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.

In vivo depletion of CD8+ T lymphocytes abrogates protective immunity to African swine fever virus.

To understand the mechanisms involved in protective immunity to African swine fever virus (ASFV) infection, the observation that infection with the avirulent Portuguese ASFV isolate OUR/T88/3 protects outbred pigs from challenge with the virulent Portuguese ASFV isolate OUR/T88/1 was exploited. It was demonstrated that pigs exposed to OUR/T88/3 and then depleted of CD8+ lymphocytes were no longer fully protected from OUR/T88/1 challenge. This indicated that CD8+ lymphocytes play an important role in the protective immune response to ASFV infection and that anti-ASFV antibody alone, from OUR/T88/3 infection, was not sufficient to protect pigs from OUR/T88/1 challenge. Inbred pigs of the cc haplotype infected with OUR/T88/3 were not always protected from OUR/T88/1 challenge and developed both viraemia and fever. Such viraemia was always correlated with increased numbers of circulating CD8beta+ lymphocytes, indicating a specific role for CD8beta+ lymphocytes in combating viraemia. These experiments indicate an important role for CD8+ lymphocytes, particularly CD8beta+ lymphocytes, in ASF protective immunity.

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.

Establishment of long-term CD154-dependent porcine B-cell cultures.

Cells of the B-cell lineage play an essential part in the immune response, not only as the producers of antigen-specific antibodies, but also as antigen-presenting cells. Unlike T cells, however, the establishment of long-term normal B-cell lines has proved to be exceedingly difficult. In this paper we demonstrate that cell membrane-expressed CD154 (CD40 ligand) is able to support the continual growth of porcine mesenteric lymph node B-cell cultures for more than 4 months without the addition of exogenous cytokines, such as interleukin-4 (IL-4). Addition of IL-4, but not interferon-gamma (IFN-gamma) or IL-13, to these cultures enhanced proliferation, as, to a lesser extent, did addition of IL-2. Interestingly, however, whilst IFN-gamma-supplemented cultures largely consisted of immunoglobulin M (IgM)-positive cells, cultures with IL-13 or IL-4 contained a significantly increased proportion of IgG-positive cells.

African swine fever virus: a B cell-mitogenic virus in vivo and in vitro.

The two major characteristics of pathogenesis in African swine fever virus (ASFV) infections of domestic pigs are massive B-cell apoptosis and haemorrhage. The effects of ASFV on porcine B cells have therefore been systematically examined in vivo, by using virus-infected pigs and SCID-Beige mice reconstituted with porcine bone marrow, and in vitro, by using porcine B-cell lines and B cells from normal and ASFV-infected pigs. Secretion of porcine Ig was stimulated by ASFV both in vivo and in bone marrow cultures in vitro, with the virulent Malawi isolate of ASFV being the most effective. Stimulation of Ig secretion in vitro depended on the presence of ASFV-infected macrophages and did not occur with supernatants from ASFV-infected macrophages. Although the virus alone did not stimulate proliferation of purified B cells in vitro, it was co-stimulatory with CD154 (CD40 ligand). The B cells recovered from ASFV-infected porcine lymphoid tissue were of activated surface marker phenotypes and, interestingly, expressed diminished levels of the B-cell co-stimulatory surface molecule CD21. In addition, they were highly sensitive to IL-4 and CD154. These results may be integrated into a model of pathogenesis in which those B cells activated indirectly as a result of virulent ASFV infection of macrophages are not rescued from apoptosis through interaction with CD154, due to the drastic depletion of T cells that occurs early in infection. The consequently diminished specific anti-ASFV antibody response would favour survival of the virus, with the non-specific hypergammaglobulinaemia being perhaps another example of pathogen-mediated immune deviation.

Expression of biologically active recombinant porcine GM-CSF by baculovirus gene expression system.

The full length porcine granulocyte/macrophage colony stimulating factor (GM-CSF) cDNA, including secretion signal peptide coding region was recloned into baculovirus transfer vector pAcYM1. The vector was then transfected with Autographica californica nuclear polyhedrosis virus (AcNPV) DNA into SF21AE cells and the recombinant virus AcPGM was recovered. Recombinant porcine GM-CSF (rpGM-CSF) was obtained from the serum-free culture medium of Tn5 cells infected with the AcPGM virus, and was shown to be a glycosylated 21 kDa protein as confirmed by tunicamycin treatment and [3H]-glucosamine uptake. The biological activities of rpGM-CSF in AcPGM-infected cell culture supernatants were demonstrated by porcine bone marrow cell proliferation and haematopoietic cell colony formation assays. The use of rpGM-CSF enabled us to culture porcine monocytes/macrophage and dendritic-like cells, derived from either porcine bone marrow or peripheral blood, for up to 4 months.

Analysis of bovine B cell reactive monoclonal antibodies.

Monoclonal antibodies (mAbs) submitted to the workshop B cell panel were screened for reactivity with bovine surface immunoglobulin positive (SIg+) cells from tonsil, mesenteric lymph nodes (MLN), ileal Peyer's patches (IPP), thymus and peripheral blood by fluorescence activated cell sorter (FACS) analysis. All mAbs within B cell panel reacted with B cells except the negative control mAb VPM61. Although most mAbs stained the majority of B cells from different tissues, 14 mAbs did not stain B cells from IPP. Detailed FACS analysis of the 24 mAbs in preliminary clusters PC17, PC28, PC29, PC30, PC35, PC41 and PC43 showed all preliminary clusters (PC) to contain mAbs with similar and variable specificities. This absence of clear-cut clusters therefore did not permit a simple classification of B cell surface antigens via the B cell mAbs and suggests considerable complexity and variability of the B cell surface.

The detection of antibodies against foot-and-mouth disease virus (FMDV) in filter paper eluates from pig sera or whole blood by ELISA.

The use of pig blood samples dried on paper discs for the detection of antibodies against FMDV by the liquid phase blocking ELISA has been evaluated. The average volume of whole heparinised blood required to fully saturate 6.0 mm discs was 7.65 microliters (range 7.2 to 8.1; variation = 0.24, P = 0.05). When 200 clinically healthy animals were assessed by virus neutralisation (VN) titres up to 1/22 were recorded against types O, A and C, 97% being 1/11 or less. Using ELISA, results were more skewed. Overall, 91% showed titres of 1/32 or less, and there were occasional high non-specific reactors with types O and A. Using small groups of sera from experimental animals, a VN titre of 1/16 was found to be equivalent to an ELISA titre of approximately 1/100 (log10 2.0 +/- 0.2) with type O and 1/56 (log10 1.75 +/- 0.1) for type A. Although some loss in sensitivity from disc dried sera was found in selected negatives on testing at a single dilution of 1/32, sera from vaccinated animals with VN titres of 1/16 to 1/708 all gave strong positive results. A monoclonal antibody (Mab) assay was successfully developed to detect different swine anti-FMDV antibody isotypes.

Characterization of long-term cultured bovine CD4-positive and CD8-positive T-cell lines and clones.

Long-term cultured CD4+ or CD8+ bovine T-cell lines and clones were established. The CD8+ T-cell line and clones had a strong lectin-dependent cytotoxicity, whereas the CD4+ T-cell line did not. Both phenotype cell lines grew in an interleukin-2 (IL-2)-dependent manner and expressed 50,000-55,000 MW and 65,000-75,000 MW proteins associated with a putative IL-2 receptor (IL-2R), as demonstrated by the cross-linking of radioiodinated recombinant human IL-2 (rhIL-2). Additional molecules of 13,000 and 27,000 MW were also observed on CD8+ T cells. The binding of rhIL-2 was blocked by crude bovine IL-2, and Scatchard plot analysis of the binding data showed that both phenotype cells expressed two different affinity IL-2R that had equilibrium dissociation constants of 12-20 pm (3000-6000 sites/cell) and 146-490 pM (16,000-25,000 sites/well). Only IL-2 stimulated DNA synthesis in these cell lines, whereas mitochondrial enzymes activity, protein synthesis and protein secretion were enhanced by IL-2, mitogens and phorbol myristate acetate. The supernatant from mitogen-stimulated CD4+ cells was unable to enhance the DNA synthesis of either the CD4+ or CD8+ lines, whereas both freshly prepared Con A blasts and anti-immunoglobulin-treated bovine B cells showed elevated DNA synthesis under the same conditions.

Cross reactions between porcine, bovine and ovine interleukin-2 preparations.

Optimum conditions for the production of porcine interleukin-2 were found to include a delay of 24 hours before the addition of mitogen. Porcine and bovine interleukin-2 responded optimally in homologous systems whereas bovine interleukin-2 gave a better response in the ovine system than homologous ovine interleukin-2. Interleukin-2 produced from a continuous gibbon cell line reacted well with porcine, ovine and bovine T cell blasts indicating that it could act as a universal growth factor for T cell clones produced from these species.

Use of indirect and competitive ELISAs to compare isolates of equine influenza A virus.

Antigenic differences within equine-1 and equine-2 isolates of influenza were studied by haemagglutination inhibition tests, indirect ELISA and competition ELISA, using the same antisera. Better differentiation was obtained with the competition ELISA than with the other two tests. All three methods produced similar relationships within the equine-1 isolates but differed in their ability to differentiate the equine-2 isolates where the competition ELISA was superior and produced epidemiologically sensible results. In all three tests, post-infection ferret and horse sera were more useful in discriminating isolates than post-vaccination sera.

Development of an enzyme-linked immunosorbent assay (ELISA) for the detection of specific antibodies against an H7N7 and an H3N8 equine influenza virus.

This paper describes a solid-phase microtitre plate enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to equine influenza viruses. Using egg-grown influenza viruses as the antigens attached to the solid phase, cross-reactions were observed between an H7N7 equine virus (designated A1) and an H3N8 equine influenza virus (designated A2) when untreated antisera were tested. Absorption of antisera with egg-grown A/Porcine/Shope/1/33 influenza virus eliminated cross-reactive antibodies so that specific detection of anti-equine influenza A1 or A2 antibodies was possible. Examination of horse sera following vaccination with A1 and/or A2 isolates showed that antibodies were produced against antigen associated with egg allantoic fluid as well as against virus. Such antibodies were eliminated following the absorption of antisera with porcine influenza virus. Results using sera from horses with known vaccination histories confirmed that the ELISA preferentially detected antibodies homologous to the antigen attached to the solid phase and methods to evaluate the current serological state of individual horses by relating the titres of specific antibodies against equine influenza A1 and A2 isolates are shown. This ELISA provides a simple and rapid method of assessing specific antibodies from horse sera and offers advantages over the 'routine' HI and SRH assessments since it gives high precision, is economical of reagents and has the capacity to handle large numbers of serum samples.

Trials of an inactivated equid herpesvirus 1 vaccine: challenge with a subtype 1 virus.

Vaccinated yearlings , two-year-old and in-foal pony mares with appropriate controls were exposed to aerosols of a subtype 1 virus one to two months after two or three vaccinations; all became infected. No obvious differences in the febrile responses, clinical signs and subsequent abortions were found between vaccinated and control mares. All vaccinated yearlings and two-year-old ponies developed a febrile respiratory disease but this was less severe than that suffered by the controls and the amounts and duration of virus shedding were reduced.