Cytotoxic T lymphocyte responses to Marek’s disease herpesvirus-encoded glycoproteins

Cytotoxic T lymphocyte responses to Marek’s diseaseherpesvirus-encoded glycoproteins

Carrie J. Markowski-Grimsrud1, Karel A. Schat*

Department of Microbiology and Immunology, Unit of Avian Health, College of Veterinary Medicine,

Cornell University, Ithaca, NY 14853, USA

Received 20 June 2002; received in revised form 26 August 2002; accepted 26 August 2002

Abstract

Cell-mediated immune responses are important for protective immunity to Marek’s disease (MD), especially because MD

herpesvirus (MDV) infection is strictly cell-associated in chickens with the exception of the feather follicle epithelium. A system

previously developed using reticuloendotheliosis (REV)-transformed cell lines stably expressing individual MDV genes allows

the determination of relevant MDV proteins for the induction of cytotoxic T lymphocyte (CTL) responses. To examine the

importance of glycoproteins for the induction of CTL, the MDV genes coding for glycoproteins (g) C, D, E, H, I, K, L, and M

were stably transfected into the REV-transformed chicken cell lines RECC-CU205 (major histocompatibility complex (MHC):

B21B21) and RECC-CU91 (MHC: B19B19). All transfected cell lines were lysed by REV-sensitized, syngeneic splenocytes

obtained from MD-resistant N2a (MHC: B21B21) and MD-susceptible P2a (MHC: B19B19) chickens, indicating that the

expression of individual MDV glycoproteins did not interfere with antigen processing pathways. Only cell lines expressing gI

were recognized by CTL from both N2a and P2a MDV-infected chickens. Cell lines expressing glycoproteins gC and gK, and to

a lesser extent, gH, gL, and gM were lysed by syngeneic MDV-sensitized splenocytes from N2a birds but not P2a birds. In

contrast, gE was recognized by MDV-sensitized effector cells from the P2a line and not the N2a line. Glycoprotein D was not

recognized by either line, with the exception of one marginally significant P2a assay. These results indicate that late viral

glycoproteins are relevant for the induction of cell-mediated immunity during MDV infection.

# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Cell-mediated immunity; Chickens; Cytotoxic T lymphocytes; Glycoprotein; Marek’s disease herpesvirus; Reticuloendotheliosis

virus

1. Introduction

Marek’s disease herpesvirus (MDV) is a 180 kbp

oncogenic herpesvirus of chickens. MDV was origin-

ally classified as a gammaherpesvirus due to its lym-

photropism but more recently has been reclassified as

an alphaherpesvirus based on its genomic structural

similarity to other alphaherpesviruses such as vari-

cella-zoster virus and herpes simplex virus (HSV)

(Buckmaster et al., 1988; Lupiani et al., 2001).

MDV is grouped into three serotypes: serotype 1

consists of all pathogenic virus strains, serotype 2

comprises the naturally occurring, non-oncogenic

strains in chickens, and serotype 3 includes the

Veterinary Immunology and Immunopathology 90 (2002) 133–144

* Corresponding author. Tel.: þ1-607-253-4032;

fax: þ1-607-253-3384.

E-mail address: [email protected] (K.A. Schat).1 Present address: Department of Immunology, Institute for

Cancer Research, Norwegian Radium Hospital, University of Oslo,

N-0310 Oslo, Norway.

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 2 9 - 5

non-pathogenic herpesvirus of turkeys (HVTs)

(Bulow and Biggs, 1975).

MDVis the causative agent of Marek’s disease (MD),

a lymphoproliferative disease of chickens prevalent in

poultry-producing countries worldwide (Purchase,

1985). The disease is characterized by mononuclear

infiltration of the peripheral nerves, viscera, and various

other organs, resulting in the formation of lymphoid

tumors and variable mortality (Payne, 1985). MD inci-

dence has largely been controlled by vaccination with

MDV serotypes 2 and 3, often in bi- and multivalent

combinations since the 1970s (Witter, 2001). However,

over the past decade new MDV serotype 1 strains of

increased virulence have emerged with greater immu-

nosuppressive potential (Calnek et al., 1998). It is

predicted that the protective ability of current vaccines

is limited (Kreager, 1997). A thorough understanding of

MDV immunity is urgently needed to meet the future

challenges of vaccine development.

Cell-mediated immune responses are important in

protection from herpesvirus infections in general

(Arvin, 1992; Rickinson and Moss, 1997; Riddell

et al., 1991; Schmid and Mawle, 1991). Cell-mediated

immunity is especially important in protection from

MDV infection due to the strictly cell-associated

nature of the virus (Schat and Markowski-Grimsrud,

2001).

Our laboratory has maintained a longstanding inter-

est in elucidating the importance of cell-mediated

immune responses to MDV infection. A system pre-

viously developed in the laboratory has utilized REV-

transformed cell lines stably transfected with eukar-

yotic expression vectors containing various MDV

genes of interest (Pratt et al., 1992). These cell lines

have been used as target cells in chromium release

assays (CRAs) to ascertain CTL responses to indivi-

dual MDV-encoded proteins in genetically resistant

and susceptible chicken lines with defined MHC

haplotypes.

Previous studies utilizing this system have shown

that CTL responses are generated to a number of

immediate early, early, and late MDV proteins (Uni

et al., 1994; Omar and Schat, 1996; Schat and Xing,

2000). Target cell lines expressing glycoprotein B

(gB), a late structural protein, were significantly lysed

by MDV serotype 1- or serotype 2-sensitized syn-

geneic but not allogeneic splenocytes (Omar and

Schat, 1996). The effector cells were characterized

as classical CTL expressing CD8 and TCRab but not

CD4 (Omar and Schat, 1997). These studies have been

extended to include the following glycoproteins

encoded by MDV: gC (Binns and Ross, 1989), gD

(Brunovskis and Velicer, 1995), gE (Brunovskis and

Velicer, 1995), gH (Scott et al., 1993), gI (Brunovskis

and Velicer, 1995), gK (Ren et al., 1994), gL (Yoshida

et al., 1994), and gM (Osterrieder, 1999). Preliminary

data suggested that some of the glycoproteins are

important in inducing CTL responses (Markowski-

Grimsrud et al., 2001). The complete findings for all

MDV glycoproteins are presented in this report.

2. Materials and methods

2.1. Experimental animals

Chickens were obtained from specific-pathogen-

free (SPF) departmental flocks with defined MHC

haplotypes, the N2a (MHC: B21B21) and P2a

(MHC: B19B19) lines (Weinstock and Schat, 1987).

The departmental flocks are maintained in a filtered-

air, positive-pressure house and are free of all patho-

gens except chicken infectious anemia virus (CAV)

(Cardona et al., 2000a,b). The majority of the chicks

hatched from these breeder flocks have maternal

antibodies against CAV. Birds were hatched either

in the SPF facility and transferred to separate experi-

mental units at 1 day of age or were hatched in the

experimental units to reduce the risk of exposure to

CAV. All experiments were conducted with birds less

than 3 weeks of age before maternal antibodies have

waned, since CAV infection in birds lacking maternal

antibodies impairs the development of CTL (Mar-

kowski-Grimsrud et al., 2001). All experimental pro-

cedures were conducted in compliance with

institutional animal use protocols.

2.2. Cell cultures and cell lines

Chick kidney cell (CKC) and chicken embryo fibro-

blast (CEF) cultures were prepared from 2-week-old

SPF chicks or 10-day-old chicken embryos, respec-

tively, as previously described (Schat and Purchase,

1998). The REV-transformed chicken cell (RECC)

(Witter et al., 1979) lines CU91 (MHC: B19B19) and

CU205 (MHC: B21B21) (Schat et al., 1992) were

134 C.J. Markowski-Grimsrud, K.A. Schat / Veterinary Immunology and Immunopathology 90 (2002) 133–144

propagated in lymphocyte medium (LM) (Schat and

Purchase, 1998) containing 10% FBS (LM10) in 5%

CO2 at 41 8C. The RECC cell lines CU371 and CU368

expressing MDV gB, derived from CU91 and CU205,

respectively, have been described (Omar and Schat,

1996), and were used as positive control target cell

lines in CRA.

2.3. Viruses and inoculations

The oncogenic MDV serotype 1 strain JM-16 (Cal-

nek et al., 1984) was propagated in CKCs and was

used for all MDV inoculations. The non-defective,

low-virulence CS-strain of REV, originally obtained

from Witter (Avian Disease and Oncology Laboratory,

East Lansing, MI), was propagated in CEF as pre-

viously described (Weinstock et al., 1989). Nine- or

ten-day-old chicks were inoculated intraabdominally

with 1000–2000 focus-forming units (FFU) of JM-16,

passage 19 (JM-16/p19) or with 103.5–4.3 tissue culture

infective doses 50% (TCID50) REV-CS, or used as

uninfected controls.

2.4. Virus isolation

For isolation of JM-16 from MDV-infected chick-

ens, 250,000 splenocytes from infected birds were

inoculated onto 24–48 h CKC in 35 mm tissue culture

plates in triplicate. The presence of FFU was deter-

mined 5–7 days post-inoculation.

2.5. Plasmid construction

The plasmid constructs pcDNA3-gD, pcDNA3-gE,

and pcDNA3-gI for expression of MDV gD, gE, and gI,

respectively, were kindly provided by the late Velicer

(Michigan State University, East Lansing, MI). The

genes for MDV glycoproteins gC, gH, gK, gL, and gM

were amplified from MDV viral DNA (RB-1B (Schat

et al., 1982a) or GA-5 strain (Calnek, 1973)) by PCR

and subcloned into the pcDNA3.1/Myc-His eukaryotic

expression vector (Invitrogen, Carlsbad, CA). The pri-

mers used for the amplification of these genes are listed

in Table 1. Constructs were verified by bi-directional

sequencing at the Biotechnology Resource Center,

Cornell University. Prior to transfection each construct

was linearized using an appropriate restriction enzyme

(PvuI or ScaI) recognizing a single site in the ampicillin

resistance gene.

2.6. Target cell line generation

RECC-CU91 and CU205 were transfected with

individual plasmid constructs by electroporation as

previously described (Pratt et al., 1992). Briefly, CU91

and CU205 cells were centrifuged over Ficoll-Paque

(Amersham Pharmacia Biotech, Inc., Piscataway, NJ)

at 500 � g for 20 min 1 day prior to transfection to

remove dead cells and resuspended at 0:5 � 106 cells/

ml in LM10. On the day of electroporation the cell lines

were centrifuged again over Ficoll-Paque, resuspended

Table 1

Primers used for amplification of MDV glycoprotein genes

Gene DNA

source

Primer Sequencea RE siteb Nucleotide

location

Amplicon

length (bp)

Accession

numberc

gC GA DNA Upper 50-(GAGAAGCTT)GAAGAGACACCAAACGTAACC-30 HindIII 268–288 1554 M20001

Lower 50-(GGGCTCGAG)CATAACAATGAGATTATAATCG-30 XhoI 1800–1821

gH RB-1B DNA Upper 50-(ACACTCGAG)CTACATATCCAACATGGGTCT-30 XhoI 194–214 2452 S62555

Lower 50-(ACATCTAGA)AAGATCGTCGTACAGGCTCAAT-30 XbaI 2624–2645

gK GA DNA Upper 50-(CATCACGCGGCCGC)CACGGTAGATATTGACTCG-30 NotI 406–424 1083 U10040

Lower 50-(TTAAGTCTCGAG)AGGCAAATAGGCACGC-30 XhoI 1473–1488

gL GA DNA Upper 50-(GCGCTCGAG)TAATCGGAGCTGCTCGGTAGC-30 XhoI 212–232 606 U04994

Lower 50-(ACATCTAGA)GGCATTGGCTCGTCGGCTGCCAGA-30 XbaI 794–817

gM GA DNA Upper 50-CTACCGGCGGTCGTCGCTATG-30 N/A 24–44 1290 AF118111

Lower 50-ATCATCCCATTCGCTCTCAG-30 N/A 1294–1313

a Parentheses designate spacer or restriction enzyme (italic) sequences. Start codons designated in bold. Those not present are located

downstream of the upper primer.b Restriction enzyme site used for subcloning after PCR amplification.c GenBank accession number.

C.J. Markowski-Grimsrud, K.A. Schat / Veterinary Immunology and Immunopathology 90 (2002) 133–144 135

at 1 � 107 cells/ml in LM base without antibiotics, and

0.4 ml of cell suspension was aliquoted into 0.4 cm

cuvettes. CU91 and CU205 were mock-electroporated

or electroporated with 10 mg of each linearized con-

struct at 300 V and 500 mF using a Gene Pulser (Bio-

Rad Laboratories, Inc., Hercules, CA). After 5 min the

cells were added to 5 ml of LM10 and plated in five

wells of a 24-well-plate (Costar, Corning, Inc., Corning,

NY). At 48 h post-transfection selection with Geneticin

(GibcoBRL Life Technologies, Inc., Gaithersburg,

MD) was initiated. Different amounts of Geneticin

were administered and increased incrementally until

the mock-transfected controls were no longer viable.

Following selection of stable transformed cells 3–4

weeks later, cell lines were maintained in LM10 con-

taining 300 mg Geneticin/ml.

2.7. RT–PCR

Total RNA was extracted from cell lines using

RNAzolB (Teltest, Inc., Friendswood, TX) according

to the manufacturer’s instructions. RNA was reverse

transcribed in 20 ml reactions using the RT Core Kit

(Perkin Elmer, Inc., Boston, MA) in a PE2400 thermal

cycler using the following parameters: 42 8C for

15 min, 99 8C for 5 min, and 5 8C for 5 min. Ten

milliliters of the RT reactions were used for PCR

amplification using 100 pmol of each primer for the

respective glycoprotein (Table 2) in 50 ml reactions.

The PCR cycling parameters consisted of a hold at

94 8C for 5 min, followed by 35 cycles of 94 8C for 45 s/

56 8C for 45 s/72 8C for 45 s, and ending with a hold at

72 8C for 10 min. Control reactions lacking reverse

transcriptase were performed to ensure RNA-specific

amplification. RT–PCR products were electorphoresed

on a 1.5% agarose gel and visualized by ethidium

bromide staining using an Eagle Eye detection system

(Stratagene, La Jolla, CA).

2.8. Indirect immunofluorescence assay (IFA)

Smears of stably transfected RECC lines were

prepared on 12-well glass slides at 5 � 104 cells per

well, air-dried, and fixed in 100% acetone for 10 min.

Cells were stained for 30 min at 37 8C with rabbit

antisera specific for the various glycoproteins gC

(kindly provided by Osterrieder), gE, gH, gI, and

gL (kindly provided by Lee) followed by an FITC-

conjugated goat anti-rabbit secondary antibody (Cap-

pel/ICN Pharmaceuticals, Inc., Costa Mesa, CA).

Antisera specific for gD, gK, and gM were unavailable

for testing. Cells were also stained separately with a

REV-specific monoclonal antibody (provided by Lee),

followed by an FITC-conjugated rabbit anti-mouse

secondary antibody (Cappel/ICN). Wells were washed

twice with PBS, pH 7.2, for 10 min after each staining.

Dabco/glycerine (Beutner et al., 1987) was used to

preserve the slides. Slides were examined in a Zeiss

Axioskop2 plus microscope under both phase-contrast

and UV light at 400� and 630� magnification power.

Photographs were taken using an AxioCam digital

camera (Carl Zeiss, Inc., Thornwood, NY) and visua-

lized using Axiovision version 3.0.6 software.

2.9. Serology

Sera were obtained from all chickens at the termi-

nation of each experiment and stored at �20 8C until

Table 2

Primers used for RT–PCR confirmation of transcribed MDV glycoprotein genes

Gene Forward primer (length) Reverse primer (length) Amplicon

length (bp)

Position Accession

numbera

gC 50-TAACAGAAACGACGGGCAAGAACG-30 (24) 50-ATACTGGAATGCGGCTGAGGAGAT-30 (24) 1047 536–1582 M20001

gD 50-AACCTCCGGGCTACTCTCAATG-30 (22) 50-TCGGTCGTTTAGTTATGTATGGTG-30 (24) 841 7105–7945 L22174

gE 50-ATCCCGCGATATCCACCAGTTG-30 (22) 50-ATCGCTAGCCTCATTTTCGTCG-30 (22) 1026 9572–10597 L22174

gH 50-ACAAAGACGACGCTCAACCTACAG-30 (24) 50-TTCCGCAAATGCATTCCAGATA-30 (22) 754 634–1387 S62555

gI 50-CAATCTGCTCTTGTTGCGTTCC-30 (22) 50-GGCCTCATCAGATCTTGGTTCAC-30 (23) 834 8354–9187 L22174

gK 50-TGGACAGTAGCCTCTATGACG-30 (21) 50-CGCAAGCAAGTATAGAATGCT-30 (21) 900 554–1453 U10040

gL 50-ACGATATTCCCACGGGCATTTTTA-30 (24) 50-TGGCATTCCCCGACTATCATCTTC-30 (24) 335 429–763 U04994

gM 50-GTGCGGCATACATGGGGAGTG-30 (21) 50-CAGCAGAACGGGATTGTGTAAGTG-30 (24) 831 418–1248 AF118111

a GenBank accession number.


use in enzyme-linked immunoabsorbant assays

(ELISA) using the commercial Flokchek CAV Anti-

body Test kit (Idexx Laboratories, Westbrook, ME) to

ensure the presence of maternal antibodies to CAV.

Sera were diluted 1:10 and tested according to the

manufacturer’s instructions. Absorbances were read at

650 nm in a Microplate EL310 Autoreader (Bio-Tek

Instruments, Inc., Winooski, VT).

2.10. CRA

For all experiments, 6 to 12 9- or 10-day-old P2a or

N2a birds per group were used. All assays contained

an uninfected control group and an MDV-infected

group, and in most cases a third group of REV-infected

birds was included. Birds were euthanized and effec-

tor cells were prepared from spleens at 7 days post-

infection (dpi) using previously described methods

(Schat et al., 1982b; Omar and Schat, 1996). Briefly,

spleens were decapsulated and forced through a

0.60 mm pore-size nylon mesh (Tetko, Inc., Kansas

City, MO) to create single cell suspensions. Cells were

resuspended in PBS and centrifuged over Ficoll-Paque

at 500 � g for 20 min to remove red blood cells.

Lymphocytes were harvested from the interface,

washed once with PBS, and resuspended at 5�107 cells/ml in LM containing 20% FBS.

For target cell preparation, the stably transfected

RECC lines were centrifuged over Ficoll-Paque 1 day

prior to the assay and adjusted to 0.5–1:0 � 106 cells/

ml in LM10. On the day of the assay, each cell line was

labelled with 150–200 mCi of Na51CrO4 (New Eng-

land Nuclear, Boston, MA) as previously described

(Schat et al., 1982b). CRAs were performed in tripli-

cate at an effector:target cell ratio of 100:1 in 96-well

round-bottomed plates (Costar) using 5 � 104 target

cells per well. Three to six target cell lines were tested

per assay depending on the number of spleen cells

available; one complete trial, therefore, represented a

composite of several assays. Each cell line was tested

2–3 times; hence a total of 2–3 trials were performed

for N2a and P2a chickens.

Following a 4 h incubation at 38 8C, the plates were

centrifuged at 500 � g for 10 min. Half of the super-

natant (100 ml) from each well was harvested, mixed

with an equal volume of SuperMix scintillation fluid

(EG&G Wallac, Turku, Finland) in Microbeta 96-well

flexible plates and cpm were determined for each well

based on 5 min counts. The pellets were resuspended

in the remaining supernatant, and half (50 ml) from

each well was transferred to a new Microbeta plate

containing 50 ml of 2% Triton X and lysed overnight.

SuperMix (100 ml) was then added to the lysate in each

well and mixed by shaking the plates gently on a

Vortex Genie 2 (Fisher Scientific, Pittsburgh, PA) for

5–10 min, and the cpm were determined in a Trilux

1450 Microbeta (EG&G Wallac) scintillation counter.

The percent specific release (% SR) was calculated

using the following formula:

% SR ¼

cpm supernatant of sample

� cpm supernatant average control

cpm pellet þ supernatant

� cpm supernatant average control

� 100

2.11. Statistical analysis

For CRA analysis, the percent releases (% release ¼cpm supernatant=total cpm) between control and

infected groups were compared using a Student’s t

test (Snedecor and Cochran, 1989). Non-responder

chickens that tested negative for CAV antibodies were

removed from analysis to eliminate any possibility of

interference of CAV with CTL development (Mar-

kowski-Grimsrud et al., 2001).

3. Results

3.1. Development of REV-transformed cell lines

expressing MDV glycoprotein genes

REV-transformed cell lines stably transfected with

MDV glycoprotein constructs were tested for tran-

scription and expression of individual MDV genes by

RT–PCR and IFA analysis, respectively. All cell lines

tested positive for the appropriate transcripts of the

transfected MDV genes (Table 3 and Fig. 1).

The IFA results are summarized in Table 3. The

specific fluorescence for selected cell lines expressing

gC, gE, gH, gI, and gL is shown in Fig. 2. While there

was a faint non-specific staining pattern present in the

parent cell lines for all glycoprotein antisera (Fig. 2A),

the glycoprotein-expressing cell lines exhibited a

brighter, granular staining throughout the cytoplasm

of virtually all cells (Fig. 2B). As expected, both the


parental cell lines and all glycoprotein-expressing cell

lines stained positive using the anti-REV monoclonal

antibodies. REV-specific staining is shown for CU205,

the non-transfected parent cell line (Fig. 2A) and

CU398 expressing MDV gE (Fig. 2B).

3.2. Lysis of transfected REV cell lines by

REV-specific CTL

REV-transformed cell lines expressing MDV gly-

coproteins were first tested in CRAs using REV-sen-

sitized splenocytes from N2a (Fig. 3A) and P2a

(Fig. 3B) chickens (trials 1–3). All cell lines were

significantly lysed by syngeneic REV effector cells

irrespectively of the presence and expression of trans-

fected MDV genes, indicating that expression of the

MDV glycoproteins did not interfere with endogenous

antigen processing and presentation mechanisms.

Within individual experiments no significant differ-

ences were observed in % SR between transfected and

parental cell lines.

3.3. Lysis of transfected REV cell lines by

CTL from MDV-infected birds

The results for CRA trials 4–6 using effector cells

from MDV-infected chickens are depicted in Fig. 4.

Effector cells from N2a (MHC: B21B21) (Fig. 4A) the

B21B21 haplotype cell consistently lysed syngeneic

target cell lines expressing gC (CU437), gK

(CU455) and the positive control cell line expressing

gB (CU368). The CTL recognition of syngeneic target

cells expressing gH (CU434), gL (CU436), and gM

(CU462) by N2a effector cells was more variable, with

significant lysis occurring in 2/3, 2/3, and 1/2 trials,

respectively (Fig. 4A). The parental, non-transfected

syngeneic cell line (CU205) was not significantly

lysed by MDV-sensitized plenocytes and served as a

negative control for the assays. The B21B21-derived

cell lines expressing gD (CU399) and gE (CU398)

were not significantly lysed in any of the trials (Fig. 4).

Effector cells obtained from MDV-infected P2a

effector cells only lysed the B19B19-derived cell lines

Table 3

REV-transformed cell lines expressing MDV glycoproteins

Glycoprotein Vector Cell line Haplotype Expression

RT–PCR IFA

N/A N/A CU91 B19B19 N/Aa N/A

CU205 B21B21 N/A N/A

gB pRc/CVM CU371 B19B19 NDb þCU368 B21B21 ND þ

gC pcDNA3.1/myc-His CU458 B19B19 þ þCU437 B21B21 þ þ

gD pcDNA3 CU394 B19B19 þ NTc

CU399 B21B21 þ NT

gE pcDNA3 CU396 B19B19 þ þCU398 B21B21 þ þ

gH pcDNA3.1/myc-His CU439 B19B19 þ þCU434 B21B21 þ þ

gI pcDNA3 CU395 B19B19 þ þCU456 B21B21 þ þ

gK pcDNA3.1/myc-His CU459 B19B19 þ NT

CU455 B21B21 þ NT

gL pcDNA3.1/myc-His CU440 B19B19 þ þCU436 B21B21 þ þ

gM pcDNA3.1/myc-His CU465 B19B19 þ NT

CU462 B21B21 þ NT

a Not applicable.b Not done.c Not tested; antisera not available.


expressing gI (CU456), gE (CU396), and the positive

control cell line expressing gB (CU371) (Fig. 4B). The

B19B19 cell lines expressing gC (CU458), gH

(CU439), gK (CU459), gL (CU440), and gM

(CU465) were not significantly lysed in any of the

trials using P2a-derived effector cells (Fig. 4B).

Finally, the B19B19 cell line expressing gD (CU394)

was not lysed by MDV-sensitized P2a effector cells,

with one exception in trial 2 with a marginally sig-

nificant % SR (Fig. 4B).

3.4. Virus isolation

MDV was reisolated from the MDV-infected chick-

ens in all experiments ensuring that negative CRA

results were not caused by the absence of infection

(data not shown).

3.5. CAV serology of infected birds

Serology analysis performed on all MDV- and REV-

infected birds failed to show a correlation between

CTL response and CAV maternal antibody status,

indicating a lack of CAV-induced CTL impairment.

4. Discussion

The generation of CTL responses to late structural

proteins might serve as an advantage to most herpes-

viruses that exist in cell-free form throughout the host,

as cell lysis at a late stage of virion packaging would

aid the release of virus and perpetuate the infectious

cycle. However, since MDV is strictly cell-associated

with the exception of the feather follicle epithelium

(Calnek et al., 1970), release of infectious virions from

CTL-lysed cells is probably minimal. The function-

ality of MDV-specific antibodies is minimal in this

context of intracellular virions. Therefore, CTL

responses are of paramount importance to the adaptive

immune response during MDV infection.

The findings presented in this study indicate the

importance of several late glycoproteins in inducing

CTL responses in MDV-infected birds. Two glyco-

proteins have now been identified that are recognized

by CTL from N2a and P2a chicken strains, gB (Omar

and Schat, 1996) and gI in this report. Interestingly,

these two glycoproteins have been shown to induce

Fig. 1. RT–PCR analysis of MDV glycoprotein gene transcription in

stably transfected REV-transformed cell lines derived from the CU91

(MHC: B19B19) and CU205 (MHC: B21B21) parental lines. Total RNA

was extracted from approximately 107 cells from each cell line, and

aliquots of each were treated with DNAse to remove contaminating

DNA. RT–PCR was performed using each corresponding primer set

as described in the materials and methods. The black arrowheads

indicate the bands of expected length for each individual glycoprotein

gene (see Table 2 for details). The three negative control lanes

corresponding to CU91, CU205, and water were performed with gC

primer pairs in this figure and are representative of results obtained

with any of the glycoprotein primer sets. This figure is a composite of

several gels aligned using the DNA standards.


Fig. 2. Indirect immunofluorescence staining of REV-transformed cell lines expressing MDV glycoprotein genes. The REV-transformed non-

transfected parental cell line CU205 (A) or cell lines CU458 (gC), CU396 (gE), CU439 (gH), CU456 (gI), CU440 (gL), and CU395 (gI)

stably transfected with various MDV glycoprotein genes (B, listed from top to bottom) were seeded on glass slides at 5 � 106 cells per well,


protective immunity when administered in a recom-

binant fowlpox virus (FPV). Chickens vaccinated with

rFPV expressing gB were protected against challenge

with virulent MDV and elicited MDV neutralizing

antibodies (Nazerian et al., 1992, 1996). The same

rFPV-gB was later shown to induce gB-specific

CTL responses (Omar et al., 1998). Recently, rFPV-

gI was also shown to protect against disease after

MDV challenge (L.F. Lee, personal communication).

The importance of gI-specific CTL has also been

demonstrated in varicella-zoster virus infection,

another cell-associated alphaherpesvirus (Arvin et al.,

1991).

Of particular interest are the MDV glycoproteins gC

and gK, and to a lesser extent gH, gL and gM, which

were recognized by syngeneic effector cells from the

MDV resistant N2a line but not the susceptible P2a

line. Previously, Omar and Schat (1996) reported that

ICP4, an immediate early MDV gene product, was

recognized by MDV-sensitized CTL from the MD

resistant N2a line but not by CTL from the MD

susceptible P2a line. This strongly implicates the

importance of cell-mediated immune responses as

key players in genetic resistance to MD.

In contrast, the finding that gE is only recognized by

CTL from the MDV susceptible P2a line suggests that

air-dried, and fixed in acetone. IFA staining was performed with

rabbit sera specific for gC, gE, gH, gI, or gL or an REV-specific

monoclonal antibody as indicated at the left of the panel, followed

by either a goat anti-rabbit or rabbit anti-mouse FITC conjugate.

Slides were preserved with a Dabco/glycerine solution and viewed

under UV light at 400�, and 630� magnification. All photographs

shown are at 400�, with the exception of the anti-gC slides (top) at

630� magnification.

Fig. 3. Lysis of REV-transformed cell lines expressing MDV

glycoproteins by REV-sensitized splenocytes. REV-transformed

cell lines derived from N2a (MHC: B21B21) (panel A) or P2a

(MHC: B19B19) chickens expressing the indicated MDV glycopro-

tein were used as target cells against syngeneic REV-sensitized

splenocytes (n ¼ 6) at 7 dpi in a standard 4 h CRA. Results are

expressed as % SR; 2–3 trials were performed for each target cell

line. The % SR was significant at �P < 0:05, ��P < 0:01,��P < 0:001 based on comparing the percent release values for

splenocytes from REV-infected and control chickens for a given

cell line. aThe absence of a bar in trial 3 for a cell line expressing a

given glycoprotein indicates that only two trials were performed for

that cell line.

Fig. 4. Lysis of REV-transformed cell lines expressing MDV

glycoproteins by MDV-sensitized splenocytes. REV-transformed

cell lines derived from N2a (MHC: B21B21) (panel A) or P2a

(MHC: B19B19) chickens expressing the indicated MDV glycopro-

tein were used as target cells against syngeneic MDV-sensitized

splenocytes (n ¼ 6) at 7 dpi in a standard 4 h CRA. Results are

expressed as % SR; 2–3 trials were performed for each target cell

line. The % SR was significant at �P < 0:05, ��P < 0:01,��P < 0:001 based on comparing the percent release values for

splenocytes from REV-infected and control chickens for a given

cell line. aThe absence of a bar in trial 6 for a cell line expressing a

given glycoprotein indicates that only two trials were performed for

that cell line.


CTL responses to gE have an insignificant role in

protective immunity. The fact that gD did not appear

to play a significant role in CTL induction was not

surprising, since gD is poorly expressed during MDV

infection and was recently reported to be undetectable

in cell culture (Tan et al., 2001). The minor relevance of

gD for cell-mediated immunity and the fact that it is a

non-essential gene for in vivo infectivity (Parcells et al.,

1994) suggests that this gene is a candidate locus for the

development of recombinant MDV vaccines expressing

genes for other poultry pathogens such as Newcastle

disease virus, infectious bursal disease virus, and others

(Hirai and Sakaguchi, 2001).

The MDV-specific % SR are generally low, albeit

significant, typically ranging from 5 to 15% (Uni et al.,

1994; Omar and Schat, 1996) compared with HSV

studies, where secondary stimulation of splenocytes is

often used to demonstrate CTL activity (Borysiewicz

and Sissons, 1994). The same cell lines were lysed to a

greater extent by REV-sensitized splenocytes (10–

35%), which is also consistent with previous findings

(Weinstock et al., 1989; Pratt et al., 1992; Omar and

Schat, 1996). The differences in % SR by REV- versus

MDV-sensitized splenocytes can be caused by the fact

that based on IFA studies all cells within a cell line

strongly express REV proteins, but not all cells

express the transfected MDV gene to the same level.

It is also plausible that more than one REV protein is

processed and presented to REV-specific CTL or that

an immunodominant epitope is present, which might

account for the higher degree of REV-specific lysis.

One notable difference between this study and

previous studies (Pratt et al., 1992; Uni et al., 1994;

Omar and Schat, 1996; Schat and Xing, 2000) is that in

this study 9- to 10-day-old birds were used in all

assays as opposed to 4–5-week-old birds in the pre-

vious studies. This change was needed because: (a) the

SPF flocks had become infected with CIAV (Cardona

et al., 2000a,b), (b) Markowski-Grimsrud et al. (2001)

have shown that MDV- and REV-specific CTL devel-

opment are impaired during concurrent CAV infection

in birds lacking virus-neutralizing maternal antibo-

dies, and (c) the possibility of CAV infection in birds

lacking maternal antibodies could not be excluded.

However, the finding that the serology data in this

study did not correlate with relative CTL-mediated

lysis strongly suggests that CAV infection was not

present in the birds hatched in the experimental units.

While the use of younger birds possessing CAV

maternal antibodies was essential for the continuation

of this study as a precautionary measure, a lower level

of immunocompetence of the younger birds may

account for the relatively low and sometimes variable

results in the MDV- and REV-specific CTL assays.

The REV CTL assays serve as an important control

in two respects. It is well known that many proteins

encoded by herpesviruses are involved in the subver-

sion of the immune system (Ploegh, 1998). In this

respect the CRAs using transfected target cells and

REV-sensitized effector cells ensured that a given

MDV protein did not interfere with the antigen pro-

cessing and presentation of the MHC class I context.

Secondly, they served as a positive control for the

assay itself, especially in light of the potential CAV

problems discussed above.

Whether the MDV-specific CTL confer protection

from disease remains to be elucidated by passive

transfer assays. Previous studies using passively trans-

ferred primary splenocytes from chickens infected

with an rFPV expressing the MDV gB gene failed

to demonstrate a reduction in viral replication upon

MDV challenge (Omar, 1997). The negative results

were likely due to the limited gB-specific CTL sub-

population(s) present in the primary splenocyte pool.

These experiments have been hindered by a lack of

means to amplify antigen-specific CTL in vitro in the

absence of MDV virus that can be present in the

lymphocytes after in vivo infection.

Although MD has been largely been curtailed by

vaccination and in fact represents the first successful

vaccine against a tumor-inducing virus in any species,

the mechanisms of vaccinal immunity have remained

elusive. Although JM-16/19, a low-passage serotype-1

MDV strain was used for all inoculations in the

experiments presented here, previous studies in the

laboratory indicated that both JM-16 and the serotype-

2 vaccine strain SB-1 induce MDV-specific CTL to

comparable levels (Omar and Schat, 1996). It is of

increasing importance to identify the antigens relevant

to vaccinal immunity. MD outbreaks in vaccinated

flocks have historically occurred at 10-year intervals

as MDV strains of increased virulence emerge (Witter,

1997). Since there are currently no new vaccine strains

available for commercial use, the development of

recombinant vaccines is of major importance to con-

trol disease in the near future.


Acknowledgements

This work was supported in part by the Cooperative

State Research, Education, and Extension Service, US

Department of Agriculture, under agreements #96-

38420-3061 and #98-35204-6425 and grants #298

and #426 from the US Poultry and Egg Association.

C.J.M. was supported by a USDA Biotechnology

Training Fellowship and a dissertation fellowship

from the American Association of University Women.

The authors are grateful to the late Dr. Leland F.

Velicer for kindly providing several plasmid con-

structs containing MDV glycoproteins, and Drs. Lucy

Lee and Nicolas Osterrieder for generously providing

the glycoprotein-specific antibodies. We also thank

Dr. Keith W. Jarosinski and Priscilla O’Connell for

technical assistance with several of the CRAs. Special

thanks are due to Dr. Ole M. Grimsrud for help with

preparing the figures and tables.

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Cytotoxic T lymphocyte responses to Marek’s disease herpesvirus-encoded glycoproteins