scholarly journals Characterization of a Lipopolysaccharide-Targeted Monoclonal Antibody and Its Variable Fragments as Candidates for Prophylaxis against the Obligate Intracellular Bacterial Pathogen Coxiella burnetii

2014 ◽  
Vol 82 (11) ◽  
pp. 4530-4541 ◽  
Author(s):  
Ying Peng ◽  
Laura Schoenlaub ◽  
Alexandra Elliott ◽  
William J. Mitchell ◽  
Guoquan Zhang
Keyword(s):  
Monoclonal Antibody ◽  
Coxiella Burnetii ◽  
Natural Infection ◽  
Bacterial Pathogen ◽  
Passive Transfer ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Fab Fragment ◽  
Content Type ◽  
Aerosol Infection

ABSTRACTOur previous study demonstrated that treatment ofCoxiella burnetiiwith the phase I lipopolysaccharide (PI-LPS)-targeted monoclonal antibody (MAb) 1E4 significantly inhibitedC. burnetiiinfection in mice, suggesting that 1E4 is a protective MAb. To determine whether passive transfer of antibodies (Abs) can provide protection againstC. burnetiinatural infection, we examined if passive transfer of 1E4 would protect SCID mice againstC. burnetiiaerosol infection. The results indicated that 1E4 conferred significant protection against aerosolizedC. burnetii, suggesting that 1E4 may be useful for preventingC. burnetiinatural infection. To further understand the mechanisms of 1E4-mediated protection and to test the possibility of using humanized 1E4 to preventC. burnetiiinfection, we examined whether the Fab fragment of 1E4 (Fab1E4), a recombinant murine single-chain variable fragment (muscFv1E4), and a humanized single-chain variable fragment (huscFv1E4) retained the ability of 1E4 to inhibitC. burnetiiinfection. The results indicated that Fab1E4, muscFv1E4, and huscFv1E4 were able to inhibitC. burnetiiinfection in mice but that their ability to inhibitC. burnetiiinfection was lower than that of 1E4. In addition, treatment ofC. burnetiiwith Fab1E4, muscFv1E4, or huscFv1E4 can blockC. burnetiiinfection of macrophages. Interestingly, treatment ofC. burnetiiwith huscFv1E4 can significantly reduceC. burnetiiinfectivity in human macrophages. This report provides the first evidence to demonstrate that the humanized variable fragments of an LPS-specific MAb can neutralizeC. burnetiiinfection and appears to be a promising step toward the potential use of a humanized MAb as emergency prophylaxis againstC. burnetiiexposure.

scholarly journals Coxiella burnetii Interaction with Neutrophils and MacrophagesIn Vitroand in SCID Mice following Aerosol Infection

2013 ◽  
Vol 81 (12) ◽  
pp. 4604-4614 ◽  
Author(s):  
Alexandra Elliott ◽  
Ying Peng ◽  
Guoquan Zhang
Keyword(s):  
Immune Response ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Natural Infection ◽  
Scid Mice ◽  
Innate Immune ◽  
Content Type ◽  
Aerosol Infection

ABSTRACTCoxiella burnetiiis an obligate intracellular bacterium that causes acute and chronic Q fever in humans. Human Q fever is mainly transmitted by aerosol infection. However, there is a fundamental gap in the knowledge regarding the mechanisms of pulmonary immunity againstC. burnetiiinfection. This study focused on understanding the interaction betweenC. burnetiiand innate immune cellsin vitroandin vivo. Both virulentC. burnetiiNine Mile phase I (NMI) and avirulent Nine Mile phase II (NMII) were able to infect neutrophils, while the infection rates were lower than 29%, suggesting thatC. burnetiican infect neutrophils, but infection is limited. Interestingly,C. burnetiiinside neutrophils can infect and replicate within macrophages, suggesting that neutrophils cannot killC. burnetiiandC. burnetiimay be using infection of neutrophils as an evasive strategy to infect macrophages. To elucidate the mechanisms of the innate immune response toC. burnetiinatural infection, SCID mice were exposed to aerosolizedC. burnetii. Surprisingly, neutrophil influx into the lungs was delayed until day 7 postinfection in both NMI- and NMII-infected mice. This result suggests that neutrophils may play a unique role in the early immune response against aerosolizedC. burnetii. Studying the interaction betweenC. burnetiiand the innate immune system can provide a model system for understanding how the bacteria evade early immune responses to cause infection.

scholarly journals Single Chain Variable Fragment Fused to Maltose Binding Protein: A Modular Nanocarrier Platform for the Targeted Delivery of Antitumorals

2021 ◽  
Author(s):  
Francisco J. Reche-Perez ◽  
Simona Plesselova ◽  
Eduardo De los Reyes-Berbel ◽  
Mariano Ortega-Muñoz ◽  
F. Javier Lopez-Jaramillo ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Targeted Delivery ◽  
Specific Binding ◽  
Protein A ◽  
Antibody Fragments ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Binding Properties ◽  
Single Chain Variable Fragments ◽  
Selective Delivery

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due...

scholarly journals Selective Inhibition of Coxiella burnetii Replication by the Steroid Hormone Progesterone

2020 ◽  
Vol 88 (12) ◽  
Author(s):  
Zachary P. Howard ◽  
Anders Omsland
Keyword(s):  
Coxiella Burnetii ◽  
Efflux Pump ◽  
Q Fever ◽  
Steroid Hormone ◽  
Natural Infection ◽  
Placental Tissue ◽  
Selective Inhibition ◽  
Inhibitory Effect ◽  
Content Type ◽  
Direct Inhibitory Effect

ABSTRACT Coxiella burnetii is a zoonotic bacterial obligate intracellular parasite and the cause of query (Q) fever. During natural infection of female animals, C. burnetii shows tropism for the placenta and is associated with late-term abortion, at which time the pathogen titer in placental tissue can exceed one billion bacteria per gram. During later stages of pregnancy, placental trophoblasts serve as the major source of progesterone, a steroid hormone known to affect the replication of some pathogens. During infection of placenta-derived JEG-3 cells, C. burnetii showed sensitivity to progesterone but not the immediate precursor pregnenolone or estrogen, another major mammalian steroid hormone. Using host cell-free culture, progesterone was determined to have a direct inhibitory effect on C. burnetii replication. Synergy between the inhibitory effect of progesterone and the efflux pump inhibitors verapamil and 1-(1-naphthylmethyl)-piperazine is consistent with a role for efflux pumps in preventing progesterone-mediated inhibition of C. burnetii activity. The sensitivity of C. burnetii to progesterone, but not structurally related molecules, is consistent with the ability of progesterone to influence pathogen replication in progesterone-producing tissues.

scholarly journals Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese

2013 ◽  
Vol 80 (2) ◽  
pp. 694-703 ◽  
Author(s):  
Milda Stuknytė ◽  
Eeva-Christine Brockmann ◽  
Tuomas Huovinen ◽  
Simone Guglielmetti ◽  
Diego Mora ◽  
...  
Keyword(s):  
Western Blotting ◽  
Lactobacillus Helveticus ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Content Type ◽  
Bacterial Surface ◽  
Protected Designation Of Origin ◽  
Antibody Libraries ◽  
Grana Padano ◽  
Detection And Localization

ABSTRACTSingle-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein ofLactobacillus helveticusMIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein ofL. helveticusMIMLh5 and one was also capable of binding to the S-layer protein ofL. helveticusATCC 15009. All five anti-S-layer scFvs were expressed inEscherichia coliXL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the S-layer protein ofL. helveticusMIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.

scholarly journals Coxiella burnetii Effector Proteins That Localize to the Parasitophorous Vacuole Membrane Promote Intracellular Replication

2014 ◽  
Vol 83 (2) ◽  
pp. 661-670 ◽  
Author(s):  
Charles L. Larson ◽  
Paul A. Beare ◽  
Daniel E. Voth ◽  
Dale Howe ◽  
Diane C. Cockrell ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Parasitophorous Vacuole ◽  
Coiled Coil ◽  
Bacterial Pathogen ◽  
Effector Proteins ◽  
Intracellular Replication ◽  
Intracellular Growth ◽  
Content Type ◽  
Cell Cytosol ◽  
Pv Generation

The intracellular bacterial pathogenCoxiella burnetiidirects biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supportsC. burnetiireplication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promoteC. burnetiiintracellular growth and PV expansion. We predict additionalC. burnetiieffectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predictedC. burnetiiT4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion byC. burnetiiduring infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termedCoxiellavacuolarprotein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins.C. burnetiiΔcvpB, ΔcvpC, ΔcvpD, and ΔcvpEmutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ΔcvpDand ΔcvpEmutants rescued intracellular growth and PV generation, whereas the growth ofC. burnetiiΔcvpBand ΔcvpCwas rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicateC. burnetiiencodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages.

scholarly journals Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody

2016 ◽  
Vol 64 (1) ◽  
Author(s):  
Róza Sawicka ◽  
Paweł Siedlecki ◽  
Barbara Kalenik ◽  
Jan P Radomski ◽  
Violetta Sączyńska ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Structural Model ◽  
Expression System ◽  
Antigenic Specificity ◽  
Hybridoma Cells ◽  
Single Chain Variable Fragment ◽  
Single Chain

Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitated epitope mapping. Based on the sequences of the affinity-selected polypeptides and the structural model of HA the epitope has been located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibition activity and its antigenic specificity. Additionally, total RNA from hybridoma cells secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed slight differences in specificity are discussed.

Heparin-Induced Thrombocytopenia and Thrombosis: Therapeutic Strategy Using a Single-Chain Variable Fragment (scFv) Antibody

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3346-3346
Author(s):  
Jaa Yien New ◽  
Jose Perdomo ◽  
Xing-Mai Jiang ◽  
Beng Chong
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Heparin Therapy ◽  
Human Platelets ◽  
Binding Activity ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Heparin Induced Thrombocytopenia ◽  
E Coli

Abstract Abstract 3346 Introduction and Aim Heparin-Induced Thrombocytopenia and Thrombosis (HIT) is a life threatening disorder that affects 1–5% of patients receiving heparin therapy. A low platelet count is usually recorded (<150,000 per cubic millimetre) with a decrease of 50% or more from the baseline. The occurrence of HIT is due to the presence of an IgG antibody that recognizes the immune complex formed between Platelet Factor 4 (PF4) and heparin. The antibody/PF4/Heparin complex binds to the FcγRIIa receptor on platelets, leading to platelet activation and thrombotic complications in patients receiving heparin. IV.3 is a murine monoclonal antibody that was raised against the FcγRIIa receptor and has been used as an inhibitor in specificity assays to confirm HIT in patients. We have developed a humanized single-chain variable fragment (scFv) antibody based on the IV.3 monoclonal antibody that binds to the FcγRIIa receptor on platelets and prevents platelet aggregation induced by HIT antibodies. Methods The variable heavy chain (VH) and light chain (VL) of the IV.3 antigen binding fragment (Fab) moiety were amplified using polymerase chain reaction (PCR). These two fragments were then coupled with a linker (Glycine4 and Serine)6. This was followed by introduction of several components including fusion tags (FLAG and c-Myc) at both termini for cloning, detection and purification purposes. The construct was transformed into E. coli (strain-BL21) for protein expression of the scFv. The presence of the protein was detected via immunostaining using anti-FLAG and anti-c-Myc antibodies. The scFv was purified by affinity chromatography and the binding activity was detected using flow cytometry and confocal microscopy. The functional activity was determined using Platelet Aggregation Assay. The scFv was then humanized to minimize potential immunogenicity. Humanization was achieved by introducing specific mutations that rendered the molecule human-like but did not affect binding specificity. The humanized scFv was also expressed in E. coli, purified and tested as before. Results The scFv protein (32kDa) was expressed, purified and confirmed via immunostaining. The created humanized scFv exhibits binding activity against the FcγRIIa on human platelets as determined by flow cytometry and confocal microscopy. In addition, the protein successfully inhibits platelet aggregation at micro molar concentrations in aggregation assays conducted in vitro in the presence of HIT antibodies. Conclusions The humanized scFv was successful in recapitulating the properties of the IV.3 murine monoclonal antibody. It demonstrated binding activity against the FcgRIIa on human platelets and exhibited functional activity by inhibiting platelet activation and aggregation in vitro. This implies that our scFv is able to stop binding of the antibody/PF4/Heparin immune complex to platelets, thus hindering one of the critical initial steps in HIT. The scFv described here may be able to ameliorate the unwanted side effects of heparin therapy and could serve as a potential therapeutic drug for HIT patients. Disclosures: No relevant conflicts of interest to declare.

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