Application of next generation phage display technology to develop virus specific peptide-based serology diagnostics for mosquito-borne and tick-borne flaviviruses
Publication Date
July 19, 2023
Creator
Varghese, Anitha
Abstract
Flaviviruses have been intensively studied since the early 20th century as a direct result of the major impacts they have on human and animal health globally. Most flaviviruses are vectored by hematophagous arthropods and the disease distribution and seasonality matches that of the specific vector and its lifecycle. Vector competence in transmitting multiple arboviruses have resulted in some flaviviruses co-circulating. Flaviviruses are structurally similar, and antibodies generated during flavivirus infections to the envelope protein are highly cross-reactive especially if the viruses belong to the same sero-group. These antibodies can also lead to negative outcomes for the host when the immune system next encounters a secondary flavivirus infection for example, in the case of Dengue Haemorrhagic Fever. Cross-reactive antibodies have also been a major challenge to developing accurate serological methods of detecting specific flaviviruses.
In this thesis, phage biopanning was used to identify virus specific peptide mimotopes using flavivirus IgG positive serum samples. The specific phage library used was a phagemid library expressing a random 16-mer peptide on the major coat protein of M13 phage. Over several rounds of biopanning, a sub-library of phage that are mimics of the target serum antibody are enriched. The strategy used included subtractive biopanning where the phage library was incubated with the control sera and the bound phage was depleted from the library and positive biopanning where binders to the target were rescued and amplified as a sub-library.
Following the phage biopanning, the peptide mimotopes from the sub-libraries are identified through sequencing. The traditionally used method of clone picking, and the modern route of next generation sequencing were used to analyse the peptide mimotopes enriched towards the target.
A novel method of data analysis, originally used to analyse peptide microarray data, was applied to the Next Generation Phage Display (NGPD) datasets from flavivirus biopanning experiments to identify target specific peptide mimotopes. Machine learning concepts of data training and testing were applied to generate a list of peptides (immunosignature) enriched only in the target sub-libraries in comparison to the control. The immunosignature was then tested against target and control samples. Area Under the Curve (AUC) values from Receiver Operating Characteristic (ROC) curve was used as the statistical method used to evaluate the sensitivity and specificity of the immunosignature as an in-silico assay.
The aim of the first study where this analysis was applied was to identify a peptide mimotope panel unique to Zika virus (ZIKV) IgG from human sera (also positive for DENV IgG). A panel of human sera from mosquito-borne flavivirus endemic region was used in the biopanning experiment. The control sera were seropositive for dengue (DENV) IgG only. NGPD was applied to develop a ZIKV immunosignature with an AUC value of 0.922 and the in-silico assay was 82.1% sensitive and 69.0% specific when tested against additional sub-libraries generated by re-screening the phage sub-library with sera not used in biopanning.
Immunosignature analysis was also applied to identify virus specific peptide mimotopes for LIV and tick-borne encephalitis viruses (TBEV) from ovine sera in a method similar to that followed in the first study. The AUC for the TBEV immunosignature was 0.930 and for 0.78 for the LIV immunosignature. Due to a smaller number of samples available, the sensitivity and specificity values against independent samples not used in the generation of the library.
The second output of this thesis was the development of a proof of principle phage peptide ELISA to detect LIV IgG from ovine sera. Currently there are no ELISA based serology assays for detecting LIV IgG. A phage sub-library enriched with binders to LIV IgG was raised by biopanning the peptide phage library with serum samples positive for LIV IgG. Following traditional phage display techniques, phage clones were picked, grown, Sanger sequenced and tested on ELISA for their ability to distinguish between LIV IgG positive and LIV IgG negative ovine sera. A phage ELISA method was developed, and the clones were screened against a panel of sera not used in the biopanning process. Statistical analysis of the results showed that one peptide had an AUC value over 0.9 and four others had AUC values >0.80. To improve the assay further, it has been proposed that the best performing clones be combined to increase sensitivity and specificity. The approaches developed in this project can be applied to other flaviviruses and potentially lead to the development of a flavivirus pan-species multiplex assay.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Veterinary Medicine and Science
eprints ID
73716
UoN Repository URI
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