Download Passive transfer of anti-herpes simplex virus type 2

Passive Transfer of Anti-Herpes Simplex Virus Type 2
Monoclonal and Polyclonal Antibodies Protect Against
Herpes Simplex Virus Type 1-Induced but Not Herpes
Simplex Virus Type 2-Induced Stromal Keratitis
Mary H. Ritchie, John E. Oakes, and Robert N. Lausch
Purpose. To investigate whether passive transfer of antibodies to viral glycoproteins would
protect against herpes simplex virus type 2-induced stromal keratitis.
Methods. Balb/c mice were infected on the scarified cornea with herpes simplex virus types 1
or 2 (HSV-1 and HSV-2, respectively), and monoclonal or polyclonal antibodies were administered intraperitoneally 24 hr later. Eyes were monitored for corneal opacity. Flow cytometry
was used to examine the expression of glycoproteins on the surface of HSV-infected cells.
Results. Passive transfer of monoclonal antibodies to viral glycoproteins gB, gD, or gE or
anti-HSV-2 hyperimmune serum were all highly effective (P < 0.005) at preventing blinding
disease induced by HSV-1. In contrast, none of the antibody preparations could prevent
stromal keratitis when the animals were challenged with various HSV-2 strains. However,
antibody treatment could prevent the development of fatal encephalitis in the majority of
HSV-2 infected hosts. Flow cytometry analysis revealed that gD and gB expression on the
membranes of HSV-2 infected corneal epithelial cells isolated from excised corneas was substantially less (P < 0.005) than that detected on HSV-1 infected cells at both 24 and 48 hours
postinfection. This antigenic difference was not due to the failure of HSV-2 to replicate in
corneal epithelial cells in vivo.
Conclusions. Decreased levels of membrane glycoprotein antigen expression may be one factor
contributing to the refractiveness of HSV-2-induced ocular disease to humoral immunotherapy. Invest Ophthalmol Vis Sci. 1993; 34:2460-2468.
Jrierpes simplex keratitis is the most common cause
of corneal disease and the second leading cause of
blindness in the United States.12 Although herpes simplex virus type 1 (HSV-1) has traditionally been associated with herpetic eye disease, herpes simplex virus
type 2 (HSV-2) eye infections are becoming increas-
From the Department of Microbiology and immunology, College of Medicine,
University of South Alabama, Mobile, Alabama.
Supported in part by grant N1H EY-07564.
Submitted for publication: September I, 1992; accepted December 21, 1992.
Proprietary interest category: N.
Reprint requests: Robert N. Uiusch, Department of Microbiology and Immunology,
College of Medicine, University of South Alabama, Mobile, AL 36688.
2460
ingly important in adults.3"8 Indeed, HSV-2 eye infections may manifest more severe and prolonged corneal lesions than those caused by HSV-1.67 The corneal opacification and scar formation that develop are
at least partly attributable to the host immune response.9
HSV-1 inoculated onto the scarified cornea of
Balb/c mice produces a blinding necrotizing stromal
keratitis. Studies of HSV-1 murine corneal infection
have indicated that this disease also has an immunopathologic basis.1011 That is, immune T cells responding to HSV-1 infection play a prominent role in the
inflammatory process. To date the humoral immune
Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8
Copyright © Association for Research in Vision and Ophthalmology
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933174/ on 05/02/2017
2461
Antibody and HSV-2 Corneal Infection
response has not been shown to contribute to the immunopathology. Instead, we1213 and others14'16 have
found that the timely administration of monoclonal
antibody to selected viral glycoproteins is highly effective at preventing the development of blinding corneal
disease.
Ten different glycoproteins have been identified
on the virion surface and the plasma membrane of the
infected host cell where they serve as targets of the
immune response.1718 Glycoproteins gB and gD are
among the most immunogenic and constitute candidates for a subunit vaccine.19"22 The genes of HSV-1
and HSV-2 display substantial homology, and so the
glycoproteins of the two serotypes share numerous
cross-reactive epitopes.2324
HSV-2 infection of the murine cornea also can
induce stromal keratitis, which is clinically indistinguishable from that induced by HSV-1. However, it is
not known whether antibody immunotherapy can prevent corneal disease induced by this serotype. To investigate this question experiments were conducted
with a number of monoclonal antibodies to HSV glycoproteins, and also with hyperimmune serum. In
contrast to our results with HSV-1, antibody failed to
protect when HSV-2 was used as the challenge virus.
Studies investigating possible reasons for the inability
of antibody to promote resistance against HSV-2 corneal infection are described.
MATERIALS AND METHODS
Animals
Three- to four-week-old female Balb/c mice were purchased from Charles River Breeding Laboratories
(Wilmington) MA. The animals were treated and
housed in accordance with the ARVO Resolution on
the Use of Animals in Research.
Viruses
HSV-1 (17) and HSV-2(186) were originally obtained
from Bernard Roizman (University of Chicago, Chicago, IL). HSV-1 (RE) was originally obtained from
Ysolina Centifanto-Fitzgerald (Tulane University, New
Orleans, LA), and HSV-2(333) was obtained from
Fred Rapp (Hershey Medical Center, Hershey, PA).
HSV-2(Av) was obtained from Richard J. Whitley (University of Alabama, Birmingham, AL). After being
plaque-purified, virus stocks were grown in Vero cells
and titrated by a plaque assay as previously described.25
Antibodies
The anti-HSV glycoprotein D (gD) monoclonal antibody (mAb) 8D2 was prepared and purified as previously described.26 Anti-gD mAbs III-l 14-4 and III174-1.2 raised via immunization with HSV-2(G)27 were
donated by Patricia G. Spear (Northwestern University Medical School, Chicago, IL). III-114-4 is of the
IgG2b isotype whereas 8D2 and III-l 74-1.2 are IgG2a
as determined by radial immunodiffusion (RID). AntigB (F3AB) and anti-gE (H7E), both IgG2a antibodies,
were prepared as described by Rector et al.26 AntiHSV-2 hyperimmune mouse sera was raised via immunization with HSV-2(333). The dosages of antibody
given were 200 /ig of III-l74-1.2 or III-l 14-4, a 1:2
dilution of a-HSV-2 serum, and 2000 /*g of F3AB and
500 ^ig of H7E. Antibodies were passively transferred
by inoculating a 0.2 ml volume intraperitoneally.
Corneal Infection
Mice were anesthetized with 0.2 ml of sodium pentobarbital at a concentration of 5 mg/ml, and one eye
was scarified by three twists of a 2 mm corneal trephine. A 2 fi\ volume containing the desired challenge
dose of HSV was dropped onto the scarified cornea
and rubbed in with the eyelids. Eye scores were read
weekly using a dissecting biomicroscope with a fiber
optic light source. Corneal opacity was graded on a
scale of 0 to +5 as described by Metcalf et al.14 The
person scoring the eyes was ignorant of the treatment
the mice received.
Isolation of Mouse Corneal Epithelial Cells
Corneas from HSV infected and uninfected mice were
excised and incubated in 5 mmol/1 ethylenediaminetetraacetic acid (EDTA) for 20 min in 5% carbon dioxide
at 37°C. Using forceps the epithelial sheets were lifted
off the stromal-endothelial portions of the cornea and
incubated with 0.1% trypsin for 5 min in 5% carbon
dioxide at 37°C. The epithelial sheets were then
passed repeatedly through a 25-g needle to dissociate
the cells. The cells were washed twice with buffer
(phosphate buffered saline with 2% fetal bovine serum
and 0.1% sodium azide) before being used in further
tests. Fourteen to twenty epithelial sheets were pooled
for each sample. Comparable numbers of epithelial
cells were recovered from HSV-1- and HSV-2-infected
corneas, and the percentage of cells recovered that
were viable was > 80% for both serotypes.
Fluorescence Activated Cell Sorter
The reactivity of mAb with cultured mouse corneal
fibroblasts or isolated corneal epithelial cells was analyzed by fluorescence activated cell sorter (FACS).
Corneal fibroblasts were cultured as previously described.28 The cells (2X106) were seeded into 75 cm2
flasks and allowed to adhere overnight. The monolayers were infected with HSV-1 (RE) or HSV-2(333) at
a multiplicity of infection of 3. After 45 min adsorption, fresh medium was added and the cells were incubated for various intervals. At the desired times postin-
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933174/ on 05/02/2017
2462
Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8
fection the cells were harvested with trypsin/EDTA
(0.25% trypsin, 1 mmol/1 EDTA-4Na, Gibco BRL,
Grand Island, NY).
The mouse corneal fibroblasts or epithelial cells
were prepared for FACS analysis according to the protocol used by Jennings et al.29 The cells were washed
twice with FACS buffer (phosphate buffered saline
with 2% fetal bovine serum and 0.1% sodium azide)
and underwent centrifugation. The resulting pellet
was suspended at 1X106 viable cells/ml and 100 pi was
separated into aliquot portions into wells of a 96-well
plate. The cells were centrifugated and the supernatant was removed. The pellet was suspended with 50 ixl
of primary antibody and incubated for 30 min at 4°C.
The cells were washed with 150 n\ of FACS buffer
containing an additional 27% fetal bovine serum.
After centrifugation the supernatant was removed and
the resulting pellet was suspended in 50 /xl of secondary antibody and incubated for 30 min at 4°C. The
secondary antibody was fluorescein isothiocyanateconjugated goat anti-mouse affinity purified F(ab)'2
fragments (Cappell Research Products, Durham, NC).
The cells were washed twice with FACS buffer and
analyzed using an FACS 440 (Becton Dickinson, San
Jose, CA). The mean fluorescence intensity (MFI) was
calculated by using the formula:
2 (Number of cells in a channel)(channel number)
Total number of cells analyzed
HSV Growth in Mouse Corneal Cells
Three to five corneas from mice infected with the desired concentration of HSV were excised at varying
time points after infection, and the epithelial layers
were separated from the stromal-endothelial layers as
described earlier. After separation the samples from
each cornea were frozen at —70°C. The samples were
subsequently thawed, sonicated for 10-20 sec using a
Sonic 300 Dismembrator (Arteck Systems Incorporated, Farmingdale, NY), and titrated for infectious
virus by plaque assay on Vero cell monolayers.
The infectious virus titer of infected isolated corneal epithelial cells was also determined from aliquot
portions of the same samples used by FACS analysis.
The corneal epithelial cells were isolated as described
above. After being washed twice with FACS buffer,
5X104 cells per sample were dispersed into test tubes
and frozen at —70°C. The samples were then thawed,
sonicated, and assayed for infectious progeny on Vero
cell monolayers.
Statistical Analysis
The Mann-Whitney U test was used to determine significant differences in the corneal opacity scores between control and test groups. Student's t test was
used to compare the MFI values between HSV-l(RE)and HSV-2(333)-infected cells.
RESULTS
The reported AMFI values represent the MFI of
the test samples minus the MFI of the IgG2a controls.
8D2 mAb Treatment Fails to Prevent HSV-2Induced Stromal Keratitis
The mAb 8D2 recognizes a type common discontinuous epitope on gD and neutralizes HSV-2 strains
TABLE l.
Effect of Anti-gD Therapy on HSV-Induced Stromal
Keratitis and Encephalitis*
Challenge
Virus
HSV-1 (17)
HSV-1 (17)
HSV-2 (186)
HSV-2 (186)
HSV-2 (AV)
HSV-2 (333)
Viral
Challenge Dose
(PFU/eye)
2 X 105
2 X 105
4 X 104
4 X 1O4
2 X 105
2 X 1O5
4 X 104
4 X 104
4 X 104
4 X 104
4 X 104
4 X 104
8D2 MAb
Treatment
No.
Survivors/
Total
6/6
3/6
6/6
5/6
6/6
6/6
6/6
6/6
4/6
0/6
7/7
6/7
Survivor
Mean Corneal
Opacity Score
(P Value)
3
° (<0.025)
0
(0.005)
4.4
4.8
(NS)
5.0
3.0
4.8 (NS)
5.0
4.8
(NS)
5.0
* Mice were infected on the trephined cornea with the indicated strain and challenge dose of HSV.
Twenty-four hours after corneal infection, the animals were given MAb 8D2 (55 ^g) or control IgG2a
(55 ng) intraperitoneally. Survivors are defined as those living at the time corneal opacity scores were
determined. Corneas were examined on days 4 or 28 post-infection.
NS = not significant.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933174/ on 05/02/2017
Antibody and HSV-2 Corneal Infection
2463
to the same high titer (103) as HSV-1 strains. Previous
studies have shown that this purified antibody is strikingly effective at preventing or significantly reducing
the incidence of HSV-1-induced stromal keratitis.12
We tested whether 8D2 would also protect mice challenged on the cornea with HSV-2. Table 1 shows that a
dose of 55 jug/mouse, which provided excellent protection against HSV-1 strain 17 corneal infection,
failed to protect animals infected with any of three
different HSV-2 strains. Moreover, administering the
antibody 30 min before HSV-2 corneal infection, or
increasing the mAb dose to 110 ^,g/mouse, that is,
tenfold higher than that shown to be effective against
HSV-112, failed to provide protection against HSV-2
challenge (data not shown).
It was possible that the failure to protect against
stromal keratitis was peculiar to mAb 8D2. Therefore,
an additional five mAbs that were specific for type
common continuous epitopes or other discontinuous
epitopes on gD were tested. None proved to be efficacious although allfivehad previously been shown to
protect against HSV-1 strain RE corneal challenge13
(data not shown).
8.0
Q
4.0
».O
*
O
S
§
o
D
A
•
#
o
t.O r
L.
Q.
«1.0
CO
O
QC
UJ
(0
D
QC
••••—ooooo
DAYS POST INFECTION
FIGURE 2. Titers of HSV-1 and HSV-2 in corneal epithelial
and stromal—endothelial layers. The right, corneas were infected with 4 X 104 plaque-forming units of HSV-1 strain 17
(•) or HSV-2 strain 333 (O) on day 0. At the indicated times
after infection three tofivecorneas were excised. The epithelial layers were separated from the stromal-endothelial portions and each was titrated for infectious virus. Panel A (top)
shows infectious virus titers of individual epithelial layers
while Panel B (bottom) depicts the virus titers observed in the
individual stromal-endothelial layers.
w
d
O
o
CO
o
<
Q.
O
<
UJ
Protection Studies with Other Monoclonal and
Polyclonal Antibodies
O
O
<
UJ
IgG2a
a-gD'
a-gD
PBS a-HSV-2 IgG2a
a-gB
a-gE
ANTIBODY TREATMENT
l. Effect of mAb and hyperimmune serum therapy
on HSV-induced stromal keratitis and encephalitis. Mice
were infected on the trephined cornea with 4 X 104 plaqueforming units of HSV-1 strain RE (Panels A, B, C) or HSV-2
strain 333 (Panels D, E, F). The desired antibody was given
intraperitoneally 24 hr later. Controls received an equivalent amount of IgG2a or phosphate buffered saline. There
were initially 6-9 mice in each group. The opacity scores
were determined on day 21 or 28 postinfection. Parentheses
indicate the number of survivors per total number challenged. *P < 0.005, fcv-gD mAb Ill-l 14-4, Ja-gD mAb III174-1.2.
FIGURE
In the forgoing experiments all of the anti-gD mAbs
tested had been produced in response to HSV-1 immunization. There are data that indicate that antibodies
raised against HSV-2 may be more therapeutically active against HSV-2 than antibodies raised against
HSV-1.30 Therefore, two anti-gD mAbs raised through
immunization with HSV-2 were tested by passive
transfer intraperitoneally 24 hr after infection. We
found that antibodies III-l 14-4 and III-174-1.2, provided excellent protection against HSV-1 strain RE
challenge (Figure 1A) but neither could protect
against HSV-2 strain 333 (Figure ID) ocular infection.
Similarly, a polyclonal hyperimmune mouse serum
raised against HSV-2 and tested at a 1:2 dilution was
therapeutically effective against HSV-1 (Figure IB)
but not against HSV-2 (Figure IE).
Additional protection studies were performed using type common mAbs to two other major glycopro-
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933174/ on 05/02/2017
Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8
2464
tein antigens, gB and gE. Anti-gB and anti-gE were
highly effective in protecting against HSV-1 challenge
(Figure 1C) but failed to prevent corneal disease induced by HSV-2 infection (Figure IF). Conversely, Figure 1 (Panels D and F) also shows that 79% (27/34) of
the niAb-treated hosts survived HSV-2 infection
whereas only 12% (2/17) of the virus-infected controls
did so. Thus,rnAbsto glycoproteins B, D, and E could
provide significant (P < 0.05) protection against type
2-induced encephalitis.
10 3
io4 10°
10'
13h
HSV-1
10°
101
10 2
10 3
10*
10°
10 1
10 2
10 3
10 4
10°
101
10 2
10 3
10*
10°
101
10 2
103
10*
Log Fluorescence Intensity
FIGURE 3. Cell surface expression of gD on HSV-infected
cultured mouse corneal fibroblasts as analyzed by FACS.
Monolayers of mouse corneal fibroblasts were infected with
HSV-1 strain RE (Panels A and B) or HSV-2 strain 333 (Panels C and D) at a multiplicity of infection of 3 and harvested
at the indicated times postinfection. The cells were stained
with anti-gD mAb III32D174-1.2 (—) or an IgG2a control
[g (—) followed by fluorescein isothiocyanate-conjugated
goat anti-mouse affinity purified F(ab')2 fragments. The
AMFI values are (A) 37, (B) 67, (C) 23, and (D) 45.
io 3 io4
Day 2
HSV-2
HSV-1 and HSV-2 Growth in Murine Corneas
Studies were initiated to determine why antibody
failed to protect against HSV-2 induced stromal keratitis. One possibility was that HSV-2 replicated to
higher titer in ocular tissue than HSV-1 and thereby
overwhelmed the therapeutic effect of antibody. To
investigate this hypothesis virus titers in epithelial (Figure 2A) and stromal-endothelial (Figure 2B) layers of
individual corneas were determined at various times
post infection. Although HSV-1 replicated to slightly
higher titers than HSV-2 the differences were not statistically significant at any time point for either corneal
layer. By day 7 postinfection only residual amounts of
HSV-1 and HSV-2 were detected. Thus, HSV-2 was
not found to replicate more extensively or persist
longer in ocular tissue than HSV-1.
io2
10*
Log Fluorescence Intensity
FIGURE 4. Cell surface expression of gD on epithelial cells
isolated from HSV-infected mouse corneas as analyzed by
FACS. Mice were infected on the scarified cornea with 1 X
106 plaque-forming units of HSV-1 strain RE (Panels A and
B) or HSV-2 strain 333 (Panels C and D). The corneal epithelial cells were isolated at the indicated times postinfection
and stained with anti-gD mAb III-l 74-1.2 (—) or an IgG2a
control Ig (—) followed by fluorescein isothiocyanate-conjugated goat anti-mouse affinity purified F(ab')2 fragments.
The AMFI values are (A) 23, (B) 21, (C) 5, and (D) 3.
Glycoprotein Antigen Expression on HSVinfected Cell Membranes
Because antibody was not administered until 24 hr
after corneal infection the target in vivo was expected
to be cell-associated virus antigen rather than cell-free
virus. Perhaps less glycoprotein antigen was being expressed on the surface of HSV-2 infected cells compared to that found on HSV-1-infected cells. To address this possibility FACS analysis was performed using III-l 74-1.2, an anti-gD mAb generated by HSV-2
immunization. We first compared the levels of gD
found on mouse corneal fibroblasts that had been
grown in culture and then infected with HSV-1 or
HSV-2. Glycoprotein antigen was readily detected on
the surface of HSV-1 (Figure 3A and 3B) and HSV-2
(Figure 3C and 3D) infected cells at 8 and 13 hr postinfection.
We next investigated whether the levels of gD on
corneal epithelial cells were also comparable after corneal infection in vivo. Single-cell preparations derived
from epithelial sheets of excised corneas were examined by FACS 1 and 2 days postinfection. As shown in
Figure 4 (Panels A and B) gD was clearly demonstrable
on the surface of HSV-1- infected cells at both time
points. However, the results with HSV-2 were quite
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933174/ on 05/02/2017
2465
Antibody and HSV-2 Corneal Infection
different. In Figure 4 (Panels C and D), little or no gD
antigen could be seen. These results were consistently
obtained in six independent experiments.
Analogous studies were conducted to assess gB
expression in vivo. Results representative of 5 independent experiments are shown in Figure 5. Although gB
could be detected on HSV-2 infected cells (Figure 5C
and D), the amount was consistently low and significantly less (P < 0.005) than that expressed on HSV-1infected cells (Figure 5A and B).
HSV-1 and HSV-2 Titers in Isolated Corneal
Epithelial Cells
It was possible that the reduced HSV-2 glycoprotein
expression observed in vivo reflected reduced HSV-2
replication in the epithelial cells analyzed. Therefore,
aliquot portions of the same preparations used for
FACS analysis were titrated for infectious virus content. The results of two such experiments are shown in
Figure 6. It is evident that HSV-2 could readily replicate in mouse corneal epithelial cells although the
titers tended to be approximately threefold lower than
those for HSV-1. Whether the slightly lower virus yield
was sufficient to account for the reduced gB and gD
antigen expression on the surface of HSV-2-infected
cells is not clear.
10'
10
10'
10 J
10'
10 u
101
10*
10'
Log Fluorescence Intensity
FIGURE 5. Cell surface expression of gB on epithelial cells
isolated from HSV-infected mouse corneas as analyzed by
FACS. Mice were infected on the scarified cornea with 1 X
106 plaque-forming units of HSV-1 strain RE (Panels A and
B) or HSV-2 strain 333 (Panels C and D). The corneal epithelial cells were isolated at the indicated times postinfection
and stained with anti-gD mAb F3AB (—) or an IgG2a control Ig (—) followed by fluorescein isothiocyanate-conjugated goat anti-mouse affinity purified F(ab')2 fragments.
The AMFI values are (A) 21, (B) 17, (C) 8, and (D) 7.
Exp. #2
Q| 4.0
1
2
3
1
2
DAYS POST INFECTION
FIGURE 6. HSV-1 and HSV-2 titers in epithelial cells derived
from infected mouse corneas. Mice were infected on the
trephined cornea with 1 X 106 plaque-forming units of
HSV-1 strain RE or HSV-2 strain 333. At the indicated times
the corneas were excised, and the epithelial cells were isolated as described in the Materials and Methods section. The
lysates from 5 X 104 cell aliquot portions were assayed for
infectious progeny.
DISCUSSION
The major observation in this report is that passively
transferred antibody failed to prevent stromal keratitis
after HSV-2 corneal infection. This was the case regardless of whether the immunoglobulin transferred
was anti-HSV-2 hyperimmune polyclonal serum or
mAbs to gD, gB, or gE. Whether antibodies to other
viral glycoproteins would be protective remains to be
determined. However, it is striking that the same
monoclonal and polyclonal antibody preparations that
failed to protect against HSV-2 were highly effective in
preventing corneal disease induced by HSV-1.
It is important to emphasize that not all tissues
infected by HSV-2 were unresponsive to antibody therapy. We found that immunoglobulin treatment consistently protected the majority of the recipients against
viral encephalitis that developed as a consequence of
HSV-2 spread into the central nervous system. This
latter observation agrees with previously published reports. Thus, antibody transferred before31 or after32
HSV-2 footpad challenge was found to protect mice
against fatal central nervous system disease. In addition, mAbs to gD33 as well as polyclonal HSV-2 specific
antibodies34 have been shown to prevent skin and retinal lesions resulting from HSV-2 subcutaneous infections.
McDermott et al35 reported that mice given antigC or anti-gD mAbs were protected against HSV-2
footpad but not intravaginal challenge. These authors
suggested that antibody ineffectiveness against the latter was attributable to a lack of transudation into vaginal secretions. However, Eis-Hiibinger et al,36 found
that antibody treatment readily protected mice inocu-
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933174/ on 05/02/2017
2466
Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8
lated intravaginally with HSV-1. Thus, vaginal tissue
may be a body site where antibody can protect against
HSV-1 but not HSV-2 lethal infection. Collectively,
our experiments in the cornea and those involving intravaginal infection suggest that the capacity of immunoglobulin to prevent disease is dependent on both
the tissue infected and the infecting HSV serotype.
There are a number of potential explanations for
the failure of antibody to prevent HSV-2 corneal disease. One possibility is that HSV-2 replicated more
rapidly and to a higher titer in ocular tissue than HSV1, thereby overwhelming the protective effect of antibody. We investigated this hypothesis and found that
the two serotypes replicated to similar levels in corneal
cells. In an earlier study in rabbits Stevens and Oh37
reported that HSV-2 titers in the eye were 100-fold
lower than those of type 1. Thus, there is no evidence
to support the theory that HSV-2 replicates more extensively in ocular tissue than HSV-1.
Stevens and Oh37 also noted that corneal inflammation in rabbits infected with HSV-2 strains was
more severe than in animals infected with HSV-1
strains. In our work blepharitis appeared to be more
intense in the HSV-2-infected mice (unpublished observation). Recent studies in our laboratory have
shown that interleukin la can be detected in HSV-1
infected murine corneal buttons, and that effective
mAb immunotherapy is accompanied by decreased
production of this pro-inflammatory cytokine38. One
can speculate that the inflammatory cytokine cascade
induced following HSV-2 infection differs either quantitatively or qualitatively from that induced by HSV-1
infection, and thus was insensitive to putative antibody-mediated down-regulation. A comparative analysis of cytokine expression after HSV-1 and HSV-2 infection merits investigation.
Through FACS analysis we discovered a definite
disparity in gD antigen expression between HSV-1 and
HSV-2 infected cells after in vivo infection. Although
gD was readily detected on epithelial cells isolated
from HSV-1 infected corneas it was barely demonstrable on epithelial cells isolated from HSV-2 infected
corneas. Moreover, surface gB was also more abundant on HSV-1 than HSV-2 infected cells. In contrast,
the amount of surface gD was similar on cultured
mouse corneal fibroblasts infected in vitro with HSV-1
or HSV-2. These results are compatible with the observations of Jennings et al.29 They reported that the
kinetics and amount of glycoprotein antigen expressed on HSV-infected cells varied according to the
host cell type. Reduced glycoprotein antigen expression on the surface of one target cell type but not
another could conceivably explain why antibody treatment prevents HSV-2-induced central nervous system
disease but not stromal keratitis. Likewise, a difference
in glycoprotein expression could account for why antibody protects against HSV-1- but not HSV-2 induced
corneal disease. Although we currently favor this explanation, our conclusions are strictly tentative because a causal relationship has not been established.
To explain the dichotomy in corneal protection it will
be important to develop an understanding of why antibody is effective against HSV-1 challenge. The failure
to detect accelerated clearance of virus from ocular
tissue of antibody-treated hosts12 suggests that conventional antibody protective mechanisms such as virus
neutralization, or antibody-mediated lysis of infected
cells, are not operative.
A multiplicity of factors in addition to the extent
of virus replication appear to influence how much
viral glycoprotein antigen will be expressed by infected cells. Gething et al.39 have shown that the
correct folding of the influenza virus hemagglutinin is
required for it to be transported from the endoplasmic reticulum to the cell surface. Similar conclusions
were reached by de Silva et al40 in studies on vesicular
stomatitis virus G protein. Therefore, it is possible that
HSV-2 glycoproteins fail to fold in a conformation
that is conducive for efficient transport to the corneal
epithelial cell surface. Johnson and Smiley41 observed
that the kinetics of intracellular transport of gD in
HSV-2 infected L cells was much slower than in transformed mouse L cells. They suggested that gD interaction with viral structural components retarded its
movement to the cell surface. In our study, HSV-1
replicated at least as well as HSV-2, which suggests
comparable levels of viral structural components
within cells infected with either of the two serotypes.
Thus, it seems unlikely that inhibition in gD transport
would be greater in type 2- than type 1-infected cells.
The kinetics of glycoprotein appearance on infected cell membranes as well as the amount made
seems to be critical. Thus, Wachsman et al.42 have
found that gD-1 expressed under the control of an
early but not a late promoter in vaccinia recombinant
viruses could protect guinea pigs from cutaneous
HSV-2 disease. Regulation of glycoprotein genes during viral infection can occur at the levels of transcription43 and posttranscription.44 Several immediate
early proteins including ICPO,45 ICP4,46-47 and ICP27
44
have been shown to play a role in regulating viral
glycoprotein gene expression. Possibly the differential
interaction of HSV-1 and HSV-2 immediate early gene
products with components of the host cell could account for the dissimilarity in virus-infected cell surface
glycoprotein expression in selected cell types. Alternatively, glycoproteins may be shed in greater abundance
by HSV-2-infected cells into the corneal interstitium
making it harder to eliminate stromal disease by passive immunization.
In summary, we have shown that passive transfer
of antibody fails to prevent HSV-2-induced stromal
keratitis, although it provides excellent protection
against HSV-1 ocular infection. However, antibody
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933174/ on 05/02/2017
Antibody and HSV-2 Corneal Infection
therapy is able to prevent encephalitis induced by
HSV-2 spread into the central nervous system. The
dissimilar protection seen in different tissues suggests
that the protective mechanism is influenced by inherent properties of the infected host cell as well as the
genetic makeup of the infecting HSV serotype.
Key Words
2467
14.
15.
HSV-1, HSV-2, monoclonal antibodies, stromal keratitis,
HSV glycoproteins
16.
Acknowledgments
The authors thank Ray Hester, PhD, for flow cytometry assistance and Nikki Nixon for manuscript preparation.
References
1. Vaughan D. Viral keratitis. In: Vaughan D, Asbury T,
Tabbara KF, eds. General Ophthalmology. Norwalk, CT:
Appleton & Lange; 1989:104-125.
2. Whitley RJ. Herpes simplex virus. In: Fields BN,
Knipe DM, et al, eds. Virology. New York: Raven Press;
1990:1843-1887.
3. Oh JO, Kimura SJ, Ostler HB. Acute ocular infection
by type 2 herpes simplex virus in adults. Report of two
cases. Arch Ophthalmol. 1975; 93:1127-1129.
4. Oh JO, Kimura SJ, Ostler HB, Dawson CR, Smolin G.
Oculogenital transmission of type 2 herpes simplex
virus in adults. Surv Ophthalmol. 1976; 21:106-109.
5. Hanna L, Ostler HB, Keshishyan H. Observed relationship between herpetic lesions and antigenic type
of Herpesvirus hominis. Surv Ophthalmol. 1976;
21:110-114.
6. Neumann-Haefelin D, Sundmacher R, Wochnik G,
Bablok B. Herpes simplex virus types 1 and 2 in ocular
disease. Arch Ophthalmol. 1978; 96:64-69.
7. Sumers KD, SugarJ, Levine R. Endogenous dissemination of genital Herpesvirus hominis type 2 to the eye. Br
J Ophthalmol. 1980;64:770-772.
8. Rosenwasser GOD, Greene WH. Simultaneous
herpes simplex types 1 and 2 keratitis in acquired
immunodeficiency syndrome. AmJ Ophthalmol. 1992;
113:102-103.
9. Pepose JS. Herpes simplex keratitis: Role of viral infection versus immune response. Surv Ophthalmol.
1991;35:345-352.
10. Doymaz MZ, Rouse BT. Herpetic stromal keratitis: An
immunopathologic disease mediated by CD4+ T lymphocytes. Invest Ophthalmol Vis Sci. 1992; 33:21652173.
11. Hendricks RL, Tao MSP, Glorioso JC. Alterations in
the antigenic structure of two major HSV-1 glycoproteins, gC and gB, influence immune regulation and
susceptibility to murine herpes keratitis. j Immunol.
1989; 142:263-269.
12. Lausch RN, OakesJE, Metcalf JF, ScimecaJM, Smith
LA, Robertson SM. Quantitation of purified monoclonal antibody needed to prevent HSV-1 induced stromal keratitis in mice. Curr Eye Res. 1989;8:499-506.
13. Lausch RN, Staats H, OakesJE, Cohen GH, Eisenberg RJ. Prevention of herpes keratitis by monoclonal
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
antibodies specific for discontinuous and continuous
epitopes on glycoprotein D. Invest Ophthalmol Vis Sci.
1991;32:2735-2740.
Metcalf JF, Koga J, Chatterjee S, Whitley RJ. Passive
immunization with monoclonal antibodies against
herpes simplex virus glycoproteins protects mice
against herpetic ocular disease. Cuir Eye Res.
1987;6:173-177.
Raizman MB, Foster CS. Passive transfer of anti-HSV1 IgG protects against stromal keratitis in mice. CurtEye Res. 1988; 7:823-829.
Shimeld C, Hill TJ, Blyth WA, Easty DL. Passive immunization protects the mouse eye from damage after
herpes simplex virus infection by limiting spread of
virus in the nervous system. J Gen Virol. 1990; 71:681687.
Hutchinson L, Browne H, Wargent V, et al. A novel
herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and surface expression of gH. J Virol.
1992;66:224O-225O.
Muggeridge MI, Roberts SR, Isola VJ, Cohen GH, Eisenberg RJ. Herpes simplex virus. In: van Regenmortel MHV, Neurath AR, eds. Immunochemistry of Viruses.
Vol 2. Amsterdam: Elsevier; 1990:459-481.
Berman PW, Gregory T, Crase D, Lasky LA. Protection from genital herpes simplex virus type 2 infection
by vaccination with cloned type 1 glycoprotein D.
Science. 1984;227:1490-1492.
Cremer KJ, Mackett M, Wohenberg C, Notkins AL,
Moss B. Vaccinia virus recombinant expressing herpes
simplex virus type 1 glycoprotein D prevents latent
herpes in mice. Science. 1985; 228:737-740.
Bernstein DI, Frenkel LM, Bryson YJ, Myers MG. Antibody response to herpes simplex virus glycoproteins
gB and gD. f Med Virol. 1990; 30:45-49.
Stanberry LR, Bernstein DI, Burke RL, Pachl C,
Myers MG. Vaccination with recombinant herpes simplex virus glycoproteins: protection against initial and
recurrent genital herpes. J Infect Dis. 1987; 155:914920.
Watson RJ. DNA sequence of the herpes simplex virus
type 2 glycoprotein D gene. Gene. 1983; 26:307-312.
Bzik DJ, Debroy C, Fox BA, Pederson NE, Person S.
The nucleotide sequence of the gB glycoprotein gene
of HSV-2 and comparison with the correspondinggene of HSV22D1. Virology. 1986; 155:322-333.
Lausch RN, Kleinschrodt WR, Monteiro C. Resolution of HSV corneal infection in the absence of delayed-type hypersensitivity. Invest Ophthalmol Vis Sci.
1985;26:1509-1515.
Rector JT, Lausch RN, OakesJE. Use of Monoclonal
antibodies for analysis of antibody-dependent immunity to ocular herpes simplex virus type 1 infection.
Infect Ivimun. 1982;38:168-174.
Para MF, Parish ML, Noble AG, Spear PG. Potent
neutralizing activity associated with anti-glycoprotein
D specificity among monoclonal antibodies selected
for binding to herpes simplex virions. / Virol.
1985;55:483-488.
Lausch RN, Su Y-H, Ritchie M, Oakes JE. Evidence
endogenous interferon production contributed to the
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933174/ on 05/02/2017
2468
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8
lack of ocular virulence of an HSV intertypic recombinant. Curr Eye Res. 1991;10(suppl):39-45.
Jennings SR, Lippe PA, Pauza KJ, Spear PG, Pereira
L, Tevethia SS. Kinetics of expression of herpes simplex virus type 1-specific glycoprotein species on the
surfaces of infected murine, simian, and human cells:
flow cytometric analysis./ Virol. 1987;61:104-112.
Brown ZA, Benedetti J, Ashley R, et al. Neonatal
herpes simplex virus infection in relation to asymptomatic maternal infection at the time of labor. N EnglJ
Med. 1991; 324:1247-1252.
Balachandran N, Bacchetti S, Rawls WE. Protection
against lethal challenge of BALB/c mice by passive
transfer of monoclonal antibodies to five glycoproteins of herpes simplex virus type 2. Infect Ivimun.
1982;37:1132-1137.
Oakes JE, Rosemond-Hornbeak H. Antibody-mediated recovery from subcutaneous herpes simplex
type 2 infection. Infect Ivimun. 1978; 21:489-495.
Inoue Y, Oh JO, Minasi P. Protective effect of antiglycoprotein D antibody on herpetic chorioretinitis in
newborn rabbits. Cwr Eye Res. 1991; lO(suppl): 159—
165.
Friedlaender RP, Oh JO, Minasi P, Ohashi Y. Protective effect of passive immunization on herpetic retinitis of newborn rabbits. Curr Eye Res. 1987;6:161—166.
McDermott MR, Brais LJ, Evelegh MJ. Mucosal and
systemic antiviral antibodies in mice inoculated intravaginally with herpes simplex virus type 2 . / Gen Virol.
1990;7l:1497-1504.
Eis-Hubinger AM, Mohr K, Schneweis KE. Different
mechanisms of protection by monoclonal and polyclonal antibodies during the course of herpes simplex
virus infection. Intervirol. 1991 ;32:351-360.
Stevens TR, Oh JO. Comparison of types 1 and 2 Herpesvirus hominis infection of rabbit eyes. II. Histopathologic and virologic studies. Arch Ophthalmol.
1973;90:477-480.
Staats HF, Oakes JE, Lausch RN. Quantitation of interleukin 1 alpha in mouse corneal buttons after HSV-
39.
40.
41.
42.
43.
44.
45.
46.
47.
1 ocular infection with and without protective antibody therapy. Invest Ophthalmol Vis Sci. 1992;33:786.
Gething M-J, McCammon K, Sambrook J. Expression
of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport.
Cell. 1986;46:939-950.
de Silva AM, Balch WE, Helenius A. Quality control in
the endoplasmic reticulum: folding and misfolding of
vesicular stomatitis virus G protein in cells and in vitro.
fCellBiol. 1990; 111:857-866.
Johnson DC, Smiley JR. Intracellular transport of
herpes simplex virus gD occurs more rapidly in uninfected cells than in infected cells./ Virol. 1985;54:
682-689.
Wachsman M, Aurelian L, Smith CC, Perkus ME, Paoletti E. Regulation of expression of herpes simplex
virus (HSV) glycoprotein D in vaccinia recombinants
affects their ability to protect from cutaneous HSV-2
disease. / Infect Dis. 1989; 159:625-634.
Smith IL, Sandri-Goldin RM. Evidence that transcriptional control is the major mechanism of regulation
for the glycoprotein D gene in herpes simplex virus
type 112Dinfected cells./ Virol. 1988;62:1474-1477.
Smith IL, Hardwicke MA, Sandri-Goldin RM. Evidence that the herpes simplex virus immediate early
protein ICP27 acts post-transcriptionally during infection to regulate gene expression. Virology. 1992;
186:74-86.
Chen J, Zhu X, Silverstein S. Mutational analysis of
the sequence encoding ICPO from herpes simplex
virus type 1. Virology. 1991; 180:207-220.
Arsenakis M, Campadelli-Fiume G, Roizman B. Regulation of glycoprotein D synthesis: does «4, the major
regulatory protein of herpes simplex virus 1, regulate
late genes both positively and negatively? / Virol.
1988;62:148-158.
Tedder DG, Pizer LI. Role for DNA-protein interaction in activation of the herpes simplex virus glycoprotein D gene./ Virol. 1988;62:4661-4672.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933174/ on 05/02/2017