Current Eye Research

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Protection from retinal necrosis by passive transfer of monoclonal antibody specific for herpes simplex virus glycoprotein D Sally S. Atherton To cite this article: Sally S. Atherton (1992) Protection from retinal necrosis by passive transfer of monoclonal antibody specific for herpes simplex virus glycoprotein D, Current Eye Research, 11:1, 45-52, DOI: 10.3109/02713689209069166 To link to this article: http://dx.doi.org/10.3109/02713689209069166

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Date: 15 March 2016, At: 22:56

Current Eye Research

Yolumc 11 number 1 1992, 45-52

Protection from retinal necrosis by passive transfer of monoclonal antibody specific for herpes simplex virus glycoprotein D Sally S.Atherton

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Departments of Microbiology and Immunology and of Ophthalmology, PO Box 016960 (R-138), University of Miami School of Medicine, Miami, FL 33101, USA

ABSTRACT Passive administration of antibody against herpes simplex virus type 1 (HSV-1) has been shown to protect against stromal keratitis and death from encephalitis. Although the exact mechanism by which passivelytransferred antibody protects is not known, one of the features of protection by passively-transferred antibody is interference with the ability of the virus to spread within the nervous system. In the experiments reported herein, studies were performed to determine if 8D2, a monoclonal antibody against a type-common epitope of glycoprotein D, could protect mice from retinal necrosis following uniocular anterior chamber inoculation of HSV1. Mice were protected from retinal necrosis when the antibody was administered 2 hours before virus inoculation or 24 hours after virus inoculation. When antibody was injected 2 hours before virus inoculation, the titer of virus at day 1 p.i. in the injected eyes of antibody-treated and control mice was the same, but by 3 days p i , the titer of virus in the antibody-treated mice was significantly lower than that recovered from control mice. The titers of virus in the brains and in the uninoculated eyes of antibody-treated mice were also significantly lower than in control mice. The results of these studies suggest that passively-transferred antibody protects against retinal necrosis by limiting spread of virus to the CNS or replication of virus within the CNS.

not significantly different from control mice, but virus replication was significantly reduced several days later in the lower flank, the site of zosteriform spread of the virus in untreated mice. These authors suggested that treatment with antibody interfered with the ability of the virus to emerge from nerve endings. In their studies, the neutralizing capability of the virus, but not the target epitope of the virus, correlated with the ability of the passively-transferred antibody to protect (1). Many investigators have studied the ability of passively-transferred anti-HSV-1 antibody to protect against disease after corneal inoculation. Davis and coworkers demonstrated that passive immunization with a polyclonal anti-HSV-1 immune serum prevented death of the mice from encephalitis following corneal inoculation (2). In these investigators’ studies, the average titer of

virus in the brains of antibody-treated mice was significantly lower than the average titer of virus in the brains of control mice suggesting that antibody treatment reduced spread of virus to the CNS from the

INTRODUCTION In HSV-1-infected mice, passive immunization with

eye or prevented replication of virus after entry into the CNS (2). Passive immunization also has been shown to

serum containing antibodies to HSV-1 can prevent

protect mice from stromal keratitis after corneal

disease and even death from encephalitis. Using a

inoculation of HSV-1 (3-6). Recent work by Shimeld

model in which HSV-1 is inoculated into the flank of

and colleagues suggested that passively-transferred

BALB/c mice, Simmons and Nash demonstrated that

anti-HSV-1 antibody protects by preventing neuronal

administration of either polyclonal or monoclonal

spread of the virus back into the eye from the CNS (7).

antibody against HSV-1 prevented zosteriform spread of

Protection against stromal keratitis is not limited to a

the virus following flank inoculation (1). Virus replication

single epitope, and monoclonal antibodies against

at the site of inoculation in antibody-treated mice was

glycoprotein B (gB), gC, gD and gE protect (4). Metcalf

Received on August 27, 1991; accepted on January 2, 1992

0 Oxford University Press

45

Current Eye Research and co-workers demonstrated that protection did not

in Dulbecco’s Modified Eagle Medium (DMEEM)

depend on the ability of the monoclonal antibody to

supplemented with 5% bovine serum and antibiotics.

neutralize the virus (4). Lausch and co-workers showed

Virus stocks were titered by plaque assay on duplicate

that passive administration of a monoclonal antibody to

cultures of Vero cells using standard methods and

gD did not affect the host’s ability to make both a

stored at -70°C as described previously (13). A fresh

cellular and a humoral immune response against the

aliquot of stock virus was thawed and used for each

virus (8). The IgG isotype appears to be important in

experiment.

protection; when protective antiserum was depleted of

Monoclonal antibody

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IgG prior to passive transfer, the ability to protect mice from stromal keratitis was abrogated (5). In BALBIc mice, uniocular anterior chamber

8D2, a monoclonal antibody of the lgG2, isotype which recognizes a type-common epitope on gD of HSV-1 and HSV-2 and which neutralizes both viruses in

inoculation of the KOS strain of HSV-1 produces retinal

the absence of complement was used in these

necrosis in the uninoculated eye within 8-10 days (9).

experiments. The preparation and characteristics of this

Following anterior chamber inoculation, virus spreads to

monoclonal antibody have been described previously

the CNS via neural routes (10,ll) and from the CNS,

(6). Antibody was injected intraperitoneally in a volume

travels via the optic nerve of the uninoculated eye to the

of 0.1 ml.

retina (1 1,12). If one mechanism by which passively-

Virus inoculation

transferred antibody confers protection from disease

Mice were anesthetized with pentobarbital (0.65

and/or death in skin and corneal models is by preventing

mg/lO 9). The right eye was proptosed, an anterior

virus transport in the nervous system, it might be

chamber paracentesis was performed, and 1-2 X lo4

predicted that passively-transferred antibody to HSV-1

plaque forming units (PFU) of the KOS strain of HSV-1

would prevent retinal necrosis in mice following anterior

contained in a volume of 2 1 were injected in the

chamber inoculation of the virus. This communication

anterior chamber.

describes protection from retinal necrosis after passive

Histopatholoaic studies

transfer of monoclonal antibody against a type common epitope of herpes simplex virus gD. MATERIALS AND METHODS Animals -~ Three- to four-week old, female, euthymic BALB/c

In experiments to determine the effect of antibody on the incidence of retinal necrosis in the uninoculated contralateral eye, animals were injected intraperitoneally with antibody or phosphate buffered saline (PBS; 0.1 ml) either 2 hours before anterior chamber inoculation of

mice (Taconic, Germantown, NY) were used in these

virus or 24 hours following anterior chamber inoculation

experiments. Mice were given unrestricted access to

of virus. On day 14 p.i., all mice were sacrificed. The

food and water, and a 12 hour light alternating with a 12

uninoculated eye was removed, fixed in buffered

hour dark cycle was maintained. All experiments were

formalin, embedded in paraffin, sectioned, and stained

performed in accordance with the Association for

with hematoxylin and ensin. Multiple sections of the

Research in Vision and Ophthalmology (ARVO)

posterior segment of each eye were examined for retinal

Resolution on the Use of Animals in Research.

necrosis characterized by retinal inflammation, hemorrhage, retinal schisis, cuffing of the retinal

The

KOS strain of WSV-1 was used in these

experiments. Virus was propagated in Vero cells grown

vasculature, and loss of the retinal architecture as described previously (9, 14-16). The retina!; of eyes

Current Eye Research classified as normal appeared to be completely

2 mice were injected with antibody 24 hours following

unremarkable when examined microscopically.

virus inoculation, and group 3 mice received only an

Virus recovery studies

intraperitoneal injection of PBS 2 hours before anterior

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For these experiments, mice were injected either with

chamber inoculation of virus. The results of this

antibody (27.5 pg) or PBS intraperitoneally 2 hours

experiment (Table 1-Experiment 1) revealed that no

before inoculation of HSV-1 into the anterior chamber.

antibody-treated mouse developed retinal necrosis in the

On days 1, 3, 5, 7-10, 12, and 14 p i , 5 mice in the

uninoculated eye; the retinas of all antibody-treated mice

PBS-treated control group and 5 mice in the antibody-

appeared to be normal when hematoxylin and eosin-

treated experimental group were selected at random

stained ocular sections were examined microscopically.

and sacrificed. Immediately after sacrifice, both eyes of

Protection from retinitis was observed irrespective of

each mouse were enucleated, and the brain was

whether the antibody was administered before anterior

removed and separated into right and left halves by a

chamber inoculation of virus or 24 hours after virus

mid-sagittal incision using a scalpel blade. Both eyes

inoculation. In contrast, the retinas of 4 of 5 (80%) of

and both halves of the brain were frozen at -70°C. At

the PBS-treated mice were necrotic, a result which is

the time of titration, the tissues were thawed and

consistent with the incidence of retinal necrosis reported

homogenized in 1.0 ml of DMEM without serum using a

previously for normal BALB/c mice injected with the

hand-held tissue homogenizer (Dremel, Racine, WI).

KOS strain of HSV-1 via the anterior chamber route (9).

The cell debris was pelleted by low-speed centrifugation,

Since there was no difference in protection from retinal

and the supernatant from each sample was serially

necrosis observed between mice in group 1 that

diluted, and the titer of infectious virus was determined

received antibody immediately before anterior chamber

by plaque assay on duplicate cultures of Vero cells.

injection of virus and mice in group 2 that received

Antibody and control group values were compared within

antibody 24 hours after virus inoculation, 8D2 was

each location (inoculated eye, uninoculated eye, right

administered 2 hours before virus inoculation in

brain and left brain) with a two-way analysis of variance

subsequent experiments.

in which group and day after inoculation were factors.

Lausch and co-workers have reported that 10 c(g of

After establishing that the group-day interaction was

8D2 administered via the intraperitoneal route protects

statistically significant at each location, groups were

mice against stromal keratitis following corneal

compared on each day using the Scheffe multiple

inoculation of the RE strain of HSV-1 (6). To determine

comparison procedure.

whether antibody doses lower than 55 pg conferred protection from retinal necrosis, three additional groups

RESULTS Effect of 8D2 on retinal necrosis in the uninoculated eve To determine whether BALB/c mice treated with 8D2,

of animals were injected with the 8D2 monoclonal antibody (Table 1-Experiment 2); mice in group 1 received 55 pg of antibody, mice in group 2 received

a monoclonal antibody against g D, were protected from

27.5 pg of antibody, and mice in group 3 received 13.75

retinal necrosis in the uninoculated eye following uniocular anterior chamber inoculation of HSV-1, three

pg of antibody. Each group was injected with virus via the anterior chamber route 2 hours following

groups of euthymic BALB/c female mice were used.

intraperitoneal administration of antibody. All mice were

Mice in group 1 were injected with 8D2 (55 pg) 2 hours

sacrificed at day 14 p i , and the uninoculated eye of

before virus inoculation into the anterior chamber, group

each mouse was examined microscopically for retinal

47

Current Eye Resear-ch TABLE 1 Protection from retinal necrosis following passive transfer of 8D2

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Time of Antibody Injection"

a

Dose of Antibody (P9)

Mice with Retinal Necrosisflotal (%)

- 8D2 was injected intraperitoneally 2 hours before or 24 hours after 1-2 X 1O4 PFU

of HSV-1

(KOS)were inoculated into the anterior chamber of one eye

- control mice received 0.1 ml of PBS 2 hours before virus inoculation

See text for additional experimental details.

--

____

-

necrosis. As shown in Table 1 (Experiment 2), there

average titer of virus recovered at day 1 was identical

was no significant difference in the ability of 8D2 to

for both the antibody-treated mice and the control mice

protect at either of the two lower doses, and 13.75 pg of

(average titer: 5.49 versus 5.46 log,, PFU/mI). By day 3

antibody protected the mice from retinal necrosis as well

p.i., the average titer of virus in the inoculated eyes of

as the 55 pg dose. An unexpected finding was that one

antibody-treated mice was significantly lower (pl0.01)

of four animals treated with the intermediate dose of

than that recovered from the injected eyes of PBS-

antibody had retinal necrosis.

treated control mice (Figure 1). These findings suggest

Virus recoverv experiments

that virus was able to replicate in the inoculated eye of

lniected eye. It has been reported previously after

antibody-treated animals within the first 24 hours but

uniocular anterior chamber inoculation of HSV-1 in

was prevented from further replication after this time.

normal BALBIc mice that the titer of virus in the injected

Replicating virus was cleared from the injected eyes of

eye reaches a peak at 3 days p i , and virus can be

both antibody-treated and control mice by day 14 p i

recovered sequentially from the injected eye, the brain

which suggests that administration of antibody did not

and the uninoculated eye (13) To determine whether

prolong clearance of virus from the injected eye.

passive administration of the monoclonal antibody 8D2

Brain. In both antibody-treated experimental mice and

affected the titer of virus at any of these sites, a time-

PBS-treated control mice, a low titer of virus was

course experiment was performed to compare

recovered from the ipsilateral (right) side of the brain at

sequentially the titer of virus at each location in

day 3 p.i. (Figure 2A). The titer of this virus was not

antibody-treated and PBS-treated control mice (see

significantly greater than the minimum level of virus

Materials and Methods). In the virus-injected eye, the

detection and was essentially identical for both treated

48

Current Eye Research 7 -

A 6 -

A

PBS

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8D2

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3

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5

-

4 -

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3 -

9

2 -

0

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c3

1 -

0

: 0

0

2 4 6 8 1 0 1 2 1 4 DAYS AFTER INOCULATION

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2

4

8

6

10

12

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DAYS AFTER I N O C U L A T I O N 41-

Figure 1: Recovery of virus from the inoculated eyes of BALB/c mice treated with 8D2 monoclonal antibody (00) or PBS (A-A) 2 hours before inoculation of 1-2 X 1O4 PFU of HSV-1 (KOS) into the anterior chamber of the right eye. Five mice from each group were sacrificed at each time point, and the amount of virus in each specimen was determined (in duplicate) by plaque assay on monolayers of Vero cells. The amount of virus at each time point is the mean virus titer (expressed as Log,, PFU/ml k S.E.M.) of all samples harvested at that time. The solid line indicates the lower limit of virus detection. *=significantly different from control group, p