Antigen-specific blastogenesis assays for duck hepatitis B virus using duck peripheral blood and splenic mononuclear cells

Veterinary

ELSEYIER

Immunology and Immunopathology 59 (1997) 349-358

Veterinary immunology and immunopathology

Antigen-specific blastogenesis assays for duck hepatitis B virus using duck peripheral blood and splenic mononuclear cells Karen Vickery ’

Department

h Department

aq*, Yvonne Cossart ‘, Xingnian Gu b, Robert Dixon b

of Infectious Diseases,

UnioersiQ cjf Sydney. Sydney NSW 2006. Australia

of Animal Health, CJnkersiry of Sydney. Weromhi Rd.. Camden NSW 2570, Australiu

Accepted

10 June 1997

Abstract

An antigen-specific lymphoblastogenesis assay for duck hepatitis B surface antigen (DHBsAg) and duck hepatitis B core antigen (DHBcAg) was developed using mononuclear cells from the peripheral blood (PBMC) or spleens (SMC) of immune ducks. Optimal culture conditions for the assay were determined by testing a number of variables, including antigen concentration, cell numbers/well, and the day of harvest. The specificity of the assay was assessed. The assay used 10% pooled duck serum supplement, and 8 X lo5 cells/well for PBMC or 5 X lo5 cells/well for SMC. The optimum antigen concentration ranged from 0.01 to 0.1 pg/ml for both DHBsAg and DHBcAg. Maximum antigen-specific blastogenesis occurred between 4 to 7 days after establishment of the culture. The use of PHA (10 pg/ml) mitogenesis could predict the optimal cell numbers/well for antigen-specific blastogenesis. The assay demonstrated specific responses by immune ducks compared with those of unexposed ducklings and adult ducks (for DHBsAg P < 0.001; DHBcAg P < 0.05). For immune ducks, PBMC from all 8 ducks responded to DHBsAg. however, cells from only 4 of 7 immune ducks responded to DHBcAg. Splenic mononuclear cells from all immune ducks responded to either DHBsAg or DHBcAg or both antigens. 0 1997 Elsevier Science B.V. Keywords:

Duck: DHBV: CM1

* Corresponding

author. Tel.: +61 2 9351 4037: fax: + 61 2 9351 4731: e-mail: [email protected].

0162427/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO165-2427197100075-5

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1. Introduction The humoral component of the duck immune system has been subjected to a number of recent investigations. Duck innnunoglobulins, particularly IgG, have been found to differ structurally from those of the domestic fowl and mammals (for review, Higgins and Warr, 1993). Nevertheless, conventional RIA and ELISA methods have been successfully exploited in studies of antibody responses to experimental duck hepatitis B (DHBV) infection (Vickery et al., 1989). The cells of the duck immune system also differ. Higgins and Chung (1986) determined that standard approaches to avian and mammalian cell markers did not readily differentiate duck lymphoid cells into T and B lymphocytes. A recent study by Bertram et al. (1996) has shown that duck T cells can be identified using antihuman CD3 antiserum. It can be assumed that cellular immunity plays a central role in the overall immune response of the duck, and there are publications on the response of duck mononuclear cells to nonspecific mitogens (Higgins and Teoh, 1988; Higgins, 1990, 1991, 1992; Vickery et al., 1995). However, there are no detailed reports in the scientific literature on specific cell-mediated immune (CMI) responses by the duck. CM1 is important in the pathogenesis of hepadnaviral infections of humans. The role of the individual hepatitis B virus (HBV) antigens in the pathogenesis of HBV infection has not been fully elucidated and may differ in acute or chronic infection. Duck hepatitis B virus (DHBV) and its animal host, the duck, have the potential to explore this as the duck immune system is capable of in-vivo manipulation. Different clinical outcomes of infection can be established in vivo (Vickery and Cossart, 1996). These outcomes may be related to the immune response, but there are no published studies on cellular immunity to DHBV. Antigen-specific blastogenesis of lymphoid cells to re-exposure by viral antigens in vitro (Elves et al., 1963) has been the starting point for numerous studies of cellular immunity to HBV infection (for review, Chisari and Ferrari, 1995). This paper reports the development of an antigen-specific blastogenesis assay for mononuclear cells from the peripheral blood and spleen of ducks immune to DHBV. A number of variables were examined and conditions were optimised for detection of responsiveness to both duck hepatitis B surface antigen (DHBsAg) and duck hepatitis B core antigen (DHBcAgl. This assay will be useful in evaluating the role of CM1 in determining the course of DHBV infection in its natural host.

2. Materials

and methods

2.1. Ducks All ducks were Pekin-Aylesbury cross birds originating from a DHBV-negative commercial supplier. Both male and female ducks were used. The use of animals was approved by the University of Sydney’s Animal Care and Ethics Committee. Ducks were made immune to DHBV by first intramuscular injections of purified DHBsAg (without adjuvant) at a rate of 25 pug/kg on days 4, 13 and 22 days post

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hatch. On day 26 post-hatch, they were challenged with 10 ID,, doses of DHBV-positive serum intravenously. All ducks remained negative for DHBV in the serum and liver to the end of the experiment. The nonimmune four-week-old ducks were obtained at one-day of age and housed separately from DHBV-positive ducks. Nonimmune adult (twelve-month-old) ducks were bled on the farm of the commercial supplier. 2.2. Viral antigens DHBsAg was purified from high titre duck serum by ultracentrifugation. This involved pelleting through a 10% sucrose cushion at 140000 g for 16 h, after which the resuspended pellet was layered on a discontinuous CsCl gradient. The gradient was centrifuged at 140000 g for 48 h and fractions collected. Fractions containing DHBV were identified by dot-blot hybridisation, pooled and centrifuged through a second discontinuous CsCl gradient also at 140000 g for 48 h. Fractions containing DHBsAg were determined by PAGE and Western blot, pooled and dialysed against PBS (Marion et al., 1983). DHBcAg was purified from the liver by the method of Summers and Mason (1982). Briefly, homogenised liver was clarified first in a preparative centrifuge followed by a second centrifugation at 155000 g for 90 min in an ultracentrifuge. The clarified supematant was then layered on a 15-30% linear sucrose gradient and centrifuged at 55 000 g for 16 h. The pellet was resuspended in a solution containing 50 mM NaCl and 5.5% PEGmoo. This was again centrifuged on a 15-30% linear sucrose gradient at 150 000 g for 165 min and the core-rich fractions identified by PAGE and Western blot analysis. The purity of both antigens was confirmed by Western blot analysis. Rabbit antiDHBsAg antibody (Vickery et al., 1989) and rabbit anti-DHBcAg antiserum raised from E. coEi expressed DHBcAg (a kind gift from Dr. Allison Jilbert, University of Adelaide) were used for detection of the 2 antigens (Jilbert et al.. 1992). 2.3. Separation

of peripheral

blood mononuclear

cells

Peripheral blood mononuclear cells (PBMC) were collected and separated by an adaptation of the method of Higgins and Chung (1986). Briefly, 7 ml of blood was collected by jugular venipuncture into an equal volume of prewarmed (30°C) PBS (pH 7.2) containing 10 IU/ml of heparin. Seven ml of the PBS/blood were layered over 3.5 ml of Ficoll-Paque (Pharmacia, Uppsala, Sweden) and centrifuged at 170 g for 25 min. Centrifugation and all remaining procedures were carried out at room temperature. The mononuclear cell-rich fractions were pooled and washed in RPM1 1640 medium (Sigma. St Louis, USA) 3 times. 2.4. Separation

of mononuclear

cells ,from the spleen

Spleen mononuclear cells @MC) were separated by the method of Vickery et al. (1995). Briefly, the aseptically removed spleen was diced, washed and gently pressed

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through a stainless steel 120 mesh sieve into 5 ml of RPM1 1640 medium. The suspended cells were made to a volume of 50 ml with medium and then layered on Ficoll-Paque (7 ml of cells:3 ml of Ficoll-Paque) prior to centrifugation in a benchtop centrifuge at 265 g for 30 min. The interface layer was harvested and washed 3 times with medium by centrifugation at 170 g for 10 min at room temperature. 2.5. Culture of mononuclear

cells

Both PBMC and SMC were counted and their viability assessed by trypan blue exclusion. Cells were cultured in 96 well flat-bottom microculture plates (Nunc) in 200 ~1 RMPI 1640 with 20 mM Hepes and 23 mM NaHCO,, 100 IU benzyl-penicillin, 100 pg dihydrostreptomycin sulphate/ml and supplemented with 10% heat-inactivated pooled duck serum (PDS) derived from DHBV-negative ducks. Mitogen or antigen was added at the same time the cultures were established. Phytohaemagglutinin (PHA) (10 pug/ml) was added to the cells cultured in three wells of each microculture plate to monitor whether culture conditions were suitable for blastogenesis. Each variable was tested in triplicate wells unless stated. Plates were incubated at 41°C in a humidified 5% COZ in air atmosphere. 2.6. Calculating

the stimulation

index

Cells were pulsed with 1 &i of 3H thymidine (ICN, Irvine, USA) in 20 ~1 of RMPI 1640 for 6 h. They were harvested onto glass-fibre mats (GF/C, Whatman, Maidstone, UK) and the uptake of 3H in dpm was measured by an LKB 1214 Rakbeta Scintillation Counter. All cultures included unstimulated labelled and unstimulated unlabelled controls. The stimulation index (SI) was calculated using the mean dpm of the triplicate wells by the formula: (stimulated (unstimulated

2.7. Optimisation

3H - labelled) 3H - labelled)

- (unstimulated

unlabelled)

- (unstimulated

of the antigen-specijk

blastogenesis

unlabelled)

assay

2.7.1. Optimisation of viral antigen concentration This was determined in two stages. A dose response study used DHBsAg concentrations of 0.001, 0.01, 0.02, 0.04, 0.1, 1, 5 and 10 Fug/ml to stimulate SMC cultures from two ducks. Cells were cultured at 5 X lo5 cells/well and harvested from day 4 to 9. For the second stage, PBMC cultures were established from 6 ducks and SMC cultures from an additional 4 ducks. These were stimulated with DHBsAg and DHBcAg at various concentrations ranging from 0.001 to I pg/ml. PBMC were cultured at their individual optimal cell numbers/well (see below) while SMC were cultured at 5 X 10’ cells/well. All cultures were harvested 4 to 9 days after being established.

K. Vicke?

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Table 1 Comparison of antigen specific and mitogen induced transformation ducks cuhured at various cell numbers/well Duck

Mitogen

R81

PHA DHBsAg PHA DHBsAg PHA DHBsAg PHA DHBsAg

R96 R8S R82”

59 (I 997) 349-35X

353

responses (in SD of PBMC from immune

Cell numbers/well 6x IO5

8X105

1x106

9.5 6.6 nd rid nd nd nd nd

21.7 12.1 5.2 5.2 5.0

15.0 9.1 7.Y X6 Y.6

I .9 nd nd

2.9 25.0

I xl

“Transformation responses to DHBsAg of PBMC cultured from duck R82 at 3 X lo6 and 5 X IOh cells/well were not significantly different from control unstimulated cultures. nd = not done. SI = stimulation index. PBMC = peripheral blood mononuclear cells. PHA = phytohaemagglutinin. DHBsAg = duck hepatitis B surface antigen.

2.7.2. Uptimisation of cell numbers/well The responsiveness to specific and nonspecific mitogens is influenced by the number of cells/well in culture and the optimum can vary from individual to indtvidual. However, there is a limit to the amount of blood that can be removed from a duck at any single time and this precludes the testing of a range of cell numbers/well for each assay. To overcome this limitation, the optimal cell numbers/weIl to give the best response by PBMC and SMC to PHA was evaluated as a predictor for the optimal cell numbers/well for antigen-specific blastogenesis. The optimal cell number could then be determined prior to the actual antigen-specific assay. PBMC were purified from 4 immune ducks and cultured at 2 or 3 different cell numbers/well (Table 1). The cells were stimulated with either PHA (10 pug/ml) or DHBsAg (0.01 and 0.1 pg/ml). Cultured cells were harvested on 4 different days between 1 and 9 days later. It is known that the optimal responsiveness of SMC to PHA mitogenesis occurs at 5 X IO5 cells/well with little animal to animal variation (Vickery et al.. 1995). A comparison was made between the responses observed to PHA or DHBV antigens by SMC evaluated at various cell numbers/well. SMC were purified from 2 DHBV immune ducks and cultured at a range of cell numbers/well (Table 2) in the presence of DHBsAg at 0.01, 0.1 and 1 pg/ml or DHBcAg at 0.01 and 0.1 pg/ml. Cells were harvested on 4 different days between 1 and 9 days after the cultures were established. 2.7.3. Optimisation of day of haruest The time to onset of blastogenesis was ascertained for PBMC from 7 immune ducks. Three of these ducks were bled twice 60 days apart. PBMC were cultured at the optimum cell number/well for each individual animal as determined before and were

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Table 2 Comparison of antigen specific and mitogen-induced transformation responses (in SI) of SMC from immune ducks cultured at various cell numbers/well and at different antigen concentrations ( pg/ml) Duck

Mitogen

Cell numbers/well

R82

PHA DHBsAg 0.01 0.1 1.0 DHBcAg 0.01 0.1 PHA DHBsAg 0.01 0.1 1.0 DHBcAg 0.01 0.1

nd nd nd nd nd nd 3.4 -0.6 -0.6 -0.1 -1.5 -1.4

1x

R91

IO5

2x10s

5x105

7.5 x to5

1x lo6

zx106

nd nd nd nd nd nd 131 -0. I 0.1 NS -0.3 -0.1

270 NS 5.0 NS 4.0 3.0 1070

29.0 I .76 2.4 NS 1.9 NS nd

2.3 NS NS NS NS 3.0 nd nd nd nd nd nd

0.7 NS NS I .4 1.9 NS nd nd nd nd nd nd

NS = not significantly different from control unstimulated nd = not done. SI = stimulation index. PBMC = peripheral blood mononuclear cells. PHA = phytohaemagglutinin. DHBsAg = duck hepatitis B surface antigen. DHBcAg = duck hepatitis B core antigen.

10.0

nd

12.8 11.0 4.0 8.3

nd nd nd nd

cultures.

Table 3 Days after establishment of PBMC cultures from immune ducks when significant tion (in SI) due to DHBsAg was detected Duck

P54a P54b P56a P56b P59a P59b R82 R85 R88 R96

antigen-specific

transfotma-

Day of Harvest 4

5

6

7

8

9

2.0 17.5 NS 4.6 NS 2.0 nd NS nd NS

2.0 NS NS NS 2.1 2.0 11.0 2.9 nd NS

NS NS 5.4 nd NS NS NS NS 16.0 5.2

4.2 NS 4.3 NS NS NS 12.0 nd NS nd

NS NS NS 7.0 NS NS NS nd 0.2 nd

NS NS NS 1.6 NS NS NS nd 8.0 nd

SI = stimulation index. NS = not significantly different from control cultures. nd = not done. a = first sample. b = second sample 60 days after first sample. DHBsAg = duck hepatitis B surface antigen.

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59 c 19971349-35X

stimulated with either DHBsAg or DHBcAg at varying concentrations ranging 0.001 to 1 pg/ml. Cells were harvested on a number of days (see Table 3). 2.8. Specijkity

35.5

from

of the assay

PBMC were purified from 10 four-week-old ducks and 10 twelve- month-old (adult) ducks from a DHBV-negative commercial farm. Cells from the four-week-old ducks were cultured at the optimum cell numbers/well previously determined by the individual responsiveness to PHA. Cells from the adult ducks were cultured only at 8 X 10’ cells/well (Higgins and Teoh, 1988). DHBsAg or DHBcAg were used at 0.01 and 0.1 pg/ml. Cells were harvested daily from day 3 to 7. PBMC from 8 immune ducks were cultured in the presence of DHBsAg or DHBcAg at varying concentrations from 0.001 to O.l/ml at their optimal cell numbers/well. SMC were purified from 2 DHBV-negative four-week-old and 4 immune ducks and cultured at 5 X lo5 cells/well with DHBsAg or DHBcAg at 0.01 and 0.1 pg/ml. Cells were harvested daily from day 5 to 9. 2.9. Statistical

analysis

The student’s t-test was used to test for significant differences in blastogenesis between antigen-stimulated and control cultures in individual ducks and to test for differences between group means.

3. Results 3.1. Optimisation

of the antigen-specific

blastogenesis

assay

3.1.1. Optimisation of viral antigen concentration The optimal DHBsAg concentration for PBMC was either 0.01 pg/ml or 0.1 pg/ml depending on the duck tested. Cells from 3 birds showed maximum transformation at the first concentration, and PBMC from the 3 other ducks responded better to the second. The same concentrations (0.01 pg/ml for 3 ducks: 0.1 pg/ml for one duck) were optimal for DHBcAg. The response by SMC to DHBsAg was optimum at 0.1 pg/ml for 4 ducks, whereas for one duck it was 0.02 Fg/ml and for another 1.0 pg/ml. For DHBcAg, either 0.01 pg/ml(2 ducks) or 0.1 pg/ml(3 ducks) was the optimal concentration. Higher antigen concentrations of 5 and 10 pg/ml did not result in increased blastogenesis. 3. I .2. Optimisation of cell numbers / well The cell numbers/well that gave the optimal response to PHA mitogenesis coincided with the cell numbers/well for the best response for antigen-specific blastogenesis (Table 1). Therefore, although there was bird to bird variation in the cell numbers/well at which maximum responsiveness to PHA occurred, this was a predictor for the optimal cell numbers for antigen-specific blastogenesis. In further experiments, ducks were bled and their PBMC were tested for PHA responsiveness through a range of cell

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numbers/well. The ducks were then rebled and their PBMC tested for antigen responsiveness only at the optimum cell number/well for that individual. In both ducks for which SMC were tested, the optimum cell concentration for maximum antigen-specific proliferation was 5 X lo5 cells/well (Table 2). Culturing of SMC at greater than 5 X IO5 cells/well induced auto-transformation (Vickery et al., 1995) which masked any antigen-specific response until day 7 (data not shown). 3.1.3. Optimisation of day of haruest Significant antigen-specific transformation by PBMC was always evident by day 7 (Table 3) although this varied between ducks and to a minor extent within a duck. In the majority of ducks, SI peaked on a single day, while in some, it remained high over several days. This was also true for SMC cultures (results not shown). 3.2. Spec$city

of the assay

The PBMC responses to stimulation with DHBsAg and DHBcAg were significantly different between immune and DHBV-negative ducks (DHBsAg, P < 0.001; DHBcAg, P < 0.05). The mean + standard error SI for the immune birds was 9.9 + 1.9 for DHBsAg and 2.3 + 0.6 for DHBcAg. All ducks responded to DHBsAg and 4 of the 7 immune ducks showed significant blastogenesis to DHBcAg. The mean f standard error SI for the 20 negative ducks was 1.5 + 0.3 for DHBsAg and 0.9 _+0.3 for DHBcAg. Only one of the four-week-old ducks demonstrated blastogenesis in response to stimulation with DHBsAg while 6 of 10 adult ducks showed some nonspecific stimulation in response to the DHBsAg (SI 2.2 + 0.4). Although this resulted in a statistically significant difference between adult and young non-immune ducks in their response to DHBsAg stimulation (P < 0.051, the difference between the immune ducks and the nonimmune adult ducks still remained highly significant (P < 0.001). There was no difference between age groups in their response to DHBcAg. Tests of significance were not applied to the responses by SMC because of the low number of animals in each group. However, SMC from immune ducks responded to either DHBsAg or DHBcAg or both antigens. SMC from 2 nonimmune birds did not respond to either antigen (data not presented).

4. Discussion Antigen-specific blastogenesis has been established using PBMC and SMC from immune ducks. Both DHBsAg and DHBcAg could stimulate blastogenesis in cells from immune ducks. For each individual animal, the PBMC numbers/well that gave the maximum response to stimulation with PHA also gave the best response to antigen stimulation. Culturing SMC at the optimum cell numbers/well for mitogen stimulation also elicited the greatest transformation to antigen. This was 5 X lo5 cells/well as we have previously reported (Vickery et al., 1995). The optimum antigen concentration was found to vary between ducks from 0.01 and 0.1 pug/ml for both DHBsAg and DHBcAg. For HBV, maximum antigen-specific

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transformation has been achieved using nanogram (ng) amounts of antigens while stimulation with higher concentrations has led to nonspecific transformation (Sylvan et al., 19851. The level of antigen-specific transformation by the duck was not as great as that obtained with stimulation by a nonspecific mitogen such as PHA, but was similar to that observed for LPS (data not presented). The time to maximum antigen-specific transformation in vitro varied from duck to duck but occurred between days 4 and 7 for both PBMC and SMC. In some ducks, the PBMC proliferative response to viral antigens was over several days, while in others, the response occurred on a single day. In humans, the responses to HBV antigens also show substantial patient to patient variation (Hellstrom et al., 1985), both in the timing of maximum response and the antigen concentrations required. Most workers report peak HBV-specific antigen stimulation on days 5-7 (Hellstrom et al., 1985; Sylvan et al.. 19871. Sylvan et al. (1987) found that the proliferative responses to the core antigen of HBV (HBcAg) were over several days of culture while proliferation in response to stimulation with surface antigens was short and usually present on only one day of culture. This trend was not evident in the duck. The poor response of duck mononuclear cells to DHBcAg contrasts with the vigorous response to HBcAg in humans (Ferrari et al., 1990). Technical factors such as the presence of duck proteins in the semi-purified native DHBcAg may have interfered with antigen recognition while culture conditions initially optimised for DHBsAg may have been suboptimal for DHBcAg. More importantly, the immune response to DHBcAg by the duck may not be strong and it is tempting to speculate that this may be reflected in the mildness of liver damage observed in DHBV infection (Freiman et al., 19881. Further studies, including the use of recombinant antigens, may clarify these issues. The specificity of the assay was demonstrated by the general lack of transformation responses in the majority of 20 nonexposed ducks despite their good response to PHA. and the significant statistical difference between the responses of the immune and nonimmune groups. The low level of blastogenesis in response to stimulation with DHBsAg in adult nonimmune and nonexposed ducks contrasts to the lack of response by younger ducks is of interest since we have confirmed the ongoing absence of DHBV in this flock. This must be a nonspecific response to some common epitope(s1 from other sources to which these older ducks have been exposed.

5. Conclusion This study has shown that antigen-specific cell-mediated immune responses can be detected in ducks. Mononuclear cells from both the peripheral blood and the spleen can be used satisfactorily and the conditions determined for this assay may be applicable to other studies of infection and immunity in the duck.

Acknowledgements The review of the manuscript by Ms. Kim Hall was greatly appreciated. The technical assistance of Ms. Aniko Pajkos and Mrs. Linda Ludlow is thankfully acknowledged.

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This work was supported by grants from the National Council and from Whiteley Industries Pty.

59 (1997) 349-358

Health and Medical

Research

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