Search Results (268)

Adenoviral vectors are widely used for gene therapy and vaccine development. To circumvent pre-existing immunity against commonly used human adenovirus type 5, vectors based on rare human serotype or animal adenoviruses have attracted increasing interest. Previously, we constructed vectors based on fowl adenovirus 4 (FAdV-4) and replaced the knob of FAdV-4 fiber2 with that of FAdV-1 fiber1 to generate FAdV4-CF1K vectors with enhanced transduction efficiency in human cells. In this study, we aimed to modify the packaging system to efficiently produce FAdV-4 vectors carrying transgenes toxic to viral replication. Chicken LMH cells failed to form colonies at low seeding densities. We collected used medium from LMH cell cultures and used it as a supplement to adapt LMH cells, generating the colony-competent subclone LMH-C3532. A lentiviral vector encoding a codon-optimized tetracycline repressor (tetR) was transduced into LMH-C3532 to establish a tetR-integrated cell line, LMH-tetR24. An adenoviral plasmid, pKFAV4-CF1K-CtG, was constructed in which a tetracycline operator (tetO)-bearing CMV promoter controlled GFP expression. The SwaI-flanked GFP in this plasmid was replaced with the HA gene from an H5N1 influenza virus to generate pKFAV4-CF1K-CtHA. Linearized adenoviral plasmids were transfected into LMH-tetR24 cells, and recombinant FAdV4-CF1K-CtG and FAdV4-CF1K-CtHA viruses were successfully rescued, amplified, and purified. When infected with FAdV4-CF1K-CtG at various multiplicities of infection (MOI), the progeny virus yield from LMH-tetR24 cells was 4–10 times higher than that from LMH-C3532 cells. For FAdV4-CF1K-CtHA, the yield difference between the two cell lines was even more pronounced, reaching 3–4 orders of magnitude. Overexpression of HA in LMH-C3532 cells negatively affected FAdV4-CF1K-CtHA replication, resulting in smaller and fewer plaques. In conclusion, by separately integrating tetR into packaging cells and TetO into the adenoviral plasmid, we established a system that can be routinely used to package FAdV-4 vectors. Notably, this system facilitates the propagation of FAdV-4 vectors carrying toxic transgenes. Full article

Peripheral arterial disease and critical limb-threatening ischemia remain major clinical challenges, particularly in patients who are not candidates for surgical or endovascular revascularization. These limitations have stimulated extensive investigation into therapeutic angiogenesis using gene therapy approaches. This review summarizes experimental and clinical studies employing non-viral and viral gene delivery systems for the transfer of angiogenic factors, including VEGF, FGF-2, HGF, HIF-1α, and SDF-1. Particular attention is given to vector platforms, study design, patient populations, clinical endpoints, and safety outcomes reported in preclinical investigations and clinical trials. Although several studies demonstrated biological activity and favorable safety profiles, randomized trials such as RAVE and TAMARIS failed to demonstrate consistent and significant clinical efficacy. These findings emphasize the translational challenges associated with therapeutic angiogenesis and highlight the persistent gap between promising preclinical data and clinical outcomes in humans. Future progress in the field will likely depend on improved vector engineering, tissue-specific and regulated gene expression systems, optimized delivery strategies, and the integration of gene therapy with emerging regenerative technologies. Full article

Porcine epidemic diarrhea (PED), caused by the PED virus (PEDV), remains one of the most devastating diseases in the swine industry, with a mortality rate approaching 90–100% in suckling piglets due to severe dehydration and electrolyte imbalances. Passive immunization with egg yolk antibodies (IgY) represents a promising therapeutic strategy. In this study, we developed a novel recombinant adenovirus, rADM-IFN-G-ped, co-expressing selected antigenic regions of the PEDV S protein and chicken interferon-gamma (ChIFN-γ) as a molecular adjuvant. Laying hens were immunized with this construct to produce PEDV-specific IgY, which was subsequently purified from eggs using a polyethylene glycol (PEG-6000) precipitation method. The induced IgY demonstrated potent neutralizing activity against PEDV in vitro, with a neutralization titer (NT₅₀) of 1:96, which was significantly higher than that of IgY derived from hens immunized with a commercial inactivated PEDV G2b vaccine (NT₅₀ = 1:52). In a passive immunization and challenge trial, piglets treated with the rADM-IFN-G-ped-derived IgY exhibited significantly reduced fecal viral RNA shedding following challenge with the virulent PEDV-NX-2022 strain, compared to control groups. Crucially, while all piglets in the challenge control group succumbed to infection within 72 h, a 50% survival rate was achieved in the IgY-treated group. Histopathological examination of intestinal tissues further confirmed the protective efficacy, showing that IgY treatment markedly alleviated villous atrophy, epithelial necrosis, and inflammatory cell infiltration in the small intestine. These findings demonstrate that vaccination of laying hens with the rADM-IFN-G-ped recombinant adenovirus elicits a robust immune response, enabling the production of protective IgY. This proof-of-concept study establishes the viability of the multi-epitope adenoviral IgY platform as a passive immunization strategy against PEDV. Full article

The VHL–HIF-1α–VEGF axis regulates angiogenesis and metabolism. Beyond oncology, pVHL is essential for pancreatic β-cell function and is reduced in hypercaloric diet (HCD)-induced type 2 diabetes mellitus (T2DM). This study aimed to overexpress pVHL in pancreatic tissue and evaluate its effects on blood glucose levels and the expression of proteins related to glucose metabolism in the pancreas. HCD-induced diabetic C57BL/6 and BALB/c mice received a single intrapancreatic injection of an adenoviral vector (1 × 10¹² viral particles) encoding the murine Vhlh gene (AdVHL) to induce transient pVHL overexpression. The glycemic delta (post-load glucose minus fasting) and net incremental area under the curve (niAUC) were determined on days 3, 6, 9, 12, and 15 post-treatment, as the peak in GFP overexpression (used as a surrogate reporter of transduction efficiency) was detected between days 9 and 12. Immunohistochemistry (IHC) and immunofluorescence (IF) were used to assess the expression of pVHL, HIF-1α, GLUT-1, GLUT-2, and insulin in pancreatic tissue. AdVHL treatment significantly decreased the glycemic delta and niAUC in mice with T2DM (p < 0.01). On day 15 after treatment, HIF-1α and GLUT-1 expression were markedly reduced in AdVHL-treated mice (p < 0.01), while GLUT-2 and insulin were significantly increased (p < 0.01). These results were reproduced in both mouse strains. Transient overexpression of pVHL in pancreatic tissue of mice with T2DM was associated with decreased glucose levels and changes in the expression of proteins related to glucose metabolism in the pancreas, resembling a healthier phenotype than that of mice with T2DM. These findings support an important functional role of the pVHL–HIF-1α axis in pancreatic physiology, provide a proof-of-concept for further mechanistic and translational studies, and implicate pVHL in the altered glucose metabolism observed in T2DM. Full article

Background: Osteoarthritis (OA) is a multifactorial disease, including inflammation, autophagy and senescence. Published work has indicated that empagliflozin (EMP) exhibits robust anti-inflammatory and anti-senescence effects, while its role in autophagy appears paradoxical. Here, we aim to identify the chondroprotective effect of EMP on OA. Methods: An OA model was established both in vitro, by stimulating primary chondrocytes (isolated from C57BL/6J mice) with IL-1β, and in vivo, by performing (Destabilized medial meniscus) DMM surgery on C57BL/6J mice. (Western blot) WB and (quantitative real-time polymerase chain reaction) qRT-PCR analysis were employed to detect the gene expression. (Immunofluorescence) IF staining was employed to detect the expression and location of target protein. SA-β-gal staining was employed to evaluate cellular senescence. Autophagic flux was assessed using a GFP-RFP-LC3 adenoviral vector. Network pharmacology was applied to identify potential pathways for experimental validation. The effects of EMP in vivo were evaluated by μ-CT, histological and (Immunohistochemistry) IHC staining. Results: EMP promoted anabolism, inhibited the inflammatory response and catabolism in IL-1β stimulated chondrocytes. EMP enhanced autophagic activity and attenuated senescent phenotype in vitro. Mechanistically, EMP regulated the PI3K/Akt/mTOR and AMPK pathways. The chondroprotective effects of EMP were reversed by (3-methyladenine) 3-MA. EMP also ameliorated OA-related phenotype in DMM models. Compared with (Kartogenin) KGN, EMP showed more pronounced suppression of inflammatory and catabolic markers, while both compounds similarly promoted anabolic marker expression. Conclusions: These in vitro and in vivo data collectively indicates that EMP can alleviate OA both in IL-1β stimulated chondrocytes and DMM induced models. Beyond its established role in diabetes management, EMP is evaluated in the context of OA, emerging as a novel and promising therapeutic agent for OA. Full article

Background/objectives: SARS-CoV-2 antibodies wane after natural infections and vaccinations. COVID-19 booster vaccination enhances the durability and functionality of antibodies against emerging SARS-CoV-2 variants. Data on booster-induced antibody durability in sub-Saharan Africa remain sparse. Comparative analysis of vaccine-induced responses between heterologous and homologous vaccination regimens remains limited. This study evaluated longitudinal RBD-specific IgG responses following homologous and heterologous COVID-19 booster vaccination in previously vaccinated adults. Methods: Adults with prior mRNA or adenoviral-vectored vaccination were boosted with either Pfizer (mRNA) or Janssen (adenoviral-vectored) vaccines. Plasma IgG binding to Wuhan, Delta, and Omicron RBDs was measured pre-booster and at 3, 6, and 9 months. A total of 181 participants were enrolled between November 2022 and October 2023. Results: More than 60% of participants had detectable pre-booster RBD- and N-antigen-specific IgG. Booster vaccination substantially increased Wuhan-specific RBD-IgG at three months, with limited boosting of Delta and Omicron responses. Antibody levels waned to pre-booster concentrations by month nine. Heterologous boosting with a viral-vectored prime followed by Pfizer mRNA significantly enhanced both peak RBD-IgG levels and durability. Conclusions: These longitudinal data provide rare real-world evidence on booster immunogenicity in African adults and demonstrate that heterologous regimens confer a short- to intermediate-term advantage in antibody magnitude compared to a homologous regimen. This benefit was most pronounced within the first six months post-boost. The findings support additional booster dosing to strengthen protection against emerging variants in sub-Saharan Africa. Full article

The SERPINE1 gene encodes the serine protease inhibitor plasminogen activator inhibitor type-1 (PAI-1), a major negative regulator of the plasmin-dependent pericellular proteolytic cascade and a crucial determinant in the program of stromal remodeling. Recent omics approaches confirmed that high tumor SERPINE1 levels are prognostic for poor disease outcomes and shorter disease-free survival in various malignancies. Kinetic analysis of biomarkers of cell cycle transit in growth-synchronized p53 dual mutant human keratinocytes confirmed that PAI-1 transcription occurred early after growth activation of quiescent (G₀) cells and prior to G1 entry. Previous evidence has confirmed that differential residence of USF family members (USF1→USF2 switch) at the PE2 region hexanucleotide E box motif (CACGTG) in the SERPINE1 proximal promoter characterizes the G₀→G₁ transition period and the transcriptional status of the SERPINE1 gene. A consensus PE2 E box motif (5′-CACGTG-3′) at nucleotides −566 to −561 is required for USF occupancy of the PE2 E box and serum-stimulated SERPINE1 transcription. Interference with USF2 occupancy of the PE2 E Box site by a double-stranded PE2 “decoy”, or induced expression of a dominant-negative USF (A-USF) construct, attenuate serum- and TGF-β1-stimulated SERPINE1 synthesis. Tet-Off activation of an A-USF insert reduced both PAI-1 and PAI-2 transcripts while increasing the fraction of proliferating (Ki-67⁺ cells). Conversely, overexpression of USF2 or adenoviral delivery of a PAI-1 vector inhibited HaCaT colony expansion. These findings are discussed in this review and collectively suggest that the USF1→USF2 transition at the PE2 E box site and subsequent SERPINE1 transcription impact serum-stimulated keratinocyte growth and, likely, cell cycle progression. Full article

Background/Objectives: Chimpanzee adenoviral-vectored vaccines have proven to be both safe and effective, with a manufacturing and distribution pipeline capable of rapid global supply, as demonstrated during the COVID-19 pandemic. Yellow fever is a mosquito-borne viral hemorrhagic disease endemic in parts of Africa and Latin America, and although an effective live attenuated vaccine exists, its use is limited by safety and eligibility restrictions. Moreover, large outbreaks continue to expose critical challenges, such as an insufficient vaccine supply, reliance on fractional dosing, and slow and difficult-to-scale manufacturing processes. Here, we report the design, development and in vivo immunogenicity of multiple yellow fever virus (YFV) antigen constructs based on the pre-membrane (prM) and envelope (E) proteins—with or without the transmembrane domain (TM or ΔTM)—delivered using the ChAdOx1 adenoviral vector. Methods: Four ChAdOx1 YF vaccines were developed, and immunogenicity was evaluated. The efficacy of the full-length YF envelope vaccine was also tested in Balb/c mice. Results/Conclusions: In contrast to previously described orthoflavivirus vaccines on the same platform, the full-length antigen elicited superior immunogenicity and conferred protection against intracranial challenge with the YF17D virus in mice. Notably, this protection was comparable to that induced by the licensed YF17D vaccine, highlighting the promise of this platform as a next-generation yellow fever vaccine candidate. Full article

Background/Objectives: Guillain–Barré syndrome (GBS) is a rare but serious immune-mediated neurological disorder monitored as an adverse event of special interest during the COVID-19 pandemic. This study aimed to estimate the incidence of GBS following COVID-19 vaccination and SARS-CoV-2 infection and to compare the risk by vaccine platform. Methods: We conducted a population-based retrospective cohort study using data from the Valencia Health System Integrated Database (Spain) between January 2018 and March 2022. Two cohorts were defined: individuals receiving COVID-19 vaccines (mRNA-based vaccines (BNT162b2 [Pfizer–BioNTech] and mRNA-1273 [Moderna]) or non-virus-vectored (NVV) adenoviral vector-based vaccines (ChAdOx1-S [AstraZeneca] and Ad26.COV2.S [Janssen])) and individuals with laboratory-confirmed SARS-CoV-2 infection. Incident GBS cases were identified within a predefined 42-day risk window following vaccination or infection. Incidence rates were calculated per 100,000 vaccine doses administered or SARS-CoV-2 infections. Results: Among 5,109,919 individuals, 4,270,610 received at least one COVID-19 vaccine dose and 920,643 experienced at least one SARS-CoV-2 infection. A total of 69 GBS cases occurred within 42 days following vaccination (incidence: 0.67 per 100,000 doses), whereas 21 cases occurred after infection (incidence: 2.20 per 100,000 infections). Incidence was lower after mRNA-based vaccines (0.55 per 100,000 doses) than after NVV vaccines (1.57 per 100,000 doses). Conclusions: This study confirms that GBS occurrence following vaccination is rare. The incidence is lower among individuals who received mRNA vaccines compared to those who received NVV vaccines. Moreover, GBS appears to be more frequent after a COVID-19 infection than after vaccination. These findings highlight the importance of integrating pharmacoepidemiological analyses with pharmacovigilance data to contextualize rare but serious adverse events. Full article

Background/Objectives: The large size of commonly used regulatory elements such as the cytomegalovirus (CMV) immediate-early promoter imposes a significant burden on the already restricted payload capacity of first-generation adenoviral vectors, potentially hindering the development of multi-antigen vaccine candidates. To address this limitation, we have engineered a panel of novel, small, semi-synthetic promoters designed to leverage the changes in transcriptomic milieu following adenoviral vector entry. Methods: Eight synthetic enhancer modules (SE1–SE8) were designed in silico, each composed of transcription factor binding sites (TFBSs) previously found in host genes that are upregulated during early adenoviral infection. These synthetic enhancers were coupled with a minimal CMV core promoter to generate a panel of compact semi-synthetic promoters (cSE1–cSE8), and their activity was evaluated in the context of ChAdOx1 viral vectors expressing GFP or a modified Plasmodium falciparum circumsporozoite (CSN) antigen. Promoter performance was characterised in vitro via flow cytometry, RT-qPCR, and Western blotting, and in vivo by quantifying antigen-specific T-cell (IFN-γ ELISpot) and IgG antibody (ELISA) responses in BALB/c mice. Results: In vitro characterisation revealed a wide range of promoter activity across the panel, with cSE3 and cSE5 driving transgene expression levels comparable to the benchmark CMV promoters despite their markedly reduced genomic footprint. In vivo, ChAdOx1 vectors incorporating cSE3 and cSE5 elicited potent antigen-specific T-cell and IgG responses that were comparable to those induced by the larger CMV control promoters. Conclusions: We have successfully developed semi-synthetic promoters that match the potency of the much larger, frequently used CMV promoters whilst simultaneously reducing genomic footprint. These novel regulatory elements will facilitate the design of next-generation vaccines, particularly those requiring large antigens or multi-antigen cassettes. Full article

Adenoviral vectors are among the most efficient platforms for gene delivery; however, the level and pattern of transgene expression in these vectors are largely shaped by the viral genomic context. This review discusses the mechanisms of adenoviral transcription and alternative splicing and how they influence the expression of inserted expression cassettes. Particular attention is given to the role of insertion orientation and transgene placement within the E1 and E3 regions, as well as to the effects of viral regulatory elements, including the E1A enhancer. We analyze evidence on the use of insulating sequences to reduce nonspecific activation and improve the controllability of transgene expression. We also consider the use of endogenous adenoviral promoters—the major late promoter (MLP) and the E3 region promoter—and their contribution to enhanced transgene expression through late viral transcription. Overall, these findings support principles for the rational design of adenoviral vectors, both for high-level protein production and for building systems with regulated or tissue-specific expression. Full article

Background/Objectives: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but severe immune-mediated adverse event associated with adenoviral vector-based SARS-CoV-2 vaccines. Beyond its clinical relevance, VITT provides a unique human model of vaccine-triggered autoimmunity and immune-thrombosis. This review critically reassesses the immunopathogenic framework of VITT in light of recent evidence. Methods: We conducted a structured narrative review of studies published between 2021 and 2025, focusing on clinical, epidemiological, and mechanistic data relevant to PF4 immunogenicity, platelet activation, and long-term outcomes. Results: Current evidence supports a multistep model in which adenoviral vector components form immunogenic PF4–polyanion complexes that induce high-affinity anti-PF4 IgG antibodies. These antibodies activate platelets via FcγRIIa, amplify complement signaling, promote neutrophil extracellular trap formation, and drive endothelial perturbation, establishing a self-sustaining thrombo-inflammatory loop. Recent longitudinal studies refine earlier interpretations by distinguishing persistent anti-PF4 seropositivity from sustained platelet-activating capacity. Epidemiological data support platform-enriched risk rather than absolute platform exclusivity, with a proposed mechanistic “border zone” for incomplete phenotypes. Conclusions: VITT represents a tractable human model of vaccine-induced autoimmunity in which innate immune activation and multivalent antigen presentation converge to break tolerance. Updated evidence clarifies antibody persistence, platform enrichment, and translational implications, while highlighting unresolved questions regarding host susceptibility and long-term immune regulation. Full article

Serum amyloid A (SAA) proteins increase significantly in the blood following inflammation. Recently, SAAs were increased in humans following stroke and in ischemic animal models. However, the impact of SAAs on whether this signal is critical in the ischemic brain remains unknown. Therefore, we investigated the role of SAA and SAA signaling in the ischemic brain. Wild-type and SAA-deficient mice were exposed to middle cerebral artery occlusion and reperfusion and examined to determine the impact of infarct volumes, behavioral changes, inflammatory markers, TUNEL staining, and BBB changes. The underlying mechanisms were investigated using SAA-deficient mice, transgenic mice, and viral vectors. SAA levels were significantly increased following MCAo, and mice deficient in SAA showed reduced infarct volumes and improved behavioral outcomes. SAA-deficient mice showed a reduction in TUNEL staining, inflammation, and decreased glial activation. Mice lacking acute phase SAAs demonstrated a reduction in the expression of the NLRP3 inflammasome, and SAA/NLRP3 KO mice showed improvement. The restoration of SAA expression via SAA tg mice or adenoviral expression re-established the detrimental effects of SAA. A reduction in BBB permeability was seen in SAA KO mice, and anti-SAA antibody treatment reduced the effects on ischemic injury. SAA signaling plays a critical role in regulating NLRP3-induced inflammation and glial activation in the ischemic brain. Blocking this signal will be a promising approach for treating ischemic stroke. Full article

The purpose of our study is to explore the potential of a transcription factor-based strategy for directly converting mouse fibroblasts into photoreceptor-like cells. The mouse cDNAs of Ascl, Crx, Ngn1, Nrl, and Otx2 were cloned into a modified commercial adenoviral vector. Mouse embryonic fibroblasts (MEFs) were isolated from E13.5 embryos, and mouse postnatal fibroblasts (MPFs) were isolated from three-day-old mice. A pool of adenoviruses containing five genes was prepared to infect MEFs or MPFs once daily for two days. The MEFs or MPFs were incubated in a specific medium supplemented with forskolin and were changed every two days. After 7 or 14 days, the photoreceptor-like cells were assayed via immunofluorescence or polymerase chain reaction with reverse transcription (RT–PCR). The photoreceptor-like cells were then transplanted into adult C57BL/6 mouse retinas and were assessed by immunofluorescence 14 days following transplantation. Screening from a pool of five candidate genes, we reported that a combination of only three factors—Crx, Nrl, and Otx2—was sufficient to convert mouse embryonic and postnatal fibroblasts into photoreceptor-like cells. The induced photoreceptor-like cells expressed photoreceptor-specific proteins such as Recoverin, Rhodopsin, and Opsin and integrated into the outer nuclear layer of the retina following transplantation. This exploratory study provides preliminary evidence that fibroblasts can be directly converted into photoreceptor-like cells, suggesting a cellular model and potential source for future transplantation strategies aimed at retinal repair. Full article

Background/Objectives: The rapid development of safe and efficacious vaccines is often hindered by extensive, mandated non-clinical safety evaluations in animals. With the aim to provide scientific evidence supporting a “vaccine platform approach”, here we present the complete non-clinical studies for two investigational vaccines, GRAd-COV2 and GRAdHIVNE1, based on GRAd, a gorilla-derived group C adenoviral vector. Methods: The biodistribution of GRAd genomes following the intramuscular administration of the vaccines was assessed in rats by a sensitive qPCR method. Local tolerance and systemic toxic effects were evaluated in single- and repeated-dose toxicity studies in rabbits. Results: GRAd-COV2 and GRAdHIVNE1 were well-tolerated. Distribution was highly confined to the injection site and draining lymph nodes, and toxicity profile consisted of transient, non-adverse inflammatory responses, while the expected immune responses to the encoded antigens were successfully induced. Notably, both vaccines demonstrated a consistent safety profile despite transgene and backbone differences, comparable to other replication-defective adenoviral vectors. Conclusions: The established non-clinical safety profile of the GRAd platform provides a robust foundation for a more efficient and streamlined regulatory pathway. By leveraging this prior knowledge, future GRAd-based vaccines can achieve accelerated clinical development while fully adhering to the ethical principles of replacement, reduction, and refinement of animal use in research. Full article