scholarly journals Single Chain Fragment Variable (scFv) Antibodies Targeting the Spike Protein of Porcine Epidemic Diarrhea Virus Provide Protection against Viral Infection in Piglets

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 58 ◽  
Author(s):  
Fanqing Zhang ◽  
Yuxue Chen ◽  
Yong Ke ◽  
Lei Zhang ◽  
Bo Zhang ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Passive Immunization ◽  
Spike Protein ◽  
Single Chain ◽  
Effective Prevention ◽  
Chain Fragment ◽  
Diarrhea Virus ◽  
Single Chain Fragment Variable ◽  
Porcine Epidemic Diarrhea ◽  
Single Chain Fragment

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe diarrhea and death in neonatal piglets. Passive immunization with neutralizing antibodies against PEDV is an effective prevention measure. In this study, single chain fragment variable (scFv) antibodies against PEDV were screened from the porcine scFv phage display library. After four rounds of biopanning, scFvs that showed higher affinity to the PEDV antigen were selected for further study. The scFv genes were cloned into the expression plasmid for recombinant protein expression. These scFvs were shown to inhibit PEDV infectivity by the plaque reduction neutralization assay. Immunofluorescence assay (IFA) revealed that the epitopes recognized by these scFvs were in the S1 region of the spike protein. The potential of scFvs to provide prevention against PEDV infections in piglets was further investigated. Piglets orally administered scFvs showed no to mild clinical symptoms, significantly less viral shedding, no mortality and no intestinal lesions. The field application also revealed that the survival rate of piglets was significantly increased by oral administration of scFvs. Our data support the potential role of scFvs in the prevention and treatment of PEDV infection.

scholarly journals Acquisition and bioactivity analysis of single-chain fragment variable antibody against CSFV

2020 ◽  
Author(s):  
Benqiang Li ◽  
Jie Tao ◽  
Jinghua Cheng ◽  
Ying Shi ◽  
Huili Liu
Keyword(s):  
B Lymphocytes ◽  
Passive Immunization ◽  
Classical Swine Fever ◽  
Scfv Antibody ◽  
Single Chain ◽  
Effective Prevention ◽  
Chain Fragment ◽  
Single Chain Fragment Variable ◽  
Neutralizing Capacity ◽  
Single Chain Fragment

Abstract Background Classical swine fever (CSF) is a highly contagious disease that threatens the pig industry. Passive immunization with neutralizing antibodies against classical swine fever virus (CSFV) is an effective prevention and therapeutic measure. Results In this study, to prepare a single-chain fragment variable (scFv) antibody against CSFV, two weanling piglets were injected twice with a CSFV attenuated vaccine. After the second immunization, peripheral blood lymphocytes were separated from the blood of the piglets by lymphocyte separation medium. After FACS, B lymphocytes with FITC labelled-E2 and FITC goat anti-swine IgG were sorted, extracted and cultivated on 96-well plates for 72 h. Then, the culture supernatants of B lymphocytes were screened by indirect ELISA with the purified CSFV-E2 antigen,and the target scFv antibody was bound to CSFV and obtained. Then, the scFv gene was inserted into the eukaryotic expression plasmid pCAGGS, and the constituted plasmid pCAGGS-scFv was transfected into ST cells. Western blot analysis showed that an approximately 31 kDa fusion protein was detected in the cell supernatant. Addition, the neutralizing capacity was measured in vitro. Indirect immunofluorescence assay (IFA) results showed that scFv-16 was able to neutralize CSFV. Conclusion Our data demonstrated that scFv-16 would be an effective diagnostic tool and potential therapeutic reagent for the CSFV infection in swine.

scholarly journals Generation and Characterization of a Spike Glycoprotein Domain A-Specific Neutralizing Single-Chain Variable Fragment against Porcine Epidemic Diarrhea Virus

Vaccines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 833
Author(s):  
Chia-Yu Chang ◽  
Yong-Sheng Wang ◽  
Jou-Fei Wu ◽  
Tzu-Jing Yang ◽  
Yen-Chen Chang ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Expression System ◽  
Single Chain ◽  
Variable Regions ◽  
Diarrhea Virus ◽  
Single Chain Fragment Variable ◽  
Neutralizing Monoclonal Antibody ◽  
Porcine Epidemic Diarrhea ◽  
Lactogenic Immunity

The emergence of the genotype (G) 2 and re-emergence of the G1 porcine epidemic diarrhea virus (PEDV) has caused severe economic impacts in the past decade. Developments of efficient vaccines against new variants of PEDV have been challenging, not least because of the difficulties in eliciting mucosal and lactogenic immunity. A single-chain fragment variable (scFv) capable of efficient antigen recognition is an alternative to vaccination and treatment of a viral infection. In the present study, the variable regions of the light chain and the heavy chain of a G2b PEDV spike domain A (S1A)-specific neutralizing monoclonal antibody (mAb) were sequenced, constructed with a (G4S) x3 linker, and produced by a mammalian protein expression system. Our results demonstrated that the PEDV S1A domain scFv was able to bind to S proteins of both G1 and G2b PEDVs. Nevertheless, the scFv was only capable of neutralizing the homologous G2b PEDV but not the G1 PEDV. The binding ability of the G2b-specific neutralizing scFv was not able to predict the neutralizing ability toward heterologous PEDV. The anti-PEDV S1A scFv presented herein serves as a potential therapeutic candidate against the virulent G2b PEDV.

scholarly journals Engineering a Live Attenuated Porcine Epidemic Diarrhea Virus Vaccine Candidate via Inactivation of the Viral 2'-O-Methyltransferase and the Endocytosis Signal of the Spike Protein

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Yixuan Hou ◽  
Hanzhong Ke ◽  
Jineui Kim ◽  
Dongwan Yoo ◽  
Yunfang Su ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Spike Protein ◽  
Economic Losses ◽  
High Dose ◽  
Type I ◽  
Viral Pathogen ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets; however, effective and safe vaccines are still not available. We hypothesized that inactivation of the 2′-O-methyltransferase (2′-O-MTase) activity of nsp16 and the endocytosis signal of the spike protein attenuates PEDV yet retains its immunogenicity in pigs. We generated a recombinant PEDV, KDKE4A, with quadruple alanine substitutions in the catalytic tetrad of the 2′-O-MTase using a virulent infectious cDNA clone, icPC22A, as the backbone. Next, we constructed another mutant, KDKE4A-SYA, by abolishing the endocytosis signal of the spike protein of KDKE4A. Compared with icPC22A, the KDKE4A and KDKE4A-SYA mutants replicated less efficiently in vitro but induced stronger type I and type III interferon responses. The pathogenesis and immunogenicities of the mutants were evaluated in gnotobiotic piglets. The virulence of KDKE4A-SYA and KDKE4A was significantly reduced compared with that of icPC22A. Mortality rates were 100%, 17%, and 0% in the icPC22A-, KDKE4A-, and KDKE4A-SYA-inoculated groups, respectively. At 21 days postinoculation (dpi), all surviving pigs were challenged orally with a high dose of icPC22A. The KDKE4A-SYA- and KDKE4A-inoculated pigs were protected from the challenge, because no KDKE4A-SYA- and one KDKE4A-inoculated pig developed diarrhea whereas all the pigs in the mock-inoculated group had severe diarrhea, and 33% of them died. Furthermore, we serially passaged the KDKE4A-SYA mutant in pigs three times and did not find any reversion of the introduced mutations. The data suggest that KDKE4A-SYA may be a PEDV vaccine candidate. IMPORTANCE PEDV is the most economically important porcine enteric viral pathogen and has caused immense economic losses in the pork industries in many countries. Effective and safe vaccines are desperately required but still not available. 2′-O-MTase (nsp16) is highly conserved among coronaviruses (CoVs), and the inactivation of nsp16 in live attenuated vaccines has been attempted for several betacoronaviruses. We show that inactivation of both 2′-O-MTase and the endocytosis signal of the spike protein is an approach to designing a promising live attenuated vaccine for PEDV. The in vivo passaging data also validated the stability of the KDKE4A-SYA mutant. KDKE4A-SYA warrants further evaluation in sows and their piglets and may be used as a platform for further optimization. Our findings further confirmed that nsp16 can be a universal target for CoV vaccine development and will aid in the development of vaccines against other emerging CoVs.

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