Isolation and Characterisation of a Human-Like Antibody Fragment (scFv) That Inactivates VEEV In Vitro and In Vivo

Abstract

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.

Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library

“…For BoNT/A1-L, VH1 and VH7 genes with Nco I restriction sites were particularly prevalent in the anti-BoNT/A immune response 25 . This had never before been reported for other macaque immune libraries 18 , 20 , 22 , 38 . It also contrasts with reports for human V genes, as Nco I sites have never been detected in VH genes (Hust, unpublished results).…”

Generation and characterization of protective antibodies to Marburg virus

“…22,28,29 In brief, the secondary PCRs were carried out for each forward oligonucleotide primers separately to keep the diversity. Each PCR was performed in a volume of 100 ml using 100 ng purified PCR reaction product of the pGemT cloned cDNA, 2.5 U Go Taq polymerase (Promega, Mannheim, Germany), 200 mM dNTPs each and 200 nM of each oligonucleotide primer for 20 cycles (30 s 94 C, 30 s 57 C, 30 s 72 C), followed by 10 min 72 C. The PCR products were separated by 1.5% (w/v) agarose gel, cut out and purified using Nucleospin Extract II Kit (Macherey-Nagel, D€ uren, Germany) according to the manufacturer's instructions.…”

Generation of Recombinant Antibodies Against Toxins and Viruses by Phage Display for Diagnostics and Therapy

“…Libraries originating from different species have been successfully employed to isolate virus specific antibodies in the past. Among others, libraries were constructed from macaque [105], mouse [68], chimpanzee [39], llama [33] and human origin [120].…”

“…Immune libraries are constructed from patients or immunized humans or animals. Also from these immune libraries, human antibodies were selected against viruses [105] or toxins [96]. Further details about antibody gene libraries are given in the chapter 3 "Selection of Recombinant Human Antibodies" by Tomszak et al or by [35].…”