Search Results (96)

The focus of this study was to explore phage display systems employing bacteriophage lambda (λ) gene fusions to its capsid decoration protein gpD as reagent tools for tackling disease. The biological activity of gpD-fusions was examined by testing for the retained antimicrobial toxicity of cathelicidins or defensins fused to gpD. Our previous finding that only COOH fusions of either cathelicidins or defensins to gpD were toxigenic was expanded to show that only the reduced form of fused defensin antimicrobial polypeptides was found to be toxigenic. Compared in review are gene-fusion lytic display systems (where the fusion-display gene is integrated within the viral genome) with a surrogate system, employed herein, that exogenously provides the fusion-display protein for addition to phage capsid. It is easily possible to produce fully coated lambda display particles (LDP) serving as single epitope vaccines (SEV), or antimicrobials, or to produce partially coated LDP without any complex bacteriophage genetic engineering, making the system available to all. The potential to build vaccine vector phage particles (LDNAP) comprising essentially sheathed DNA vaccines encapsulated within an environmentally protective capsid is described. LDNAP are produced by introducing a cassette into the phage genome either by phage–plasmid recombination or cloning. The cassette carries a high-level eukaryotic expression promoter driving transcription of the vaccine candidate gene and is devoid of plasmid resistance elements. Full article

In this study, sucrase A (SacA) from Bacillus subtilis was successfully displayed on the outer membrane of Escherichia coli via fusion with the AIDA-I autotransporter from E. coli. The pAIDA-sacA plasmid was constructed by fusing sacA with the ctxB signal sequence and the β-barrel domain of aida gene, enabling surface expression under both aerobic and anaerobic conditions. Functional expression of AIDA–SacA was confirmed by the appearance of reducing sugars in enzymatic assays of sucrose hydrolysis and by acid production on phenol red agar. Structural prediction suggested correct localisation of the catalytic domain on the extracellular surface. Enzymatic characterisation revealed that AIDA-SacA exhibits optimal activity at 40 °C and pH 7. The calculated Km for sucrose was 1.18 mM, while the corresponding Vmax was 2.32 U mL⁻¹. Thermal stability assays showed that the enzyme retained over 80% of its activity after 60 min at 45 °C, indicating notable resistance to moderate temperatures. Metal ion assays indicated that K⁺ enhanced enzymatic activity, while Zn²⁺, Cu²⁺, and Mg²⁺ were inhibitory. SDS-PAGE analysis confirmed the expression of the recombinant fusion protein, with a distinct band at approximately 114 kDa corresponding to the expected size. These results demonstrate the feasibility of employing the AIDA-I system for the surface display of SacA in E. coli, providing a functional platform for future applications in whole-cell biocatalysis. Full article

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine with therapeutic applications in oncology and neurodegenerative diseases. However, its clinical use is limited by the high cost of eukaryotic production systems. Here, we developed a cost-effective Escherichia coli-based platform for high-yield production of biologically active recombinant human GM-CSF (rhGM-CSF) using SUMO fusion technology. The engineered pET-SUMO-GM plasmid enabled expression of a 33 kDa fusion protein, accounting for 23–25% of total cellular protein, though it primarily accumulated in inclusion bodies. A multi-step purification strategy—including nickel affinity chromatography, Ulp protease cleavage, and hydrophobic chromatography—yielded >99.5% pure rhGM-CSF. In vitro functional assays demonstrated equivalent activity to the WHO international standard (ED50: 0.045 vs. 0.043 ng/mL in TF-1 cell proliferation). In vivo, the preparation significantly restored neutrophil counts (3.4-fold increase, p ≤ 0.05) in a murine cyclophosphamide-induced myelosuppression model. Our results establish a scalable, prokaryotic-based method to produce functional rhGM-CSF, overcoming solubility and folding challenges while maintaining therapeutic efficacy. This approach could facilitate broader clinical and research applications of GM-CSF, particularly in resource-limited settings. Full article

Background/Objectives: Developing next-generation mRNA-based seasonal influenza vaccines remains challenging, primarily because of the relatively low immunogenicity of influenza B hemagglutinin (HA) antigens. We describe a systematic vaccine development strategy that combined vector and antigen design optimization. Methods: Novel untranslated region (UTR) sequences and a hybrid poly(A) tail were used to increase plasmid stability and mRNA expression. Fusion proteins containing HA antigens linked by T4 foldon domains were engineered to enhance the immune responses against influenza B HA antigens and to permit the expression of multiple HA ectodomains from a single mRNA species. The vaccine performance was verified in a traditional encapsulated lipid nanoparticle (LNP) formulation that requires long-term storage at temperatures below −15 °C as well as in a proprietary thermo-stable LNP formulation developed for the long-term storage of the mRNA vaccine at 2–8 °C. Results: In preclinical studies, our next-generation seasonal influenza vaccine tested alone or as a combination influenza/COVID-19 mRNA vaccine elicited hemagglutination inhibition (HAI) titers significantly higher than Fluzone HD, a commercial inactivated influenza vaccine, across all 2024/2025 seasonal influenza strains, including the B/Victoria lineage strain. At the same time, the combination mRNA vaccine demonstrated superior neutralizing antibody titers to 2023/2024 Spikevax, a commercial COVID-19 comparator mRNA vaccine. Conclusions: Our data demonstrate that the multimerization of antigens expressed as complex fusion proteins is a powerful antigen design approach that may be broadly applied toward mRNA vaccine development. Full article

Background and Objectives: Myricetin, a flavonoid compound, was demonstrated to effectively arrest the cell-to-cell fusion and syncytial development triggered by Nipah virus (NiV) fusion (F) and attachment (G) envelope glycoproteins in vitro involving two permissive mammalian cell lines. Methods: Time-of-addition assays were carried out using codon-optimized NiV wild type (WT) F and G plasmids followed by a challenge with the addition of myricetin 1 h and 6 h post-transfection in HEK 293T and Vero cells. Results: Upon evaluating different myricetin concentrations, it was determined that a 100 μM concentration of myricetin effectively inhibited 64–80% of syncytia in HEK and Vero cells. Interpretation & Conclusions: In this study, we concluded that myricetin mitigated the syncytial development in HEK and Vero cell lines. Given the flavonoid attributes of myricetin which is widely present in fruits, vegetables, tea, and wine, it may be regarded as a phytonutrient and a safer antiviral alternative against Nipah virus infections. Due to the BSL-4 nature of the virus, further research involving live virus culture is necessary to confirm myricetin as a potential antiviral compound for the mitigation of pathological effects of NiV infections. Full article

The influenza A viruses (IAV) are the principal pathogens for annual (seasonal) influenza, which cause world-wide outbreaks in poultry and pose a persistent threat to public health. The Hemagglutinin protein (HA) of IAV promotes virus infection by binding the host membrane receptor and mediating virus–host membrane fusion. Immunoprecipitation–mass spectrometry (IP-MS) provides global insights into IAV HA–host protein interactions. However, various experimental conditions might affect the identification of interactions. Here, we performed a serial IP-MS to compare interactors of IAV HA in accidental host human, chicken and reservoir host duck cells. We found that the positive ratio of interactors identified by the IP-MS was improved when the transfected HA plasmid had a similar expression level to HA proteins found in IAV virus infection. Comparing interactors in human, chicken and duck cells, we found that HA–interacting host factors might play a role in the susceptibility of accidental hosts (human and chicken) to IAV infection compared to reservoir hosts (duck). We then focused on the function of two heat shock proteins (HSPA5 and HSPA8), which interacted with IAV HA proteins in all three species (human, chicken and duck). We found that both HSPA5 and HSPA8 promoted the IAV replication by enhancing the viral attachment and internalization. These findings extend our knowledge about the mechanisms of IAV entry to host cells and provide target genes to create chickens resistant to avian influenza. Full article

Considering the absence of a widely utilized EBV vaccine, we have developed an EBV gL-gH344-Ferritin nanoparticle vaccine utilizing ferritin as a carrier. The gL-gH344-Ferritin fusion gene was synthesized and inserted into the pET30a plasmid. The expression of the fusion protein in the recombinant plasmid was verified by Western blot. Then, the gL-gH344-Ferritin subunit nanoparticle vaccine was obtained by purification of the fusion protein. BALB/c mice were immunized using a two-dose protocol. The titers of EBV specific antibodies were determined using enzyme-linked immunosorbent assay at 4, 8, and 12 weeks after the initial immunization. Moreover, the levels of EBV gL-gH344 specific splenocytes secreting interferon (IFN)-γ and interleukin (IL)-6 were determined using an enzyme-linked immunospot assay. The pET30a-gL-gH344-Ferritin prokaryotic expression plasmid was successfully constructed. gL-gH344-Ferritin was efficiently expressed in E. coli. Following immunization with gL-gH344-Ferritin, the mice sera demonstrated elevated titers of EBV specific immunoglobulin G. Moreover, after stimulating with EBV gL-gH344 specific peptides, the splenocytes of the immunized mice showed a marked tendency to secrete large amounts of IFN-γ and IL-6. The gL-gH344-Ferritin nanoparticle vaccine carrying the EBV gL-gH344 fusion protein induced robust and sustained specific humoral and cellular immune responses in mice. Full article

tet(X4)-positive IncHI1 plasmids are widely prevalent in various bacteria. To understand their transmission characteristics, we analyzed two extensively drug-resistant (XDR) Escherichia coli strains isolated from pet dog feces in Henan Province, China. Strain T28R harbored tet(X4)-positive IncHI1, IncF18:A-:B-, and mcr-1-positive IncI2 plasmids, while T16R carried tet(X4)-positive IncHI1, F16:A-:B-, and mcr-1-positive IncX4 plasmids. Four representative fusion plasmids, pT28R-F1, pT28R-F2, pT28R-F3, and pT16R-F1, in transconjugants were analyzed using WGS and PCR mapping. The results showed that IS26 from the IncF18:A-:B--plasmid attacked the conjugative transfer-associated genes trhc and rsp on the IncHI1 plasmid, generating pT28R-F1 and pT28R-F2. pT28R-F3 was generated through ISCro1- and ISCR2-mediated homologous recombination, deleting the Tra1 region of the IncHI1 plasmid. T16R-F1 emerged from ISCR2- and IS1B-mediated homologous recombination, losing transfer regions of parental plasmids. Notably, fusion plasmids lost the temperature sensitivity of the IncHI1 plasmid, with conjugation frequencies between 1.57 × 10⁻⁴ and 3.84 × 10⁻⁵ at 28 °C and 37 °C. The findings suggest that tet(X4)-positive IncHI1 plasmids could be mobilized with the assistance of conjugative helper plasmids and that fusion events enhance the adaptability of these plasmids, thus facilitating the spread of antibiotic resistance, posing a growing public health threat. Full article

Background: Next-generation vaccines against coronavirus disease 2019 (COVID-19) focus on inducing a long-lasting immune response against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its emerging variants. To achieve this, antigens other than spike proteins have been proposed, and different platforms have been evaluated. Nucleic acid-based vaccines are fundamental for this process. Preclinical data have shown that the SARS-CoV-2 nucleocapsid protein induces a protective cellular immune response, and when combined with the spike protein, the resulting humoral and cellular immune responses are effective against some SARS-CoV-2 variants. Methods: We designed a DNA vaccine against the spike and nucleocapsid proteins of SARS-CoV-2 to generate fusion proteins based on the Delta and Omicron B.5 strains. The most immunogenic regions of the spike and nucleocapsid proteins of the Delta and Omicron B strains were selected using bioinformatics. The nucleotide sequences were cloned into pcDNA3.1, and named pcDNA3.1/D-S1, pcDNA3.1/D-S1N, and pcDNA3.1/O-SN. The immunogenicity of the generated fusion proteins was evaluated by analyzing the humoral and cellular responses elicited after the immunization of BALB/c mice. Results: DNA immunization induced antibody production, neutralization activity, and IFN-γ production. The inclusion of the nucleocapsid regions in the plasmid greatly enhanced the immune response. Moreover, cross-reactions with the variants of interest were confirmed. Conclusions: Plasmids-encoding fusion proteins combining the most immunogenic regions of the spike and nucleocapsid proteins present a promising strategy for designing new and effective vaccines against SARS-CoV-2. Full article

Rahnella aquatilis is an emerging pathogen in fish that poses a potential risk to human and public health. However, its pathogenicity and molecular interaction mechanism with the fish host are still poorly understood. For this study, we conducted analyses into the artificial infection, bacterial load, histopathological observation, and molecular characterization of T6SS, as well as its mediated host immune response to R. aquatilis infection. The results showed that the R. aquatilis KCL-5 strain had high pathogenicity in teleosts, such as the cyprinid fish crucian carp Carassius auratus and the zebrafish Danio rerio, as well as a macrophage infection model that was successfully established, both in vivo and in vitro. A significant time-dependent increase in bacterial distribution in the infected tissues of crucian carp was examined using real-time qPCR and immunohistochemical analysis. The recombinant plasmid pET32a-hcp of T6SS was constructed and the fusion protein was of the expected size of 35.9 kD, as shown by SDS-PAGE and Western blot analysis. Moreover, the single-cell identification of kidney-derived Mφ/Mo cells was achieved, defined with the potential cellular marker gene expression in each cell and the genes’ expression of bacterial chemotaxis and flagellar assembly, inflammation, and PRRs, as well as the T6SS-mediated interaction between fish host cells and KCL-5, which was verified by multi-omics analysis. To our knowledge, this is the first report of T6SS/PAMPs-PRRs pathways related to the emerging R. aquatilis pathogen–host interaction mechanism in fish. Full article

This study aimed to explore the interactions among genetic determinants influencing ciprofloxacin resistance in Salmonella. Treatment with PAβN, an efflux pump inhibitor, resulted in a 4–32-fold reduction in the minimum inhibitory concentration (MIC) across all 18 ciprofloxacin-resistant Salmonella isolates. Notably, isolates without point mutations reverted from resistance to sensitivity. The efflux pump played a crucial role in resistance development, particularly in serovar Enteritidis, where PAβN treatment caused a more significant MIC reduction (16–32-fold) in five strains carrying the GyrA (Asp87Tyr) mutation, which initially exhibited high MICs (8 μg/mL). Several resistance genes were identified on transferable plasmids: oqxAB and aac(6′)-Ib-cr were associated with IncF plasmids in S. Enteritidis, IncA/C plasmids in S. Typhimurium, and IncHI2 plasmids in S. Virchow. Additionally, qnrS1 and/or qepA were carried by IncA/C plasmids in S. Thompson. Whole-genome sequencing revealed the presence of an oqxAB module integrated into the chromosomal DNA of S. Derby. Although the MICs of ciprofloxacin in transconjugants and transformants remained low (1–4 μg/mL), they exceeded the clinical breakpoint for susceptibility. These findings highlight the synergistic impact of efflux pumps and plasmid-mediated resistance mechanisms, contributing to the increasing prevalence of ciprofloxacin resistance and posing a significant threat to food safety. Full article

Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway. Full article

Recently, low pathogenic avian influenza virus (LPAIV), including H9N2 subtype, has been common clinical epidemic strains, and is widely distributed globally. The PB1 protein is a key component of the viral RNA polymerase complex (vRNP), and is vital to viral transcription and translation. In this study, to investigate the antigenic determinants in the PB1 protein, the truncated PB1 sequence (1bp-735bp) from H9N2 subtype AIV was amplified with PCR, and expressed in plasmid pET-28a (+). After purification, the recombinant PB1 protein was used to immunize BALB/c mice. Following immunization, hybridoma cells producing PB1-specific monoclonal antibodies were generated through the fusion of splenic lymphocytes with SP2/0 cells. Then, four stable hybridoma cell lines (5F12, 5B3, 2H9, and 3E6) were screened using indirect ELISA and Western blotting. Furthermore, two antigenic sites, 67NPIDGPLPED76 and 97ESHPGIFENS106, were identified through the construction of truncated overlapping fragments of the PB1 protein. These sites were conserved among 28 AIV strains, and were located on the PB1 protein surface. The findings offer a theoretical reference for the development and improvement of H9N2 vaccines and offer biological materials for virus detection during AIV infection mechanisms. Full article

Peptides from heptad repeat (HR1 and HR2) regions of gp41 are effective inhibitors of HIV-1 entry that block the fusion of viral and cellular membranes, but the generation of antibodies highly specific for these peptides is challenging. We have previously described a mouse hybridoma that recognizes MT-C34-related peptides derived from HR2. It was used for the selection of HIV-1-resistant CD4 lymphocytes engineered to express the MT-C34 peptide via a CRISPR/Cas9-mediated knock-in into the CXCR4 locus. In this study, we cloned variable domains of this antibody and generated a recombinant chimeric antibody (chAb) by combining it with the constant regions of the humanized antibody Trastuzumab. The new chAb displayed a high specificity and two-fold higher level of affinity than the parental mouse monoclonal antibody. In addition, chAb mediated up to 27–43% of the antibody-dependent cellular cytotoxicity towards cells expressing MT-C34 on their surface. The anti-MT-C34 chAb can be easily generated using plasmids available for the research community and can serve as a valuable tool for the detection, purification, and even subsequent elimination of HIV-1-resistant CD4 cells or CAR cells engineered to fight HIV-1 infection. Full article

Hepatitis B virus (HBV) infection remains a major health threat with limited treatment options. One of various new antiviral strategies is based on a fusion of Staphylococcus aureus nuclease (SN) with the capsid-forming HBV core protein (HBc), termed coreSN. Through co-assembly with wild-type HBc-subunits, the fusion protein is incorporated into HBV nucleocapsids, targeting the nuclease to the encapsidated viral genome. However, coreSN expression was based on transfection of a plasmid vector. Here, we explored whether introducing protein transduction domains (PTDs) into a fluorescent coreSN model could confer cell-penetrating properties for direct protein delivery into cells. Four PTDs were inserted into two different positions of the HBc sequence, comprising the amphiphilic translocation motif (TLM) derived from the HBV surface protein PreS2 domain and three basic PTDs derived from the Tat protein of human immunodeficiency virus-1 (HIV-1), namely Tat4, NP, and NS. To directly monitor the interaction with cells, the SN in coreSN was replaced with the green fluorescent protein (GFP). The fusion proteins were expressed in E. coli, and binding to and potential uptake by human cells was examined through flow cytometry and fluorescence microscopy. The data indicate PTD-dependent interactions with the cells, with evidence of uptake in particular for the basic PTDs. Uptake was enhanced by a triplicated Simian virus 40 (SV40) large T antigen nuclear localization signal (NLS). Interestingly, the basic C terminal domain of the HBV core protein was found to function as a novel PTD. Hence, further developing cell-permeable viral capsid protein fusions appears worthwhile. Full article