The role of T cells in protection by an inactivated infectious bursal disease virus vaccine

Veterinary Immunology and Immunopathology 89 (2002) 159–167

The role of T cells in protection by an inactivated infectious bursal disease virus vaccine Silke Rautenschlein1, H.-Y. Yeh, J.M. Sharma* Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, USA Received 4 February 2002; received in revised form 16 July 2002; accepted 16 July 2002

Abstract The current belief is that the humoral immune response plays the principal role in defense against virulent infectious bursal disease virus (IBDV). In this study we used a model, in which chickens were compromised in functional T cells by neonatal thymectomy and Cyclosporin A (TxCsA) treatment, to demonstrate the role of T cells in protective immunity against IBDV. We demonstrated that T cells were necessary to achieve full protection against virulent IBDV. When T cell compromised TxCsAtreated chickens were vaccinated with an inactivated IBDV (iIBDV) vaccine, 91% were not protected against IBDV challenge in comparison to T cell-intact chickens, which had a protection rate of 91%. The iIBDV vaccine induced virus neutralizing (VN) and ELISA antibodies, respectively, in 65 and 5% of TxCsA-treated, and in 100 and 58% of T cell-intact birds. These observations provide evidence that the stimulation of T helper cells is needed for the production of protective antibody levels in iIBDV-vaccinated chickens. Passive administration of VN anti-IBDV antibodies inducing a circulating antibody level of log2 8 in chickens revealed that the levels of antibodies that protected T cell-intact chickens against virulent IBDV challenge were not protective for TxCsA chickens. These results indicated that antibody alone was not adequate in inducing protection against IBDV in chickens and that T cell-involvement was critical for protection. We propose that the inability of iIBDV to protect TxCsA chickens was due to compromised T cell immunity, functional T helper cells and most likely also cytotoxic T cells are needed in iIBDV vaccine protection. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Vaccination; T cells; IBDV; Chicken

1. Introduction Abbreviations: CPM, counts per minute; CsA, Cyclosporin A; iIBDV, inactivated IBDV; IBDV, infectious bursal disease virus; IBD, infectious bursal disease; Tx, thymectomy; VN, virus neutralizing * Corresponding author. Tel.: þ1-612-625-5276; fax: þ1-612-625-5203. E-mail addresses: [email protected] (S. Rautenschlein), [email protected] (J.M. Sharma). 1 Present address: Clinic for Poultry, Veterinary School Hannover, Bu¨nteweg 17, 30559 Hannover, Germany. Tel.: þ49-511-953-8763.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family whose genome consists of two segments of double stranded RNA, causes an acute, highly contagious disease in young chickens (Lukert and Saif, 1997). Because of the emergence of highly virulent viral variant strains in several countries (Jackwood and Saif, 1987; Snyder et al., 1988) IBDV has become a major problem in the intensive poultry industries worldwide. These strains cause

0165-2427/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 2 ) 0 0 2 0 2 - 7

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very high incidence of acute disease associated with high mortality, often exceeding 50% (Nunoya et al., 1992). Immunization of chickens is the principal method used for the control of IBD in chickens. Despite vaccination, IBDV continues to cause outbreaks in commercial flocks. Chickens infected with IBDV mount vigorous antibody response (Lukert and Saif, 1997; Tsukamoto et al., 1995). This antibody response is believed to play a major role in the defense against the disease. Current attenuated live and inactivated IBDV (iIBDV) vaccines are selected on the basis of their ability to induce antibodies, and protection by inactivated vaccines is attributed to humoral immunity. Whether cell-mediated immunity (CMI) contributes to protection induced by non-replicating IBDV is not know. Studies with several inactivated viral vaccines have revealed that CMI, particularly T cells, may play an important role in protection. (Christensen et al., 2000; Hedge and Srikumaran, 2000; Woodland et al., 2001). McNeal et al. (2002) demonstrated that CD4þ T cells were the only lymphocytes needed to protect mice against rotavirus shedding after administration of a non-replicating vaccine. Tripp and Anderson (1998) showed that a formalin-inactivated respiratory syncytial virus (RSV) vaccine-induced MHC class II restricted cytotoxic T-lymphocytes (CTLs) in mice. There is also evidence that an inactivated influenza virus vaccine may induce MHC class I restricted CTLs (Mbawuike and Wyde, 1993). Our objective was to examine the role of T cells in protection by an iIBDV vaccine. We speculated that T cell-deficient birds would not be sufficiently protected against IBDV-challenge. The results showed that only 9% of the TxCsA birds were protected by iIBDV in comparison to 91% protection in the T cell-intact chickens. This lack of protection in the TxCsA group could not only be attributed to reduced antibody levels in TxCsA birds. Passive transfer of protective levels of VN anti-IBDV antibodies to TxCsA birds did protect them significantly less against IBDVchallenge than T cell-intact birds indicating the involvement of cytotoxic T cells in IBDV protection. We propose that the inability of iIBDV to protect TxCsA chickens was due to compromised T cell immunity, most likely mediated by T helper and cytotoxic T cells.

2. Material and methods 2.1. Chickens Specific pathogen-free (SPF) chickens or embryonated eggs from HyVac (Gowrie, IA) were used. Chickens were hatched and reared in Horsfall– Bauer-type isolation units for the duration of the study. The birds were given food and water ad libitum. In each experiment, different test groups were housed in separate isolation units following the guidelines of the Minnesota Animal Care Committee. 2.2. Virus We used a virulent strain of IBDV (IBDV-IM) which was propagated and titrated in embryonated chicken eggs (Tanimura and Sharma, 1997; Kim et al., 1999). For the preparation of iIBDV, IBDV-IM was propagated in chickens. At 5 days post-IBDV inoculation, bursae from infected birds were harvested, homogenized and titrated in embryonated chicken eggs following previously published procedures (Winterfield et al., 1972). To inactivate IBDV, formaldehyde (Sigma) was added to 5 ml of 106.8 embryo lethal dose (ELD50)/ml of IBDV in phosphate buffered saline (PBS) to a final concentration of 0.2% (v/v) and incubated at 37 8C following previously described procedures (Hoshi et al., 1995). After 48 h of incubation iIBDV was free of live virus. Preliminary in vivo experiments confirmed that the inoculation of chickens with an inactivated dose of previously 105.8 ELD50 of IBDV per bird did not result in detectable replication of residual live virus in the bursa of Fabricius by immunohistochemical procedure. In the presented experiments, birds were inoculated with iIBDV (105–106 ELD50/bird of virus dose before inactivation) with Freund’s incomplete adjuvant. 2.3. Reduction of T cell responsiveness by thymectomy (Tx) and Cyclosporin A (CsA) At 1 day post-hatch, chickens were surgically thymectomized following previously published procedures (Sharma et al., 1975; Cihak et al., 1991). Briefly, the chickens were anesthetized by intramuscular injection of 0.1–0.15 ml of sodium pentobarbital solution (16 mg/ml). The neck was entered via a

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dorsal incision. Thymus lobes running beside the carotid arteries were removed with forceps. The wound was closed with surgery clamps. Starting-point 7 days post-operation and lasting through the duration of the experiments, the birds were treated intramuscularly every 3–4 days with 100 mg/kg body weight of CsA (Nowak et al., 1982; Kim et al., 2000). One or two days following the third CsA injection, chickens were examined for the numbers of circulating T cells and mitogenic responses. During the first 2 weeks of the experiment, chickens were given antibiotics in the drinking water to prevent bacterial infections. 2.4. H&E staining For the detection of histopathological lesions, the bursa of Fabricius was collected, fixed in 10% buffered formalin and stained with hematoxylin and eosin (H&E). Lesions were observed microscopically and lesion scores were determined and compared between groups (Sharma et al., 1989; Kim et al., 1999). 2.5. Immunohistochemistry assay to detect IBDV The procedures have been described (Tanimura and Sharma, 1997; Kim et al., 1999). Bursae of Fabricius were collected, snap frozen, sectioned and processed for immunohistochemical staining. A polyclonal rabbit anti-IBDV serum obtained from Dr. K. Tsukamoto, National Institute of Animal Health, Japan, was used for IBDV detection (Tanimura and Sharma, 1997). The number of IBDV-positive cells were counted in 10 microscopic fields per bird at a magnification of 400. The group means of these numbers were calculated and statistically compared between groups. 2.6. Proliferation assay Mitogenic responses of peripheral blood leukocytes were carried out as described previously (Sharma and Belzer, 1992). Briefly, 1:10 dilutions of heparinized whole blood samples were stimulated ex vivo with 100 mg of ConA/ml at 41 8C. After 43 h of incubation, the cells were pulsed with 1 mCi/well [3 H]-thymidine for 5 h, harvested and counted in a Matrix 9600TM Direct Beta Counter (Packard Instrument Company, Meriden, CT). The results are expressed as stimulation indices (SI) which equals counts per minute

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(CPM; CMI) after ConA stimulation/CPM without stimulation. 2.7. Flow cytometric analysis of lymphoid cell populations Single cell suspensions of peripheral blood leukocytes were separated in a discontinuous density gradient using Ficoll-Hypaque (Karaca et al., 1995). For flow cytometric evaluation we followed the protocol by Kim et al. (2000). We used directly FITC-labeled monoclonal anti-chicken CD4, antichicken CD8, or anti-chicken IgM antibodies (Southern Biotechn.). 2.8. Serology Serum samples were collected at 14 and 21 days post-iIBDV inoculation. Anti-IBDV antibody titers were determined by using a kit from KPL (ProFlok1 plus, Gaithersburg, MA). VN antibody titers were determined following standard procedures (Winterfield et al., 1972). 2.9. Hyperimmune-anti-IBDV-antiserum For the preparation of a chicken anti-IBDV hyperimmune serum, 2-week-old SPF chickens were inoculated with 103 EID50 of an intermediate strain of IBDV per birds intraocular. Two-and-a-half weeks later, they received IBDV-IM (103 ELD50 per bird intraocular). Fourteen days later, chicken serum was collected and anti-IBDV-antibody titers determined. The hyperimmune serum used had an anti-IBDV antibody titer of log10 3.82 and log2 15.6 determined in the ELISA (ProFlok1, KPL) and virus neutralization test, respectively. 2.10. Experimental procedure In three repeat experiments, 1-day-old SPF chickens were Tx. At 1-week of age, groups of 5–15 Tx birds were treated with repeat injections of 100 mg/kg body weight of CsA intramuscularly every 3–4 days throughout the experiments. At 2 days following the third injection of CsA, TxCsA and T cell-intact groups were examined for the presence of CD4þ, CD8þ and IgMþ cells by flow cytometric analysis as well as for

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T cell mitogenesis. At 2–3 weeks of age, the birds were vaccinated with iIBDV by intramuscular injection. At 10–14 days post-vaccination, the serum antiIBDV antibody levels were determined by ELISA and VN assay. At the same time, the birds were challenged with 103 ELD50/bird of IBDV-IM. At 5 days postchallenge, the pathological lesions, histopathological lesions, and IBDV antigen distribution in the bursa were observed. It was confirmed by necropsy that surgical Tx did not leave behind intact thymus lobes. Chickens that had remaining intact lobes were excluded from the experiment. In Experiment 4, groups of seven chickens were treated intramuscularly with 100 mg/kg BW of CsA every 3–4 days through the experiment. After the third treatment the depletion of functional T cells was confirmed using the whole blood mitogenic assay and the number of circulating CD4þ and CD8þ cells was determined by flow cytometric analysis. At 14 days post-hatch, groups of 7 T cell-depleted and intact birds were treated with chicken hyperimmune antiIBDV antiserum intravenously. Chickens were given 50 and 100 ml of the hyperimmune serum to achieve virus neutralizing (VN) serum antibody levels of log2 6.5 and 8 per ml, respectively. A serum neutralizing antibody titer of log2 8 was protective against IBDV-challenge virus-induced mortality, pathological lesions, and histopathological lesions with scores >2 as determined in preliminary experiments (data not shown). At 15 days post-hatch the antiserum inoculation was repeated and birds were inoculated 2 h later with virulent IBDV-IM (103 EID50 intraocular; Kim et al., 1998). The passively transferred circulating anti-IBDV antibody titers were determined at the time of IBDV-inoculation in serum samples using the VN test (Winterfield et al., 1972). After infection, the mortality rate was determined and at 5 days PI the remaining chickens were sacrificed and bursa histopathological lesions recorded. Further, the number of IBDV-positive cells in the bursa were determined using immunohistochemical methods (Tanimura and Sharma, 1997). 2.11. Statistical analysis Group responses within experiments were analyzed by one-way analysis of variance (ANOVA) and pairwise multiple comparison procedure by the Tukey test,

Student’s t-test or w2-test as indicated in the table and figure legends.

3. Results 3.1. T cell depletion regimens reduced the number of circulating T cells and the mitogenic responsiveness of residual T cells As expected, TxCsA- and CsA-treatment induced a dramatic reduction (P < 0:001) in the mitogenic response of circulating T cells, which lasted through the duration of the vaccine experiments (Table 1) (Suresh and Sharma, 1995; Kim et al., 2000). The flow cytometric analysis indicated a similar reduction in the number of circulating CD4þ and CD8þ cell populations in TxCsA and CsA-treated birds. The CD4þ cells were reduced by 62  20%, and the CD8þ cells by 47  6:2% in comparison to the untreated control birds (Kim et al., 2000). TxCsA did not affect the number of circulating IgMþ cells, the ability of B cells to produce antibodies or LPSinduced NO production by splenic macrophages (Karaca et al., 1996; Rautenschlein et al., 2002) (data not shown).

Table 1 Effect of T cell depletion regimens on T cell mitogenesisa Groups

T cell-intact TxCsA CsA

Stimulation indexb (whole blood, ConA 100 mg/ml) at experimental days 20 (n ¼ 1020)

36 (n ¼ 5)

64.0  7.9 a 3.2  1.6 b 3.0  2.3 b

58.7  24.8 a 2.4  0.8 b 7.3  9.9 b

a Intact or Tx birds were treated with 100 mg/kg body weight of CsA every 3–4 days during the course of the experiment. Three days following the third CsA treatment (experimental day 20) and at experimental day 36 the whole blood mitogenic assay was performed as described. Different letters within a column indicate significant differences between T cell-intact and T cell-compromised birds (Student’s t-test, P < 0:01). b Stimulation index ¼ CPM after ConA stimulation/CPM following incubation in medium only. The average CPM values for cultures without ConA-stimulation were as follows: T cellintact chickens—80 CPM, TxCsA chickens 96 CPM and CsAchickens—73 CPM.

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Table 2 Anti-IBDV antibody development in TxCsA birds following vaccination with iIBDVa Treatment

Vaccine

Number of birds positive in VN/number tested (mean titer of positive birds for VN log2 titer > 1  S:D:)

Number of birds positive in ELISA/number tested (mean log10 titer of positive birds  S:D:)

None TxCsA None TxCsA

– – iIBDV iIBDV

0/15 (0  0 a) 0/10 (0  0 a) 28/28 (9.8  1.0 b) 23/35 (4.4  3.8 c)

0/15 b (0  0) 0/10 b (0  0) 18/31 a (3.5  0.5) 2/38 b (3.1  0.2)

a Tx birds were treated with 100 mg/kg body weight of CsA every 3–4 days during the course of the experiment. At 2–3 weeks post-hatch, T cell-compromised and T cell-intact birds were vaccinated with iIBDV intramuscularly. At 10–14 days post-vaccination, sera were tested for anti-IBDV antibodies by ELISA (IgG class, KPL, Gaithersburg, MA) or VN assay. Different letters indicate significant differences within a column (ANOVA or w2-test, P < 0:01). S.D. is the standard deviation. Presented is summary of three experiments.

3.2. TxCsA-treated chickens developed lower circulating anti-IBDV antibody levels than T-cell-intact birds TxCsA treatment reduced the ability of chickens to produce anti-IBDV VN antibodies. TxCsA birds of 65% developed VN antibodies in comparison to 100% of the T cell-intact birds (P < 0:05; Table 2). T cell-intact birds had VN antibody titers of mean log2 9.8 versus log2 4.4 in TxCsA birds (P < 0:05; Table 2). Anti-IBDV IgG antibodies, indicating immunoglobulin class switch, were detected in the ELISA using anti-chicken IgG as secondary antibodies. iIBDV

induced in 58% of T cell-intact birds and 5% of the TxCsA birds anti-IBDV ELISA antibodies (Table 2). 3.3. TxCsA treatment reduced the protective ability of iIBDV in chickens Results presented in Table 3 demonstrated that 100% of the iIBDV-vaccinated T cell-intact birds were protected against mortality after challenge with virulent IBDV-IM while 23% of the vaccinated TxCsA birds died after challenge. Vaccinated T cell-intact birds of 91% were protected against microscopical bursa lesions in comparison to 9% protection in the

Table 3 Protection against virulent IBDV induced by iIBDV in T cell-intact and T cell-compromised birdsa Groups

T cell-intact T cell-intact T cell-intact þ iIBDV TxCsA TxCsA þ iIBDV a

Challenge with IBDV-IM

 þ þ þ þ

Response to IBDV-IM (n ¼ 1021) Mortality (%)

Percentage of birds with pathological lesions in bursa

Mean pathohistological lesion score  S:D:

0 63 0 44 23

0 100 9 100 76

0.0 4.0 0.09 3.8 3.0

a b a bc c

a b a b b

    

0.0 0.0 0.3 0.4 1.7

a b a bc c

Protection (%)b NAc 0a 91 b 0a 9a

Presented is the summary of two repeat experiments. Tx birds were treated with 100 mg/kg body weight of CsA every 3–4 days during the course of the experiment. At 2–3 weeks post-hatch T cell-compromised and T cell-intact birds were vaccinated with iIBDV. Fourteen days post-vaccination, the birds were challenged with virulent live IBDV (IBDV-IM, 103 ELD50/bird intraocular). The mortality rate was determined and the birds were sacrificed at 5 days post-challenge. Pathological bursa lesions are expressed in the table as percentage of birds positive for bursa gelatination, hemorrhage and/or necrosis. Sections of the bursa of Fabricius were histologically evaluated based on the previously described scoring system (Kim et al., 1999; Sharma et al., 1989). Different letters indicate significant differences between groups (P < 0:05, w2-test or ANOVA). b Protection (%) is defined as % of birds/group with an IBDV-lesion score of <1. c Not applicable.

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Fig. 1. IBDV replication following challenge with virulent IBDV of vaccinated and unvaccinated birds with intact or compromised T cell functions. Presented is the result of one of three repeat experiments. At 2 weeks post-hatch, TxCsA- and T cell-intact birds were intramuscularly vaccinated with iIBDV. At 14 days post-vaccination, the birds were challenged with virulent IBDV-IM (103 EID50/bird intraocular). At 5 days post-challenge, bursae were harvested, snap-frozen and immunohistochemically analyzed for IBDV-antigen. Ten randomly chosen fields were evaluated for the number of IBDV-positive cells. The group average of IBDV-positive cells/ field at 400 is presented. Different letters indicate significant differences between groups (ANOVA, P < 0:05).

vaccinated TxCsA birds (Table 3). Vaccination with iIBDV protected T cell-intact but not TxCsA chickens from vigorous replication of the challenge virus as presented in Fig. 1. 3.4. Functional T cells were necessary to complete protection in the presence of VN antibodies The lack of protection in TxCsA birds may be due to low antibody production by these birds. In order to examine this possibility we determined minimal circulating VN-anti-IBDV antibody levels needed for

protection in T cell-intact chickens against bursal destruction by virulent IBDV. We correlated the presence of circulating neutralizing antibody levels with protection against challenge with virulent virus (Fig. 2). Data in Fig. 2 shows that VN antibody levels of log2 8 and above were protective against mortality and a bursal lesion score of 2 or higher. Data from two repeat experiments revealed that over 90% of the TxCsA birds failed to produce antibody levels of log2 8 and above. Most of the intact birds had VN levels well above the minimum protective level of log2 8 (Fig. 2).

Fig. 2. Correlation between anti-IBDV-VN antibody level and protection against virulent IBDV in TxCsA and T cell-intact birds. Each filled circle indicates one bird. % IBD indicates the percentage of birds with a microscopical lesion score >1. Summary of two repeat experiments.

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Table 4 Effect of functional T cell depletion on protection against IBDV in the face passively transferred neutralizing anti-IBDV antibodiesa IBDV

Mortality (%)

IBDV-mean bursa score  S:D: (number positive/number tested)

0 0 0.7 0.5

 þ þ þ

0 60 0 0

0 4 0.6 2.9

   

0 a (0/5) 0 b (5/5) 0.9 c (2/7) 1.7 d (6/7)

0 68 13 67

   

0 a (0/5) 26 b (5/5) 12 c (4/7) 41 b (6/7)

0 0 0 0.5

 þ þ þ

0 43 0 0

0 4 2.1 3.9

   

0 a (0/5) 0 b (5/5) 1.1 d (7/7) 0.4 d (7/7)

0 61 30 78

   

0 a (0/5) 16 b (5/5) 8 d (7/7) 11 b (7/7)

Groups

Circulating VN titer (mean log2  S:D:) after passive antibody treatment

T cell-intact

0 0 8 6.6

   

CsA

0 0 8 6.4

   

Number of IBDV þ cells/ fieldb (number positive/ number tested)

a Groups of seven chickens were treated intramuscularly with 100 mg/kg BW of CsA every 3–4 days through the experiment. After the third treatment the depletion of functional T cells was confirmed using the whole blood mitogenic assay. At 14 days post-hatch, T cell-depleted and intact birds were treated with 50 or 100 ml of a chicken hyperimmune anti-IBDV antiserum intravenously. At 15 days post-hatch the antiserum inoculation was repeated and birds were inoculated with virulent IBDV-IM (103 EID50/bird intraocular; Kim et al., 1998). The passively transferred circulating anti-IBDV titers were determined in serum samples using the VN test. After infection, the mortality rate was determined and at 5 days PI the remaining chickens were sacrificed and bursa histopathological lesions recorded. Different letters indicate significant differences between groups (ANOVA, P < 0:05). S.D. is the standard deviation. b The number of IBDV-positive cells in the bursa were determined using immunohistochemical methods (Tanimura and Sharma, 1997).

We passively transferred VN antibody levels to naı¨ve T cell-intact and TxCsA SPF chickens. The birds given passive transfer were divided into two groups: those attaining a titer of at least log2 8 (protective level in T cell-intact chickens) and those attaining a titer of lower than log2 8. There was a significant difference in the susceptibility to IBDV infection between T cell-intact and T cell-depleted birds that had a circulating VN antibody titer of log2 8 (P < 0:05). Two of 7 T cell-intact birds had minor IBDV-induced histological bursa lesions with an average lesion score of 0.6 and low viral replication with 13 IBDV-positive cells/microscopic field. Seven of 7 TxCsA birds had advanced bursa lesion scores with an average score of 2.1. The average number of IBDVpositive cells was 30 per microscopic field (400). When the transferred antibody levels were reduced by approximately 100 there was no difference between T cell-intact and T cell-compromised birds in protection against viral replication and virus-induced bursa lesions (Table 4).

4. Discussion The current belief is that the humoral immune response plays the principal role in defense against

virulent IBDV (Lukert and Saif, 1997; Tsukamoto et al., 1995). The possible involvement of cellular immunity, particularly T cell immunity, in vaccine protection against IBDV is not known. An adequate T cell response seems to be necessary for the development of protective immunity against most viruses (Zinkernagel, 1996). We used a T cell compromised chicken model and an iIBDV vaccine for examining the role of T cells in protection against virulent IBDV. In this study we demonstrated for the first time that T cell immunity was critical for protection induced by iIBDV. When T cell-compromised (TxCsA) chickens were vaccinated with iIBDV, 9% were protected against virulent IBDV challenge in comparison to T cell-intact chickens, which had a protection rate of 91%. The iIBDV vaccine induced VN and ELISA antibodies, respectively, in 65 and 5% of TxCsAtreated, and in 100 and 58% of T cell-intact birds. These observations provide evidence that the stimulation of T helper cells is needed for the production of protective antibody levels in iIBDV-vaccinated chickens. These observations concur with previous studies in mammals demonstrating that T helper cells play a critical role in the induction of protection using nonreplicating HIV-vaccines (Heeney et al., 1998; Sarobe et al., 1994). High levels of neutralizing antibodies and high levels of T helper cell responses correlated with

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protection against simian/human immunodeficieny virus (Heeney et al., 1998). To support this indirect evidence that CD4þ T cells are important in protection against IBDV, transfer experiments are needed in the future. IBDV-specific CD4þ T memory cells should be passively administered to T cell immunodeficient chickens and their protective ability tested. Passive administration of VN anti-IBDV antibodies inducing a circulating antibody level of log2 8 in chickens revealed that the levels of antibodies that protected T cell-intact chickens against virulent IBDV challenge were not protective for TxCsA chickens. These results indicated that antibody alone was not adequate in inducing protection against IBDV in chickens and that T cell involvement was critical for protection. T cells may be involved in two ways. They may be needed for additional anti-IBDV antibodies levels to complete protection in T cell-intact birds even in the face of passively transferred antibodies. Alternatively, cytotoxic T cell functions may be important. How cytotoxic T cells may be stimulated by inactivated vaccines and mediate protection is not known. In vitro studies have suggested mature dendritic cells may play a role as antigen-presenting cells by complexing their MHC class I molecules with epitopes of inactivated virus (Subklewe et al., 1999). Alternative TAP-independent pathways may contribute to the ability of inactivated vaccines to stimulate CTLs in the absence of an active infection of the antigen presenting cells (Liu et al., 1997; Snyder et al., 1997). Adoptive transfer experiments using anti-IBDV memory cytotoxic CD8þ cells or T cell depletion models using in vivo anti-CD8 antibody treatment (Morimura et al., 1999) are needed to substantiate the observations obtained in this study. This study clearly shows that T cells are important for protection against virulent IBDV. T helper cells and most likely also cytotoxic T cells are involved in iIBDV vaccine protection because passive transfer of VN antibodies did not induce protection in TxCsA birds. Future IBDV vaccine development should be directed to support T cell stimulation to induce adequate protection. T cell-mediated protection may be of special importance if the humoral immune response may not have been induced efficiently by a vaccine as may be the case in maternal antibody positive chicken.

Acknowledgements We thank Edwin Pereira, Angela Rezin and Anton Yerich, for their excellent technical assistance.

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