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Journal of Virology, April 2000, p. 3832-3841, Vol. 74, No. 8
Molecular Neurosurgery Laboratory,
Departments of Neurosurgery1 and
Microbiology & Immunology,2
Georgetown University Medical Center, Washington, D.C. 20007
Received 9 September 1999/Accepted 6 January 2000
Herpes simplex virus type 1 (HSV-1) mutants that are attenuated for
neurovirulence are being used for the treatment of cancer. We have
examined the safety of G207, a multimutated replication-competent HSV-1
vector, in mice. BALB/c mice inoculated intracerebrally or
intracerebroventricularly with 107 PFU of G207 survived for
over 20 weeks with no apparent symptoms of disease. In contrast, over
80% of animals inoculated intracerebrally with 1.5 × 103 PFU of HSV-1 wild-type strain KOS and 50% of animals
inoculated intracerebroventricularly with 104 PFU of
wild-type strain F died within 10 days. Similarly, after intrahepatic
inoculation of G207 (3 × 107 PFU) all animals
survived for over 10 weeks, whereas no animals survived for even 1 week
after inoculation with 106 PFU of KOS. After
intracerebroventricular inoculation, LacZ expression was initially
observed in the cells lining the ventricles and subarachnoid space;
expression decreased until almost absent within 5 days postinfection,
with no apparent loss of ependymal cells. G207 DNA could be detected by
PCR in the brains of mice 8 weeks after intracerebral inoculation;
however, no infectious virus could be detected after 2 days. As a model
for latent HSV in the brain, we used survivors of an intracerebral
inoculation of HSV-1 KOS at the 50% lethal dose. Inoculation of a high
dose of G207 at the same stereotactic coordinates did not result in
reactivation of detectable infectious virus or symptoms of disease. We
conclude that G207 is safe at or above doses that were efficacious in
mouse tumor studies.
Herpes simplex virus type 1 (HSV-1)
is a neurotropic DNA virus which infects a wide range of cell types in
different animals. Natural infections either follow a lytic,
replicative cycle or establish latency. Latency is characterized by the
long-term persistence of viral DNA in latently infected neurons, the
lack of infectious or replicating virus, the lack of viral gene
expression except for the latency-associated transcripts (LATs), and in
some situations episodic reactivation of infectious virus
(74). In humans, the natural host, HSV-1 causes a number of
diseases, including gingivostomatitis and pharyngitis after primary
oral-facial infection, recurrent herpes labialis, genital herpes,
keratitis after eye infection, disseminated visceral infections in
immunocompromised patients, hepatitis, and encephalitis after spread to
the central nervous system (CNS) (13).
HSV encephalitis is the most common CNS viral infection, occurring in
two to three persons per million (13). Brain pathology is
usually localized to the temporal lobe and limbic system, with asymmetric necrotizing encephalitis, inflammation, and hemorrhage (19, 33). The majority of cases of encephalitis occur in
patients with recurrent HSV who are seropositive at onset of disease.
Even with acyclovir therapy, mortality is about 20%, and about 20% of
survivors have long-term morbidity, including cognitive abnormalities (23, 51).
A number of animal models of HSV encephalitis involving spread from
peripheral infection (3, 80) or direct inoculation in the
CNS (11, 39, 50) have been developed. Direct inoculation into the olfactory bulb in rabbits and rats leads to encephalitis, with
virus isolatable from the temporal cortex (69) and surviving animals exhibiting learning deficits (3, 53). In addition, encephalitis has been induced in rabbits by reactivation of latent HSV
established by intranasal inoculation of a neurovirulent strain (75). About a quarter of the reactivated rabbits developed
behavioral changes (lethargy and unresponsiveness), and a similar
proportion had gross necrotic lesions in the cortex within 1 to 2 weeks
postreactivation (75).
Considerable progress has been made in understanding latency in the
sensory ganglia of the peripheral nervous system (PNS). Unfortunately,
latency in the CNS is much less characterized. The role of CNS latency
in pathogenesis has become an important issue with the demonstration of
HSV DNA in human brain tissue from patients without encephalitis. HSV
DNA was detected in brains from 14 of 40 HSV-seropositive patients,
mostly in the olfactory bulb, pons, and medulla, (2), and
from 6 of 22 patients with nonneurologic disease and 5 of 22 patients
with Alzheimer's disease (68). PCR primers in these studies
could not differentiate HSV-1 from HSV-2. In a study of 109 human
corpses at forensic postmortem, 16% were positive for HSV-1 DNA in the
olfactory bulbs, whereas 72% of the trigeminal ganglia were positive
(43). In animal models, HSV DNA can often be detected in the
CNS of animals surviving HSV-induced encephalitis (17, 67).
Attenuated replication-competent mutants of HSV-1 have been shown to be
efficacious in the treatment of a variety of experimental tumor models
(1, 5, 6, 30, 49, 56, 63, 65, 88). We have created a
second-generation, multimutated HSV-1, G207, that is currently in
clinical trial for the treatment of recurrent gliomas. G207 has
deletions of both copies of the Viruses.
HSV-1 G207 (57), parental wild-type
strain F (obtained from B. Roizman, University of Chicago, Chicago,
Ill.), wild-type strain 17syn+ (obtained from J. Subak-Sharpe, Institute of Virology, Glasgow, United Kingdom), and
wild-type strain KOS1.1 (obtained from D. Knipe, Harvard Medical
School, Boston, Mass.) were propagated on mycoplasma-free Vero (African
green monkey kidney) cells (American Type Culture Collection). Virus
stocks were prepared by infecting subconfluent monolayers of Vero cells
cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented
with 1% heat-inactivated fetal calf serum (IFCS) (HyClone, Logan,
Utah) at a multiplicity of infection of 0.01. Infected cells were
incubated at 34 or 37°C and harvested when total cytopathic effect
was observed. After a freeze-thaw/sonication regimen, cell debris was
removed by low-speed centrifugation (2,000 × g at 4°C for
10 min) and virus (F and G207) was concentrated by high-speed
centrifugation (45,000 × g at 4°C for 60 min). The
viral pellet was resuspended in virus buffer (VB; 150 mM NaCl, 20 mM
Tris [pH 7.5]) and titered by plaque assay on Vero cells. Viral
stocks were stored at Intracerebral inoculation of HSV-1.
Three-week-old BALB/c
female mice, obtained from the National Cancer Institute (Frederick,
Md.), were anesthetized with a 250-µl intraperitoneal (i.p.)
injection of sodium pentobarbital solution (84% bacteriostatic saline,
10% sodium pentobarbital [50 mg/ml; Abbott Laboratories, Chicago,
Ill.], 6% ethyl alcohol) and placed in a KOPF stereotactic frame. A
small burr hole was made 1 mm rostral of the bregma and 2 mm right of
midline, and a beveled 29-gauge Hamilton needle was used to inoculate 2 to 5 µl of virus at a depth of 2 mm from the skull surface over a period of 10 min. After another 3 min, the needle was slowly withdrawn (26). Animals were monitored for 6 weeks to 1 year
postinfection (p.i.). All procedures involving animals were approved by
the Georgetown University Animal Care and Use Committee.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Attenuated, Replication-Competent Herpes Simplex
Virus Type 1 Mutant G207: Safety Evaluation in Mice
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
34.5 gene (RL1) and an
Escherichia coli lacZ insertion that inactivates the ICP6
gene (UL39) (57). Mutations in 34.5 causes a large decrease in neurovirulence, so that after intracerebral (i.c.) inoculation in mice, the 50% lethal dose (LD50) is
>107 PFU within the standard 3-week observation period
(10, 47). In prior studies, we were unable to detect an
LD50 for G207 after i.c. inoculation in mice or Aotus
nancymae primates (25, 57). However, as part of the
preclinical evaluation of these vectors for use in tumor therapy, we
expanded on these studies and examined other potential pathological
consequences of G207 inoculation. Other safety concerns include
inoculation into the ventricles of the brain, leakage of virus into the
bloodstream, or infection of susceptible peripheral organs. Another
issue to consider is whether i.c. inoculation of G207 could lead to
reactivation of, or complementation or recombination with, a patient's
latent virus to cause disease. It has been shown in mice that mixed
infections with two nonlethal nonneuroinvasive HSV-1 strains can result
in a lethal infection and spread to the brain (29). To
address this, we used a mouse model involving i.c. inoculation of
wild-type HSV-1 strain KOS and subsequent challenge of the survivors
with a high dose of G207 at the same i.c. location. The present studies found that G207 caused no detectable disease in mice at titers many
logs above the lethal dose of the parental virus, HSV-1 strain F.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
80°C and thawed rapidly for use. Mock
extracts were prepared identically, except that VB was used in place of
virus during the infection step.
Intracerebroventricular (i.c.v.) inoculation of HSV-1. Ten- to twelve-week-old BALB/c female mice were anesthetized by i.p. injection of 250 µl of sodium pentobarbital solution and placed in a KOPF stereotactic frame. A 2-mm burr hole was made 0.8 mm anterior and 1 mm off midline. A Hamilton microliter syringe containing G207 virus (107 PFU) or mock extract in 10 µl was introduced to a 2-mm depth, and virus was injected slowly over 10 min. To confirm that an i.c.v. injection occurred, two mice in each group were injected with Evans blue (Sigma) dye (2% in saline) and sacrificed at the end of surgery.
For 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside
(X-Gal) histochemistry, mice were anesthetized with 500 µl of sodium pentobarbital solution and perfused transcardially with cold 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains were removed, postfixed for 2 h in 4% paraformaldehyde, cut in half coronally, and stained with X-Gal substrate solution (56).
Brain fragments were washed in PBS, cryoprotected overnight in 30%
sucrose, sectioned on a cryostat at 10 or 40-µm thickness, mounted on
gelatin-coated slides, and counterstained with carmalum or hematoxylin
and eosin.
Intravenous (i.v.) inoculation of G207. Three-week-old BALB/c female mice were anaesthetized by i.p. injection of 250 µl of sodium pentobarbital solution. Virus (100 µl) was administered by percutaneous puncture of the lateral tail vein.
Intrahepatic (i.h.) inoculation of G207. Four- to five-week-old BALB/c mice were inoculated with 30 µl of virus or PBS into the right hepatic lobe as described elsewhere (58).
Titration of virus from the brain. Mice were sacrificed at different times following G207 challenge (1, 2, 4, 7, 14, and 30 days; two mice per time point) by lethal injection (500 µl of sodium pentobarbital solution). Brains were removed and quick-frozen. Brain homogenates were made in DMEM-10% FCS at a 10% wt/vol) ratio, using a Thomas pestle tissue grinder (Thomas Scientific, Swedesboro, N.J.). After centrifugation at 700 × g at 4°C for 10 min, the supernatants were serially diluted in PBS-1% IFCS, and viral titer on Vero cells was determined.
PCR amplification.
Brain tissue (~25 mg) was isolated from
the right frontal cortex (inoculation site), and DNA was purified using
a QIAamp tissue kit (QIAGEN, Chatsworth, Calif.) as described by the
manufacturer. Amplification reactions were performed in 50-µl volumes
containing DNA, 2.0 mM MgCl2, 200 µM each
deoxyribonucleoside triphosphate (Perkin-Elmer), 1 µM each primer
(Gibco BRL, Gaithersburg, Md.), and 2.5 U of Taq polymerase
(Perkin-Elmer) with PCR buffer II (Perkin-Elmer). Primer sequences and
their expected product sizes are shown in Table
1. The conditions for PCR amplification
were 94°C (denaturation) for 90 s, annealing at 55°C for
60 s, and extension at 72°C for 120 s (35 cycles) on a
PTC-100 thermal controller (MJ Research Inc., Watertown, Mass.).
Amplification products were separated by electrophoresis on a 2.5%
NuSieve agarose (FMC) gel in Tris-acetate-EDTA (TAE) buffer and
visualized by ethidium bromide fluorescence. To determine the
sensitivity of the PCR assay, known amounts of virus was mixed with
uninfected mouse brain, and DNA was purified and amplified as described
above.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Safety of G207 in BALB/c mice.
The safety of G207 inoculation
in young BALB/c mice was determined after four different routes of
inoculation: i.c., i.c.v., i.v., and i.h. The first three are likely
sites of viral spread after inoculation of brain tumors in situ.
Intracerebral inoculation of 107 PFU of G207 or mock
extract resulted in no symptomatic evidence of disease during the
1-year observation period (Table 2). In an earlier set of studies from this laboratory (57), we
found that 50% of mice died after an i.c. inoculation of
103 PFU of strain F, similar to the reported
LD50 (10). The dose for i.c. injection of G207
was the maximum that could be administered in the small volumes
required. No HSV-related mortality or morbidity was noted. Ten-week-old
BALB/c mice were injected i.v. with G207 (107 PFU) or mock
extract and followed for 1 year with no evidence of disease (Table 2).
HSV infection of the liver can cause a lethal necrotic hepatitis in
both mice and humans (22, 32, 81). Direct injection of G207
into the liver at a very high dose (3 × 107 PFU)
caused no disease, whereas injection of 106 PFU of KOS was
uniformly lethal.
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Intracerebroventricular injection of G207.
Brain tumors are
very often located near ventricles, and therefore it was important to
determine the toxicity of G207 inoculation into the ventricles, where
the virus could spread throughout the CNS. The accuracy of the i.c.v.
injections was confirmed by injecting the first and last animal in a
group with Evans blue dye, sacrificing the animal, and visualizing
bilateral ventricular staining (see Fig. 2D). No symptoms of disease
were noted in any of the animals inoculated with G207 (107
PFU) or mock extract, whereas 50% of the strain F (104
PFU)-inoculated animals died within 10 days (Table 2). The spread of
G207 in the ventricular system was examined by sacrificing G207-injected mice on days 1, 3, and 5 p.i. and histochemically staining for LacZ expression (Fig. 1 and
2). In G207, the lacZ transgene is driven by the HSV-1 ICP6 promoter. The most intense X-Gal
staining was seen 1 day p.i., in the bilateral ventricles and
subarachnoid spaces (Fig. 1, d1; Fig. 2A). By day 3, the contralateral, left ventricle (Fig. 1, d3, left) is much less intensely stained, as
are the subarachnoid spaces (Fig. 1, d3, right). There is almost no
staining visible by day 5 (Fig. 1, d5; Fig. 2C) and no apparent loss of
ependymal cells (Fig. 2F). Sectioning of these brains further
demonstrated that LacZ expression was limited to cells in contact with
cerebrospinal fluid (CSF) (Fig. 2). No X-Gal-stained cells of obvious
neuronal morphology were detected. We have found that X-Gal staining is
more sensitive than immunohistochemical staining for -galactosidase
(60).
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Persistent HSV-1 KOS in the brain.
Groups of eight 3-week-old
female BALB/c mice were inoculated i.c. with doses of HSV-1 KOS ranging
from 2.5 × 102 to 6 × 103 PFU per
mouse (Table 3). Most of the mice
exhibited signs of CNS disease or encephalitis, such as ruffled fur,
hunched posture, hind limb paralysis, and lethargy. Clinical symptoms
in mice usually resolved within 48 h of onset, or death occurred.
At the highest inoculation doses (3 × 103 and 6 × 103 PFU), HSV-1 infection was fatal in all of the mice.
The LD50 of i.c.-inoculated KOS was approximately 1.5 × 103 PFU (Table 3). It should be noted that in a separate
experiment, 100% of animals died after i.c. inoculation of 1.5 × 103 PFU KOS (Table 2). At death, infectious virus could be
recovered from brain tissue homogenates. The titer of infectious KOS
virus recovered after i.c. inoculation was about 3 logs greater than the inoculated dose (3 × 106 PFU after 3 × 103-PFU inoculation and 5 × 106 PFU after
6 × 103-PFU inoculation on day 3 or 4 p.i.). The
persistence of viral DNA in brain tissue was detected by PCR
amplification, using primers from ICP6 (ribonucleotide reductase
[RR]), thymidine kinase (TK), and glycoprotein E (gE), which amplify
sequences of 221, 273, and 320 bp, respectively (Table 1). In all
brains from KOS-inoculated survivors analyzed at 56 and 112 days p.i.,
viral DNA was detectable (Fig. 3A; Table
4).
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Attempts to reactivate KOS by superinfection with G207. We attempted to reactivate i.c. KOS virus or generate neurovirulent recombinants by superinfection with a high dose of G207 (107 PFU). At 2 months after KOS i.c. inoculation, a time when HSV-1 DNA was detectable in the brain by PCR, surviving mice were inoculated with G207 at the same stereotactic coordinates as the prior KOS injection. Some of these animals were followed for over 1 year with no sign of disease, suggesting that neurovirulent recombinants were not generated. The persistence of viral genomes after G207 superinfection was determined by PCR (Table 4). The LacZ primer (Table 1) was used to differentially identify G207 sequences, with the RR, TK, and gE primers common to both G207 and KOS DNA (Fig. 3B). We have been unable to generate useful PCR primers that would uniquely identify KOS DNA and not G207. Amplification of the cellular fatty acid binding protein gene (Fx) served as internal control and to normalize the amount of tissue DNA in all experiments. G207 and likely KOS DNA sequences were detected from 8 to 24 weeks after the second injection with G207 (Table 4). To determine the sensitivity of the PCR assay, we mixed known amounts of KOS or G207 virus with normal mouse brain tissue and extracted the DNA as was done with the inoculated brain samples. We could detect approximately 10 PFU equivalents of KOS and G207 with the ICP6 and LacZ primers, respectively (Fig. 3C).
It was possible that KOS reactivated but was unable to cause detectable disease. Therefore, we assayed brain homogenates for infectious virus on days 1, 2, 4, 7, 14, and 30 after G207 challenge by plaque assay (Fig. 4). G207 plaques could be distinguished from KOS plaques because of the expression of-galactosidase which is histochemically stained with X-Gal.
Infectious G207 could be detected 1 day after injection (4 × 105 PFU/brain), with titers declining by day 2 and no
plaques detectable at day 4 or later (Fig. 4). We were unable to detect
any KOS plaques (white after X-Gal staining) at any of these time
points, in contrast to the results obtained after the initial i.c.
inoculation of KOS.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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G207 is a second-generation, attenuated replication-competent HSV vector that was constructed for the original purpose of treating brain tumors. We have shown that G207 is efficacious in the treatment of brain tumors, inhibiting the growth and/or prolonging survival in animal models of human glioma (57), meningioma (88), and breast cancer metastatic to the brain (79). G207 is also effective in treating a variety of solid peripheral human tumors, such as head and neck squamous cell carcinoma, breast adenocarcinoma, prostate adenocarcinoma, and colorectal cancer, after intraneoplastic injection (9, 37, 79, 82). Hepatic portal vein injection of G207 was shown to reduce the number of liver metastases in a syngeneic hepatoma model (37). Recently, we found that i.v. injection of G207 in nude mice was able to induce tumor regression in distant subcutaneous LNCaP and DU-145 human prostate adenocarcinomas (82). In humans, HSV infection of the CNS often causes a rapidly debilitating or fatal encephalitis (13). Blood-borne virus concentrates in the liver and HSV infections of the liver can cause a fulminant hepatitis (22). It is therefore important to demonstrate that inoculation with G207 in the brain and possible leakage to CSF or blood will not pose a significant risk to any patient undergoing therapy.
G207 is derived from HSV-1 strain F, one of the most attenuated
laboratory strains (16), and contains engineered mutations in two viral genes affecting neurovirulence, 34.5 (10, 77, 78) and ICP6 (8, 28, 87). These features were
incorporated into G207 in order to maximally decrease neurovirulence
and yet maintain the ability of the virus to replicate in tumor cells. We found no morbidity associated with injection of 107 PFU
of G207 into the cerebral cortex, ventricles, liver, or tail vein of
BALB/c mice. There was no or minimal G207 replication after i.c.
inoculation, so that the titer of recoverable infectious virus
decreased from the input of 107 PFU to 2 × 102 PFU on day 2 p.i. to undetectable on day 4. This
recovered G207 could be remaining input virus or newly synthesized.
Intracerebral injection of 106 PFU R3613, the strain F
34.5
parent of G207, was found to cause no deaths, and only
102 PFU/g of brain tissue could be recovered
(10).
In contrast, other attenuated, replication-competent HSV vectors being
used for cancer therapy are derived from the strain 17+
background, and 34.5 mutations in this background are more
neurovirulent than those in the strain F background (35, 47, 52,
54). Intracerebral injection of 106 PFU of 1716, 34.5 in strain 17+, in both AO rats and BALB/c mice,
caused a severe inflammatory response in the brain that was accompanied
by excessive weight loss and clinical illness (54).
Interestingly, C3H/He mice remained healthy after similar injections
(54), even though C3H/He mice are similar to BALB/c mice in
sensitivity to wild-type HSV infection (44). After i.c.
inoculation of R3616 or 1716, virus is widely distributed via
retrograde transport in neurons, and there is some expression of viral
antigens and possible viral replication within the first week, which
then ceases (48, 55). In studies using G207 as a helper
virus for defective HSV vectors, we found that inoculation in the rat
substantia nigra resulted in LacZ expression only in cells surrounding
the site of inoculation and not at retrograde sites that were
identified by expression of the alkaline phosphatase reporter gene on
the defective vector (59).
A feature common to both R3616 and 1716 is a predilection to replicate
in and destroy ependymal cells in the ventricles. When R3616 was
injected into the striatum of mice, virus spread to the ventricles,
presumably through leakage into the CSF, so that within 7 days the
ependymal cells in the lateral ventricles had disappeared
(48). Direct inoculation of 1716 into the ventricles of
BALB/c mice also led to a loss of ependymal cells and hydrocephalus but
no mortality (34). When 1716 was injected into the
ventricles of nude mice, it caused high mortality even at low doses
(103 PFU) (41). Intrathecal injection of
hrR3 (KOS backbone with same ICP6
mutation as
G207) into rats caused significant neurologic morbidity and some
mortality (38). In contrast, animals receiving i.c.v. inoculations of G207 exhibited no symptoms of disease. The virus spread
throughout the ventricular system, with large numbers of cells
bilaterally expressing LacZ within the first day. LacZ expression then
rapidly decreased, so that by day 5 postinoculation very few positive
cells were detected, with no apparent loss of ependymal cells. LacZ
expression is a marker for active viral transcription. In G207, LacZ is
driven by the ICP6 promoter, a leaky early (E) or ß promoter, that is
activated by VP16 (
TIF) and ICP0 but not ICP4 (14, 24,
76).
Mutations in a number of nonessential viral genes, including the
34.5 (61, 66, 71, 86), ICP6 (27, 28), TK
(12), uracil DNA glycosylase (UL2) (62), and ICP0
(42) genes, affect the ability of HSV to establish latency
in the PNS and to be reactivated. In many cases the degree of
reactivation and/or establishment of latency is dependent on the in
vivo model, animal species, route of infection (e.g., corneal
infection/latent trigeminal ganglia, footpad infection/latent dorsal
root ganglia, or vaginal infection/latent sacral ganglia)
(71), and means of inducing reactivation (e.g., explant
cocultivation or in vivo stimuli) (20, 62). Based on the
ability of HSV to establish latency, replication-defective mutants of
HSV have been used for gene delivery to the CNS, usually by injection
into the hippocampal region (4, 21, 64). Unfortunately, in
most cases the delivered gene is only transiently expressed. However,
viral DNA transcripts and LATs can be detected for long periods of time
in the region of injection in the brain (4, 21, 64),
suggesting that a latent infection has been established. Intracerebral
inoculation of strain 1716 was similarly found to lead to the
establishment of a latent infection in large numbers of neurons, as
detected by LAT expression (35). We found that G207 DNA was
present in the brains of mice 6 months after i.c. inoculation.
With the recent findings of HSV DNA in the CNS of patients dying without encephalitis (2, 43, 68), it became important to determine whether i.c. injection of G207 might lead to reactivation of or recombination with latent virus, leading to a neurovirulent phenotype. Mixed infections in the periphery with two nonneuroinvasive HSV-1 strains can lead to a lethal infection that spreads to the brain at doses logs less than for each of the viruses alone (29). This is due to both complementation and generation of neuroinvasive recombinants (70) and occurs with mixtures of HSV-1 KOS plus F (parental strain of G207) (73). There have been only a few reports of attempts to establish HSV-1 latency models in the CNS, most unable to demonstrate reactivation (35, 45, 72, 75, 85). For example, using a variety of explant protocols with mouse brainstem, Steiner et al. were unable to demonstrate any evidence of reactivation in 44 samples where reactivation from trigeminal ganglia was readily apparent (72). In an HSV-1 latency model established in motor neurons of the spinal cord, explanting spinal cord did not lead to viral reactivation, in contrast to 100% reactivation from latently infected dorsal root ganglia (85). In the two studies where latent HSV-1 could be reactivated from the mouse CNS, virus was identified in explants of 1 of 20 brain hemispheres and none of 20 brainstems, whereas it could be isolated from 38 of 40 trigeminal ganglia (7), and in 4 of 53 explanted spinal cords, whereas 36 of 53 spinal ganglia were positive (36). The difficulty in demonstrating HSV reactivation from the CNS as opposed to the PNS may be due to the ability to explant and maintain viable ganglia from the adult PNS, whereas it is not readily possible to maintain viable tissue from the adult brain in vitro.
To establish a mouse model with persistent HSV DNA in the brain, we
i.c. inoculated mice with approximately 1 LD50 of wild-type HSV-1 KOS 1.1, which was about 103.2 PFU. This can be
compared to previously determined LD50s of
102.2 PFU for KOS 321 in DBA/2 mice (11) and
101.7 PFU for KOS-63 in BALB/c mice (16). Large
amounts of infectious virus (>106 PFU) could be isolated
from the brains of animals that did not survive KOS i.c. injection. It
seems likely that in those animals that survived, significant numbers
of cells were infected. Superinfection with a high dose of G207
(107 PFU), at the same stereotactic coordinates as the
initial inoculation with HSV-1 KOS, did not lead to detectable
reactivation of infectious virus, nor did it result in disease through
the generation of neurovirulent recombinants. While this work was in
progress, Wang et al. (83) similarly used an i.c.
inoculation of a sublethal dose of HSV-1 KOS to establish latency in
the brains of rats. They were unable to detect ICP6 transcripts from
this virus, as a marker of reactivation, after i.c. inoculation of
hrR3 (ICP6) at the same stereotactic
coordinates (83).
These studies provide strong support for the safety of G207 when inoculated into the brain, liver, or systemic venous circulation of mice, even at high doses. In light of the serious neurologic complications that HSV infection causes, it is rather remarkable that mutations in two viral genes can have such a dramatic effect on the pathogenesis of the virus. It will be important to determine what aspects of viral replication or virus-host interactions are blocked in the CNS or peripheral organs that permit G207 replication and destruction of brain and other solid tumors but not normal tissue.
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ACKNOWLEDGMENTS |
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We thank Bernard Roizman, John Subak-Sharpe, and David Knipe for providing viruses, Anu Iyer for generating HSV stocks, Masahiro Toda and Tomoki Todo for technical assistance, Joseph T. Newsome and the Georgetown University Research Resource Facility staff for assistance with the animals, and Herbert J. Manz for assistance with pathology.
This study was supported in part by grants from the National Institutes of Health (NS32677) and NeuroVir, Inc. Samuel D. Rabkin and Robert L. Martuza are consultants to NeuroVir, Inc., which has a license from Georgetown University to commercialize G207.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Neurosurgery, Georgetown University Medical Center, 3970 Reservoir Rd., NW, Washington, DC 20007. Phone: (202) 687-8047. Fax: (202) 687-3046. E-mail: rabkins{at}odrge.odr.georgetown.edu.
Present address: Aravind Eye Hospital, Madurai-625 020, Tamilnadu, India.
Present address: Department of Neurological Surgery, University of
Wisconsin, Madison, Wis.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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