Simon Graham

Dr Simon Graham

Reader in Veterinary Immunology
+44 (0)1483 682314
01 VSM 02

Academic and research departments

School of Veterinary Medicine.


Areas of specialism

Immunology; Virology; Vaccinology

University roles and responsibilities

  • Year 2 Lead, BVMSci Veterinary Medicine and Science Programme
  • Module Coordinator, MSc Veterinary Microbiology Programme


    Research interests


    Postgraduate research supervision

    Completed postgraduate research projects I have supervised

    Courses I teach on


    Postgraduate taught

    My publications


    Londt BZ, Brookes SM, Nash BJ, Núñez A, Kelly MD, Garçon F, Graham SP, Brown IH (2013) Enhanced infectivity of H5N1 highly pathogenic avian influenza (HPAI) virus in pig ex vivo respiratory tract organ cultures following adaptation by in vitro passage, Virus Research
    Pigs are thought to play a role in the adaptation of avian influenza (AI) viruses to mammalian hosts. To better understand this mechanism and to identify key mutations two highly pathogenic AI (HPAI) viruses (H5N1 and H7N7) were grown in pig cells, To mimic the pressure of an immune response, these viruses were grown in the presence of antiserum to the homologous virus or porcine IFN-³. Mutations were identified in both viruses grown in vitro in the presence and absence of antisera or IFN-³ and included the PB2 mutations, E627K or 627E,D701N, described previously as requirements for the adaptation of AI viruses to mammalian species. Additional mutations were also identified in PB1, HA, NP and M genes for viruses passaged in the presence of immune pressure. The infectivity of these viruses was then assessed using ex vivo pig bronchi and lung organ cultures. For lung explants, higher levels of virus were detected in organ cultures infected with H5N1 HPAI viruses passaged in pig cell lines regardless of the presence or absence of homologous antisera or IFN-³ when compared with the wild-type parental viruses. No infection was observed for any of the H7N7 HPAI viruses. These results suggest that the mutations identified in H5N1 HPAI viruses may provide a replication or infection advantage in pigs in vivo and that pigs may continue to play an important role in the ecology of influenza A viruses including those of avian origin. Crown Copyright © 2013.
    Akoolo L, Pellé R, Saya R, Awino E, Nyanjui J, Taracha EL, Kanyari P, Mwangi DM, Graham SP (2008) Evaluation of the recognition of Theileria parva vaccine candidate antigens by cytotoxic T lymphocytes from Zebu cattle., Vet Immunol Immunopathol121(3-4)pp. 216-221
    East Coast fever (ECF) is a highly fatal lymphoproliferative disease of cattle caused by Theileria parva, a tick-borne intracellular apicomplexan parasite. Parasite antigens that are targets of protective cytotoxic T lymphocyte (CTL) responses are required to formulate a sub-unit vaccine against ECF. A number of CTL target antigens have recently been identified and initial evaluation has shown their vaccine potential. This study aimed to evaluate whether these antigens were recognised by CTL obtained from six genetically diverse Zebu cattle immunized with a cocktail of T. parva stocks. T. parva Muguga specific polyclonal CD8(+) CTL lines were generated and confirmed to specifically lyse autologous infected cells. CTL recognition of autologous skin fibroblasts (iSF) transduced with recombinant modified vaccinia virus Ankara strain (MVA) expressing previously identified T. parva Muguga vaccine candidate antigens was evaluated using an IFN-gamma ELISpot assay. CTL lines from one of the four calves, BY120, responded specifically to cells infected with MVA expressing the antigen Tp2 and synthetic peptides were employed to map a new CTL epitope on this antigen. Immunoscreening of the T. parva genome with these CTL lines should identify novel antigens that will constitute valuable additions to the vaccine candidates currently being evaluated.
    Walters AA, Somavarapu S, Riitho V, Stewart GR, Charleston B, Steinbach F, Graham SP (2015) Assessment of the enhancement of PLGA nanoparticle uptake by dendritic cells through the addition of natural receptor ligands and monoclonal antibody, VACCINE33(48)pp. 6588-6595 ELSEVIER SCI LTD
    Targeting of specific receptors on antigen-presenting cells is an appealing prospect in the production of novel nanoparticulate vaccines. In particular, the targeting of vaccines to dendritic cell (DC) subsets has been shown in models to significantly improve the induction of immune responses. This paper describes the evaluation of natural ligands, mannan and chitosan, and monoclonal antibodies as targeting motifs to enhance uptake of PLGA nanoparticle carriers by bovine DCs. To assess enhancement of uptake after the addition of natural ligands a bovine monocyte derived DC (MoDC) model was used. For the assessment of monoclonal antibody targeting, the model was expanded to include afferent lymph DCs (ALDCs) in a competitive uptake assay. Mannan, proved unsuccessful at enhancing uptake or targeting by MoDCs. Chitosan coated particle uptake could be impeded by the addition of mannan suggesting uptake may be mediated through sugar receptors. Inclusion of monoclonal antibodies specific for the DEC-205 (CD205) receptor increased the number of receptor expressing DCs associated with particles as well as the number of particles taken up by individual cells. These results support the further evaluation of active targeting of nanovaccines to DCs to enhance their immunogenicity in cattle and other large mammalian species including humans.
    Franzoni G, Kurkure NV, Edgar DS, Everett HE, Gerner W, Bodman-Smith KB, Crooke HR, Grahama SP (2013) Assessment of the phenotype and functionality of porcine cd8 t cell responses following vaccination with live attenuated classical swine fever virus (CSFV) and virulent CSFV challenge, Clinical and Vaccine Immunology20(10)pp. 1604-1616
    Vaccination with live attenuated classical swine fever virus (CSFV) induces solid protection after only 5 days, which has been associated with virus-specific T cell gamma interferon (IFN-³) responses. In this study, we employed flow cytometry to characterize T cell responses following vaccination and subsequent challenge infections with virulent CSFV. The CD3 + CD4- CD8hi T cell population was the first and major source of CSFV-specific IFN-³. A proportion of these cells showed evidence for cytotoxicity, as evidenced by CD107a mobilization, and coexpressed tumor necrosis factor alpha (TNF-±). To assess the durability and recall of these responses, a second experiment was conducted where vaccinated animals were challenged with virulent CSFV after 5 days and again after a further 28 days. While virus-specific CD4 T cell (CD3+ CD4+ CD8±+) responses were detected, the dominant response was again from the CD8 T cell population, with the highest numbers of these cells being detected 14 and 7 days after the primary and secondary challenges, respectively. These CD8 T cells were further characterized as CD44hi CD62L- and expressed variable levels of CD25 and CD27, indicative of a mixed effector and effector memory phenotype. The majority of virus-specific IFN-³+ CD8 T cells isolated at the peaks of the response after each challenge displayed CD107a on their surface, and subpopulations that coexpressed TNF-± and interleukin 2 (IL-2) were identified. While it is hoped that these data will aid the rational design and/or evaluation of next-generation marker CSFV vaccines, the novel flow cytometric panels developed should also be of value in the study of porcine T cell responses to other pathogens/vaccines. Copyright © 2013, American Society for Microbiology. All Rights Reserved.
    Nichani AK, Campbell JD, Glass EJ, Graham SP, Craigmile SC, Brown CG, Spooner RL (2003) Characterization of efferent lymph cells and their function following immunization of cattle with an allogenic Theileria annulata infected cell line., Vet Immunol Immunopathol93(1-2)pp. 39-49
    Immunization of cattle with in vitro propagated bovine mononuclear cells infected with Theileria annulata induces a protective immune response. Activation and effector function of T cells exiting the lymph node draining the site of cell line immunization were investigated to understand the mechanisms involved in the generation of immunity. Immunized animals exhibited a biphasic immune response in efferent lymph as well as peripheral blood. The first phase corresponded to allogenic responses against MHC antigens of the immunizing cell line and the second was associated with parasite specific responses. An increase in the output of CD2(+) cells and MHC class II(+) cells in efferent lymph was observed after cell line immunization with a corresponding decrease in WC1(+) cells. Although the percentage of CD4(+) T cells did not change significantly over the course of the experiment, they became activated. Both CD25 and MHC class II expressing CD4(+) T cells were detected from day 7 onwards, peaking around day 13. Efferent lymph leukocytes (ELL) exhibited sustained responses to IL-2 in vitro following cell line immunization. Antigen specific proliferation was also detected first to the immunizing cell line and then to parasite antigens. The two peaks of CD2(+) cells were observed, which corresponded to similar peaks of CD8(+) cells. The increase in CD8(+) cells was more pronounced during the second parasite specific phase than the first allogenic phase. Activated CD8(+) T cells mainly expressed MHC class II and some expressed CD25. Significantly the peak of activated CD4(+) T cells preceded the peak of activated CD8(+) T cells, highlighting the role of T. annulata specific CD4(+) T cells in inducing parasite specific CD8(+) cytotoxic responses. A biphasic cytotoxic response also appeared in efferent lymph and peripheral blood, the first directed against MHC antigens of the immunizing cell line followed by MHC class I restricted parasite specific cytotoxicity. The cytotoxic responses in efferent lymph appeared earlier than peripheral blood, suggesting that activated CD8(+) cells exiting the draining lymph node following immunization with T. annulata infected schizonts play an important role in the development of protective immune responses.
    MacHugh ND, Connelley T, Graham SP, Pelle R, Formisano P, Taracha EL, Ellis SA, McKeever DJ, Burrells A, Morrison WI (2009) CD8+ T-cell responses to Theileria parva are preferentially directed to a single dominant antigen: Implications for parasite strain-specific immunity., Eur J Immunol39(9)pp. 2459-2469
    Although immunodominance of CD8(+) T-cell responses is a well-recognised feature of viral infections, its role in responses to more antigenically complex pathogens is less clear. In previous studies we have observed that CD8(+) T-cell responses to Theileria parva exhibit different patterns of parasite strain specificity in cattle of different MHC genotypes. In the current study, we demonstrated that animals homozygous for the A10 and A18 MHC haplotypes have detectable responses to only one of 5 T. parva antigens. Over 60% of the responding T cells from the A18(+) and A10(+) animals recognised defined epitopes in the Tp1 and Tp2 antigens, respectively. Comparison of T-cell receptor beta chain expression profiles of CD8(+) T-cell lines and CD8(+) T cells harvested ex vivo confirmed that the composition of the T-cell lines was representative of the in vivo memory CD8(+) T-cell populations. Analysis of the Tp1 and Tp2 antigens revealed sequence polymorphism, which was reflected by differential recognition by T-cell lines. In conclusion, we have demonstrated a profound immunodominance in the CD8(+) T-cell response to T. parva, which we propose is a major determinant of the parasite strain specificity of the response and hence immune protection.
    Graham SP, Everett HE, Johns HL, Haines FJ, La Rocca SA, Khatri M, Wright IK, Drew T, Crooke HR (2010) Characterisation of virus-specific peripheral blood cell cytokine responses following vaccination or infection with classical swine fever viruses.,Vet Microbiol.142(1-2)pp. 34-40 Elsevier
    Existing live attenuated classical swine fever virus (CSFV) vaccines provide a rapid onset of complete protection but pose problems in discriminating infected amongst vaccinated animals. With a view to providing additional information on the cellular mechanisms that may contribute to protection, which in turn may aid the development of the next generation of CSFV vaccines, we explored the kinetics of the cytokine responses from peripheral blood cells of pigs vaccinated with an attenuated C-strain vaccine strain and/or infected with a recent CSFV isolate. Peripheral blood cells were isolated over the course of vaccination/infection and stimulated in vitro with C-strain or UK2000/7.1 viruses. Virus-specific responses of peripheral blood cells isolated from C-strain vaccinated pigs were dominated by the production of IFN-³. IFN-³ production in response to the C-strain virus was first detected in vaccinates 9 days post-vaccination and was sustained over the period of observation. In contrast, cells from challenge control animals did not secrete IFN-³ in response to stimulation with C-strain or UK2000/7.1 viruses. Supernatants from UK2000/7.1 infected animals contained significant levels of pro-inflammatory cytokines from day 8 post-infection and these cytokines were present in both virus and mock stimulated cultures. The results suggest that the C-strain virus is a potent inducer of a type-1 T cell response, which may play a role in the protection afforded by such vaccines, whereas the pro-inflammatory cytokine responses observed in cultures from infected pigs may reflect a pathological pro-inflammatory cascade initiated in vivo following the replication and spread of CSFV.
    García-Nicolás O, Rosales RS, Pallarés FJ, Risco D, Quereda JJ, Graham SP, Frossard JP, Morgan SB, Steinbach F, Drew TW, Strickland TS, Salguero FJ (2015) Comparative analysis of cytokine transcript profiles within mediastinal lymph node compartments of pigs after infection with porcine reproductive and respiratory syndrome genotype 1 strains differing in pathogenicity, Veterinary Research46(1)
    © 2015 Garcia-Nicolas et al.; licensee BioMed Central.Porcine reproductive and respiratory syndrome virus (PRRSV) induces a weak immune response enabling it to persist in different organs of infected pigs. This has been attributed to the ability of PRRSV to influence the induction of cytokine responses. In this study, we investigated the cytokine transcriptional profiles in different compartments of the mediastinal lymph node of pigs infected with three genotype 1 PRRSV strains of differing pathogenicity: the low virulence prototype Lelystad virus (LV), and UK field strain 215-06 and the highly virulent subtype 3 SU1-Bel isolate from Belarus. We have used a combination of laser capture micro-dissection (LCM) followed by real time quantitative PCR (RT-qPCR) and immunohistochemical (IHC) detection of immune cell markers (CD3, CD79a and MAC387) and RT-qPCR quantification of PRRSV and cytokine transcripts. Compared to mock infected pigs, we found a significant downregulation of TNF-± and IFN-± in follicular and interfollicular areas of the mediastinal lymph node from 3 days post-infection (dpi) in animals infected with all three strains. This was accompanied by a transient B cell depletion and T cell and macrophage infiltration in the follicles together with T cell depletion in the interfollicular areas. A delayed upregulation of IFN-³ and IL-23p19 was observed mainly in the follicles. The PRRSV load was higher in all areas and time-points studied in the animals infected with the SU1-Bel strain. This paper describes the first application of LCM to study the cytokine transcript profiles and virus distribution in different compartments of the lymph node of pigs.
    Amarilla S, Gómez-Laguna J, Carrasco L, Rodríguez-Gómez I, Caridad y Ocerín J, Graham S, Frossard J, Steinbach F, Salguero Bodes F (2017) Porcine reproductive and respiratory syndrome type 1 viruses induce hypoplasia of erythroid cells and myeloid cell hyperplasia in the bone marrow of experimentally infected piglets independently of the viral load and virulence,Veterinary Microbiology201pp. 126-135 Elsevier
    Porcine reproductive and respiratory syndrome viruses (PRRSV) present a wide phenotypic and genetic diversity. Experimental infections have demonstrated viral replication, including highly pathogenic strains (HP-PRRSV), in primary lymphoid organs such as the thymus. However, studies of the bone marrow are scarce but necessary to help elucidate the immunobiology of PRRSV strains of differing virulence. In this study, whereas viral RNA was detected within the bone marrow of animals experimentally infected with both low virulent Lelystad (LV) and 215-06 PRRSV-1 strains and with the highly virulent SU1-bel strain, PRRSV positive cells were only occasionally detected in one SU1-bel infected animal. PRRSV RNA levels were associated to circulating virus with the highest levels detected in LV-infected pigs. At 3 dpi, a decrease in the proportion of haematopoietic tissue and number of erythroid cells in all infected groups was associated with an increase in TUNEL or cleaved caspase 3 labelling and higher counts of myeloid cells compared to control. The expression of IL-1± and IL-6 was elevated at the beginning of the infection in all infected animals. The expression of TNF-± was increased at the end of the study in all infected groups with respect to control. Different PRRSV-1 strains induced, presummably by indirect mechanisms and independently of viral load and strain virulence, moderate and sustained hypoplasia of erythroid cells and myeloid cell hyperplasia at early stages of infection. These changes were paralleled by a peak in the local expression of IL-1±, IL-6 and TNF-± in all infected groups.
    Everett H, Salguero FJ, Graham SP, Haines F, Johns H, Clifford D, Nunez A, La Rocca SA, Parchariyanon S, Steinbach F, Drew T, Crooke H (2010) Characterisation of experimental infections of domestic pigs with genotype 2.1 and 3.3 isolates of classical swine fever virus., Vet Microbiol142(1-2)pp. 26-33
    The early identification of classical swine fever epizootics is hampered by difficulties in recognising early signs of infection, due to a lack of specific clinical signs. In addition many textbook descriptions of CSF are based on observations of disease caused by historic, mainly genotype 1, strains. Our objective was to improve our knowledge of the diverse range of signs that different CSFV strains can cause by characterising the experimental infection of domestic pigs with both a recent strain of CSFV and a divergent strain. Conventional pigs were inoculated with a genotype 2.1 isolate, that caused an outbreak in the UK in 2000, and a genotype 3.3 strain that is genetically divergent from European strains. This latter strain is also antigenically distinct as it is only poorly recognised by the CSFV-specific monoclonal antibody, WH303. Transmission was monitored by use of in-contact animals. Clinical, virological and haematological parameters were observed and an extended macro- and histopathological scoring system allowed detailed characterisation of pathological lesions. Infection with the genotype 2.1 isolate resulted in a similar outcome to other recent genotype 2 European strains, whereas the genotype 3.3 strain produced fewer and delayed clinical signs, notably with little fever. This strain would therefore be particularly difficult to detect in the early stages of infection and highlights the importance of encouraging early submission of samples for laboratory diagnosis. As representatives of recent and divergent CSFV isolates, these strains are good candidates to study the pathogenesis of current CSFV isolates and as challenge models for vaccine development.
    Singleton H, Everett H, Graham S, Bodman-Smith K, Steinbach F (2013) Characterisation of porcine monocytes, macrophages and dendritic cells, IMMUNOLOGY140pp. 122-122 WILEY-BLACKWELL
    Graham SP, Pellé R, de Villiers EP, Yamage M, Mwangi DM, Honda Y, Ellis SA, MacHugh ND, Ivan Morrison W, Taracha ELN (2009) Identification of bovine cytotoxic T lymphocyte epitopes on the intracellular parasite Theileria Parva, Veterinary Immunology and Immunopathology128(1?3)
    Salguero FJ, Frossard JP, Rebel JM, Stadejek T, Morgan SB, Graham SP, Steinbach F (2015) Host-pathogen interactions during porcine reproductive and respiratory syndrome virus 1 infection of piglets., Virus Res202pp. 135-143
    Porcine reproductive and respiratory syndrome (PRRS) is a major disease affecting pigs worldwide and resulting in considerable economic losses. While PRRS is a global phenomenon, the causative viruses PRRSV-1 (first detected in Europe) and PRRSV-2 (isolated in North America) are genetically and biologically distinct. In addition, the disease outcome is directly linked to co-infections associated with the porcine respiratory disease complex and the host response is variable between different breeds of pigs. It is therefore warranted when studying the pathogenesis of PRRS to consider each viral genotype separately and apply careful consideration to the disease model studied. We here review the respiratory pig model for PRRSV-1, with a focus on a recent set of studies conducted with carefully selected virus strains and pigs, which may serve as both a baseline and benchmark for future investigation.
    Forberg H, Hauge AG, Valheim M, Garcon F, Nunez A, Gerner W, Mair KH, Graham SP, Brookes SM, Storset AK (2014) Early Responses of Natural Killer Cells in Pigs Experimentally Infected with 2009 Pandemic H1N1 Influenza A Virus,PLOS ONE9(6)ARTN e100619 PUBLIC LIBRARY SCIENCE
    Amarilla SP, Gómez-Laguna J, Carrasco L, Rodríguez-Gómez IM, Caridad y Ocerín JM, Morgan SB, Graham SP, Frossard JP, Drew TW, Salguero FJ (2015) A comparative study of the local cytokine response in the lungs of pigs experimentally infected with different PRRSV-1 strains: Upregulation of IL-1± in highly pathogenic strain induced lesions, Veterinary Immunology and Immunopathology164(3-4)pp. 137-147
    © 2015.Porcine reproductive and respiratory syndrome viruses (PRRSV) show high genetic differences both among and within genotypes. Recently, several highly pathogenic PRRSV (HP-PRRSV) strains have been described. This study compares and characterizes the production of cytokines by pulmonary macrophages in pigs experimentally infected with four different PRRSV-1 strains: two low-virulent strains, Lelystad (LV) and a British field strain (215-06); a HP strain (SU1-bel) from Belarus and the attenuated vaccine strain DV (Porcilis® PRRS). Animals were clinically monitored and post-mortem examinations were performed at 3, 7 and 35 days post-infection (dpi). Lung samples were processed for histopathological and immunohistochemical studies by using specific antibodies against PRRSV, IL1-±, IL-6, TNF-±, IL-10 and IFN-³. SU1-bel infected animals presented the highest mean scores for clinical observations, gross and microscopic lesions as well as for PRRSV expression compared with the other infected groups (pd0.027). These animals displayed the highest expression of IL1-± at 7dpi, together with the highest score for lung pathology, whereas LV, 215-06 and DV inoculated animals only showed a transient enhancement in some of these cytokines. SU1-bel-infected pigs showed a positive correlation between the amount of PRRSV antigen and IL-1± expression (r=0.645, p<0.001). The highest expression of IL-10 was detected in 215-06-infected animals (pd0.004), with a positive correlation with the numbers of virus-infected cells (r=0.375, pd0.013). In conclusion, the HP-PRRSV SU1-bel strain replicated more efficiently in the lung of infected animals and induced a higher expression of IL-1± than the other PRRSV-1-infected groups, which may have played a key role in the onset of the clinical signs and interstitial pneumonia.
    Graham SP, Everett HE, Johns HL, Haines FJ, La Rocca SA, Crooke HR (2008) Characterization of virus-specific T cell cytokine responses following vaccination and challenge with classical swine fever viruses, IMMUNOLOGY125pp. 93-94 WILEY-BLACKWELL PUBLISHING, INC
    Graham SP, Pellé R, Honda Y, Mwangi DM, Tonukari NJ, Yamage M, Glew EJ, de Villiers EP, Shah T, Bishop R, Abuya E, Awino E, Gachanja J, Luyai AE, Mbwika F, Muthiani AM, Ndegwa DM, Njahira M, Nyanjui JK, Onono FO, Osaso J, Saya RM, Wildmann C, Fraser CM, Maudlin I, Gardner MJ, Morzaria SP, Loosmore S, Gilbert SC, Audonnet JC, van der Bruggen P, Nene V, Taracha EL (2006) Theileria parva candidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes., Proc Natl Acad Sci U S A103(9)pp. 3286-3291
    East Coast fever, caused by the tick-borne intracellular apicomplexan parasite Theileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8(+) cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted by T. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8(+) CTL from immune cattle. CD8(+) T cell responses to these antigens were boosted in T. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.
    Vaccination with live attenuated classical swine fever virus (CSFV) vaccines induces a rapid onset of protection which has been associated with virus-specific CD8 T cell IFN-³ responses. In this study, we assessed the specificity of this response, by screening a peptide library spanning the CSFV C-strain vaccine polyprotein to identify and characterise CD8 T cell epitopes. Synthetic peptides were pooled to represent each of the 12 CSFV proteins and used to stimulate PBMC from four pigs rendered immune to CSFV by C-strain vaccination and subsequently challenged with the virulent Brescia strain. Significant IFN-³ expression by CD8 T cells, assessed by flow cytometry, was induced by peptide pools representing the core, E2, NS2, NS3 and NS5A proteins. Dissection of these antigenic peptide pools indicated that, in each instance, a single discrete antigenic peptide or pair of overlapping peptides was responsible for the IFN-³ induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core241-255 PESRKKLEKALLAWA and NS31902-1912 VEYSFIFLDEY, or minimal length antigenic peptides: E2996-1003 YEPRDSYF, NS21223-1230 STVTGIFL and NS5A3070-3078 RVDNALLKF. The epitopes are highly conserved across CSFV strains and variable sequence divergence was observed with related pestiviruses. Characterisation of epitope-specific CD8 T cells revealed evidence of cytotoxicity, as determined by CD107a mobilisation, and a significant proportion expressed TNF-± in addition to IFN-³. Finally, the variability in the antigen-specificity of these immunodominant CD8 T cell responses was confirmed to be associated with expression of distinct MHC class I haplotypes. Moreover, recognition of NS 21223-1230 STVTGIFL and NS31902-1912 VEYSFIFLDEY by a larger group of C-strain vaccinated animals showed that these peptides could be restricted by additional haplotypes. Thus the antigenic regions and epitopes identified represent attractive targets for evaluation of their vaccine potential against CSFV. © 2013 Franzoni et al.
    Mwangi DM, Honda Y, Graham SP, Pelle R, Taracha EL, Gachanja J, Nyanjui JK, Bray J, Palmer GH, Brown WC, Mwangi W (2011) Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-³ responses but not CTL responses., Vet Immunol Immunopathol140(3-4)pp. 244-251
    Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-³-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-³-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.
    Graham SP, Haines FJ, Johns HL, Sosan O, La Rocca SA, Lamp B, Ruemenapf T, Everett HE, Crooke HR (2012) Characterisation of vaccine-induced, broadly cross-reactive IFN-gamma secreting T cell responses that correlate with rapid protection against classical swine fever virus, VACCINE30(17)pp. 2742-2748 ELSEVIER SCI LTD
    Tchakouté VL, Graham SP, Jensen SA, Makepeace BL, Nfon CK, Njongmeta LM, Lustigman S, Enyong PA, Tanya VN, Bianco AE, Trees AJ (2006) In a bovine model of onchocerciasis, protective immunity exists naturally, is absent in drug-cured hosts, and is induced by vaccination., Proc Natl Acad Sci U S A103(15)pp. 5971-5976
    Onchocerciasis (river blindness) is a major parasitic disease of humans in sub-Saharan Africa caused by the microfilarial stage of the nematode Onchocerca volvulus. Using Onchocerca ochengi, a closely related species which infects cattle and is transmitted by the same black fly vector (Simulium damnosum sensu lato) as O. volvulus, we have conducted longitudinal studies after either natural field exposure or experimental infection to determine whether, and under what circumstances, protective immunity exists in onchocerciasis. On the basis of the adult worm burdens (nodules) observed, we determined that cattle reared in endemic areas without detectable parasites (putatively immune) were significantly less susceptible to heavy field challenge than age-matched, naïve controls (P = 0.002), whereas patently infected cattle, cured of infection by adulticide treatment with melarsomine, were fully susceptible. Cattle immunized with irradiated third-stage larvae were significantly protected against experimental challenge (100% reduction in median nodule load, P = 0.003), and vaccination also conferred resistance to severe and prolonged field challenge (64% reduction in median nodule load, P = 0.053; and a significant reduction in microfilarial positivity rates and density, P < 0.05). These results constitute evidence of protective immunity in a naturally evolved host-Onchocerca sp. relationship and provide proof-of-principle for immunoprophylaxis under experimental and field conditions.
    MacHugh ND, Weir W, Burrells A, Lizundia R, Graham SP, Taracha EL, Shiels BR, Langsley G, Morrison WI (2011) Extensive polymorphism and evidence of immune selection in a highly dominant antigen recognized by bovine CD8 T cells specific for Theileria annulata., Infect Immun79(5)pp. 2059-2069
    Although parasite strain-restricted CD8 T cell responses have been described for several protozoa, the precise role of antigenic variability in immunity is poorly understood. The tick-borne protozoan parasite Theileria annulata infects leukocytes and causes an acute, often fatal lymphoproliferative disease in cattle. Building on previous evidence of strain-restricted CD8 T cell responses to T. annulata, this study set out to identify and characterize the variability of the target antigens. Three antigens were identified by screening expressed parasite cDNAs with specific CD8 T cell lines. In cattle expressing the A10 class I major histocompatibility complex haplotype, A10-restricted CD8 T cell responses were shown to be focused entirely on a single dominant epitope in one of these antigens (Ta9). Sequencing of the Ta9 gene from field isolates of T. annulata demonstrated extensive sequence divergence, resulting in amino acid polymorphism within the A10-restricted epitope and a second A14-restricted epitope. Statistical analysis of the allelic sequences revealed evidence of positive selection for amino acid substitutions within the region encoding the CD8 T cell epitopes. Sequence differences in the A10-restricted epitope were shown to result in differential recognition by individual CD8 T cell clones, while clones also differed in their ability to recognize different alleles. Moreover, the representation of these clonal specificities within the responding CD8 T cell populations differed between animals. As well as providing an explanation for incomplete protection observed after heterologous parasite challenge of vaccinated cattle, these results have important implications for the choice of antigens for the development of novel subunit vaccines.
    Graham SP, Brown DJ, Vatansever Z, Waddington D, Taylor LH, Nichani AK, Campbell JD, Adamson RE, Glass EJ, Spooner RL (2001) Proinflammatory cytokine expression by Theileria annulata infected cell lines correlates with the pathology they cause in vivo., Vaccine19(20-22)pp. 2932-2944
    Control of Theileria annulata is currently best achieved by the use of live attenuated cell line vaccines. However, the mechanisms underlying attenuation are unclear and there is a need to rapidly produce new cell line vaccines, which could safely and effectively vaccinate cattle against tropical theileriosis. There is increasing evidence to suggest that proinflammatory cytokines produced by T. annulata infected cells play a central role in both pathology and immune evasion. This study aimed to test this hypothesis and to evaluate cytokine expression as a marker of virulence. The pathogenicity and protective efficacy of cloned T. annulata cell lines that expressed different levels of proinflammatory cytokines were compared. In two independent trials using different stocks of T. annulata, cell lines that expressed higher levels of proinflammatory cytokines induced severe reactions, and in some cases death, when used to vaccinate groups of cattle. In contrast, low cytokine expressing lines induced low post-vaccinal reactions. The results clearly demonstrated that cytokine expression by T. annulata infected cells could be used as a marker of virulence and provided strong evidence to support a role for cytokines in the induction of pathology. Both high and low cytokine expressing cell lines protected cattle against heterologous challenge infection, offering the possibility of using cytokine expression to rapidly select new safe, potent vaccines against tropical theileriosis without the need for culture attenuation.
    Franzoni G, Edwards JC, Kurkure NV, Edgar DS, Sanchez-Cordon PJ, Haines FJ, Salguero FJ, Everett HE, Bodman-Smith KB, Crooke HR, Graham SP (2014) Partial Activation of natural killer and ³´ T cells by classical swine fever viruses is associated with type I interferon elicited from plasmacytoid dendritic cells., Clin Vaccine Immunol21(10)pp. 1410-1420
    Vaccination with live attenuated classical swine fever virus (CSFV) vaccines can rapidly confer protection in the absence of neutralizing antibodies. With an aim of providing information on the cellular mechanisms that may mediate this protection, we explored the interaction of porcine natural killer (NK) cells and ³´ T cells with CSFV. Both NK and ³´ T cells were refractory to infection with attenuated or virulent CSFV, and no stimulatory effects, as assessed by the expression of major histocompatibility complex (MHC) class II (MHC-II), perforin, and gamma interferon (IFN-³), were observed when the cells were cultured in the presence of CSFV. Coculture with CSFV and myeloid dendritic cells (mDCs) or plasmacytoid dendritic cells (pDCs) showed that pDCs led to a partial activation of both NK and ³´ T cells, with upregulation of MHC-II being observed. An analysis of cytokine expression by infected DC subsets suggested that this effect was due to IFN-± secreted by infected pDCs. These results were supported by ex vivo analyses of NK and ³´ T cells in the tonsils and retropharyngeal lymph nodes from pigs that had been vaccinated with live attenuated CSFV and/or virulent CSFV. At 5 days postchallenge, there was evidence of significant upregulation of MHC-II but not perforin on NK and ³´ T cells, which was observed only following a challenge of the unvaccinated pigs and correlated with increased CSFV replication and IFN-± expression in both the tonsils and serum. Together, these data suggest that it is unlikely that NK or ³´ T cells contribute to the cellular effector mechanisms induced by live attenuated CSFV.
    Bishop RP, Odongo DO, Mann DJ, Pearson TW, Sugimoto C, Haines LR, Glass E, Jensen K, Seitzer U, Ahmed JS, Graham SP, de Villiers EP (2009) Theileria, In: Nene V, Kole C (eds.), Genome Mapping and Genomics in Animal-Associated Microbes.pp. 191-231 Springer-Verlag
    Rhodes SG, Palmer N, Graham SP, Bianco AE, Hewinson RG, Vordermeier HM (2000) Distinct response kinetics of gamma interferon and interleukin-4 in bovine tuberculosis., Infect Immun68(9)pp. 5393-5400
    This study shows that gamma interferon (IFN-gamma) and interleukin-4 (IL-4) cytokine responses are produced by peripheral blood cells in cattle infected with Mycobacterium bovis. The different kinetics of the IFN-gamma and IL-4 responses to bovine tuberculin and to ESAT-6 following experimental intratracheal infection with M. bovis are described. An early increase in IFN-gamma was observed that was maintained throughout the period studied. In contrast, the IL-4 response was delayed and confined to a peak of activity lasting 6 to 8 weeks. Interestingly, an experimental challenge of cattle with a lower dose of M. bovis which did not result in the development of lesions, positive DTH skin test, or substantial IFN-gamma responses nevertheless generated strong specific IL-4 responses. Investigation of naturally infected M. bovis field reactors showed increased IFN-gamma and IL-4 responses compared to uninfected cattle and that both of these cytokines were equally able to differentiate infected from uninfected animals. The magnitude of the M. bovis-induced IL-4 responses were found to be similar to the antigen-specific IL-4 responses of cattle infected with the parasitic nematode Onchocerca ochengi, further supporting the presence of this type 2 cytokine in bovine tuberculosis.
    Mokhtar H, Pedrera M, Frossard JP, Biffar L, Hammer SE, Kvisgaard LK, Larsen LE, Stewart GR, Somavarapu S, Steinbach F, Graham SP (2016) The Non-structural Protein 5 and Matrix Protein Are Antigenic Targets of T Cell Immunity to Genotype 1 Porcine Reproductive and Respiratory Syndrome Viruses.,Front Immunol7
    The porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of one of the most economically important diseases affecting swine worldwide. Efforts to develop a next-generation vaccine have largely focused on envelope glycoproteins to target virus-neutralizing antibody responses. However, these approaches have failed to demonstrate the necessary efficacy to progress toward market. T cells are crucial to the control of many viruses through cytolysis and cytokine secretion. Since control of PRRSV infection is not dependent on the development of neutralizing antibodies, it has been proposed that T cell-mediated immunity plays a key role. Therefore, we hypothesized that conserved T cell antigens represent prime candidates for the development a novel PRRS vaccine. Antigens were identified by screening a proteome-wide synthetic peptide library with T cells from cohorts of pigs rendered immune by experimental infections with a closely related (subtype 1) or divergent (subtype 3) PRRSV-1 strain. Dominant T cell IFN-³ responses were directed against the non-structural protein 5 (NSP5), and to a lesser extent, the matrix (M) protein. The majority of NSP5-specific CD8 T cells and M-specific CD4 T cells expressed a putative effector memory phenotype and were polyfunctional as assessed by coexpression of TNF-± and mobilization of the cytotoxic degranulation marker CD107a. Both antigens were generally well conserved among strains of both PRRSV genotypes. Thus, M and NSP5 represent attractive vaccine candidate T cell antigens, which should be evaluated further in the context of PRRSV vaccine development.
    Entrican G, Brown J, Graham S (1998) Cytokines and the protective host immune response to Chlamydia psittaci., Comp Immunol Microbiol Infect Dis21(1)pp. 15-26
    The immunobiology of enzootic abortion of ewes (EAE) is incompletely understood. The causative agent is Chlamydia psittaci, which infects many ruminant species and has zoonotic potential. The organism can survive in the ovine host for many months without causing clinical symptoms but does not generate a sterile immunity during this time. It has been postulated that the organism persists in the host entering at a latent phase, possibly mediated by host cytokine production. The effects of cytokines on chlamydial multiplication vary between host species, between different cell types within those species and also vary between chlamydial species and strains. The multiplication of the EAE strain of C. psittaci in ovine ST-6 cells can be restricted by interferon-gamma (IFN-gamma) but not with comparable concentrations of IFN-alpha. Altering the nutrient composition of the cultures by addition of tryptophan partially reverses the antichlamydial effects of the IFN-gamma. This offers a potential mechanism by which C. psittaci can persist in sheep. The implications of these observations for the pathogenesis of EAE are discussed.
    Graham SP, Lustigman S, Trees AJ, Bianco AE (2000) Onchocerca volvulus: comparative analysis of antibody responses to recombinant antigens in two animal models of onchocerciasis., Exp Parasitol94(3)pp. 158-162
    Experimental infections of chimpanzees with Onchocerca volvulus and cattle with Onchocerca ochengi provide model systems for research in human onchocerciasis. These infections share many similarities from the standpoint of parasite biology, but little is known about the comparability of immune responses in the two systems. To make a direct comparison between the models in terms of immune responsiveness to defined parasite products, three recombinant antigens of O. volvulus (Ov7, Ov103, and B20) were used to analyze the kinetics of antibody production following experimental infection. Each of the antigens was derived from adult cDNA libraries following immunoscreening with sera from chimpanzees (Ov7, Ov103) or cattle (B20). All chimpanzees (n = 12) and cattle (n = 8) displayed responses to Ov7 and Ov103, and all cattle, but only 33% of chimpanzees, showed responses to B20. The dynamics of the response to individual antigens showed further similarities between the chimpanzees and the cattle, with responses to Ov7 and Ov103 peaking after, and B20 before, the onset of patent infections. We conclude that there is good preliminary evidence of concordance in the kinetics of serological responses in the two models. However, individual antigens many be more or less immunogenic in the two systems, making it inadvisable to extrapolate between models concerning the relative immunodominance of specific parasite products.
    Rhodes SG, Graham SP (2002) Is 'timing' important for cytokine polarization?, Trends Immunol23(5)pp. 246-249
    Graham SP, Wu Y, Henkle-Duehrsen K, Lustigman S, Unnasch TR, Braun G, Williams SA, McCarthy J, Trees AJ, Bianco AE (1999) Patterns of Onchocerca volvulus recombinant antigen recognition in a bovine model of onchocerciasis., Parasitology119 ( Pt 6)pp. 603-612
    The antibody responses of 8 cattle experimentally infected with Onchocerca ochengi to 18 recombinant O. volvulus antigens were measured by ELISA. In addition to establishing antigenic cross-reactivity between the species, the dynamics of antigen-specific responses were examined to assess how the recognition of the antigens compared to the known stage-specificity of expression. Six cattle responded to all of the antigens and 2 animals responded to all but 1. The dynamics of the recognition of 4 antigens (B20, MOv-2, MOv-14 and OvNHR2 02E1) were characterized by rapid seroconversion following infection. Antibody levels to 2 antigens (Ov7 and OvALT-1) increased gradually over the course of infection. Antibody levels to 4 antigens (OvTPX-2, OvL3Chitinase, Ov103 and Ov9m) reached maximum levels coincident with the onset of patency. The levels to 3 antigens (OvProalf C50, OvAldolase, Ov39) varied little over the course of infection. Responses to antigens with functional similarities (OvSOD1, OvSOD2 and OvSOD3 or OvGST1 and OvGST2) showed comparable temporal profiles. This study demonstrates the high degree of immunological cross-reactivity between the antigens of O. volvulus and O. ochengi. The immunogenicity of antigens varied over the course of infection in an antigen-specific manner, which not always reflected developmentally regulated expression of the corresponding gene, possibly owing to cross-reactive epitopes on distinct parasite products.
    Amarilla SP, Gómez-Laguna J, Carrasco L, Rodríguez-Gómez IM, Caridad Y Ocerín JM, Morgan SB, Graham SP, Frossard JP, Drew TW, Salguero FJ (2015) A comparative study of the local cytokine response in the lungs of pigs experimentally infected with different PRRSV-1 strains: upregulation of IL-1± in highly pathogenic strain induced lesions., Vet Immunol Immunopathol164(3-4)pp. 137-147
    Porcine reproductive and respiratory syndrome viruses (PRRSV) show high genetic differences both among and within genotypes. Recently, several highly pathogenic PRRSV (HP-PRRSV) strains have been described. This study compares and characterizes the production of cytokines by pulmonary macrophages in pigs experimentally infected with four different PRRSV-1 strains: two low-virulent strains, Lelystad (LV) and a British field strain (215-06); a HP strain (SU1-bel) from Belarus and the attenuated vaccine strain DV (Porcilis(®) PRRS). Animals were clinically monitored and post-mortem examinations were performed at 3, 7 and 35 days post-infection (dpi). Lung samples were processed for histopathological and immunohistochemical studies by using specific antibodies against PRRSV, IL1-±, IL-6, TNF-±, IL-10 and IFN-³. SU1-bel infected animals presented the highest mean scores for clinical observations, gross and microscopic lesions as well as for PRRSV expression compared with the other infected groups (pd0.027). These animals displayed the highest expression of IL1-± at 7dpi, together with the highest score for lung pathology, whereas LV, 215-06 and DV inoculated animals only showed a transient enhancement in some of these cytokines. SU1-bel-infected pigs showed a positive correlation between the amount of PRRSV antigen and IL-1± expression (r=0.645, p<0.001). The highest expression of IL-10 was detected in 215-06-infected animals (pd0.004), with a positive correlation with the numbers of virus-infected cells (r=0.375, pd0.013). In conclusion, the HP-PRRSV SU1-bel strain replicated more efficiently in the lung of infected animals and induced a higher expression of IL-1± than the other PRRSV-1-infected groups, which may have played a key role in the onset of the clinical signs and interstitial pneumonia.
    Bishop R, Shah T, Pelle R, Hoyle D, Pearson T, Haines L, Brass A, Hulme H, Graham SP, Taracha EL, Kanga S, Lu C, Hass B, Wortman J, White O, Gardner MJ, Nene V, de Villiers EP (2005) Analysis of the transcriptome of the protozoan Theileria parva using MPSS reveals that the majority of genes are transcriptionally active in the schizont stage.,Nucleic Acids Res33(17)pp. 5503-5511
    Massively parallel signature sequencing (MPSS) was used to analyze the transcriptome of the intracellular protozoan Theileria parva. In total 1,095,000, 20 bp sequences representing 4371 different signatures were generated from T.parva schizonts. Reproducible signatures were identified within 73% of potentially detectable predicted genes and 83% had signatures in at least one MPSS cycle. A predicted leader peptide was detected on 405 expressed genes. The quantitative range of signatures was 4-52,256 transcripts per million (t.p.m.). Rare transcripts (<50 t.p.m.) were detected from 36% of genes. Sequence signatures approximated a lognormal distribution, as in microarray. Transcripts were widely distributed throughout the genome, although only 47% of 138 telomere-associated open reading frames exhibited signatures. Antisense signatures comprised 13.8% of the total, comparable with Plasmodium. Eighty five predicted genes with antisense signatures lacked a sense signature. Antisense transcripts were independently amplified from schizont cDNA and verified by sequencing. The MPSS transcripts per million for seven genes encoding schizont antigens recognized by bovine CD8 T cells varied 1000-fold. There was concordance between transcription and protein expression for heat shock proteins that were very highly expressed according to MPSS and proteomics. The data suggests a low level of baseline transcription from the majority of protein-coding genes.
    Sosan O, Graham S, Everett H, Crudgington B, Bodman-Smith K, Crooke H (2012) Differential antiviral effect of porcine interferon alpha subtypes on classical swine fever virus infection of porcine monocytes, CYTOKINE59(3)pp. 552-552 ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
    Graham SP, Trees AJ, Collins RA, Moore DM, Guy FM, Taylor MJ, Bianco AE (2001) Down-regulated lymphoproliferation coincides with parasite maturation and with the collapse of both gamma interferon and interleukin-4 responses in a bovine model of onchocerciasis., Infect Immun69(7)pp. 4313-4319
    Onchocerciasis is a debilitating parasitic infection caused by the filarial nematode Onchocerca volvulus. Infections are chronic, and persistence of the parasites for several years argues for highly adapted mechanisms of immune evasion. Due to the restricted host repertoire of O. volvulus, we have used the cattle parasite Onchocerca ochengi to investigate the nature of immunomodulation underpinning these long-term infections. Cattle were infected with a single inoculation of 350 infective-stage larvae under laboratory conditions (n = 6). Intradermal nodules containing immature adult worms were detected from 110 days postinfection, and microfilariae in skin were detected from day 280 postinfection. Parasite-specific responses during early infection were nonpolarized with respect to the major Th cytokines (interleukin-4 [IL-4], IL-2, and gamma interferon [IFN-gamma]) produced by antigen-stimulated peripheral blood mononuclear cells (PBMC) or serum antibody isotypes. Antigen-induced proliferation of PBMC peaked shortly after exposure and remained high during the prepatent infection. As the parasites matured and animals developed patent infections, there was a profound down-regulation of lymphoproliferation, accompanied by sharp falls in the expression of both IL-4 and IFN-gamma and a gradual decline in IL-2. Levels of immunoglobulin G2 (IgG2) fell, while those of IgG1 remained high. We conclude that neither a classical Th2 response nor a simple Th1-to-Th2 switch is sufficient to explain the immunomodulation associated with patent Onchocerca infections. Instead, there is an initial Th0 response, which matures into a response with some, but not all of the features of a Th2 response. The natural host-parasite relationship of O. ochengi in cattle may be useful as both a descriptive and predictive tool to test more refined models of immunomodulation in onchocerciasis.
    Franzoni G, Bonelli P, Graham SP, Anfossi A, Dei Giudici S, Pilo G, Pittau M, Nicolussi P, Oggiano A (2017) Comparative phenotypic and functional analyses of the effects of autologous plasma and recombinant human macrophage-colony stimulating factor (M-CSF) on porcine monocyte to macrophage differentiation,Veterinary Immunology and Immunopathology187pp. 80-88 Elsevier
    Porcine monocyte-derived macrophages (moM¦) have been employed as a model cell in numerous studies of the porcine immune system. However, the lack of a standardized method for moM¦ differentiation hampers the comparison of results coming from the use of different laboratory protocols. In this study we compared the use of varying concentrations of autologous plasma (10, 20 and 30% v/v) or recombinant human macrophage-colony stimulating factor (hM-CSF; 50, 100, and 200 ng/ml) to differentiate porcine monocytes into macrophages. Changes in cell morphology and surface marker expression were assessed by confocal microscopy and flow cytometry. Macrophage differentiation was evaluated by analysing TNF-± response to LPS stimulation and determining cytokine secretion patterns under both basal conditions and after classical and alternative activation. The effects of the differentiation methods on metabolic activity and susceptibility to infection with the myelotropic African swine fever virus (ASFV) were also evaluated. Monocytes cultured using the different culture conditions tested augmented in dimension and cellular complexity, but increasing porcine plasma concentrations resulted in a dose dependent enhancement in granularity and a marked pleomorphism. As expected, CD163, MHC class II DR and CD203a expression were up-regulated in both hM-CSF (M-CSF-moM¦) and autologous plasma cultured macrophages (AP-moM¦), although a lower percentage of CD163+ cells were found following differentiation with high percentages of porcine plasma. We observed enhanced number of viable cells using high concentration of hM-CSF compared to porcine plasma, suggesting a proliferative effect. Irrespective of differentiation conditions, monocyte differentiation into macrophages resulted in an increased susceptibility to ASFV and yielded larger amounts of LPS-induced TNF-±. AP-moM¦ showed a higher basal release of IL-1RA compared to those cultured with hM-CSF and displayed a reduced ability to respond to classical activation, suggesting that the use of high percentages of porcine plasma led to the acquisition of a M2-like phenotype. We conclude that all the protocols tested in this study can be considered as suitable to produce porcine moM¦, although the use of hM-CSF provides high responsiveness to M1 polarization. Since a higher phenotypic and functional inter-animal variability was observed in AP-moM¦, we propose that the use of low concentration of hM-CSF should be adopted as the method
    Trees AJ, Graham SP, Renz A, Bianco AE, Tanya V (2000) Onchocerca ochengi infections in cattle as a model for human onchocerciasis: recent developments., Parasitology120 Supplpp. S133-S142
    The bovine parasite Onchocerca ochengi is a nodule-dwelling filarial nematode, closely related to O. volvulus, the causal agent of human River Blindness, and, sharing with it, the same vector. This brief review, based on a presentation at the BSP Autumn Symposium 1999, describes recent work supported by the WHO Drug Development Research Macrofil programme and the Edna McConnell Clark Foundation vaccine development programme, to research the chemotherapy and immunology of onchocerciasis utilising this model system, with experimental infections in Liverpool and field infections in northern Cameroon. In a series of chemotherapeutic trials involving 10 compounds in 20 treatment regimes, the comparability of drug efficacy against O. ochengi with that described against O. volvulus has been demonstrated. Repeated, long-term treatment with oxytetracycline has been shown to be macrofilaricidal and the effect is hypothesized to be related to action on Wolbachia endobacteria, abundant in O. ochengi. Avermectins/milbemycins are not macrofilaricidal (even in high and repeated long-term treatments) but induce sustained abrogation of embryogenesis. In prospective, field exposure experiments with naive calves, prophylactic treatments with ivermectin and moxidectin prevented the development of adult worm infection, raising the possibility that drug-attenuated larval challenge infections may induce immunity. Putatively immune adult cattle exist in endemically exposed populations, and these have been shown to be significantly less susceptible to challenge than age-matched naive controls, whereas radically drug-cured, previously patently-infected cattle were not. Experimental infections with O. ochengi have revealed the kinetics of the immune response in relation to parasite development and demonstrate analogous responses to those reported in O. volvulus infection in humans and chimpanzees. In an immunization experiment with irradiated L3 larvae, cattle were significantly protected against experimental challenge--the first such demonstration of the experimental induction of immunity in a natural Onchocerca host-parasite system. Taken collectively, these studies not only demonstrate the similarity between the host-parasite relationships of O. ochengi in cattle and O. volvulus in humans, but promise to advance options for the control of human onchocerciasis.
    Mokhtar H, Eck M, Morgan SB, Essler SE, Frossard JP, Ruggli N, Graham SP (2014) Proteome-wide screening of the European porcine reproductive and respiratory syndrome virus reveals a broad range of T cell antigen reactivity., Vaccine32(50)pp. 6828-6837
    The porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving and diversifying pathogen necessitating the development of improved vaccines. Immunity to PRRSV is not well understood although there are data suggesting that virus-specific T cell IFN-³ responses play an important role. We therefore aimed to better characterise the T cell response to genotype 1 (European) PRRSV by utilising a synthetic peptide library spanning the entire proteome and a small cohort of pigs rendered immune to PRRSV-1 Olot/91 by repeated experimental infection. Using an IFN-³ ELISpot assay as a read-out, we were able to identify 9 antigenic regions on 5 of the viral proteins and determine the corresponding responder T cell phenotype. The diversity of the IFN-³ response to PRRSV proteins suggests that antigenic regions are scattered throughout the proteome and no one single antigen dominates the T cell response. To address the identification of well-conserved T cell antigens, we subsequently screened groups of pigs infected with a closely related avirulent PRRSV-1 strain (Lelystad) and a divergent virulent subtype 3 strain (SU1-Bel). Whilst T cell responses from both groups were observed against many of the antigens identified in the first study, animals infected with the SU1-Bel strain showed the greatest response against peptides representing the non-structural protein 5. The proteome-wide peptide library screening method used here, as well as the antigens identified, warrant further evaluation in the context of next generation vaccine development.
    Graham SP, Everett HE, Haines FJ, Johns HL, Sosan OA, Salguero FJ, Clifford DJ, Steinbach F, Drew TW, Crooke HR (2012) Challenge of pigs with classical swine fever viruses after C-strain vaccination reveals remarkably rapid protection and insights into early immunity., PLoS One7(1)
    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® 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-³ 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-³ 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.
    Immunity against Theileria parva is associated with CD8 T-cell responses that exhibit immunodominance, focusing the response against limited numbers of epitopes. As candidates for inclusion in vaccines, characterization of responses against immunodominant epitopes is a key component in novel vaccine development. We have previously demonstrated that the Tp249?59 and Tp1214?224 epitopes dominate CD8 T-cell responses in BoLAA10 and BoLA-18 MHC I homozygous animals, respectively. In this study, peptide?MHC I tetramers for these epitopes, and a subdominant BoLA-A10-restricted epitope (Tp298?106), were generated to facilitate accurate and rapid enumeration of epitope-specific CD8 T cells. During validation of these tetramers a substantial proportion of Tp249?59-reactive T cells failed to bind the tetramer, suggesting that this population was heterogeneous with respect to the recognized epitope. We demonstrate that Tp250?59 represents a distinct epitope and that tetramers produced with Tp50?59 and Tp49?59 show no cross-reactivity. The Tp249?59 and Tp250?59 epitopes use different serine residues as the N-terminal anchor for binding to the presenting MHC I molecule. Molecular dynamic modelling predicts that the two peptide?MHC I complexes adopt structurally different conformations and Tcell receptor b sequence analysis showed that Tp249?59 and Tp250?59 are recognized by non-overlapping T-cell receptor repertoires. Together these data demonstrate that although differing by only a single residue, Tp249?59 and Tp250?59 epitopes form distinct ligands for T-cell receptor recognition. Tetramer analysis of T. parva-specific CD8 T-cell lines confirmed the immunodominance of Tp1214?224 in BoLA-A18 animals and showed in BoLA-A10 animals that the Tp249?59 epitope response was generally more dominant than the Tp250?59 response and confirmed that the Tp298?106 response was subdominant.
    Weesendorp E, Morgan S, Stockhofe-Zurwieden N, Popma-De Graaf DJ, Graham SP, Rebel JMJ (2013) Comparative analysis of immune responses following experimental infection of pigs with European porcine reproductive and respiratory syndrome virus strains of differing virulence, VETERINARY MICROBIOLOGY163(1-2)pp. 1-12 ELSEVIER SCIENCE BV
    Graham SP, Everett HE, Johns HL, Haines FJ, La Rocca SA, Khatri M, Wright IK, Drew T, Crooke HR (2010) Characterisation of virus-specific peripheral blood cell cytokine responses following vaccination or infection with classical swine fever viruses,VETERINARY MICROBIOLOGY142(1-2)pp. 34-40 ELSEVIER SCIENCE BV
    Morgan SB, Graham SP, Salguero J, Steinbach F, Frossard JP (2012) Increased pathology during infection with an atypical European porcine Arterivirus correlates with an enhanced adaptive immune response, IMMUNOLOGY137pp. 641-641 WILEY-BLACKWELL
    Graham SP, Pelle R, Yamage M, Mwangi DM, Honda Y, Mwakubambanya RS, de Villiers EP, Abuya E, Awino E, Gachanja J, Mbwika F, Muthiani AM, Muriuki C, Nyanjui JK, Onono FO, Osaso J, Riitho V, Saya RM, Ellis SA, McKeever DJ, MacHugh ND, Gilbert SC, Audonnet J-C, Morrison WI, van der Bruggen P, Taracha ELN (2008) Characterization of the fine specificity of bovine CD8 T-Cell responses to defined antigens from the protozoan parasite Theileria parva, INFECTION AND IMMUNITY76(2)pp. 685-694 AMER SOC MICROBIOLOGY
    Macdonald IK, Harkiolaki M, Hunt L, Connelley T, Carroll AV, MacHugh ND, Graham SP, Jones EY, Morrison WI, Flower DR, Ellis SA (2010) MHC Class I Bound to an Immunodominant Theileria parva Epitope Demonstrates Unconventional Presentation to T Cell Receptors,PLOS PATHOGENS6(10)ARTN e1001149 PUBLIC LIBRARY SCIENCE
    Salguero FJ, Frossard JP, Rebel JMJ, Stadejek T, Morgan SB, Graham SP, Steinbach F (2015) Host-pathogen interactions during porcine reproductive and respiratory syndrome virus 1 infection of piglets, Virus Research202pp. 135-143
    © 2015.Porcine reproductive and respiratory syndrome (PRRS) is a major disease affecting pigs worldwide and resulting in considerable economic losses. While PRRS is a global phenomenon, the causative viruses PRRSV-1 (first detected in Europe) and PRRSV-2 (isolated in North America) are genetically and biologically distinct. In addition, the disease outcome is directly linked to co-infections associated with the porcine respiratory disease complex and the host response is variable between different breeds of pigs. It is therefore warranted when studying the pathogenesis of PRRS to consider each viral genotype separately and apply careful consideration to the disease model studied. We here review the respiratory pig model for PRRSV-1, with a focus on a recent set of studies conducted with carefully selected virus strains and pigs, which may serve as both a baseline and benchmark for future investigation.
    Graham SP, Jones GE, MacLean M, Livingstone M, Entrican G (1995) Recombinant ovine interferon gamma inhibits the multiplication of Chlamydia psittaci in ovine cells., J Comp Pathol112(2)pp. 185-195
    The local production of interferon-gamma (IFN-gamma) in sheep in response to Chlamydia psittaci was measured by cannulation of the efferent lymph duct draining the site of challenge inoculation. Peak production of IFN-gamma (256 U/ml) was detected 24 h after challenge. Based on these physiological data, functional studies were carried out in vitro to determine the effect of recombinant ovine (rOv) IFN-gamma on the multiplication of C. psittaci in ovine fibroblasts. IFN-gamma inhibited the multiplication of C. psittaci in ovine cells over a range of concentrations (250 U/ml to 2.5 U/ml) in a dose-dependent manner. The inhibition of chlamydial multiplication was most pronounced when cells were treated with rOvIFN-gamma for 24 h before infection. The addition of exogenous L-tryptophan (500 micrograms/ml) to cultures within 48th of infection abrogated the anti-chlamydial effect of rOvIFN-gamma thus suggesting that tryptophan deprivation is an anti-chlamydial mechanism induced by rOvIFN-gamma in these ovine cells.
    Singleton H, Frossard J-P, Bodman-Smith KB, Graham SP, Steinbach F (2011) The effect of cytokines on porcine monocytes and alveolar macrophages, IMMUNOLOGY135pp. 90-90
    Franzoni G, Graham SP, Giudici SD, Bonelli P, Pilo G, Anfossi AG, Pittau M, Nicolussi PS, Laddomada A, Oggiano A (2017) Characterization of the interaction of African swine fever virus with monocytes and derived macrophage monocytes and derived macrophage subsets.,Veterinary Microbiology198pp. 88-98 Elsevier
    African swine fever (ASF) is a devastating disease for which there is no vaccine available. The ASF virus (ASFV) primarily infects cells of the myeloid lineage and this tropism is thought to be crucial for disease pathogenesis. A detailed in vitro characterization of the interactions of a virulent Sardinian isolate (22653/14) and a tissue culture adapted avirulent strain (BA71V) of ASFV with porcine monocytes, un-activated (moM¦), classically (moM1) and alternatively (moM2) activated monocyte-derived macrophages was conducted in an attempt to better understand this relationship. Using a multiplicity-of-infection (MOI) of 1, both viruses were able to infect monocytes and macrophage subsets, but BA71V presented a reduced ability to infect moM1 compared to 22653/14, with higher expression of early compared to late proteins. Using an MOI of 0.01, only 22653/14 was able to replicate in all the macrophage subsets, with initially lowest in moM1 and moM2Å No differences were observed in the expression of CD163 between ASFV infected and uninfected bystander cells. ASFV down-regulated CD16 expression but did not modulate MHC class II levels in monocytes and macrophage subsets. BA71V-infected but not 22653/14-infected moM¦ and moM2 presented with a reduced expression of MHC class I compared to the mock-infected controls. Higher levels of IL-18, IL1-² and IL-1± were released from moM1 after infection with BA71V compared to 22653/14 or mock-infected control. These results revealed differences between these ASFV strains, suggesting that virulent isolates have evolved mechanisms to counteract activated macrophages responses, promoting their survival, dissemination in the host and so ASF pathogenesis.
    Morgan SB, Graham SP, Salguero FJ, Sánchez Cordón PJ, Mokhtar H, Rebel JMJ, Weesendorp E, Bodman-Smith KB, Steinbach F, Frossard JP (2013) Increased pathogenicity of European porcine reproductive and respiratory syndrome virus is associated with enhanced adaptive responses and viral clearance, Veterinary Microbiology163(1-2)pp. 13-22
    Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases of swine worldwide. Since its first emergence in 1987 the PRRS virus (PRRSV) has become particularly divergent with highly pathogenic strains appearing in both Europe and Asia. However, the underlying mechanisms of PRRSV pathogenesis are still unclear. This study sets out to determine the differences in pathogenesis between subtype 1 and 3 strains of European PRRSV (PRRSV-I), and compare the immune responses mounted against these strains. Piglets were infected with 3 strains of PRRSV-I: Lelystad virus, 215-06 a British field strain and SU1-bel from Belarus. Post-mortem examinations were performed at 3 and 7 days post-infection (dpi), and half of the remaining animals in each group were inoculated with an Aujeszky's disease (ADV) vaccine to investigate possible immune suppression resulting from PRRSV infection. The subtype 3 SU1-bel strain displayed greater clinical signs and lung gross pathology scores compared with the subtype 1 strains. This difference did not appear to be caused by higher virus replication, as viraemia and viral load in broncho-alveolar lavage fluid (BALF) were lower in the SU1-bel group. Infection with SU1-bel induced an enhanced adaptive immune response with greater interferon (IFN)-³ responses and an earlier PRRSV-specific antibody response. Infection with PRRSV did not affect the response to vaccination against ADV. Our results indicate that the increased clinical and pathological effect of the SU1-bel strain is more likely to be caused by an enhanced inflammatory immune response rather than higher levels of virus replication. © 2012.
    Graham SP, Saya R, Awino E, Ngugi D, Nyanjui JK, Hecker R, Taracha EL, Nene V (2007) Immunostimulatory CpG oligodeoxynucleotides enhance the induction of bovine CD4+ cytotoxic T-lymphocyte responses against the polymorphic immunodominant molecule of the protozoan parasite Theileria parva., Vet Immunol Immunopathol115(3-4)pp. 383-389
    Enhancement of the induction of cytotoxic T-cell responses by immunostimulatory CpG oligodeoxynucleotides has been described in humans and mouse models. The present study attempted to address whether CpG has a similar effect in cattle. Immunisation of cattle with a recombinant form of the polymorphic immunodominant molecule from Theileria parva emulsified with immunostimulatory CpG oligodeoxynucleotides in adjuvant had no effect on the induction of antibody responses including the isotype profile, but significantly enhanced the induction of cytolytic responses that were mediated by CD4+CD3+ T cells utilizing the perforin-granzyme pathway.
    Graham SP, Honda Y, Pellé R, Mwangi DM, Glew EJ, de Villiers EP, Shah T, Bishop R, van der Bruggen P, Nene V, Taracha EL (2007) A novel strategy for the identification of antigens that are recognised by bovine MHC class I restricted cytotoxic T cells in a protozoan infection using reverse vaccinology.,Immunome Res3pp. 2-2
    BACKGROUND: Immunity against the bovine protozoan parasite Theileria parva has previously been shown to be mediated through lysis of parasite-infected cells by MHC class I restricted CD8+ cytotoxic T lymphocytes. It is hypothesized that identification of CTL target schizont antigens will aid the development of a sub-unit vaccine. We exploited the availability of the complete genome sequence data and bioinformatics tools to identify genes encoding secreted or membrane anchored proteins that may be processed and presented by the MHC class I molecules of infected cells to CTL. RESULTS: Of the 986 predicted open reading frames (ORFs) encoded by chromosome 1 of the T. parva genome, 55 were selected based on the presence of a signal peptide and/or a transmembrane helix domain. Thirty six selected ORFs were successfully cloned into a eukaryotic expression vector, transiently transfected into immortalized bovine skin fibroblasts and screened in vitro using T. parva-specific CTL. Recognition of gene products by CTL was assessed using an IFN-gamma ELISpot assay. A 525 base pair ORF encoding a 174 amino acid protein, designated Tp2, was identified by T. parva-specific CTL from 4 animals. These CTL recognized and lysed Tp2 transfected skin fibroblasts and recognized 4 distinct epitopes. Significantly, Tp2 specific CD8+ T cell responses were observed during the protective immune response against sporozoite challenge. CONCLUSION: The identification of an antigen containing multiple CTL epitopes and its apparent immunodominance during a protective anti-parasite response makes Tp2 an attractive candidate for evaluation of its vaccine potential.
    Walters A, Riitho V, Somavarapu S, Rumenapf T, Krey T, Oviedo-Orta E, Steinbach F, Graham S (2011) Assessment of poly (Lactic:glycolic acid) nanoparticles as an antigen delivery system for cattle, IMMUNOLOGY135pp. 59-59 WILEY-BLACKWELL
    Adamson R, Lyons K, Sharrard M, Kinnaird J, Swan D, Graham S, Shiels B, Hall R (2001) Transient transfection of Theileria annulata., Mol Biochem Parasitol114(1)pp. 53-61
    We have developed a method to transiently transfect infective, uninucleate, Theileria annulata sporozoites. Transfection vectors have been constructed using a number of T. annulata 5' gene flanking sequences linked to the enhanced green fluorescence protein (eGFP) reporter gene. Sporozoites were transfected with these constructs using the lipid transfection agent SuperFect, then allowed to infect purified bovine mononuclear cells (PBMs). Green fluorescence was observed in developing trophozoites, 36-40 h post infection, using constructs containing the upstream regions of the T. annulata Hsp70, T. annulata merozite surface antigen 1 (TamS1) and T. annulata macroschizont-specific AT hook-containing protein2 (TashAT2) genes. A construct with the 5' TamS1 upstream sequence in reverse orientation gave no detectable fluorescence indicating fluorescence was derived by expression from the T. annulata promoter. A cytomegalovirus (CMV) promoter construct showed no activity in this stage of the parasite. However, when this construct was introduced directly into schizont-infected cells by electroporation, fluorescence was observed in the bovine cells but not the schizont. We describe the significance of these results in relation to novel control strategies and the fundamental biology of Theileria parasites.
    Singleton H, graham SP, Bodman-Smith KB, Frossard JP, Steinbach F (2016) Establishing porcine monocyte-derived macrophage and dendritic cell systems for studying the interaction with PRRSV-1,Frontiers in Microbiology Frontiers Media
    Monocyte-derived macrophages (MoMØ) and monocyte-derived dendritic cells (MoDC) are two model systems well established in human and rodent systems that can be used to study the interaction of pathogens with host cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect myeloid cells, such as macrophages (MØ) and dendritic cells (DC). Therefore, this study aimed to establish systems for the differentiation and characterization of MoMØ and MoDC for subsequent infection with PRRSV-1. M-CSF differentiated monocyte-derived macrophages (MoMØ) were stimulated with activators for classical (M1) or alternative (M2) activation. GM-CSF and IL-4 generated monocyte-derived dendritic cells (MoDC) were activated with the well established maturation cocktail containing PAMPs and cytokines. In addition, MoMØ and MoDC were treated with dexamethasone and IL-10, which are known immuno-suppressive reagents. Cells were characterized by morphology, phenotype and function and porcine MØ subsets highlighted some divergence from described human counterparts, while MoDC, appeared more similar to mouse and human DCs. The infection with PRRSV-1 strain Lena demonstrated different replication kinetics between MoMØ and MoDC and within subsets of each cell type. While MoMØ susceptibility was significantly increased by dexamethasone and IL-10 with an accompanying increase in CD163/CD169 expression, MoDC supported only a minimal replication of PRRSV These findings underline the high variability in the susceptibility of porcine myeloid cells towards PRRSV-1 infection.
    Pelle R, Graham SP, Njahira MN, Osaso J, Saya RM, Odongo DO, Toye PG, Spooner PR, Musoke AJ, Mwangi DM, Taracha EL, Morrison WI, Weir W, Silva JC, Bishop RP (2011) Two Theileria parva CD8 T cell antigen genes are more variable in buffalo than cattle parasites, but differ in pattern of sequence diversity.,PLoS One6(4)pp. e19015-e19015
    BACKGROUND: Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8(+) T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8(+) T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8(+) T-cell epitopes, and to analyse the sequences for evidence of selection. METHODOLOGY/PRINCIPAL FINDINGS: Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (<12%) in Tp1 and in 320 positions (<61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle. CONCLUSIONS/SIGNIFICANCE: The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.
    Morgan SB, Frossard JP, Pallares FJ, Gough J, Stadejek T, Graham SP, Steinbach F, Drew TW, Salguero FJ (2014) Pathology and Virus Distribution in the Lung and Lymphoid Tissues of Pigs Experimentally Inoculated with Three Distinct Type 1 PRRS Virus Isolates of Varying Pathogenicity., Transbound Emerg Dis63(3)pp. 285-295
    Porcine reproductive and respiratory syndrome (PRRS) continues to be the most economically important disease of swine worldwide. The appearance of highly pathogenic PRRS virus (PRRSV) strains in Europe and Asia has raised concerns about this disease and initiated increased efforts to understand the pathogenesis. In this study, we have compared the pathology and the virus distribution in tissues of pigs experimentally inoculated with three different genotype 1 PRRSV isolates. Sixty 5-week-old pigs were inoculated intranasally with a) the Lelystad virus (LV), b) a field strain from the UK causing respiratory clinical signs (UK) or c) a highly pathogenic strain from Belarus (BE). Sixteen animals were mock-infected and used as controls. The animals were euthanized at 3, 7 and 35 days post-infection (dpi), and lung and lymphoid tissues collected for histopathological examination and PRRSV detection by immunohistochemistry (IHC). Histopathological lesions consisted of interstitial pneumonia with mononuclear cell infiltrates in the lungs, lymphoid depletion, apoptosis and follicular hyperplasia in the spleen, lymph nodes and tonsil and lymphoid depletion in the thymus. Porcine reproductive and respiratory syndrome virus was detected mainly in monocytes-macrophages. BE-infected animals showed the highest pathological scores and the highest presence of virus at 3 and 7 dpi, followed by the UK field strain and then LV. Moderate lesions were observed at 35 dpi with lesser detection of PRRSV by IHC in each infected group. The highly pathogenic BE strain induced more severe pathology in both lungs and lymphoid organs of pigs compared with the classic field isolate and the prototype LV. The increased severity of pathology was in correlation with the presence of a higher number of PRRSV-infected cells in the tissues.
    Jansen CA, de Geus E, van Haarlem DA, van de Haar PM, Loendt BZ, Graham SP, Goebel TW, van Eden W, Brookes SM, Vervelde L (2013) Differential lung NK cell responses in avian influenza virus infected chickens correlate with pathogenicity,SCIENTIFIC REPORTS3ARTN 2478 NATURE PUBLISHING GROUP
    Hemmink JD, Weir W, MacHugh ND, Graham SP, Patel E, Paxton E, Shiels B, Toye PG, Morrison WI, Pelle R (2016) Limited genetic and antigenic diversity within parasite isolates used in a live vaccine against Theileria parva, International Journal for Parasitology46(8)pp. 495-506
    An infection and treatment protocol is used to vaccinate cattle against Theileria parva infection. Due to incomplete cross-protection between different parasite isolates, a mixture of three isolates, termed the Muguga cocktail, is used for vaccination. While vaccination of cattle in some regions provides high levels of protection, some animals are not protected against challenge with buffalo-derived T. parva. Knowledge of the genetic composition of the Muguga cocktail vaccine is required to understand how vaccination is able to protect against field challenge and to identify the potential limitations of the vaccine. The aim of the current study was to determine the extent of genetic and antigenic diversity within the parasite isolates that constitute the Muguga cocktail. High throughput multi-locus sequencing of antigen-encoding loci was performed in parallel with typing using a panel of micro- and minisatellite loci. The former focused on genes encoding CD8+ T cell antigens, believed to be relevant to protective immunity. The results demonstrate that each of the three component stocks of the cocktail contains limited parasite genotypic diversity, with single alleles detected at many gene/satellite loci and, moreover, that two of the components show a very high level of similarity. Thus, the vaccine incorporates very little of the genetic and antigenic diversity observed in field populations of T. parva. The presence of alleles at low frequency (<10%) within vaccine component populations also points to the possibility of variability in the content of vaccine doses and the potential for loss of allelic diversity during tick passage. The results demonstrate that there is scope to modify the content of the vaccine in order to enhance its diversity and thus its potential for providing broad protection. The ability to accurately quantify genetic diversity in vaccine component stocks will facilitate improved quality control procedures designed to ensure the long-term efficacy of the vaccine.
    Thi KAN, Koets AP, Vordermeier M, Jervis PJ, Cox LR, Graham SP, Santema WJ, Moody DB, van Calenbergh S, Zajonc DM, Besra GS, Van Rhijn I (2013) The bovine CD1D gene has an unusual gene structure and is expressed but cannot present -galactosylceramide with a C26 fatty acid, INTERNATIONAL IMMUNOLOGY25(2)pp. 91-98 OXFORD UNIV PRESS
    Tinega AN, Pelle R, Kang'a S, Gicheru MM, Taracha ELN, Nene V, Graham SP (2009) Fusion of a cell penetrating peptide from HIV-1 TAT to the Theileria parva antigen Tp2 enhances the stimulation of bovine CD8(+) T cell responses, VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY130(1-2)pp. 107-113 ELSEVIER SCIENCE BV
    Amarilla S, Gomez-Laguna J, Carrasco L, Rodriguez-Gomez I, Ocerin J, Graham SP, Frossard J, Steinbach F, Salguero Bodes FJ (2016) Thymic depletion of lymphocytes is associated with the virulence of PRRSV-1 strains,Veterinary Microbiology188pp. 47-58 Elsevier
    Porcine reproductive and respiratory syndrome virus (PRRSV) exists as two distinct viruses, type 1 (PRRSV-1) and type 2 (PRRSV-2). Atrophy of the thymus in PRRSV-2 infected piglets has been associated with a loss of thymocytes. The present study aimed to evaluate the impact of PRRSV-1 strains of differing virulence on the thymus of infected piglets by analysing the histomorphometry, the presence of apoptotic cells and cells producing cytokines. Thymic samples were taken from animals experimentally infected (with LV, SU1-bel, and 215-06 strains) or mock inoculated animals at 3, 7 and 35 days post-infection (dpi) and processed for histopathological and immunohistochemical analyses. PRRSV antigen was detected in the thymus from 3dpi until the end of the study in all virus-infected animals with the highest numbers of infected cells detected in SU1-bel group. The histomorphometry analysis and counts of CD3+ thymocytes in the thymic cortex displayed significant differences between strains at different time-points (p d 0.011), with SU1-bel group showing the most severe changes at 7dpi. Cell death displayed statistically significant increase in the cortex of all infected animals, with SU1-bel group showing the highest rate at 3 and 7dpi. The number of cells immunostained against IL-1±, TNF-± and IL-10 were predominantly detected in the medulla (p d 0.01). An increase in the number of TNF-± and IL-10 positive cells was observed in LV and SU-1bel groups. Our results demonstrate that different PRRSV-1 strains induced depletion of the thymic cortex due to apoptosis of thymocytes and that the most severe depletion was associated with the highly virulent SU1-bel strain.
    Edwards J, Everett H, Pedrera M, Mokhtar H, Marchi E, Soldevila F, Kaveh D, Hogarth P, Johns H, Nunez-Garcia J, Steinbach F, Crooke H, Graham SP (2017) CD1- and CD1+ porcine blood dendritic cells are enriched for the orthologues of the two major mammalian conventional subsets,Scientific Reports740942 Nature Publishing Group
    Conventional dendritic cells (cDC) are professional antigen-presenting cells that induce immune activation or tolerance. Two functionally specialised populations, termed cDC1 and cDC2, have been described in humans, mice, ruminants and recently in pigs. Pigs are an important biomedical model species and a key source of animal protein; therefore further understanding of their immune system will help underpin the development of disease prevention strategies. To characterise cDC populations in porcine blood, DC were enriched from PBMC by CD14 depletion and CD172a enrichment then stained with a lineage mAbs (Lin; CD3, CD8±, CD14 and CD21) and mAbs specific for CD172a, CD1 and CD4. Two distinct porcine cDC subpopulations were FACSorted CD1- cDC (Lin-CD172+CD1-CD4-) and CD1+ cDC (Lin- CD172a+CD1+CD4-), and characterised by phenotypic and functional analyses. CD1+ cDC were distinct from CD1- cDC, expressing higher levels of CD172a, MHC class II and CD11b. Following TLR stimulation, CD1+ cDC produced IL-8 and IL-10 while CD1- cDC secreted IFN-±, IL-12 and TNF-±. CD1- cDC were superior in stimulating allogeneic T cell responses and in cross-presenting viral antigens to CD8 T cells. Comparison of transcriptional profiles further suggested that the CD1- and CD1+ populations were enriched for the orthologues of cDC1 and cDC2 subsets respectively.
    Mokhtar H, Biffar L, Somavarapu S, Frossard J, Mcgowan S, Pedrera M, Strong R, Edwards J, Garcia-Durán M, Rodriguez M, Stewart G, Steinbach F, Graham S (2017) Evaluation of hydrophobic chitosan-based particulate formulations of porcine reproductive and respiratory syndrome virus vaccine candidate T cell antigens,Veterinary Microbiology209pp. 66-74 Elsevier
    PRRS control is hampered by the inadequacies of existing vaccines to combat the extreme diversity of circulating viruses. Since immune clearance of PRRSV infection may not be dependent on the development of neutralising antibodies and the identification of broadly- neutralising antibody epitopes have proven elusive we hypothesised that conserved T cell antigens represent potential candidates for development of a novel PRRS vaccine. Previously we had identified the M and NSP5 proteins as well-conserved targets of polyfunctional CD8 and CD4 T cells. To assess their vaccine potential, peptides representing M and NSP5 were encapsulated in hydrophobically-modified chitosan particles adjuvanted by incorporation of a synthetic multi-TLR2/TLR7 agonist and coated with a model B cell PRRSV antigen. For comparison, empty particles and adjuvanted particles encapsulating inactivated PRRSV-1
    Riitho V, Walters A, Somavarapu S, Lamp B, Rümenapf T, Krey T, Rey F, Oviedo-Orta E, Stewart G, Locker N, Steinbach F, Graham S (2017) Design and evaluation of the immunogenicity and efficacy of a biomimetic particulate formulation of viral antigens,Scientific Reports713743 Nature Publishing Group
    Subunit viral vaccines are typically not as efficient as live attenuated or inactivated vaccines at inducing protective immune responses. This paper describes an alternative ?biomimetic? technology; whereby viral antigens were formulated around a polymeric shell in a rationally arranged fashion with a surface glycoprotein coated on to the surface and non-structural antigen and adjuvant encapsulated. We evaluated this model using BVDV E2 and NS3 proteins formulated in poly-(D, L-lactic-co-glycolic acid) (PLGA) nanoparticles adjuvanted with polyinosinic:polycytidylic acid (poly(I:C) as an adjuvant (Vaccine-NP). This Vaccine-NP was compared to ovalbumin and poly(I:C) formulated in a similar manner (Control-NP) and a commercial adjuvanted inactivated BVDV vaccine (IAV), all inoculated subcutaneously and boosted prior to BVDV-1 challenge. Significant virus-neutralizing activity, and E2 and NS3 specific antibodies were observed in both Vaccine-NP and IAV groups following the booster immunisation. IFN-³ responses were observed in ex vivo PBMC stimulated with E2 and NS3 proteins in both vaccinated groups. We observed that the protection afforded by the particulate vaccine was comparable to the licenced IAV formulation. In conclusion, the biomimetic particulates showed a promising immunogenicity and efficacy profile that may be improved by virtue of being a customisable mode of delivery.
    Franzoni G, Graham S, Sanna G, Angioi P, Fiori M, Anfossi A, Amadori M, Giudici S, Oggiano A (2018) Interaction of porcine monocyte-derived dendritic cells with African swine fever viruses of diverse virulence,Veterinary Microbiology216pp. 190-197 Elsevier
    African swine fever (ASF) is a devastating disease for which there is no vaccine. The ASF virus (ASFV) can infect dendritic cell (DC), but despite the critical role these cells play in induction of adaptive immunity, few studies investigated their response to ASFV infection. We characterized the in vitro interactions of porcine monocyte-derived DCs (moDC) with a virulent (22653/14), a low virulent (NH/P68) and an avirulent (BA71V) ASFV strain. At a high multiplicity of infection (MOI/=/1), all three strains infected immature moDC. Maturation of moDC, with IFN-±/TNF-±, increased susceptibility to infection with 22653/14 and other virulent strains, but reduced susceptibility to NH/P68 and BA71V. The reduced moDC susceptibility to BA71V/NH/P68 was IFN-± mediated, whereas increased susceptibility to 22653/14 was induced by TNF-±. Using an MOI of 0.01, we observed that BA71V replicated less efficiently in moDC compared to the other isolates and we detected increased replication of NH/P68 compared to 22653/14. We observed that BA71V and NH/P68, but not 22653/14, downregulated expression of MHC class I on infected cells. All three strains decreased CD16 expression on moDC, whereas ASFV infection resulted in CD80/86 down-regulation and MHC class II DR up-regulation on mature moDC. None of the tested strains induced a strong cytokine response to ASFV and only modest IL-1± was released after BA71V infection. Overall our results revealed differences between strains and suggest that ASFV has evolved mechanisms to replicate covertly in inflammatory DC, which likely impairs the induction of an effective immune response.
    Strong Rebecca, Graham Simon, La Rocca Anna, Raue Rudiger, Vangeel Ilse, Steinbach Falko (2018) Establishment of a bovine viral diarrhoea virus type 2 intranasal challenge model for assessing vaccine efficacy,Frontiers in Veterinary Science5(24) Frontiers Media
    The objective of this study was to develop a bovine viral diarrhoea virus type 2 (BVDV-2) challenge model suitable for evaluation of efficacy of BVDV vaccines; a model that mimics natural infection and induces clear leucopoenia and viraemia. Clinical, haematological and virological parameters were evaluated after infection of two age groups of calves (3 months and 9 months) with two BVDV-2 strains (1362727 and 502643). Calves became pyrexic between 8-9 days post inoculation and exhibited symptoms such as nasal discharge, mild depression, cough and inappetance. Leukopenia with associated lymphopenia and neutropenia was evident in all groups with lowest leukocyte and lymphocyte counts reached 8dpi and granulocyte counts between 11 and 16 dpi, dependent on the strain and age of the calves. A more severe thrombocytopeania was seen in those animals inoculated with strain 1362727. Leukocyte and nasal swab samples were positive by virus isolation, as early as 3 dpi and 2 dpi respectively, independent of the inocula used. All calves seroconverted with high levels of BVDV-2 neutralising antibodies. BVDV RNA was evident as late as 90 dpi and provides the first evidence of the presence of replicating virus long after recovery from BVDV-2 experimental infection. In summary, moderate disease can be induced after experimental infection of calves with a low titre (104.8 or 105.0 TCID50) of virulent BVDV-2, with leucopoenia, thrombocytopoenia, viraemia and virus shedding. These strains represent an attractive model to assess the protective efficacy of existing and new vaccines against BVDV-2.
    Charleston B, Graham Simon (2018) Recent advances in veterinary applications of structural vaccinology,Current Opinion in Virology29pp. 33-38 Elsevier
    The deployment of effective veterinary vaccines has had a major impact on improving food security and consequently human health. Effective vaccines were essential for the global eradication of Rinderpest and the control and eradication of foot-and-mouth disease in some regions of the world. Effective vaccines also underpin the development of modern intensive food production systems such as poultry and aquaculture. However, for some high consequence diseases there are still significant challenges to develop effective vaccines. There is a strong track record in veterinary medicine of early adoption of new technologies to produce vaccines. Here we provide examples of new technologies to interrogate B cell responses and using structural biology to improve antigens.
    Strong Rebecca, Graham Simon P., La Rocca S. A., Raue Rudiger, Vangeel Ilse, Steinbach Falko (2018) Establishment of a Bovine Viral Diarrhea Virus Type 2 Intranasal Challenge Model for Assessing Vaccine Efficacy,Frontiers in Veterinary Science524pp. 24-1 Frontiers Media
    The objective of this study was to develop a bovine viral diarrhea virus type 2 (BVDV-2) challenge model suitable for evaluation of efficacy of BVDV vaccines; a model that mimics natural infection and induces clear leukopenia and viremia. Clinical, hematological and virological parameters were evaluated after infection of two age groups of calves (3 and 9 months) with two BVDV-2 strains (1362727 and 502643). Calves became pyrexic between 8 and 9 days post inoculation and exhibited symptoms, such as nasal discharge, mild depression, cough, and inappetence. Leukopenia with associated lymphopenia and neutropenia was evident in all groups with lowest leukocyte and lymphocyte counts reached 8 dpi and granulocyte counts between 11 and 16 dpi, dependent on the strain and age of the calves. A more severe thrombocytopenia was seen in those animals inoculated with strain 1362727. Leukocyte and nasal swab samples were positive by virus isolation, as early as 3 dpi and 2 dpi respectively, independent of the inocula used. All calves seroconverted with high levels of BVDV-2 neutralizing antibodies. BVDV RNA was evident as late as 90 dpi and provides the first evidence of the presence of replicating virus long after recovery from BVDV-2 experimental infection. In summary, moderate disease can be induced after experimental infection of calves with a low titer of virulent BVDV-2, with leukopenia, thrombocytopenia, viremia, and virus shedding. These strains represent an attractive model to assess the protective efficacy of existing and new vaccines against BVDV-2.
    Singleton Helen, Graham Simon P., Frossard Jean-Pierre, Bodman-Smith Katherine B., Steinbach Falko (2018) Infection of monocytes with European porcine reproductive and respiratory syndrome virus (PRRSV-1) strain Lena is significantly enhanced by dexamethasone and IL-10,Virology517pp. 199-207 Elsevier
    Monocytes are considered refractory to porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) infection. However, monocytes are only short-lived in blood, being able to differentiate into macrophages and dendritic cells (DC). It was therefore merited to revisit PRRSV-1 interaction with monocytes, particularly those treated with cytokines influencing monocyte biology. Thus, several factors were screened, particularly those modulating monocyte differentiation and expression of putative PRRSV-1 receptors (CD169 and CD163). M-CSF, known to stimulate macrophage differentiation, did not increase their susceptibility to PRRSV-1. Nor did GM-CSF or IL-4, known drivers for monocyte-derived DC (MoDC) differentiation. In contrast, monocyte treatment with IL-10 or the corticosteroid, dexamethasone, known to be potent suppressors of monocyte differentiation, was correlated with increased susceptibility to PRRSV-1 infection. While this effect was strongly correlated to CD163 and CD169 expression, our data suggest that receptor expression is not the only factor driving successful infection of PPRSV-1 in monocytes.
    Schwartz J, Hemmink J, Graham Simon, Tchilian E, Charleston B, Hammer S, Ho C-S, Hammond J (2018) The major histocompatibility complex homozygous inbred Babraham pig as a resource for veterinary and translational medicine,HLA92(1)pp. 40-43 Wiley
    The Babraham pig is a highly inbred breed first developed in the United Kingdom approximately 50 years ago. Previous reports indicate a very high degree of homozygosity across the genome, including the major histocompatibility complex (MHC) region, but confirmation of homozygosity at the specific MHC loci was lacking. Using both direct sequencing and PCR?based sequence?specific typing, we confirm that Babraham pigs are essentially homozygous at their MHC loci and formalise their MHC haplotype as Hp?55.6. This enhances the utility of the Babraham pig as a useful biomedical model for studies in which controlling for genetic variation is important.
    Riitho Victor, Larska Magdalena, Strong Rebecca, La Rocca Anna, Locker Nicolas, Alenius Stefan, Steinbach Falko, Liu Lihong, Uttenthal Åse, Graham Simon P. (2018) Comparative analysis of adaptive immune responses following experimental infections of cattle with bovine viral diarrhoea virus-1 and an Asiatic atypical ruminant pestivirus,Vaccine36(30)pp. 4494-4500 Elsevier
    Atypical ruminant pestiviruses are closely related to the two bovine viral diarrhoea virus (BVDV) species, BVDV-1 and BVDV-2. While there is evidence of cross-protective immune responses between BVDV-1 and BVDV-2, despite antigenic differences, there is little information on the antigenic cross-reactivity with atypical ruminant pestiviruses. The aim of this study was therefore to assess the specificity of antibody and T cell responses induced by experimental infection of calves with BVDV-1 strain Ho916, Th/04_KhonKaen (TKK), an Asiatic atypical ruminant pestivirus, or co-infection with both viruses. Homologous virus neutralization was observed in sera from both single virus infected and co-infected groups, while cross-neutralization was only observed in the TKK infected group. T cell IFN-³ responses to both viruses were observed in the TKK infected animals, whereas Ho916 infected calves responded better to homologous virus. Specifically, IFN-³ responses to viral non-structural protein, NS3, were observed in all infected groups while responses to viral glycoprotein, E2, were virus-specific. Broader antigen-specific cytokine responses were observed with similar trends between inoculation groups and virus species. The limited T cell and antibody immune reactivity of Ho916 inoculated animals to TKK suggests that animals vaccinated with current BVDV-1-based vaccines may not be protected against atypical ruminant pestiviruses.
    Soldevila Ferran, Edwards Jane C., Graham Simon P., Stevens Lisa M., Crudgington Bentley, Crooke Helen R., Werling Dirk, Steinbach Falko (2018) Characterisation of the myeloid cell populations? resident in the porcine palatine tonsil,Frontiers in Immunology91800pp. 1-16 Frontiers Media
    The palatine tonsil is the portal of entry for food and air, and is continuously subjected to environmental challenges including pathogens which use the tonsil and pharynx as a primary site of replication. In pigs, this includes the viruses causing porcine respiratory and reproductive syndrome, and classical and African swine fever; diseases which have impacted the pig production industry globally. Despite the importance of tonsils in host defence, little is known regarding the phenotype of the myeloid cells resident in the porcine tonsil. Here, we have characterised five myeloid cell populations that align to orthologous populations defined in other mammalian species: a CD4+ plasmacytoid DC (pDC) defined by expression of the conserved markers E2.2 and IRF-7, a conventional dendritic cell (cDC1) population expressing CADM1highCD172alow and high levels of XCR1 able to activate allogeneic CD4 and CD8 T cells; a cDC2 population of CADM1dim cells expressing FLT3, IRF4 and CSF1R with an ability to activate allogeneic CD4 T cells; CD163+ macrophages (MŸs) defined by high levels of endocytosis and responsiveness to LPS and finally a CD14+ population likely derived from a myelo-monocytic lineage, which showed the highest levels of endocytosis, a capacity for activation of CD4+ memory cells, combined with lower relative expression of FLT3. Increased knowledge regarding the phenotypic and functional properties of myeloid cells resident in porcine tonsil, will enable these cells to be targeted for future vaccination strategies to current and emerging porcine viruses.
    Hodgson Sophia, Moffat Katy, Hill Holly, Flannery John T., Graham Simon, Baron Michael D., Darpel Karin E. (2018) Comparison of the immunogenicity and cross-lineage efficacy of live attenuated peste des petits ruminants virus vaccines PPRV/Nigeria/75/1 and PPRV/Sungri/96.,Journal of Virology American Society for Microbiology
    Peste des petits ruminants (PPR) is a severe disease of goats and sheep that is widespread in Africa, the Middle East and Asia. Several effective vaccines exist for the disease, based on attenuated strains of the virus (PPRV) that causes PPR. While the efficacy of these vaccines has been established by use in the field, the nature of the protective immune response has not been determined. In addition, while the vaccine derived from PPRV/Nigeria/75/1 (N75) is used in many countries, those developed in India have never been tested for their efficacy outside that country. We have studied the immune response in goats to vaccination with either N75 or the main Indian vaccine, which is based on isolate PPRV/India/Sungri/96 (S96). In addition, we have compared the ability of these two vaccines, in parallel, to protect animals against challenge with pathogenic viruses from the four known genetic lineages of PPRV, representing viruses from different parts of Africa as well as Asia. These studies showed that, while N75 elicited a stronger antibody response than S96, as measured by both ELISA and virus neutralisation, S96 resulted in more pronounced cellular immune responses, as measured by virus antigen-induced proliferation and interferon gamma production. While both vaccines induced comparable numbers of PPRV-specific CD8+ T cells, S96 induced a higher number of CD4+ T cells specifically responding to virus. Despite these quantitative and qualitative differences in the immune responses following vaccination, both vaccines gave complete clinical protection against challenge with all four lineages of PPRV.
    Bovine viral diarrhoea virus (BVDV) is an important pathogen that causes infectious disease of cattle worldwide and results in significant economic losses. Vaccination has long been used as a tool for control of BVDV but inadequacies of existing vaccines have hampered eradication efforts. Attempts to develop sub-unit vaccines have focused on the structural envelope protein E2, which is a dominant target of neutralising antibodies and as well as CD4 T cell responses. This study aimed to rationally address the development of more efficacious vaccines by characterising the kinetics and specificity of T cell responses to a BVDV type 1 peptide library in calves rendered immune to BVDV following recovery from experimental infection. Upon identification of E2 and NS3 as the dominant targets of CD4 T cell responses, we assessed whether T cells induced by one virus genotype were capable of responding to a heterologous virus genotype and to identified E2 and NS3 as targets of genotype-specific and genotype transcending responses, respectively. This finding strengthened the argument for inclusion of both antigens in a subunit vaccine formulation. A nanoparticulate formulation of E2 and NS3 adjuvanted with poly(I:C) was shown to induce protective responses comparable to a commercial available BVDV vaccine in a vaccination and challenge experiment. It is hoped that the data generated will have implications for the design of improved vaccines against BVD.
    McLean Rebecca K., Graham Simon P. (2019) Vaccine development for Nipah virus infection in pigs,Frontiers in Veterinary Science Frontiers Media
    Nipah virus (NiV) causes a severe and often fatal neurological disease in humans. Whilst fruit bats are considered the natural reservoir, NiV also infects pigs and may cause an unapparent or mild disease. Direct pig-to-human transmission was responsible for the first and still most devastating NiV outbreaks in Malaysia and Singapore in 1998-99, with nearly 300 human cases and over 100 fatalities. Pigs can therefore play a key role in the epidemiology of NiV by acting as an ?amplifying? host. The outbreak in Singapore ended with the prohibition of pig imports from Malaysia and the Malaysian outbreak was ended by culling 45% of the country?s pig population with costs exceeding US$500 million. Despite the importance of NiV as an emerging disease with the potential for pandemic, no vaccines or therapeutics are currently approved for human or livestock use. In this mini-review, we will discuss current knowledge of NiV infection in pigs; our ongoing work to develop a NiV vaccine for use in pigs; and the pig as a model to support human vaccine development.
    Goldeck David, Perry Dana, Hayes Jack, Johnson Luke, Young Jordan, Roychoudhury Parimal, McLuskey Elle, Moffat Katy, Bakker Arjen, Kwakkenbos Mark, Frossard Jean-Pierre, Rowland Bob, Murtaugh Michael, Graham Simon P. (2019) Establishment of systems to enable isolation of porcine monoclonal antibodies broadly neutralizing the porcine reproductive and respiratory syndrome virus,Frontiers in Immunology Frontiers Media
    The rapid evolution of porcine reproductive and respiratory syndrome viruses (PRRSV) poses a major challenge to effective disease control since available vaccines show variable efficacy against divergent strains. Knowledge of the antigenic targets of virus-neutralizing antibodies that confer protection against heterologous PRRSV strains would be a catalyst for the development of next-generation vaccines. Key to discovering these epitopes is the isolation of neutralizing monoclonal antibodies (mAbs) from immune pigs. To address this need, we sought to establish systems to enable the isolation of PRRSV neutralizing porcine mAbs. We experimentally produced a cohort of immune pigs by sequential challenge infection with four heterologous PRRSV strains spanning PRRSV-1 subtypes and PRRSV species. Whilst priming with PRRSV-1 subtype 1 did not confer full protection against a subsequent infection with a PRRSV-1 subtype 3 strain, animals were protected against a subsequent PRRSV-2 infection. The infection protocol resulted in high serum neutralizing antibody titers against PRRSV-1 Olot/91 and significant neutralization of heterologous PRRSV-1/-2 strains. Enriched memory B cells isolated at the termination of the study were genetically programmed by transduction with a retroviral vector expressing the Bcl-6 transcription factor and the anti-apoptotic Bcl-xL protein, a technology we demonstrated efficiently converts porcine memory B cells into proliferating antibody-secreting cells. Pools of transduced memory B cells were cultured and supernatants containing PRRSV-specific antibodies identified by flow cytometric staining of infected MARC-145 cells and in vitro neutralization of PRRSV-1. Collectively, these data suggest that this experimental system may be further exploited to produce a panel of PRRSV-specific mAbs, which will contribute both to our understanding of the antibody response to PRRSV and allow epitopes to be resolved that may ultimately guide the design of immunogens to induce cross-protective immunity.
    Franzoni Giulia, Graham Simon P., Dei Giudici Silvia, Oggiano Annalisa (2019) Porcine Dendritic Cells and Viruses: An Update,Viruses11(5) MDPI
    Several viral infections of swine are responsible for major economic losses and represent a threat to the swine industry worldwide. New tools are needed to prevent and control endemic, emerging, and re-emerging viral diseases. Dendritic cells (DC) play a central role in linking the innate and adaptive arms of the immune system, so knowledge regarding their interaction with pathogens is necessary to understand the mechanisms underlying diseases pathogenesis and protection. In the first part of this review, we provide an update on the heterogeneous cell subsets that comprise the porcine DC family. In the second part of this review, we provide an overview of how three viruses, affecting pork production at a global level, African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine circovirus 2 (PCV2), modulate DC function.
    McCarthy Ronan R., Everett Helen E., Graham Simon P., Steinbach Falko, Crooke Helen R. (2019) Head Start Immunity: Characterising the early protection of C strain vaccine against subsequent classical swine fever virus infection,Frontiers In Immunology Frontiers Media
    Classical Swine Fever Virus (CSFV) is an ongoing threat to the pig industry due to its high transmission and mortality rates associated with infection. Live attenuated vaccines such as the CSFV C strain vaccine are capable of protecting against infection within 5 days of vaccination, but the molecular mechanisms through which this early protection is mediated have yet to be established. In this study, we compared the response of pigs vaccinated with the C strain to non-vaccinated pigs both challenged with a pathogenic strain of CSFV. Analysis of transcriptomic data from the tonsils of these animals during the early stages after vaccination and challenge reveals a set of regulated genes that appear throughout the analysis. Many of these are linked to the ISG15 antiviral pathway suggesting it plays a key role in the rapid and early protection conferred by C strain vaccination.
    Porcine reproductive and respiratory syndrome viruses (PRRSV) cause the most economically significant disease of swine. The emergence of highly pathogenic strains and the limitations of current vaccines pose significant challenges to PRRS control. An improved understanding of pathogenesis is needed to support the development of improved vaccines. Experimental infection with virulent PRRSV-1 subtype 3 strains is associated with enhanced immune responses, which have been attributed to the more effective viral clearance compared to ?typical? PRRSV-1 subtype 1 strains. Previous ex vivo data has also shown that this increased virulence correlates with a broader cell tropism for cells of the myeloid lineage. The causation of this correlation was investigated by assessing whether European PRRSV-1 strains can infect dendritic cells (DC), which are not canonically a target cell. A staining strategy was optimised to enrich and phenotype blood-derived DC (BDC) subsets. Infection experiments demonstrated that BDC were not susceptible, and that cytokine responses in enriched BDC culture were suppressed by inoculation of subtype 3 strain Lena. Further characterisations of host responses instead used monocyte-derived macrophages (M¦) and DC. Functional assays determined that pro-inflammatory responses were increased by subtype 3 strain SU1-Bel more than subtype 1 strains, including modulation of surface activation markers, ability to prime allogeneic T cell proliferation, and secretion of inflammatory cytokines; Lena, however, did not induce pro-inflammatory cytokine responses but decreased endocytic and phagocytic capacity. This trend was validated by transcriptomic analysis of infected monocyte-derived M¦, which identified putative genes of interest for future studies, including pattern recognition receptors. Inflammasome treatments suggested that SU1-Bel primed inflammasome activation and that subtype 3 strain replication was decreased by inflammasome inhibition, proportionately increasing M¦ viability. Finally, preliminary trials of a novel macrophage cell line demonstrated its utility for further investigation of the mechanistic basis for the enhanced virulence of PRRSV-1 subtype 3 strains.
    Culicoides biting midges are biological vectors of arthropod-borne viruses (arboviruses) of international importance. The emergence of bluetongue virus (BTV) and Schmallenberg virus (SBV) in Europe has highlighted the vulnerability of the UK to incursions. Persistence of Culicoides-borne viruses through adverse conditions in the cold winter months (?overwintering?) contributes to their economic and welfare impact and is a major source of uncertainty in policy response. Understanding the role of temperature in adult Culicoides activity, particularly in colder months, will inform policy on the likely risk of transmission. Reducing exposure to Culicoides biting is an important factor in controlling the spread of Culicoides-borne viruses following an incursion and immunological responses to the Culicoides bite may provide a novel approach to measuring the exposure of susceptible hosts to Culicoides. This thesis investigates adult Culicoides seasonal activity and biting in the UK using both field and laboratory-based studies, focusing on the impact of winter. Culicoides activity, examined directly using ultraviolet-baited light-suction traps, was limited to days when maximum temperatures were >7°C in a field study. An experimental system that tested movement of adult Culicoides within a controlled environment further demonstrated the effect of temperature, with the minimum temperature for flight activity varying according to collection location, time of year of collection and species. Furthermore, immunoassays were developed to detect the presence of antibodies against Culicoides salivary proteins in host sera and to indirectly infer Culicoides blood-feeding activity. This work demonstrated that horses, cattle and sheep in the UK produce detectable levels of antibodies recognising Culicoides salivary protein antigens for the first time, as a result of natural biting exposure in the field environment. These findings have direct implications in future surveillance programs and policy decision making, particularly when defining vector-free periods of adult activity.