scholarly journals Engineered Recombinant Single Chain Variable Fragment of Monoclonal Antibody Provides Protection to Chickens Infected with H9N2 Avian Influenza

Vaccines ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 118
Author(s):  
Deimante Lukosaityte ◽  
Jean-Remy Sadeyen ◽  
Angita Shrestha ◽  
Joshua E. Sealy ◽  
Sushant Bhat ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Avian Influenza ◽  
Influenza Viruses ◽  
H9n2 Virus ◽  
Control Measures ◽  
Mitigation Measures ◽  
Passive Immunisation ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Recombinant Scfv

Passive immunisation with neutralising antibodies can be a potent therapeutic strategy if used pre- or post-exposure to a variety of pathogens. Herein, we investigated whether recombinant monoclonal antibodies (mAbs) could be used to protect chickens against avian influenza. Avian influenza viruses impose a significant economic burden on the poultry industry and pose a zoonotic infection risk for public health worldwide. Traditional control measures including vaccination do not provide rapid protection from disease, highlighting the need for alternative disease mitigation measures. In this study, previously generated neutralizing anti-H9N2 virus monoclonal antibodies were converted to single-chain variable fragment antibodies (scFvs). These recombinant scFv antibodies were produced in insect cell cultures and the preparations retained neutralization capacity against an H9N2 virus in vitro. To evaluate recombinant scFv antibody efficacy in vivo, chickens were passively immunized with scFvs one day before, and for seven days after virus challenge. Groups receiving scFv treatment showed partial virus load reductions measured by plaque assays and decreased disease manifestation. These results indicate that antibody therapy could reduce clinical disease and shedding of avian influenza virus in infected chicken flocks.

scholarly journals Avian influenza virus transmission is suppressed in chickens fed Lactobacillus paracasei expressing the 3D8 single-chain variable fragment protein

2019 ◽  
Vol 67 (4) ◽  
pp. 610-618
Author(s):  
Hoonsung Choi ◽  
Sang In Lee ◽  
Shanmugam Sureshkumar ◽  
Mi-Hyang Jeon ◽  
Jeom Sun Kim ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Nuclease Activity ◽  
Lactobacillus Paracasei ◽  
Post Inoculation ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Contact Transmission ◽  
3D8 Scfv

The 3D8 single-chain variable fragment (scFv) is a mini-antibody sequence with independent nuclease activity that shows antiviral effects against all types of viruses in chickens and mice. In this study, chickens were treated daily with an oral dose of 109 CFU Lactobacillus paracasei (L. paracasei) expressing either a secreted or anchored 3D8 scFv for three weeks. After L. paracasei administration, the chickens were challenged with avian influenza virus (AIV). From each experimental group, three chickens were directly infected with 100 µL of 107.5 EID50/mL H9N2 AIV and seven chickens were indirectly challenged through contact transmission. oropharyngeal and cloacal swab samples were collected at 3, 5, 7, and 9 days post-inoculation (dpi) from AIV-challenged chickens, AIV Shedding titres were measured by quantitative real-time PCR. Contact transmission in the chickens that were fed 3D8 scFv-secreting L. paracasei showed a significant reduction in viral shedding when compared with other groups. These results suggest that L. paracasei secreting 3D8 provides a basis for the development of ingestible antiviral probiotics with activity against AIV.

scholarly journals Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Sung June Byun ◽  
Seong-su Yuk ◽  
Ye-Jin Jang ◽  
Hoonsung Choi ◽  
Mi-Hyang Jeon ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Influenza Transmission ◽  
Transgenic Chickens

scholarly journals Endothelial targeting of a recombinant construct fusing a PECAM-1 single-chain variable antibody fragment (scFv) with prourokinase facilitates prophylactic thrombolysis in the pulmonary vasculature

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4191-4198 ◽  
Author(s):  
Bi-Sen Ding ◽  
Claudia Gottstein ◽  
Andrea Grunow ◽  
Alice Kuo ◽  
Kumkum Ganguly ◽  
...  
Keyword(s):  
Antibody Fragment ◽  
Pulmonary Vasculature ◽  
Single Chain Variable Fragment ◽  
Wild Type ◽  
Single Chain ◽  
Recombinant Scfv ◽  
Platelet Endothelial Cell Adhesion ◽  
Limited Effectiveness ◽  
Recombinant Molecule ◽  
Cell Adhesion Molecule 1

Means to prevent thrombus extension and local recurrence remain suboptimal, in part because of the limited effectiveness of existing thrombolytics. In theory, plasminogen activators could be used for this purpose if they could be anchored to the vascular lumen by targeting stably expressed, noninternalized determinants such as platelet-endothelial-cell adhesion molecule 1 (PECAM-1). We designed a recombinant molecule fusing low-molecular-weight single-chain prourokinase plasminogen activator (lmw-scuPA) with a single-chain variable fragment (scFv) of a PECAM-1 antibody to generate the prodrug scFv/lmw-scuPA. Cleavage by plasmin generated fibrinolytically active 2-chain lmw-uPA. This fusion protein (1) bound specifically to PECAM-1-expressing cells; (2) was rapidly cleared from blood after intravenous injection; (3) accumulated in the lungs of wild-type C57BL6/J, but not PECAM-1 null mice; and (4) lysed pulmonary emboli formed subsequently more effectively than lmw-scuPA, thereby providing support for the concept of thromboprophylaxis using recombinant scFv-fibrinolytic fusion proteins that target endothelium.

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.

scholarly journals Contribution of Segment 3 to the Acquisition of Virulence in Contemporary H9N2 Avian Influenza Viruses

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Anabel L. Clements ◽  
Joshua E. Sealy ◽  
Thomas P. Peacock ◽  
Jean-Remy Sadeyen ◽  
Saira Hussain ◽  
...  
Keyword(s):  
Middle East ◽  
Avian Influenza ◽  
Mortality Rates ◽  
Influenza Viruses ◽  
H9n2 Virus ◽  
Morbidity And Mortality ◽  
Single Amino Acid ◽  
Economic Damage ◽  
Poultry Production ◽  
Avian Influenza Viruses

ABSTRACT H9N2 avian influenza viruses (AIVs) circulate in poultry throughout much of Asia, the Middle East, and Africa. These viruses cause huge economic damage to poultry production systems and pose a zoonotic threat both in their own right and in the generation of novel zoonotic viruses, for example, H7N9. In recent years, it has been observed that H9N2 viruses have further adapted to gallinaceous poultry, becoming more highly transmissible and causing higher morbidity and mortality. Here, we investigate the molecular basis for this increased virulence, comparing a virus from the 1990s and a contemporary field strain. The modern virus replicated to higher titers in various systems, and this difference mapped to a single amino acid polymorphism at position 26 of the endonuclease domain shared by the PA and PA-X proteins. This change was responsible for increased replication and higher morbidity and mortality rates along with extended tissue tropism seen in chickens. Although the PA K26E change correlated with increased host cell shutoff activity of the PA-X protein in vitro, it could not be overridden by frameshift site mutations that block PA-X expression and therefore increased PA-X activity could not explain the differences in replication phenotype. Instead, this indicates that these differences are due to subtle effects on PA function. This work gives insight into the ongoing evolution and poultry adaptation of H9N2 and other avian influenza viruses and helps us understand the striking morbidity and mortality rates in the field, as well as the rapidly expanding geographical range seen in these viruses. IMPORTANCE Avian influenza viruses, such as H9N2, cause huge economic damage to poultry production worldwide and are additionally considered potential pandemic threats. Understanding how these viruses evolve in their natural hosts is key to effective control strategies. In the Middle East and South Asia, an older H9N2 virus strain has been replaced by a new reassortant strain with greater fitness. Here, we take representative viruses and investigate the genetic basis for this “fitness.” A single mutation in the virus was responsible for greater fitness, enabling high growth of the contemporary H9N2 virus in cells, as well as in chickens. The genetic mutation that modulates this change is within the viral PA protein, a part of the virus polymerase gene that contributes to viral replication as well as to virus accessory functions—however, we find that the fitness effect is specifically due to changes in the protein polymerase activity.

scholarly journals The PB2 and M genes of genotype S H9N2 virus contribute to the enhanced fitness of H5Nx and H7N9 avian influenza viruses in chickens

Virology ◽  
2019 ◽  
Vol 535 ◽  
pp. 218-226 ◽  
Author(s):  
Xiaoli Hao ◽  
Xiaoquan Wang ◽  
Jiao Hu ◽  
Min Gu ◽  
Jiongjiong Wang ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Influenza Viruses ◽  
H9n2 Virus ◽  
Avian Influenza Viruses

Screening and Identification of Recombinant Anti-Idiotype Antibodies against Gastric Cancer and Colon Cancer Monoclonal Antibodies by a Phage-Displayed Single-Chain Variable Fragment Library

2010 ◽  
Vol 15 (3) ◽  
pp. 308-313 ◽  
Author(s):  
Li Zhikui ◽  
Guo Changcun ◽  
Nie Yongzhan ◽  
He Fengtian ◽  
Ren Xingling ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Gastric Cancer ◽  
Monoclonal Antibodies ◽  
High Sensitivity ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Fragment Library ◽  
Colorectal Cancer Cells ◽  
Screening And Identification ◽  
Antibody Titers

Several monoclonal antibodies (McAbs) have been developed that show high sensitivity and specificity to gastric cancer and colorectal cancer. However, few of the antigens recognized by these antibodies have been identified. The authors now report the selection of anti-idiotype (anti-id) antibodies of MGb1 McAb against gastric cancer and MC5 McAb against colorectal cancer using phage-displayed single-chain variable fragment (ScFv) libraries. After purification, the anti-id antibodies were approximately 30 kd and could be recognized by MGb1/MC5 McAb. Anti-id antibodies significantly blocked the binding of MGb1 and MC5 to gastric cancer/colorectal cancer cells, respectively, suggesting that the antibodies were specific to MGb1 and MC5. Antibodies against gastric and colorectal cancer could be detected in mice at 6 weeks after immunization with the anti-id antibodies. At week 8, antibody titers reached 1:400. The anti-id antibodies may be useful as novel reagents for developing vaccines against gastric cancer and colorectal cancer.

scholarly journals Novel reassortant of H9N2 avian influenza viruses isolated from chickens and quails in Egypt

2021 ◽  
pp. 2142-2149
Author(s):  
Moataz Elsayed ◽  
AbdelSatar Arafa ◽  
Shahira Abdelwahab ◽  
Amro Hashish ◽  
Ahmed Youssef
Keyword(s):  
Avian Influenza ◽  
Influenza Pandemic ◽  
Phylogenetic Analyses ◽  
Influenza Viruses ◽  
H9n2 Virus ◽  
Avian Influenza Viruses ◽  
Genetic Changes ◽  
Genome Variability ◽  
Commercial Chicken ◽  
Amino Acid Mutations

Background and Aim: Poultry infections with H9N2 avian influenza viruses (AIVs) are endemic in Egypt. This study determined the genetic changes in the sequences of H9N2 AIVs isolated from chicken and quails in Egypt, including determining genetic reassortment and detecting the main genetic changes in hemagglutinin (HA) and neuraminidase (NA) genes. Materials and Methods: Swab samples were collected from chicken and quails, examined through reverse transcription-polymerase chain reaction, and AIVs from positive samples were isolated in embryonated chicken eggs. Complete genome sequencing and phylogenetic analyses were conducted for two H9N2 AIV isolates, and sequences of HA and NA gene segments were analyzed in another two isolates. Results: A novel reassortant virus was identified from a commercial chicken flock (A/chicken/Egypt/374V/2016) and quails from a live bird market (A/quail/Egypt/1253V/2016). The reassortant viruses acquired four genome segments from the classic Egyptian H9N2 viruses (HA, NA, NP, and M) and four segments from Eurasian AIVs (PB2, PB1, PA, and NS). Many genetic changes have been demonstrated in HA and NA genes. The isolated novel reassortant H9N2 virus from quails showed amino acid mutations in the antigenic sites on the globular head of the mature HA monomer matched with the parent Egyptian H9N2 virus. Conclusion: This work described the genetic characterization of a novel reassortment of the H9N2 virus in Egypt. The emergence of new reassorted AIV viruses and genome variability raises the concern of an influenza pandemic with zoonotic potentials.

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