Abstract
Equine influenza virus (EIV) is the major cause of respiratory tract infection in horses worldwide. The most effective preventative and control measure against EIV infections is by vaccination. The currently commercialized EIV vaccines are propagated in chicken embryonated eggs. However, vaccine production in this manner has several well-recognised drawbacks (supply of chicken eggs, contamination by egg protein and yield in eggs, cost, disposal of waste, working directly with the highly pathogenic EIV etc.). These bottlenecks necessitate the development of an alternative EIV vaccine production strategy. Like other influenza viruses, EIV comprises of two major surface glycoproteins named hemagglutinin (HA) and neuraminidase (NA). The HA protein is the principal antigen against which neutralizing antibodies are produced, making it an obvious target for vaccine formulation against EIV infections. The NA protein, although lessimportant in contrast to the HA protein, is also used in some instances for vaccine formulation. The addition of the NA protein with the HA component is known to boost antibodyresponse. The aim of this present study was to use recombinant DNA (rDNA) technology for heterologous production of recombinant proteins(HA/NA) to be used in the development of HA, a combination of HA and NA EIV vaccine candidates formulated with and without an adjuvant. The Yarrowia lipolytica yeast system was used in this present study for heterologous expression of HA and NA EIV major surface glycoproteins. This system was chosen for its multifaceted advantages it provides (safe, costeffective, high yield and, scalable). The HA/NA genes were sub-cloned into a yeast expression vector (pKOV410) following their release from a donor vector (pUC57) by restriction enzyme digest. Recombinant pKOV410-HA/NA vectors were subsequently linearized with NotI restriction enzyme to separate the expression cassettes from the DNA moiety to facilitate the transformation of the expression cassettes into the yeast cell genome. The sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the expression of the recombinant HA/NA proteins. The produced vaccine candidates were tested for their immunogenicity in clinical studies using guinea pigs as animal models. The vaccine types were able to induce a positive antibodyresponse in the first and second week post-vaccination (PV) but were seronegative following booster dose. Therefore, intensive research is needed towards the improvement of boosting strategies, for example, the alternative route of administration and purification of proteins should be the center of focus in the improvement of a better alternative yeast-based vaccine platform.
M.Sc. (Biochemistry)