
[Cancer Research 60, 1663-1670, March 15, 2000]
© 2000 American Association for Cancer Research
Preclinical Evaluation of "Whole" Cell Vaccines for Prophylaxis and Therapy Using a Disabled Infectious Single Cycle-Herpes Simplex Virus Vector to Transduce Cytokine Genes1
S. A. Ali,
C. S. McLean,
M. E. G. Boursnell,
G. Martin,
C. L. Holmes,
S. Reeder,
C. Entwisle,
D. M. Blakeley,
J. G. Shields,
S. Todryk,
R. Vile,
R. A. Robins and
R. C. Rees2
Department of Life Sciences, Nottingham Trent University, Nottingham NG11 8NS, United Kingdom [S. A. A., S. R., R. C. R.]; Cantab Pharmaceuticals Research Limited, Cambridge CB4 4GN, United Kingdom [C. S. M., M. E. G. B., G. M., C. L. H., E. E., D. M. B., J. G. S.]; Imperial Cancer Research Fund Laboratory of Molecular Therapy, Imperial College of Science and Medicine, Hammersmith Hospital, London W12 0NN, United Kingdom [S. T., R. V.]; Division of Immunology, Queens Medical Centre, Nottingham, United Kingdom [R. A. R.]; and Molecular Medicine Program, Mayo Clinic, Rochester, Minnesota 55905 [R. V.]
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ABSTRACT
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The development of genetically modified "whole" tumor cell vaccines
for cancer therapy relies on the efficient transduction and expression
of genes by vectors. In the present study, we have used a disabled
infectious single cycle-herpes simplex virus 2 (DISC-HSV-2) vector
constructed to express cytokine or marker genes upon infection.
DISC-HSV-2 is able to infect a wide range of tumor cells and
efficiently express the ß-galactosidase reporter
gene, granulocyte-macrophage colony-stimulating factor (GM-CSF),
or IL-2 genes. Gene expression occurred rapidly after
infection of tumor cells, and the level of production of the gene
product (ß-galactosidase, GM-CSF, or IL-2) was shown to be both
time-and dose-dependent. Vaccination with irradiated DISC-mGM-CSF or
DISC-hIL-2-infected murine tumor cells resulted in greatly enhanced
immunity to tumor challenge with live parental tumor cells compared
with control vaccines. When used therapeutically to treat existing
tumors, vaccination with irradiated DISC-mGM-CSF-infected tumor cells
significantly reduced the incidence and growth rates of tumors when
administered locally adjacent to the tumor site, providing up to 90%
protection. The prophylactic and therapeutic efficacy of
DISC-mGM-CSF-infected cells was shown initially using a murine renal
cell carcinoma model (RENCA), and the results were confirmed in two
additional murine tumor models: the M3 melanoma and 302R sarcoma.
Therapy with DISC-infected RENCA "whole" cell vaccines failed to
reduce the incidence or growth of tumor in congenitally T-cell
deficient (Nu+/Nu+) mice or
mice depleted of CD4+ and/or CD8+
T-lymphocytes, confirming that both T-helper and T-cytotoxic effector
arms of the immune response are required to promote tumor rejection.
These preclinical results suggest that this "novel" DISC-HSV vector
may prove to be efficacious in developing genetically modified
whole-cell vaccines for clinical use.
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INTRODUCTION
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Cancer vaccination strategies have focused on the use of
autologous and allogeneic tumor cells genetically modified to express a
range of different immunomodulatory genes which include cytokines,
costimulatory molecules, and tumor antigens. Studies using animal
models have shown that inoculation/immunization with tumor cells
engineered to express
IL3
-2, IFN-
, IL-4, tumor necrosis factor
, GM-CSF, IL-7, or IL-6
enhances antitumor immunity (1, 2, 3, 4, 5, 6)
. In most tumor
models, this results not only in the rejection of the genetically
modified tumor cells but also the induction of systemic immunity
capable of mediating the rejection of a subsequent challenge with
parental, unmodified tumor cells.
A major drawback and limitation in using autologous cellular vaccines
to treat cancer patients is the need to establish in vitro
tumor cell lines prepared from biopsy tumor tissue for the transduction
of immuno-modulatory genes. Difficulties associated with establishing
cell lines from human tumor biopsy material and the relative
inefficiency of many of the transfection methodologies have led to
renewed efforts to establish alternative strategies for the efficient
delivery of genes into freshly prepared/isolated tumor cells. Vectors
that efficiently deliver genes into tumor cells either in
vivo or ex vivo are required, and several viral and
nonviral vector systems have been investigated for their suitability in
this regard (7)
. Viral vectors represent the most
efficient means of transducing genes into tumor cells, and many
replication-competent and replication-defective viruses have been used
to deliver genes of interest to in vitro and in
vivo targets. HSVs have been used recently for cancer therapy and
gene transduction studies. Intratumoral injection of
replication-competent attenuated mutants of HSV-1 were shown to be
effective in killing malignant gliomas (8
, 9)
, and Toda
et al. (10)
have reported recently that
immunization with a defective HSV-1 vector encoding the
IL-12 gene in combination with a HSV helper virus can induce
local and systemic antitumor immunity to the CT26 murine colon
carcinoma. Similarly, systemic therapy using a recombinant adenovirus
encoding both subunits of IL-12 inhibited the formation of 3-day
hepatic metastasis of murine tumors (11)
.
We have reported previously the development of a genetically
inactivated HSV-2 vector that is restricted to a single cycle of
replication, DISC, for use as a vaccine against genital herpes
infection (12, 13, 14)
. We have used this virus to deliver
cytokine genes to tumor cells, and there are several reasons why this
vector is potentially useful for cancer immunotherapy: (a)
DISC-HSV-2 is unable to spread from cell to cell; replication of the
virus is genetically restricted by deletion of the gH gene,
which is essential for the production of infectious progeny; and
(b) HSV-2 has a broad host cell range, making the DISC
variant an appropriate vehicle for the delivery of genes to a variety
of tumors. In addition, DISC-HSV infects nondividing as well as
dividing cells and has been shown to rapidly and efficiently infect
primary human leukemia and neuroblastoma cells (15)
, human
carcinoma cells (16)
, and cultured murine tumor cells
(17)
. In the present study, the DISC-HSV-2 vector has been
used to deliver genes encoding murine GM-CSF, human IL-2, or the
lacZ reporter genes to murine tumor cells and the efficacy
of DISC-HSV-2-infected "whole" tumor cell vaccines for
prophylactic immunization prior to tumor challenge and for the therapy
evaluated in three murine tumor models. The results show that
DISC-HSV-2 is an efficient vector for cytokine gene delivery into tumor
cells, and that the expression of mGM-CSF or hIL-2 enhances the
immunogenicity of whole-cell vaccines. In this study, the therapeutic
response was shown to be depend on the functionality of
CD4+ and CD8+ lymphocytes.
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MATERIALS AND METHODS
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Tumors.
RENCA-3 is a BALB/c renal carcinoma cell line of spontaneous origin and
was generously provided by Dr. Robert Wiltrout (National Cancer
Institute, Bethesda, MD). The immunogenicity of RENCA has been
determined as being low to moderate. RENCA-3 cells were maintained by
serial in vitro passage in RPMI 1640 supplemented with 10%
FCS, sodium pyruvate, and NEAA. The M3 cell line is a DBA/2
melanoma cell line that was obtained from American Type Culture
Collection. M3 cells were grown and maintained in Hams F-12 media
supplemented with 15% FCS. The 302R is C57Bl/6 mouse sarcoma cell
line, derived through repeated in vivo passage in mice, and
was kindly supplied by Dr. B. Fox (Portland, OR). 302R cells were grown
and maintained in DMEM media supplemented with 10% FCS.
Animals.
Female DBA/2, C57Bl/6, BALB/c, and BALB/c
Nu+/Nu+ mice were
purchased from Harlan (UK) Ltd. and were maintained in accordance with
the Home Office Codes of Practice for housing and care of animals.
Infection of Tumor cells with DISC-HSV lacZ Virus.
Tumor cells were either cultured on glass
slides precoated with fibronectin to increase the attachment of the cells or
in 24-well plates at 1 x 105 cells/well and
infected with DISC-HSV lacZ virus at a MOI of 1.2510 pfu/cell. At
various times postinfection, the cells were either fixed in acetone and
stained for the presence of HSV-2 antigen using a polyclonal anti-HSV-2
antibody (Dako) or fixed in glutaraldehyde and stained for ß-gal by
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining
(Promega).
Cytokine Assays.
Expression of cytokines after infection of tumor cells with
DISC-mGM-CSF and DISC-hIL-2 was determined by ELISA (R&D
Systems, United Kingdom). Tumor cells were cultured in 24-well
plates at a concentration of 1 x 105 cells/well overnight. The medium was removed,
and cells were infected with 1.2510 pfu/cell of each virus in a total
volume of 100200 µl for 1 h at 37°C. The medium was removed
and replaced by 1 ml of serum-free medium, and the plates were
reincubated at 37°C for various times, up to 48 h. Supernatants
were collected and stored at -20°C and assayed for mGM-CSF or hIL-2.
Apoptosis and HSP Expression.
To determine whether DISC-HSV infection of tumor cells induced cell
death by apoptosis, 5 x 105 cells were
cultured in T25 flasks and infected with DISC lacZ virus at 10 pfu/cell
for 24 h. Floating and adherent (trypsinized) cells were pooled
together for analysis. An ABO-BrdUrd kit from PharMingen (San Diego,
CA) was used according to the manufacturers instructions. Briefly,
the cells were prefixed in 1% paraformaldehyde and then stored in 70%
ethanol for 24 h. The cells were then washed and incubated with
terminal deoxynucleotidyl transferase and bromo-dUTP, followed by
FITC-anti- BrdUrd and propidium iodide. DNA breaks are indicative of
apoptosis.
Prophylactic Immunization and Therapy with DISC-HSV-infected
Cells.
Tumor cells were infected with 510 pfu/cell with either DISC-mGM-CSF,
DISC-hIL-2, or DISC-lacZ viruses for 1 h. The virus inoculum was
removed, and cells were washed two times in medium. Fresh serum-free
medium was added, and the cells were then irradiated (15,000 rads)
using a Gammacell cesium-137 source; uninfected tumor cells were
prepared in similar manner and used as control. Cells (1 x 106) cells infected with DISC-mGM-CSF or
DISC-hIL-2 viruses (before and after irradiation) were cultured in
24-well plates to assess cytokine production (as detailed above). To
assess the effect of prophylactic vaccination using RENCA cells,
animals were immunized s.c. two times on the right flank at 2-week
intervals with irradiated, noninfected RENCA cells or irradiated,
DISC-infected RENCA cells in a volume of 200 µl (see individual
experiments for details). Unless otherwise stated, animals were
challenged s.c 7 days after the second inoculation, with 5 x 104 (10 times TD50)
parental RENCA cells on the opposite flank.
To assess the efficacy of DISC-HSV infected whole-cell vaccines in
therapy, mice received injections in the right flank with 5 x 104 tumor cells and were vaccinated with
1 x 106 DISC-HSV
infected-irradiated RENCA cells at the same site or contralaterally on
day 0 or 3. Mice then received two additional immunizations at 3-day
intervals. Similar protocols using the M3 melanoma and 302R sarcoma
were used to confirm the findings obtained with the RENCA model.
Construction of the DISC-HSV Viruses.
Construction of the basic vector DISC-HSV-2 (DISC-HSV) by plasmid
recombination was described previously (17)
. A similar
process was used to construct dH2B (DISC-mGM-CSF), which required a
two-stage recombination strategy. For the first stage, sodium
iodide-purified, wild-type DNA and plasmid DNA (pIMMB56) were
transfected into gH expressing complementing CR1 cells. The plasmid
pIMMB56 contains the lacZ gene under the control of the SV40
promoter; the expression cassette is flanked by HSV sequences to enable
recombination into viral genome and is similar in construction to
pIMMB47+ (17)
. The resulting virus is designated dH2D. For
the second stage, sodium iodide-purified dH2B viral DNA and plasmid DNA
pIMR3 were transfected into CR1 cells as described above. Plasmid pIMR3
was constructed by ligation of the mGM-CSF gene from plasmid
pJL3.2 (received as a gift; Ref. 18
) into the
shuttle vector pIMMB46. Plasmid pIMMB46 had been adapted previously to
contain the CMV promoter and bovine growth hormone poly(A) addition
signal from the plasmid PPRC/CMV (R&D Systems). The resulting virus was
passaged three times on BHK gH+/TK- cells in the presence of
methotrexate to select for TK+ virus. The final virus was designate
dH2B.
dH2J (DISC hIL-2) was constructed by ligation. This process is simpler
than traditional recombinant techniques and yields a high frequency of
recombinant viruses. A linker sequence containing a unique
PacI restriction site was engineered into the basic vector
during construction to allow subsequent manipulation. dH2J was
constructed by a ligation process similar to basic plasmid
construction. The IL-2 gene was excised from the plasmid
BBG30 (R&D systems); the plasmid was engineered to construct the native
signal sequence and insert a Kosak consensus sequence upstream of the
gene. The modified IL-2 gene was ligated into the "Pac
ligation" vector (pIMJ2), which contains two PacI sites,
before insertion into the virus. The vector pIMJ2 was digested with
PacI to release a fragment containing the modified
IL-2 gene downstream of the CMV promoter. The expression
cassette was ligated with PacI-digested HSV in a similar
process to basic plasmid construction. The ligated DNA was transfected
into CR2 cells, and the resulting virus was designated DH2J.
Depletion of CD4+ and CD8+ Cells.
The effect of the in vivo depletion of
CD4+ or CD8+ T-cells or
both CD4+ and CD8+ T-cells
on the therapeutic efficacy of the DISC vaccine was investigated.
Groups of 10 mice were given three i.p. injections of 1 mg of anti-CD4
(YTS191.1.2), anti-CD8 (YTS 169.4.2.1), control isotype antibody
(YTH24), or a combination of anti-CD4+ and
anti-CD8+antibodies over a period of 1 week
(19)
. Ten days after the last injection, three
representative animals from each group were tail bled to determine the
efficiency of depletion by flow cytometric analysis of the blood cells
using anti-CD4 and anti-CD8 antibodies (Serotec Ltd. Oxford, United
Kingdom). Mice received injections of RENCA cells (5 x 104 cells/mouse) 7 days after the last antibody
injection. Three vaccinations, with irradiated DISC-infected RENCA
cells, were given 3 days apart at an adjacent body site, commencing on
day 3 after the injection of tumor cells.
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RESULTS
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Reporter and Cytokine Gene Expression in RENCA Cells.
DISC-HSV-2 viruses have been constructed to express the genes for
LacZ, mGM-CSF, and hIL-2, respectively, after infection.
In vitro studies were performed to confirm the ability of
these viruses to infect murine tumor cells and to express the gene of
interest. Expression of the LacZ gene was observed in RENCA
cells coexpressing HSV viral glycoproteins, as shown by dual staining
for ß-gal and HSV protein expression. RENCA cells infected with
DISC-LacZ (0.510 pfu) demonstrated an increase in immunostaining for
the virus using anti-HSV fluorescent-labeled antibody only in cells
expressing the ß-gal gene (Fig. 1A)
. The
infectability of RENCA cells using a MOI ranging from 0.3 to 10
pfu/cell was determined. A MOI of 1.25 pfu/cell resulted in 50% of the
cells staining for ß-gal 24 h after infection, whereas a MOI of
2.5 pfu/cell or greater resulted in ß-gal protein expression in
virtually all of the cells (Fig. 1B)
3 h after
infection (Fig. 1C)
. Similar results were obtained for 302R
and M3 cells (results not shown).

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Fig. 1. Infectability of DISC-lacZ and reporter gene expression in
RENCA cells. A, 2 x 104
RENCA cells were cultured in wells on fibronectin-coated glass
microscope slides (MIC3412; Scientific Laboratory Supplies Ltd.,
Nottingham, United Kingdom) in 100 µl of culture medium at 37°C/5%
CO2 for 3 h. The attached cells were then infected
with 0.5, 1, 5, and 10 pfu/cell of DISC-lacZ virus for 3 h. The
cells were then fixed and stained for both ß-gal and HSV-2 antigen
expression and examined by light and fluorescent microscopy to
determine the percentage of stained cells. Bars, SD.
B, 1 x 105 cells were
cultured in 24-well plates overnight. Culture medium was removed, and
the virus inoculum was added for 1 h (experiments were performed
in triplicate). Residual virus was removed, and fresh serum-free medium
was added, and the cells were reincubated for 24 h. Thereafter,
the cells were fixed by adding 500 µl of 1% glutaraldehyde solution
and stained for ß-gal expression. At least 100 cells were evaluated,
and the percentage of stained cells was determined.
Bars, SD. C, experiments were performed
as outlined in B. RENCA cells were stained for ß-gal
expression at time intervals of 0.5 to 24 h after infection with
DISC-lacZ virus at a MOI of 10 pfu.
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RENCA cells infected with DISC-mGM-CSF released up to 400 pg of
m-GM-CSF/ml/105 cells in 24 h (Fig. 2A)
, which was the maximum amount detected in this time
course. The mGM-CSF release from RENCA cells increased proportionally
with virus MOI (0.3 to 10 pfu/cell). Similar results were obtained for
RENCA cells infected with DISC-hIL-2 virus, with the maximum release
occurring 48 h after infection (Fig. 2B)
. For RENCA
cells infected with DISC-hIL-2 virus at a MOI of 10 pfu/cell, 548 x
pg/ml/105 cells of hIL-2 was released into the
supernatant at 24 h. In comparison, the mouse sarcoma 302R cells
released 440 pg/ml/105 cells 24 h after
infection with 10pfu/cell of DISC-hIL2, and the melanoma cell line M3
released 600 pg/ml/105 cells at 24 h.
Cytokine production and release into culture medium by
DISC-HSV-infected cells peaked at 24 and 48 h for mGM-CSF and
IL-2, respectively, and thereafter and by day 5 declined to negligible
levels; this was demonstrated using transwells when culture medium was
replaced with fresh medium at 24-h intervals (results not shown). The
level of cytokine production by DISC-HSV-2-infected tumor cells is high
in comparison to conventional gene transfection is transitional but of
limited duration (72 h) because of the cytopathic effect of the virus.

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Fig. 2. Time course for the production of mGM-CSF and hIL-2 by
cells infected with DISC-mGM-CSF and DISC-hIL-2 viruses. RENCA cells
were cultured in 24-well plates overnight and infected with 10 pfu/cell
of DISC-mGM-CSF virus (A) and DISC-hIL-2 virus
(B) for 1 h, washed twice in serum-free medium, and
incubated for up to 72 h. Supernatant from individual wells was
collected at each indicated time points and stored at -20°C for
cytokine analysis. Bars, SD.
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Because it was necessary to use irradiated tumor cells for in
vivo vaccination studies, the effect of the irradiation on
cytokine release was determined. The release of mGM-CSF varied,
depending on the cell line. For RENCA cells, the release of mGM-CSF
remained at the same level after irradiation (Table 1)
, whereas 302R cells showed an increase in the amount of mGM-CSF
release of
65% and M3 cells a decrease of
40% (results not
shown). A significant (P
0.01) increase in
hIL-2 release occurred after irradiation of DISC-IL-2-infected RENCA
cells (Table 1
; mean of three experiments). Treatment of RENCA cells
with the protein synthesis inhibitor cycloheximide resulted in a >95%
inhibition of the reporter (LacZ) and cytokine gene expression (results
not shown), confirming that virus replication is essential for gene
expression.
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Table 1 Effect of irradiation on cytokine production by RENCA cells infected
with DISC mGM-CSF virus and DISC-IL-2 virus
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Apoptosis and Heat Shock Protein Expression in RENCA Cells Infected
with DISC-HSV Virus.
Cell death by apoptosis was investigated in RENCA cells infected with
the DISC-lacZ virus. RENCA cells infected with DISC-lacZ at a MOI of 10
pfu/cell for 24 h induced apoptotic cell death in 7.8% of all
cells compared with 2.1% of noninfected control cells (Fig. 3)
. Analysis of the DNA profile of cells showed that infected cells
contained more DNA than uninfected cells, indicative of a block in the
S or G2-M phases of the cell cycle, together with
an increase in sub-G1 cells, which is suggestive
of an increase in necrotic cell death (Fig. 3)
. Furthermore, infecting
RENCA cells with DISC-lacZ did not induce the expression of the HSP
protein HSP70, as shown by quantitative mRNA expression (results not
shown), inferring that virus infection does not induce the early onset
of stress response proteins that has been reported previously to
influence the immunogenicity of tumor cells (20)
.

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Fig. 3. Necrosis versus apoptosis in RENCA cells
infected with DISC-lacZ virus. RENCA cells (5 x 105) were seeded into T25 tissue culture flasks and
infected with 10 pfu/cell of DISC-lacZ for 24 h. Floating and
adherent (trypsinized) cells were pooled and analyzed using an
APO-BrdUrd kit to determine the apoptosis-indicative DNA breaks. The
DNA profile (a and b) and DNA breaks
(c and d) for noninfected
(a and c) and infected (b
and d) tumor cells are shown.
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Prophylactic Immunization Using Irradiated Tumor Cells
Infected with DISC-HSV.
Having demonstrated that RENCA cells release mGM-CSF and hIL-2
after infection with DISC-mGM-CSF and DISC-hIL-2 viruses, respectively,
experiments were performed to compare the immunogenicity of
DISC-infected whole-cell vaccines with standard vaccines prepared from
irradiated, uninfected RENCA cells. Cultured RENCA cells were infected
with DISC-HSV-2-containing either the lacZ, mGM-CSF, or
hIL-2 gene for 1 h, irradiated, and injected into
groups of up to 10 mice. Two immunizations were performed at 2-week
intervals, and the animals were challenged s.c. with 5 x 104 live RENCA cells (10 x TD50) 7 days after the second
immunization. Results representative of several experiments are shown
in Fig. 4
. The degree of protective immunity was increased after immunization
with 1 x 104 DISC-infected cells
expressing mGM-CSF compared with nonimmunized control mice or mice
receiving irradiated RENCA cells or irradiated RENCA cells infected
with DISC-lacZ (Fig. 4A)
. In addition, mice immunized with
1 x 105 DISC-mGM-CSF-infected
RENCA cells showed an increased level of protection to tumor challenge
compared with mice immunized with irradiated (noninfected) RENCA cells
and control (nonimmunized) mice (Fig. 4B)
. It was observed
that immunization with 1 x 105
irradiated RENCA cells infected with DISC-lacZ could also increase
resistance to tumor challenge compared with nonimmunized control mice,
although the degree of protective immunity was consistently lower than
that observed for mice immunized with DISC-mGM-CSF-infected cells.

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Fig. 4. Prophylactic immunization of DISC-mGM-CSF-infected RENCA
cells. RENCA cells were infected with 5 pfu/cell of either DISC-mGM-CSF
virus or DISC-lacZ (ß-gal) virus for 1 h and irradiated (15,000
rads). Groups of up to 10 mice were injected with 1 x 104 or 1 x 105 irradiated
virus-infected or irradiated noninfected RENCA cells, and cells were
injected s.c. on the right flank on two occasions 2 weeks apart;
control mice were untreated. One week after the second
immunization, the mice were challenged s.c with 5 x 104 (10 x TD50) parental RENCA
cells on the opposite flank, and tumor growth and incidence were
monitored on a regular basis. Animals immunized with 104
cells and challenged with 5 x 104 RENCA
cells (A), immunized with 105 cells and
challenged with 5 x 104 RENCA cells
(B), immunized with 104 cells and challenged
with 105 RENCA cells (C), or immunized with
104 cells and challenged with 2 x 105 RENCA cells (D) are shown.
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The level of protective immunity was decreased after immunization with
whole-cell vaccines (1 x 104
cells/inoculum) and challenge with 20 x TD50 or 40 x TD50 RENCA cells (Fig. 4, C and D
, respectively). Vaccination with DISC-hIL-2-infected RENCA
cells gave results that were similar to those obtained using
DISC-mGM-CSF-infected RENCA cells (results not shown). These
experiments were repeated in a second tumor model. Prophylactic
immunization with irradiated DISC- mGM-CSF- or DISC-hIL-2-infected 302R
cells enhanced immunity to tumor challenge and confirmed the results
obtained using the RENCA model (results not shown).
Tumor Therapy Using DISC-HSV-infected Tumor Cells.
The ability of DISC-infected tumor cells to influence the growth
of established tumors was investigated in three tumor models: RENCA,
302R, and M3. Groups of 10 mice were injected s.c. on the right flank
with 5 x 104 (10 x TD50) viable RENCA cells prior to
vaccination with irradiated DISC-infected or irradiated noninfected
RENCA cells. Three immunizations (each containing 1 x 106 irradiated tumor cells) were given s.c. on
the same or contralateral flank on days 3, 6, and 9, and the tumor
incidence and tumor size were recorded during a 9-week observation
period. By day 3 after inoculation of live tumor cells, defined tumor
foci were detected by H&E histological analysis (results not shown),
and for most of the experiment performed, this was the start date for
initiation of therapy. The results (Fig. 5A)
demonstrate a slight but insignificant delay in the onset
of tumors in mice receiving the irradiated (noninfected) RENCA cell
vaccine compared with control mice; this difference was not apparent
when the average tumor sizes of the groups were compared (results not
shown). Mice receiving DISC-mGM-CSF-infected RENCA cells showed a
significant delay in the onset of tumors, and a high proportion of mice
remained tumor free up to 9 weeks after challenge. In addition,
vaccination with DISC-hIL2-infected cells significantly inhibited tumor
growth; 60% of mice remained free of tumor throughout the observation
period (Fig. 5A)
. These results were reproducible and
occurred when immunization was initiated on day 0 or day 3 after tumor
cell inoculation. Up to 80% of mice immunized with either DISC-mGM-CSF
or DISC-hIL-2 vaccines remained free of tumor throughout the study
period (data from several experiments, not shown).

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Fig. 5. Treatment of established RENCA tumors with DISC-infected
cells. A and B, animals were implanted
with either 5 x 104 RENCA
(A) or 1 x 104 M3
(B) tumor cells on the right flank (RF). Three
vaccinations (1 x 106 cells/inoculum;
arrows) were given into the RF, 3 days apart,
starting on day 3 and day 0 for RENCA and M3, respectively, with either
irradiated noninfected (tumor cells) or irradiated tumor cells infected
with either DISC-lacZ, DISC-mGM-CSF, or DISC-IL-2 virus. Tumor growth
was monitored regularly throughout the observation period.
C, animals were implanted with 5 x 104 RENCA cells on the right flank (RF). Three vaccination
(1 x 106 cells/inoculum;
arrows) were given 3 days apart, starting on day
3 with irradiated RENCA cells infected with DISC-mGM-CSF injected
either on the right flank (DISC-mGM-CSF RHS) or on the
left flank (DISC-mGM-CSF LHS). Tumor growth was
monitored throughout the observation period.
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Similar results were obtained with the M3 melanoma (Fig. 5B)
model. Vaccination, beginning on day 0 after live tumor
cell implantation, with DISC-mGM-CSF or DISChIL-2-infected M3 cells
inhibited tumor growth in 60 and 20% of vaccinated animals,
respectively (Fig. 5C)
, and caused a delay in tumor growth
in the remaining mice (results not shown). Inhibition of established M3
tumor growth also occurred in 40% of mice treated with DISC-lacZ
M3-infected cells.
One important feature of this immunotherapy model was the development
of local immunity in vaccinated mice. Vaccination at a site adjacent to
the tumor implantation site with the DISC-mGM-CSF RENCA cell vaccine
was effective in delaying the onset and growth of tumors; however,
immunization on the contralateral flank was less effective (Fig. 5C)
. These results demonstrate that an increased therapeutic
benefit can be derived by local administration of tumor cells infected
with DISC-HSV-2 engineered to express either mGM-CSF or hIL-2 and were
confirmed using the 203R tumor model (results not given).
Vaccine Therapy in T-Cell-deficient Mice.
To determine that T-lymphocytes were required for effective
immunotherapy using DISC whole tumor-cell vaccines, BALB/c nude mice
(Nu+/Nu+) received
injections s.c. with 10 x TD50 of
RENCA cells on the right flank and vaccinated (starting on day 0) three
times (3 days apart) on the same flank with irradiated noninfected
RENCA cells or irradiated DISC-mGM-CSF-infected RENCA cells. The tumor
incidence and growth rate were similar in control and vaccinated
Nu+/Nu+ mice,
indicating that T lymphocytes play a pivotal role in promoting tumor
rejection (results not given). To establish the involvement of
CD4+ and CD8+ T lymphocytes
in immunotherapy, mice were depleted of the respective T-cell
populations by the administration of Mabs raised to either CD4 or CD8
antigens. Seven days after antibody treatment, mice were injected with
5 x 104 RENCA cells and vaccine
therapy (irradiated RENCA cells infected with DISC-mGMCSF) given on
days 3, 6, and 9. The results demonstrate that CD4, CD8, and CD4/CD8
"knock out" mice failed to respond to whole-cell vaccine therapy,
whereas mice inoculated with isotype control serum or untreated mice
were successfully treated by vaccination with DISC-mGM-CSF-infected
RENCA cells (Fig. 6)
. The abrogation of therapeutic efficacy was proportional to the
reduction in the subpopulations of T lymphocytes; the administration of
CD8 Mab caused a 50% depletion of circulating CD8+ T cells and
abrogated the effect of immunotherapy in 60% of mice. Administration
of CD4 Mab reduced circulating CD4+ T cells by <95% and completely
abrogated the effect of vaccine therapy. Collectively, these results
demonstrate an absolute requirement for both CD4+
and CD8+ T lymphocytes for effective
immunotherapy using DISC-mGM-CSF-infected whole-cell vaccination.

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Fig. 6. Immunotherapy using a DISC-mGM-CSF-infected RENCA vaccine
in mice depleted of CD4+ or CD8+ T lymphocytes.
Treatment of mice with anti-CD4 monoclonal antibody, given 3 times i.p.
over a period of 1 week, reduced the circulating CD4+
lymphocytes by 95% (CD4 depl, CD4+
depleted); anti-CD8+ antibody treatment reduced the
circulating CD8+ lymphocyte population by 50% (CD8
depl, CD8+ depleted); as determined by antibody
staining and flow cytometry. Control mice were similarly inoculated
with medium or with an isotype control antibody
(isotype). A group of mice was also treated with both
anti-CD4+ and anti-CD8+ antibody
(CD4 + CD8 depl, CD4++
CD8+ depleted). One week after depletion treatment, all
mice were inoculated with RENCA cells (5 x 104 cells/animal) on the right flank and vaccinated
(1 x 106cells/inoculum) on the right flank.
Three vaccinations were given 3 days apart starting on day 3.
mGM-CSF, mice inoculated with medium and vaccinated with
DISC-mGM-CSF-infected RENCA cells; CD4 depl,
CD4-depleted mice receiving vaccination; CD8 depl,
CD8-depleted mice receiving vaccination; CD4 + CD8
depl, mice depleted of CD4 and CD8 T lymphocytes receiving
vaccination; isotype, mice inoculated with isotype
control antibody and receiving vaccination; medium, mice
inoculated with medium and not vaccinated. Tumor growth was monitored
throughout the observation period.
|
|
 |
DISCUSSION
|
|---|
Murine tumor models have been used to evaluate whole tumor cell
vaccines, genetically modified by gene transfection to produce
cytokines, for their ability to promote anticancer immunity. These
vaccines have been shown to elicit systemic immunity against tumor
challenge and in some instances induce the regression of small tumors
when given therapeutically (1, 2, 3, 4, 5, 6)
. Thus, a number of viral
and nonviral vector systems have been investigated as vehicles for gene
delivery into tumor cells (21)
. In the present study, a
DISC-HSV-2 was used as a vector for gene transfection of tumor cells in
preclinical studies to assess its potential for human application.
We previously constructed gH-deleted HSV-2 to be used as a
vaccine for the prevention of HSV-induced disease. This virus, which we
term DISC, can only complete one replication cycle in normal cells and
was shown to stimulate broad humoral and cell-mediated antiviral immune
responses (12)
. DISC-HSV offers advantages as a vector
system for gene transfer; they are safe because they are unable to
spread from cell to cell within the patient, and they have a broad host
cell range, making them suitable for the delivery of genes to a variety
of tumors. Initial studies have shown that the DISC-HSV-2 will infect a
wide range of murine and human tumor cells, including primary human
leukemia and neuroblastoma cells (15
, 16
, 22)
. Here we
show that DISC-HSV is able to infect murine carcinoma, sarcoma, and
melanoma cells.
We have used three murine tumors, RENCA, 302R, and M3, as models
to assess the ability of DISC-HSV-2 to deliver cytokine genes into
tumor cells and undertaken preclinical evaluation of whole-cell
vaccines expressing the cytokines mGM-CSF or hIL-2 to assess their
ability to promote protective and therapeutic immunity. The RENCA tumor
model was used extensively in this study, and the results were
confirmed using 302R and M3 tumors. The relationship between the
expression of the reporter ß-gal gene and viral proteins
after in vitro infection with the DISC-HSV-lacZ virus was
established by dual staining for the expression HSV glycoprotein and
the ß-gal protein. Infection and reporter gene expression were time
and dose dependent. A correlation between ß-gal expression and the
MOI was shown and confirmed that the virus was incapable of lateral
spread. In a study by Lowstein et al. (23)
,
recombinant HSV type 1 mutant tsk vectors containing ß-gal were shown
to infect neurocortical cells; ß-gal expression was directly related
with the MOI of the virus.
Infecting RENCA cells with DISC-HSV-2 encoding mGM-CSF and
recombinant hIL-2 genes resulted in the release of cytokines
into the culture supernatant in a time-dependent manner. After
infection with DISC-HSV-ß-gal, an increase in necrotic cell death
versus apoptosis occurred. RENCA cells undergoing death by
necrosis may provide addition activation of the immune system in
vivo by promoting tumor antigen processing and presentation by
professional antigen-presenting cells, leading to an increase in T-cell
activation (24
, 25)
. In situ killing of tumor
cells using suicide gene transfer to induce cell death through a
nonapoptotic pathway is associated with enhanced immunogenicity and may
in some cases require the induction of HSP expression
(20)
, although in the present study RENCA cells infected
with DISC-HSV failed to show elevated expression of HSP.
On the basis of these in vitro results and because of the
potential of these cytokines to activate effector T cells
(26)
, DISC-mGM-CSF and DISC-hIL-2 vectors were chosen for
in vivo studies. In animal models, mGM-CSF expression by
tumor cells results in potent systemic antitumor immunity, which can
potentiate the rejection of weakly immunogenic murine tumors
(27)
. IL-2 is also a potent mediator of antitumor immunity
and can promote CTL activation and T-cell differentiation, enhance the
activation status of natural killer cells, and induce
lymphokine-activated killer cell activity (28
, 29)
.
Interestingly, hIL-2 production and release by DISC-hIL-2-infected
cells were significantly increased after irradiation, an effect
observed previously by Simova et al. (30)
,
where administration of irradiated IL-2-secreting plasmacytoma cells
was shown to be more effective than nonirradiated cells in promoting
tumor immunity. Here, we demonstrate that immunization with irradiated
RENCA cells infected with DISC encoding either mGM-CSF or
hIL-2 cytokine genes protects mice against challenge with
parental tumor cells in a dose-related manner.
DISC-mGM-CSF vaccine therapy prevented tumor growth in a high
percentage of mice. The response to therapy was T-lymphocyte dependent
and required the participation of both CD4+ and
CD8+ T lymphocytes. One important consideration
in this therapy model is the relative contribution of HSV infection
versus cytokine production. Partial protection was observed
after immunization with tumor cells infected with the DISC ß-gal
virus (used as a control for cells expressing cytokine), indicating
that protection against tumor challenge may, in part, be a consequence
of viral infection of the tumor cells; immunization with the ß-gal
protein alone does not illicit a measurable antitumor immune response
(10)
. We suggest that DISC-HSV infection can act as an
additional stimulus to enhance the immunogenicity of the tumor cells.
HSV infection of mice has been shown to lead to the up-regulation of
IL-12 expression (31)
and to have potent antitumor effects
in animal models (32)
, most probably by promoting a Th1
response to tumor antigen(s). Preexisting immunity to HSV infection did
not seem to affect the efficacy of this vaccine because no significant
difference was shown when animals were preimmunized with HSV prior to
tumor implantation and subsequent therapy (data not shown). Irradiated
RENCA cell vaccination also induced a degree of protection against
rechallenge with the parental tumor line. These data are consistent
with previous observations demonstrating that RENCA cells are weakly to
moderately immunogenic (33)
, and where irradiation itself
may affect the immunogenicity of tumor cells through the up-regulation
of H-2Kd class I MHC antigens (34)
. For the reasons
outlined, there is a precedent for using DISC-HSV to deliver immune
response genes to tumor cells, in the present study by ex
vivo infection of the tumor cells, but additionally by direct
in vivo injection into the tumor using a murine colon
carcinoma model, where 40% of tumors regressed completely
(16)
; we have shown that both approaches are efficacious
for therapy in animal models.
GM-CSF has been used to potentiate antitumor immunity by
promoting the maturation and function of professional
antigen-presenting cells (35
) and by recruiting
additional antigen-specific and nonspecific effector cells. One
noticeable feature of the immune response after inoculation of
whole-cell vaccines expressing GM-CSF is the prevalence of a
delayed-type hypersensitivity response at the site of vaccination and
at the site of tumor rejection. Infiltration of tumors by eosinophils
in response to GM-CSF has been reported in preclinical and clinical
studies (27
, 36)
, and a similar response is also observed
after immunization with IL-4 gene-transduced vaccines (37
, 38)
. There is also evidence that patients treated with an
autologous GM-CSF gene-transduced vaccine can undergo an objective
clinical response (36)
, although it is unclear to what
extent eosinophils and effector cells other than CD8+ and CD4+
lymphocytes actually contribute to tumor rejection. Eosinophil
infiltration of small established RENCA tumors occurs within 24 h
after vaccine therapy with irradiated DISC-mGM-CSF RENCA
cells4
and may represent a response associated with the production of Th2
cytokines IL-4 and IL-5 (36)
. In conclusion, the results
obtained in this study allow us to propose a clinical approach to
cancer immunotherapy based on the use of a novel DISC-HSV vector for
the efficient delivery of cytokine genes to tumor cells.
 |
ACKNOWLEDGMENTS
|
|---|
We are grateful to Glenda Kill for typing the manuscript.
 |
FOOTNOTES
|
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Funded by Cantab Pharmaceuticals. 
2 To whom requests for reprints should be
addressed, at Department of Life Sciences, Nottingham Trent University,
Clifton Lane, Nottingham NG11 8NS, United Kingdom. 
3 The abbreviations used are: IL, interleukin;
hIL, human IL; CSF, colony-stimulating factor; GM-CSF,
granulocyte-macrophage CSF; mGM-CSF, murine GM-CSF; HSV, herpes simplex
virus; DISC, disabled infectious single cycle; pfu, plaque-forming
unit(s); HSP, heat shock protein; BrdUrd, bromodeoxyuridine;
CMV, cytomegalovirus; ß-gal, ß-galactosidase; MOI, multiplicity of
infection; TD50, tumor dose 50%; Mab, monoclonal
antibody; gH, glycoprotein H. 
4 Unpublished results. 
Received 8/23/99.
Accepted 12/16/99.
 |
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