RESUMO
The resurgence of influenza viruses as a significant global threat emphasizes the urgent need for innovative antiviral strategies beyond existing treatments. Here, we present the development and evaluation of a novel super-multivalent sialyllactosylated filamentous phage, termed t-6SLPhage, as a potent entry blocker for influenza A viruses. Structural variations in sialyllactosyl ligands, including linkage type, valency, net charge, and spacer length, were systematically explored to identify optimal binding characteristics against target hemagglutinins and influenza viruses. The selected SLPhage equipped with optimal ligands, exhibited exceptional inhibitory potency in in vitro infection inhibition assays. Furthermore, in vivo studies demonstrated its efficacy as both a preventive and therapeutic intervention, even when administered post-exposure at 2 days post-infection, under 4 lethal dose 50% conditions. Remarkably, co-administration with oseltamivir revealed a synergistic effect, suggesting potential combination therapies to enhance efficacy and mitigate resistance. Our findings highlight the efficacy and safety of sialylated filamentous bacteriophages as promising influenza inhibitors. Moreover, the versatility of M13 phages for surface modifications offers avenues for further engineering to enhance therapeutic and preventive performance.
Assuntos
Antivirais , Animais , Antivirais/farmacologia , Antivirais/química , Humanos , Cães , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/fisiologia , Células Madin Darby de Rim Canino , Inovirus/efeitos dos fármacos , Oseltamivir/farmacologia , Oseltamivir/química , Camundongos , Influenza Humana/virologia , Influenza Humana/tratamento farmacológico , Camundongos Endogâmicos BALB C , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , FemininoRESUMO
Filamentous bacteriophages (bacterial viruses) are semiflexible proteinous nanofilaments with high aspect ratios for which the surface chemistry can be controlled with atomic precision via genetic engineering. That, in addition to their ability to self-propagate and replicate a nearly monodisperse batch of biologically and chemically identical nanofilaments, makes these bionanofilaments superior to most synthetic nanoparticles and thus a powerful tool in the bioengineers' toolbox. Furthermore, filamentous phages form liquid crystalline structures at high concentrations; these ordered assemblies create hierarchically ordered macro-, micro-, and nanostructures that, once cross-linked, can form hierarchically ordered hydrogels, hydrated soft material with a variety of physical and chemical properties suitable for biomedical applications (e.g., wound dressings and tissue engineering scaffolds) as well as biosensing, diagnostic assays. We provide a critical review of these hydrogels of filamentous phage, and their physical, mechanical, chemical, and biological properties and current applications, as well as an overview of limitations and challenges and outlook for future applications. In addition, we present a list of design parameters for filamentous phage hydrogels to serve as a guide for the (bio)engineer and (bio)chemist interested in utilizing these powerful bionanofilaments for designing smart, bioactive materials and devices.
Assuntos
Antivirais/farmacologia , Materiais Biocompatíveis/farmacologia , Hidrogéis/farmacologia , Inovirus/efeitos dos fármacos , Antivirais/química , Bandagens , Materiais Biocompatíveis/química , Humanos , Hidrogéis/química , Teste de Materiais , Tamanho da Partícula , Engenharia Tecidual , Alicerces Teciduais/química , Cicatrização/efeitos dos fármacosRESUMO
Interaction of fd or M13 filamentous phage with a chloroform/water interface induces morphological change, contracting the filaments sequentially into shortened rods (I-forms), and then into spheroidal particles (S-forms). To further investigate this phage contraction, 34 and 26 chloroform-resistant isolates of fd and M13, respectively, were selected after chloroform treatment of wild-type phages at pH 8. 2 and 4 degrees C. DNA sequencing of gene VIII of the 34 fd isolates revealed five different mutants: these were D5H, M28L, V31L, I37T, and S50T. All 26 M13 isolates were I37T. These mutants exhibited variable sensitivity to chloroform, but all contracted much more slowly than wild-type phage during treatment at 4 degrees C. They all contracted like wild-type phage at 37 degrees C. Site-directed mutagenesis showed that the indicated single mutations carried the chloroform resistance. In structural models of the phage, the D5H locus is on the outside and the S50T locus is on the inside. The M28L and I37T loci are buried in a mostly hydrophobic region in the middle. Although these four mutants are spread out radially, they are localized in the axial direction into a thin disk in the model. The last mutant locus, V31L, is out of this disk, but this locus is proximal to the M28L and I37T loci and also in contact with the surface via a deep hydrophobic hole or depression. These five mutants, their locations, and their variable affects on contraction suggest that chloroform-induced contraction involves a specific mechanism rather than a generalized solvent-induced denaturation and that the critical structural changes occur in a localized level in the phage. These results add weight to suggestions that the sequential contraction of filaments-->I-forms-->S-forms mimic corresponding steps in phage penetration, and, in the reverse order, for phage assembly.
Assuntos
Bacteriófago M13/genética , Bacteriófago M13/ultraestrutura , Inovirus/genética , Inovirus/ultraestrutura , Mutação , Bacteriófago M13/efeitos dos fármacos , Capsídeo/química , Capsídeo/ultraestrutura , Clorofórmio/farmacologia , Resistência Microbiana a Medicamentos/genética , Escherichia coli/virologia , Inovirus/efeitos dos fármacos , Microscopia Eletrônica , Modelos Biológicos , Modelos Moleculares , Fenótipo , Virulência/efeitos dos fármacosRESUMO
Contact with a chloroform/water interface at 2 degrees C induces contraction of fd filamentous phage into rodlike I-forms; this contraction is accompanied by a decrease in the magnitude of circular dichroism spectral intensity near 222 nm and an increase near 210 nm. Comparisons with circular dichroism spectra of 100% helical poly-L-lysine and N-bromosuccinimide-oxidized fd phage indicate that the spectral change accompanying the fd to I-forms transition is due primarily to a change in the contributions from the single tryptophan (W26) of the major coat protein, with probably no significant change in the alpha-helix content. Further contraction of the rodlike I-forms to spherical S-forms at 25 degrees C is accompanied by a substantial general decrease in the magnitude of the ellipticity throughout the 230-210-nm region, which is indicative of a decrease in the alpha-helix content of the major coat protein. The similarity of the circular dichroism spectrum of S-forms with that of coat protein in detergents suggests that the S-form coat protein resembles the coat protein in lipid bilayers. The intrinsic fluorescence of W26 is quenched without red-shift (but perhaps a barely detectable blue-shift) following fd contraction to I-forms and S-forms. The accessibility of W26 to aqueous quenchers does not change significantly upon contraction. However, interaction with hydrophobic quenchers is dramatically altered in the contracted forms in a manner suggesting that the environment surrounding the tryptophan changes from native-protein-like in the fd filament to molten globule-like in the I-form rods and S-form spheroids. As discussed herein, certain features of these data support previous suggestions that chloroform-induced filamentous phage contraction may provide information about phage penetration and assembly in vivo.