Yonago Acta medica 1996;39:99–107

Decreased Expression of Early Antigens in P3HR-1-EBV SuperinfectedRaji Cells Cultured in EBV-Seropositive Human Sera
Takeshi Sairenji, Patricia S. Reisert*, Robert C. Spiro* and Robert E.
Humphreys*
Department of Biosignaling, Faculty of Medicine, Tottori University, Yonago 683, Japanand *Department of Pharmacology, University of Massachusetts Medical School, Worcester,
MA 01655, United States
Antibody-induced change in the expression of Epstein-Barr virus (EBV) antigens wasstudied after P3HR-1-EBV superinfection of Raji cells. The superinfected Raji cells
were cultured in medium containing fetal calf serum (FCS) and either EBV antibodypositive or -negative human sera which had been selected for low natural cytotoxic
antibodies to Raji cells. At 24 postsuperinfective (psi) h the percentages of early antigen(EA)-positive cells were similar for cultures with FCS only or also with EBV antibodypositive or -negative sera. However, at 48 psi h decreased expression of EA-positive
cells was observed in cultures with EBV antibody-positive sera, but not with EBV
antibody-negative sera or with FCS only. This EA suppressing effect was dependent
upon concentration of EBV antibody-positive human serum and related with anti-viralcapsid antigen (VCA) or membrane antigen (MA) titers among effective sera. Antibodymediated suppression of EA at 48 psi h was inhibited by treatment with phosphonoacetic
acid which inhibits herpesvirus DNA replication and blocks late viral functions. These
results are consistent with hypotheses that suppression of intracellular EA follows frommodulation (or alteration) of MA by an anti-MA antibody and that a late viral function
is required for the antibody-mediated inhibition of EA expression in this system.
Key words: Epstein-Barr virus; early antigen; membrane antigen; modulation; anti-EBV antibodies

infection and transformation. Cytotoxic T cells
attack virally infected cells in vivo and retard
the outgrowth of virally transformed cells in
vitro (Thorey-Lawson et al., 1977; Moss et al.,
1979; Klein, 1994). Virus-neutralizing antibodies can protect against reinfection or recurrent viremia (Sairenji et al., 1984, 1985a, 1985b).
Antibody-dependent cellular cytotoxicity(ADCC) effectors can kill cells expressing
antibody-recognized, EBV-induced antigens(Takaki et al., 1980). Natural killer cells can
attack cells with other EBV neoantigens(Patarroyo et al., 1980). EBV antigens of these
immune responses have also been identified

To understand how the immune system controls
infections with Epstein-Barr virus (EBV), we
have analyzed the role of antibodies in modulating expression of EBV genome functions. EBV
causes infectious mononucleosis (IM) and isassociated with the pathogenesis of Burkitt’s
lymphoma (BL), nasopharyngeal carcinoma(NPC) and the X-linked lymphoproliferative
syndrome (Purtilo et al., 1976; Epstein andAchong, 1979; Henle and Henle, 1979).
Chronic EBV infections can occur in some patients with nonmalignant diseases (Virelizier etal., 1978; Sairenji et al., 1995). Cellular andhumoral immune mechanisms influence EBV

Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; BL, Burkitt’s lymphoma; EBV, EpsteinBarr virus; EA, early antigen; EA-SI, EA suppression index; EBNA, EBV-nuclear antigen; FCS, fetal calf
serum; IM, infectious mononucleosis; MA, membrane antigen; NPC, nasopharyngeal carcinoma; PAA,
phosphonoacetic acid; psi, postsuperinfective; VCA, viral capsid antigen.

99

T. Sairenji et al.

Table 1. Anti-EBV characteristics of human sera
Patient’s No.

Diagnosis

Anti-EBV titer
EA

VCA

MA

60
Normal adult
<5
<5
<5
61
Normal adult
<5
<5
<5
72
Normal adult
<5
<5
<5
73
Normal adult
<5
<5
<5
70
Normal adult
80
<5
80
58
Normal adult
80
<5
80
64
Normal adult
80
<5
40
53
Normal adult
160
< 10
160
59
Normal adult
160
< 10
160
63
Normal adult
320
< 10
160
74
Normal cord blood
160
< 10
160
71
NPC
320
80
640
EA, early antigen; EBV, Epstein-Barr virus; MA, membrane antigen; NPC, nasopharyngeal carcinoma; VCA,
viral capsid antigen.

with immunofluorescence assays: EBV-nuclear
antigen (EBNA) (Reedman and Klein, 1973),
early antigen (EA) (Henle et al., 1970), viralcapsid antigen (VCA) (Henle and Henle, 1966),
and MA (Klein et al., 1966).
The components of MA can be considered
to play a central role in many of these mechanisms for attack upon EBV-infected or transformed cells. MA determinants were found in
the virus envelope and could participate inneutralization (Sairenji et al., 1984, 1985a,
1988). MA appeared to be a target for ADCC
killing (Takaki et al., 1980). The presence ofhigh antibody titers to ADCC-determined MA
correlates with survival in patients with NPC
and with tumor response to chemotherapy inpatients with BL (Chan et al., 1979; Pearson etal., 1979). We demonstrated that EBV releasefrom EBV producer cells was inhibited by antiMA antibodies (Sairenji et al., 1985a, 1988). In
order to explore further how anti-EBV antibodies in human serum might influence EBVinfected cells, we sought to determine whether
an antibody might modulate or change theexpression of EBV-associated antigens. We
found that the expression of EA in P3HR-1EBV superinfected Raji cells was decreased
when culturing the cells with EBV-seropositive
human serum and that this phenomenon is
phosphonoacetic acid (PAA)-sensitive.

Materials and Methods
Cell lines and medium

The cell line Raji (Pulvertaft, 1965) was thetarget for superinfection with EBV derived
from P3HR-1 cells (Hinuma et al., 1967).
RPMI 1640 medium (GIBCO, Grand Island,NY), containing 2% fetal calf serum (FCS) and
8% newborn calf serum with 100 µg/mL
streptomycin and 100 IU/mL penicillin, wasused for cell growth and for EBV production.
EBV preparation

Methods for optimal yields in P3HR-1 virus
production have been described (Sairenji and
Hinuma, 1975). P3HR-1 EBV was obtained
from 14-day-old, 33˚C cultures. Cell cultures
were frozen at –70˚C, thawed, centrifuged at2,000 × g for 20 min to remove cellular debris
and centrifuged again at 26,000 × g for 90 min.
The pellets were resuspended in medium at a100-fold concentration relative to the original
culture medium. This virus suspension was
passed through a membrane filter (0.45 µM
pore size) and stored at –70˚C.
EBV superinfection

Raji cells in logarithmic growth phase (viability
100Antibody-mediated suppression of EBV antigen98%) were used for the EA suppression assay.
An amount of virus to induce 20% to 40% EA+
cells at 48 postsuperinfective (psi) h was mixed
with target cells to yield a final concentration of
2 × 106 cells/mL which were incubated at 37˚C
for 90 min (Sairenji and Hinuma, 1973). Afterbeing washed twice with fresh culture medium,the cells were resuspended in fresh medium(RPMI 1640 medium containing 5% FCS andwith varying concentrations of human sera) at
10 6 cells/mL and incubated in 5% CO2 at 37˚C.

human IgG (gamma-specific; Hyland Laboratory,
Deerfield, IN). EA-positive (EA+) and -negative
cells were counted, in a group of at least 500
cells, under a fluorescence microscope and thepercentages of EA+ cells were determined. The
EA-SI was calculated from the percentages of
EA+ cells at 48 psi h according to the following
formula.
EA-SI = (control% without) – (test% with)(control% without)where, (control% without) stands for (control %EA+ cells without human serum) and (test%with) for (test % EA+ cells with human serum).

Human sera

Human sera were heat-inactivated and stored at–20˚C. The sera were screened for natural
cytotoxic antibodies to Raji cells (Yang andHewetson, 1974) by culture at 37˚C for 48 h in
RPMI 1640 medium containing 10% humanserum. Cell viability was assayed by trypan
blue dye exclusion and sera which maintained
viability at greater than 90% were used in thisstudy (Table 1).

Results
Effect of anti-EBV antibody -positive or negative human sera on EA expression

Titration of antibodies to EBV antigens

EBV-superinfected Raji cells were cultured in
medium (RPMI 1640 with 5% FCS) with different concentrations of EBV antibody-positive(Nos. 70 and 71) or -negative (Nos. 72 and 73)
sera (Fig. 1). The percentages of EA+ cells
were determined at 24 psi h and 48 psi h. At 24
psi h the percentages of EA+ cells were similarin all cultures with different concentrations ofeach serum regardless of whether or not theserum contained anti-EBV antibodies. However, at 48 psi h the expression of EA+ cells
varied significantly among the cultures, withfewer EA+ cells being observed in the cultureswith EBV antibody-positive sera, but nodepression in numbers of EA+ cells being foundin cultures with EBV antibody-negative sera. In
each assay showing inhibition of EA expression
with a given EBV antibody-positive serum, thedecrease in percentage of EA+ cells was dependent upon the concentration of the serum. The
concentration of Raji cells in the culture did notaffect significantly the percentages of EA+ cells
at any concentration of human serum. Death of
EA expressing, superinfected cells was apparently not a highly significant factor in thiseffect. In a control experiment with serum No.

Anti-VCA titers were determined with acetonefixed smears of P3HR-1 cells which had been
cultured at 33˚C, using an indirect immunofluorescence technique (Henle and Henle,
1966). Anti-EA titers were determined byindirect immunofluorescence on acetone-fixedsmears of P3HR-1-EBV superinfected Rajicells (Sairenji et al., 1991). Anti-MA titers were
determined by indirect membrane immunofluorescence on living P3HR-1 cells cultured at
33˚C (Harada et al., 1980).
EA detection and EA suppression index (SI)P3HR-1-EBV superinfected Raji cells were
smeared and fixed with acetone for 10 min atroom temperature. EA-positive cells weredetected with indirect immunofluorescence(Henle et al., 1970). Serum (anti-EA and antiVCA titers each 1:320) from a patient with NPC
was used as the anti-EA indicator antibody. The
fluorescent second antibody was a goat anti-101

T. Sairenji et al.

Anti-VCA titers
80
30

EA+ cells (%)

70

20

C
H
G
B
E
J

G 0.6
HB1.2 71
0

C

2.5

<5

320

G
E

G 0.6 72
B0

B

H 1.2

J
H
C
J5

<5

73

E

10
10

0

B

C 2.5

C
J
E
H

B 5
J

C
H 2.5
1.2

B

E
J
C

E0
B
J
C5

2.5

J5

10

E 10
10

E
24

48

24

48

24

48

24

48

Time after superinfection (h)Fig. 1. Effect of anti-Epstein-Barr virus (EBV)-positive (Nos. 70 and 71) or -negative (Nos. 72 and 73)
human sera on early antigen (EA) expression. EBV-superinfected Raji cells were cultured in medium
containing 5% fetal calf serum and different concentrations (0, 0.6, 1.2, 2.5, 5 or 10%) of human serum.
Percentages of EA+ cells were determined 24 and 48 h after superinfection.

53, for example, high levels of EA suppression
were observed at 48 h, but cell viability in 10%
serum was 90% (data not shown). Cell viability
in FCS or EBV antibody-negative serum was 94
and 92%, respectively. These data were consistent with the view that EA suppression was
induced by an antibody(ies) to EBV-induced
determinants. In order to test this EBV antibodyspecificity further we analyzed the relationshipbetween antibody titers to EBV antigens andeffect in suppressing EA induction.
Relationship between EA suppression andantibody titers to EBV antigens

Normal adults’ sera with high anti-VCA titers(1:320 or 1:160; Nos. 59, 53 and 63), with moderate anti-VCA titers (1:80; Nos. 58 and 64) andwithout detected VCA titers (<1:5; Nos. 60 and61) were tested for effectiveness in suppressing
EA at 48 psi h (Fig. 2). Effectiveness in EA

suppression was quantitated by EA-SI (presented in Materials and Methods). The EA-SI
for each serum correlated, in general, with its
anti-VCA antibody titer.
A general concordance exists between titers
to VCA and MA in our tested sera (Table 1).
However, EA suppression activity was notalways parallel to the immunofluorescence titers
of VCA or MA. The EA suppressing antibody
titers at 0.5 EA-SI were 1:160 for serum No. 74
or 1:20 for sera Nos. 70 and 71, respectively(Fig. 3). Cord blood serum (No. 74), with amodest anti-VCA titer (1:160) had the highest
titer of EA-SI (1:160). This serum also had ahigh anti-MA reactivity as reflected in: (i) ADCC
antibody titer (1:10,000); (ii) EBV neutralizing
antibody titer; (1:160) and (iii) strong immunoprecipitation reactivity to membrane-associated,
EBV-induced antigens (data not shown).

102

Antibody-mediated suppression of EBV antigen

Anti-VCA titers
320, 160

<5

80

1.0

EA-SI

53
0.5

63

Fig. 2. Relationshipof anti-viral capsid
antigen (VCA)
antibody titer toearly antigen (EA)
suppression. The
EA suppression
index (EA-SI)
reflects the frequency of EA+ cells
in the cultures 48 h
after superinfection.

G

59

H
C

G
H
C

0
5.0

2.5

C 58
G
H
C

1.25

H

64

5.0

C
H

60
H
C
HC 61

H
C

2.5

1.25

2.5

5.0

H
C

1.25

Percentage of serum
1.0

HC

Effect of PAA on EA suppression by antiEBV antibody

H

0.8Time course studies of EA suppression showedthat the suppression was observed at 48 psi hbut not at 24 psi h (Fig. 1). This result wasconsistent with the hypothesis that the EA suppression was dependent upon expression of alate gene function(s) of EBV. PAA inhibits viralspecific DNA polymerase and therefore, blocks
expression of late EBV functions (Sairenji etal., 1978; Takagi et al., 1991; Tsurumi et al.,1994). If EA suppression is mediated by lateviral function, it could be hypothesized thattreatment of cells with PAA inhibits EA suppression. We tested varying doses of PAA on
the EA suppressing activity of 2 sera (Nos. 53
and 63) (Fig. 4). The decrease in EA-SI wasdependent upon PAA concentration. At a PAA
concentration of 100 µg/mL, EA suppression
was almost completely negated. No significant
toxic effect of PAA (as judged with trypan blue
dye exclusion) was observed on either virus
infected or uninfected cells treated with any
concentrations of PAA. These results are consistent with the view that suppression of intracellular EA expression may follow the modulation or alteration of a late EBV antigen(s) by an

H

G
0.6

EA-SI

G
C

74 (CB)

H
H
71 (NPC)

0.4

G
0.2

G

70 (N)

C
0
10

20

40

C
80

GC
160

Serum concentration–1

103

Fig. 3. Suppression index (SI) related to serum
dilution. Cord blood serum (No. 74) with high antimembrane antigen (MA) reactivity reveals a high
EA-SI in comparison to nasopharyngeal carcinoma(NPC) serum (No. 71) and EBV antibody-positive
serum from a normal person (No. 70).

T. Sairenji et al.

anti-EBV antibody. Alternatively, it is also
possible that a late viral function is required for
antibody-mediated inhibition of EA expression.

Discussion
In order to understand one aspect of the immune
response to EBV, we have tried to define therole of antibodies in affecting the course of antigen expression following EBV infection. In
particular we have looked for modulation of
EBV antigen expression by anti-EBV antibodies.
EBV-determined MA are expressed on the cellsurface of EBV-infected cells. The anti-MA antibody, like the anti-VCA antibody, is present in
the sera of nearly all persons who have been
infected with EBV or who have an EBVassociated malignancy. MA was originallydemonstrated in BL biopsies (Klein et al.,1966). It seems likely that one of the important

H

EA-SI

0.6

C

63

0.4

0.2

53

0

0

H H
C C
25

50

H
H
C C

100

200

PAA concentration ( µg/mL)
Fig. 4. Effect of phosphonoacetic acid (PAA) on
early antigen (EA) suppression. Superinfected Rajicells were cultured in the medium containing 10%
serum with EA suppressing activity (No. 53 or 63)
and different concentrations of PAA. The EA suppression index (EA-SI) was determined 48 h after
superinfection.

events leading to the growth of a BL tumor isthe escape of the tumor clone from an alreadydeveloped host immune response (Klein, 1978).
Aoki and Old reported that antigenic modulation by the anti-MA antibody could occur inincubation of cultured BL cells in human serum
containing BL antibodies to escape from such
immune responses (Aoki et al., 1971). In ourstudy we demonstrated that EBV release from
EBV producer cell lines was inhibited by monoclonal antibodies against MA and we proposed
it as a mechanism of immune defense for thecontrol of EBV infection (Sairenji et al., 1985a,
1988, 1991).
For this study human sera with low levels ofnatural cytotoxic antibodies to Raji cells wereselected (Yang and Hewetson, 1974). Thus, we
could observe alteration of EA antigen expression mediated by anti-EBV antibodies during
the 48 h assay period following P3HR-1-EBV
superinfection of Raji. We conclude that suppression of EA expression at 48 h in EBVsuperinfected cells was, in fact, mediated byanti-EBV antibodies for the following reasons:first, suppression was observed in the cultureswith only EBV antibody-positive sera but notwith EBV antibody-negative sera (Figs. 1 and2); second, suppression was inhibited with PAA
treatment which inhibits EBV DNA polymerase (Fig. 4); and third, suppression wasmediated by an immunoglobulin fraction of an
EA-suppressing serum, separated by DEAE
cellulose ion exchange chromatography (datanot shown).
We do not know about the mechanism of
EA supression in cells cultured with EBV
antibody-positive serum. It may be hypothesized that an anti-MA antibody was responsible
for the EA suppression. MA is a major antigen
expressed on the cell surface of EBV-superinfected Raji cells to which EBV-infectedpatients generate a substantial antibody response (Casareale et al., 1983). MA is a target
antigen(s) for ADCC (Pearson et al., 1978;
Takaki et al., 1980) and for complementdependent cytolysis (Strnad et al., 1982). It was
not likely that anti-EA antibodies reacted
directly with EA because EA is an intracellularantigen, and because most of the sera we tested

104

Antibody-mediated suppression of EBV antigen

with the suppressing activity had negligible orundetectable levels of anti-EA antibodies.
EBNA and VCA are also intracellular antigens,which would not react directly with the antibodies in culture fluids. However, there is no
exact concordance between EA suppression
and MA titers in some sera. For example, one
cord blood serum (No. 74 with a moderate antiMA titer of 1:160) had higher EA suppressing
activity than the other sera (Nos. 70 and 71)
with the same level of anti-MA titer (Fig. 3).
On the other hand, serum from an NPC patient(No. 71) had a high anti-MA (1:640) titer but
showed an EA suppression titer at the samelevel as did a normal adult’s serum with an antiMA titer at lower levels (1:80). For thisdiscrepancy we noticed that No. 74 had a high
ADCC titer (1:10,000) and a high neutralization
titer (1:160), while No. 71 had a low neutralization titer (1:20) (data not shown). These datasuggest that EA suppression was induced byanti-MA antibody to a specific MA component(s). Studies of anti-MA immunopreciptation have shown that MA is actually complex
with at least 3 distinguishable molecules forhuman anti-MA antibodies (Sairenji et al.,
1988; Bertoni et al., 1989).
MA is a product of an EBV late gene after
EBV DNA replication and the expression is
inhibited with PAA (Sairenji et al., 1978). The
EA suppression was completely inhibited by
the addition of 100 µg/mL of PAA to the cell
culture (Fig. 4). These results are consistent
with the view that suppression of intracellular
EA might follow modulation or alteration of
MA by an anti-MA antibody or that a late viralfunction is required for antibody-mediatedinhibition of EA expression in this system. In
addition to this study we observed that cell
culturing with EBV-seropositive sera reduced
both production of infectious virus, amounts ofvirus DNA in the culture fluids and also intracellular EBV DNA in EBV producing P3HR-1
cells (Sairenji et al., 1991).
In conclusion, we can consider the potential
significance of EA suppression to EA-positive
cells by an anti-EBV antibody in human serumin vivo. EA suppression should be associatedwith inhibition of productive infection of EBV-

infected cells and, thus, per se could constitutean immune defense against EBV.

Acknowledgments: This work was supported by
grants CA-25873 and AI-23061 from the National
Institute of Health, and in part by Grants-in-Aid for
Scientific Research (B) and for Scientific Research
on Priority Areas from The Ministry of Education,
Science and Culture, Japan. We thank Dr. Jen-Yang
Chen for reading this manuscript and Ms. Lena
Desantis and Ms. Sayuri Matsumoto for typing.
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(Received April 22, Accepted June 3, 1996)

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