| dc.description.abstracteng | Horses are exposed to a large variety of pathogens and diseases. Equine herpesvirus Type 1 (EHV-1) is one of the most prevalent viral pathogens infecting horses. Approximately 60 % of the horse population worldwide are infected with this virus. EHV-1 infection symptoms range from mild respiratory disease to potentially fatal neonatal pneumonia, abortion during late gestation or the severe neurological disease equine myeloencephalopathy (EHM). Not only is it a highly contagious virus but the most critical form of transmission are the silent shedders once the virus has undergone latency. Viral infection can be reactivated at any time and cause severe diseases and contribute to transmission among horses. Current prevention methods consist of vaccination regimes, horse husbandry adjustments or symptomatic treatment. Vaccination programs aim to eliminate viremia and prevent subsequent diseases. However, these methods have not proven to alter duration of viremia (1, 2) nor protect against latent infection. Therapeutics against EHV-1 infection have only been trialled in vitro at this point and their efficacy has not been proven to be ground breaking. It is suggested that protection and treatment will require equine specific neutralizing antibodies. A modern approach to resolve this issue, is the generation of recombinant antibodies in antibody libraries. Recombinant antibodies are produced synthetically and manipulated according to a specific host or antigen. As a result, these antibodies can be used as preventative or therapeutic drugs. My dissertation aims to construct recombinant equine antibodies in the single-chain-Fragment-variable format (scFv) in an antibody library since to date, no such recombinant antibody therapeutics exist for horses. As proof-of-principle, this library was screened against EHV-1.
Equine scFv antibodies were constructed by using a primer set designed to amplify the whole equine genomic immunoglobulin diversity. Primers were tested and PCR conditions optimised before scFv construction. For primer establishment and scFv construction, PBMCs were isolated from a gelding that has been immunized against EHV-1. RNA was extracted and reversely transcribed into cDNA. Variable antibody regions of antibody heavy and light chains were amplified separately and connected during SOE PCR by a linker sequence, thereby generating scFv sequences. In preparation of library assembly, all PCR products were confirmed to contain desired sequences by cloning and sequencing. ScFv sequences were ligated into a phagemid vector and transformed into phage-competent E. coli cells. This process of ligation and transformation was repeated until a library diversity of 4.8x108 individual clones was reached.
The library was screened for an anti-EHV-1 scFv antibody by phage display technique. During the phage display technique scFv sequences of the library are incorporated into bacteriophages. These bacteriophages will express the scFv sequences on their genotype as well as on their phenotype. All bacteriophages are exposed to the antigen of interest (EHV-1) and those bacteriophages that are phenotypically expressing scFv sequences binding to EHV-1 are eluted. The exposure to antigen occurs during biopanning rounds. Throughout each panning rounds, conditions such as concentration of antigen or washing steps, are adjusted for more stringency, aiming to separate weak binding scFv sequences from strong binding scFv sequences. ELISA will determine strong binders, when OD signals are five times higher than OD signals of the negative control. Prior to the screening, the antigen of interest, EHV-1 was purified by sucrose gradient centrifugation. The generated antibody library was packaged into M13KO7 bacteriophages and exposed to EHV-1 during three rounds of biopanning. After the third round, a specific scFv antibody was isolated. The scFv-3H2 shows strong binding ability and sequencing revealed it to be a promising scFv antibody candidate, which will be characterised further to prove pharmacokinetic potential for future therapeutic application.
My thesis contributes to the development of novel, alternative solutions in the prevention and treatment of equine pathogens and diseases. The equine scFv antibody library presents a starting point for the broader establishment of recombinant, equine antibodies and phage display method in equine and veterinary drug development improving animal health and welfare. | de |