The Pirbright Institute publication directory contains details of selected publications written by our researchers.

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Fowler V, Robinson L, Bankowski B, Cox S, Parida S, Lawlor C, Gibson D, O'Brien F, Ellefsen B, Hannaman D, Takamatsu H H, Barnett P V (2012)

A DNA vaccination regime including protein boost and electroporation protects cattle against foot-and-mouth disease

Antiviral Research 94 (1), 25-34


Protection against foot-and-mouth disease (FMD) using DNA technology has been documented for sheep and pigs but not for the highly susceptible species of cattle. Twenty-five Holstein Friesian cross-bred cattle were vaccinated twice, 21 days apart, with a DNA vaccine containing the capsid coding region (P1) along with the non-structural proteins 2A, 3C and 3D (pcDNA3.1/P1-2A3C3D) of O-1 Kaufbeuren alone or coated onto PLG (D,L-lactide-co-glycolide) microparticles. In some pcDNA3.1/P1-2A3C3D was also combined with an adjuvant plasmid expressing bovine granulocyte macrophage colony stimulating factor (GM-CSF). DNA vaccinations were administered intramuscularly with, or without, the use of electroporation and at 42 days post primary vaccination cattle received a protein boost of 146S FMD virus (FMDV) antigen and non-structural protein 3D. For comparison, four cattle were vaccinated with a conventional FMD vaccine and two more included as unvaccinated controls. Apart from those immunised with PLC microparticles all cattle were challenged with 10(5) TCID50 cattle adapted O-1 Lausanne FMDV virus at day 93 post primary vaccination. All DNA vaccinated cattle regardless of regime developed good humoral and cell mediated responses prior to challenge. The best overall virus neutralising antibody, IFN-gamma and clinical protection (75%) were seen in the cattle whereby the DNA was delivered by electroporation. In contrast, only 25% of cattle vaccinated with the DNA vaccine without electroporation were clinically protected. The addition of GM-CSF in combination with electroporation further improved the efficacy of the vaccine, as demonstrated from the reduction of clinical disease and virus excretions in nasal swabs. We thus demonstrate for the first time that cattle can be clinically protected against FMDV challenge following a DNA prime-protein boost strategy, and particularly when DNA vaccine is combined with GM-CSF and delivered by electroporation.


DNA vaccines are, in principle, the simplest yet most versatile methods of inducing protective humoral and cellular immune responses. Research involving this type of vaccine against veterinary diseases began in the early 1990s and has since seen the evaluation of more than 30 important viral pathogens, including the economically important foot-and-mouth disease. With the demonstration that DNA vaccines protect against foot-and-mouth disease in sheep and pigs, and the advantages these DNA vaccines have over the conventional formulations, this approach may provide a better solution to the control of this disease. In this review, we provide a comprehensive overview of DNA vaccination strategies for foot-and-mouth disease reported in the literature, in which we highlight the studies that have reported protection in the key target species.
Fraile L, Crisci E, Cordoba L, Navarro M A, Osada J, Montoya M (2012)

Immunomodulatory properties of Beta-sitosterol in pig immune responses

International Immunopharmacology 13 (3), 316-321


The ability to control an immune response for the benefit and production efficiency of animals is the objective of immunomodulation in food-producing animals; substances that exert this control are called immunomodulators. A Spanish product (Inmunicin MAYMO(R)), based on food plant phytosterols, is being commercialized as complementary feed. The main component of this product is Beta-sitosterol (BSS). BSS and its glycoside (BSSG) have been shown to exhibit anti-inflammatory, anti-neoplasic, anti-pyretic and immune-modulating activity demonstrated by in vitro and in vivo experiments. The objective of the present study was to characterize the effect of BSS on the pig immune system using in vitro cell cultures first and to elucidate whether BSS possesses any in vivo activity in fattener pigs after vaccination with porcine reproductive and respiratory syndrome virus (PRRSV) modified life vaccine (MLV). Firstly, our in vitro results showed that BSS increased viable peripheral blood mononuclear cell (PBMC) numbers and it activated swine dendritic cells (DCs) in culture. Secondly, pigs treated with phytosterols prior to vaccination with PRRSV-MLV vaccine exhibited some changes in immunological parameters at different times post-vaccination, such as the proliferation ability of PBMC after phytohemaglutinin stimulation and increased apolipoprotein A1 plasma concentration which may contribute to enhance PRRSV vaccine response. In conclusion, the data in this report show that BSS can be considered an immunomodulator in pigs.
Gale P, Stephenson B, Brouwer A, Martinez M, de la Torre A, Bosch J, Foley-Fisher M, Bonilauri P, Lindström A, Ulrich R G, de Vos C J, Scremin M, Liu Z, Kelly L, Muñoz M J (2012)

Impact of climate change on risk of incursion of Crimean-Congo haemorrhagic fever virus in livestock in Europe through migratory birds

Journal of Applied Microbiology 112 (2), 246-257


Aims: To predict the risk of incursion of Crimean-Congo haemorrhagic fever virus (CCHFV) in livestock in Europe introduced through immature Hyalomma marginatum ticks on migratory birds under current conditions and in the decade 2075–2084 under a climate-change scenario. Methods and Results: A spatial risk map of Europe comprising 14 282 grid cells (25 × 25 km) was constructed using three data sources: (i) ranges and abundances of four species of bird which migrate from sub-Saharan Africa to Europe each spring, namely Willow warbler (Phylloscopus trochilus), Northern wheatear (Oenanthe oenanthe), Tree pipit (Anthus trivialis) and Common quail (Coturnix coturnix); (ii) UK Met Office HadRM3 spring temperatures for prediction of moulting success of immature H. marginatum ticks and (iii) livestock densities. On average, the number of grid cells in Europe predicted to have at least one CCHFV incursion in livestock in spring was 1·04 per year for the decade 2005–2014 and 1·03 per year for the decade 2075–2084. In general with the assumed climate-change scenario, the risk increased in northern Europe but decreased in central and southern Europe, although there is considerable local variation in the trends. Conclusions: The absolute risk of incursion of CCHFV in livestock through ticks introduced by four abundant species of migratory bird (totalling 120 million individual birds) is very low. Climate change has opposing effects, increasing the success of the moult of the nymphal ticks into adults but decreasing the projected abundance of birds by 34% in this model. Significance and Impact of the Study: For Europe, climate change is not predicted to increase the overall risk of incursion of CCHFV in livestock through infected ticks introduced by these four migratory bird species.


Six breeds of swine were used to study the structure of swine leukocyte antigen class I (SLA-I). SLA-I complexes were produced by linking SLA-2 genes and beta(2)m genes via a linker encoding a 15 amino acid glycine-rich sequence, (G4S)3, using splicing overlap extension (SOE)-PCR in vitro. The six recombinant SLA-2-linker-beta(2)m genes were each inserted into p2X vectors and their expression induced in Escherichia coli TB1. The expressed proteins were detected by SDS-PAGE and western blotting. The maltose binding protein (MBP)-SLA-I fusion proteins were purified by amylose affinity chromatography followed by cleavage with factor Xa and separation of the SLA-I protein monomers from the MOP using a DEAE Ceramic Hyper D F column. The purified SLA-I monomers were detected by circular dichroism (CD) spectroscopy and the 3-dimensional (3D) structure of the constructed single-chain SLA-I molecules were analyzed by homology modeling. Recombinant SLA-2-Linker-beta(2)m was successfully amplified from all six breeds of swine by SOE-PCR and expressed as fusion proteins of 84.1 kDa in pMAL-p2X, followed by confirmation by western blotting. After purification and cleavage of the MBP-SLA-I fusion proteins. SLA-I monomeric proteins of 41.6 kDa were separated. CD spectroscopy demonstrated that the SLA-I monomers had an a-helical structure, and the average alpha-helix, beta-sheet, turn and random coil contents were 21.6%, 37.9%, 15.0% and 25.5%, respectively. Homology modeling of recombinant single-chain SLA-I molecules showed that the heavy chain and light chain constituted SLA-I complex with an open antigenic peptide-binding groove. It was concluded that the expressed SLA-I proteins in pMAL-p2X folded correctly and could be used to bind and screen nonameric peptides in vitro


The human IL-1 family contains 11 genes encoded at three separate loci. Nine, including IL-1R antagonist (IL-1RN), are present at a single locus on chromosome 2, whereas IL-18 and IL-33 lie on chromosomes 11 and 9, respectively. There are currently only two known orthologs in the chicken, IL-1 beta and IL-18, which are encoded on chromosomes 22 and 24, respectively. Two novel chicken IL-1 family sequences were identified from expressed sequence tag libraries, representing secretory and intracellular (icIL-1RN) structural variants of the IL-1RN gene, as seen in mammals. Two further putative splice variants (SVs) of both chicken IL-1RN (chIL-1RN) structural variants were also isolated. Alternative splicing of human icIL-1RN gives three different transcripts; there are no known SVs for human secretory IL-1RN. The chicken icIL-1RN SVs differ from those found in human icIL-1RN in terms of the rearrangements involved. In mammals, IL-1RN inhibits IL-1 activity by physically occupying the IL-1 type I receptor. Both full-length structural variants of chIL-1RN exhibited biological activity similar to their mammalian orthologs in a macrophage cell line bioassay. The four SVs, however, were not biologically active. The chicken IL-1 family is more fragmented in the genome than those of mammals, particularly in that the large multigene locus seen in mammals is absent. This suggests differential evolution of the family since the divergence of birds and mammals from a common ancestor, and makes determination of the full repertoire of chicken IL-1 family members more challenging. The Journal of Immunology, 2012, 189: 539-550.


The human IL-1 family contains eleven genes encoded at three separate loci. Nine, including IL-36 receptor antagonist (IL-36RN), also known as IL-1F5, are present at a single locus on chromosome 2, whereas IL-18 and IL-33 lie on chromosomes 11 and 9 respectively. There are currently only three known orthologues in the chicken - IL-1 beta, IL-18 and IL-1RN - which are encoded on chromosomes 22, 24 and unplaced, respectively. A novel chicken IL-1 family sequence representing IL-36RN (IL-1F5) was initially identified from an expressed sequence tag (EST) library by its similarity to both chicken IL-1 RN and chicken IL-1 beta. Following isolation of the cDNA from the liver of an uninfected bird, a number of unique sequence features were identified. The predicted protein has a longer NH2-terminus than the human protein; however, as in mammals, this region contains neither a prodomain nor a signal peptide. A putative nuclear export sequence is also apparent, yet a similar motif is absent in mammalian IL-36RN. Although chIL-36RN exhibits low homology with its mammalian orthologues, it encodes a predicted beta-trefoil structure whose beta-strands are conserved with those of the mouse sequence. Unlike in mammals, chIL-36RN expression was constitutive in all tissues and cell subsets examined. In response to viral infection, expression was significantly downregulated in a line of birds which are susceptible to the virus. Chicken IL-36RN, like chIL-1RN, is not encoded at the chIL-1 beta locus, further emphasising the genomic fragmentation of the large IL-1 gene cluster found in mammals. This suggests differential evolution of this cytokine family since the divergence of birds and mammals from a common ancestor, and underlines the difficulty of determining the full repertoire of chIL-1 family members.
Goodwin R, Schley D, Lai K, Ceddia G M, Barnett J, Cook N (2012)

Interdisciplinary approaches to zoonotic disease

Infectious Disease Reports 4 (2), 146-151


Zoonotic infections are on the increase worldwide, but most research into the biological, environmental and life science aspects of these infections has been conducted in separation. In this review we bring together contemporary research in these areas to suggest a new, symbiotic framework which recognises the interaction of biological, economic, psychological, and natural and built environmental drivers in zoonotic infection and transmission. In doing so, we propose that some contemporary debates in zoonotic research could be resolved using an expanded framework which explicitly takes into account the combination of motivated and habitual human behaviour, environmental and biological constraints, and their interactions.


Pre-emptive culling is becoming increasingly questioned as a means of controlling animal diseases, including classical swine fever (CSF). This has prompted discussions on the use of emergency vaccination to control future CSF outbreaks in domestic pigs. Despite a long history of safe use in endemic areas, there is a paucity of data on aspects important to emergency strategies, such as how rapidly CSFV vaccines would protect against transmission, and if this protection is equivalent for all viral genotypes, including highly divergent genotype 3 strains. To evaluate these questions, pigs were vaccinated with the Riemser (R) C-strain vaccine at 1, 3 and 5 days prior to challenge with genotype 2.1 and 3.3 challenge strains. The vaccine provided equivalent protection against clinical disease caused by for the two challenge strains and, as expected, protection was complete at 5 days post-vaccination. Substantial protection was achieved after 3 days, which was sufficient to prevent transmission of the 3.3 strain to animals in direct contact. Even by one day post-vaccination approximately half the animals were partially protected, and were able to control the infection, indicating that a reduction of the infectious potential is achieved very rapidly after vaccination. There was a close temporal correlation between T cell IFN-gamma responses and protection. Interestingly, compared to responses of animals challenged 5 days after vaccination, challenge of animals 3 or 1 days post-vaccination resulted in impaired vaccine-induced T cell responses. This, together with the failure to detect a T cell IFN-gamma response in unprotected and unvaccinated animals, indicates that virulent CSFV can inhibit the potent antiviral host defences primed by C-strain in the early period post vaccination.


Live attenuated C-strain classical swine fever viruses (CSFV) provide a rapid onset of protection, but the lack of a serological test that can differentiate vaccinated from infected animals limits their application in CSF outbreaks. Since immunity may precede antibody responses, we examined the kinetics and specificity of peripheral blood T cell responses from pigs vaccinated with a C-strain vaccine and challenged after five days with a genotypically divergent CSFV isolate. Vaccinated animals displayed virus-specific IFN-gamma responses from day 3 post-challenge, whereas, unvaccinated challenge control animals failed to mount a detectable response. Both CD4(+) and cytotoxic CD8(+) T cells were identified as the cellular source of IFN-gamma. IFN-gamma responses showed extensive cross-reactivity when T cells were stimulated with CSFV isolates spanning the major genotypes. To determine the specificity of these responses, T cells were stimulated with recombinant CSFV proteins and a proteome-wide peptide library from a related virus, BVDV. Major cross-reactive peptides were mapped on the E2 and NS3 proteins. Finally, IFN-gamma was shown to exert potent antiviral effects on CSFV in vitro. These data support the involvement of broadly cross-reactive T cell IFN-gamma responses in the rapid protection conferred by the C-strain vaccine and this information should aid the development of the next generation of CSFV vaccines.


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