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

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.

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

Acta Biochimica Polonica ◽

10.18388/abp.2016_1292 ◽

2016 ◽

Vol 64 (1) ◽

Cited By ~ 2

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.

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