Prospects for development of African swine fever virus vaccines.

African swine fever virus is a large DNA virus which can cause an acute haemorrhagic fever in pigs resulting in high mortality. No vaccine is available, limiting options for control. The virus encodes up to 165 genes and virus particles are multi-layered and contain more than 50 proteins. Pigs immunised with natural low virulence isolates or attenuated viruses produced by passage in tissue culture and by targeted gene deletions can be protected against challenge with virulent viruses. CD8+ cells are required for protection induced by attenuated strain OURT88/3. Passive transfer of antibodies from immune to naïve pigs can also induce protection. Knowledge of the genome sequences of attenuated and virulent strains and targeted gene deletions from virulent strains have identified a number of virus genes involved in virulence and immune evasion. This information can be used to produce rationally attenuated vaccine strains. Virus antigens that are targets for neutralising antibodies have been identified and immunisation with these recombinant proteins has been shown to induce partial protection. However knowledge of antigens which encode the dominant protective epitopes recognised by CD8+ T cells is lacking.

Clinical signs and pathology shown by British sheep and cattle infected with bluetongue virus serotype 8 derived from the 2006 outbreak in northern Europe.

Four poll Dorset sheep and four Holstein-Friesian cattle were infected with the northern European strain of bluetongue virus (BTV), BTV-8, to assess its pathogenicity in UK breeds. The time course of infection was monitored in both species by using real-time reverse transcriptase-PCR (RT-PCR), conventional RT-PCR and serology. Two of the sheep developed severe clinical signs that would have been fatal in the field; the other two were moderately and mildly ill, respectively. The cattle were clinically unaffected, but had high levels of viral RNA in their bloodstream. Real-time RT-PCR detected viral RNA as early as one day after infection in the cattle and three days after infection in the sheep. Antibodies against BTV were detected by six days after infection in the sheep and eight days after infection in the cattle. Postmortem examinations revealed pathology in the cattle that was more severe than suggested by the mild clinical signs, but the pathological and clinical findings in the sheep were more consistent.

Porcine gammadelta T cells: possible roles on the innate and adaptive immune responses following virus infection.

gammadelta T cells recognise different types of antigen in alternative ways to alphabeta T cells, and thus appear to play a complementary role in the immune response. However, unlike alphabeta T cells, the role or function of gammadelta T cells is still unclear. As pigs possess a high proportion of circulating gammadelta T cells, they are suitable large animal model to study gammadelta T cell functions. This as yet has not been fully exploited, leaving porcine gammadelta T cell biology and its role in immunity in its infancy. Foot-and-mouth disease (FMD) high potency "emergency" vaccines are able to induce early protection from challenge and it has been suggested that, in part, there is some involvement of innate immune responses. The antigen component of the vaccine is able to stimulate purified naive pig gammadelta T cells and induce the mRNA of various cytokines and chemokines. This observation suggests that gammadelta T cells probably contribute to the early phase of the immune responses to FMD vaccination, and perhaps infection. A subset of these circulating gammadelta T cells display a phenotype similar to professional antigen presenting cells and are able to take up and present soluble antigen to CD4(+) T cells in a direct cell-cell interaction via MHC class II. This direct interaction between gammadelta T cells and CD4(+) T cells is likely to have a significant influence on the out come of the adaptive immune response.

Immunization of African Indigenous Pigs with Attenuated Genotype I African Swine Fever Virus OURT88/3 Induces Protection Against Challenge with Virulent Strains of Genotype I.

The attenuated African swine fever virus genotype I strain OURT88/3 has previously been shown to induce protection of European breeds of domestic pigs against challenge with virulent isolates. To determine whether protective immune responses could also be induced in indigenous breeds of pigs from the Kinshassa region in Democratic Republic of Congo, we immunized a group of eight pigs with OURT88/3 strain and challenged the pigs 3 weeks later with virulent genotype I strain OURT88/1. Four of the pigs were protected against challenge. Three of the eight pigs died from African swine fever virus and a fourth from an unknown cause. The remaining four pigs all survived challenge with a recent virulent genotype I strain from the Democratic Republic of Congo, DRC 085/10. Control groups of non-immune pigs challenged with OURT88/1 or DRC 085/10 developed signs of acute ASFV as expected and had high levels of virus genome in blood.

A sub-population of circulating porcine gammadelta T cells can act as professional antigen presenting cells.

A sub-population of circulating porcine gammadelta T cells express cell surface antigens associated with antigen presenting cells (APCs), and are able to take up soluble antigen very effectively. Functional antigen presentation by gammadelta T cells to memory helper T cells was studied by inbred pig lymphocytes immunised with ovalbumin (OVA). After removing all conventional APCs from the peripheral blood of immunised pigs, the remaining lymphocytes still proliferated when stimulated with OVA. When gammadelta T cells were further depleted, OVA specific proliferation was abolished, but reconstitution with gammadelta T cells restored proliferation. The proliferation was blocked by monoclonal antibodies (mAb) against MHC class II or CD4, and by pre-treatment of gammadelta T cells with chloroquine. These results indicate that a sub-population of circulating porcine gammadelta T cells act as APCs and present antigen via MHC class II.

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

Protection against foot-and-mouth disease (FMD) using DNA technology has been documented for sheep and pigs but not for the highly susceptible species of cattle. Twenty-five Holstein Friesian cross-bred cattle were vaccinated twice, 21 days apart, with a DNA vaccine containing the capsid coding region (P1) along with the non-structural proteins 2A, 3C and 3D (pcDNA3.1/P1-2A3C3D) of O(1) Kaufbeuren alone or coated onto PLG (d,l-lactide-co-glycolide) microparticles. In some pcDNA3.1/P1-2A3C3D was also combined with an adjuvant plasmid expressing bovine granulocyte macrophage colony stimulating factor (GM-CSF). DNA vaccinations were administered intramuscularly with, or without, the use of electroporation and at 42 days post primary vaccination cattle received a protein boost of 146S FMD virus (FMDV) antigen and non-structural protein 3D. For comparison, four cattle were vaccinated with a conventional FMD vaccine and two more included as unvaccinated controls. Apart from those immunised with PLG microparticles all cattle were challenged with 10(5) TCID(50) cattle adapted O(1) Lausanne FMDV virus at day 93 post primary vaccination. All DNA vaccinated cattle regardless of regime developed good humoral and cell mediated responses prior to challenge. The best overall virus neutralising antibody, IFN-γ and clinical protection (75%) were seen in the cattle whereby the DNA was delivered by electroporation. In contrast, only 25% of cattle vaccinated with the DNA vaccine without electroporation were clinically protected. The addition of GM-CSF in combination with electroporation further improved the efficacy of the vaccine, as demonstrated from the reduction of clinical disease and virus excretions in nasal swabs. We thus demonstrate for the first time that cattle can be clinically protected against FMDV challenge following a DNA prime-protein boost strategy, and particularly when DNA vaccine is combined with GM-CSF and delivered by electroporation.

A gamma delta T cell specific surface receptor (WC1) signaling G0/G1 cell cycle arrest.

Three monoclonal antibodies (mAb; SC-6, SC-12, and SC-29) reactive with the gammadelta T cell-restricted antigen WC1 were obtained immunizing mice with an ovine interleukin (IL)-2-dependent gammadelta T cell line. These mAb strongly inhibited DNA synthesis in IL-2-dependent gammadelta T cell lines with cell cycle arrest in G0/G1 phase, but did not induce apoptosis. The mAb-induced growth arrest was reversible, either by removing the mAb or by co-culture with mitogen or anti-CD3 in the presence of IL-2. In contrast, addition of phorbol ester, ionomycin and IL-2 had no effect on the mAb-induced growth arrest. The observations define a biologically important role for the cell surface molecule WC1 in the regulation of gammadelta T cell proliferation and also provide a suitable system to study the relevant signal transduction events.

Growth arrest of gammadelta T cells induced by monoclonal antibody against WC1 correlates with activation of multiple tyrosine phosphatases and dephosphorylation of MAP kinase erk2.

WC1 is a 215-kDa type 1 transmembrane glycoprotein, the expression of which is restricted to gammadelta T lymphocytes. The binding of an anti-WC1 monoclonal antibody (mAb) (SC-29) induces reversible growth arrest in proliferating interleukin (IL)-2-dependent gammadalta T lymphocytes and this study has examined the relevant biochemical mechanisms. WC1 binding activates multiple protein tyrosine phosphatases causing specific tyrosine dephosphorylation in the absence of calcium mobilization. One of the dephosphorylated proteins was identified as the MAP kinase erk2. Another phosphotyrosine protein of 70 kDa, found to coprecipitate with p85 phosphoinositol (PI)3-kinase was either dephosphorylated or uncoupled from the p85 PI 3-kinase immunoprecipitate after WC1 receptor binding by mAb SC-29. The anti-WC1-induced tyrosine dephosphorylation was reversed by stimulation of gammadelta T cells with concanavalin A or anti-CD3 mAb, demonstrating that at the biochemical level, mitogen stimulation is dominant to the growth-arresting effects of anti-WC1. It is therefore proposed that the activation of tyrosine phosphatases by WC1 binding and the resultant dephosphorylation of certain key signaling protein such as erk2 correlates with and may cause the induction of growth arrest in IL-2-dependent gammadelta T cells, without affecting the cells ability to respond to antigen. Possible mechanisms, which include the inhibition of IL-2 signal transduction pathways, are discussed.

Ligation of the WC1 receptor induces gamma delta T cell growth arrest through fumonisin B1-sensitive increases in cellular ceramide.

Ceramide is a powerful regulator of cell fate, inducing either apoptosis or growth arrest. We have previously shown that an Ab to the gammadelta T cell-specific orphan receptor, WC1, is able to induce growth arrest in proliferating IL-2-dependent gammadelta T cells. We now show that this WC1-mediated growth arrest is associated with an increase in cellular ceramide, in the absence of any measurable changes in acidic/neutral sphingomyelinase activity. Moreover, cell-permeable analogues of ceramide also mimicked WC1-induced growth arrest along with an associated decrease in pocket protein expression and phosphorylation status. An important role for ceramide in WC1-induced growth arrest was confirmed by demonstrating that the specific ceramide synthase inhibitor fumonisin B1 blocked WC1-induced growth arrest and the associated molecular effects on the pocket proteins. Finally, we observed constitutive expression of both antiapoptotic factors bcl-2 and bcl-X, the former having increased expression upon WC1 stimulation. It is therefore proposed that ligation of WC1 leads to an accumulation in cellular ceramide through activation of ceramide synthase. This in turn results in a decreased overall expression of the pocket proteins pRb and p107, their hypophosphorylation, and an eventual growth arrest of the gammadelta T cell. To our knowledge, these results demonstrate for the first time that cell surface receptor-mediated ceramide synthase activation can affect cell fate through increases in cellular ceramide and provide further evidence that the orphan receptor WC1 regulates gammadelta T cell biology through a novel signaling pathway.

A potential overwintering mechanism for bluetongue virus--recent findings.

Bluetongue virus (BTV) is transmitted between its mammalian hosts almost exclusively via bites from the adults of certain species of Culicoides biting midges. Theoretically, the spread of BTV into the more northerly areas of Europe should therefore be terminated by the harsh winters experienced in these regions, when adult midges disappear for extended periods of time. However, it has been shown that BTV can survive for periods as long as 9 to 12 months in such locations in the absence of adult insect vectors, with no detectable cases of viraemia, overt disease or seroconversion in the host species. Virus survival in this manner throughout the winter is called 'overwintering' but the mechanism involved has not been satisfactorily explained. With knowledge currently available and results from a series of preliminary experiments, the authors discuss a possible overwintering mechanism.

Transcription factor E2F controls the reversible gamma delta T cell growth arrest mediated through WC1.

IL-2-stimulated expansion of T cells requires continued and sequential passage of the dividing cells through a major cell cycle check point in the G1 phase. We have previously shown that a gamma delta T cell-specific surface receptor, WC1, induces G0/G1 growth arrest, reversible with Con A, in proliferating IL-2-dependent gamma delta T cells. We now show that this reversible WC1-induced cell cycle arrest is correlated with induction of the cyclin kinase inhibitor p27kip1 and an associated down-regulation in cyclins A, D2, and D3 expression, along with dephosphorylation of pocket proteins p107, p130, and pRb. Together with diminished pocket protein phosphorylation, p107 expression levels are significantly down-regulated in response to WC1 stimulation. This coordinated sequence of signaling events is focused on E2F regulation so that, downstream of the pocket proteins, WC1 stimulation results in a diminished DNA binding activity for free E2F as a consequence of reduced E2F1 expression, whereas E2F4 expression is unaffected. Consistent with this interpretation, overexpression of E2F1 overcomes the growth-arresting effects induced by WC1 stimulation. Finally, in accordance with our previous observations at both the cellular and molecular level, subsequent mitogen stimulation can reverse all the above changes induced by WC1. These results, focused on E2F regulation, therefore provide a first insight into the effects of both positive (mitogen) and negative (anti-WC1) stimuli on cell cycle control in IL-2-dependent gamma delta T cells.