scholarly journals Arsenal of Nanobodies for Broad-Spectrum Countermeasures against Current and Future SARS-CoV-2 Variants of Concerns

2021 ◽  
Author(s):  
Martin A Rossotti ◽  
Henk van Faassen ◽  
Anh Tran ◽  
Joey Sheff ◽  
Jagdeep A Sandhu ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Cross Reactivity ◽  
Antibody Fragments ◽  
Domain Specificity ◽  
Spike Glycoprotein ◽  
Viral Burden ◽  
Cryptic Epitopes

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The panel of nanobodies were shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across many VoCs; wide-ranging epitopic and mechanistic diversity; high and broad in vitro neutralization potencies; and high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to tackle current and future SARS-CoV-2 variants and SARS-related viruses. Furthermore, the high aerosol-ability of nanobodies provides the option for effective needle-free delivery through inhalation.

Specific Labeling of Protein Domains with Antibody Fragments

1981 ◽  
Vol 39 ◽  
pp. 416-417
Author(s):  
U. Aebi ◽  
L.E. Buhle ◽  
W.E. Fowler
Keyword(s):  
Image Processing ◽  
Specific Protein ◽  
Antibody Fragments ◽  
Supramolecular Organization ◽  
Two Dimensional ◽  
Ordered Structures ◽  
Virus Capsids ◽  
Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

Design, Synthesis, and Pharmacokinetic Evaluation of O-Carbamoyl Tizoxanide Prodrugs

2020 ◽  
Vol 16 ◽  
Author(s):  
Xi He ◽  
Wenjun Hu ◽  
Fanhua Meng ◽  
Xingzhou Li
Keyword(s):  
Plasma Concentration ◽  
Broad Spectrum ◽  
Plasma Concentrations ◽  
Antiviral Drug ◽  
Systemic Exposure ◽  
Pharmacokinetic Profile ◽  
Hydroxyl Group ◽  
First Pass

Background: The broad-spectrum antiparasitic drug nitazoxanide (N) has been repositioned as a broad-spectrum antiviral drug. Nitazoxanide’s in vivo antiviral activities are mainly attributed to its metabolitetizoxanide, the deacetylation product of nitazoxanide. In reference to the pharmacokinetic profile of nitazoxanide, we proposed the hypotheses that the low plasma concentrations and the low system exposure of tizoxanide after dosing with nitazoxanide result from significant first pass effects in the liver. It was thought that this may be due to the unstable acyloxy bond of nitazoxanide. Objective: Tizoxanide prodrugs, with the more stable formamyl substituent attached to the hydroxyl group rather than the acetyl group of nitazoxanide, were designed with the thought that they might be more stable in plasma. It was anticipated that these prodrugs might be less affected by the first pass effect, which would improve plasma concentrations and system exposure of tizoxanide. Method: These O-carbamoyl tizoxanide prodrugs were synthesized and evaluated in a mouse model for pharmacokinetic (PK) properties and in an in vitro model for plasma stabilities. Results: The results indicated that the plasma concentration and the systemic exposure of tizoxanide (T) after oral administration of O-carbamoyl tizoxanide prodrugs were much greater than that produced by equimolar dosage of nitazoxanide. It was also found that the plasma concentration and the systemic exposure of tizoxanide glucuronide (TG) were much lower than that produced by nitazoxanide. Conclusion: Further analysis showed that the suitable plasma stability of O-carbamoyl tizoxanide prodrugs is the key factor in maximizing the plasma concentration and the systemic exposure of the active ingredient tizoxanide.

scholarly journals Antibacterial Mechanisms and Efficacy of Sarecycline in Animal Models of Infection and Inflammation

Antibiotics ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 439
Author(s):  
Christopher G. Bunick ◽  
Jonette Keri ◽  
S. Ken Tanaka ◽  
Nika Furey ◽  
Giovanni Damiani ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Bacterial Resistance ◽  
Antibiotic Use ◽  
In Vivo Studies ◽  
Infection Model ◽  
Severe Acne ◽  
Rat Paw Edema ◽  
Infection And Inflammation

Prolonged broad-spectrum antibiotic use is more likely to induce bacterial resistance and dysbiosis of skin and gut microflora. First and second-generation tetracycline-class antibiotics have similar broad-spectrum antibacterial activity. Targeted tetracycline-class antibiotics are needed to limit antimicrobial resistance and improve patient outcomes. Sarecycline is a narrow-spectrum, third-generation tetracycline-class antibiotic Food and Drug Administration (FDA)-approved for treating moderate-to-severe acne. In vitro studies demonstrated activity against clinically relevant Gram-positive bacteria but reduced activity against Gram-negative bacteria. Recent studies have provided insight into how the structure of sarecycline, with a unique C7 moiety, interacts with bacterial ribosomes to block translation and prevent antibiotic resistance. Sarecycline reduces Staphylococcus aureus DNA and protein synthesis with limited effects on RNA, lipid, and bacterial wall synthesis. In agreement with in vitro data, sarecycline demonstrated narrower-spectrum in vivo activity in murine models of infection, exhibiting activity against S. aureus, but reduced efficacy against Escherichia coli compared to doxycycline and minocycline. In a murine neutropenic thigh wound infection model, sarecycline was as effective as doxycycline against S. aureus. The anti-inflammatory activity of sarecycline was comparable to doxycycline and minocycline in a rat paw edema model. Here, we review the antibacterial mechanisms of sarecycline and report results of in vivo studies of infection and inflammation.

scholarly journals Previous Humoral Immunity to the Endemic Seasonal Alphacoronaviruses NL63 and 229E Is Associated with Worse Clinical Outcome in COVID-19 and Suggests Original Antigenic Sin

Life ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 298
Author(s):  
Daniele Focosi ◽  
Angelo Genoni ◽  
Ersilia Lucenteforte ◽  
Silvia Tillati ◽  
Antonio Tamborini ◽  
...  
Keyword(s):  
Humoral Immunity ◽  
Cross Reactivity ◽  
Clinical Severity ◽  
World Health ◽  
Laboratory Findings ◽  
Cellular And Humoral Immunity ◽  
Rate Ratios ◽  
Original Antigenic Sin

Antibody-dependent enhancement (ADE) of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) infection has been hypothesized. However, to date, there has been no in vitro or in vivo evidence supporting this. Cross-reactivity exists between SARS CoV-2 and other Coronaviridae for both cellular and humoral immunity. We show here that IgG against nucleocapsid protein of alphacoronavirus NL63 and 229E correlate with the World Health Organization’s (WHO) clinical severity score ≥ 5 (incidence rate ratios was 1.87 and 1.80, respectively, and 1.94 for the combination). These laboratory findings suggest possible ADE of SARS CoV-2 infection by previous alphacoronavirus immunity.

scholarly journals In VitroandIn VivoCharacterization of the Novel Oxabicyclooctane-Linked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase

2016 ◽  
Vol 60 (8) ◽  
pp. 4830-4839 ◽  
Author(s):  
Christopher M. Tan ◽  
Charles J. Gill ◽  
Jin Wu ◽  
Nathalie Toussaint ◽  
Jingjun Yin ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Antibacterial Agents ◽  
Cell Activity ◽  
Dna Gyrase ◽  
Topoisomerase Iv ◽  
Bacterial Dna ◽  
Whole Cell ◽  
Content Type

ABSTRACTOxabicyclooctane-linked novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of recently described antibacterial agents with broad-spectrum activity. NBTIs dually inhibit the clinically validated bacterial targets DNA gyrase and topoisomerase IV and have been shown to bind distinctly from known classes of antibacterial agents directed against these targets. Herein we report the molecular, cellular, andin vivocharacterization of AM-8722 as a representative N-alkylated-1,5-naphthyridone left-hand-side-substituted NBTI. Consistent with its mode of action, macromolecular labeling studies revealed a specific effect of AM-8722 to dose dependently inhibit bacterial DNA synthesis. AM-8722 displayed greater intrinsic enzymatic potency than levofloxacin versus both DNA gyrase and topoisomerase IV fromStaphylococcus aureusandEscherichia coliand displayed selectivity against human topoisomerase II. AM-8722 was rapidly bactericidal and exhibited whole-cell activity versus a range of Gram-negative and Gram-positive organisms, with no whole-cell potency shift due to the presence of DNA or human serum. Frequency-of-resistance studies demonstrated an acceptable rate of resistance emergencein vitroat concentrations 16- to 32-fold the MIC. AM-8722 displayed acceptable pharmacokinetic properties and was shown to be efficacious in mouse models of bacterial septicemia. Overall, AM-8722 is a selective and potent NBTI that displays broad-spectrum antimicrobial activityin vitroandin vivo.

scholarly journals Allergenic Characterization of New Mutant Forms of Pru p 3 as New Immunotherapy Vaccines

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
C. Gómez-Casado ◽  
M. Garrido-Arandia ◽  
P. Gamboa ◽  
N. Blanca-López ◽  
G. Canto ◽  
...  
Keyword(s):  
Food Allergy ◽  
Ex Vivo ◽  
Binding Capacity ◽  
Cross Reactivity ◽  
T Cell Epitopes ◽  
Native Form ◽  
Ige Binding ◽  
Pru P 3

Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named asPru p 3.01, Pru p 3.02, andPru p 3.03.Pru p 3.01showed very similar allergenic activity as the wild type byin vitroassays. However,Pru p 3.02andPru p 3.03presented reduced IgE binding with respect to the native form, byin vitro,ex vivo,and in vivo assays. In addition,Pru p 3.03had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, bothPru p 3.02andPru p 3.03maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus,Pru p 3.02andPru p 3.03could be good candidates for potential immunotherapy in peach-allergic patients.

scholarly journals Active immunization against the proregions of GDF9 or BMP15 alters ovulation rate and litter size in mice

Reproduction ◽  
2012 ◽  
Vol 143 (2) ◽  
pp. 195-201 ◽  
Author(s):  
C Joy McIntosh ◽  
Steve Lawrence ◽  
Peter Smith ◽  
Jennifer L Juengel ◽  
Kenneth P McNatty
Keyword(s):  
Litter Size ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Cross Reactivity ◽  
Full Length ◽  
Ovulation Rate ◽  
Corpora Lutea ◽  
Immunization Group

The transforming growth factor β (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.

scholarly journals Non-Toxic Virucidal Macromolecules Show High Efficacy Against Influenza Virus Ex Vivo and In Vivo

2020 ◽  
Author(s):  
Ozgun Kocabiyik ◽  
Valeria Cagno ◽  
Paulo Jacob Silva ◽  
Yong Zhu ◽  
Laura Sedano ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Viral Infections ◽  
Ex Vivo ◽  
Public Health Problem ◽  
Major Public Health Problem ◽  
New Class ◽  
Influenza Strain ◽  
Low Toxicity

AbstractInfluenza is one of the most widespread viral infections worldwide and represents a major public health problem. The risk that one of the next pandemics is caused by an influenza strain is very high. It is very important to develop broad-spectrum influenza antivirals to be ready for any possible vaccine shortcomings. Anti-influenza drugs are available but they are far from ideal. Arguably, an ideal antiviral should target conserved viral domains and be virucidal, i.e. irreversibly inhibit viral infectivity. Here, we describe a new class of broad-spectrum anti-influenza macromolecules that meets these criteria and displays exceedingly low toxicity. These compounds are based on a cyclodextrin core modified on its primary face with long hydrophobic linkers terminated in 6’sialyl-N-acetyllactosamine (6’SLN) or 3’SLN. SLN enables nanomolar inhibition of the viruses while the hydrophobic linkers confer irreversibility to the inhibition. The combination of these two properties allows for efficacy in vitro against several human or avian influenza strains, as well as against a 2009 pandemic influenza strain ex vivo. Importantly, we show that, in mice, the compounds provide therapeutic efficacy when administered 24h post-infection allowing 90% survival as opposed to no survival for the placebo and oseltamivir..

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