Vaccination against foot-and-mouth disease virus using peptides conjugated to nano-beads
Introduction
Foot-and-mouth disease (FMD) was the first animal disease to be identified in the 1500s. The causative agent, FMD virus (FMDV, genus Aphthovirus, family Picornaviridae) is a single-stranded positive sense RNA virus of 8500 bp and is enclosed by an icosahedral capsid [1]. The capsid contains four structural proteins, namely VP4, VP2, VP3 and VP1 [2], of which the surface of the viral particle is produced by complex folding of VP1-3 forming seven antigenically distinct serotypes (O, A, C, Asia 1 and SAT 1, 2, 3). The virus affects artiodactyls, importantly cattle and swine and is one of the most contagious of all animal diseases. Recent outbreaks of FMD in Taiwan, South Korea and United Kingdom resulted in the slaughter of millions of animals and huge economic losses, due to embargoes on export of animals and meat and milk products [3]. In countries in which FMD is endemic, eradication has been attempted using chemically (by treatment with 10 mM ethylenimine, 37 °C 72 h) inactivated virus vaccines. These vaccines are generally not utilized in FMDV-free countries as their use makes it difficult to differentiate vaccinated from infected animals. As a result FMDV-free status is lost until all vaccinated animals are slaughtered and in many countries where livestock are a major food source this becomes impractical.
While inactivated FMDV vaccines have been used effectively to control outbreaks, there are a number of limitations including: limited cross protection between virus types; slow onset of immunity; persistent infection in vaccinated ruminants (carrier state) and an inability to serologically differentiate infected from vaccinated animals [4]. In an attempt to avoid the use of live virus, a number of other vaccines have been reported such as DNA vaccines, cytokine-enhanced DNA vaccines, recombinant subunit vaccines, chimeric viral vaccines, genetically engineered attenuated vaccines, recombinant viral vector vaccines, self-replicating genetic vaccines, transgenic plants with expressed FMDV proteins and peptide-based vaccines [5]. Peptide-based vaccines have been explored and have a number of key advantages such as: lack of infectivity, high stability, defined composition, standardized production method and design to stimulate appropriate immune responses [6]. In addition, since peptide vaccines induce immune responses against only a small part of the virus, it might be possible to use the presence of immunity to other epitopes to differentiate infected from vaccinated animals. Peptide-based vaccines have been used successfully to identify a number of immunogenic sequences in the capsid protein VP1. The amino acid sequence 141–160 of VP1, has been found to elicit protective neutralizing antibody (Ab) in several species [7]. One way of achieving maximal immunogenicity with minimal reactogenicity is to use Ag conjugated to inert nano-beads [8], [9]. These studies demonstrated in mice that Ag covalently linked to inert nano-beads with a size range of 40–50 nm are preferentially taken up by DCs and induce high antibody titres, as well as CM immune responses including cytotoxic T cell responses. Subsequent studies in sheep have demonstrated that this approach was appropriate for livestock [10]. Improved immunogenicity of a FMDV peptide has been observed when attached to part of a core protein of hepatitis B virus, forming 27 nm complexes identified by sucrose-gradient centrifugation [11]. Here, we explore the cell-mediated and humoral immune responses induced in sheep by this novel nano-bead-based adjuvant conjugated to FMDV-specific peptides. We also investigate whether conjugation of several peptides to the same nano-bead improves immune responses compared to mixing nano-beads each conjugated with individual peptides.
Section snippets
FMDV peptide conjugation to nano-beads
The peptide-coated nano-beads were prepared as described previously [9]. In short, carboxylated polystyrene nano-beads of 49 nm (1%, w/v, solids final) (Polysciences, Warrington, PA, USA) in 0.05 M 2-N-morpholino-ethanesulfonic acid (ICN, OH, USA) buffer pH 6.2 were preactivated with 0.05 M N-hydroxysulfosuccinamide (Pierce, Rockford, USA) and 4 mg/ml 1-ethyl-(3-dimethylaminopropyl) carbodiimide (Sigma, St. Louis, USA) for 2 h at room temperature. The peptide was then added to a final concentration
Statistical analysis
Statistical analysis was performed using a Student's t-test, using paired two samples of mean (Microsoft Excel).
Results
To generate a vaccine effective across a range of genotypes it is anticipated that any peptide-based vaccine will need to include a combination of peptides. Additionally, to induce optimal B and T cell responses, a mixture of peptides representing B and T cell epitopes is expected to be beneficial. We therefore compared immunizing with the individual peptides (Groups A–C) to immunizing with mixed individually conjugated FMDV peptides to nano-beads (Group D) or multiple FMDV peptides conjugated
Discussion
Protective immunity to FMDV has been achieved using synthetic peptide vaccines which have been found to provide protection in pigs and limited protection in cattle after challenge with virus (see Table 2). While most of the studies have focused on humoral responses it has been suggested that the CM immunity is also important [17]. Considering that previously nano-beads have been shown to induce good CM responses in both mice [8], [9] and sheep [10], we have investigated whether FMDV synthetic
Acknowledgments
D.L.V.G. and J.P.S. received support from the Australian Research Council. M.P. receives support from the NH&MRC. This work was funded by Prima Biomed Ltd. (Australia) as part of an Australian Research Council Linkage project.
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Equal contribution.