Local Immune Stimulation by Intravesical Instillation of Baculovirus to Enable Bladder Cancer Therapy

Abstract

Intravesical instillation of Bacillus Calmette-Guérin is currently used as adjuvant therapy for superficial, non-muscle invasive bladder cancer (NMIBC). However, nearly 40% of patients with NMIBC will fail Bacillus Calmette-Guérin therapy. In an attempt to investigate the feasibility of using insect baculovirus-based vectors for bladder cancer therapy, we observed that intravesical instillation of baculoviruses without transgene up-regulated a set of Th1-type of cytokines and increased the survival rate of mice bearing established orthotopic bladder tumors. When baculoviral vectors were used to co-deliver the mouse CD40 ligand and IL-15 genes through intravesical instillation, the immunogene therapy triggered significantly increased bladder infiltrations of inflammatory monocytes, CD4+, CD8+ and γδ T lymphocytes. All treated animals survived beyond 12 months whereas control animals died around 2 months after tumor inoculation. We conclude that direct intravesical instillation of baculoviral gene transfer vectors holds the potential to be a novel therapeutic modality for NMIBC.

Full text

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Scientific RepoRts | 6:27455 | DOI: 10.1038/srep27455
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Local Immune Stimulation
by Intravesical Instillation of
Baculovirus to Enable Bladder
Cancer Therapy
Wei Xia Ang1,2, Ying Zhao2, Timothy Kwang3, Chunxiao Wu2,3, Can Chen3, Han Chong Toh 4,
Ratha Mahendran5, Kesavan Esuvaranathan5 & Shu Wang1,2
Intravesical instillation of Bacillus Calmette-Guérin is currently used as adjuvant therapy for supercial,
non-muscle invasive bladder cancer (NMIBC). However, nearly 40% of patients with NMIBC will
fail Bacillus Calmette-Guérin therapy. In an attempt to investigate the feasibility of using insect
baculovirus-based vectors for bladder cancer therapy, we observed that intravesical instillation of
baculoviruses without transgene up-regulated a set of Th1-type of cytokines and increased the survival
rate of mice bearing established orthotopic bladder tumors. When baculoviral vectors were used to
co-deliver the mouse CD40 ligand and IL-15 genes through intravesical instillation, the immunogene
therapy triggered signicantly increased bladder inltrations of inammatory monocytes, CD4+, CD8+
and γδ T lymphocytes. All treated animals survived beyond 12 months whereas control animals died
around 2 months after tumor inoculation. We conclude that direct intravesical instillation of baculoviral
gene transfer vectors holds the potential to be a novel therapeutic modality for NMIBC.
Adjuvant therapy is oen given following transurethral resection of supercial, non-muscle invasive bladder
cancer (NMIBC) to control tumor recurrence and progression1,2. Intravesical immunotherapy with Bacillus
Calmette-Guérin (BCG) is currently recommended for patients with intermediate- or high-risk of bladder can-
cer recurrence. BCG is produced from attenuated live bovine tuberculosis bacterium and can activate innate
immune responses mediated by cytokines such as interleukin-2 (IL-2), IL-12, IL-18 and interferon-gamma
(INF-γ ). Unfortunately, up to 40% of patients with NMIBC will fail BCG immunotherapy within the rst year
due to BCG refractory, resistance, relapsing, or intolerance1–3. Currently, there is still no gold standard for salvage
therapy aer BCG failure, highlighting the urgent need to develop new adjuvant therapies to improve treatment
outcomes for patients with NMIBC.
e bladder is a hollow organ, allowing nonsurgical intravesical drug administration through a urethral cathe-
ter and evaluation of treatment ecacy by the means of endoscopy. Taking advantage of the fact that intravesically
delivered therapeutics act locally with limited systemic exposure and that supercial bladder cancer is easily
accessible, viruses, either replication-competent viruses or replication-decient recombinant viral vectors, have
been tested for bladder cancer therapy in tumor models3,4. ese viral therapeutics include vaccinia virus5–8,
adenovirus7,9–15, canarypox virus7, reovirus16, retrovirus17–20, lentivirus21, and vesicular stomatitis virus22. At least
6 clinical trials have been reported on the use of vaccinia virus and adenoviral vectors to treat bladder can-
cer4,6,11,12,14,15. However, the hurdles to translating the current success of these viral therapeutics to a broad clinical
application appear high, which include the lack of gene delivery vectors with active immunostimulatory function
and inecient expression of a therapeutic gene in bladder tumor cells and in the organ.
Recombinant vectors derived from the insect baculovirus Autographa californica multiple nucleopolyhedro-
virus (AcMNPV) hold the ability to enter mammalian cells without replicating or causing toxicity to the trans-
duced cell. Since baculoviral vectors with a mammalian expression cassette could be highly eective in mediating
1Department of Biological Sciences, National University of Singapore, 117543 Singapore. 2Institute of Bioengineering
and Nanotechnology, 138669 Singapore. 3Tessa Therapeutics, 239351 Singapore. 4Division of Medical Oncology,
National Cancer Centre, 169610 Singapore. 5Department of Surgery, Yong Loo Lin School of Medicine, National
University of Singapore, 119074 Singapore. Correspondence and requests for materials should be addressed to S.W.
(email: dbsws@nus.edu.sg)
Received: 24 August 2015
Accepted: 19 May 2016
Published: 08 June 2016
OPEN

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transient expression and usually do not integrate into the genome of the transduced cells23, they are ideally suited
for applications requiring short-term, high level transgene expression and pose much less risk of insertional
mutagenesis. From an application point of view, baculoviral vectors are easy to manipulate, able to carry large
(at least 38 kbp) and multiple DNA inserts, and can be readily produced in serum-free cell culture medium and
puried at high titers in Biosafety Level 1 laboratories. us, recombinant baculoviruses have been suggested as a
novel type of vectors for cancer gene therapy23–27.
In preclinical animal studies, baculoviral vectors have been shown to possess a strong adjuvant activity in
inducing humoral and cellular immune responses against co-administered antigens, possibly through promoting
maturation of dendritic cells (DCs) and producing pro-inammatory cytokines, chemokines and type I IFNs28,29
and activating natural killer (NK) cell-dependent antitumor immunity30,31. e “adjuvant” eect of baculoviral
vectors is attributed to the high frequency of CpG motifs in the viral genome, which is similar to that in bacterial
DNA and signicantly higher than that of mammalian and adenovirus DNA32,33. However, a critical obstacle
toward in vivo baculoviral transduction is the inactivation of systemically delivered baculoviral vectors as a con-
sequence of virus recognition by serum complement proteins, a major component of the innate immune system34.
Hence, in immunocompetent animals systemically delivered baculoviral vectors failed to transduce target cells.
Bladder cancer therapy can be performed with intravesical catheterization through the urethra and easily
avoids many barriers to systemic virus administration, thereby presenting a unique opportunity to explore in vivo
applications of baculoviral vectors. Herein we report that intravesical treatment with baculoviruses alone is able
to prolong survival of mice with established orthotopic bladder cancer. We further demonstrate that baculoviral
vectors are eective in delivering therapeutic genes into normal and malignant urothelial cells and can be used for
immunogene therapy for bladder cancer in a mouse model.
Materials and Methods
Baculoviral vectors. Recombinant baculoviral vectors, CMV-Luc, CMV-Luc-WPRE and CMV-RU5-
Luc-WPRE, were constructed using BAC-to-BAC baculovirus expression system (Invitrogen, Carlsbad, CA).
BV-CD40 ligand (CD40L) virus and BV-IL15 virus which contain the mouse CD40L gene and mouse IL15 gene
respectively were produced by homologous recombination aer co-transfection of Sf9 insect cells with pBacPAK6
transfer vector containing the expression cassette and linearized AcMNPV viral DNA (Clontech, Mountain View,
CA). BacPAK6, the parental virus with the lacZ gene driven by viral polyhedrin promoter, was obtained from
Clontech.
Mouse tumor model. Adult female C57BL/6 mice were used to generate orthotopic bladder tumors on the
luminal surface of the bladder by intravesical instillation of syngeneic MB49 cells with a 24-gauge catheter. Aer
a 30-minute PLL pre-treatment, 100 μ l of MB49 cells in PBS was instilled and retained in the bladder for 1 hour
by leaving the catheter in situ and clamped. A dose of 1 × 105 cancer cells per animal was used in all experiments.
ereaer, the catheter was removed and the bladder was evacuated by spontaneous voiding. MB49 cells used
for tumor inoculation were pre-labeled with a lipophilic, near-infrared uorescent dye DiR (20 ng/ml) (Caliper
Life Sciences) overnight. One week aer intravesical instillation of MB49 cells, mice were imaged with IVIS100
in vivo imaging system using 710 nm excitation and 760 nm emission lter set to examine tumor implantation.
Animals successfully implanted with DiR-labeled MB49 cells and with similar tumor burden were kept for exper-
iments designed to evaluate therapeutic ecacy. In the current study, the uorescent signal of the DiR-labelled
MB49 cells persisted without substantial change for at least 7 days. e signal began to decay thereaer, although
remaining weakly detectable even on day 14. Hematuria, an indicator for the tumour formation in the bladder,
was also monitored in some experiments to further conrm the tumor implantation and growth.
To test therapeutic eects of baculoviral vectors, mice bearing bladder tumors were randomized to control
or treatment groups, re-anaesthetized, and re-catheterized. Baculoviruses (1 × 108 pfu in 100 μ l), PBS (100 μ l), or
BCG (1.35 mg = 2.3 × 107 colony forming units in 100 μ l, Aventis Pasteur, Canada) were intravesically instilled
and retained in the bladders for 2 hours. In some experiments, mice were given up to three weekly intravesical
instillations. Animals were observed for up to 12 months for signs and symptoms of bladder cancer (hematuria
and weight loss) and viability status.
e animal study protocol was reviewed and approved by Institutional Animal Care and Use Committee
(IACUC), the Biological Resource Centre, the Agency for Science, Technology and Research (A*STAR), Singapore
(Permit Number: BRC IACUC 110612). e methods were carried out according to the guidelines for the Care
and Use of Animals for Scientic Purposes issued by the National Advisory Committee for Laboratory Animal
Research, Singapore.
Cytokine/chemokine expression. Mice were euthanized 48 hours aer intravesical instillation of PBS or
BacPAK6. Bladders were harvested and weighed. Bladder homogenates were obtained by adding 1 ml of a tis-
sue lysis buer (Fermentas, Maryland, USA) with a protease inhibitor cocktail (Merck, Darmstadt, Germany)
to 50 mg of tissue sample and homogenizing by sonication. Bladder homogenates were then centrifuged at
16,000 × g for 30 min at 4 °C and the supernatants collected. Protein concentrations of the supernatants were
determined by the Biorad protein assay method (Biorad, California, USA). An aliquot of the supernatant con-
taining 80 μ g of total protein concentration was loaded onto the Mouse Cytokine Array C3 (Raybiotech, Norcross,
GA) to measure expression levels of cytokines and chemokines. RayBio® Analysis Tool (Raybiotech) was used to
correlate the average signal intensities to relative expression levels of cytokines.
Analysis of inltrated immune cells. For ow cytometry analysis of inltrated immune cells, the col-
lected bladder cells were pre-incubated with Fc block (CD16/CD32, Clone 2.4G2, BD), washed, and incubated
with appropriate uorescent-conjugated antibodies. For immunohistochemistry analysis, the tissue sections were

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incubated with 0.3% H2O2 for 10 min to block the endogenous peroxidase activity. e tissue sections were then
incubated in 5% BSA for 1 h to block non-specic binding sites before incubation with appropriate primary anti-
bodies and HRP-conjugated secondary antibodies. Staining was developed by 3,3′ -diaminodbenzidine substrate
and the nuclei were counterstained by hematoxylin.
Statistical analysis. For survival rates, statistical analysis was performed using the log-rank test. For others,
statistical signicance was assessed by Student’s t-test; p < 0.05 was considered statistically signicant.
Supplemental methods. Details of methods for baculoviral vector construction, cell culturing, in vitro &
in vivo baculoviral transduction, ow cytometry analysis, histological analysis, and immunostaining are provided
in the Supplementary Materials and Methods.
Results
Baculoviral vectors eectively transduce the mouse bladder and bladder tumors after intra-
vesical instillation. We rst assessed whether baculovirus could transduce the bladder in immunodecient
BALB/c nude mice. For that, we constructed three dierent recombinant baculoviral vectors containing a rey
luciferase gene and compared their in vivo transduction eciency aer intravesical instillation into the bladder
at a dose of 107 viral particles per mouse. e bladders were pre-treated with PLL before viral vector instillation
as we found that this pre-treatment signicantly enhanced the uptake of baculoviral vectors (Fig. S1A). All three
vectors were able to transduce the mouse bladder as evidenced by using the IVIS living animal imaging system
(Fig.1A). One of the baculoviral vectors, BV-RU5-Luc-WPRE, that contains two viral transcriptional regulatory
elements WPRE and RU5, provided the highest transgene expression level in the bladder. While decreasing over
time, the expression levels provided by the three vectors remained signicantly higher than a background level
for at least 35 days (Fig.1B).
We next tested baculoviral transduction in the bladder of immunocompetent C57BL/6 mice aer intravesical
instillation of BV-RU5-Luc-WPRE. e in vivo transduction eciency was dosage-dependent with the luciferase
expression level at day 1 in C57BL/6 mice treated with 108 viral particles per mouse being approximately 5-fold
greater than that provided by treatment with 107 viral particles per mouse (Fig.1C). Although the initial expres-
sion level provided by 108 viral particles of BV-RU5-Luc-WPRE in C57BL/6 mice was similar to that observed in
immunodecient nude mice treated with 107 viral particles, the level dropped quickly and the detectable trans-
gene expression lasted for approximately 2 weeks only. e dierence in transgene expression between immuno-
decient nude mice and immunocompetent mice indicates a strong immune response to baculoviral transduction
that might eliminate the viral vectors in the transduced organ. Aer intravesical instillation with 108 viral par-
ticles in C57BL/6 mice we observed no behavioral abnormalities and hepato- and nephro-toxicities (Fig. S2).
Hence, this dose was used for all following animal experiments.
We then tested whether BV-RU5-Luc-WPRE, following intravesical instillation, could transduce bladder tum-
ors in an orthotopic tumor model generated by implantation of syngeneic MB49 bladder cancer cells into the
bladders of C57BL/6 mice (Fig.1D). Immunohistological staining with an antibody against the luciferase protein
conrmed that the viral vectors transduced the tumors eciently, penetrating deep into the tumor mass and
distributing extensively throughout the whole tumor bed (Fig.1E). However, in tumor-free areas, as well as in the
normal bladder, baculovirus-mediated transgene expression was conned to the supercial bladder epithelium,
suggesting a restricted regional transgene delivery by intravesically instilled baculoviral vectors in the normal
bladder (Fig.1E; Fig. S1B). Baculoviral transduction of MB49 mouse bladder cancer cells was further conrmed
using BV-RU5-eGFP-WPRE (Fig.1F). is viral vector could also transduce several of human bladder cancer cell
lines, including T24, HBC1, HTB2 and HTB5 (Fig. S3).
Baculoviral transduction alone is capable of retarding bladder tumor growth. As baculoviral
transduction in the bladder possibly stimulates local immune responses, we investigated expression of cytokines
and chemokines in the organ upon intravesical instillation of baculovirus in C57BL/6 mice. We used BacPAK6,
a baculoviral vector without a mammalian gene expression cassette, for this purpose to avoid possible inter-
ference by transgene expression. Using an antibody array method, we detected the up-regulation (> 3-fold
increase in expression) of 61% of the cytokines and chemokines in a murine array (38 out of 62) in the bladder
that received intravesical instillation of BacPAK6 two days before as compared to the expression levels in the
bladder that received PBS instillation (Fig.2A). e top 5 up-regulated proteins were granulocyte-macrophage
colony-stimulating factor (GM-CSF, 154-fold increase), interleukin-6 (IL-6, 101-fold increase), interleukin-1 beta
(IL1-beta, 48-fold increase), granulocyte colony-stimulating factor (G-CSF, 42-fold increase), and leptin receptor
(Leptin R, 33-fold increase). A comparison between the normal mouse bladder receiving no treatment and the
bladder receiving PBS instillation showed no signicant dierence in the expression levels of the cytokines and
chemokines (data not shown).
e above antibody array results led us to hypothesize that the up-regulation of cytokines and chemokines
upon baculoviral transduction would aect bladder tumor growth. To test this, C57BL/6 mice were inoculated
with MB49 tumor cells and one week later randomly distributed into two groups (n = 10 per group): one group
received single intravesical instillation of BacPAK6 and another group received PBS as an instillation control.
Bladders were harvested on day 21 post-tumor inoculation for weight measurement. Indeed, reduced bladder
weight as compared with the PBS group, an indication of retarded tumor growth, was observed in the BacPAK6
group (Fig.2B). We further observed signicantly prolonged survival of the bladder tumor-bearing mice aer
BacPAK6 instillation in the bladder. While all animals in the PBS control group died within 70 days, 50% of the
animals in the treatment group were still alive on day 95 post-tumor inoculation (Fig.2C, p < 0.01 in log-rank
test). Furthermore, no therapeutic eects were observed aer baculovirus instillation in immunodecient nude

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mice (data not shown). ese ndings indicate that BacPAK6-triggered immune responses are responsible for
the observed antitumor eects.
To further test the impact of baculovirus-triggered immune responses on tumor growth, BacPAK6-treated
mice that survived from the above animal experiment in Fig.2C were re-challenged with MB49 tumor cells intra-
vesically. All mice survived for at least 180 days aer the 2nd tumor challenge (Fig.2D). As a control, age-matched
naïve mice were challenged with the same number of MB49 cells and all animals died by day 60. us, baculovirus
instillation in the bladder is able not just to trigger local immune responses, but also promote systemic, adaptive
anti-tumor immunity against MB49 tumor cells.
Baculoviral transduction-mediated immunogene therapy for bladder cancer in mice. To eval-
uate the potential of using baculovirus as a gene therapy vector, two recombinant baculoviral vectors (BV-CD40L
and BV-IL15) armed with either the mouse CD40L or the mouse IL-15 genes were constructed by replacing the
luciferase gene in BV-RU5-Luc-WPRE. Baculovirus-mediated CD40L and IL-15 expressions were conrmed
by in vitro transduction in MB49 cells, followed by Western blot analysis 48 hours post transduction (Fig. S4).
rough subcutaneous injection of original MB49 cells, in vivo selection/enrichment, and primary tumor cell
culturing, we collected faster growing MB49 tumor cells to evaluate the therapeutic eects of BV-CD40L and
BV-IL15 (see Supplementary Information). In the orthotopic model generated with aggressive MB49 cells,
Figure 1. In vivo baculoviral transduction aer intravesical instillation of baculoviral vectors in mice.
(A) Bioluminescence images of luciferase reporter gene expression in immunodecient BALB/c nude mice.
Images of representative animals transduced with 3 dierent baculoviral vectors (107 viral particles per mouse)
are shown. Heat map represents the transgene expression area and color represents the intensity. e schematic
structures of baculoviral vector expression cassettes are shown on the le. Abbreviations: CMV: the human
cytomegalovirus immediate-early gene promoter and enhancer; WPRE: the woodchuck hepatitis virus post-
transcriptional regulatory element; RU5: R segment and part of the U5 sequence of the long terminal repeat
from the human T-cell leukemia virus type 1. (B) Time course analysis of luciferase reporter gene expression
in BALB/c nude mice. In vivo gene expression levels are quantied by measuring bioluminescence signals. e
data represent the mean + s.d., n = 5 per group. (C) Time course analysis of luciferase reporter gene expression
in C57BL/6 mice. Mice were transduced with the baculoviral vector BV-RU5-Luc-WPRE at a dose of 108 or
107 viral particles per mouse. e data represent the mean + s.d., n = 5 per group. (D) MB49 tumor growth in
the bladder of C57BL/6 mice. MB49 mouse bladder tumor cells (105 per mouse) were implanted into poly-L-
lysine pre-treated bladder and allowed to establish tumors for one week. A bright eld image of an H&E stained
section showing mouse bladder architecture and a tumor inside the bladder (arrow). (E) Immunostaining to
demonstrate baculoviral transduction in bladder tumors. Tumor-bearing bladders were collected 24 hours
aer transduction with BV-RU5-Luc-WPRE for immunostaining with antibodies against the luciferase protein.
A uorescence image is shown. (F) Flow cytometric analysis to demonstrate transduction of MB49 tumor
cells with BV-RU5-eGFP-WPRE. Phase contrast and uorescence images of transduced cells are shown in the
bottom panels.

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hematuria, an early sign of tumor growth, was observed in most of the mice as early as one week aer tumor cell
inoculation. We treated these tumor-bearing mice with one, two or three repeated instillations of viral vectors
or control reagents on days 7, 14 and 21 post-tumor implantation respectively. Since BCG treatment is the gold
standard for immunotherapy of bladder cancer, it was included for comparison with baculoviral vectors.
In the rst set of the gene therapy experiments, animals were sacriced on day 35 post-tumor inoculation
and bladders were harvested for weight measurement (Fig.3A). erapeutic eects were obvious aer one instil-
lation of viral vectors or BCG and became increasingly pronounced with increase in the number of instilla-
tions. Aer giving two or three instillations of both BV-CD40L and BV-IL15, the weight of the bladders from the
tumor-inoculated mice was almost same as that in the normal mice group, indicating that multiple instillations
of therapeutic gene-expressing baculoviral vectors may promote complete tumor regression. is was supported
by a long-term survival study in which all MB49 cell-inoculated mice that were administered three instillations
of both BV-CD40L and BV-IL15 survived for at least 12 months. Figure3B depicts the survival rates of dierent
groups at day 125 post-tumor inoculation, with 100% survival in the group treated with both BV-CD40L and
BV-IL15, followed by 90% in the mice receiving BV-CD40L or BV-IL15, 75% in the BacPAK6 group, and 60%
in BCG-treated mice. e results of statistical analysis of survival rates are shown in Table S1. Complete tumor
regression aer three instillations of both BV-CD40L and BV-IL15 was conrmed by histological examination
(Fig.3C). In the mice sacriced on day 35 post-tumor inoculation bladder tumors were almost undetectable and
distinct normal transitional epithelium structure of the bladder mucosa was seen. Taken together, our results
Figure 2. Baculovirus as an immunostimulatory agent for bladder cancer therapy. (A) Baculovirus-
mediated cytokine/chemokine expression in mice treated with PBS or BacPAK6, a baculoviral vector without
mammalian gene expression cassette. Top: Bladders were harvested 48 hours aer the respective instillation,
homogenized and extracts were used to probe the cytokine/chemokine arrays. Aer densitometric analysis,
the top 5 up-regulated cytokines/chemokines are labeled on the blots. e pictures shown are representative.
Bottom: Densitometric analysis of the cytokine/chemokine arrays. e RayBio® Analysis Tool was used to relate
average signal intensity to cytokine/chemokine expression level. e average signal intensity of 3 mice per group
and a > 3-fold change as a baseline was used for analysis. (B) erapeutic eects of BacPAK6 as demonstrated
by bladder weight measurement. Mice were inoculated with MB49 tumor cells intravesically and 7 days later
treated with PBS or BacPAK6. Survived mice were sacriced on day 21 post-tumor inoculation and bladders
were collected for weight measurement. e data represent mean + s.d., n = 6 or 10 per group. (C) BacPAK6
transduction prolongs survival of bladder tumor-bearing mice. Survival curves till day 95 are shown. n = 10
in the PBS group and n = 20 in the BacPAK6 group. e statistical analysis was performed using the log rank
test. (D) Induction of protective immunity in mice survived in BacPAK6 group in Fig. 2C. ese mice were re-
challenged by intravesical tumor inoculation with MB49 cells (n = 10). Age-matched naïve mice were used as
controls and inoculated with the same number of MB49 cells (n = 10). Survival curves till day 180 post-tumor
inoculation are shown.

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conrmed that three instillations of both BV-CD40L and BV-IL15 could eectively suppress bladder cancer
growth in vivo.
Baculoviral transduction in the bladder is associated with robust infiltration of immune
cells. To elucidate the cellular mechanisms underlying the immune response activated by baculoviral trans-
duction, we used ow cytometric analysis to characterize immune cell inltrates in the bladder aer repeated
instillations. Sixteen-hours aer the third instillation as illustrated in Fig.3A, C57BL/6 mouse bladders were
harvested, digested and stained for analysis. Neutrophils were dened as live CD45.2+Ly-6G+ cells and inam-
matory monocytes as live CD45.2+Ly-6G−Ly-6ChighCD11b+ cells. Results were expressed as % of a lymphocyte
subset among live leukocytes (Fig.4). We observed signicantly elevated inltration of inammatory monocytes
in the bladder aer repeated intravesical instillations of BCG, BacPAK6, or baculoviral gene therapy vectors
as compared to PBS instillation. e percentage of inammatory monocytes among live leukocytes increased
from 0.2% in the PBS group to approximately 20 to 30% aer treatment with BCG, BacPAK6, and baculoviral
gene therapy vectors, with the highest inltration rate (34.3%) aer co-instillations of BV-CD40L and BV-IL15.
Neutrophil inltration increased modestly, from 10% in the PBS group to 12 to 18% aer treatment with BCG,
BacPAK6, or baculoviral gene therapy vectors. us, inammatory monocytes could be one type of eector cells
for eliminating bladder tumors by baculoviral transduction in our mouse model.
T lymphocytes were gated as live CD45.2+CD3ε
+NK1.1− cells, and then as γ δ T-cell receptor (TCR) positive
or negative cells. From the γ δ TCR negative population CD4 and CD8α expression were assessed. As illustrated
in Fig.5, both BCG and BacPAK6 increased the accumulation of T lymphocytes in the bladder, with a slightly
higher number of CD8+ T lymphocytes aer BacPAK6 instillation. Using BV-CD40L and BV-IL15 for repeated
instillations, total T cell populations increased by 2-fold and 1.5-fold respectively as compared with BacPAK6.
Co-instillations of BV-CD40L and BV-IL15 increased the accumulation of CD4+ T cells and CD8+ T cells by
approximately 4-fold. Furthermore, co-instillations with BV-CD40L and BV-IL15 generated a profound stimu-
latory eect on the accumulation of γ δ T cells, providing an approximately 10-fold increase in γ δ T cell inltra-
tion in the bladder. We further analyzed local T lymphocyte subsets based on the expression of T-cell memory
Figure 3. Baculovirus as a gene transfer vector for bladder cancer therapy. (A) Bladder weight measurement.
Le: e animal experiment protocol used. Mice were inoculated with MB49 tumor cells intravesically and
subsequently treated three times with baculoviral vectors (days 7, 14, and 21). Right: Mice were treated with
baculoviral vectors expressing CD40L, IL-15, or both. Control animals were treated with PBS, BCG, or empty
baculoviral vector BacPAK6. Mice were sacriced on day 35 post-tumor inoculation and bladders were
collected for weight measurement. e data represent mean + s.d., n = 5 per group. (B) Survival curves to
demonstrate anti-tumor eects of baculoviral gene therapy aer 3 instillations in the mouse bladder. e tumor
inoculation and virus/BCG instillations were performed as shown in (A). Survival curves till day 125 are shown.
e statistical analysis was performed using the log rank test. n = 20 for PBS, BacPAK6, and BCG groups and
10 for BV-CD40L, BV-IL-15 and BV-CD40L+ BV-IL-15 groups. (C) H&E staining shows MB49 bladder tumor
development. Bladders were collected on day 35 post-tumor inoculation.

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markers CD44 and CD62L (Fig.6A). Upon repeated intravesical instillations with baculoviral vectors or BCG, the
naïve T cells dened as CD44lowCD62Lhigh were markedly decreased, accompanied by increased percentages of
CD44highCD62Llow eector T cells and CD44highCD62Lhigh memory T cells in both CD4+ and CD8+ com-
partments (Fig.6B). ese changes were most obvious aer co-instillations with BV-CD40L and BV-IL15. e
percentages of eector and memory T cells, which play a pivotal role in the development of immune responses,
increased from 12% in the PBS group to 49% in the CD40L+ IL15 group in the CD4+ T cell population and 11%
in the PBS group to 44% in the CD40L+ IL15 group in the in the CD8+ T cell population. Lymphocytic inltra-
tions aer intravesical instillation of BacPAK6 and BV-CD40L/BV-IL15 were also conrmed by immunostaining
(Fig. S5). Such pronounced lymphocyte accumulation triggered by baculoviral transduction might play impor-
tant roles in the above observed bladder tumor regression.
Discussion
Bladder cancer is the most common form of malignancy in the urinary tract. At initial diagnosis the majority
of bladder cancer are NMIBC. e recurrence rate of NMIBC aer transurethral resection could be as high as
70%, necessitating adjuvant therapy to control recurrence and progression1,2. e current study demonstrated for
the rst time that baculoviral transduction is a possible new approach for adjuvant treatment of bladder cancer.
Baculoviruses can be used not only for immune stimulation, but also for therapeutic gene delivery, providing
a new combinatorial approach that harnesses the power of immunotherapy and gene therapy in a single viral
ve ctor.
Given their intrinsic potent immunostimulatory property and ecient cell transduction capacity, baculovi-
ral vectors oer an unprecedented advantage over many other viral vectors in immunotherapy against bladder
cancer. We observed that intravesical instillation of empty baculoviral vectors, without the use of any therapeutic
genes, was eective in prolonging the life of mice with established orthotopic bladder cancer. is nding is
consistent with previous studies that found that aer being injected into the animal body, baculovirus can elicit
protective innate immune responses30,35–38. is property has been exploited to protect animals from lethal chal-
lenges with encephalomyocarditis virus35 and inuenza virus37, suppress liver cirrhosis induced by dimethylni-
rosamine38, and inhibit tumor growth30.
Presumably, the initial step aer baculovirus instillation in the bladder should be the binding of the virus to
the urothelium, aer which the virus can enter both normal and malignant cells, resulting in urothelial activation
Figure 4. Inltration of innate immune cells into the bladder aer intravesical instillation with PBS, BCG,
or baculoviral vectors. ree bladder samples per group were collected 16 hours aer the third instillation for
ow cytometric analysis. Neutrophils were dened as live CD45.2+Ly-6G+ cells; inammatory monocytes were
dened as live CD45.2+Ly-6G−Ly-6ChighCD11b+ cells. Representative FACS plots of two repeated experiments
are shown.

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and subsequent inammatory responses within the bladder. Virus antigens can be presented on the surfaces
of transduced urothelial and antigen-presenting cells in the context of MHC class II to stimulate CD4+ T cells.
Intracellular viral antigens will predominantly induce 1 immune response, resulting in secretion of 1-type
cytokines. Indeed, we have observed the induction of an array of cytokines by intravesical instillation of bacu-
lovirus. ese baculovirus-induced cytokines include 1-type of cytokines such as IFN-gamma, IL-2, TNF-α ,
and IL12 as well as 2-type of cytokines IL-13 and IL-10. Although the specic role of each of these cytokines in
orchestrating baculovirus-induced anti-tumor immunity is not clear at this point, a high-level expression of 1
cytokines has been observed to be associated with BCG responders and eective BCG therapy of bladder cancer
depends largely on proper induction of the 1 immune pathway. Baculovirus DNA contains abundant CpG
motifs32,33, and CpG stimulates host production of IL-12 and drives 1 immune responses, providing potential
therapeutic value in treating cancer.
Previous studies have further demonstrated that baculovirus potentiates adaptive immune responses by trans-
ducing professional antigen-presenting cells and inducing INF-α and INF-β
28,29,31,39. e initiation of an adaptive
immune response is critically dependent on the activation, functional maturation and migration of dendritic
cells. Baculovirus can transduce dendritic cells, activate these cells via the interaction with intracellular toll-like
receptor 9, and promote their maturation28,29,31,32,37,39. ese immune stimulatory eects can possibly be har-
nessed to promote systemic anti-tumor immunity. is hypothesis is supported by the current study using a
syngeneic model system that provides intact immune functions and allows the study of therapeutic vaccines
against bladder cancer. With this mouse model, we demonstrated that tumor-bearing animals that were cured by
baculovirus instillation survived aer the 2nd tumor challenge.
To test the idea of using wild-type baculovirus as an adjunct in the design of antitumor therapies, Takaku
et al. have examined the eects of intravenously injected baculoviruses that do not express foreign genes on
anti-tumor immunity in immunocompetent mice with B16 mouse melanoma and demonstrated the activation of
NK cell-dependent antitumor immunity by baculovirus30. Further studies from the same laboratory indicate that
baculovirus-induced antitumor action possibly also involves acquired immunity by enhancing tumor-specic
cytotoxic T lymphocyte (CTL) responses and tumor-specic antibody production31,39. ese ndings highlight
Figure 5. Inltration of T cells into the bladder aer intravesical instillation with PBS, BCG or baculoviral
vectors. ree bladder samples per group were collected 16 hours aer the third instillation for ow cytometric
analysis. T cells were gated as live CD45.2+CD3ε
+NK1.1− cells. e gated T cells were further gated as γ δ TCR
positive or negative cells, and the latter population was assessed for CD4 or CD8a expression. Representative
FACS plots of two repeated experiments are shown. Bottom: Average numbers of T cell subpopulations are
shown.

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that the intrinsic immunostimulatory property of baculovirus can possibly be favorably exploited for cancer
immunotherapy. However, since baculoviruses are highly sensitive to the inactivation by serum complement pro-
teins34, systemically delivered baculoviral vectors usually do not transduce target cells. In the above studies from
Takaku’s lab, high titers of baculoviruses were used to overcome the ability of the complement to neutralize the
viruses, yet there was no demonstration of baculovirus-mediated transgene expression30,31,39.
As shown in the current study, the ecacy of baculovirus-mediated bladder cancer therapy was further
improved by including therapeutic genes into baculoviral vectors. In this regard, we showed the strong anti-tumor
eects of baculoviral vector-mediated CD40L or IL-15 expression in an animal model with aggressively growing
bladder cancer. Local transduction, either in normal or malignant bladder cells, that suciently activates only
targeted cytokines would be an attractive strategy to improve the eciency of immunotherapy and to diminish
possible side eects associated with systemic exposure. Using an intravesical liposomal gene delivery approach in
a mouse bladder cancer model, IL-15 gene therapy has been demonstrated to be a new promising approach for
bladder cancer treatment40. Adenoviral vectors encoding IL-15 were also found to be able to abolish tumorigenic-
ity of murine bladder tumor MB4941. Following preclinical testing in experimental bladder cancer animal models,
a Phase I/IIa clinical trial using adenoviral vector expressing CD40L for immunogene therapy of bladder cancer
has been performed and demonstrated a boosted immune activation14.
CD40L, also called CD154, is a potent 1 immune stimulator. CD40L is mainly expressed on activated CD4+
T cells and interacts with CD40 expressed on a wide range of antigen-presenting cells (APCs) and malignant
cells42. CD40/CD40L ligation may activate APCs, stimulating their maturation to present antigens to T cells and
ensuring the generation of antigen-specic CTLs. CD40L stimulation can also activate secretion of cytokines,
such as IL-12 and IFN-γ , leading to a 1 response, and abrogate the suppressive eect of T regulatory cells.
Moreover, CD40 expression has been found on various tumor cells, and CD40/CD40L ligation can inhibit pro-
liferation and induce apoptosis directly in tumor cells by activation of NF-κ B, AP-1, CD95, or caspase-depended
Figure 6. T lymphocyte subset analysis upon repeated intravesical instillations with PBS, baculoviral
vectors or BCG. ree mouse bladder samples per group were collected 16 hours aer the third instillation
and cut into small pieces for enzyme digestion. e collected bladder cells were stained with CD4, CD8, CD62L
and CD44 antibodies conjugated with uorescence, and the results were analyzed by ow cytometry. (A)
CD4+ (le) and CD8+ (right) cells were gated for T lymphocyte subset analysis. Naïve T cells were dened as
CD44lowCD62Lhigh, memory T cells as CD44highCD62Lhigh and eector T cells as CD44highCD62Llow.
FACS plots shown are representative of two independent experiments. (B) Average numbers of CD4+ (le) and
CD8+ (right) T lymphocyte subsets.

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pathways43. Acting both as an activator of immune cells and cancer cell death inducer, CD40L is currently under
intensive investigation for its potent anti-tumor eects.
IL-15 is a 15-kDa cytokine member in the IL-2 family. While IL-15 has pleiotropic immune-enhancing
activities, it plays a pivotal role in the generation and maintenance of memory CD8+ T cells and NK cells44. A
rst-in-human Phase I clinical trial that involved recombinant IL-15 in patients with refractory metastatic malig-
nant melanoma and metastatic renal cell cancer has just completed45. e study revealed that NK cells and γ δ T
lymphocytes in blood were most dramatically aected, followed by CD8+ memory cells, by IL-15 administration.
Since IL-15 activates anti-tumor eectors such as NK cells, γ δ T lymphocytes, and memory phenotype CD8+ T
cells and exerts more long-lasting antitumor eects, it might be especially suitable for immunotherapy of NMIBC.
When viewed from the interaction perspective, IL-15 can induce CD40 expression on conventional DCs
and interaction between CD40 on conventional DCs and CD40L on plasmacytoid DCs leads to IL-12 produc-
tion by conventional DCs, which is essential for CpG-induced immune activation46. On the other hand, stim-
ulation of monocytes or DCs with the ligation of CD40 with CD40L or an agonistic anti-CD40 monoclonal
antibody can coordinately induce IL-15 and IL-15Ra expression. IL-15Ra on the cell surfaces of monocytes
or DCs can then present IL-15 in trans to cells such as CD8+ memory T cells and NK cells that express IL-2/
IL-15Rb47. Also, IL-15 can cooperate with CD40L to increase growth of normal and follicular lymphoma
B-cells48. Although the mechanism of their interaction is still unclear, the baculoviral transduction-mediated
co-expression of IL-15 and CD40L, as demonstrated in the current study, exhibits potent therapeutic ecacy
for bladder cancer.
e bladder is a conned compartment in which an immunotherapy agent can be given in a high concentra-
tion to eectively recruit immune cells and activate them locally, thus serving as an ideal target organ for immu-
notherapy. Normally, the bladder can be regarded as an organ that is not inltrated by large numbers of immune
cells3,49. is study shows that following intravesical baculoviral transduction, the bladder is inltrated by mono-
nuclear leukocytes, including inammatory monocytes, CD4+, CD8+, and γ δ T lymphocytes. We focused mainly
on immune cell inltration aer repeated instillations since this is a clinically relevant scheme. is inltration
pattern of immune cells aer repeated instillations of baculoviral vectors is in general similar to the one observed
following intravesical BCG instillation3,49. However, instillation with baculoviral vectors expressing CD40L
and IL-15 signicantly increased the inux of CD3+ T lymphocytes with higher absolute numbers of CD4+,
CD8+, and γ δ T lymphocytes. is increase is much more pronounced when looking at γ δ T lymphocytes, the
non-MHC-restricted cytotoxic cells that can initiate cytotoxicity aer infection, even though BCG is known to
be able to induce the proliferation of γ δ T lymphocytes50. Also noteworthy, intravesical instillation of baculovirus
alone was able to increase the number of γ δ T lymphocytes in the bladder. While the antitumor activity of bacu-
loviral transduction in the current bladder cancer model possibly depends on the interplay of dierent immune
cells, the correlation between increased numbers of γ δ T lymphocytes in the bladder and anti-cancer ecacies
achieved with dierent therapeutics used for bladder instillation indicates that this type of cytotoxic eector cells
play an important role in treatment success.
In conclusion, we have demonstrated in a syngeneic orthotopic animal model of bladder cancer that insect
baculovirus can be used as a new agent for bladder cancer therapy with such functions as stimulating local innate
immune reactions, promoting systemic anti-tumor immune responses, and delivering therapeutic genes. When
tested as an adjuvant therapy for urothelial malignancies, the success of the approach will benet bladder cancer
patients who fail conventional BCG immunotherapy or who are intolerant of BCG treatment. When used as a
single therapy, this intravesical immunotherapy approach may improve bladder preservation and the quality of
life for patients, thus delivering better health outcomes. Hence, intravesical instillation of recombinant baculoviral
vectors holds potential to develop into an advanced therapy for NMIBC.
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Acknowledgements
is work was supported by the Singapore Ministry of Health’s National Medical Research Council (NMRC/
CIRG/1367/2013; NMRC/CIRG/1406/2014) and Institute of Bioengineering and Nanotechnology (Biomedical
Research Council, Agency for Science, Technology and Research, Singapore).
Author Contributions
W.X.A, Y.Z., T.K., C.W. and C.C. collection and assembly of data, data analysis and interpretation, and manuscript
writing; H.C.T., R.M. and K.E. conception, design, and manuscript preparation. S.W. conception, design,
manuscript preparation, and grant support. All authors reviewed the manuscript.

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Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Ang, W. X. et al. Local Immune Stimulation by Intravesical Instillation of Baculovirus
to Enable Bladder Cancer erapy. Sci. Rep. 6, 27455; doi: 10.1038/srep27455 (2016).
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