Use of baculovirus surface display for characterization of AHSV4-VP2 antigenic structure

African horse sickness is an infectious, non-contagious, insect vector-borne disease of equids. It is highly lethal and mortality rates can exceed 90% in susceptible populations. The disease is caused by African horse sickness virus (AHSV), which is mostly endemic in sub-Saharan Africa. However, occasional outbreaks have occurred outside Africa, such as the devastating AHSV-9 outbreak in 1959 reaching India and Pakistan or the European outbreaks caused by serotype 4 (AHSV4) between 1987 and 1993. AHS is an OIE (World Organisation for Animal Health) listed disease for its impact on animal health economy and international trade of equids. The prevention of disease is mainly dependent on an efficient vaccination regime. However, the current live-attenuated vaccines are not considered safe for use in non­endemic regions. Sub-unit protein vaccines expressed from recombinant baculoviruses are an option for generating safe vaccines due to the absence of pre-existing immunity, non-toxicity in mammalian systems and their inability to replicate in mammalian cells. In addition, the fact that AHSV replicates in insect cells during its transmission to a mammalian host, makes the baculovirus insect cell expression system very advantageous for the production of correctly-folded AHSV proteins. Baculoviruses can also be used to display foreign antigens on the baculovirus envelope surface, which is very beneficial for studying protein-protein interactions. The AHSV major outer capsid protein, VP2, is a good target for subunit vaccine studies as it has been shown to be the main-serotype specific antigen, carrying virus neutralising (VN) epitopes. This thesis focused on investigating the antigenic structure of AHSV4-VP2. Full-length AHSV-VP2 and eleven subdomains thereof were expressed using standard baculovirus expression systems and a baculovirus surface display approach. The antigenic properties of these proteins were studied using a range of techniques including; immunoblotting and ELISA in order to gain further understanding of the properties of VN epitopes. Understanding the antigenic structure of this major immunogenic protein is paramount for generating more efficient, safe vaccines. This thesis demonstrated the benefits of using TnHi5 cells for the production of full-length VP2. In addition, polyhistidine-tag affinity-purified VP2 was shown to retain biological functions, generating VNAbs in mice, unlike the purified VP2 indicated in the literature. All eleven VP2 subdomains were successfully produced by baculoviruses, however, the surface display constructs greatly improved the yield of these proteins when compared to their synthesis as individual proteins. The antigenicity of VP2 subdomains was investigated by several in vitro binding assays using AHSV4-VP2-specific antibodies and a vaccination study in mice. Although previous studies found the amino terminal half of the protein to be more antigenic, this thesis provided evidence that the carboxy terminus might play a role in the formation of important antigenic epitopes, including those involved in virus neutralisation. It was also demonstrated that VN epitopes of VP2 are highly conformational and difficult to mimic using individual subdomains. These results raised the question whether AHSV neutralisation is a multifunctional process and whether trimerisation is a critical factor in the formation of VNAb epitopes and the induction of VNAb when VP2-based vaccines are used. Additional studies aimed at advancing further our knowledge of AHSV-VP2 were suggested.

Grantor dc:publisher

Oxford Brookes University

Author dc:creator

• Aksular, Mine

Contributors dc:contributor

• King, Linda
• Castillo-Olivares, Javier

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/54dc-yf60

OAI identifier oai:identifier

tle:75336310-1eb2-4d24-8433-b99ba29cab7e:d6bd9758-527a-46cd-bfe2-c433766e8fca:1