Division of Immunology1 and Division of Molecular Biology2, Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK
Division of Immunology, Institute for Animal Health, Compton Laboratory, Compton, Nr Newbury, Berkshire RG16 0NN, UK3
Author for correspondence: Tom Barrett. Fax +44 1483 232448. e-mail tom.barrett{at}bbsrc.ac.uk
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Abstract |
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Introduction |
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Very little is known about the nature or specificity of T cell responses to RPV, their role in protective immunity, or indeed if RPV vaccination results in immunosuppression. RPV is known to be highly lymphotropic, with virulent strains causing a severe leukopenia with destructive pathology of lymphoid tissues (Brown & Torres, 1994 ; Wohlsein et al., 1993
, 1995
). The related morbillivirus MV has been reported by many authors to cause immunosuppression (Schnorr et al., 1997
; Hirsch et al., 1984
; Dagan et al., 1987
; Tamashiro et al., 1987
; McChesney et al., 1989
; McChesney & Oldstone, 1989
; Gans et al., 1999
), with some effects seen following vaccination with otherwise attenuated strains (Smedman et al., 1994
; Pala et al., 1998
). It has been suggested that RPV vaccination could result in transient immunosuppression (Jeggo et al., 1987
) which, if true, would be of great practical importance since vaccination could lead to an increase in mortality in the field due to flare-up of intercurrent infections as a consequence of vaccination.
As a first step to addressing these issues, a study was undertaken to examine the effects of RPV vaccination and infection on bovine T cell responses and to define the viral antigen-specificity of any responding T cells. Vaccination was found to induce a strong CD4+ T cell response, with an equivalent response to all of the major structural proteins. The RPV vaccine virus did not cause a detectable suppression of the immune system in the target host animals as judged by their ability to respond to a T cell mitogen.
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Methods |
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Viruses.
The RBOK rinderpest vaccine virus was propagated in Vero cells and the Saudi 1/81 RPV used for challenge was obtained as freeze-dried spleen extract from a previously infected animal. Lamb testes (LT) cells up to the 16th passage were used to propagate the KS-1 strain of capripox. These cells were maintained in Dulbeccos minimal essential medium (DMEM) supplemented with 10% (v/v) foetal calf serum (FCS), containing 100 U/ml benzylpenicillin and 100 µg/ml streptomycin (DMEM-complete). Other cell lines, namely 293, BHK, MDBK, MDCK, HeLa, HT-COS, HEP, BSC, RK-13, RS-2 and bovine skin fibroblasts, which were used to test the growth of adenovirus, were grown in the same medium. A marmoset lymphoblastoid cell line, B95a (Kobune et al., 1991 ), was used to propagate RPV Saudi 1/81 for antigen preparation. These cells, along with CHO cells, which were also used to test growth of adenovirus, were grown and maintained in RPMI 1640 with HEPES buffer containing the same amounts of antibiotics and FCS. Cells were grown at 37 °C in an atmosphere of 5% CO2. RPV stocks were titrated by determining the TCID50 in Vero cells. Tenfold dilutions of virus were seeded on semi-confluent monolayers in 96-well tissue culture plates and the titre calculated using the method of Reed & Muench (1938)
.
Antibody assays.
Serum was collected at weekly intervals following vaccination, and stored at -20 °C. The titre of antibody produced was assayed in a microneutralization test, on Vero cells for rinderpest-specific antibody or LT cells for capripox-specific antibody (Romero et al., 1994a ).
Isolation of peripheral blood mononuclear cells (PBMC) and CD4+ T cells.
PBMC were isolated from heparinized venous blood. Blood was diluted 1:3 with PBS and then centrifuged at room temperature at 250 g for 12 min to obtain buffy coat cells. PBMC were purified from buffy coats by centrifugation at 400 g over Histopaque 1083 (Sigma) for 35 min at room temperature. Cells collected at the interface were washed three times in PBS supplemented with 1% (v/v) FCS prior to being used in the assays.
CD4+ T cells were purified from PBMC by magnetic cell sorting (Miltenyi Biotec). PBMC were incubated with the anti-CD4+ monoclonal antibody (MAb) CC8 (IgG2a: Bensaid & Hadam, 1991 ) for 30 min at 4 °C, washed twice with PBS and then incubated with paramagnetic bead-coupled anti-mouse IgG for 20 min at 4 °C. Cells were washed with PBS and antibody-labelled cells were purified according to the manufacturers instructions.
Flow cytometry.
PBMC were harvested from in vitro assays and washed twice in PBS supplemented with 1% (v/v) FCS and 0·1% (w/v) sodium azide (PBA) prior to FACS analysis. The cells were analysed by single-colour fluorescence for CD4+, CD8+ and the T cell marker WC1 using MAbs CC8 (IgG2a: Bensaid & Hadam, 1991
), CC63 (IgG2a: MacHugh & Sopp, 1991
) and CC15 (IgG2a: Clevers et al., 1990
), respectively, and by two-colour fluorescence for these markers and CD25, detected by MAb IL-A111 (IgG1: Naessens et al., 1992
). For single-colour immunofluorescence, 104cells were incubated with MAbs, at the predetermined optimal concentrations, for 30 min at 4 °C. The cells were washed twice with PBA and then incubated with phycoerythrin-labelled F(ab)2 goat anti-mouse whole immunoglobulin at predetermined optimal concentrations, for 30 min at 4 °C. The cells were then washed twice and resuspended in 100 µl of PBA prior to analysis. For two-colour immunofluorescence, 105 cells were incubated with the MAbs, at the predetermined optimal concentrations, for 30 min at 4 °C. Cells were washed twice and then incubated with both phycoerythrin-labelled F(ab)2 goat anti-mouse IgG2a and fluorescein-labelled F(ab)2 goat anti-mouse IgG1 at predetermined optimal concentrations, for 30 min at 4 °C. The cells were then washed twice and resuspended in 100 µl of PBA prior to analysis.
Preparation of rinderpest antigen.
The Saudi 1/81 strain of RPV was passaged three times in B95a cells to prepare a high titre stock and this stock was used as for subsequent antigen preparation. Cells infected with the virus were harvested when cytopathic effect became extensive. The cells were lysed by freezethawing and the cell debris was pelleted at 1500 g for 10 min. The supernatant was recovered, the virus pelleted at 18000 g for 60 min at 4 °C, and then resusupended in TE buffer (10 mM TrisHCl, pH 7·6; 1 mM EDTA). The resuspended pellet was layered over a 1560% (w/v) sucrose gradient in TE and centrifuged at 18000 g for 90 min at 4 °C. The band of virus protein was collected and resuspended in TE before being pelleted once more at 18000 g for 60 min at 4 °C. The final virus pellet was resuspended in TE and used as the rinderpest antigen preparation. Control antigen was prepared in an identical manner from B95a cells that had not been infected with RPV.
Preparation of adenovirus recombinants.
Recombinant adenoviruses were produced using a slight modification of previously published techniques (McGrory et al., 1988 ; Wilkinson & Akrigg, 1992
). We created a combined adenovirus transfer and expression plasmid (pAH1) by ligating the HindIIIHindIII fragment from pMV100 (Wilkinson & Akrigg, 1992
), which contains the HCMV immediate early promoter, to HindIII-digested pMV31 (Wilkinson & Akrigg, 1992
), which contains the adenovirus sequences required for recombination. The open reading frames for the various RPV proteins (Baron et al., 1993
, 1994
; Baron & Barrett, 1995
; Evans et al., 1994
) were excised from their respective plasmids, blunt-ended, and cloned into the unique BamHI site just downstream of the CMV promoter in pAH1. Inserts present in the correct orientation were identified by restriction analysis. Recombinant adenoviruses were produced in 293 cells as described by McGrory et al. (1988)
. Recombinant adenoviruses were checked for expression of the appropriate RPV protein by immunoprecipitation and immunofluorescence and further purified by growth at limiting dilution in 293 cells for three more passages. PCR, using primers specific for the different RPV genes, was used to confirm the presence of viral genes at each stage of the process. The final virus preparations were again checked for expression of the expected proteins before preparing recombinant virus stocks. The titres were then determined by measuring the TCID50 in 293 cells.
Preparation of adenovirus-derived rinderpest antigens.
B95a cells were infected with recombinant adenoviruses expressing the different RPV genes, or a recombinant adenovirus expressing bacteriophage T7 RNA polymerase, at an m.o.i. of 500. The cells were grown for 72 h following infection, at which time crude preparations of rinderpest antigens were made. For this, B95a cells from each infection were harvested and resuspended in 2 ml of buffered sucrose (8% sucrose, 1 mM EDTA, 20 mM TrisHCl pH 7·8). Cells were passed 20 times through a 28 gauge needle and cell debris, remaining whole cells and nuclei were pelleted by centrifugation at 6000 r.p.m. for 1 min in a microcentrifuge. The whole process was repeated on the supernatant and the final post-nuclear supernatant was centrifuged at 14000 r.p.m. for 2030 min at 4 °C in a microcentrifuge. Preliminary experiments with the resulting pellet and supernatant fractions indicated that the supernatant, comprising the cytosol fraction plus plasma membrane, and most non-nuclear internal membrane fractions, contained the majority of rinderpest antigen in all infections with recombinant adenoviruses, irrespective of the RPV gene expressed. Therefore, this supernatant fraction of each different rinderpest or control recombinant antigen preparation was used in assays of antigen-specific T cell proliferation.
Proliferative and cellular responses to antigen.
Antigen-specific proliferation of PBMC and purified CD4+ T cells was assessed in a 96 h assay measuring [3H]thymidine incorporation. Purified PBMC were resuspended at 106/ml in RPMI medium supplemented with 10% (v/v) FCS, 100 U/ml benzylpenicillin, 100 µg/ml streptomycin and 10-5 M -mercaptoethanol (RPMI-complete); 100 µl of the PBMC suspension was added to the wells, in triplicate assays, in the presence of optimal concentrations of the different antigens, 5 µg/ml ConA, or medium alone, in flat-bottomed 96-well plates. In cultures with CD4+ T cells the proliferative response of 104 cells per well was measured in the presence of the various antigens, with 105 gamma-irradiated (3000 rads) PBMC as antigen-presenting cells. Cultures were pulsed with 1 µCi per well of [3H]thymidine during the last 1215 h of the proliferation assay and the cells were harvested on glass-fibre filter paper and counted in a scintillation counter.
Optimal dilutions of whole rinderpest antigens, and all recombinant adenovirus-expressed rinderpest antigen preparations, were determined in preliminary experiments using PBMC from rinderpest-vaccinated animals. The proliferative response of PBMC was determined, in triplicate, over a wide range of serial dilutions of the antigen preparations and the optimal dilution was determined as that which gave the maximal proliferative response. In all experiments using recombinant adenovirus-expressed rinderpest antigens, the antigen-specific proliferative response was determined using the optimal dilution. This response was compared to that obtained using the same dilution of the supernatant fraction from B95a cells infected with a recombinant adenovirus expressing the bacteriophage T7 RNA polymerase.
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Results |
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The various RPV antigens were produced in B95a cells since preliminary experiments showed that expression of RPV H was higher in these cells than in any other of a range of cell lines tested (Vero, MDCK, Cos1, Rs-2. HeLa, BSC 40, Hep2, MDBK, CHO, BHK bovine skin fibroblasts and bovine lymphoblasts) (data not shown). Expression of RPV H on the cell surface was increased by increasing the m.o.i. of the infecting adenovirus; however, only a marginal increase in expression was found when the m.o.i. was raised from 500 to 1000. All other antigens were therefore produced from cells infected at an m.o.i. of 500.
Proliferative responses to rinderpest antigens following vaccination
Antigen was prepared from B95a cells infected with RPV H, F, N and M adenovirus recombinants and control antigen from cells infected with a recombinant adenovirus expressing bacteriophage T7 RNA polymerase. Fig. 6 shows that a proliferative response could be detected to all of the different rinderpest antigens in one animal on day 7 and in all animals from day 14 onwards following vaccination. A response to each rinderpest antigen was detected at every time-point tested. This response was maintained throughout the course of the experiment. In contrast, no response was detected to antigen prepared from uninfected cells (data not shown) or to the control antigen (bacteriophage T7 RNA polymerase). Animals vaccinated with KS-1 showed no response to any of the rinderpest antigens at any time (data not shown).
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Discussion |
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Immune suppression has been noticed following immunization with the vaccine strain of MV (Smedman et al., 1994 ; Pala et al., 1998
). It has also been suggested that RPV vaccination could result in transient immunosuppression and hence to an increase in mortality following RPV vaccination in the field (Jeggo et al., 1987
). In the case of RPV vaccination, the present study showed a rapid in vitro CD4+ T cell proliferative response to RPV antigen, together with unaltered responses to the mitogen ConA, despite the transient leukopenia caused by the RPV vaccine. This leukopenia is presumably caused by the low-level replication of the virus necessary for triggering the immune response. These data indicate that the RPV vaccine does not result in biologically significant immunosuppression. The results are in agreement with a recent field investigation into the frequency of infection with trypanosomosis in vaccinated and unvaccinated cattle in Kenya, where no increased risk of infection was noted in the vaccinated cattle (Stevenson et al., 1999
). This contrasts with the immunosuppression observed following MV vaccination in humans. It may therefore be possible to develop other morbillivirus vaccines, including MV vaccines, which cause less immunosuppression in the host. Further studies are required to investigate the cytokine profile of the rinderpest-specific CD4+ T cell responses to determine whether or not they are similar to those induced by MV, and if these responses have the potential to subvert cellular responses to other pathogens.
There was also no evidence of immunosuppression following challenge of RBOK vaccinated animals with the highly virulent RPV: in vitro proliferative responses to rinderpest antigen and ConA were maintained at normal levels. In addition, no increase was seen in the T cell proliferative responses to RPV antigen. This is likely to be a consequence of the solid immunity induced by this vaccine, as reflected by the absence of any clinical signs following challenge. However, an anamnestic response in serum antibody titres was seen in all animals following challenge, indicating that the virus must have replicated to some extent in the vaccinated animals.
A T cell proliferative response to crude RPV antigen was readily detected in animals vaccinated with the attenuated RPV strain. The response was detectable as early as 7 days in some animals and was consistently high from 28 days onwards. As anticipated, when using an inactivated antigen in the in vitro assay, this response was mediated mainly by CD4+ T cells, as demonstrated by immunofluorescence staining of the responding cells and testing the responses of purified CD4+ T cells. This finding does not exclude the possibility that RPV also stimulates a CD8+ T cell response, as the inactivated antigen used in the assay is unlikely to undergo processing by the endogenous route, which is required for stimulation of CD8+ T cells. Examination of cattle vaccinated with an unrelated vaccine (capripox KS-1) was carried out as a control and these animals showed a similar immune response to crude KS-1 antigen.
Studies of the antigen-specificity of the CD4+ T cell response focussed on the F, H, N and M structural proteins of the virus. The first three proteins have each been implicated in stimulating protective T cell responses following infections by MV and other paramyxoviruses (Bellini et al., 1981 ; Rose et al., 1984
; Ilonen et al., 1990
; Muller et al., 1993
; Pette et al., 1993
; Hickman et al., 1997
) and the M protein of MV has also been identified as a target for T cell responses (Bellini et al., 1981
). In the case of RPV, the N protein has been shown to induce a low level of protection and synthetic peptides based on the N protein sequence have been shown to stimulate T cells in vaccinated cattle and in cattle recovered from infection with a mild strain of the virus (Ohishi et al., 1999a
, b
). The aim of the present experiments was to determine whether the response in animals is biased to particular proteins and, if so, whether the bias in specificity varies between animals. Such data would be valuable when designing new recombinant vaccines for RPV or other morbilliviruses. Adenovirus recombinants were used to produce individual RPV proteins for analysis of the specificity of virus-specific CD4+ T cell responses. This expression system allows the production of relatively high levels of antigen with protein folding and post-translational modification properties similar to those of the native viral proteins. Uptake and processing of the proteins by antigen-presenting cells should be similar and any biological effects the molecules might have on immune cell types should be retained. The T cell responses detected using these recombinants showed a high degree of specificity for RPV antigens, as there were no detectable responses to either the adenovirus or host cell proteins present in the preparations. This was demonstrated by the lack of responses to adenovirus-derived bacteriophage T7 polymerase antigen prepared in an identical manner to that of the individual rinderpest antigens. No bias in the response was observed to any of the rinderpest antigens; responses of similar magnitude and kinetics were detected in all the animals examined. A similar broad specificity of the CD4+ T cell response to gel-purified MV proteins has been reported (Bellini et al., 1981
).
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Acknowledgments |
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Footnotes |
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c Present address: Moyne Institute of Preventive Medicine, Trinity College, University of Dublin, Dublin 2, Republic of Ireland.
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References |
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Received 3 March 2000;
accepted 24 May 2000.