Search Results (2,360)

Background/objectives: RSV is a major cause of mortality in infants, and despite recent progress to prevent RSV in the very young, an RSV vaccine for this population is still highly warranted. Clinical studies in infants in the 1960s using formalin-inactivated RSV (FI-RSV) led to life-threatening enhanced respiratory disease (ERD). Therefore, a thorough safety assessment of RSV vaccine candidates intended for RSV seronegative infants is crucial. Methods: Prior to clinical pediatric development of Ad26.RSV.preF, an adenovirus type 26 vector-encoding RSV F protein stabilized in its prefusion conformation, predisposition to ERD was extensively assessed in cotton rat and mouse models. Results: Cotton rats intramuscularly immunized with a wide dose range of Ad26.RSV.preF, including low and sub-protective vaccine doses, and challenged with vaccine homologous RSV A2 or heterologous RSV B Wash 18537, did not show signs of predisposition to ERD. Histopathology scores for alveolitis, peribronchiolitis, interstitial pneumonia, and perivasculitis after challenge were significantly lower for Ad26.RSV.preF-immunized cotton rats compared to FI-RSV-immunized cotton rats and comparable to or lower than scores in cotton rats intranasally pre-exposed to RSV prior to challenge to mimic natural repeated infection. These results were observed in animals with or without viral replication in the lung after RSV challenge, in the presence or absence of vaccine-induced antibodies. Similar results were observed in mice, where more extensive assessment of mono- and polymorphonuclear cell alveolitis, mucus cell hyperplasia, and mucus accumulation was performed. Conclusions: Based on these extensive analyses, we conclude that there are no indications of ERD predisposition after Ad26.RSV.preF vaccination in rodent models, irrespective of the vaccine dose, challenge virus strain, or presence of viral replication in the lung. These results are crucial for the pediatric development of this vaccine. Full article

Hepatitis E virus (HEV) is a zoonotic pathogen that can infect pregnant women and cause adverse pregnancy outcomes, including miscarriage and preterm delivery. The previous study demonstrated that HEV genotype 3 (HEV-3) inhibits complete autophagic flux in both mouse placental tissue and human trophoblast cells (JEG-3), evidenced by reduced expression of ATG proteins (including LC3, Beclin1, ATG4B, ATG5, and ATG9A) and accumulation of p62. However, the specific regulatory pathway involved remains unclear. Thus, eukaryotic expression vectors for HEV open reading frames (ORFs) were constructed, and ORF2 and ORF3 proteins were transiently overexpressed in JEG-3 cells via liposome transfection. While both ORF2 and ORF3 significantly reduced LC3B protein levels (p < 0.01), only ORF2 induced p62 accumulation (p < 0.01), indicative of autophagic inhibition, which indicates that ORF2 was the key viral protein mediating autophagy suppression in JEG-3. The results of WB and RT-qPCR showed that ORF2 suppressed the PI3K/Akt/mTOR pathway while enhancing nuclear translocation of TFEB (p < 0.01) and AMPK phosphorylation (p < 0.01), suggesting paradoxical activation of upstream autophagy regulators. Through co-transfection of mCherry-LC3 with ORF2, co-localization studies, and AlphaFold 3-based intermolecular interaction predictions, we propose that ORF2 directly binds LC3B to block autophagosome formation. Finally, co-immunoprecipitation confirmed physical interaction between HEV ORF2 and LC3B, elucidating the molecular mechanism of HEV-induced autophagy suppression in trophoblasts. These findings reveal the molecular mechanism by which HEV inhibits autophagy leading to miscarriage in mice, providing new insights into HEV-induced reproductive damage. Full article

The cornea is an avascular, immune-privileged tissue critical to maintaining transparency, optimal light refraction, and protection from microbial and immunogenic insults. Corneal neovascularization (CoNV) is a pathological sequela of multiple anterior segment diseases and presents a major cause for reduced visual acuity and overall quality of life. Various aetiologies, including infection (e.g., herpes simplex), inflammation (e.g., infective keratitis), hypoxia (e.g., contact lens overuse), degeneration (e.g., chemical burns), and trauma, disrupt the homeostatic avascular microenvironment, triggering an overactive compensatory response. This response is governed by a complex interplay of pro- and anti-angiogenic factors. This review investigates the potential for these mediators to serve as therapeutic targets. Current therapeutic strategies for CoNV encompass topical corticosteroids, anti-VEGF injections, fine-needle diathermy, and laser modalities including argon, photodynamic therapy and Nd:YAG. Emerging therapies involve steroid-sparing immunosuppressants (including cyclosporine and rapamycin), anti-fibrotic agents and advanced drug delivery systems, including ocular nanosystems and viral vectors, to enhance drug bioavailability. Adjunctive therapy to attenuate the protective corneal epithelium prior to target neovascular plexi are further explored. Gene-based approaches, such as Aganirsen (antisense oligonucleotides) and CRISPR/Cas9-mediated VEGF-A editing, have shown promise in preclinical studies for CoNV regression and remission. Given the multifactorial pathophysiology of CoNV, combination therapies targeting multiple molecular pathways may offer improved visual outcomes. Case studies of CoNV highlight the need for multifaceted approaches tailored to patient demographics and underlying ocular diseases. Future research and clinical trials are essential to elucidate optimal therapeutic strategies and explore combination therapies to ensure better management, improved treatment outcomes, and long-term remission of this visually disabling condition. Full article

Hepatic ischemia–reperfusion injury (IRI) is a critical clinical condition associated with liver transplantation and acute liver injury. This study investigated the role of sulfide quinone oxidoreductase (SQOR) and its downstream product, supersulfides, in hepatic IRI. C57BL/6NJ mice were subjected to 45 min of partial hepatic ischemia, followed by reperfusion lasting 4 h. Control of shRNA mediated knockdown of SQOR expressing adeno-associated viral vectors were administered 3 weeks prior to liver ischemia. In the shRNA-mediated knockdown of SQOR group, the hydro-trisulfide donor sodium trisulfide was administered daily for 1 week prior to the induction of liver ischemia. SQOR played a crucial protective role during hepatic IRI by facilitating electron transport to the mitochondrial respiratory chain and maintaining the oxidized and reduced nicotinamide adenine dinucleotide ratio. Administration of sodium trisulfide, exhibited protective effects against hepatic IRI. Sodium trisulfide restored the oxidized and reduced nicotinamide adenine dinucleotide ratio, reduced oxidative stress, and preserved the expression of key enzymes involved in the sulfide oxidation pathway. SQOR and supersulfides contribute to hepatic protection against IRI, likely through their potent antioxidative and redox-regulating functions, and highlight sodium trisulfide as a potential therapeutic agent. Full article

Plant viruses are useful tools for quickly and easily producing recombinant proteins in plants. Compared to systems that use genetically modified plants, viral vectors are easier to work with and can produce recombinant proteins faster and in larger amounts. Recently, there has been growing interest in using plant viruses as vectors to make vaccines, either as whole proteins or as small parts displayed on plant virus particles. The best examples for this purpose are tobacco mosaic virus, cowpea mosaic virus and potato virus X. Vaccines made using these viruses target various human and animal diseases and have often triggered immune responses and provided protection against infections. This review looks at the benefits of using plant virus vectors, the progress in developing different viral vector systems, and immune studies that support the idea of vaccines made from plant viruses. Full article

Arthropod-borne orthoflaviviruses, including dengue, Zika, Japanese encephalitis, yellow fever and West Nile viruses, pose a significant global public health threat, causing hundreds of millions of infections annually with severe clinical symptoms. However, the lack of effective vaccines and antiviral drugs, coupled with the biosafety risks associated with handling live highly pathogenic strains, hinders progress in antiviral research. Pseudovirus technology, which uses single-round infectious viral particles lacking replication competence, has thus gained prominence as a safe and versatile tool for antiviral research. This review systematically summarizes the construction, optimization, and applications of orthoflavivirus pseudoviruses in antiviral research. The primary construction strategies of orthoflavivirus pseudoviruses rely on multi-plasmid co-transfection of viral replicons and structural protein expression vectors, leveraging the host cell secretory pathway to mimic natural viral assembly and maturation. The core applications of pseudovirus technology are highlighted, including high-throughput screening and detection of neutralizing antibodies, identification of antiviral drugs targeting viral entry or replication, and evaluation of vaccine immunogenicity. Despite these strengths, the approach still faces limitations, such as incomplete simulation of native viral structures and batch-to-batch titer variability, which may affect the physiological relevance of findings. In summary, orthoflavivirus pseudovirus technology has become an essential platform in both basic virology research and translational medicine, providing critical insights and tools in the ongoing fight against arthropod-borne orthoflaviviruses diseases. Full article

Background: Feline panleukopenia virus (FPV) causes acute and frequently fatal disease in cats, underscoring the urgent need for safe, rapidly effective, and scalable vaccines. While virus-like particle (VLP) vaccines are inherently safe and immunogenic, their development is constrained by low yields of recombinant protein in insect cell expression systems. Methods: An optimized baculovirus expression vector system (BEVS) incorporating the hr1-p6.9-p10 transcriptional enhancer and the Ac-ie-01 anti-apoptotic gene was employed to enhance recombinant protein production. VP2 expression levels, viral titers, and hemagglutination activity were quantified using qPCR, SDS-PAGE/Western blotting, transmission electron microscopy (TEM), and functional assays. Immunogenicity and protective efficacy were assessed in both mice and cats through serological analysis, neutralizing antibody detection, and post-challenge clinical monitoring. Results: The optimized BEVS enhanced recombinant protein transcription by 1.5-fold, viral titers by 3.7-fold, and hemagglutination activity by 15-fold. The purified protein self-assembled into uniform 25 nm virus-like particles (VLPs). Immunization elicited earlier responses compared to commercial vaccines. Vaccinated cats maintained normal body temperature, stable leukocyte counts, and minimal viral shedding following FPV challenge. Conclusions: This study validates an enhanced BEVS that effectively overcomes VP2 yield constraints and generates highly immunogenic FPV VLPs. The platform enables rapid-onset protection and offers a scalable strategy for next-generation FPV vaccine development. Full article

Cancer immunotherapy increasingly relies on nucleic acid-based vaccines, yet achieving efficient and safe delivery remains a critical limitation. Polyplexes—electrostatic complexes of cationic polymers and nucleic acids—have emerged as versatile carriers offering greater chemical tunability and multivalent targeting capacity compared to lipid nanoparticles, with lower immunogenicity than viral vectors. This review summarizes key design principles governing polyplex performance, including polymer chemistry, architecture, and assembly route—emphasizing microfluidic fabrication for improved size control and reproducibility. Mechanistically, effective systems support stepwise delivery: tumor targeting, cellular uptake, endosomal escape (via proton-sponge, membrane fusion, or photochemical disruption), and compartment-specific cargo release. We discuss therapeutic applications spanning plasmid DNA, siRNA, miRNA, mRNA, and CRISPR-based editing, highlighting preclinical data across multiple tumor types and early clinical evidence of on-target knockdown in human cancers. Particular attention is given to physiological barriers and engineering strategies—including size-switching systems, charge-reversal polymers, and tumor-penetrating peptides—that improve intratumoral distribution. However, significant challenges persist, including cationic toxicity, protein corona formation, manufacturing variability, and limited clinical responses to date. Current evidence supports polyplexes as a modular platform complementary to lipid nanoparticles in selected oncology indications, though realizing this potential requires continued optimization alongside rigorous translational development. Full article

Background: Developing an effective vaccine is crucial for the prevention and control of AIDS. Viral vector-based vaccines, particularly those utilizing homologous or heterologous prime-boost strategies, represent an important direction in current HIV vaccine research. Methods: In this study, replication-defective chimeric adenovirus Ad5F35 and modified vaccinia virus Ankara (rMVA) vector vaccines expressing the HIV-1 AEgp145 were successfully constructed, designated as Ad5F35-AEgp145 and rMVA-AEgp145, respectively. Sixty BALB/c mice were randomly divided into three groups: Ad5F35 alone, rMVA prime/Ad5F35 boost, and PBS control. The mice were immunized intramuscularly at weeks 0 and 3, and humoral and cellular immune responses were assessed at 4, 8, 12, and 16 weeks after the initial immunization. Results: The homologous Ad5F35 and heterologous rMVA/Ad5F35 vaccination regimens elicited comparable levels of HIV Env-specific cellular immune responses, peaking at 2100 ± 222 SFCs/million splenocytes and 2200 ± 619 SFCs/million splenocytes, respectively (p > 0.05). Compared to the heterologous regimen, the homologous Ad5F35 regimen induced significantly higher levels of gp120-binding antibodies at weeks 4 and 8 post-initial immunization, with geometric mean titers of 1:25,600 ± 7011 versus 1:1280 ± 150.7 and 1:10,240 ± 4048 versus 1:2560 ± 391.9, respectively. Furthermore, neutralizing activity at week 8 was significantly higher in the homologous group, with a 50% neutralization titers of 1:45 compared to 1:12 in the heterologous group (p < 0.01). Conclusion: This study demonstrates that the Ad5F35-AEgp145 vaccine, whether administered alone or in combination with rMVA-AEgp145, effectively induces strong and comparable cellular immune responses targeting HIV-1 Env in mice. While both regimens are effective, homologous immunization elicits moderately higher levels of antibody responses. These findings provide an important foundation for the further investigation of vector-based HIV vaccine formulations. Full article

The mammary gland is a valuable model in cancer research and developmental biology. Gene delivery techniques are crucial for mammary tissue research to understand how genes function and study on diseases such as cancer. Viral vector-based approaches provide a high degree of transduction efficiency, but they raise safety and immunogenicity concerns, whereas non-viral vector-based approaches are considered safer and have lower immunogenicity than viral methods. Unfortunately, non-viral gene delivery has rarely been applied to the mammary glands because it is technically challenging. Here, we developed a novel method for in vivo transfection of epithelial cells lining murine mammary glands via intraductal injection of plasmid DNA using a breath-controlled glass capillary and subsequent electroporation (EP) of the injected area. Female mice were transfected with plasmids harboring the enhanced green fluorescent protein (EGFP) gene. Widespread EGFP fluorescence was observed in the mammary epithelial cells of the ducts and adipocytes adjacent to the ducts. As this in vivo gene delivery method is simple, safe, and efficient for gene transfer to the mammary glands, we named this technique “Mammary Intraductal Gene Electroporation” (MIGE). The MIGE method is a useful experimental tool for studies on mammary gland development and differentiation as well as breast cancer research. Full article

Background: Emerging immune-evasive viral variants threaten the efficacy of current vaccines, underscoring the need for strategies that elicit broad and durable protection. Heterologous prime–boost regimens combining distinct vaccine platforms can enhance humoral and cellular immunity through complementary mechanisms. Methods: Using an intramuscular immunization scheme aligned with clinical vaccination practice, CD-1 mice received homologous or heterologous prime–boost regimens combining a replication-deficient Orf virus (Parapoxvirus orf, ORFV)-based spike vaccine (ORFV-S) with the licensed adjuvanted recombinant protein vaccine VidPrevtyn Beta. Spike-specific humoral and cellular immune responses were assessed. Results: ORFV-S alone induced potent and broad spike-specific IgG responses and achieved the strongest ACE2-binding inhibition across variants of concern. ORFV-S priming followed by VidPrevtyn Beta boosting markedly enhanced the magnitude and cross-variant breadth of antibody responses compared with homologous protein vaccination. Both homologous ORFV-S and heterologous regimens incorporating ORFV-S elicited strong CD4⁺ and CD8⁺ T-cell responses, whereas VidPrevtyn Beta alone induced only modest T-cell activity, demonstrating that ORFV-S effectively complements protein-based vaccines. Conclusions: The ORFV-S vector represents a potent vaccine platform capable of inducing broad humoral and cellular immunity. Its use in heterologous prime–boost combinations enhances both antibody magnitude and breadth beyond homologous protein vaccination, supporting its application in vaccination strategies against evolving viral pathogens. Full article

Foot-and-mouth disease virus (FMDV) infection elicits sustained, high-level interleukin-10 (IL-10) secretion in cattle and pigs, which correlates with lymphopenia and immunosuppression. We previously showed that macrophages are the principal source of IL-10 during FMDV infection in mice, but the viral trigger and host pathways remained unknown. In the present study, we examined whether the FMDV structural protein VP3 regulates IL-10 expression. To this end, a eukaryotic VP3 expression vector was transfected into porcine alveolar macrophages (3D4/21 cells), and IL-10 expression together with related signaling pathways was interrogated by qRT-PCR, ELISA, Western blot, co-immunoprecipitation (Co-IP), confocal microscopy, and luciferase reporter assays. The results showed that VP3 significantly increased IL-10 mRNA and protein levels (p < 0.001) in a time-dependent manner. Mechanistically, VP3 promoted phosphorylation of PI3K, AKT, and mTOR; this effect was abolished by the PI3K inhibitor LY294002, which also abrogated VP3-induced IL-10 secretion (p < 0.05). Furthermore, VP3 upregulated mRNA expression of STAT3, ATF1, and CREB (p < 0.05) and enhanced IL-10 promoter activity. The STAT3 inhibitor Stattic reduced IL-10 secretion by 22% (p < 0.05). Co-IP and confocal microscopy confirmed direct binding of VP3 to PI3K in the cytoplasm. In conclusion, FMDV VP3 induces IL-10 overexpression by directly activating the PI3K/AKT-mTOR signaling pathway, thereby elucidating a key mechanism of FMDV-induced immunosuppression. Full article

Recombinant adeno-associated viral vectors (rAAVs) are the leading gene delivery vehicles in clinical development, yet efficient nuclear delivery remains a major barrier to effective transduction. This limitation is partly due to the incomplete understanding of rAAV’s complex subcellular trafficking dynamics. Here, we establish a longitudinal confocal live-cell imaging workflow that tracks rAAV2 from 4 to 12 h post-transduction, paired with an automated 3D analysis pipeline that quantifies spatiotemporal vector distribution, cytoplasmic trafficking, nuclear accumulation, and transgene expression at single-cell resolution. We use this platform to evaluate the effects of vector dose, cell cycle progression, and the behavior of empty particles. We identify previously undescribed trafficking features associated with high transgene expression. Higher rAAV2 doses enhanced cytoplasmic trafficking and nuclear delivery, while cell cycle progression facilitated both trafficking efficiency and transgene expression. We also characterize empty rAAV2 particles, revealing distinct trafficking patterns and markedly reduced nuclear accumulation compared to genome-containing vectors. By uncovering new bottlenecks in rAAV transduction, this platform provides mechanistic insights and potential strategies to improve AAV-based gene therapy. Its generalizable design further supports broad applicability to other non-enveloped viruses. Full article

Dengue fever remains a major mosquito–borne disease worldwide, with over 400 million infections annually and a high risk of severe complications such as dengue hemorrhagic fever. The disease is prevalent in tropical and subtropical regions, where population density and limited vector control accelerate transmission, making early and reliable diagnosis essential for outbreak prevention and disease management. Conventional diagnostic methods, including virus isolation, reverse transcription polymerase chain reaction (RT–PCR), enzyme–linked immunosorbent assays (ELISA), and serological testing, are accurate but often constrained by high cost, labor–intensive procedures, centralized laboratory requirements, and delayed turnaround times. This review examines current dengue diagnostic technologies by outlining their working principles, performance characteristics, and practical limitations, with emphasis on key target analytes such as viral RNA; nonstructural protein 1 (NS1), including DENV–2 NS1; and host antibodies. Diagnostic approaches across commonly used biofluids, including whole blood, serum, plasma, and urine, are discussed. Recent advances in biosensing technologies are reviewed, including optical, electrochemical, microwave, microfluidic, and CRISPR–based platforms, along with the integration of artificial intelligence for data analysis and diagnostic enhancement. Overall, this review highlights the need for accurate, scalable, and field–deployable diagnostic solutions to support early dengue detection and reduce the global disease burden. Full article

Dengue is a mosquito-borne viral disease transmitted by Aedes aegypti, the main vector in the Americas. The lack of effective antiviral treatments, limited vaccine coverage, and the increasing resistance of mosquitoes to conventional insecticides emphasize the need for alternative vector control strategies. Plant-derived larvicides represent a promising and eco-friendly approach. This study characterized the phytochemical profile of Persea americana Mill. (var. Lorena) and evaluated its larvicidal activity against Ae. aegypti (Rockefeller strain). The phytochemical profile was assessed through qualitative screening, UV-Vis spectrophotometry, and UHPLC analysis. Larvicidal activity was evaluated against third-instar larvae of Ae. aegypti (Rockefeller strain) and the median lethal concentration (LC₅₀) values were determined. Preliminary screening of ethanolic extracts revealed the presence of various secondary metabolites of pharmacological relevance, including alkaloids, coumarins, tannins, flavonoids, saponins, triterpenes/sterols, and quinones. UV-Vis spectra displayed distinct absorption patterns, with a prominent peak near 260 nm, consistent with the presence of aromatic compounds. UHPLC profiling revealed high chemical diversity across different plant parts, with 70, 98, 71, and 52 peaks (above 1 × 10⁵ intensity) detected in seed, flower, pulp, and leaf extracts, respectively. Larvicidal bioassays showed significant activity, particularly in the seed extract, with LC₅₀ values (µg/mL) of 3.8 (3.3–4.1) for seeds, 22.4 (21.8–23.9) for flowers, 23.0 (21.5–24.6) for pulp, and 29.7 (28.1–31.2) for leaves. This study highlights the larvicidal potential of ethanolic extracts from P. americana (var. Lorena), with the seed extract exhibiting the highest chemical diversity and bioactivity against Ae. aegypti larvae. The detection of key secondary metabolites, including flavonoids, alkaloids, and saponins, supports the development of an effective, plant-based larvicide for sustainable vector control strategies. Full article