The thermostability of vaccines is of crucial importance in Africa, where the
logistical process to get the vaccine from the manufacturer to the animal may take months,
and in many remote regions transport and storage is in the absence of a cold-chain.
Vaccines with improved stability and less reliance on a cold-chain are needed and could
improve the longevity of immune responses elicited in vaccinated animals. In South Africa,
cattle in the vaccination zone neighbouring the Kruger National Park have to be
vaccinated thrice annually because of declining antibody responses at three months postvaccination.
FMDV is known to be unstable, especially for O and SAT2 serotypes in mildly
acidic pH conditions or at elevated temperatures, leading to dissociation of the capsid
(146S particle) and loss of immunogenicity. The link between rapidly declining antibody
responses and capsid stability have been reported by Doel and Baccarini, 1981. We
hypothesized that more stable viruses, especially thermostability, will not only improve the
protective immune response in animals but also require less frequent booster
vaccinations.
The residues at the capsid inter-pentamer interfaces, and their interactions, are
important for the infectivity and stability of the virion and mutations adjacent to these
interfaces have an effect on the conformational stability of FMDV. However, experimental
studies on the relative importance of residues and molecular interactions in viral capsid
assembly, disassembly, and/or stability are still very limited, especially for the SAT
serotypes of FMDV. This study investigated the effects of potential residues at the
pentameric interfaces that are responsible for increased thermostability and potentially
improved stability candidates were tested in small (guinea pigs) and large (cattle) animal
vaccination trials to understand the role of stabilised antigens on immune responses. The
biological variation in biophysical stability in SAT2 viruses in the southern Africa region
was investigated to determine if any naturally occurring viruses have greater capsid
thermostability. Naturally occurring stable viruses could be used as prospective
candidates in vaccine production and therefore potentially result in increased duration of
immune responses.
Our first aim was to investigate the role of different amino acid changes at the
interface and their effect on capsid stability using models derived by Oxford University.
These changes were introduced by mutating the SAT2 ZIM7/83 infectious genome-length
clone (pSAT2) to derive mutated chimeric SAT2 viruses. We quantified the stabilizing effects of these mutations by using various stability assays. We established the novel
thermofluor shift assay that is able to quantify the capsid stability of viruses. The growth
kinetics, antigenicity, genetic stability, pH and salt sensitivity were investigated for each of
the genetically engineered viruses (Chapters 2 and 3).
The second aim was to further our understanding on the correlation between
improved stability and immune responses by performing small animal (Chapter 2) and
large animals trials in cattle (Chapter 4) and comparing stabilised and wild-type antigens.
This study for the first time for SAT vaccines, determined differences in IgG1 and IgG2
profiles, interferon gamma (IFN-γ) responses and differences in total and neutralising
antibodies of stabilised and wild-type antigens over a six month period in cattle (Chapter
4). Animals were intra-dermolingually challenged with live virus to determine levels of
protection the antigens have afforded.
In addition, a third aim will be to better understand the inherent thermostability
variation of SAT2 viruses in the Southern African region (Chapter 5) by establishing a
protocol for screening field isolates as potential vaccine strains and correlating their
stability to amino acid residues at the interface of the 146S particles.
