Search Results (62)

Background/Objectives: Vesicular Stomatitis Virus (VSV)-vectored vaccines represent a versatile platform for the development of vaccines against infectious diseases. Replication-competent recombinant VSV vectors in which the native glycoprotein (G) is replaced with a heterologous antigen (rVSVΔG) are widely used and have demonstrated clinical success. In addition to antigen-specific responses, these vaccines induce humoral immunity directed against vector components, which primarily reflects vector exposure and contributes to the overall characterization of vaccine-induced immunity. Standardized assays for quantifying such vector-directed responses are therefore of increasing importance. Methods: We developed and qualified a quantitative enzyme-linked immunosorbent assay (ELISA) for the detection of human IgG antibodies against VSV-Nucleoprotein (N). Assay development included optimization of antigen coating, blocking conditions, and detection reagents. A 10-point standard curve was established using pooled human serum, and assay performance was evaluated by assessing dynamic range, sensitivity, cut point, dilutional linearity, precision, robustness, and sample stability. Results: The optimized assay utilized a coating concentration of 2 μg/mL VSV-N antigen (100 ng/well) and 1% casein as the blocking buffer. The assay demonstrated a dynamic range of 0.33–41.66 arbitrary units per milliliter (AU/mL) with excellent curve fit (R² > 0.996). The cut point was established at an OD₄₅₀ of 0.286. Precision across intra-assay, inter-assay, and inter-operator evaluations met predefined acceptance criteria (≤25% CV). The assay was robust across different plate washers and readers and maintained performance following up to three freeze–thaw cycles. Conclusions: This study describes a robust and reproducible ELISA for quantifying anti-VSV-N IgG responses. The assay provides a fit-for-purpose tool for assessing vector-directed humoral immunity and supports standardized immunogenicity evaluations across VSV-vectored vaccine studies. Full article

Background/Objectives: Eliciting robust immune responses against the hepatitis B virus (HBV) through therapeutic vaccination holds promise for curing chronic hepatitis B. We previously developed the heterologous protein prime/viral vector boost clinical vaccine candidate, TherVacB. Here, we evaluated a replication-competent chimeric vesicular stomatitis virus vector (VSV-GP) as an alternative viral vector boost vaccine. Methods: A recombinant VSV-GP vector co-expressing HBV surface and core antigens (VSV-GP-HBs/c) was generated and characterized for antigen expression. Its immunogenicity, antiviral efficacy, and durability were assessed in HBV-naïve and HBV-carrier mice, using protein primed, viral vector-primed, and multi-viral vector boost regimens. Results: VSV-GP-HBs/c efficiently expressed both HBV antigens in vitro. A single immunization with VSV-GP-HBs/c induced only weak HBV-specific immune responses in vivo. Replacing protein priming with VSV-GP-HBs/c resulted in modest immune activation and limited antiviral effects in HBV-carrier mice. In contrast, substituting the modified vaccinia virus Ankara (MVA)-HBs/c boost in the TherVacB regimen with VSV-GP-HBs/c elicited robust HBV-specific antibody responses and strong CD4 and CD8 T-cell immunity, assessed by intracellular IFN-γ staining after peptide stimulation. This regimen achieved a substantial reduction in serum HBsAg levels, numbers of HBV-positive hepatocytes, and intrahepatic HBV-DNA, with antiviral efficacy comparable to that of the classical TherVacB regimen. Notably, a second viral vector boost did not enhance HBV-specific immunity or antiviral efficacy; instead, it promoted dominant vector-specific CD8 T-cell responses. Long-term analyses performed 10 weeks after the last vaccination further demonstrated that a single protein-prime/VSV-GP-HBs/c boost was sufficient to achieve sustained antiviral control. Conclusions: These findings identify VSV-GP-HBs/c as an effective boost vector for therapeutic hepatitis B vaccination and establish protein priming followed by a single viral vector boost as an optimal strategy for sustained antiviral immunity. Full article

Background/Objectives: Lentiviral vectors (LVs) are most commonly pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), which lends LVs a wide tropism as it uses the low-density lipoprotein receptor (LDLR) as the main receptor for cell entry. In some gene therapy and research applications, however, alternative pseudotypes can be useful. In this work, we characterized LVs pseudotyped with lymphocytic choriomeningitis virus (LCMV) glycoprotein, particularly in gene transfer to an LDLR-deficient mouse strain used to model cardiovascular disease, Ldlr^−/−ApoB^100/100. Methods: LCMV-LVs were used in vitro to test their transduction efficiency across a variety of cell types. In vivo, the gene transfer efficiency, LV-specific immune responses and biodistribution of VSV-G-LVs and LCMV-LVs were compared after systemic gene transfer. Results: In vitro, LCMV-LVs transduced all tested cell types at high efficiency without the use of transduction enhancers. In vivo, VSV-G-LVs showed a higher gene transfer efficiency at the same LV dose, but increasing the LCMV-LV dose enhanced the measured vector copy numbers. With both pseudotypes, most of the vector accrued in the liver, but with LCMV-LVs, a larger portion of the measured vector copies were found in the lungs. VSV-G-LVs also generated a higher titer of LV-specific IgG antibodies. The gene transfer efficiency of LCMV-LVs was affected by the mouse diet, with a high-fat diet decreasing the transduction. Conclusions: LCMV-LVs can be used as a substitute for VSV-G-LVs if an alternative pseudotype is required; however, they may require the use of a higher LV dose. Full article

Selective gene delivery to defined cell populations remains one of the key challenges in lentiviral vector-based gene therapy. The vesicular stomatitis virus glycoprotein (VSV-G) confers high infectivity but lacks cell-type specificity because of the ubiquitous expression of its receptor, LDLR. To enable modular, receptor-specific targeting while retaining the production efficiency of VSV-G-pseudotyped vectors, we designed a bispecific adapter, 929-B6, comprising a VSV-G-binding nanobody and an ERBB2-binding DARPin 9.29. Anti-VSV-G nanobodies were isolated from an alpaca immune library and screened in cell-based pseudoreceptor assays to identify the optimal binder (VSVG-B6). The resulting adapter was evaluated with receptor-ablated (VSV-G_mut) and wild-type VSV-G-pseudotyped LVs across ERBB2-positive and -negative cell lines and in a mouse xenograft model. 929-B6 enabled efficient, receptor-specific transduction of ERBB2-expressing cells without increasing infection of ERBB2-negative controls. Pre-incubation of VSV-G_mut-pseudotyped LVs with 1–2 µg/mL 929-B6 increased transduction up to eight-fold in ERBB2⁺ cells, with similar but smaller effects for VSV-G and VSV-G_mut + 929R pseudotypes. Across breast cancer lines, transduction enhancement correlated with ERBB2 surface density, and co-culture experiments confirmed selective entry into ERBB2⁺ populations. In vivo imaging of ERBB2⁺ tumors revealed a visible tumor-localized luminescent signal following administration of 929-B6-treated vectors. The 929-B6 adapter provides a rapid, scalable means to retarget standard LV stocks toward chosen receptors without re-engineering the envelope or co-packaging pseudoreceptor plasmids. Its modularity suggests a generalizable platform for both gene therapy and oncolytic applications requiring flexible, receptor-defined tropism. Full article

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

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/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

Over the past several decades, viral vector-based vaccines have emerged as some of the most versatile and potent platforms in modern vaccinology. Their capacity to deliver genetic material encoding target antigens directly into host cells enables strong cellular and humoral immune responses, often superior to what traditional inactivated or subunit vaccines can achieve. This has accelerated their application to a wide array of pathogens and disease targets, from well-established threats like HIV and malaria to emerging infections such as Ebola, Zika, and SARS-CoV-2. The COVID-19 pandemic further highlighted the agility of viral vector platforms, with several adenovirus-based vaccines quickly authorized and deployed on a global scale. Despite these advances, significant challenges remain. One major hurdle is pre-existing immunity against commonly used vector backbones, which can blunt vaccine immunogenicity. Rare but serious adverse events, including vector-associated inflammatory responses and conditions like vaccine-induced immune thrombotic thrombocytopenia (VITT), have raised important safety considerations. Additionally, scaling up manufacturing, ensuring consistency in large-scale production, meeting rigorous regulatory standards, and maintaining equitable global access to these vaccines present profound logistical and ethical dilemmas. In response to these challenges, the field is evolving rapidly. Sophisticated engineering strategies, such as integrase-defective lentiviral vectors, insect-specific flaviviruses, chimeric capsids to evade neutralizing antibodies, and plug-and-play self-amplifying RNA approaches, seek to bolster safety, enhance immunogenicity, circumvent pre-existing immunity, and streamline production. Lessons learned from the COVID-19 pandemic and prior outbreaks are guiding the development of platform-based approaches designed for rapid deployment during future public health emergencies. This review provides an exhaustive, in-depth examination of the historical evolution, immunobiological principles, current platforms, manufacturing complexities, regulatory frameworks, known safety issues, and future directions for viral vector-based vaccines. Full article

During the current outbreak of Marburg virus disease (MVD) in Rwanda, we synthesized evidence from the literature to improve case management. Accordingly, experimental treatment was offered to patients under close follow-up. Remdesivir alone or in combination with monoclonal antibody treatment (MBP091) complemented with supportive care has improved the clinical outcomes of patients. Additionally, we have identified several experimental therapies currently under investigation, including antiviral drugs such as favipiravir, galidesivir, obeldesivir, and remdesivir, along with monoclonal and polyclonal antibodies (e.g., polyclonal IgG, monoclonal antibody MR-78-N; MR82-N; MR191-N; monoclonal antibodies MR186-YTE and MBP091). Furthermore, substantial progress is being made in vaccine development, with promising candidates including adenovirus-vectored vaccines, DNA vaccines, and the recombinant vesicular stomatitis virus (rVSV) vaccine. Moreover, innovative preventive and treatment strategies—such as synthetic hormones like estradiol benzoate, small interfering RNA (siRNA), interferon-β therapy, and phosphorodiamidate morpholino oligomers—are emerging as potential options for MVD management. Further investment is needed to accelerate research and optimize these therapeutics and preventive modalities. Additional epidemiological, preclinical, and clinical studies are warranted to generate the evidence required to inform policymaking, resource mobilization, and the implementation of cost-effective interventions for the prevention, control, and treatment of MVD. Full article

The vesicular stomatitis virus (VSV)-vectored African swine fever virus (ASFV) vaccine can induce efficient immune response, but the potential mechanism remains unsolved. In order to investigate the efficacy of recombinant viruses (VSV-p35, VSV-p72)-mediated dendritic cells (DCs) maturation and the mechanism of inducing T-cell immune response, the functional effects of recombinant viruses on DC activation and target antigens presentation were explored in this study. The results showed that surface-marked molecules (CD80, CD86, CD40, and MHC-II) and secreted cytokines (IL-4, TNF-α, IFN-γ) were highly expressed in the recombinant virus-infected DCs. In addition, the co-culture results of recombinant virus-treated DCs with naive T cells showed that the Th1- and Th17-type responses were effectively activated. Taken together, the study indicated that the VSV-vectored ASFV vaccine activated the maturation of DCs and the Th1- and Th17-type immune response, which provided a theoretical basis for the development of novel ASF vaccines. Full article

The Junín virus (JUNV) is one of the New World arenaviruses that cause severe hemorrhagic fever. Human transferrin receptor 1 (hTfR1) has been identified as the main receptor for JUNV for virus entry into host cells. To date, no treatment has been approved for JUNV. Herein, we investigated 12 anti-hTfR1 VHH (variable domain of the heavy chain of heavy-chain antibody) antibodies and confirmed their interaction with hTfR1. Most of them could bind to the hTfR1 apical domain, which is the glycoprotein 1 (GP1) binding domain of JUNV. Among them, 18N18 exhibited neutralizing activity against both the human immunodeficiency virus (HIV)-vectored lentiviral Junín pseudoviruses and the recombinant vesicular stomatitis virus (VSV)-vectored Junín pseudoviruses. We also verified that 18N18 blocked the interaction between hTfR1 and JUNV GP1. In addition, 18N18 could neutralize another New World arenavirus, the Machupo virus. Using AlphaFold 3-based simulation of 18N18–hTfR1 docking, we determined that 18N18’s binding epitope was located at the JUNV GP1 binding epitope. 18N18 represents a candidate for JUNV treatment and provides a potential approach that could be applied to New World arenaviruses. Full article

Vesicular stomatitis virus (VSV), comprising vesicular stomatitis New Jersey virus (VSNJV) and vesicular stomatitis Indiana virus (VSIV), emerges from its focus of endemic transmission in Southern Mexico to cause sporadic livestock epizootics in the Western United States. A dearth of information on the role of potential arthropod vectors in the endemic region hampers efforts to identify factors that enable endemicity and predict outbreaks. In a two-year, longitudinal study at five cattle ranches in Chiapas, Mexico, insect taxa implicated as VSV vectors (blackflies, sandflies, biting midges, and mosquitoes) were collected and screened for VSV RNA, livestock vesicular stomatitis (VS) cases were monitored, and serum samples were screened for neutralizing antibodies. VS cases were reported during the rainy (n = 20) and post-rainy (n = 2) seasons. Seroprevalence against VSNJV in adult cattle was very high (75–100% per ranch) compared with VSIV (0.6%, all ranches). All four potential vector taxa were sampled, and VSNJV RNA was detected in each of them (11% VSNJV-positive of 874 total pools), while VSIV RNA was only detected in four pools of mosquitoes. Our findings indicate that VSNJV is the dominant serotype across our sampling sites with a variety of potential insect vectors involved in its transmission throughout the year. Although no livestock cases were reported in Chiapas during the dry season, VSNJV was detected in insects during this period, suggesting that mechanisms other than transmission from livestock support VSV endemicity. Full article

Liquid formulations have been successfully used in many viral vector vaccines including influenza (Flu), hepatitis B, polio (IPV), Ebola, and COVID-19 vaccines. The main advantage of liquid formulations over lyophilized formulations is that they are cost-effective, as well as easier to manufacture and distribute. However, studies have shown that the liquid formulations of enveloped viral vector vaccines are not stable over extended periods of time. In this study, we explored the development of the liquid formulations of an enveloped recombinant Vesicular Stomatitis Virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. To do so, we used a design of experiments (DOE) method, which allowed us to assess the stability dynamics of the viral vector in an effective manner. An initial stability study showed that trehalose, gelatin, and histidine were effective at maintaining functional viral titers during freeze–thaw stress and at different temperatures (−20, 4, 20, and 37 °C). These preliminary data helped to identify critical factors for the subsequent implementation of the DOE method that incorporated a stress condition at 37 °C. We used the DOE results to identify the optimal liquid formulations under the selected accelerated stress conditions, which then guided the identification of long-term storage conditions for further evaluation. In the long-term stability study, we identified several liquid formulations made of sugar (sucrose, trehalose, and sorbitol), gelatin, and a histidine buffer that resulted in the improved stability of rVSV-SARS-CoV-2 at 4 °C for six months. This study highlights an effective approach for the development of liquid formulations for viral vector vaccines, contributing significantly to the existing knowledge on enveloped viral vector thermostability. Full article

Baculoviral vectors (BVs) derived from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) are an attractive tool for multigene delivery in mammalian cells, which is particularly relevant for CRISPR technologies. Most applications in mammalian cells rely on BVs that are pseudotyped with vesicular stomatitis virus G-protein (VSV-G) to promote efficient endosomal release. VSV-G expression typically occurs under the control of the hyperactive polH promoter. In this study, we demonstrate that polH-driven VSV-G expression results in BVs characterised by reduced stability, impaired morphology, and VSV-G induced toxicity at high multiplicities of transduction (MOTs) in target mammalian cells. To overcome these drawbacks, we explored five alternative viral promoters with the aim of optimising VSV-G levels displayed on the pseudotyped BVs. We report that Orf-13 and Orf-81 promoters reduce VSV-G expression to less than 5% of polH, rescuing BV morphology and stability. In a panel of human cell lines, we elucidate that BVs with reduced VSV-G support efficient gene delivery and CRISPR-mediated gene editing, at levels comparable to those obtained previously with polH VSV-G-pseudotyped BVs (polH VSV-G BV). These results demonstrate that VSV-G hyperexpression is not required for efficient transduction of mammalian cells. By contrast, reduced VSV-G expression confers similar transduction dynamics while substantially improving BV integrity, structure, and stability. Full article