Search Results (81)

Persistent infection with high-risk human papillomavirus (HPV), particularly HPV16, is a major driver of HPV-associated cancers; however, strategies for treating established HPV-induced tumors remain scarce. Here, we developed a DNA-based vaccine linking the SARS-CoV-2 spike (S) protein with an HPV16 E7 epitope (aa 49-57) to simultaneously induce antiviral humoral immunity and antitumor cellular responses. We generated 2 constructs, S-E7 and S-RE7, with the latter incorporating a furin cleavage site (R) to enhance antigen processing. In vitro, S-RE7 significantly enhanced E7-specific CD8⁺ T cell activation compared to S-E7, highlighting the importance of the furin sequence. In vivo, both S-linked vaccines elicited robust E7-specific CD8⁺ T cell responses and provided complete protection against TC-1 tumor challenge in a prophylactic murine model, with long-lasting immunity upon tumor rechallenge. In therapeutic settings, vaccination with S-E7 or S-RE7 significantly suppressed tumor growth, extended survival, and reduced circulating myeloid-derived suppressor cells (MDSCs), indicating alleviation of systemic immunosuppression. Notably, S-RE7 demonstrated faster antitumor effects overall in early tumor progression. In addition to cellular immunity, both constructs induced high levels of anti-spike antibodies, with S-RE7 eliciting approximately fourfold higher responses than S-E7. Furthermore, S-RE7 effectively boosted pre-existing anti-spike immunity in mice that were previously vaccinated. This “two-in-one” strategy represents a promising and versatile platform for the prevention and treatment of HPV-associated cancers while maintaining preparedness against potential SARS-CoV-2. Full article

Dengue virus (DENV) and SARS-CoV-2 are emerging viral pathogens that share overlapping clinical features, including fever, fatigue, and respiratory symptoms, complicating differential diagnosis in endemic regions. Their co-circulation has increased the risk of co-infections, which may result in unpredictable disease progression, increased morbidity, and mortality. This overlap presents a significant challenge in managing outbreaks, as both viruses pose a major public health threat. Vaccines and direct-acting antivirals may be rendered ineffective by viral mutations, making it difficult to address evolving strains. Host-directed antivirals offer a promising alternative, potentially maintaining efficacy against a multitude of variants. Both DENV and SARS-CoV-2 rely on host proteases for viral maturation and entry, with furin playing a crucial role in viral glycoprotein cleavage. In DENV, furin cleaves the prM protein, facilitating virion maturation, while in SARS-CoV-2, the polybasic furin cleavage site in the spike protein enhances viral entry. This makes furin a compelling pan-viral target, where inhibiting furin could reduce viral fitness without relying on viral mutations. This review highlights the therapeutic rationale for targeting furin and discusses luteolin, a furin inhibitor showing antiviral activity against both viruses. Furin-targeted therapies may offer a durable antiviral strategy effective across DENV serotypes, SARS-CoV-2 variants, and co-infection settings. Full article

A 19 nt fragment that spans the SARS-CoV-2 furin cleavage site (FCS) is identical to the reverse complement of a proprietary human DNA repair gene sequence. Rather than interpreting this overlap as evidence of a laboratory event, this article uses it as a theoretical springboard to explore underappreciated biorisk concerns, specifically in the context of cancer research. Although they are RNA viruses, coronaviruses are capable of hijacking host DNA damage response (DDR) pathways, exploiting nuclear functions to enhance replication and evade innate immunity. Under selective pressures (antivirals, DDR antagonists, or large-scale siRNA libraries designed to silence critical host genes), escape mutants may arise with fitness advantages. Parallel observations involving in vivo RNA interference via chimeric viruses lend plausibility to some of the key aspects underlying unappreciated biorisks. The mechanistic insights that incorporate DNA repair mechanisms, CoVs in the nucleus, specifics of viruses in cancer research, anticancer drugs, automated gene silencing experiments, and gene sequence overlaps identify gaps in biorisk policies, even those unaccounted for by the potent “Sequences of Concern” paradigm. Key concerning attributes, including genome multifunctionality, such as NLS/FCS in SARS-CoV-2, antisense sequences, and their combination, are further described in more general terms. The article concludes with recommendations pairing modern technical safeguards with enduring ethical principles. Full article

Monomer-to-excimer transition has become a valuable technique in fluorescence imaging because of its ability to enhance imaging contrast. However, from a practical perspective, the accuracy of excimer formation at target sites warrants further exploration. Enzyme-triggered peptide self-assembly provides a promising solution to this limitation. As a proof-of-concept, in this study, we developed a furin-triggered peptide self-assembling fluorescent probe RF-Cou by coupling a coumarin dye 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid (Cou) with a furin-responsive peptide scaffold for precision live-cell imaging. Upon entering furin-overexpressing 4T1 tumor cells, RF-Cou underwent enzymatic cleavage, releasing an amphiphilic peptide motif and self-assembling into nanoparticles largely concentrated in the Golgi apparatus to confine the diffusion of Cou. During this process, the Cou excimers were formed and induced a red shift in the fluorescence emission, validating the feasibility of RF-Cou in efficient excimer imaging of furin-overexpressing tumor cells. We expect that our findings will highlight the potential of stimuli-responsive small molecular peptide probes to advance excimer-based imaging platforms, particularly for enzyme-specific cell imaging and therapeutic monitoring. Full article

The proteolytic processing of the SARS-CoV-2 spike glycoprotein by host cell membrane-associated proteases is a key step in both the entry of the invading virus into the cell and the release of the newly generated viral particles from the infected cell. Because of the critical importance of this step for the viral infectivity cycle, it has been a target of extensive efforts aimed at identifying highly specific protease inhibitors as potential antiviral agents. An alternative strategy to disrupt the pre-fusioviden processing of the SARS-CoV-2 S glycoprotein aims to protect the substrate rather than directly inhibit the proteases. In this work, we focused on furin, a serine protease located primarily in the Golgi apparatus, but also present on the cell membrane. Its cleavage site within the S glycoprotein is located within the stalk region of the latter and comprises an arginine-rich segment (SPRRARS), which fits the definition of the Cardin–Weintraub glycosaminoglycan recognition motif. Native mass spectrometry (MS) measurements confirmed the binding of a hexadecameric peptide representing the loop region at the S1/S2 interface and incorporating the furin cleavage site (FCS) to heparin fragments of various lengths, as well as unfractionated heparin (UFH), although at the physiological ionic strength, only UFH remains tightly bound to the FCS. The direct LC/MS monitoring of FCS digestion with furin revealed a significant impact of both heparin fragments and UFH on the proteolysis kinetics, although only the latter had IC50 values that could be considered physiologically relevant (0.6 ± 0.1 mg/mL). The results of this work highlight the importance of the long-range and relatively non-specific electrostatic interactions in modulating physiological and pathological processes and emphasize the multi-faceted role played by heparin in managing coronavirus infections. Full article

Objective: We developed a targeted integration-based CHO cell platform for simultaneous antibody display and secretion, enabling a streamlined transition from antibody library screening to production without requiring the re-cloning of antibody genes. Methods: The platform consists of a CHO master cell line with a single-copy landing pad, a helper vector expressing FLPe recombinase, and bi-functional targeting vectors. Recombinase-mediated cassette exchange was utilized to integrate targeting vectors into the landing pad. Bi-functional vectors were designed by incorporating a minimal furin cleavage sequence (mFCS), RRKR, and various 2A peptides between the heavy chain (HC) and a membrane anchor. Results: Incomplete cleavage at the mFCS and 2A sites facilitated the expression of both membrane-bound and secreted antibodies, while mutations in the 2A peptide produced a range of display-to-secretion ratios. However, a fraction of secreted antibodies retained 2A residues attached to the HC polypeptides. Further analysis demonstrated that modifying the first five amino acids of the 2A peptide significantly influenced furin cleavage efficiency, resulting in different display-to-secretion ratios for targeting vectors containing mFCS-2A variant combinations. To overcome this, we designed nine-amino-acid FCS variants that, when placed between the HC and membrane anchor, provided a range of display-to-secretion ratios and eliminated the issue of attached 2A residues in the secreted antibodies. Vectors with lower display levels proved more effective at distinguishing cells expressing high-affinity antibodies with closely matched binding affinities. The platform also demonstrated high sensitivity in isolating high-affinity antibody-expressing cells and supported robust antibody production. Conclusion: This targeted integration-based CHO platform enables efficient, in-format screening and production of antibodies with tunable display-to-secretion profiles. It provides a powerful and scalable tool for accelerating the development of functional, manufacturable therapeutic antibodies. Full article

This retrospective study analyzed SARS-CoV-2 Omicron variability since its emergence, focusing on immunocompromised (IPs) and non-immunocompromised adult people (NIPs). Phylogenetic analysis identified at least five major Omicron lineage groups circulating in Central Italy, from December 2021 to December 2023: (a) BA.1 (34.0%), (b) BA.2 + BA.4 (25.8%), (c) BA.5 + BF (10.8%), (d) BQ + BE + EF (9.2%), and (e) Recombinants (20.2%). The BA.2 + BA.4 lineages were more common in IPs compared to NIPs (30.9% vs. 17.8%, respectively; p = 0.011); conversely, Recombinants were less prevalent in IPs than in NIPs (16.0% vs. 27.1%, respectively; p = 0.018). High-abundant single nucleotide polymorphisms (SNPs; prevalence ≥ 40%) and non-synonymous SNPs (prevalence ≥ 20%) increased during the emergence of new variants, rising from BA.1 to Recombinants (54 to 92, and 43 to 70, respectively, both p < 0.001). Evaluating the genetic variability, 109 SNPs were identified as being involved in significant positive or negative associations in pairs (phi > 0.70, p < 0.001), with 19 SNPs associated in 3 distinct clusters (bootstrap > 0.96). Multivariate regression analysis showed that hospitalization was positively associated with one specific cluster, including S686R and A694S in Spike and L221F in Nucleocapsid (AOR: 2.74 [95% CI: 1.13–6.64, p = 0.025]), and with increased age (AOR:1.03 [95% CI: 1.00–1.06], p = 0.028). Conversely, negative associations with hospitalization were observed for female gender and previous vaccination status (AORs: 0.34 [95% CI: 0.14–0.83], p = 0.017 and 0.19 (95% CI: 0.06–0.63, p = 0.006, respectively). Interestingly, the S686R SNP located in a furin cleavage site suggests its potential pathogenetic role. The results show how Omicron genetic diversification significantly influences disease severity and hospitalization, together with age, sex, and vaccination status as key factors. Full article

Bovine coronavirus (BCoV) exhibits dual tissue tropism, infecting both the respiratory and enteric tracts of cattle. Viral entry into host cells requires a coordinated interaction between viral and host proteins. However, the specific cellular receptors and co-receptors facilitating BCoV entry remain poorly understood. Similarly, the roles of host proteases such as Furin, TMPRSS2, and Cathepsin-L (CTS-L), known to assist in the replication of other coronaviruses, have not been extensively explored for BCoV. This study aims to identify novel BCoV receptors and host proteases that modulate viral replication and tissue tropism. Bovine cell lines were infected with BCoV isolates from enteric and respiratory origins, and the host cell gene expression profiles post-infection were analyzed using next-generation sequencing (NGS). Differentially expressed genes encoding potential receptors and proteases were further assessed using in-silico prediction and molecular docking analysis. These analyses focused on known coronavirus receptors, including ACE2, NRP1, DPP4, APN, AXL, and CEACAM1, to identify their potential roles in BCoV infection. Validation of these findings was performed using the qRT-PCR assays targeting individual genes. We confirmed the gene expression profiles of these receptors and enzymes in some BCoV (+/−) lung tissues. Results revealed high binding affinities of 9-O-acetylated sialic acid and NRP1 to BCoV spike (S) and hemagglutinin-esterase (HE) proteins compared to ACE2, DPP4, and CEACAM1. Additionally, Furin and TMPRSS2 were predicted to interact with the BCoV-S polybasic cleavage site (RRSRR|A), suggesting their roles in S glycoprotein activation. This is the first study to explore the interactions of BCoV with multiple host receptors and proteases. Functional studies are recommended to confirm their roles in BCoV infection and replication. Full article

Background/Objectives: The ectodysplasin A (EDA) gene, a member of the tumor necrosis factor ligand superfamily, is involved in the early epithelial–mesenchymal interaction that regulates ectoderm-derived appendage formation. Numerous studies have shown that mutations in the EDA gene can cause X-linked ectodermal dysplasia (ED) and non-syndromic oligodontia (NSO). Accordingly, this study aimed to identify the causative genetic mutations of the EDA gene. Methods: We investigated EDA gene mutations in two X-linked oligodontia families using candidate gene sequencing and whole-exome sequencing, with a single proband identified and studied for each family. The first family included a patient with NSO, while the second family had a patient exhibiting variable expression of ED. Results: Mutational analysis identified two missense mutations in the EDA gene (NM_001399.5): one novel mutation, c.787A>C p.(Lys263Gln), in family 2; and one previously reported mutation, c.457C>T p.(Arg153Cys), in family 1. All mutated residues are evolutionarily highly conserved amino acids. The p.(Arg153Cys) mutation would destroy the furin recognition site and affect the cleavage of EDA. The p.(Lys263Gln) mutation in a TNF homology domain would interfere with the binding of the EDA receptor. The p.(Lys263Gln) mutation was associated with NSO, while the other mutation demonstrated ED. Conclusions: This study helps to better understand the nature of EDA-related ED and NSO and their pathogenesis, and it expands the mutational spectrum of EDA mutations. Full article

Antibodies are complex protein structures, and producing them using eukaryotic expression systems presents significant challenges. One frequently overlooked aspect of expression vectors is the nucleotide sequence encoding the signal peptide, which plays a pivotal role in facilitating the secretion of recombinant proteins. This study presents the development of an integrative vector, pVEAL3, for expressing full-length recombinant monoclonal antibodies in mammalian cells. The vector features a distinctive nucleotide sequence that encodes an artificial chimeric signal peptide with the following amino acid sequence: MMRTLILAVLLVYFCATVHC. Additionally, the vector incorporates several regulatory elements to enhance antibody expression, including the Gaussia luciferase signal sequence, internal ribosome entry site (IRES), P2A peptide, and a furin cleavage site. These elements coordinate to regulate the synthesis levels of the antibody chains. The analysis of clones obtained via transfection with the developed vector showed that over 95% of them secreted antibodies at levels significantly higher than those of the control. The immunochemical analysis of the chimeric antibody produced by the CHO-K1-10H10ch cell line confirmed the preservation of its functional activity. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza, and respiratory syncytial virus (RSV) are significant global health threats. The need for low-cost, easily synthesized oral drugs for rapid deployment during outbreaks is crucial. Broad-spectrum therapeutics, or pan-antivirals, are designed to target multiple viral pathogens simultaneously by focusing on shared molecular features, such as common metal cofactors or conserved residues in viral catalytic domains. This study introduces a new generation of potent sartans, known as bisartans, engineered in our laboratories with negative charges from carboxylate or tetrazolate groups. These anionic tetrazoles interact strongly with cationic arginine residues or metal cations (e.g., Zn²⁺) within viral and host target sites, including the SARS-CoV-2 ACE2 receptor, influenza H1N1 neuraminidases, and the RSV fusion protein. Using virtual ligand docking and molecular dynamics, we investigated how bisartans and their analogs bind to these viral receptors, potentially blocking infection through a pan-antiviral mechanism. Bisartan, ACC519TT, demonstrated stable and high-affinity docking to key catalytic domains of the SARS-CoV-2 NSP3, H1N1 neuraminidase, and RSV fusion protein, outperforming FDA-approved drugs like Paxlovid and oseltamivir. It also showed strong binding to the arginine-rich furin cleavage sites S1/S2 and S2′, suggesting interference with SARS-CoV-2’s spike protein cleavage. The results highlight the potential of tetrazole-based bisartans as promising candidates for developing broad-spectrum antiviral therapies. Full article

The collaboration between cellular proteases and host cells is pivotal in mounting an effective innate immune defense. Of particular interest is the synergistic interaction between cathepsin G (CatG) and neutrophil elastase (NE), which are proteases secreted by activated neutrophils, and the human alveolar basal epithelial cell line (A549) and the human lung epithelial-like cell line (H1299), because of the potential implications for viral infection. Our study aimed to investigate the binding capacity of CatG and NE on the surface of A549 and H1299 cells through preincubation with purified CatG and NE; thereby, the proteolytic activity could be detected using activity-based probes. Both CatG and NE were capable of binding to the cell surface and exhibited proteolytic activity, leading to increased cell surface levels of MHC I molecules, which is crucial for displaying the endogenous antigenic repertoire. In addition, CatG cleaved the S2′ site of the SARS-CoV-2 spike protein at two specific sites (₈₁₅RS₈₁₆ and ₈₁₇FI₈₁₈) as well as NE (₈₁₃SK₈₁₄ and ₈₁₈IE₈₁₉), which potentially leads to the destruction of the fusion peptide. Additionally, furin required the presence of Ca²⁺ ions for the distinct cleavage site necessary to generate the fusion peptide. Overall, the findings suggest that CatG and NE can fortify target cells against viral entry, underscoring the potential significance of cell surface proteases in protecting against viral invasion. Full article

Host restriction factor GBP2 suppresses the replication of the ecotropic Moloney murine leukemia virus (E-MLV) by inhibiting furin protease, which cleaves the viral envelope glycoprotein (Env) into surface (SU) and transmembrane (TM) subunits. We analyzed the impacts of GBP2 on the infection efficiency mediated by MLV Envs of different strains of ecotropic Moloney, polytropic Friend, amphotropic, and xenotropic MLV-related (XMRV) viruses. Interestingly, the Envs of ecotropic Moloney and polytropic Friend MLV were sensitive to the antiviral activity of GBP2, while XMRV and amphotropic Envs showed resistance. Consistent with the sensitivity to GBP2, the amino acid sequences of the sensitive Envs at the SU-TM cleavage site were similar, as were the sequences of the resistant Envs. SU-TM cleavage of the GBP2-sensitive Env protein was inhibited by furin silencing, whereas that of GBP2-resistant Env was not. The substitution of the ecotropic Moloney cleavage site sequence with that of XMRV conferred resistance to both GBP2 and furin silencing. Reciprocally, the substitution of the XMRV cleavage site sequence with that of the ecotropic sequence conferred sensitivity to GBP2 and furin silencing. According to the SU-TM cleavage site sequence, there were sensitive and resistant variants among ecotropic, polytropic, and xenotropic MLVs. This study found that the dependence of MLV Env proteins on furin cleavage and GBP2-mediated restriction is determined by the amino acid sequences at the SU-TM cleavage site. Full article

Ad26.COV2.S vaccination can lead to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare but severe adverse effect, characterized by thrombocytopenia and thrombosis. The mechanism of VITT induction is unclear and likely multifactorial, potentially including the activation of platelets and endothelial cells mediated by the vaccine-encoded spike protein (S protein). Here, we investigated the biodistribution of the S protein after Ad26.COV2.S dosing in three animal models and in human serum samples. The S protein was transiently present in draining lymph nodes of rabbits after Ad26.COV2.S dosing. The S protein was detected in the serum in all species from 1 day to 21 days after vaccination with Ad26.COV2.S, but it was not detected in platelets, the endothelium lining the blood vessels, or other organs. The S protein S1 and S2 subunits were detected at different ratios and magnitudes after Ad26.COV2.S or COVID-19 mRNA vaccine immunization. However, the S1/S2 ratio did not depend on the Ad26 platform, but on mutation of the furin cleavage site, suggesting that the S1/S2 ratio is not VITT related. Overall, our data suggest that the S-protein biodistribution and kinetics after Ad26.COV2.S dosing are likely not main contributors to the development of VITT, but other S-protein-specific parameters require further investigation. Full article

SARS-CoV-2 is the pathogen responsible for the most recent global pandemic, which has claimed hundreds of thousands of victims worldwide. Despite remarkable efforts to develop an effective vaccine, concerns have been raised about the actual protection against novel variants. Thus, researchers are eager to identify alternative strategies to fight against this pathogen. Like other opportunistic entities, a key step in the SARS-CoV-2 lifecycle is the maturation of the envelope glycoprotein at the RARR685↓ motif by the cellular enzyme Furin. Inhibition of this cleavage greatly affects viral propagation, thus representing an ideal drug target to contain infection. Importantly, no Furin-escape variants have ever been detected, suggesting that the pathogen cannot replace this protease by any means. Here, we designed a novel fluorogenic SARS-CoV-2-derived substrate to screen commercially available and custom-made libraries of small molecules for the identification of new Furin inhibitors. We found that a peptide substrate mimicking the cleavage site of the envelope glycoprotein of the Omicron variant (QTQTKSHRRAR-AMC) is a superior tool for screening Furin activity when compared to the commercially available Pyr-RTKR-AMC substrate. Using this setting, we identified promising novel compounds able to modulate Furin activity in vitro and suitable for interfering with SARS-CoV-2 maturation. In particular, we showed that 3-((5-((5-bromothiophen-2-yl)methylene)-4-oxo-4,5 dihydrothiazol-2-yl)(3-chloro-4-methylphenyl)amino)propanoic acid (P3, IC₅₀ = 35 μM) may represent an attractive chemical scaffold for the development of more effective antiviral drugs via a mechanism of action that possibly implies the targeting of Furin secondary sites (exosites) rather than its canonical catalytic pocket. Overall, a SARS-CoV-2-derived peptide was investigated as a new substrate for in vitro high-throughput screening (HTS) of Furin inhibitors and allowed the identification of compound P3 as a promising hit with an innovative chemical scaffold. Given the key role of Furin in infection and the lack of any Food and Drug Administration (FDA)-approved Furin inhibitor, P3 represents an interesting antiviral candidate. Full article