Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK
Correspondence
C. A. L. Oura
chris.oura{at}bbsrc.ac.uk
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ABSTRACT |
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Present address: Instituto Gulbenkian de Ciencia, 2780-156 Oeiras, Portugal.
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MAIN TEXT |
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In this study, we directly addressed the question of whether cellular components of immunity, in addition to anti-ASFV antibodies, are required to protect pigs from ASFV challenge. As a model for protective immunity we exploited the fact that outbred pigs infected with the avirulent Portuguese non-haemadsorbing tick ASFV isolate OUR/T88/3 (Boinas et al., 2004) are immune to subsequent challenge with the virulent Portuguese tick isolate of ASFV, OUR/T88/1 (Boinas et al., 2004
). Using this model, we carried out a series of in vivo depletion experiments. In the first experiment, 12 healthy outbred pigs of similar body weight (2025 kg) were inoculated intramuscularly with 104 TCID50 of the avirulent OUR/T88/3 and the pigs were then divided into three groups of four according to treatment. Group A received anti-CD8 (IgG2a) monoclonal antibody (mAb) [a mixture of anti-CD8 mAb 76-2-11 (Pescovitz et al., 1985
) and anti-CD8 mAb 11-295-33 (Saalmüller, 1996
)]. Group B received isotype control IgG2a anti-bovine WC1 mAb (CC15), which does not cross-react in pigs. Group C received no mAb treatment (protected control group). An additional group (group D) consisting of four naive pigs (without previous exposure to OUR/T88/3) was an unprotected control group set up in order to check challenge virus efficacy. As pigs infected with OUR/T88/3 do not exhibit viraemia or any evidence of persistence post-infection (p.i.) (Boinas et al., 2004
), pigs in groups A and B were injected intravenously with clarified mouse ascitic fluid (10 ml per animal per day) daily for 5 days from day 31 to day 35 after OUR/T88/3 infection. On the second day of ascitic fluid inoculation (day 32), all four groups of pigs were challenged intramuscularly with a 50 % haemadsorbing dose (HAD50) of 104 of the virulent OUR/T88/1. All animals were then monitored daily for clinical signs, viraemia (using a haemadsorption assay; Malmquist & Hay, 1960
) and temperature, and the phenotype of circulating lymphocytes was analysed by two-colour flow cytometry with the following mouse mAbs: anti-porcine CD4 (74-12-4, IgG2b; Saalmüller, 1996
), anti-porcine CD8
(11-295-33, IgG2a; Saalmüller, 1996
), anti-porcine
T cells (PPT27, IgG1, and PPT16, IgG2b; Yang & Parkhouse, 1996
, 2000
), anti-porcine CD8
(PPT22, IgG1; Yang & Parkhouse, 1997
). These mAbs were used in conjunction with fluorescent-labelled goat anti-mouse isotype-specific secondary antibodies (Southern Biotechnology) and analysed on a FACSCalibur (Becton Dickinson). Serum samples were taken every 7 days throughout the course of the experiment and antibody titres were measured by ELISA (Office International des Epizooties, 1996
). The second in vivo depletion experiment was identical to the first except that the isotype control IgG2a mAb used was anti-bovine CD8 (CC63), which does not cross-react with pigs, and the ascitic fluid was given for 6 rather than 5 days from day 31 to 36 days after infection with OUR/T88/3. Also, in the second experiment only three pigs were used in the protected control group.
In the first experiment, clear depletion of CD8+ lymphocytes was observed in only two of the four pigs (Fig. 1a, Experiment 1). The two pigs (C96 and C99) in which depletion of CD8+ lymphocytes was observed developed high viraemia (Fig. 1b
) and raised body temperatures, but survived challenge with the virulent ASFV isolate OUR/T88/1. The remaining two pigs in the group (C97 and C98) were not depleted of CD8+ lymphocytes, exhibited no viraemia (Fig. 1b
) or clinical signs and were completely protected from challenge with OUR/T88/1. This experiment showed that CD8+ lymphocytes play an important role in the protective immune response to ASFV infections, although as the two CD8+ lymphocyte-depleted viraemic pigs survived challenge with the virulent OUR/T88/1 isolate, this indicated that either the depletion of CD8+ lymphocytes was not complete or other factors such as antibodies are playing a role in the protective immune response to ASFV.
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In this paper, we have demonstrated the importance of CD8+ lymphocytes in the protective immune response to challenge with ASFV isolate OUR/T88/1. As a number of porcine lymphocyte subpopulations express CD8 on their surface, the next question was which CD8+ lymphocyte subpopulation(s) plays a role in the protective immune response to ASFV infection. In young pigs, a large proportion of CD8+ lymphocytes are NK cells (Yang & Parkhouse, 1996, 1997
) and a possible contribution of NK cells in the immune response to ASFV infection was suggested by Leitao et al. (2001)
. However, very low levels of NK cells were seen in these experiments, as the combined number of CD8
+ and CD4+CD8+ lymphocytes was roughly the same as the total CD8+ lymphocyte population (Figs 2 and 3
). In addition, porcine NK cells do not express CD8
and therefore NK cells were not responsible for the increased CD8+ lymphocyte populations seen as a response to viraemia. Unique to the pig, porcine memory helper T cells also express CD8 (Saalmüller et al., 2002
). As we did not perform specific depletion of helper T-cell subsets in this study, we could not eliminate the possibility that the total CD8+ lymphocyte depletion, including the depletion of memory CD4+CD8+ helper T cells, may have resulted in failure of the secondary antibody response, which could abrogate protective immunity to ASFV challenge. However, due to the following reasons, we could conclude that the involvement of CD4+CD8+ helper T cells or secondary antibody responses in the protective immune response in this system was unlikely. Firstly, the anti-ASFV antibody titres in pigs in all OUR/T88/3-infected groups (CD8 depleted, protected control and isotype control groups) were raised before OUR/T88/1 challenge (28 days p.i. with OUR/T88/3) and remained at a similar level during ascitic fluid inoculation and after the challenge with OUR/T88/1 (data not shown). The pigs in the CD8+ lymphocyte-depleted group developed viraemia and clinical symptoms very soon after the challenge infection (by 3 days post-challenge) and importantly there were no differences in antibody titres seen in the CD8+ lymphocyte-depleted unprotected group compared with the isotype-protected and control-protected groups at this time point (data not shown). If an effective secondary antibody response was protecting the pigs, one would expect to see increased amounts of antibody in the protected compared with the unprotected groups of pigs, which was not the case. Also, viraemic pigs without CD8+ lymphocyte depletion (Figs 2 and 3
) all showed an increased number of circulating CD8
+ lymphocytes after challenge with OUR/T88/1, whereas memory helper T cells of the CD4+CD8+ phenotype remained at a low steady level and did not increase after challenge. An increase in the number of CD4+CD8+ memory T cells associated with activation would be expected if this cell population were involved in the development of a secondary antibody response. Therefore, we could conclude that anti-ASFV antibodies alone, from OUR/T88/3 infection, were not sufficient to protect pigs from OUR/T88/1 challenge and there was no evidence of a protective secondary antibody response at 3 days post-challenge when the pigs developed the disease.
Observations from these experiments and others (M. S. Denyer, T. Wileman, C. Stirling & H. Takamatsu, unpublished data) indicate that the population of CD8+ lymphocytes observed in this paper is likely to belong to a CD3+CD4CD5+CD6+CD8
+CD8
+ T-cell subset expressing the cytotoxic granule perforin internally and this T-cell subset is therefore likely to be cytotoxic. If this is the case, this cytotoxic T-cell population may contribute to the elimination of ASFV-infected cells, resulting in reduced levels of viraemia. Although further phenotypic and functional analyses of CD8+ lymphocyte subpopulations involved in protection from ASFV infections are needed in the future, this paper demonstrates an important role for CD8+ lymphocytes in the protective immune response to ASFV infection. This information, combined with work showing a role for antibodies in ASFV protection, will aid in the development of new strategies aimed at the production of an effective ASF vaccine. Finally, the experiments with cc haplotype inbred pigs have provided a first insight into the possible involvement of MHC in resistance to ASF.
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ACKNOWLEDGEMENTS |
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Received 16 March 2005;
accepted 3 June 2005.
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