Our results confirmed that rVSV-M2-JN.1 and rVSV-M4-JN.1 retain strong immunogenicity while enhancing safety in hamsters. In summary, the rVSV variants with M protein mutations represent promising candidate vectors for mucosal vaccines and warrant further investigation. Full article

Background: An effective vaccine against Nipah virus (NiV) is crucial due to its high fatality rate and recurrent outbreaks in South and Southeast Asia. Vaccine development is challenged by the lack of validated accessible neutralization assays, as virus culture requires BSL-4 facilities, restricting implementation in resource-limited settings. To address this, we standardized and validated a pseudotyped virus neutralization assay (PNA) for assessing NiV-neutralizing antibodies in BSL-2 laboratories. Methods: The NiV-PNA was validated following international regulatory standards, using a replication-defective recombinant Vesicular stomatitis virus (rVSV) backbone dependent pseudotyped virus. Assessments included sensitivity, specificity, dilutional linearity, relative accuracy, precision, and robustness. The assay was calibrated using the WHO International Standard for anti-NiV antibodies and characterized reference sera to ensure reliable performance. Findings: Preliminary evaluation of the developed NiV-PNA showed 100% sensitivity and specificity across 10 serum samples (5 positive, 5 negative), with a positive correlation to a calibrated reference assay (R² = 0.8461). Dilutional linearity (R² = 0.9940) and accuracy (98.18%) were confirmed across the analytical titer range of 11-1728 IU/mL. The assay also exhibited high precision, with intra-assay and intermediate precision geometric coefficients of variation of 6.66% and 15.63%, respectively. Robustness testing demonstrated minimal variation across different pseudotyped virus lots, incubation times, and cell counts. Conclusions: The validated NiV-PNA is a reproducible and scalable assay platform for quantifying NiV neutralizing antibodies, offering a safer alternative to virus culture. Its validation and integration into the CEPI Centralized Laboratory Network will enhance global capacity for vaccine evaluation and outbreak preparedness. Full article

Background/Objectives: Baculoviruses represent promising gene delivery vectors for mammalian systems, combining high safety profiles with substantial cargo capacity. While pseudotyping with vesicular stomatitis virus G-protein (VSV-G) enhances transduction efficiency, optimal expression strategies during the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection cycle remain unexplored. This study investigates how VSV-G expression timing affects pseudotype incorporation into budded virions (BVs) and subsequent transduction efficacy. Methods: Three recombinant AcMNPV constructs were generated, each expressing VSV-G under distinct baculoviral promoters (ie1, gp64, and p10) and GFP via a CMV promoter. VSV-G incorporation was verified by Western blot, while transduction efficiency was quantified in mammalian cell lines (fluorescence microscopy/flow cytometry) and rat hind limbs. Viral productivity was assessed through production kinetics and plaque assays. Results: All the pseudotyped viruses showed significantly enhanced transduction capacity versus controls, strongly correlating with VSV-G incorporation levels. The p10 promoter drove the highest VSV-G expression and transduction efficiency. Crucially, BV production yields and infectivity remained unaffected by VSV-G expression timing. The in vivo results mirrored the cell culture findings, with p10-driven constructs showing greater GFP expression at low doses (10⁴ virions). Conclusions: Strategic VSV-G expression via very late promoters (particularly p10) maximizes baculoviral transduction without compromising production yields. This study establishes a framework for optimizing pseudotyped BV systems, demonstrating that late-phase glycoprotein expression balances high mammalian transduction with preserved insect-cell productivity—a critical advancement for vaccine vector development. Full article

Background/Objectives: CD47 is a cell surface glycoprotein moderately expressed in healthy cells and upregulated in cancer and viral infected cells. CD47’s interaction with signal regulatory protein alpha (SIRPα) inhibits phagocytic cells and its interaction with thrombospondin-1 inhibits T cell response. Experimental evidence has revealed that the blockade of CD47 resulted in the increased activation and function of both innate and adaptive immune cells, therefore exerting antitumoral and antiviral effects. Recent studies have shown that the combination of vaccines and immune checkpoint inhibitors could be a promising approach to increasing vaccine immunogenicity. Here, we investigated the vaccinal effect of anti-CD47 antibodies and discussed the possibilities of combining anti-CD47 treatments with vaccines. Methods: Using vesicular stomatitis virus (VSV), a widely used replication-competent vaccine vector, we evaluated the impact of the immunotherapeutic blockade of CD47 on cellular, humoral, and protective immunity. We infected C57BL/6 mice with VSV, treated them with anti-CD47 antibodies or an isotype, and evaluated the total immunoglobulin (Ig), IgG neutralizing antibodies, B cell activation, CD8+ T cell effector function, and survival of the mice. Results: We found that the treatments of anti-CD47 antibodies led to significantly increased Ig and IgG neutralizing antibody levels compared to the isotype treatment. Flow cytometric analysis of B cells revealed no difference in the number of circulating B cells; however, we observed an increased surface expression of CD80 and CD86 in B cells among anti-CD47-treated mice. Further analysis of the impact of CD47 blockade on T immunity revealed a significantly higher percentage of IFN-γ⁺ CD4 and IFN-γ⁺ CD8 T cells in anti-CD47-treated mice. Upon infecting mice with a lethal VSV dose, we observed a significantly higher survival rate among the anti-CD47-treated mice compared to control mice. Conclusions: Our results indicate that anti-CD47 treatment induces a stronger cellular and humoral immune response, leading to better protection. As such, immunotherapy by CD47 blockade in combination with vaccines could be a promising approach to improve vaccine efficacy. Full article

This review provides an updated overview of oncolytic virotherapy as a promising therapeutic strategy for colorectal cancer (CRC), focusing on six key viral platforms: adenovirus, herpes simplex virus (HSV), reovirus, vesicular stomatitis virus (VSV), vaccinia virus (VV), and measles virus (MV). These viruses exhibit tumor-selective replication and exert their effects through mechanisms such as direct oncolysis, the delivery of immunostimulatory genes (e.g., IL-12, IL-15, GM-CSF), the activation of innate and adaptive immune responses, and the remodeling of the tumor microenvironment. Preclinical and early clinical studies suggest that oncolytic viruses can enhance the efficacy of existing treatments, particularly in immunologically “cold” tumors such as microsatellite stable CRC, when used in combination with chemotherapy or immune checkpoint inhibitors. Despite encouraging results, several challenges remain, including antiviral immune clearance, tumor heterogeneity, and limitations in systemic delivery. Current research focuses on improving viral engineering, enhancing tumor targeting, and designing combinatorial strategies to overcome resistance and maximize clinical benefits. Overall, oncolytic viruses represent a versatile and evolving therapeutic class with the potential to address unmet clinical needs in CRC. Full article

Background/Objectives: Patients suffering from inflammatory bowel diseases (IBDs) undergoing treatment with anti-TNF antibodies mount a diminished humoral immune response to vaccination against SARS-CoV-2 compared to healthy controls. The characterization of variant-specific immune responses is particularly warranted among immunosuppressed patients, where reduced responses may necessitate further medical interventions. Methods: This pilot study investigated the humoral immune response of vaccinated IBD patients on anti-TNF medication and a comparable group of healthy individuals against the viral variants Alpha, Beta, Gamma, Delta, and Omicron BA.1 and BA.5. While total IgG antibodies targeting the receptor binding site of the spike protein of SARS-CoV-2 were quantified using a chemiluminescence microparticle immunoassay (CMIA), their potential neutralizing capacity was determined using commercial and variant-specific in-house surrogate virus neutralization tests (sVNTs) against a variant-specific in-house VSV-pseudotyped virus neutralization test (pVNT) as the gold standard. Results: Employing variant-specific assays recapitulated the immune escape functions of virus variants. Conspicuously, antibody reactivity against Alpha and Omicron BA.1 and BA.5 was strikingly poor in IBD patient sera post-initial vaccination compared to healthy individuals. A comparison of the diagnostic performance of assays with the pVNT revealed that identification of patients with inadequate humoral responses by CMIA and sVNT may require adjustments to cut-off values and end-point titration of sera. Following adaptation of cut-off values, patient sera exhibited reduced reactivity against all tested variants. The assay panel used substantiated the impact of anti-TNF therapy in IBD patients as to reduced strength, function, and breadth of the immune response to several SARS-CoV-2 variants. The immune response measured following the second vaccination was comparable to the antibody response observed in healthy individuals following the first vaccination. Conclusion: Variant-specific sVNTs and pVNTs have the potential to serve as valuable tools for evaluating the efficacy of adapted vaccines and to inform clinical interventions in the care of immunosuppressed patients. Anti-TNF-treated individuals with antibody levels below the optimized CMIA threshold should be considered for early booster vaccination and/or close immunological monitoring. Full article

Zaire Ebolavirus (EBOV) is one type of filovirus that causes the deadly EBOV disease, with an average fatality rate of around 50%. EBOV outbreaks are devastating and unpredictable and may emerge as the next global pandemic. As a BSL-4 pathogen, EBOV is inaccessible to regular biological laboratories. Therefore, EBOV virus-like particles (EBOV-VLPs) and EBOV pseudoviruses (EBOV-PVs) are utilized in the initial development of many potential therapies, for safety reasons and ease of procurement, as opposed to using infectious viruses. To investigate the host cell entry of EBOV and develop viral entry blockers, the EBOV model virions must accurately represent the morphological and mechanical properties of infectious EBOV virions. Due to the nanometer scale and irregular shape of EBOVs, these properties are challenging to characterize. In this research, state-of-the-art nanoscale characterization techniques are employed to examine the mechanical and structural elements of a selection of commonly used EBOV-approximating model virions. This study comprehensively determines the accuracy of EBOV approximation with a variety of model virions and the uniformity of mechanical and structural traits across different model virion types and preparation methods. This provides important implications for developing therapeutic treatments against EBOV using these model virions. Full article

Porcine teschovirus (PTV) circulates in pig populations, causing clinical diseases such as poliomyelitis, reproductive disorders, and pneumonia. However, the molecular mechanisms underlying the pathogenesis of PTV infection have not been fully elucidated. Here, we found that PTV infection does not activate the promoters of NF-κB or IFN-β. The expression of PTV 3C^pro inhibits the promoter activity of NF-κB and IFN-β stimulated by SeV and inhibits the downstream transcription of NF-κB and IFN-β by blocking the phosphorylation and nuclear translocation of NF-κB. Coimmunoprecipiation (co-IP) experiments demonstrated that 3C^pro and NF-κB interact. The degradation of NF-κB was unaffected by inhibitors targeting lysosomes (NH4Cl), proteasomes (MG132), or caspases (Z-VAD-FMK). The protease activity of 3C^pro, which relies on its catalytic active site, is vital for NF-κB cleavage and degradation. Loss of proteolytic activity in mutants abolished NF-κB degradation, impairing the ability of 3C^pro to suppress SeV-induced innate immunity and restore VSV-GFP replication, thereby underscoring its critical role in immune evasion by targeting NF-κB. This study reveals novel mechanisms underlying PTV-mediated suppression of host innate immunity. Full article

Hantaviruses, including the Sin Nombre virus (SNV) and Andes virus (ANDV), are associated with severe global health risks, causing high mortality rates in hantavirus pulmonary syndrome (HPS) patients. Neutralizing antibodies are essential for virus clearance and survival, making neutralization assays critical for understanding immunity and evaluating therapeutic strategies. In this study, we developed a recombinant vesicular stomatitis virus (VSV)-based surrogate system expressing SNV and ANDV glycoproteins (GPCs), enabling neutralization studies under biosafety level 2 conditions. The neutralization titers obtained with the VSV-based system closely matched the findings from authentic hantavirus assays performed under biosafety level 3 conditions, confirming its potential as a useful tool for determining immune responses and advancing hantavirus research. Full article