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The Oncologist, Vol. 2, No. 5, 300–310, October 1997
© 1997 AlphaMed Press

Vaccine Trials for the Clinician: Prospects for Viral and Non-Viral Vectors

Peter S. Goedegebuure, Timothy J. Eberlein

Laboratory of Biologic Cancer Therapy, Division of Surgical Oncology, Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA

Correspondence: Peter S. Goedegebuure, Ph.D., Division of Surgical Oncology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA. Telephone: 617-732-6247; Fax: 617-278-6914; e-mail: psgoedegeb{at}bics.bwh.harvard.edu


   ABSTRACT
Top
 Abstract
Introduction
 Vectors
 Genes
 References
 
Recent progress in tumor genetics, tumor biology, and tumor immunology has renewed interest in the development of tumor vaccines. Unlike the previous generation of vaccines that consisted of the patient’s own tumor cells in some form, the new vaccines contain defined peptides or genes with a known function. In order to induce a potent and long-lasting cell-mediated antitumor response, viral as well as nonviral vectors have been explored as vehicles for gene delivery. Both types of vectors have shown encouraging results in animal models. However, because of the many possible vectors that have been designed, it may be too early to say which type of vector is most efficient in the human. Clearly, viral vectors have a higher transduction efficiency than most nonviral delivery systems. A drawback is that viral vectors may be toxic or immunogenic. Current research focuses on enhancing the targeting and specificity of both viral and nonviral vectors and control of transgene expression levels. Clinical studies using a variety of both viral and nonviral vectors have begun, and the results are forthcoming.

Key Words. Human • Cancer • Vaccine • Vector • T lymphocyte • Immune response


   INTRODUCTION
Top
 Abstract
 Introduction
Vectors
 Genes
 References
 
Historically, vaccines have been used for immunization against viral, bacterial, and other infectious diseases. Characterized by the landmark experiments of Dr. Edward Jenner [1], vaccines have been used for the past 200 years. The overwhelming success achieved with several vaccines (smallpox, diphtheria, tetanus, etc.) sparked interest to exploit vaccines in the battle against cancer. The concept that vaccines may be effective against tumors dates back only a few decades. The initial cancer vaccines were similar in make-up to the vaccines against infectious diseases. Crude preparations of homogenized tumor cells were mixed with immune-stimulating adjuvants such as viral or bacterial particles. Some of these early vaccines were tested in clinical trials and induced objective clinical responses in about 25% of the cancer patients, the majority of whom had malignant melanoma [2]. Encouraged by the initial success, investigators attempted to make more effective vaccines, but the field moved forward very slowly due to a lack of understanding of the underlying mechanisms. However, the past 10 years have shown a renewed interest in cancer vaccines. Some of the fundamental differences between immunity to infectious organisms and cancer are now better understood, and many investigators currently believe that there is a viable future for cancer vaccines.

One of the differences between immunity to infectious organisms and cancer is that unlike an effective humoral response to many infectious diseases, a humoral response against cancer has appeared to be ineffective. Immunity to cancer is characterized by a cell-mediated immune response almost exclusively mediated through T lymphocytes. Consequently, antigenic epitopes have to be presented in a major histocompatibility complex (MHC) class I-restricted and MHC class II-restricted fashion. A second major difference is that the pool of potentially antigenic epitopes derived from tumor cells is virtually unlimited because of the complexity of the genome in contrast to the relatively simple genomes of viruses and bacteria. For this reason, the early vaccines were made up of the patient’s own tumor cells. However, progress in the identification of tumor antigens has demonstrated the presence of multiple shared tumor antigens, even among tumors of different histologic type [2, 3].

Several additional developments are associated with the renewed focus on tumor vaccines, not the least of which is the development of the gene therapy technology. This technology was first clinically applied in 1990 [4], and investigators have attempted to overcome genetic disorders by inserting healthy copies of mutated genes. Disorders that are caused by a single-gene mutation such as adenosine deaminase (ADA) deficiency or cystic fibrosis are perhaps the most likely candidates for gene therapy. However, cancer researchers have built on this concept and designed vaccines to treat human tumors. The second major development is an improved understanding of how tumors arise, grow, and manage to escape from the immune system. In addition, a considerable number of tumor-associated antigens (TAA) have been identified that are inducers of an antitumor cellular immune response [5]. Thus, considerable progress has been made in the fields of tumor biology, tumor genetics, and tumor immunology. As a result, new vaccines have been designed based on single or multiple genes instead of whole cells. In this overview, the various vaccine strategies will be discussed as well as their potential as anticancer agents.

To better understand the rationale behind some of the strategies, a schematic overview of a cellular antitumor immune response is shown in Figure 1Go [6]. A central role in this response is played by antigen-presenting cells (APC) in presenting TAA to T cells. Fragments of tumor cells are taken up by APC such as dendritic cells, processed intracellularly, and presented by HLA molecules. Generally, exogenous antigens are processed in the endosomal compartment, which leads to presentation by HLA class II molecules. Endogenous antigens, on the other hand, are routinely combined with HLA class I molecules in the endoplasmic reticulum. However, some reports suggest that exogenous TAA may also be presented by HLA class I molecules on APC [7]. The HLA class II-TAA complex on APC is recognized by tumor-specific CD4+ T cells. Activation of CD4+ T cells results in secretion of cytokines (Th1- or Th2-like cytokines) that may directly affect the efficiency of tumor-specific CD8+ T cells. Because of the restriction by HLA class I molecules rather than HLA class II molecules, CD8+ T cells can directly interact with tumor cells presenting TAA. Since most solid tumors do not express HLA class II molecules, CD4+ T cells are dependent on APC for stimulation.



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  		Figure 1. Schematic overview of the general activation pathways of tumor-specific CD4+ and CD8+ T cells. CD4+ T cells recognize TAA presented by HLA class II molecules on APC. Costimulation is provided through the CD28-B7 interaction. Activated CD4+ T cells produce cytokines that regulate CD8+ T cell activity. The CD8+ cells directly interact with HLA class I molecules presenting TAA on the tumor cells. Lysed tumor cells eventually fragment, and these fragments are taken up by APC and are processed for recognition by CD4+ T cells. Reprinted, with modifications, with permission [6].

 
Investigators have demonstrated that several events in this schema are susceptible to failure. Adequate activation of T cells requires two signals, one of which is provided by the T cell receptor that interacts with the HLA-TAA complex. The second signal is provided through the interaction of costimulatory molecules on the target cell and their receptors expressed on the T cell. Known costimulatory molecules for T cells are CD80 (B7.1), CD86 (B7.2), CD54 (ICAM-1), and CD58 (LFA-3) that interact with the T cell molecules CD28, CTLA-4, CD11a/CD18, and CD2, respectively. In addition, T cell proliferation requires the presence of cytokines such as interleukin 2 (IL-2), IL-4, IL-12 and others. It is important to note that the schema presented in Figure 1Go is a simplification of the in vivo situation. It does not take into account the kinetics of an antitumor response (induction phase, expansion/effector phase, and termination phase), nor does it include the effects of other cells and soluble factors present in the tumor environment. This, in fact, may in part be the explanation for the lack of spectacular clinical responses through immune intervention. The schema does include those elements investigators have focused on most: enhancement of the number and quality of APC, improvement of costimulation, improvement of the cytokine milieu, and enhancement of the concentration of immunogenic TAA.


   VECTORS
Top
 Abstract
 Introduction
 Vectors
Genes
 References
 
It should be noted that the list of available methods of gene transfer as given below is far from complete. Investigators have expanded the repertoire of vectors by making specific modifications demanded by specific models. Instead of listing all these options, we will outline the basic characteristics of the most common methods. Although the general strategy of vaccine treatment applied to different aspects of cancer research is more or less the same (to induce or enhance an immune response that causes eradication of tumor cells without causing damage to normal cells, and to prevent recurrence), the individual vaccines may differ widely. Important differences are the vector of choice and the identity of the genetic material that is being transferred. Because of the relatively efficient delivery of genes into cells, viral vectors are preferred over nonviral delivery systems [8]. A number of the most commonly used recombinant viral vectors are listed in Table 1Go. Ideally, the recombinant viral vector is safe, does not integrate into the genomic DNA, does not replicate, is genetically stable and easy to engineer, and mediates sufficient expression of the transgene. None of the currently available viral vectors fulfill all these criteria. Nonetheless, many viral vectors have been successfully used in animal models and are currently tested in clinical trials. Viral vectors are "packaged" in specialized cell lines termed "packaging cell lines" that permit the production of high-titer, replication-defective viral vectors. Since no genetic information for virus replication is transferred from the packaging cells, transduced cells are unable to replicate the recombinant viruses [9].


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  		Table 1. Recombinant viral vectors
 
Retroviral vectors have the advantage that a high transduction efficiency is obtained ex vivo. However, their target cell spectrum is limited, and successful transduction is dependent on cell division of the target cells. Moreover, retroviruses integrate into the host genome, but the site of insertion is random, thus posing a small risk of turning on or off neighboring genes that could be oncogenes or tumor suppressor genes [9]. A potential limitation for the treatment of large tumors may be the relatively low titer (about 107) that is usually obtained. (Titers of viral stocks are determined by plaque-forming assay and are expressed in PFU. The infectivity of the recombinant virus is usually determined by titrating the number of viruses onto target cells in vitro for about 24 hours followed by analysis of transgene expression. The ratio of the number of viruses to a target cell is expressed as a multiplicity of infection [MOI]. In general, ratios range from 1 to 100). Examples of retroviruses used for gene therapy studies are human immunodeficiency virus (HIV) [4] and murine leukemia viruses or MLV [10]. Envelope proteins on the outside of the viruses interact with specific receptors on the host cells. Based on their tropism, five groups of MLV are distinguished. Current research focuses on the replacement of the env gene with that of another MLV to alter the tropism of the vector and create vectors with an extended host cell range [10]. In addition, investigators are focusing on the development of new packaging cell lines to enhance viral titers [9]. Retroviral vectors have also been used to transduce T lymphocytes prior to adoptive immunotherapy [11, 12].
Although the general strategy of vaccine treatment applied to different aspects of cancer research is more or less the same, the individual vaccines may differ widely.

Alternatively, adenoviral vectors efficiently transduce nondividing cells, and the vector usually does not integrate into chromosomal DNA. The target cell spectrum of adenoviral vectors is larger than that of the retroviral vector. Another advantage is the high virus titer (>1011) that can be obtained. Drawbacks of the adenoviral vector include the expression of adenovirus gene products that provoke immune responses that eventually may neutralize infected cells. Therefore, the effects of adenoviral vectors may last only several weeks.

Other viral vectors are derived from adeno-associated virus (AAV), herpes virus, or poxviruses such as vaccinia virus, fowl poxvirus, and avian poxvirus. The AAV integrates into the host genome at a specific site, although less efficient and less precise than the retrovirus [13]. AAV infects a wide range of different cell types and stably expresses the transgene. Similar to adenoviral vectors, AAV transduces quiescent cells. A potential downside is that AAV is difficult to produce in large quantities (titers of about 104). The herpes virus targets the central nervous system and is capable of replicating in both neurons and glia [14]. However, because the virus DNA is difficult to manipulate, the generation of a replication-defective recombinant virus has been problematic. Finally, poxviral vectors are preferred when large genes need to be transferred [15]. Poxviruses transiently transduce a wide range of different host cell types. The downside is that only low virus titers can be obtained, thereby limiting the expression levels of the transgene in vivo. Of the poxviruses, vaccinia virus is the most commonly used. Ongoing research focuses on vector targeting and specificity, and reduction of immunogenicity.

Several alternative, nonviral methods are exploited by investigators (Table 2Go). Instead of a virus, genetically modified intact mammalian cells are used as carriers of the transgene. Most commonly used are autologous tumor cells genetically modified in vitro with a viral vector to express the gene of interest [16, 17]. Results in many animal models have been encouraging, as outlined in the Table. Alternatively, modified fibroblasts [18] or APC such as dendritic cells [19, 20] have been successfully used. Even T cells have been tested as vectors [11], but with limited success related to the difficulty of genetically transforming T cells.


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  		Table 2. Nonviral vectors
 
Finally, investigators have started exploring the use of bacterium-based vectors. An example is the intracellular bacterium Listeria monocytogenes [21]. This bacterium has several unique characteristics. One of these is that infection with Listeria induces a Th1 response [22]. More importantly, Listeria can escape from the phagolysosome and live in the cytoplasm of the host cell. Thus, once the bacterium is phagocytosed, the bacterium is either lysed in the lysozomal compartment or escapes into the cytoplasm. In the first case, bacterial peptides will be presented in the context of HLA class II molecules. In the second case, bacterial proteins can be processed by proteosomes and transported into the endoplasmic reticulum where peptides are combined with HLA class I molecules. This latter feature could be exploited for the design of a cancer vaccine using a recombinant Listeria vector. Recombinant Listeria has been shown to be effective in several murine models in which the effective delivery and synthesis of the transgene product led to regression of macroscopic tumors [23].

Other methods include the incorporation of DNA into lipid vesicles or liposomes [24, 25]. The challenge with liposomes is to accomplish efficient fusion of the liposome and cell membrane of the target cell, and the delivery of undegraded DNA to the nucleus. To avoid degradation of liposomes, cationic liposomes are used that cause direct fusion of the liposome with the cell membrane [10]. Incorporation of viral surface glycoproteins into liposomes improved cell attachment considerably [24]. In spite of these improvements, the efficiency of genetic transfer is lower than that obtained with viral vectors.

In the past several years, DNA vaccines have received a lot of attention [26]. Unexpectedly, the uptake of "naked DNA" by cells in vivo occurs with greater efficiency than one would expect based on in vitro experiments. The best target cells for the direct injection of plasmid DNA are muscle cells, in particular skeletal and cardiac muscle cells [27]. Both HLA class I- and II-mediated responses are elicited, which may make DNA vaccination an effective method. Recently, it was demonstrated in a murine model that dendritic cells in the skin play an important role in DNA-based immunization [28]. Dendritic cells in the skin were transfected by the DNA, localized in draining lymph nodes, and induced protective immunity mediated by CD8+ T cells. Experiments with a human carcinoembryonic antigen (CEA) encoding plasmid administered in murine tongues resulted in the induction of both a humoral and cellular immune response to human CEA [29]. Improved efficiency may be obtained with a DNA gun to inject DNA-coated particles into cells [26]. Coupling of the DNA to a protein or synthetic ligand may improve the specific targeting [30], a strategy that is also explored to improve the targeting of viral vectors [31].

An alternative approach is based on vaccination with MHC class I-binding peptides instead of with DNA. This strategy is used to immunize against either virus-induced tumors such as Burkitt’s lymphoma or cervical cancer induced by papilloma virus or nonvirus-induced tumors [32]. TAA that have been shown to induce an immune response are combined in peptide form with adjuvant and injected subcutaneously. Now that more TAA have been identified recently, this approach is gaining attention. The advantage of using peptides as opposed to DNA is that the processing and presentation events are bypassed. A potential downside is that the application is HLA type-dependent. Nonetheless, promising results were obtained in a vaccination trial using melanoma peptides [33].

As illustrated by the large variety of vectors, there is no consensus yet as to which delivery system is the most effective for the patient. Both viral and nonviral delivery systems have shown promise, but better vector targeting and specificity are required. In addition, current viral vectors may pose some risks with regard to toxicity and immunogenicity. Nonviral vectors such as liposomes, peptides, and naked DNA could be improved by enhancing the transduction efficiency.


   GENES
Top
 Abstract
 Introduction
 Vectors
 Genes
References
 
The choice of the transgene to be used in a vector is codetermined by the type of response one wants to obtain. In general, elements of an optimal immune response include the cytokine milieu, adequate stimulation of immune cells through antigen and costimulatory molecules, and the availability of immune cell subsets required to mount an efficient immune response. In a simplified classification, one can distinguish transgenes coding for cytokines, costimulatory molecules, tumor antigens, and alloantigens (Table 3Go). Within the category of cytokines, practically all interleukins have been tested for their ability to induce or enhance a cellular immune response [2, 34]. Most effective thus far were IL-2, IL-4, and IL-12. The first result of successful induction of a host antitumor response by transfection of tumor cells was obtained with the IL-4 gene [35] followed shortly by a similar report using IL-2-transfected tumor cells [16]. Although both cytokines induced effector T cells reactive against tumor cells, the mechanisms were different. In the case of IL-4, an inflammatory infiltrate was observed composed of eosinophils and macrophages shortly after injection of transfected tumor cells. Since these experiments were carried out in athymic (nu/nu) or beige (bg/bg; no natural killer cells or T cells) mice, this response was independent of T cells.


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  		Table 3. Transgenes
 
A later report expanded on these observations [17]. It was found that IL-4-transfected renal cancer cells were rejected after transfection in a T cell-independent fashion through macrophages and eosinophils. In addition, systemic immunity was induced against rechallenges with wild type tumor in immunocompetent mice but not in immunoincompetent mice. The rejection was CD8+ T cell-mediated. Likewise, in the report on IL-2 transfection [16], IL-2-transfected colon tumor cells induced a CD8+ T cell-mediated antitumor response even in the absence of CD4+ T cells and induced systemic immunity.

Following these reports, a large variety of similar studies were performed in animal models using histologically different tumors and different cytokines. In summary, almost all cytokines seem to be able to induce complete or partial regression of tumor growth and a certain degree of immunity to subsequent challenge with wild type tumor [36, 37]. Unfortunately, the response seems to be dependent on many factors, such as tumor load, immunogenicity of the parental tumor, histotype and extracellular matrix, susceptibility of the tumor cells to various immune cell types, host immune cell repertoire, and the amount of cytokine released. Of interest are those models in which a direct comparison was made between various cytokines using the same parental tumor. Allione et al. [36] used a genetically engineered mouse mammary adenocarcinoma cell line to secrete IL-2, IL-4, IL-6, IL-7, IL-10, TNF-{alpha}, GM-CSF, or IFN-{gamma}. If replicating tumor cells were used for immunization, 80%-100% of mice were protected against wild type challenge if IL-2, IL-4, IL-7, IL-10, or IFN-{gamma} was secreted by the immunizing tumor cells. No cure was observed in the case of IL-6, GM-CSF, or control tumor. Survival was less than 30% when nonreplicating tumor cells were used for immunization. In contrast, nonreplicating B16 melanoma cells transduced to express GM-CSF and, to a lesser extent, IL-4 and IL-6 induced specific and long-lasting immunity. IL-2, IL-5, IFN-{gamma}, IL-1 receptor antagonist, TNF-{alpha}, ICAM-1, or CD2 did not induce specific immunity when tested in this system [37]. In a recently published study, GM-CSF-producing autologous irradiated renal cell carcinoma vaccines were tested in a phase I clinical trial [38]. An objective partial response was observed in one patient out of seven who received the GM-CSF vaccine (nine patients received the GM-CSF-negative vaccine and did not show a clinical response). No replication-competent retrovirus, dose-limiting toxicities or autoimmune disease were detected. As illustrated by these examples, there is no consensus on which strategy is optimal, but there may be more than one option available.

A second group of genes that is currently used either alone or in combination with a cytokine gene or tumor antigen is formed by those genes that code for costimulatory molecules. Most common are the B7.1 (CD80) and B7.2 (CD86) molecules. B7.1 and B7.2 are generally expressed on APC such as macrophages, dendritic cells, and activated B cells, and are usually absent on tumors other than those of hematopoietic origin. Both molecules bind to T cell ligands CD28 and CTLA-4 and provide costimulatory signals for T cell activation [39]. Although recent studies suggest that B7.1 and B7.2 costimulatory signals may be functionally distinct [40], transfection of B7.2 into tumors [41] or vaccination with a viral vector containing the B7.2 gene [42] induced antitumor responses. Delivery of B7.1 in combination with a tumor antigen either on the same vaccinia vector [42] or on different vaccinia vectors [43] induced antitumor immunity in an animal model using the colon carcinoma CT26 and colonic adenocarcinoma MC38, respectively. Similarly, B7.1 and cytokine genes, in particular IL-12, because of their synergistic effect in stimulating proliferation and cytokine production [44], have been successfully tested in experimental tumor models [45].

Another category is the use of alloantigens. The objective is to induce an immune response against a foreign HLA molecule [46]. This response may at the same time induce a response against tumor antigens expressed on unmodified (wild type) tumor cells. This strategy has been shown to be efficacious in animal models and has been applied to humans as well [47].

Almost all cytokines seem to be able to induce complete or partial regression of tumor growth and a certain degree of immunity to subsequent challenge with wild type tumor.

With the rapidly expanding list of newly identified TAA, investigators have more possibilities for vaccine design. Particularly in malignant melanoma, since the initial discovery of MAGE-1 [48], numerous TAA have been identified. Some of these are mutated peptides or viral peptides (cervical cancer) that provide unique opportunities for immunization. However, others, perhaps even the majority of TAA, are nonmutated peptides including differentiation antigens (MART-1/MelanA, gp100, tyrosinase) as well as peptides that are overexpressed in tumor cells but not in normal cells (oncogenes such as Her2/neu and K-Ras, and tumor suppressor genes such as p53) [49]. Immunization against nonmutated or overexpressed antigens may complicate vaccine design because of the potential risk of inducing autoimmune reactions such as vitiligo in melanoma patients [50, 51]. It is conceivable that in case of overexpression of a normal antigen, tolerance to this antigen is broken because of its high concentration on tumor cells resulting in T cell activation. In normal cells, the antigen concentration would be too low to induce T cell activation. This would imply that the overall avidity between T cells and antigen is quite low. Supporting this hypothesis is the observation that most of the TAA derived from normal antigens have a low binding affinity for HLA molecules [5]. The Her2/neu peptide that was recently identified by us [3] is an example of such an overexpressed normal peptide. This peptide is an HLA-A2.1-binding peptide, but this peptide does not express any of the dominant anchor motifs, and therefore the affinity for HLA-A2.1 is very low. Nonetheless, a number of clinical trials have begun using TAA encoded on a viral vector, or in peptide form with or without APC (Table 4Go) [52]. Dendritic cells are by far the most commonly used APC for presentation of TAA. In preclinical models, a number of investigators have shown that autologous dendritic cells pulsed ex vivo with model or natural tumor antigens induce a potent immune response and protective immunity [53–56]. Encouraging results were obtained in patients with B cell lymphoma treated with autologous dendritic cells pulsed in vitro with idiotype proteins [57]. As was the case with the choice of vectors, it is not clear at this point which transgene should be used, although TAA seem to attract most attention, especially for treatment of patients with malignant melanoma [51].


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  		Table 4. Overview of current vaccine trials
 
An active area of research focuses on the regulation of the transgene expression level. To better control transgene expression levels, investigators are exploiting tissue- or cell-specific regulatory elements such as the cytomegalovirus promoter and enhancer that are primarily active in dividing cells [58], inducible promoters, or temperature-sensitive mutant sequences [59] (Fig. 2Go). Elegant studies were performed using tissue-specific promoters such as the {alpha}-fetoprotein promoter expressed in hepatocellular carcinoma [60], the erbB2 promoter [61], and the CEA promoter [62, 63]. In each case, a viral vector was designed containing the promoter of a gene preferentially expressed in tumor cells. The promoter regulated transcription of a suicide gene such as cytosine deaminase, an enzyme that converts the prodrug 5-fluorocytosine into cytotoxic 5-fluorouracil [61, 62] or the thymidine kinase gene of herpes simplex virus to confer sensitivity to ganciclovir [60, 63]. It should be noted that most of these systems are still experimental.



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  		Figure 2. Two types of generic viral vectors are presented. Most commonly, vectors contain a promoter to induce transcription of the transgene followed by the transgene itself. Currently, investigators are exploring the use of tissue- or cell-specific promoters to promote better targeting of the vectors to specific tissues or cells. The second generic construct contains two transgenes, each regulated by a promoter. Some viruses, such as the poxviruses, have the ability to take up large DNA fragments. In that case, two genes could be inserted coding for different transgenes. The most common transgenes are listed in Table 3Go.

 
In summary, the design of vaccines for cancer has come a long way in a relatively short period of time. Early reports on vaccine trials indicate that in general the vectors appear to be safe vehicles for gene transfer without any major toxicity. More importantly, responses ranging from immune responses against the transgene to objective clinical responses were induced in a variety of cancers. Once the ongoing clinical trials have been completed (Table 4Go) [51, 52], it may be better possible to evaluate the role of immunotherapy with cancer vaccines against more conventional treatments.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Vectors
 Genes
 References
 
Supported by NIH grants R01 CA60662, R01 CA68500, and T32 CA09535, and a grant from the Massachusetts Department of Public Health.


   REFERENCES
Top
 Abstract
 Introduction
 Vectors
 Genes
 References
 

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accepted for publication June 11, 1997.





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