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GENE THERAPY
From the Departments of Medicine, Neurology,
Microbiology and Immunology, University of Rochester Cancer Center; and
the Center for Aging and Developmental Biology, University of Rochester
School of Medicine and Dentistry, Rochester, NY.
Herpes simplex virus (HSV)-based vectors have favorable biologic
features for gene therapy of leukemia and lymphoma. These include high
transduction efficiency, ability to infect postmitotic cells, and large
packaging capacity. The usefulness of HSV amplicon vectors for the
transduction of primary human B-cell chronic lymphocytic leukemia (CLL)
was explored. Vectors were constructed encoding Chronic lymphocytic leukemia (CLL) is a
malignancy of mature-appearing small B lymphocytes that closely
resemble those in the mantle zone of secondary lymphoid
follicles.1 CLL remains a largely incurable disease of the
elderly; its incidence of more than 20 per 100 000 persons older than
70 makes it the most common leukemia in the United States and Western
Europe. CLL, which arises from an antigen-presenting B cell that has
undergone a nonrandom genetic event Application of these insights to gene therapy for CLL has been
hampered by difficulties in developing a safe and reliable vector
capable of transducing primary leukemia cells. In contrast to tumor
cell lines, CLL cells are effectively postmitotic, and only a small
fraction actively enter the cell cycle.14 Although both
retroviral and adenoviral vectors have been used in different clinical
trials for cancer gene therapy, both systems exhibit limitations.15-19 For example, the low levels of integrin
receptors for adenovirus on CLL cells mandates the use of high
adenovirus titers, preactivation of the CLL cell with IL-4 or
anti-CD40/CD40L,20,21 or adenovirus modification with
polycations to achieve clinically meaningful levels of transgene
expression.22
Gene therapy vectors based on the herpes simplex virus (HSV) offer a
number of advantages for gene therapy of CLL.23,24 These
include broad cellular tropism, large DNA packaging capacity that
allows for expression of multiple genes, high transduction efficiency,
and episomal vector genome maintenance which should be less prone to
insertional mutagenesis. Historically, the development of HSV vectors
for clinical applications has been slowed by the complexities inherent
in manipulating a large viral genome (150 kb) coupled with toxicities
attributable to HSV-encoded regulatory and structural viral
proteins.25
One type of HSV vector, the amplicon, is essentially a eukaryotic
expression plasmid that contains the following genetic elements: (1)
HSV-derived origin of DNA replication (ori) and packaging sequence
("a" sequence); (2) transcriptional unit driven typically by the
HSV-1 immediate early (IE) 4/5 promoter or an alternative promoter
followed by an SV-40 polyadenylation site; and (3) bacterial origin of
replication and antibiotic resistance gene for propagation in
Escherichia coli.25,26 Amplicon plasmids are
dependent on helper virus function to provide the replication machinery
and structural proteins necessary for packaging amplicon vector DNA into viral particles. Helper packaging function is usually provided by
a replication-defective virus that lacks an essential viral regulatory
gene. The final product of helper virus-based packaging contains a
mixture of varying proportions of helper and amplicon virions.
Recently, helper virus-free amplicon packaging methods were developed
by providing a packaging-deficient helper virus genome through a set of
5 overlapping cosmids27 or by using a bacterial artificial
chromosome (BAC) that encodes the entire HSV genome minus its cognate
cleavage/packaging signals.28,29
The HSV-based amplicon represents an attractive gene delivery platform
for CLL given the known expression of the HSV receptor Hve-A (formerly
HVEM/TR2 and ATAR) on B cells.30 In this report, we
compared 2 amplicon vector preparations as potential modalities for
immunotherapy of CLL Cell culture and CLL cell transduction using HSV amplicon
vectors
CLL cells (106) purified by density centrifugation as
described above were suspended in 100 µL RPMI with 10% human AB
serum in a 1.5-mL Eppendorf tube. An aliquot of 10 to 20 µL HSV
amplicon vector was added to the cell suspension and mixed well before incubation at 37°C for 2 to 3 hours. After this initial incubation, 900 µL medium was added to each vial, and the cells were maintained in culture for another 24 hours before they were washed and tested by
flow cytometry or used as stimulators in a mixed lymphocyte tumor
reaction (MLTR).
Amplicon construction
Helper virus-based amplicon packaging Amplicon DNA was packaged into HSV-1 particles by transfecting 5 µg plasmid DNA into RR1 cells with Lipofectamine as recommended by the manufacturer (Gibco-BRL). After 24-hour incubation, the transfected monolayer was superinfected with the HSV strain 17-derived IE3 deletion mutant virus D30EBA34 at a multiplicity of infection of 0.2. Once cytopathic changes were observed in the infected monolayer, the cells were harvested, freeze-thawed, and sonicated using a cup sonicator (Misonix). Viral supernatants were clarified by centrifugation at 5000g for 10 minutes before repeat passage on RR1 cells. This second viral passage was harvested as above and was concentrated for 2 hours by ultracentrifugation on a 30% sucrose cushion as previously described.35 Viral pellets were resuspended in phosphate-buffered saline (PBS; Ca++ and Mg2+ containing) and were stored at 80°C for
future use.
Helper virus-free amplicon packaging Amplicon stocks were also prepared using a modified helper virus-free packaging method. The packaging system uses BAC (kindly provided by C. Strathdee) that contains the HSV genome without its cognate pac signals as a cotransfection reagent with amplicon DNA. Because the amplicon vector has pac signals, only the amplicon genome is packaged. Briefly, on the day before transfection, 2 × 107 baby hamster kidney cells were seeded in a T-150 flask and incubated overnight at 37°C. On the day of transfection, 1.8 mL Opti-MEM (Gibco-BRL), 25 µg pBAC-V2 DNA,28 7 µg pBS(vhs), and 7 µg amplicon vector DNA were combined in a sterile polypropylene tube. Seventy microliters Lipofectamine Plus Reagent (Gibco-BRL) was added to the DNA mix over a period of 30 seconds and allowed to incubate at 22°C for 20 minutes. In a separate tube, 100 µL Lipofectamine (Gibco-BRL) was mixed with 1.8 mL Opti-MEM and also incubated at 22°C for 20 minutes. After the incubations, the contents of the 2 tubes were combined over a period of 30 seconds and incubated for an additional 20 minutes at 22°C. During this second incubation, the media in the seeded T-150 flask were removed and replaced with 14 mL Opti-MEM. The transfection mix was added to the flask and allowed to incubate at 37°C for 5 hours. Then the transfection mix was diluted with an equal volume of DMEM plus 20% FBS, 2% penicillin-streptomycin, and 2 mM hexamethylene bis-acetamide and was incubated overnight at 34°C. The next day, the media were removed and replaced with DMEM plus 10% FBS, 1% penicillin-streptomycin, and 2 mM hexamethylene bis-acetamide. The packaging flask was incubated 3 additional days before the virus was harvested and stored at80°C
until purification. Viral preparations were subsequently thawed,
sonicated, clarified by centrifugation, and concentrated by
ultracentrifugation through a 30% sucrose cushion. Viral pellets were
resuspended in 100 µL PBS (Ca++ and Mg2+
containing) and stored at 80°C for future use.
Virus titering Helper virus-containing stocks were titered for helper virus by standard plaque assay methods.36 Amplicon titers for both helper virus-based and helper-free stocks were determined as follows: NIH 3T3 cells were plated in a 24-well plate at a density of 1 × 105 cells/well and infected with the virus. Twenty-four hours after viral infection, the monolayers were washed twice in PBS and either fixed with 4% paraformaldehyde and stained by X-gal histochemistry (HSVlac, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 0.02% NP-40, 0.01% sodium deoxycholic acid, 2 mM MgCl2, and 1 mg/mL X-gal dissolved in PBS) or harvested for total DNA using lysis buffer (100 mM NaCl, 10 mM Tris, pH 8.0, 25 mM EDTA, 0.5% SDS) followed by phenol-chloroform extraction and ethanol precipitation. Real-time quantitative PCR was performed on duplicate samples using primers corresponding to the -lactamase gene
present in the amplicon plasmid, according to a previously published
method.37 Total DNA was quantitated, and 50 ng DNA was
analyzed in a PE7700 quantitative PCR reaction using a designed
-lactamase-specific primer-probe combination multiplexed with an
18S rRNA-specific primer-probe set. The -lactamase probe sequence
was 5'-CAGGACCACTTCTGCGCTCGGC-3'; the -lactamase sense primer
sequence was 5'-CTGGATGGAGGCGGATAAAGT-3'; and the -lactamase
antisense primer sequence was 5'-CGGCTCCAGATTTATCAGCAAT-3'. The 18S
rRNA probe sequence was 5'-TGCTGGCACCAGACTTGCCCTC-3'; the 18S sense
primer sequence was 5'-CGGCTACCACATCCAAGGAA-3'; and the 18S antisense
primer sequence was 5'-GCTGGAATTACCGCGGCT-3'. Helper virus titers
(pfu/mL), amplicon expression titers (bfu/mL), and amplicon
transduction titers (TU/mL) obtained from these methods were used to
calculate amplicon titer and thus standardize experimental viral
delivery. Amplicon titers of the various virus preparations ranged from
4 to 5 × 108 bfu/mL, whereas helper titers were in the
range of 5 to 15 × 107 pfu/mL.
Mixed lymphocyte tumor reaction assay CLL cells were transduced with equal transduction units of helper virus-containing or helper virus-free amplicon stocks, irradiated (20 Gy), and used as stimulators (2.5 or 5 × 104 cells/well) with allogeneic normal donor T cells (2 × 105 cells in a final volume of 200 µL) in 96-well flat-bottom plates. All cultures were grown in triplicate. Cells were incubated for 5 days at 37°C in 5% CO2. Cells were pulsed with 1 µCi [3H]-thymidine for the last 18 hours of the culture period before they were transferred to a glass fiber filter, and radioactive counts were measured by liquid scintillation counting. To determine the requirement for MHC class I-tumor antigen and T-cell receptor interaction (Signal 1), CLL cells were infected with equivalent transduction units of HSVlac, HSVB7.1, hf-HSVlac, or hf-HSVB7.1 and were used as stimulators as described above, with or without phorbol 12-myristate 13-acetate (PMA) added to a final concentration of 10 ng/mL.ELISA for IL-2 and Generation of anti-CLL-specific cytotoxic T-lymphocyte activity T cells were isolated from peripheral blood mononuclear cells (PBMCs) cells using T-cell enrichment column (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions and cultured at 1 × 106 cells/mL in RPMI with 10% human AB serum in a 6-well plate. Autologous CLL cells either were infected with hf-HSVlac or hf-HSVCD40L or were left uninfected before they were added to the T cells in the 6-well plate at a ratio of 4:1 T cells-CLL cells. IL-2, at a concentration of 100 U/mL, was added to the wells after 3 days in culture. After 1 week in culture, 1 × 106 freshly thawed CLL cells (uninfected, hf-HSVlac or hf-HSVCD40L infected) were added to the T cells, and the culture was maintained for a second week of priming. IL-2 (100 U/mL) was again added to the T-cell culture 3 days later (day 10 of priming). After 2 weeks of priming, the T cells were harvested, washed twice in PBS, and counted. T cells were subsequently tested in a standard chromium-release cytotoxic T lymphocyte (CTL) assay as described below.Cytotoxic T-lymphocyte assay After 2 weeks of in vitro priming, T cells were incubated with freshly thawed autologous CLL cells either without or with anti-MHC class I (W6/32) at 10 µg/mL. A cytotoxicity assay was performed by incubating primed T cells with 1 × 104 chromium 51Cr-labeled CLL cells in a V-shaped 96-well plate at varying effector-target ratios. Spontaneous release was measured by incubating 51Cr-labeled CLL cells alone, and maximum release was calculated by lysing the cells with 2% Triton-X. After a 6-hour incubation, the supernatant was collected and radioactivity was measured using a -counter (Packard Instrument). Mean values were
calculated for triplicate wells, and the results were expressed as
percentage specific lysis according to the formula: [experimental
counts spontaneous counts]/[total counts spontaneous
counts × 100].
HSV amplicon-mediated gene transfer into CLL cells The usefulness of HSV-based amplicon vectors for the transduction of primary human B-cell chronic lymphocytic leukemia (CLL) was examined. HSV amplicon vectors encoding-galactosidase
(LacZ), CD80 (B7.1), or CD154 (CD40L) were packaged using
either a standard helper virus (designated HSVlac, HSVB7.1, and
HSVCD40L) or a helper virus-free method (designated hf-HSVlac,
hf-HSVB7.1, and hf-HSVCD40L).
CLL cells were isolated by density gradient centrifugation and 97% or
more of the cells stained for CD19, a cell-surface marker for B
lymphocytes. The cells were transduced with either HSVlac, HSVB7.1,
hf-HSVlac, or hf-HSVB7.1. X-gal histochemistry was performed to detect
the
Effect of helper virus on host cell MHC class I expression Although both vector preparations were able to drive high-level expression of B7.1 in CLL cells, there existed the possibility that helper virus-containing amplicon preparations disrupted MHC class I-mediated antigen presentation. ICP-47, a gene present in the D30EBA helper virus, encodes a protein that blocks TAP-1-mediated peptide loading into MHC class I. Inhibition of MHC class I expression would reduce the usefulness of HSV amplicon vectors for immunotherapeutic strategies. To examine this possibility, CLL cells were transduced with HSVB7.1 or hf-HSVB7.1 and examined by flow cytometry for levels of B7.1 and MHC class I expression. Significant down-regulation of MHC class I in CLL cells transduced with HSVB7.1 was observed compared with MHC class I expression in uninfected cells (Figure 2). In contrast, transduction with hf-HSVB7.1 resulted in high levels of B7.1 expression and maintenance of MHC class I surface expression on B7.1-transduced cells. MHC class II levels were unaffected after HSV infection, as detected by flow cytometry (data not shown). These data highlight the role of HSV-encoded factors in the modulation of host immunity and underscore a fundamental difference in the immunotherapeutic potential between helper virus-based and helper virus-free amplicon preparations.
Allogeneic T-cell activation by HSV amplicon-transduced CLL cells To assess functional differences in antigen presentation after transduction with helper virus-containing or helper virus-free amplicon stocks, the effects of B7.1 transduction on the ability of CLL cells to stimulate T-cell proliferation in an allogeneic MLTR were analyzed. CLL cells were transduced with either HSVlac, HSVB7.1, hf-HSVlac, or hf-HSVB7.1, and transduced cells served as stimulators in an allogeneic MLTR using T cells from a normal donor. hf-HSVB7.1-transduced CLL cells were able to directly stimulate T-cell proliferation (Figure 3). In spite of amplicon-directed expression of B7.1 on 70% or more of the CLL cells, HSVB7.1-transduced CLL cells failed to elicit a T-cell proliferative response, suggesting that the antigen-presenting capacity of the infected CLL cells had been seriously impaired. This could have occurred through the loss of MHC class I expression, as shown in Figure 2, or through some other mechanism mediated by the helper virus. PMA was used to provide an extrinsic Signal One to potentially compensate for the adverse effect elicited by the helper virus on CLL cells, thereby allowing transduced B7.1 to elicit a costimulatory signal to T cells. The addition of PMA resulted in significant proliferation in HSVB7.1-infected cells relative to nontransduced or HSVlac-transduced CLL cells. PMA treatment also augmented proliferation in hf-HSVB7.1-transduced CLL cells, suggesting that the full stimulatory potential of T-cell activation by these transduced cells was not fully achieved by helper virus-free vector delivery alone.
IL-2 secretion serves as another correlate to T-cell activation.
Supernatants collected from the MLTR samples described above were
analyzed using IL-2 ELISA. IL-2 levels were highest when hf-HSVB7.1-transduced CLL cells were utilized as T-cell stimulators (Table 1) in comparison with HSVB7.1 or
HSVlac-transduced cells. In other mLTR assays using HSVB7.1-transduced
CLL cells, IL-2 secretion was dependent on the provision of Signal One
through PMA (data not shown), as was observed with the PMA-mediated
rescue of T-cell stimulation (Figure 3).
Up-regulation of costimulatory molecules on CLL cells transduced by HSV amplicons Engagement of the CD40 receptor on APCs is a critical step in the initiation of an immune response. Up-regulation of costimulatory molecules on CLL cells induced by CD40 receptor signaling correlates with a cell's ability to function as an APC.38,39 We selected the induction of B7.1 expression as a surrogate marker for the morphologic changes induced by CD40 receptor engagement in CLL cells. To test for paracrine and autocrine induction of B7.1, CLL cells were transduced with either hf-HSVCD40L or hf-HSVlac, incubated for 6 days, and subsequently analyzed for the expression of endogenous B7.1. As shown in Figure 4, transduction with hf-HSVCD40L resulted in the up-regulation of B7.1 on CLL cells compared with untransduced and hf-HSVlac-transduced cells. In some experiments, minimal but detectable up-regulation of B7.1 was observed in hf-HSVlac-transduced cells compared with untransduced cells (Figure 4A-B).
The percentage of CLL cells expressing B7.1, CD40L, or both was
quantitated by 2-color flow cytometry (Table
2). Although infection of CLL cells with
HSVCD40L resulted in more than 70% of the cells expressing CD40L, the
percentage of cells expressing endogenous B7.1 did not increase
appreciably over background levels observed in cells transduced with
control HSVlac vector. In contrast, CLL cells transduced with
hf-HSVCD40L exhibited a marked enhancement of B7.1 expression
relative to either hf-HSVlac- or HSVCD40L-transduced CLL cells. The
discrepancy at the level of endogenous B7.1 expression between CLL
cells transduced with HSVCD40L and hf-HSVCD40L cannot be attributed to
different efficiencies of transduction because both groups expressed
similar levels of CD40L. Similar experiments using CD19 expression as
an endogenous cell marker confirmed that an inverse relation existed
between surface CD19 expression and CD40L expression in cells
transduced with helper virus-containing HSVCD40L (data not shown), but
not in cells transduced with hf-HSVCD40L. These data suggested that
transduction with HSVCD40L resulted in a decrease in expression level
of endogenous B7.1.
The ability of CLL cells transduced by CD40L to serve as stimulators in
an allogeneic MLTR was examined. CLL cells were transduced with
hf-HSVlac, hf-HSVCD40L, HSVlac, or HSVCD40L, and they were incubated
for 4 to 6 days to allow for the up-regulation of costimulatory molecules and then were used as stimulators in an allogeneic mLTR. Although similar levels of CD40L expression were observed after transduction with either HSVCD40L or hf-HSVCD40L, cells transduced with
hf-HSVCD40L were more potent T-cell stimulators than those transduced
with HSVCD40L or control vectors (Figure
5).
Stimulation of autologous CTL activity by hf-HSVCD40L-transduced CLL cells Because CLL cells transduced with helper-containing HSV amplicons down-regulated MHC class I expression and were inefficient at stimulating T-cell proliferation in an allogeneic MLTR, we restricted our efforts to generate autologous CTL response to cells transduced with the helper virus-free amplicon stocks. T cells were purified from patients' PBMCs, as described in "Materials and methods," and were cocultured for 1 week with either freshly isolated CLL cells or with CLL cells transduced with either hf-HSVlac or hf-HSVCD40L at a T-cell-CLL cell ratio of 4:1. IL-2 (100 U/mL) was added for the last 4 days of culture. This protocol was repeated for a second cycle of priming (week 2), after which the T cells were washed and tested for CTL activity in a 4-hour chromium release assay. Three parallel CTL assays were performed using either freshly thawed autologous CLL cells as target, freshly thawed autologous CLL cells with anti-MHC class I blocking antibody (W6/32 at 10 µg/mL), or CLL cells obtained from an unrelated donor. Specific killing was observed using the patient's own CLL cells (Figure 6A). This activity was blunted when anti-MHC class I blocking antibody was added to the assay (Figure 6B). T-cell-mediated killing was essentially undetectable using CLL cells derived from a different donor as targets (Figure 6C), further confirming the specific reactivity of the primed T cells. Supernatant from an autologous T-cell-CLL coculture was assayed for-interferon levels by ELISA (Table 3).
Higher levels of -interferon were detected in supernatants derived
from coculture with hf-HSVCD40L-transduced CLL cells compared to
nontransduced and hf-HSVlac-transduced control cells.
The development of safe, reliable, and effective vectors for gene delivery into tumors is prerequisite to translate advances in tumor biology and immunology into clinical practice. The HSV amplicon was chosen as a vector for gene transfer into CLL cells for a number of reasons, central among which is the virus' wide tropism of infection. The mechanism by which HSV gains entry into the target cell has been studied in detail. HSV cell entry requires an initial binding step mediated by viral glycoproteins gC and gB interacting with heparan sulfate chains on the cell surface.40-42 This is followed by penetration through the viral glycoproteins gB and gD and the hetero-oligomer gH-gL.43-46 Cellular receptors for some of these HSV surface glycoproteins have
been cloned, including the herpesvirus entry mediator A
(Hve-A),30,47,48 Hve-B,49 Hve-C, and
Hve-D.50 Hve-A, a 230-amino acid type I transmembrane
glycoprotein, is a member of the tumor necrosis factor receptor (TNFR)
family, whereas Hve-B, Hve-C, and Hve-D are members of the
immunoglobulin superfamily. Hve-A is expressed on T cells, B cells, and
monocytes and has recently been identified by flow cytometry on the
surfaces of CLL cells.51 Similar to CD40, Hve-A lacks the
death domain resident in Fas and TNFR-I and is believed to stimulate
T-cell proliferation. The native ligand for Hve-A (HVEM/TR2) has
recently been identified as LIGHT,52,53 a type II membrane
protein member of the TNF family. Signaling through Hve-A activates
NF- In this paper, data are presented on 2 HSV amplicon vector preparations for potential use in immune therapy of leukemia and lymphoma. Helper virus-containing and helper virus-free amplicon stocks were equivalent at transducing primary CLL cells. However, we found the conventional amplicon packaging methods using helper virus to have a number of potential disadvantages, including exposing the target cell to cytotoxic effects of the helper virus induced by the proapoptotic effects of the immediate-early proteins.56-58 Loss of MHC class I molecules because of helper virus-encoded ICP47 is counterproductive for immune therapy-based strategies. As a result, the ability of tumor cells to provide tumor-specific antigen necessary for the generation of specific CTLs can be impaired.59,60 We used an ICP-4 mutant virus (D30EBA) to provide helper function for
amplicon packaging. D30EBA exhibits significantly reduced expression of
both early and late viral genes; however, the remaining nondisrupted,
immediate early (IE) genes accumulate in the infected cell, and their
gene products likely cause the observed problems. One of these IE
genes, ICP0, has been shown to arrest cell cycle in the G1/S and G2/M
junctions through both p53-dependent and -independent pathways that
involve the induction of p21, Gadd45, and mdm-2.61 Loss of
MHC class I expression in cells infected with helper virus-containing
amplicon stocks is attributable to the interaction of HSV ICP47 gene
product with the transporter associated with antigen processing (TAP)
protein.59,60 Peptide fragments generated in the cytosol
by the proteasome complex are transported by TAP into the endoplasmic
reticulum (ER) where they are loaded into MHC class I heavy chain and
Loss of MHC class I expression was observed in our studies using helper virus-containing stocks, and our attempts to stimulate allogeneic T-cell proliferation using CLL cells transduced with HSVB7.1 were unsuccessful despite high expression of B7.1 on stimulator cells (Figure 1). We hypothesize that the loss of MHC class I on infected cells was partially responsible for the decreased proliferative response despite the costimulatory signal provided by transduced B7.1. Provision of Signal One extrinsically through PMA restored the T-cell proliferative response and IL-2 production. Transduction of CLL cells with helper virus-free HSV amplicon stocks maintained preinfection levels of MHC class I surface expression on transduced cells and was sufficient to stimulate T-cell proliferation even in the absence of PMA. Helper virus-expressed proteins can additionally derail host cell
protein synthesis. Vhs is a 58-kd tegument protein that has an
RNase-like activity. In cells infected with HSV-1 or HSV-2, 90% of
cellular mRNA and protein synthesis are lost within 3 hours after
infection, which leads to a shift in translational preference to
virus-encoded transcripts. This grants the virus full access to the
host protein machinery without competition from host
cell-encoded mRNA species. The vhs protein plays a critical role
in allowing HSV to evade the host immune response and subsequently
establish latency.64,65 This is supported by multiple
lines of evidence. First, dendritic cells (DCs) infected with HSV
strains that express a wild-type version of vhs have significantly
reduced capacity to differentiate into mature DCs, stimulate allogeneic
T cells, release several cytokines (TNF- The HSV life cycle involves long periods of latency. To that end, the
virus has evolved a number of elaborate and highly efficient mechanisms
to avoid detection and elimination by immune cells.71 The
same features that allow HSV to avoid detection by the host immune
response would be counterproductive with an HSV-based vector to help
eradicate an already established tumor with its own immune evasive
strategies. This observation has led to our efforts to eliminate the
helper virus and was subsequently borne out by our experience using
helper virus-containing amplicon preparations. Our findings using
helper virus-containing amplicon stocks parallel those reached by 2 other laboratories using HSV vectors to target DCs.66,72
Both groups showed that the APC function of DCs was seriously impaired
by infection with either replication-deficient or wild-type virus.
Deletion of vhs from the viral genome was necessary for restoration of
DC-mediated cytokine secretion (IL-6, IL-10, and TNF- When considering gene delivery vectors, one must take into account the efficiency of transduction and the potential biologic interactions of the vector proteins with the host cell. The functional discrepancies observed between helper virus-containing and helper virus-free amplicon stocks in spite of comparable transduction efficiency illustrate this point. The use of helper virus-free stocks addresses most of the potential disadvantages seen with helper virus-containing stocks by eliminating the need for helper virus during the packaging process. Extensive safety and efficacy assessments are undoubtedly required before translation to the clinical arena. However, one can envision the potential use of helper virus-free amplicon preparations as tumor-specific vaccines through the transduction of leukemia cells ex vivo with immune effector molecules such as B7.1 and CD40L. An alternative approach would involve direct in situ injection into accessible tumors. Given the wide tissue tropism characteristic of HSV, this approach is equally applicable for nonhematologic malignancies. The large packaging capacity of the HSV amplicon would allow the codelivery of multiple effector molecules for combination immune therapy. Another advantage of the helper virus-free system is that its lack of expression of viral proteins after transduction minimizes the risk for generating anti-HSV immune responses that could abrogate efficacy on repeated vector delivery. This is particularly advantageous when designing clinical applications for a chronic disease such as CLL or non-Hodgkin lymphoma for which treatment would likely require repeated vector administration over long periods of time. In aggregate, helper virus-free HSV amplicon preparations are promising vectors for use in immune-based gene therapy.
We thank Dr Mark Gilbert (Immunex, Seattle, WA) for providing the murine CD40L cDNA and Dr Lewis Lanier (DNAX, Palo Alto, CA) for providing human B7.1 cDNA.
Submitted October 19, 2001; accepted March 22, 2001.
Supported by an LRFA fellowship and a Wilmot Foundation for Cancer Research fellowship (K.A.T.), National Institutes of Health grants 1RO1CA87978-01 (J.D.R.) and R01-NS36420A (H.J.F.), and a Leukemia/Lymphoma Society Translational Research Award (J.D.R.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Joseph D. Rosenblatt, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Rochester, NY 14642; e-mail: joe_rosenblatt{at}urmc.rochester.edu.
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© 2001 by The American Society of Hematology.
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  S. Daubeuf, D. Singh, Y. Tan, H. Liu, H. J. Federoff, W. J. Bowers, and K. Tolba HSV ICP0 recruits USP7 to modulate TLR-mediated innate response Blood, April 2, 2009; 113(14): 3264 - 3275. [Abstract] [Full Text] [PDF]
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 K. H. Yi, H. Nechushtan, W. J. Bowers, G. R. Walker, Y. Zhang, D. G. Pham, E. R. Podack, H. J. Federoff, K. A. Tolba, and J. D. Rosenblatt Adoptively Transferred Tumor-Specific T Cells Stimulated Ex vivo Using Herpes Simplex Virus Amplicons Encoding 4-1BBL Persist in the Host and Show Antitumor Activity In vivo Cancer Res., October 15, 2007; 67(20): 10027 - 10037. [Abstract] [Full Text] [PDF]
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A. Liu, A. Guardino, L. Chinsangaram, M. J. Goldstein, D. Panicali, and R. Levy Therapeutic Vaccination against Murine Lymphoma by Intratumoral Injection of Recombinant Fowlpox Virus Encoding CD40 Ligand Cancer Res., July 15, 2007; 67(14): 7037 - 7044. [Abstract] [Full Text] [PDF]
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N. P. Singh, E. S. Yolcu, D. D. Taylor, C. Gercel-Taylor, D. S. Metzinger, S. K. Dreisbach, and H. Shirwan A Novel Approach to Cancer Immunotherapy: Tumor Cells Decorated with CD80 Generate Effective Antitumor Immunity Cancer Res., July 15, 2003; 63(14): 4067 - 4073. [Abstract] [Full Text] [PDF]
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J. Briones, J. Timmerman, and R. Levy In Vivo Antitumor Effect of CD40L-transduced Tumor Cells as a Vaccine for B-Cell Lymphoma Cancer Res., June 1, 2002; 62(11): 3195 - 3199. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
del(13)(q14q23.1), trisomy 12, del(11)(q22q23), and del(6)(q21q23)
indicates PMA was added to the reaction at a final
concentration of 10 ng/mL, whereas 
indicates the absence of PMA.
[3H]-thymidine uptake data are expressed as counts per
minute (cpm). Error bars represent standard deviation. Asterisks
indicate statistically significant differences (P < .001)
as determined by single-factor analysis of variance. Data are
representative of 4 different patient samples.
), hf-HSVlac-infected (
) autologous CLL cells. Cytotoxicity assays were performed by
incubating these primed T cells with 51Cr-labeled
autologous CLL cells in the absence (A) or the presence (B) of anti-MHC
monoclonal antibody, W-6.32, or with 51Cr-labeled
allogeneic CLL cells (C) at varying effector-target (E/T) ratios.
Supernatant was collected, and radioactivity was measured using a 
B and AP-1 through interaction with TNF receptor-associated
factors (TRAF1, 2, 3, and 5)
, IL-6, IL-10, IL-12), and
express several surface markers (CD40, CD80, CD83, CD86).