J Cancer Res Clin Oncol 98, 165-172 (1980) aoumal of

Cancer Research Clinical Oncology �9 Springer-Verlag 1980

Herpesvirus SaimirMnduced Malignant Lymphoma in Inbred Strain 11I/3 Rabbits (Oryetolagus Cunieulus)

D.V. Ablashi 1, K.S. Sundar 1, G. Armstrong 1, p. Golway2, M. Valerio 2, Z. Bengali 2, j. Lemp 3, and R.R. Fox 4

1 National Cancer Institute, Bethesda, MD 20205, USA 2 Litton Bionetics, Kensington, MD, USA 3 Electro-Nucleonics Laboratories, Inc., Bethesda, MD, USA 4 Jackson Laboratory, Bar Harbor, ME, USA

Summary. Four young strain III/J rabbits of both sexes were inoculated with a single dose of prototype partially purified Herpesvirus saimiri (HVS) via IV and IM routes. All inoculated animals had enlarged lymph nodes, and signif- icant levels of antibodies to HVS early, late, and membrane antigens were de- tectable during the infection. The animals died or were killed and HVS was iso- lated from the peripheral blood mononuclear cells and the various lymph nodes but not from the kidney. Microscopic examination showed that these animals had poorly differentiated lymphomas. The response of mononuclear cells to PHA from peripheral blood of infected animals showed depressed cell medi- ated immune responses. Humoral and cellular immunity responses during tu- morigenesis were comparable to those reported in nonhuman primates with HVS-induced tumors. Thus, the inbred strain III/J appears to be an inexpensive suitable model for studies of oncogenic herpesvirus-induced cancers.

Key words: Inbred rabbits - Herpesvirus sa imir i - Malignant lymphoma

Daniel et al. (1970, 1974, 1975) and Hunt et al. (1975) reported the induction of malignant lymphoma in New Zealand white rabbits with single or multiple inocu- lations of HVS by intravenous (IV), intramuscular (IM), intraperitoneal (IP), sub- cutaneous (SC), or intradermal (ID) routes. In 17-149 days, approximately 56~ of the rabbits developed lymphoma but without detectable neutralizing antibody. Subsequently, Rangan et al. (1976) reported induction of malignant lymphoma in two of nine New Zealand white rabbits between 40 and 50 days. One lymphoma- tous animal tested for HVS early antigen (EA) and late antigen (LA) showed no detectable antibodies. Furthermore, only low titers of neutralizing antibodies were found in the tested animal. No cell-mediated immune response studies were carried out in any of the aforementioned studies. Moreover, titers to HVS EA, LA, and membrane antigen (MA) were not fully investigated.

Offprint requests to: D.V. Ablashi (address see above)

0171-5216/80/0098/0165/$l.60

166 D.V. Ablashi et al.

The present invest igat ions were unde r t aken (a) to increase the t u m o r induc t ion by using inbred rabbi t s and high t i tered HVS and (b) to fol low the humora l and cellular immune responses in HVS- inocu la ted animals dur ing tumorigenesis . Spe- cifically, these invest igat ions were unde r t aken to develop an inexpensive l abora - tory animal mode l system for immunovi ro log ica l , b iochemical , biological , chemo- therapeut ic , and prevent ion studies o f p r ima te herpesvirus induced cancers.

Material and Methods To remove any PPLO contamination, HVS grown in PPLO free owl monkey kidney (OMK) was par- tially purified on sucrose gradient (Sottong et al. 1975) by continuous flow centrifugation. This was concentrated to 100 x using MEM containing 2% FCS. This virus titered 108s TCIDso/ml in OMK cells. One milliliter of 10 ~'' TCID~o/ml of this virus was inoculated IV into the marginal ear vein and 1 ml of 10 ~~ TCID,o/ml was inoculated IM into each of four rabbits (Table 1). Previously, this HVS strain had induced malignant lymphoma with and without lymphocytic leukemia in owl monkeys (Ablashi et aI. 1971).

Experimental Animals

Less than 1-year-old rabbits of both sexes of the III/J strain from the rabbit colony of the Jackson Lab- oratory (Table 1) were used. This strain, which achieved full inbred status in 1976, was derived by P. B. Sawin from the New Zealand white stock of W. E. Castle in the early 1930's. Detailed strain characteris- tics of these rabbits have been described (Fox 1975; Fox et al. 1979).

Cells and Attempts to Isolate HVS

To isolate HVS, an OMK cell line was used. For virus recovery, lymphocytes from peripheral blood or tumor tissue or from kidney (Table 1)were used for assays according to Ablashi et al. (1971) and Klein et al. (1973).

HVS-EA, LA, and MA

The sera from control (uninoculated) and HVS inoculated animals were examined by indirect im- munofluorescence for HVS antigens according to methods described by Klein et al. (1973) and Ablashi et al. (1971). Assays for MA antibody to HVS were done according to the method described by Pearson et al. (1973).

Cell-mediated Immune Response

The cell-mediated immune (CMI) response to nonspecific mitogen was carried out by the lymphocyte stimulation test according to Sundar et al. (1980).

Results

Clinical Observations

Thir ty-s ix days pos t - inocu la t ion rabb i t no. 5558 exhibi ted slight resp i ra tory con- gestion. N o ocular or nasal discharge was observed, and food and water consump- t ion was normal . Both precapsu la r l ymph nodes were enlarged. By day 59 the ani- mal had general ized per iphera l l y m p h o a d e n o p a t h y and the rabb i t was found dead on the 60th day. R a b b i t no. 5552 was found depressed, anorexic, and dyspneic at 46 days after HVS inocula t ion and was killed. He sneezed f requent ly and had a co- p ious bi lateral , mucopuru len t , ocular , and nasal discharge. Both eyelids were in- fected and swollen and be had severe b i la tera l conjunctivit is . Genera l ized per iph- eral l y m p h o a d e n o p a t h y was present. This an imal was though t to have a bac ter ia l resp i ra tory disorder , p r o b a b l y similar to pasteurel losis .

Malignant Lymphoma in Inbred Rabbits 167

By the 55th day post-inoculation, the remaining two rabbits were exhibiting signs of respiratory disorders. They had mucopurulent ocular and nasal dis- charges, raspy respiration, conjunctivitis, and frequent sneezing and coughing. Animal no. 5556 developed a raw, scaly lesion on the penis and had peripheral lym- phoadenopathy. Since the condition of animal no. 5556 and animal no. 5555 be- came worse, they were killed on the 60th day post-inoculation.

The control rabbits (5554 and 5551) remained clinically normal up to the time they were killed (82 days post-inoculation).

Immuno-virologieal Investigations

Table 1 shows the isolation of HVS from whole peripheral blood from rabbits 5558, 5556, and 5552 during the infection. The peripheral blood mononuclear cells separated from blood samples of all experimental rabbits prior to HVS inoculation failed to show any previous infection of HVS. Lymph nodes of HVS-inoculated animals yielded HVS after co-cultivation with OMK, but their kidney cells did not. Control rabbits (Nos. 5554 and 5551) were negative for HVS. Virus isolates were neutralized by goat anti-HVS (strain S-295C). The OMK cells infected with virus isolated from the animals also showed immunofluorescence staining for HVS-LA. All pre-HVS inoculation serum samples from these animals (Table 1) showed no HVS-EA, LA, or MA antibodies. Moreover, no HVS-neutralizing antibody was detected in these pre-HVS inoculation samples. By day 33, all HVS infected ani- mals showed significant levels of EA, LA, and MA antibody; however, animal 5556 demonstrated relatively low levels of EA and LA. Sera from all four HVS inocu- lated rabbits prior to their termination contained considerable levels of HVS neu- tralizing antibody (1 : 10 dilution of serum neutralized 1000 TCID 50 of HVS). How- ever, only rabbit 5552 had higher EA and LA antibody 45 days postinfection.

Cell-mediated Immunity

The response of mononuclear cells obtained on the same day from one uninocu- lated animal (no. 5554) and HVS inoculated animals to PHA-P are presented in Table 1. All experimental animals showed depressed cell-mediated immune re- sponses. However, the PHA-P response was markedly lower in animal 5552. The PHA-P response of experimental animal 5555 could not be evaluated due to tech- nical difficulties.

Pathology

All experimental animals at time of necropsy showed a varying degree of ocular and nasal discharge. Animals 5556, 5558, and 5553 had enlarged submandibular and cervical lymph nodes, whereas in animal 5552 cervical, axillary and inguinal lymph nodes were enlarged. No other significant lesions were observed. Microscopic findings were essentially those described by Hunt et al. (1975). The cell type was predominately a poorly differentiated lymphocyte. There was diffuse involvement of all tissues examined (trachea, esophagus, thyroid, heart, lung, spleen, liver, kidney, lymph nodes). Lymph nodes (Fig. 1 a), liver (Fig. 1 b), and

Tab

le 1

. Im

mun

o-vi

rolo

gica

l an

d hi

stop

atho

logi

c da

ta o

n co

ntro

l an

d H

VS

-ino

cula

ted

inbr

ed I

II/J

str

ain

rabb

its

t

Ani

mal

In

ocul

atio

n an

d T

issu

es f

rom

whi

ch

Ser

um

iden

t,

rout

e of

vi

rus

was

iso

late

d co

llec

ted

(Sex

) in

ocul

atio

n"

days

pos

t-

inoc

.

HV

S a

ntib

ody

(IgG

) ti

ters

b

CM

I re

spon

se t

o P

HA

-P g

g/m

l (%

A C

PM

) in

hibi

tion

a

EA

L

A

MA

l0

50

10

0

5554

(M

) M

EM

-I/V

, I/

M

5551

(F)

M

EM

-I/V

, I/

M

5552

(M

) H

VS

-2 x

106

s I/

V, I

/M

5555

(F)

H

VS

-2 x

106

.5

I/V,

I/M

5556

(M

) H

VS

-2 x

106

.5

i/v,

VM

5558

(F)

H

VS

-2 x

106

s ~/

v, I/

M

No

HV

S w

as d

etec

ted

in t

he

33 a

nd 7

3 <

1 : 5

<

1 : 5

<

1 :

5 98

,561

90

,842

98

,899

pe

riph

eral

blo

od l

ymph

ocyt

es

(0)

(0)

No

HV

S w

as d

etec

ted

in t

he

33 a

nd 7

3 <

1 :

5 <

1 :

5 <

1 :

5 N

ot t

este

d pe

riph

eral

blo

od l

ymph

ocyt

es

Lym

ph n

odes

(+

) 33

1:

80_

1:16

0_+

1:20

7,

897

8,52

2 8,

998

(cer

vica

l, a

xill

ary

ingu

inal

) 45

1 :

80

1 : 1

60

1:20

(9

1.9)

(9

0.6)

(9

0.9)

sp

leen

(+

), k

inde

y (-

)

Lym

ph n

odes

(+

) 33

1:

40

1 :

80

1:20

N

ot t

este

d su

bman

dibl

e, c

ervi

cal

( + )

59

1:

80_+

1:

160+

1:

40

peri

pher

al b

lood

(+

), k

idne

y (-

)

Lym

ph n

odes

(+

) (c

ervi

cal,

33

1:

20

1:4

0

1:10

15

,121

15

.981

14

,586

su

bman

dibl

e) m

onon

ucle

ar

(84.

2)

(82.

4)

(84.

2)

cell

s fr

om p

erip

hera

l bl

ood

(+)

kidn

ey (

-)

Die

d-de

com

pose

d, m

onon

ucle

ar

33

1:40

1

:80

1:

40

11,2

46

13,5

72

14,9

28

ceil

s fr

om p

erip

hera

l bl

ood

(88.

6)

(85.

5)

(84.

9)

prio

r to

dea

th (

+)

a T

he e

xper

imen

tal

anim

als

rece

ived

1 m

l of

vir

us I

V a

nd 1

ml

IM.

The

con

trol

ani

mal

rec

eive

d th

e sa

me

amou

nt o

f M

EM

. b

HV

S i

sola

tion

was

con

firm

ed b

y (a

) IF

usi

ng a

nti-

HV

S s

era

(EA

and

LA

) fr

om a

n ow

l m

onke

y de

velo

ping

HV

S t

umor

and

(b)

ser

um n

eutr

aliz

atio

n us

ing

goat

-ant

i H

VS

ser

um.

All

ani

mal

s w

ere

sero

-neg

ativ

e fo

r H

VS

ant

igen

s an

d by

ser

um n

eutr

aliz

atio

n as

say.

Neu

tral

izat

ion

tite

rs a

re b

ased

on

the

dila

tion

of

<~

seru

m w

hich

neu

tral

ized

100

0 T

CID

5~

of H

VS

;~

c

The

his

topa

thol

ogic

dia

gnos

is o

f all

HV

S-i

nocu

late

d an

imal

s sh

owed

the

y ha

d m

alig

nant

lym

phom

a, w

here

as r

abbi

ts N

o. 5

551

and

5554

(co

ntro

ls)

had

no

sign

of

dise

ase

d T

he n

et c

ount

per

min

ute

(AC

PM

) gi

ven

was

cal

cula

ted

by s

ubtr

acti

ng m

ean

cont

rol

CP

M f

rom

mea

n te

st C

PM

Fig. 1 a. Lymph node. Replacement of cortical areas with a diffuse sheet of lymphocytes and extension into perinodal tissue. HE x 140. Inset. Higher magnification illustrating poorly differentiated lymphocytes. HE x 560. b. Liver. Cellular infiltrate in the periportal tissue and sinusoids. HE x 56

170 D.V. Ablashi et al.

spleen were most consistently involved and to the greatest extent. Animal no. 5556 also had extensive diffuse cellular infiltrates of the testes, epididymis, penis, and prepuce.

Discussion

Present data extend the tidings of Daniel et al. (1970, 1974, 1975), Hunt et al. (1975), and Rangan et al. (1976) who induced some lymphomas in New Zealand white rabbits. Our data also show that the inbred strain III/J of New Zealand white origin is highly susceptible to HVS (almost 100%) since all the rabbits inoculated developed lymphomas. The animals did not exhibit other clinical signs (significant- ly increased levels of white cells or lymphocytes) except generalized peripheral lym- phadenopathy and enlarged lymph nodes. The studies reported by Daniel et al. (1974) and Hunt et al. (1975) indicatedthat animals which died or were killed be- fore 46 days post-inoculation showed no clinical evidence of disease. The animals dying after this period had severe conjunctivitis, nasal discharge, and dyspnea. These same symptoms were observed in our rabbits as early as 33 days. Like Hunt et al. (1975), we also thought that the respiratory symptoms might be due to pas- teurellosis. The pathologic findings in III/J strain rabbits are similar to those re- ported by Hunt et al. (1975).

HVS was not isolated from the kidneys of experimental animals. This finding was consistent in our studies and those of Daniel et al. (1970, 1974, 1975) and Rangan et al. (1976). However, HVS was isolated from all the lymph nodes tested for virus isolation. Our animals had higher neutralizing antibody than those of Da- niel et al. (1970) and Rangan et al. (1976). In contrast to their studies, all of our HVS inoculated animals had significant levels of MA antibody. The MA antibody titers were lower than had been reported by Pearson et al. (1973) in owl monkeys which developed HVS tumors. However, those virus infected owl monkeys which remained free of tumors showed low titer MA antibodies. It has been postulated that the anti-HVS-MA titers in marmosets are quite low because of the short in- cubation period and lower severity of the disease. Thus, anti-MA titers in our rab- bits are significant since these animals lived only approximately 60 days.

Antibody patterns to HVS antigens reported by Pearson et al. (1973) and Klein et al. (1973) suggest that there is a wide spectrum of antibody titers in natural HVS carriers (squirrel monkeys) and those which developed tumors upon inoculation with HVS (owl monkeys and marmosets). In treated owl monkeys which remained free of disease, low EA titers were found. The titers of EA generally were higher in diseased animals. The lower EA and LA titers may be due to species variation or (a) degree of immunological response of the rabbits to HVS, and (b) the short duration. The studies of Klein et at. (1973) showed that in general owl monkeys had higher EA antibodies than white-lipped and cotton-topped marmosets. So, the EA and LA titers obtained from our rabbits are comparable to those reported by Klein et al. (1973) in owl monkeys.

The decreased in vitro PHA response of mononuclear cells in the affected rab- bits has not been reported in previous studies. The depressed CMI in HVS infected rabbits may be due to immunosuppression. Immunosuppression was marked in two animals with diffuse malignant lymphoma resulting in death 4 days later. Ira-

Malignant Lymphoma in Inbred Rabbits 171

munosuppress ion was also evident in two other rabbits which developed lym- phoma 17-18 days pr ior to terminat ion. Wal len et al. (1975a, b) reported de- pressed C M I in HVS-inocula ted owl monkeys. Our CMI data confirm these find- ings. Thus, the pathologic humora l and cellular immune responses to HVS in in- bred rabbits are comparable to those observed in n o n - h u m a n primates.

In view of the relative unavai labi l i ty of n o n h u m a n primates and their high ex- pense, the inbred rabbits offer an inexpensive al ternative model for i mmuno- chemotherapy and prevent ion studies for HVS and perhaps for other oncogenic pr imate herpesviruses, such as EBV. The humora l and cell-mediated immuni ty studies in rabbi ts may be potent ial ly useful for appl icat ion to EBV evaluat ion stud- ies in Burki t t ' s l ymphoma and nasopharyngeal carcinoma, where EBV plays a ma- jo r role in the mult i factoral etiology of these cancers (de Th6 et al. 1978; Epstein and Achong 1977).

Acknowledgement. This study was supported in part by grant no. RR-00251 from the Divison of Research Resources, National Institute of Health, and contract no. NOI CP 81025 from the National Cancer Institute. The authors also thank Dr. Gary Pearson, Mayo Clinic, Rochester, Minnesota (USA) for his suggestions.

References

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Ablashi DV, Armstrong GR, Fellows C, Easton JM, Pearson G, Twardzik D (1977) Evaluation of the effects of phosphonoacetic acid (PAA) and 2-deoxyd-ghicose on Herpesvirus saimiri and Epstein- Barr virus. In: Nieburg HE (ed) Detection and prevention of cancer. Marcel Dekker, New York, pp 245-262

Boyum A (1968) Isolation ofmononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 21:77-89

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Klein G, Pearson G, Rabson A, Ablashi DV, Falk L, Wolfe L, Deinhardt F, Rabin H (1973) Antibody reactions to Herpesvirus saimiri (HVS)-induced early and late antigens (EA and LA) in HVS-iufected squirrel, marmoset and owl monkeys. Int J Cancer 12:270-289

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Pearson G, Ablashi D, Orr T, Rabin H, Armstrong GR (1972) Cytoplasmic and membrane im- munofluorescence investigation on cells infected with Herpesvirus saimiri. J Natl Cancer Inst 49: 141%1424

Pearson GR, Orr T, Rabin H, Cicmanec J, Ablashi D, Armstrong G (1973) Antibody pattern to Her- pesvirus saimiri-induced antigen in owl monkeys. J Natl Cancer Inst 51:1939-1943

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Sottong P, Hill P, Feeney M, Klecker, J, Johnson K, Harris R, Bell C, Stafford K (1975) Recovery of murine leukemia virus from large volumes of freshly harvested culture fluids by using a single den- sity gradient. Appl Microbiol 29:102-105

Sundar SK, Ablashi DV, Bengali ZH, Levine PH, Nonoyama M (1980) Mitogenic effect of 12-0 te- tradecanoyl-phorbol-13-acetate on non-human mononuclear cells and in vitro interaction with Ep- stein-Barr virus transformation. Arch Virol 64:141-153

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Received May 3, 1980/Aceepted July 25, 1980