Herpes simplex virus type-1 amplicon vectors for vaccine generation in acute lymphoblastic leukemia

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Abstract

For leukemia vaccine generation, high-efficiency gene transfer is required to express immunomodulatory molecules that stimulate potent antileukemic immune responses. In this context, herpes simplex virus type-1 (HSV-1)-derived vectors have proven to be a promising tool for genetic modification of lymphoblastic leukemia cells. Yet, vector-associated viral protein expression might inadvertently modulate vaccine efficacy facilitating both immune evasion and immune stimulation. To explore the issue of immune-stimulation versus immune-suppression in immature lymphoblastic leukemia cells, two types of HSV-1 amplicon vectors, helper virus-dependent and helper virus-free that express the immunomodulatory molecules CD70 and IL-2, were compared with regard to their vector-associated immunomodulatory potential. We first established that lymphoblastic cell lines and primary acute lymphoblastic leukemia (ALL) cells express HSV receptor genes. Lymphoblastic cell lines were transduced with high efficiency, and in primary ALL cells high gene transfer rates of 47±15 and 42±14% were obtained with helper virus-dependent and -free HSV-1 amplicon vectors, respectively. The efficacy of the two amplicon vectors to induce antineoplastic responses was assessed in a vaccine setting in mice with pre-existing highly malignant lymphoblastic disease. Treatment of mice with vaccine cells transgenically expressing CD70+IL2 significantly suppressed lymphoblastic cell proliferation and improved survival. Of note, when helper virus-dependent HSV-1 amplicon vectors were used for vaccine preparation, the high immunogenic potential of the vector itself, in the absence of transgenic CD70+IL2 expression, seemed to be sufficient to mediate protection comparable to the antineoplastic response achieved by expression of immunomodulatory molecules. Thus for vaccine generation in B lymphoblastic leukemia, the immunogenic potential of HSV-1 helper virus-dependent amplicon vectors does provide additional benefit to the high transduction efficiency of HSV-1-derived vectors.

Introduction

Over the last years, herpes simplex virus (HSV)-based vectors have evolved as an attractive gene transfer system for a variety of applications reaching beyond gene therapy of nervous system diseases for which these vectors were originally developed.1 The versatility of herpesviral vectors is based on three major characteristics including (i) the capacity to carry large size inserts of foreign DNA, (ii) high infectivity and broad cellular tropism which also extends to quiescent target cells and (iii) high, albeit transient levels of transgene expression.2 While HSV-based vectors have been extensively explored in antineoplastic gene-therapy approaches for solid tumors, we and others have previously shown that for all of these reasons HSV vectors are also particularly suitable for high-efficiency gene transfer into hematopoietic cells.3, 4, 5 Also, due to the ability to hold large size inserts,6 HSV vectors facilitate combined expression of two or more immunomodulatory molecules, an essential requirement for vaccine efficacy in leukemia.7, 8, 9, 10, 11 Here, we explore the use of HSV-1 amplicon vectors for gene transfer into acute lymphoblastic leukemia (ALL) cells for vaccine generation.

Having previously described the utility of HSV-2-derived recombinant vectors for high-efficiency transduction in acute leukemia for which alternative vectors are not readily available for clinical application,3 we now assess the scope of HSV-1 amplicon infectivity in lymphoblastic B-cell lines. With regard to primary human cells, we focused our analysis on ALL, the single most common childhood malignancy. The entry of HSV into mammalian cells involves HSV envelope glycoproteins B and/or C.12, 13, 14, 15 Additional receptors are critical for viral entry into target cells termed herpes virus entry mediators (Hve). HveA is a member of the tumor necrosis factor receptor family,16, 17 whereas HveB and HveC belong to proteins of the immunoglobulin superfamily.14, 18 Thus, in addition to gene transfer rates, we examined expression of all three HSV receptor genes HveA, HveB and HveC14, 16, 17, 18 following infection of primary ALL blasts with HSV-1 amplicon vectors.

In contrast to recombinant HSV vectors, for generation of HSV-1 amplicon vectors, a transgene-carrying plasmid is employed which contains only minute sequences of the HSV genome, most importantly the HSV origin of replication and the HSV packaging signal.19 Owing to a rolling mechanism of replication, multiple copies of the amplicon plasmid up to a maximum of 150 kb, the size of HSV-1 wild-type genome, can be packaged into viral particles. For HSV-1 helper virus-dependent amplicon vector generation, producer cells are infected with a replication-defective HSV-1 helper virus in addition to transduction with a transgene-carrying amplicon plasmid. Generation of replication-defective helper virus-free HSV-1 amplicon vectors is facilitated by cotransfection of the transgene-carrying amplicon plasmid with an artificial bacterial chromosome (BAC) expressing the required herpesviral helper functions in trans.20, 21, 22

When generating HSV-1 helper virus-dependent amplicon vectors, variable amounts of ‘empty’ helper virus particles are contained in the vector preparation that although not carrying the transgene, are capable of viral protein expression.23 Thus, even in the absence of viral replication, a potential disadvantage of helper virus-dependent HSV-1 amplicon vectors are target cell toxicity due to herpesviral protein expression. HSV-protein expression facilitates immune activation as well as immune escape depending on the cell type infected. In primary infection, the characteristic ability of HSV to avoid immune eradication allows the virus to establish a state of latency and to persist long term. These highly evolved immune evasion mechanisms of HSV are specifically targeted to antigen-presenting cells of both myeloid and B-cell origin and include impairment of MHC/antigen complex assembly in the endoplasmatic reticulum by the herpesviral protein ICP47 as well as downregulation of costimulatory molecules by the virion host shut-off protein (vhs).5, 24, 25 As a result, T-cell stimulatory capacity of HSV-infected antigen-presenting cells is reduced. The availability of HSV-1 amplicons entirely free of helpervirus has opened the avenue for evaluation of gene transfer efficiency in relation to immune activation and -evasion.

We chose a highly proliferative and rapidly disseminating pre-B lymphoblastic malignancy to assess the efficacy of vaccine cells genetically modified by HSV-1 helper virus-free versus helper virus-dependent amplicons. For vaccine generation, lymphoblasts were infected with both types of HSV-1 amplicons expressing the costimulatory surface molecule CD70 and the cytokine interleukin-2 (IL-2) in trans. Antineoplastic protection mediated by both amplicon vectors differing with regard to herpesviral protein expression were compared in a vaccine approach to delineate the characteristic dichotomy of HSV with regard to immune activation and escape in a model of pre-established immature lymphoblastoid disease to mimic the situation of relapsed childhood ALL.

Results

Expression of herpesviral entry receptors HveA, -B and -C in lymphoblastic cell lines

First, expression of the three characterized Hve receptors, HveA, HveB, and HveC, was investigated. Total RNA was extracted, and subjected to reverse transcription and polymerase chain reaction (RT-PCR) using HveA-, -B-, and -C-specific primers and primers for the housekeeping gene β-actin in parallel. Expression of all three Hve receptor genes were detected in the lymphoid cell lines of both B (AD and Daudi) and T-cell origin (Jurkat), as well as in the myeloid cell line HL60 (Figure 1).

Figure 1
figure1

HVE genes are expressed by lymphoblastic cell lines. RNA obtained from 2.5 × 105 of the indicated cell lines was subjected to RT-PCR using primers specific for HveA, -B and -C, respectively. As an internal reference, the primers specific for the β-actin gene were used in the same reaction. The position of Hve- and β-actin-specific PCR products is indicated. Controls were carried out without template. Results of one out of three experiments are shown.

HSV-1 amplicon-mediated expression of the surface molecule CD70 in lymphoblastic cell lines

Next, gene transfer efficiency of HSV-1 amplicon vectors was assessed. The helper virus-dependent amplicon vector HSV.CD70 encoding the murine variant of the costimulatory molecule CD70 was employed. At 24 h post transduction (p.t.), gene transfer rates were determined by flow-cytometry analysis of cell surface expression of murine CD70 (Figure 2). All B-cell lines revealed high transduction rates independent of B-cell maturity with mean values of 31±12 and 56±8% at MOI <5 and 10–20, respectively (n=3). The T-cell line Jurkat exhibited the highest transduction rates with 62±6 and 73±16% following transduction with the same viral doses. Transduction rate in the myeloblastic cell line HL60 was lower with gene transfer rates of 14±7% at MOI <5 and 32±14% at MOI 10–20.

Figure 2
figure2

HSV-1 amplicon vector transduces lymphoblastic cell lines with high efficiency. Helper virus-dependent amplicon vector HSV.CD70 was incubated with 105 cells at the indicated MOIs. At 24 h p.t., the percentage of CD70-positive cells was determined by flow-cytometry. Means and s.d. of three experiments are shown.

HSV-1 amplicon-mediated secretion of IL-2 in lymphoblastic cell lines

Next, expression of a secreted transgene after HSV-1 amplicon-mediated transduction was assessed. The lymphoblastic cell lines AD, Daudi, Jurkat and the myeloid cell line HL60, which do not constitutively express IL-2, were transduced with helper virus-dependent amplicon vector HSV.IL2 encoding the IL-2 gene. Secretion of IL-2 into the medium of transduced cells was determined by ELISA. As expected from the results obtained with transgenic CD70 expression, high level secretion of the transgene product IL-2 was observed in all cell lines 24 h following transduction with HSV-1 amplicon (Figure 3). Mean IL-2 concentrations in supernatants of transduced target cells were 2.000–4.000 pg/ml/106 cells at an MOI of 10. IL-2 expression declined thereafter, but remained >600 pg/ml/106 cells up to 72 h p.t., and is still detectable at 96 h p.t. (n=2).

Figure 3
figure3

High levels of IL-2 are secreted from HSV-1 amplicon vector-transduced lymphoblastic cell lines. Cell lines were transduced by helper virus-dependent amplicon vector HSV.IL2. The concentration of IL-2 was determined by ELISA in the supernatant of transduced cells at different time points p.t. One of two experiments using an MOI of 10 of HSV.IL2 is shown.

Expression of Hve receptor genes in primary isolates of ALL

Having established high gene-transfer rates mediated by HSV-1 amplicon vectors in different lymphoblastic cell lines, we next examined primary blasts obtained from patients with ALL. First, expression of Hve receptor genes was assessed by RT-PCR in primary isolates of 15 ALL patients diagnosed with pre-B or c-ALL. HveA, -B and -C gene expression was detected in all of the ALL isolates (Figure 4) with an Hve/β-actin ratio consistently above 0.4 for all three Hve receptors.

Figure 4
figure4

Primary isolates of ALL cells express all three Hve genes. RNA obtained from primary isolates of ALL cells were subjected to RT-PCR using PCR primers specific for HveA, -B and -C. Primers specific for the β-actin gene were used in the same PCR reaction. Quantitative determination of specific PCR product band intensity after agarose gel separation was performed by photoimaging. The ratio of Hve gene expression relative to the β-actin gene expression is shown for individual ALL cell isolates.

High HSV-1 amplicon vector-mediated gene transfer rates into primary ALL cells

As expected from the results of Hve expression, helper virus-dependent HSV-1 amplicon vector HSV.CD70 as well as helper virus-free amplicon vector BAC.CD70 led to the expression of transgene CD70 in all primary ALL cell isolates analyzed. Transduction rates as high as 65% were achieved (Figure 5). At MOIs of 10–30, mean transduction rates of 47±15 and 42±14% were observed with HSV.CD70 and BAC.CD70, respectively, indicating that both amplicon vectors can transduce primary lymphoblastic leukemia cells with equal efficiency.

Figure 5
figure5

Primary human ALL cells are transduced with equal efficiency by helper virus-dependent and helper virus-free HSV-1 amplicon vectors. Helper virus-dependent HSV-1 amplicon vector HSV.CD70 and helper virus-free amplicon vector BAC.CD70 were used for transduction of primary ALL cells. Identical MOI of helper virus-free amplicon and helper virus-dependent HSV-1 amplicon were used for transduction of cells. The percentage±s.d. of CD70-positive cells was determined by flow-cytometry 24 h p.t. (n=3–6).

Preservation of MHC class I expression in ALL cells

A potential concern when using HSV-1-based vectors in immunotherapy is impairment of MHC class I assembly by the HSV protein ICP47.26 As HSV-1 helper virus-dependent but not helper virus-free amplicon vectors have been reported to downregulate MHC class I expression in CLL blasts,5 both vectors, HSV.CD70 and BAC.CD70, were assessed in comparison at identical vector doses. Of note, in ALL blasts, even with helper virus-dependent vector HSV.CD70 only minor downregulation of MHC class I expression was observed and with helper virus-free vector BAC.CD70 MHC class I expression remained entirely unaffected (Figure 6), suggesting that ALL cells are not sensitive to HSV-1-mediated MHC class I molecule downregulation. Also, MHC class I expression was preserved after HSV-1 amplicon transduction of the pre-B lymphoblastic murine cell line A20, which was used for experiments in the leukemic vaccination model described below (data not shown).

Figure 6
figure6

MHC class I expression is sustained following transduction of ALL cells with HSV-1 amplicon vectors. Human ALL cells were transduced with helper virus-dependent amplicon vector HSV.CD70 or helper virus-free vector BAC.CD70. Identical MOIs of 20 were used and resulted in gene expression rates ranging from 55 to 67%. Expression of CD70 (a) and MHC class I molecules (b) following transduction was determined by flow-cytometry. One representative experiment out of four is shown.

Treatment of pre-established leukemia/lymphoma with an HSV-1 amplicon-transduced vaccine expressing CD70 and IL-2

High transduction rates and preservation of MHC class I expression after HSV-1 helper virus-dependent and helper virus-free amplicon vector transduction of ALL cells prompted us to compare the efficacy of both amplicon vectors in a vaccine approach. As A20 cells lack constitutive CD70 surface expression, and previous experience suggests that immunomodulatory molecules need to be combined to achieve vaccine efficacy in highly proliferative B-cell malignancies,8, 9, 10, 11 the costimulatory molecule CD70 was combined with the cytokine IL-2. Vaccine efficacy was assessed in mice with a pre-established lymphoblastic malignancy to mimic the clinical situation of residual disease. For challenge, 105 nonirradiated blasts of the murine pre-B lymphoblastic cell line A20 were first injected subcutaneously (s.c.) followed by two vaccinations at days 4 and 14. Vaccine cells consisted of 2 × 105 A20 cells transduced with either helper virus-dependent (HSV.CD70 or HSV.IL2) or helper virus-free (BAC.CD70 or BAC.IL2) amplicon vectors, each expressing either CD70 or IL-2. For generation of the combination vaccines, transduced A20 cells expressing CD70 or IL-2 were mixed and irradiated immediately before s.c. injection. To allow for differentiation of vaccination effects mediated by vector alone, A20 cells were also transduced with ‘empty’ helper virus-dependent vector (HSV.vec) or ‘empty’ helper virus-free vector (BAC.vec) that do not carry any of the immunomodulatory transgenes. The control group was vacccinated with nontransduced, irradiated A20 vaccine cells alone. To monitor disease progression, s.c. lymphoblastic tumor growth was assessed by serial measurements.

In mice with pre-established lymphoblastic disease, vaccine cells expressing CD70+IL-2 significantly (P<0.05) suppressed lymphoblastic tumor growth (Figure 7) regardless of whether helper virus-dependent or helper virus-free HSV-1 amplicon vectors were used for vaccine modification. Pronounced inhibition of disease progression was also observed in mice vaccinated with A20 cells transduced by ‘empty’ HSV-1 helper virus-dependent amplicon vector HSV.vec alone. In contrast, this protective effect was not observed when the ‘empty’ helper virus-free amplicon vector BAC.vec was used for vaccine generation.

Figure 7
figure7

Lymphoblastic tumor growth is suppressed following treatment with HSV-1 amplicon vector-transduced vaccine cells. Mice were challenged s.c. with 105 A20 leukemic cells followed by two vaccinations at days 4 and 14. Data from 2 to 4 experiments with 5–7 mice per experimental group were pooled. Vaccines were irradiated and consisted of 2 × 105 A20 cells, which were either untransduced (A20, n=19), or transduced with HSV-1 helper virus-dependent or helper virus-free empty vectors HSV.vec (n=27) and BAC.vec (n=14), respectively, or by helper virus-dependent (HSV.CD70+IL2; n=27) and helper virus-free (BAC.CD70+IL2; n=14) amplicon vectors expressing CD70 and IL-2. In one experiment, an additional control with mice that were challenged with A20 leukemia cells but did not receive any vaccine cells was included (no vaccine; n=5). Serial tumor measurements (mean±s.e. in mm2) at individual days post leukemic challenge are presented.

Also, survival was significantly (P<0.03) improved following treatment with A20 vaccine cells expressing CD70+IL2 irrespective of transduction with helper virus-dependent or -free amplicon vectors with survival rates of 48 and 57%, respectively (Figure 8). Vaccine cells transduced with ‘empty’ helper virus-free vector BAC.vec did not significantly enhance survival in comparison to control. In contrast, vaccine transduction with ‘empty’ helper virus-dependent vector HSV.vec afforded the same degree of protection as observed with both CD70+IL2 vaccines, suggesting that helper virus-dependent vector by itself enhances the antineoplastic response.

Figure 8
figure8

Treatment with HSV-1 amplicon vector-transduced vaccine cells improves survival of mice with pre-existing lymphoblastic disease. Survival of mice after leukemic challenge is shown in a Kaplan–Meier plot. Each line represents the probability of survival within one experimental group. Mice were challenged s.c. with 105 A20 leukemic cells followed by two vaccinations at days 4 and 14. Survival data were obtained from the same experiments depicted in Figure 7.

Characterization of immune cells important for generation of antineoplastic responses

The mechanism underlying the observed antileukemic effects following injection of HSV-1 amplicon-infected vaccine cells was analyzed. First, the role of antigen-presenting as well as cytotoxic effector cell populations was characterized by flow-cytometric analysis of splenocytes obtained at autopsy (Figure 9). In mice vaccinated with A20 cells genetically modified by helper virus-dependent HSV vector, activated T as well as NK cells were significantly expanded in comparison to mice in which helper virus-free HSV vectors were used for vaccine generation. Also, activated antigen-presenting cells were significantly increased in mice vaccinated with A20 cells transduced with helper virus-dependent HSV vector independent of CD70+IL2 expression. The data suggest that in vaccines modified by helper virus-dependent HSV amplicon vector, crosspriming events might dominate the antineoplastic immune responses.

Figure 9
figure9

Activated T, NK and antigen-presenting cells are expanded in mice treated with a leukemia vaccine modified by HSV-1-derived helper virus-dependent vector. Splenocytes were obtained from mice vaccinated with untransduced A20 leukemic cells (A20), or vaccine cells transduced with HSV-1 helper virus-dependent (HSV.vec) and helper virus-free (BAC.vec) empty vectors, or helper virus-dependent (HSV.CD70+IL2) and helper virus-free (BAC.CD70+IL2) amplicon vectors expressing CD70+IL-2. Distribution of CD4+ and CD8+ T cells and DX5+ natural killer cells as well as activated T cells (CD3+MHCII+) in the vaccination groups are shown in percent per total splenocytes (a; mean and s.d.; n=4). Activation of CD11b+ antigen-presenting cells was determined by coexpression of CD80, CD86 and MHC class II (b). Significance of difference in comparison to the A20 vaccination control group is indicated by an asterisk.

The role of T lymphocytes in mediating antineoplastic immune response induced by CD70+IL2 expression following helper virus-free HSV-1 amplicon vector transduction was assessed in a second set of experiments. CD4+ and CD8+ cells were depleted in vivo by injection of monoclonal antibodies directed against respective T-cell subsets. Antibodies were injected prior to leukemia/lymphoma challenge and then weekly thereafter. Mice were vaccinated with A20 cells transduced with helper virus-free HSV-1 amplicon vectors expressing CD70+IL2. While tumor growth was significantly reduced by vaccine cells expressing CD70+IL2 (P<0.05) as shown above, in vivo depletion of T-cell subsets could abrogate the antileukemic effect (Figure 10), suggesting that both CD4+ and CD8+ T cells are required for induction of antileukemic responses.

Figure 10
figure10

Antileukemic protection afforded by vaccination with CD70+IL2-expressing tumor cells is mediated by CD4+ and CD8+ T cells. Mice were challenged s.c. with 105 A20 leukemic cells followed by two vaccinations at days 4 and 14 with the irradiated vaccine consisting of 2 × 105 A20 cells, which were either untransduced (A20), or transduced with an helper virus-free amplicon vector that expresses CD70 and IL-2 (BAC.CD70+IL2). Subgroups of mice were in vivo depleted from CD4+ or CD8+ T cells by serial i.p. injection of T cell-specific monoclonal antibodies (anti-CD4 and anti-CD8). Results of serial tumor measurements (mean+s.e. in mm2) at individual days post leukemic challenged are presented from one experiment with 6–8 mice per experimental group.

Discussion

In the past, the immunostimulatory properties of HSV and other viral vectors have been exploited to enhance systemic antineoplastic immune responses. Immunostimulation in this setting is mostly based on viral replication resulting in tumor cell lysis and a virus-associated activation of a pleiotropic immune response. On the other hand, sophisticated mechanisms of immune evasion have been described for many viruses including HSV that are potentially detrimental to the immunostimulatory effects of the respective vectors used for vaccine generation. The immune evasion mechanisms of HSV are specifically targeted to antigen-presenting cells and include impairment of MHC class I molecule peptide transport by HSV ICP47 protein as well as downregulation of costimulatory molecules and reduced T-cell stimulatory capacity by HSV vhs protein.25, 26 In vaccine development for hematological malignancies utilizing herpesviral vectors for genetic modification, careful investigation of the consequences of HSV infection is warranted. This is particularly true for the development of vaccination strategies for ALL of B-cell origin, as HSV has been shown to inhibit the capacity of lymphoblastoid B cells to stimulate an antigen-specific immune response.24 Here, we analyzed two replication-defective HSV-1 vectors that differ in their capacity to sustain immunostimulation/immune evasion. Our study extends previous findings with oncolytic or xenogenizing vectors such as Newcastle Disease Virus (reviewed in Ring27) that were used for generation of antineoplastic responses mostly in solid tumors. We demonstrate that both HSV-1 helper virus-dependent and helper virus-free amplicon vectors are highly effective in transducing B-cell ALL, and are excellent candidates for generation of a leukemia vaccine although the underlying mechanism of the induced antileukemic responses differs between vectors.

We further show that pre-B and c-ALL cells express all three Hve genes. For many HSV-1 types, HveA and HveC seem to be the predominant receptor, whereas entry of most HSV-2 involves HveB.14, 16, 18 We have previously demonstrated that HSV-2-based recombinant vectors can infect primary human ALL with high efficiency.3 In accordance with high level expression of all three Hve receptors, we have now extended this observation to HSV-1-derived amplicon vectors. It has previously been reported that HSV-1-derived recombinant28 or HSV-1 amplicon vectors5 effectively transduce human B cells obtained from patients with chronic lymphocytic leukemia (CLL). In contrast to our observation of uniformly high expression of all three Hve genes in ALL cells, CLL cells were reported to express high levels of HveA but not HveC.28 Thus, high expression of HveA and HveC predisposes ALL cells as ideal targets for HSV-1-based gene transfer strategies.

Vaccine development for B-cell malignancies is governed by different considerations than vaccine design for solid tumors due to the intrinsic B-cell capacity for antigen-presentation. Leukemia and lymphoma cells generally express MHC class I molecules to high levels and in most cases leukemia/lymphoma-specific or associated antigens have been identified. Thus in B-cell malignancies, downregulation of MHC class I molecule expression, an inherent escape mechanism of herpesvirus, is of particular concern. One of the immediate early herpesviral proteins, ICP47, is known to impair MHC/antigen complex assembly in the endoplasmatic reticulum.26, 29 In malignant B cells, significant downregulation of MHC class I molecule expression was observed in CLL following gene transfer with helper virus-dependent amplicon vectors.5 In contrast, MHC class I expression in ALL blasts is only marginally affected by herpesviral protein expression as shown here. Thus with regard to choosing between helper virus-free and -dependent HSV-1 amplicon vectors for vaccine generation, MHC class I molecule modulation is not a decisive factor in human ALL.

In B-cell malignancies, reduced T-cell stimulatory capacity in spite of adequate MHC class I expression is largely attributed to deficient costimulation and consecutive failure to deliver the required second signal to prevent antigen-specific anergy.30 Human ALL of B-cell origin,31, 32 as well as the murine pre-B lymphoblastic A20 cell line in our model33 express only low levels of costimulatory surface molecules. Vaccine approaches employing autologous gene-modified neoplastic cells aim to compensate for the reduced immunogenicity by providing in trans the required costimulatory signals. To explore the role of herpesviral protein expression for vaccine efficacy in B-cell malignancies, we examined the influence of transgenic CD70+IL2 expression following transduction with helper virus-free versus helper virus-dependent amplicon in mice with pre-established pre-B lymphoblastic disease. In acute leukemia and other highly proliferative hematopoietic malignancies, combinations of costimulatory surface molecules and cytokines/chemokines are required to induce an effective vaccine-mediated antineoplastic response in the setting of residual disease. Thus in leukemia vaccines, the costimulatory surface molecules CD80 and CD40 ligand have demonstrated superior efficacy when coexpressed with cytokines such as GM-CSF, IL-2 or IL-12.8, 9, 10, 11, 34 The T-cell costimulatory molecule CD70, a member of the tumor necrosis factor (TNF) family,35, 36 is critical in B- cell–T-cell interactions. Like CD80, stimulation via CD70 induces the proliferation of cytolytic T cells as well as the production of T-cell inflammatory cytokines.37, 38, 39 When employed in vaccination strategies, transgenic expression of CD70 does induce antitumor immunity in solid tumor models.40, 41 In contrast in mice with residual lymphoblastic disease, transgenic expression of CD70 alone is not sufficient to induce an antineoplastic immune response, while the combination of CD70 and IL-2 expressed in vaccine cells affords significant but not complete protection.42 Therefore, coexpression of CD70 with IL-2 in our current study seemed a highly suitable vaccine combination for the purpose of exploring potentially additive effects mediated by the HSV-1 amplicon vector itself. Indeed, in the murine A20 model, a highly malignant and rapidly disseminating lymphoblastic neoplasm, the immune response induced by vaccine cells expressing CD70+IL-2 significantly inhibited outgrowth of a subcutaneous lymphoblastic mass and improved survival in mice with pre-established disease. The protection achieved was comparable whether HSV-1 helper virus-dependent or helper virus-free amplicon vector was used for vaccine generation. The effector mechanism underlying immune system stimulation via the combination of CD70+IL2 was assessed in mice injected with leukemia vaccine cells genetically modified by helper virus-free amplicon vector. As shown by in vivo depletion of CD4+ and CD8+ cells, both T-cell subsets are required to mount an effective antileukemic response.

Of note, vaccination with A20 cells transduced with ‘empty’ helper virus-dependent amplicon vectors alone was equally protective as transgenic CD70+IL2 expression. Thus, de novo synthesis of immunogenic HSV proteins2 expressed in vaccine cells transduced with HSV-1 helper virus-dependent amplicon seems to provide activating signals to the immune system enhancing the antineoplastic response.43 This is in accordance with the observation that following intratumoral injection of so called oncolytic herpesvirus vectors for local tumor control, systemic antitumor activity with rejection of metastases at distant sites has been observed.44 In this setting HSV vector-induced lysis of solid tumor cells is thought to initiate crosspriming events,45, 46, 47 resulting in uptake of tumor-cell-derived proteins by professional antigen-presenting cells.48 The observed expansion of activated T, NK and antigen-presenting cells in mice splenocytes supports a similar scenario underlying the antineoplastic effects induced by the replication-defective HSV-1 helper virus-dependent vectors. In our model, following transduction of vaccine cells with HSV-1 helper virus-dependent amplicon vector, efficacy of herpesviral protein expression and CD70+IL2-mediated costimulation is not additive, although each clearly enhances the antileukemic response. One explanation for this observation might be that effector cell activation by transgenically expressed CD70+IL2 is limited by vector-mediated destruction of the vaccine cells. Thus for synergistic costimulation of vaccines transduced by HSV-1 helper virus-dependent vector, cytokines such as GM-CSF3, 48, 49 or costimulatory molecule CD40L50 with stimulatory effects on professional antigen-presenting cells enhancing the crosspriming events might be a promising choice.

In summary, we have shown that human ALL blasts expressing all three of the herpesviral entry receptors readily lend themselves to high-efficiency transduction with HSV-1-based amplicon vectors. In these immature leukemia cells, MHC class I expression is not downregulated by HSV-1 amplicon vector transduction maintaining the antigen-presentation capacity of leukemic blasts. HSV-1 amplicon vectors with their capacity to accommodate large size inserts effectively modify ALL blasts allowing for combined transgenic expression of several different costimulatory molecules. Helpervirus-free amplicons transduce ALL cells with the same efficieny as helper virus-dependent vectors. Yet in vivo, induction of the antineoplastic response differs in that in malignant B lymphoblasts, immunogenicity of herpesviral proteins themselves can mediate vaccine efficacy. Thus in contrast to corrective gene transfer strategies where one seeks to avoid expression of viral protein in hematopoietic target cells, in vaccine generation one might opt for HSV-1 helper virus-dependent amplicon vectors for transduction of lymphoblasts, taking advantage of the immunogenic potential of the vector itself.

Materials and methods

Cultivation of cells

The human pre-B-leukemic cell lines AD and RS were established from clinical ALL samples as described previously.3 The human B-cell lymphoma lines Daudi and Raji, the T-cell leukemia cell line Jurkat and the myeloid cell line HL60 were obtained from the American Type Culture Collection (Manassa, VA, USA). The cells were cultured in RPMI-1640 (Biochrom KG, Berlin, Germany) supplemented with 10% fetal calf serum (Biochrom KG, Berlin, Germany), 100 IU/ml penicillin, 100 μg/ml streptomycin (Gibco, Paisley, UK) and 2 mmol/l L-glutamine (Biochrom KG, Berlin, Germany). For cultivation of the highly proliferative murine pre-B lymphoblastic A20 cell line originally derived from a BALB/C mouse with a spontaneously growing reticulum tumor, 0.05 mM β-mercapthoethanol (Sigma, Deisenhofen, Germany) was added to the medium.51, 52 BHK-derived RR1 that stably express the HSV-IE3 gene,53 and 2-2 cells that stably express HSV ICP2754 were cultured in DMEM containing supplements as described above except that geneticin (G418; 500 μg/ml) was included into the medium.

Bone marrow or peripheral blood cells were obtained after informed consent from patients satisfying the diagnostic criteria for ALL. Leukemia cells were isolated from patients with >80% blast via density gradient centrifugation using Ficoll-Hypaque (Amersham Pharmacia Biotech AB, Uppsala, Sweden). ALL cells were cultured in RPMI-1640 with supplements as described above at 37°C in 5% CO2.

Amplicon plasmid generation

For preparation of HSV-1 amplicon vectors, the amplicon plasmid pHSVPrPUC55 containing the HSV-1 pac- and ori-sequences as well the transgene under the control of the HSV I.E 4/5 promoter was employed. The cDNA for murine CD70 kindly provided by Dr van Lier (CLB, Amsterdam, The Netherlands) was cloned into pHSVPrPUC resulting in the plasmid pHSV.CD70. For transgeneic expression of human IL-2, the plasmid pHSV.IL-2 was generated by amplification of IL-2 cDNA with the primers IndexTerm5′-TGCTGTCGACTCAACTCCTGCCA-3′ and IndexTerm5′-TCTGGGATCCTGATATGTTTTAAG-TG-3′ for insertion into pHSVPrPUC at SalI and BamHI restriction sites.

HSV-1 helper virus-dependent amplicon vector packaging

HSV-1 helper virus-dependent packaging of the plasmids pHSV.CD70, pHSV.IL2 or pHSVPrPUC as an ‘empty’ vector control was performed in RR1 cells complementing the IE3 deletion of the recombinant HSV-1 helper virus d120.56 106 RR1 cells were seeded in a 60-mm dish in 3 ml complete DMEM 24 h prior to Lipofectamine-mediated transfection with 5 μg of the amplicon plasmids according to the manufacturer's instruction (Gibco-BRL, Paisley, UK). After 24 h at 37°C, the supernatant was removed, and cells were infected with recombinant helper virus d120 at a multiplicity of infection (MOI) of 0.2. Infected RR1 cells were harvested after 48 h into the medium by scraping and subjected to three freeze–thaw cycles followed by sonication using a cup sonicator (Branson Sonifier 450, Danbury, USA). The vector preparation was clarified from cellular debris by centrifugation at 5000 rpm in a Sorvall SS34 rotor (Kendro, New Town, USA) for 10 min. RR1 cells seeded at 4.5 × 106 cells per 60-mm dish 24 h before were infected with the viral supernatant for a second viral passage. Virus was harvested after 48 h as described above and the suspension was layered on a 25% sucrose cushion. Virus was purified by 2 h ultracentrifugation at 25 000 rpm in an SW.41 rotor (Beckmann, Unterschleissheim-Lohhof, Germany). The viral pellet was resuspended in 100 μl of phosphate-buffered saline (PBS) containing Ca2+ and Mg2+, aliquoted and stored at −80°C. HSV-1 helper virus-dependent vector stock preparations were termed HSV.CD70, HSV.IL2 and HSV.vec.

HSV-1 helper virus-free amplicon vector packaging

HSV-1 helper virus-free amplicon vector stocks were generated using an bacterial artificial chromosome (BAC) fHSVΔpacΔ2757 containing the complete HSV-1 genome to provide the required helper functions except for the pac signal and the ICP27 gene. Packaging was performed in 2-2 cells that stably express ICP27 essential for HSV replication.54 2-2 cells (1.2 × 106) were seeded on a 60-mm dish 24 h prior to Lipofectamine transfection with 2 μg of pHSV.CD70, pHSV.IL2 or plasmid pHSVPrPUC and 2 μg of fHSVΔpacΔ27. After 3 days, cells were scraped into the medium. Virus was harvested, purified and aliquoted essentially as described above. HSV-1 helper virus-free vector stocks, termed BAC.CD70, BAC.IL2 and BAC.vec, were repeatedly analyzed on susceptible cells without detection of any plaque formation (<1 PFU per 108 transducing units), suggesting that the viral preparations are free of recombinant HSV particles.

Titering of HSV-1 amplicon vectors

HSV-1 helper virus-dependent amplicon stocks were titered by standard plaque assay58 in HSV IE3-expressing target cells supporting viral replication. Viral titers of helper virus-dependent amplicon stocks were consistently determined to be between 1–2 × 108 PFU/ml. As helper virus-free amplicon vectors do not replicate, titres of these vectors cannot be determined by plaque assay. Instead, the concentration of transducing particles was determined by transgene expression following infection of 105 A20 cells with serial dilutions of both helper virus-free and helper virus-dependent amplicon vectors in parallel. The amount of helper virus-free amplicon vector that produced identical expression levels on A20 cells was defined to contain identical transducing units. CD70 expression was analyzed by flow-cytometry and IL-2 expression was determined by ELISA (Eli-pair, Diaclone, Besancon, France). As judged by plaque assay in Vero cells, replication of the vector was not observed in the viral stocks used for transduction. Furthermore, HSV-transduced A20 cells as well as an array of freeze–thaw tissue specimens such as blood, brain, heart, spleen, liver obtained from mice vaccinated with HSV-1 transduced cells were screened by plaque assay to document absence of viral replication.

Flow-cytometry

The expression of CD70 or appropriate lineage markers on the surface of the transduced cells was analyzed by dual staining and flow-cytometry. The cell suspension was incubated with fluorescence-coupled antibodies directed against CD10 or CD19 (Immunotech, Krefeld, Germany) for identification of ALL blasts, or anti-HLA-A, -B, -C to investigate the expression of MCH class I molecule and/or anti-mCD70 (Pharmingen, San Diego, USA) at 4°C for 30 min. Flow-cytometry was performed in a FACS-Calibur (Becton Dickinson, San Jose, CA, USA).

RT-PCR

Total RNA from 106 primary leukemic blasts or cells derived from cell lines were prepared by the RNeasy Mini protocol according to the manufacturer's instructions (RNeasy® Mini Kit, Qiagen, Hilden, Germany). cDNA was prepared using the poly-T bifunctional primer according to standard protocol (First-strand cDNA Synthesis Kit, Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany). For PCR amplification with Taq-polymerase (Qiagen, Hilden, Germany), primer pairs (MWG Biotech, Ebersberg, Germany) specific for the herpes virus entry mediator HveA, -B or -C were each used in the same reaction with primers specific for the β-actin gene. The following sequences for sense and antisense Hve-specific primers were used: HveA IndexTerm5′-GCATGGAGCCTCCTGGAGAC-3′ and IndexTerm5′-CTCCTTGCAGGACGGCAGAG-3′, HveB: IndexTerm5′-ATGGAACCAGATGGCAAGGATGAG-3′ and IndexTerm5′-ACTGCCCGCCGTGAGATGAG-3′ and HveC: IndexTerm5′-CGGCCCTACTTCACCGTGGA-3′ and IndexTerm5′-CTTGGAAATGAAAGACCCGTCGTTC-3′. PCR reaction was carried out in a Trio ThermblockTM (Biometra®, Göttingen, Germany). Relative ethidium bromide-stained band intensities were determined by the Fluor-S MultiImager and the Multi-Analyst software (Bio-Rad, Munich, Germany). Hve-specific intensities were normalized to the intensity of the β-actin gene amplified in the same reaction, and expressed as ratio of Hve-specific intensity relative to the β-actin control.

Assessment of vaccination effects

Assessment of antileukemic protection by CD70 and IL-2-secreting A20 vaccine cells was performed in syngeneic 9–14-week-old female BALB/cBYJ mice (Animal Center of the University of Düsseldorf, Germany). For leukemia challenge, 105 trypan-blue-negative nonirradiated A20 cells were resuspended in 0.2 ml PBS and injected s.c. into the flanks of mice. At days 4 and 14 post challenge, two s.c. injections of the vaccine cells were given. A20 cells have been shown to disseminate rapidly in vivo to lymphoid organs as well as bone marrow.59 For vaccine preparation, A20 cells were incubated with HSV-1 amplicon vectors at MOI 10, resulting in >80% CD70-positive cells or to secretion of IL-2 between 10 and 14 ng/ml/106 cells 24 h p.t. Transduced A20 cells vaccine cells were washed twice with PBS, and adjusted to a cell concentration of 106/ml for each transduced cell line. Cells were irradiated at 10 Gy thereafter. In total, 0.2 ml of the cell suspension corresponding to 2 × 105 transduced cells was injected s.c. to mice. As a control, irradiated A20 cells that were untreated were also injected. Tumor growth at the site of leukemic challenge was determined by measuring the largest perpendicular diameters, and reported as mm2. The diameters of the smallest tumor nodules detected by palpation ranged from 2 to 4 mm2.

Animals were killed when tumor growth exceeded 450 mm2, their body weight increased by more than 30% or when ulcerations or other signs of physical distress were noted. Experiments were performed with 5–8 mice per vaccination group. Data from two to four experiments were pooled per group for calculation of tumor growth and survival: A20 (n=3), HSV.vec (n=4), BAC.vec (n=2), HSV.CD70+IL2 (n=4) and BAC.CD70+IL2 (n=2).

Determination of splenocyte subsets in vaccinated mice by flow-cytometry

Spleens were obtained from mice at autopsy and single cell suspensions were prepared. To determine expansion of cellular subsets, splenocytes were analyzed by flow-cytometry employing fluorescein-labeled antibodies anti-CD4 (L3T4), anti-CD8 (Ly-2), anti-CD80, anti-CD86 (B7–2), anti-MHCII (I-AD), anti-CD11b and anti-CD49b/Pan-NK (DX5) (Pharmingen, Hamburg, Germany).

In vivo T-cell depletion

Mice were depleted of effector cells by intraperitoneal (i.p.) injection of monoclonal antibodies directed against CD4 (CD4.1) or CD8 (CD8.2).60 In total, 100 μg of the monoclonal antibodies were injected 1 day prior to A20 challenge and twice weekly thereafter. Depletion efficacy was consistently >98% as verified by weekly flow-cytometric analysis of peripheral blood cells. Other cellular subsets were not affected.

Statistical analysis

Statistical analysis for comparison between experimental groups was performed by Wilcoxon test for in vivo experiments, and by Mann–Whitney test for flow-cytometry analysis. A value of P<0.05 was considered to indicate a significant difference.

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Acknowledgements

We thank D Klostermann for excellent technical assistance. The work was supported by the multicenter grant of the German National Ministry for Research and Technology ‘Strategies for Somatic Gene-Transfer’ (Research alliance of the university medical centers ‘Düsseldorf, Essen, Halle; Coordinator: U Göbel), project 4 ‘Cytokine and Chemokine Gene-Transfer into Malignant Hematopoietic Cells for the Generation of a Leukemia Vaccine’ and the Elterninitiative Kinderkrebsklinik Düsseldorf e.V.

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Correspondence to R Meisel or D Dilloo.

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Zibert, A., Thomassen, A., Müller, L. et al. Herpes simplex virus type-1 amplicon vectors for vaccine generation in acute lymphoblastic leukemia. Gene Ther 12, 1707–1717 (2005) doi:10.1038/sj.gt.3302577

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Keywords

  • herpesvirus
  • HSV
  • vector
  • tumor vaccination
  • leukemia/lymphoma

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