Structure-based energetics of protein interfaces guides FMDV  SAT2 vaccine design

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Structure of FMD capsid

FMDV belongs to the family Picornaviridae, and is the type species of the genus Aphtovirus. The virion comprises a single-stranded positive-sense RNA genome, of approximately 8 kb in length, capped at the 5’-end by a virus encoded VPg protein and contained in a non-enveloped, icosahedral capsid of ca. 22 – 25 nm in diameter (Acharya et al., 1989). The capsid consists of a symmetrical arrangement of 60 copies of each of four structural viral proteins (VP), i.e. VP1, VP2, VP3 and VP4 (Fig. 1.1). Each structural protein interacts to form protomer subunits with a sedimentation coefficient of 5S; five of these protomers come together to form a pentamer of 12S, and 12 pentamers assemble into the complete capsid. This can be a non-infectious empty capsid with a sedimentation coefficient of 70S, or a complete virion of 146S with viral RNA content, which can be infectious (Fry et al., 2005). Cell entry in infected hosts is followed immediately by translation of the viral RNA, which yields a single polyprotein precursor that has to be processed into fourteen distinct capsid and non-structural proteins for virus replication. The majority of this processing is done by the virus-encoded 3C protease (3Cpro), which cleaves the precursor at ten distinct sites (Curry et al., 2007). The four capsid proteins originate from the post-translationally cleaved P1 region of the polyprotein and are designated 1A/VP4, 1B/VP2, 1C/VP3 and 1D/VP1.

Alternative vaccine strategies: subunit and live viral-vectors

The development of potent new generation vaccines for FMD has been the subject of intense research during the last few decades. In line with other medical and veterinary viral vaccines, research and development on FMD vaccines has focused mostly on subunit and vectored vaccines. Peptides or purified proteins, recombinant DNA, viral vectors and plants expressing FMDV structural proteins, with or without immunopotentiators, have been demonstrated to elicit humoral and cell-mediated immune responses in experimental animals and shown to protect natural hosts to varying degrees in enclosed settings (Balamurugan et al., 2005; Du et al., 2007; Wang et al., 2008; Su et al., 2008; Cubillos et al., 2008; Li et al., 2008; Choudary et al., 2008; Yang et al., 2008; Greenwood et al., 2008). However, while these approaches show promise for use in FMD-free zones, none of them have so far been subjected to largescale field trials in endemic countries. Due to the limitations of inactivated vaccines, alternative strategies for vaccine development have focused on the use of VP1-proteins and peptides either isolated from FMDV or produced by recombinant DNA (Bachrach et al., 1975; Kleid et al., 1981), VP1-derived peptides (Strolmaier et al., 1982) or chemically synthesized VP1 peptides (DiMarchi et al., 1986; Francis et al., 1991; Nargi et al., 1991; Bittle et al., 1992), vectors expressing VP1 fusion proteins (Kit et al., 1991; Kitson et al., 1991; Clarke et al., 1997), inoculation with DNA expressing VP1 epitopes alone (Wong et al., 2000) or with DNA encoding IL-2 (Wong et al., 2002), and transgenic plants or recombinant tobacco mosaic virus expressing VP1 (Mulcahy et al., 1991; Taboga et al.,1997). However, they rarely achieved protection against virus challenge in livestock (Dimarchi et al., 1986; Mulcahy et al., 1990 and 1991), or as a result of a limited subset of epitopes, selected for antigenic variants that escaped from protection (Taboga et al., 1997). The reduced level of protection may be due to a lack of T-cell epitopes (Rodriquez et al., 1994). Whereas, peptides and purified proteins have failed to command an important place in vaccinology due to various reasons, the use of recombinant DNA in large domestic and wild animals may be difficult due to the necessity of injecting large and multiple doses, the period needed to attain optimal responses (compared to the rapidity with which FMD spreads through herds), delivery issues, and the potential for vaccinated animals to be carriers (e.g., suboptimal responses may suppress superinfection with a field virus but may not be able to prevent infection or virus shedding).

CHAPTER 1: Literature Review
1.1 Introduction
1.2 FMD capsid structure
1.3 Stability of FMD
1.4 Control of FMD by vaccination
1.5 Stability of FMD vaccines
1.6 Design of improved inactivated vaccines and immunity
1.7 Alternative vaccine strategies: subunit and live viral vectors
1.8 References
CHAPTER 2: Structure-based energetics of protein interfaces guides FMDV  SAT2 vaccine design
2.1 Abstract
2.2 Introduction
2.3 Materials and methods
2.4 Results
2.5 Discussion
2.6 References
CHAPTER 3: SAT2 FMDV structurally modified for increased thermostability
3.1 Abstract
3.2 Importance
3.3 Introduction
3.4 Materials and methods
3.5 Results
3.6 Discussion
3.7 Funders
3.8 Acknowledgements
3.9 References
CHAPTER 4: Evaluation of immune responses of stabilised SAT2 antigens of FMDV in cattle
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.4 Results
4.5 Discussiodn
4.6 Acknowlegments
4.7 References
CHAPTER 5: Inherent biophysical stability of FMDV SAT2 viruses
5.1 Abstract
5.2 Introduction
5.3 Materials and methods
5.4 Result
5.5 Discussion
5.6 References
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