Download Therapeutic vaccination with vhsN herpes simplex virus reduces the

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of General Virology (2002), 83, 2361–2365. Printed in Great Britain
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SHORT COMMUNICATION
Therapeutic vaccination with vhsN herpes simplex virus
reduces the severity of recurrent herpetic stromal keratitis in
mice
Tammie L. Keadle, Keith A. Laycock, Jessica L. Morris, David A. Leib, Lynda A. Morrison,†
Jay S. Pepose and Patrick M. Stuart
Washington University School of Medicine, Department of Ophthalmology and Visual Sciences, Box 8096, 660 South Euclid, St Louis,
MO 63110, USA
Virion host shutoff (vhs)-deficient herpes simplex
virus (HSV) was tested as a therapeutic vaccine in a
mouse model of UV light-induced recurrent herpetic
stromal keratitis. Four weeks after primary corneal
infection, mice were vaccinated intraperitoneally
with vhsN vaccine or control. Four weeks after
vaccination, the eyes of latently infected mice were
UV-B irradiated to induce recurrent virus shedding
and disease. Post-irradiation corneal opacity in
latently infected, vhsN-vaccinated mice was significantly reduced compared to control-vaccinated
mice (P l 0n007 to 0n035). The incidence and
duration of recurrent virus shedding were the same
in both groups. Antibody titres were increased (P l
0n05) and delayed type hypersensitive responses
were unaffected by vhsN vaccination. Combined
with studies using different vaccination timing and
vhsN genotypes, these data suggest that deletion of
vhs is a useful strategy in the development of a
therapeutic HSV vaccine, and that temporal and
genetic factors influence vaccination outcome.
When herpes simplex virus (HSV) infects the eye, the
potential for harm is great. The resulting inflammation of the
corneal stroma, known as herpetic stromal keratitis (HSK), may
lead to permanent scarring and vision changes. Indeed, HSV is
the most common cause of infectious corneal blindness in the
United States (Pepose et al., 1996). After primary ocular
infection, the virus becomes latent within sensory neurons
innervating the cornea. From this location, HSV periodically
Author for correspondence : Patrick Stuart.
Fax j1 314 362 6985. e-mail stuart!vision.wustl.edu
†Permanent address : Saint Louis University School of Medicine,
Department of Molecular Microbiology and Immunology, 1402 South
Grand Blvd, St Louis, MO 63104, USA
0001-8410 # 2002 SGM
reactivates and again replicates in the eye, causing additional
HSK lesions. Signs of recurrent ocular HSV infection in humans
include focal stromal opacities and neovascularization of the
cornea (Pepose et al., 1996). The accumulation of such lesions
over time leads to corneal clouding and blindness, and thus
recurrent HSK presents the greatest danger to normal sight.
HSK is thought to be immune-mediated, with key participation by CD4+ T cells (Russell et al., 1984 ; Niemialtowski
& Rouse, 1992). Post-infection vaccination may change
immune responses from pathological to protective, and is
therefore a reasonable approach to disease control. Desirable
characteristics of a therapeutic vaccine for HSK include the
ability to suppress virus reactivation and\or virus-induced
corneal lesions, but reports of effective therapeutic vaccines for
recurrent ocular herpes infections are scant (Walker et al.,
1998 ; Keadle et al., 1997 ; Pivetti-Pezzi et al., 1999 ; Nesburn et
al., 1998). In two such studies using different animal models,
post-infection vaccination decreased spontaneous and inducible ocular virus shedding (Walker et al., 1998 ; Nesburn et al.,
1998). In other work, vaccine treatment decreased the incidence
and duration of recurrent herpetic keratitis or reduced the
length of virus shedding (Keadle et al., 1997 ; Pivetti-Pezzi et al.,
1999 ; Nesburn et al., 1998). The severity of resulting corneal
stromal disease in all cases was unaffected.
Recently, HSV mutants deficient in virion host shutoff (vhs)
activity have been examined for efficacy as vaccines for ocular
herpes (Walker et al., 1998 ; Geiss et al., 2000 ; Walker & Leib,
1998). Vhs− mutants are attenuated for both replication and
pathogenesis following primary infection of the mouse eye
(Strelow & Leib, 1995). Overexpression of immunogenic viral
proteins, coupled with normal host MHC class I expression in
infected vhs− cells, could account for the reduced virulence of
vhs− mutants in vivo (Strelow & Leib, 1995 ; Kwong & Frenkel,
1987 ; Tigges et al., 1996), as well as the immunogenicity of
vhs− vaccine strains. Accordingly, prophylactic vaccination of
mice with vhs− virus decreased challenge virus replication in
the cornea, acute and latent infection of the trigeminal ganglia,
blepharitis and keratitis (Geiss et al., 2000 ; Walker & Leib,
1998). In previously infected mice, vaccination with vhs− virus
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T. L. Keadle and others
Fig. 1. Corneal stromal opacity following UV-B-stimulated virus recurrence
in therapeutically vaccinated, latently infected mice. Four weeks after
primary ocular infection with HSV-1, mice were vaccinated
intraperitoneally with control vaccine or vhs− HSV. Four weeks after
vaccination, recurrent corneal HSV infection was induced by UV-B
irradiation, and opacity was scored as described. Eyes of uninfected mice
were also irradiated (UV-B control) to serve as a control for transient
corneal clouding associated with UV. Values are mean corneal
opacitypSEM for all UV-B-irradiated latent or control mice. Opacity in
latently infected mice exceeded that of UV-B controls on all days (ttest ; P 0n001 to P l 0n035, n l 10 for control mice). *Opacity of
latently infected, control-vaccinated mice was greater than vhs− vaccinated
mice on days 14–35 (t-test ; P l 0n007 to 0n035, n l 11 and 20
respectively). Control vaccine consisted of uninfected cell lysate. Results
are representative of three experiments.
protected against inducible HSV reactivation and replication in
the cornea (Walker et al., 1998).
A model of recurrent HSK has been developed in which the
eyes of mice latently infected with HSV are exposed to UV-B
irradiation (Shimeld et al., 1989 ; Laycock et al., 1991). Irradiated
eyes subsequently shed virus into the tears and develop
corneal lesions that clinically and histologically mimic the
human disease (Pepose et al., 1996 ; Miller et al., 1996). Because
one of the principal clinical signs of recurrent HSK in humans
is opacification of the cornea, we investigated whether
therapeutic vaccination of latently infected mice with a vhs−
HSV vaccine virus would protect them from developing
corneal opacity after UV-induced virus reactivation.
All investigations conformed to the Association for
Research in Vision and Ophthalmology (ARVO) Statement for
the Use of Animals in Ophthalmic and Vision Research. Sixweek-old National Institutes of Health (NIH) inbred mice
(Harlan Olac, Oxford, UK) were infected on the scarified right
cornea with 1i10' p.f.u. HSV-1 McKrae, with concurrent
administration of anti-HSV serum to protect corneas from
damage during primary infection (Laycock et al., 1991). A
latency rate of 80–100 % is expected (Keadle et al., 1997). Four
weeks after primary infection, latently infected mice were
vaccinated intraperitoneally with a cell lysate stock containing
2i10' p.f.u. of vhs− strain UL41NHB (vhs deletion
mutant ; two experiments) or BGS41 (lacZ insertion into the
CDGC
vhs locus ; one experiment) in 200 µl volume. Latently infected
control mice received the same amount of uninfected cell lysate
(control vaccine). Cell lysate stocks of vhs− mutants UL41NHB
and BGS41 were prepared as described and viruses were
propagated on Vero cells (Geiss et al., 2000 ; Strelow & Leib,
1995). Four weeks after vaccination, clear eyes of latently
infected, vhs− and control-vaccinated mice, and age-matched
uninfected mice were exposed to 250 mJ\cm# UV-B irradiation
(90 s) in order to stimulate recurrent virus shedding and
disease. Before (day 0), and on days 1 to 7 post-UV-B
irradiation, eyes were swabbed, and the swab material was
cultured on Vero cells for 4 days to detect recurrent virus
shedding (reactivation). After irradiation, a masked observer
scored stromal opacity on a scale from 0 (clear) to 4 (total
opacity with no posterior view) using a binocular-dissecting
microscope (Laycock et al., 1991).
First, we observed that corneal opacity of both groups of
latently infected mice was greater than that of mock-infected,
UV-B-irradiated animals (UV-B control) on days 7 through 35
post-irradiation (Fig. 1 ; P 0n001 to P l 0n035, t-test),
indicating that irradiation itself caused significantly less severe
opacity than recurrent virus infection. Second, corneal opacity
in latently infected, control-vaccinated mice significantly
exceeded that of vhs− vaccinees on days 14 through 35 postirradiation (P l 0n007 to 0n035, t-test). The incidence of
stromal opacity in each group did not vary on any observation
day (85 %, vhs− vaccine ; 100 %, control vaccine). Third, the
proportion of eyes shedding virus (average 56 %, vhs−
vaccine ; 59 %, control vaccine) and the number of virusshedding days per mouse (average 2n8 days) were equivalent
between vaccine groups over three separate experiments.
Finally, all eyes shedding virus developed HSK. Latently
infected, non-shedding eyes also developed opacity, but UVinduced opacity could not be differentiated from virusstimulated disease because of the potential for virus recurrence
in deep, swab-inaccessible layers of the corneal stroma (Miller
et al., 1996). The results indicated that systemic therapeutic
vaccination with vhs− HSV significantly decreased the severity
of recurrent HSK without affecting virus shedding.
To assess virus-specific immune responses after vaccination,
a sampling of vhs− and control-vaccinated mice was bled 2
weeks after vaccination for determination of HSV-specific IgG
levels using ELISAs as previously described (Geiss et al., 2000).
Briefly, serial 2-fold dilutions of serum from each mouse were
incubated for 2 h in duplicate wells of a 96-well plate coated
with purified HSV-1 glycoproteins. Biotinylated goat antimouse IgG and streptavidin–HRP were subsequently added in
a colorimetric assay to determine specific IgG amounts based
on comparison to standards with known HSV-specific IgG
content. As shown in Fig. 2(A), serum antibody titres in vhs−vaccinated mice were greater than those in control-vaccinated
mice (P l 0n05, t-test of geometric means), indicating that preexisting virus-specific immunity was augmented by vhs−
vaccination.
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Vaccine therapy for herpetic stromal keratitis
Fig. 2. Anti-HSV antibody titres and DTH responses in therapeutically
vaccinated, latently infected mice. (A) Antibody titres were determined
2 weeks after therapeutic vaccination of latently infected mice with vhs−
virus or control vaccine. Values for antibody data are geometric mean
titrepSEM. *Antibody titres in vhs−-vaccinated mice significantly exceeded
that of control-vaccinated mice (t-test of geometric means ; P l 0n05, n l
5 and 8 respectively). (B) DTH activity in therapeutically vaccinated,
latently infected mice, and naive (non-vaccinated, uninfected) mice was
measured at the time of UV-B irradiation. Values are mean HSV-specific
foot pad swellingpSEM. *DTH responses in control (n l 5) and vhs−vaccinated (n l 6) mice were significantly increased compared to naive
(n l 4) mice (t-test ; P l 0n02 and 0n003 respectively) but were not
different from each other.
In addition to serum antibody determinations, delayed type
hypersensitivity (DTH) responses were measured at the time
of UV-B irradiation (Fig. 2B). Accordingly, just before corneal
UV-B irradiation, mice from each group (excluded from further
experiments) were injected in the right rear foot pad with
5i10' p.f.u. UV-inactivated HSV-1 McKrae strain in 30 µl of
medium (Keadle et al., 1997). The left rear footpad was injected
with the same amount of virus-free tissue culture medium.
Footpad swelling was measured with a micrometer (Mitutoyo
Manufacturing, Tokyo) immediately before and 24 h after
injection. HSV-specific footpad swelling was determined by
the formula (right footpad swelling at 24 hkright footpad
swelling before injection)k(left footpad swelling at 24 h after
injectionkleft footpad swelling before injection). While both
groups of latently infected mice had significant DTH responses
compared to naive controls (Fig. 2B ; P l 0n02 and 0n003, ttest), vhs− vaccination did not significantly change DTH
responses compared with control vaccination.
The data in this study show that immunotherapy of
recurrent HSK can be accomplished using a single dose of a
live-attenuated HSV-1 vaccine. Since corneal pathology resulting from HSK is thought to be an immune-mediated process,
we evaluated representative protective (antibody) and destructive (DTH) immune responses. Higher antibody levels
were detected in mice vaccinated with vhs− HSV (Fig. 2A). A
change in the type or amount of HSV-specific antibodies could
account for some disease amelioration in this group, possibly
by limiting virus spread or decreasing the amount of antigen in
corneas available for local induction or activation of pathological responses (Shimeld et al., 1990).
T cell-mediated DTH activity has been associated with
deleterious effects on corneal clarity during primary (Lausch et
al., 1985) and recurrent (T. L. Keadle, unpublished data) ocular
HSV infection. In this work, however, DTH levels were
unchanged by vaccination (Fig. 2B), suggesting that systemic
suppression of DTH activity was not responsible for vaccineinduced protection against recurrent HSK. Even so, evidence
indicates that cell-mediated responses, including cytotoxic T
cell activity and interferon-γ (IFN-γ) production, play a central
role in controlling recurrent HSV infections (Deshpande et al.,
2000 ; Stanberry et al., 2000), and CD8+ T cells can block HSV1 reactivation from latency in sensory neurons (Liu et al., 2000).
Thus, the effect of vhs− vaccination on other forms of cellmediated immunity requires investigation.
As reported by us (Keadle et al., 2001) and others (Stumpf
et al., 2001), a mix of T helper 1 (IFN-γ) and T helper 2
(interleukins 4, 10) cytokines is present in mouse corneas
throughout the course of recurrent HSK (Keadle et al., 2001). A
vaccine with protective effects might alter the balance between
such cytokines, favouring, for example, production of IL-10
with its attendant palliative effects on corneal opacity (Tumpey
et al., 1994 ; Daheshia et al., 1997). We are currently testing this
hypothesis with regard to therapeutic vhs− vaccination.
In recent work from our laboratory, a lacZ+ vhs− vaccine
strain protected mice from recurrent HSK when administered 4
months after primary ocular infection (Keadle et al., 2002).
Combined with the present work, these data confirm the
efficacy of therapeutic vhs− vaccination in controlling the
severity of recurrent herpetic corneal lesions. To our knowledge, these are the first reports of therapeutic vaccines with a
positive influence on the severity of recurrent HSK. Although
different vaccination schedules (4 months vs 4 weeks postinfection) yielded similar amelioration of virus-induced corneal
opacity, recurrent virus shedding was reduced only in mice
receiving vhs− vaccination 4 months after primary infection
(Keadle et al., 2002). Earlier studies also indicated that
vaccination at 4 months with vhs− virus decreased the
reactivation rate (Walker et al., 1998). These findings suggest
that the therapeutic effects of vhs− vaccines on recurrent virus
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T. L. Keadle and others
shedding are time dependent, whereas the effects on recurrent
HSK are not. To determine the clinical usefulness of vhs−
vaccines, further studies of the temporal effects on vaccine
efficacy are clearly needed.
In addition to temporal effects, it is possible that differences
in the vhs− virus itself play a role in vaccine efficacy. Thus,
evidence suggests that lacZ+ vhs− viruses are not as pathogenic
in vivo as gene-deleted vhs− viruses (Smith et al., 2002), and
may elicit β-galactosidase-specific immune responses that alter
HSV responses in a bystander fashion (Brubaker et al., 1996).
This might explain differential effects on corneal opacity of
lacZ− vhs− (Walker et al., 1998) and lacZ+ vhs− (Keadle et al.,
2002) vaccination at 4 months post-infection as reported in
other work. Hence, both the type and timing of vhs−
vaccination may affect clinical outcome and should be
important considerations in vaccine design.
Despite their attenuation, vhs− mutants retain the potential
to replicate and cause disease in vaccinated hosts (Strelow &
Leib, 1995). Such an adverse outcome may be avoided through
the creation of new, immunogenic and replication-defective
modified live virus vaccines (Geiss et al., 2000). Deletion of vhs
function augments protective immunity in vaccinees, and may
be an appropriate mutation to be used in combination with
other virus defects that negate replication, for therapeutic
vaccination.
This work was supported by PHS award CA75052 and Fight for Sight
Foundation grant FGA97011 to L. A. M. and National Eye Institute grant
numbers EY11885 to P. M. S. and EY10707 to D. A. L.
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