Abstract:
African horse sickness is an infectious, insect-borne but non-contagious disease of
equids responsible for more than 95% mortality in naïve horses. The African horse
sickness virus has ten segments of double stranded RNA encoding seven structural
and three non-structural proteins. There is currently no cure for the disease and one
of the effective ways to control or prevent new infection is by vaccination. Although
successful, the currently used vaccine is not registered for use outside the sub-
Saharan region because of fears of reversal to virulence. Inactivated and subunit
vaccines have been tested but are not commercially available. Research towards
recombinant vaccines that will offer protection without these limitations is thus
ongoing. Identification and characterisation of antigenic regions on proteins has
always been essential in the development of vaccines and immunodiagnostic
reagents. AHSV proteins have been shown to confer protection to model animals in one form or another. This study was undertaken to investigate the global humoral
immune reaction during immunisation in horses. This was done using sera from
immunised horses and a genome-targeted phage display library. A phage library
expressing a repertoire of AHSV-4 peptides large enough to represent the whole
genome was constructed. The library was affinity selected with purified naïve (day 0)
and immunised (day 28, day 52) horse IgGs and a pool of phages expressing binding
peptides were isolated. The DNA inserts of the pool of phages were subjected to high
throughput sequencing and sequences identified by matching them to the AHSV-4
genome. Analysis of the extensive data output selected by the naive and immunised
IgGs enabled a thorough examination of the panning process. Sequences were
normalised by subtracting the naïve from the immunised sequences. Comparing the
sequences selected with day 28 IgG enabled identification of 16 potentially antigenic
regions recognised by most horses. Some of these regions could easily have been
missed with the traditional clone picking approach. Some of the regions were also
selected by most horses with day 52 IgG, indicating persistent antibodies. Potentially
antigenic regions were identified without the functional re-testing which is common
part of traditional phage display. This was due to lack of access to the single clones in
the library. One way to confirm binding of selected regions is by peptide ELISA, which
was started in this study. This study improved on all the phage display work already
done on orbiviruses and offered more information on the immunogenicity of AHSV-4
in horses. Future work should focus on confirming antigenicity of the selected regions.
The potential antigenic regions could be fused with T-cell epitopes, identified in a
parallel study, to form a construct that might induce production of humoral and cellular
immune responses.
