Introduction

Vaccinia virus (VV) is a member of the genus Orthopoxvirus of the family Poxviridae ^56,133. The true origin of VV remains obscure. When Edward Jenner, an English country physician, used the material that he isolated from a milkmaid as a vaccine for smallpox ⁹³, he was in fact using cowpox virus. The vaccination material was passaged from one individual to another in the next 130 years. In the 1930s, it became clear that the strain being used at that time for smallpox vaccination was distinct from the cowpox virus. This new strain, subsequently named "vaccinia virus," is speculated to have either derived from the cowpox virus through serial passages under artificial conditions or represented a laboratory survivor of a virus that is extinct in nature ^14,78. In any case, VV gained popularity in the medical community as the choice for smallpox vaccination, superseding cowpox virus, on the basis that it produces a milder vaccination reaction.

Due to its role in the eradication of smallpox, VV has the longest and most extensive history of use in humans of any virus and has been studied extensively in the laboratory. It was the first animal virus seen microscopically, grown in tissue culture, accurately titered, physically purified, and chemically analyzed. A wealth of clinical experience has been gained with this virus. Many strains of VV exist, largely due to the evolution of the virus in different areas of the world during smallpox vaccination history. These strains are different in characteristics, pathogenicity, and host range. The New York City Board of Health (NYCBH) strain was originally used for smallpox vaccination in the United States. The Western Reserve (WR) strain is a particularly virulent laboratory derivative of NYCBH and has been used in most laboratory/preclinical studies. The Wyeth strain has been used extensively for experimental vaccines in clinical trials. Other strains, such as Copenhagen, Lister, IHD-W, and IHD-J, are also frequently used for various applications. Several attenuated strains have also been developed (such as the modified vaccinia Ankara, discussed below). Many strains have been partially or completely sequenced, for example, see ^9,67,187.

Beginning in the early 1980s, utility of VV has been extended beyond its role in smallpox vaccination. It has become an excellent research tool as a vector for expressing foreign genes in the target cells and as a means to study mammalian immune responses to virus infection. It has demonstrated effectiveness as a vaccine carrier to induce immunity against infectious diseases (for example, wildlife rabies). VV has also been increasingly explored in cancer therapy. In the context of cancer therapy, VV has been used mainly in three ways: (1) as a vector for tumor-specific delivery of cancer therapeutic genes, (2) as a carrier for tumor antigens and/or immunostimulatory molecules to develop cancer vaccines, and (3) as a replication-selective, tumor-specific oncolytic virus. In this review, we will first describe the biology of VV and then focus our discussion on the use of VV in cancer therapies.

An overview of VV biology

VV is a double-stranded DNA virus whose entire life cycle takes place within the cytoplasm of host cells. VV produces three forms of infectious particles: intracellular mature virus (IMV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). Vaccinia IMV particles are brick-shaped, approximately 300 240 120 nm in size, with a lipoprotein shell surrounding a complex core structure ¹³³. The core structure contains a linear, double-stranded DNA genome of approximately 192 kb associated with a number of virus-encoded proteins, including RNA polymerase; enzymes for RNA capping, methylation, and polyadenylation; and a transcription factor. These proteins are packaged within the virus core to enable early viral protein synthesis after entry into cells. CEVs and EEVs are IMVs with an additional lipoprotein envelope and have a lower buoyant density than IMVs ¹⁹⁰.

The VV genome is a linear, double-stranded DNA molecule with hairpin loop at each end: the two strands are joined at the ends, essentially resulting in a single-stranded circular DNA molecule ¹³. Like many other viruses, VV has inverted terminal repeats (ITRs), which are identical but oppositely oriented sequences at both ends of the genome. ITRs are important features that are required for VV DNA replication. The 192-kb genome encodes some 200 genes that are largely nonoverlapping. Genes that are highly conserved or concerned with essential replication functions are usually located in the central region of the genome, while those that are variable and concerned with host interactions are often located in the end regions.

The process of cell entry by VV is not well understood and is complicated by the fact that there are multiple forms of viral particles. The receptor for VV infection has not been unequivocally identified. VV can enter virtually all cell lines, suggesting that either it can use many different receptors or the receptor is ubiquitous. IMVs and EEVs have different cell surface binding sites ²¹⁰ and enter cells by different mechanisms ^152,208. IMVs enter cells by fusion with the plasma membrane ⁵⁰. In contrast, EEVs enter cells by endocytosis followed by low pH disruption of the EEV outer membrane and fusion of the released IMV with endosomal membranes ²⁰⁸.

A feature that distinguishes poxviruses from other classes of DNA viruses is that the virus remains in the cell cytoplasm for the duration of the infectious cycle, from the time the virus enters the cell until the progeny viruses exit through the plasma membrane ¹³³. Vaccinia mostly relies on its own encoded proteins for its life activities, especially for processes involved in DNA replication and mRNA synthesis. Minimal interaction with host proteins allows the virus to replicate in many different cell types and to avoid host defense mechanisms. After entry into the cytoplasm, the viral cores are transported to juxtanuclear locations where they start to synthesize early class viral mRNA. All the proteins required for RNA synthesis and maturation are packaged within the virion core along with the DNA genome. These include RNA polymerase, transcription factors, mRNA capping and methylating enzymes, and poly(A) polymerase ^21,133. About half of the VV genome is transcribed before DNA replication (early class viral genes) ^19,145, including those genes that encode proteins involved in DNA replication, nucleotide biosynthesis, intermediate viral gene expression, and host interactions (reviewed in ²⁰). VV early class mRNA appears within minutes after virus entry into the cell. No evidence of splicing has been reported. Subsequently, the viral cores undergo a second uncoating step, in which nucleoprotein complexes escape the core through breaches in the core wall. Meanwhile (within 2 h of infection), host cell syntheses (DNA, RNA, and protein) are completely shut down, setting the stage for viral DNA replication.

Replication of vaccinia viral DNA occurs very efficiently within the infected cells. The time of onset of DNA synthesis varies to some extent with multiplicity of infection (m.o.i.) and cell type. It typically begins 1–2 h after infection and results in the generation of about 10,000 genome copies per cell within hours of infection, of which half are ultimately packaged into infectious virions ^94,171. Proteins required for viral DNA replication are produced from the early transcription and translation process. DNA replication occurs at sites in cytoplasm termed viral factories ^37,115 and begins with the introduction of a nick near one or both ends of the viral genome, followed by nucleotide addition to the free 3' end, strand displacement, and concatemer resolution (reviewed in ¹³³). Viral DNA replication is followed by expression of intermediate class gene products, which encode for late transactivators, leading to late class mRNA synthesis. Late mRNAs encode enzymes and structural proteins that are assembled into the final viral particles. Promoters for early, intermediate, and late viral genes have distinctive sequence elements ^11,39,40 that are recognized by specific viral proteins ²⁰, providing the basis for a programmed cascade mechanism of gene regulation. The progeny viral DNA molecules serve as templates for the successive expression of intermediate and late genes.

Upon synthesis of the late structural proteins, infectious virus particles are assembled, a process that eventually leads to lysis of the infected cell. Initial stages of virion formation take place in virus factories ³⁷. Within these factories the newly synthesized viral DNAs are wrapped in a complex scaffold of proteins and lipids to form the first infectious viral particles, IMVs (reviewed in ¹⁹²). The majority of the IMVs remain within the cell until lysis. However, a small subset of IMVs does leave the factory in a microtubule-dependent manner ^155,175 and these particles become wrapped by a double layer of membrane derived either from the early endosomes or from the trans-Golgi network to form intracellular enveloped viruses (IEVs), an intermediate between the IMVs and the CEVs/EEVs ¹⁹⁰. IEVs then move along microtubules to the cell surface ^{62,88,214,215}, where the outer envelopes of the IEVs fuse with the plasma membrane, exposing enveloped virions on the cell surface. Some of them are retained on the cell surface to become CEVs, while others dissociate from the cell and disseminate systemically as EEVs.

The production of several different virus particles in the VV replication cycle represents a coordinated strategy to exploit cell biology to promote virus spread and to aid virus evasion of antibody and complement. IMVs represent the most abundant form of the virus and are retained in cells until lysis. They are robust, stable virions and are well suited to transmit infection between hosts. CEVs and EEVs are important for virus dissemination ^150,190. Actin tails can form underneath the cell surface below CEVs and continue to grow for considerable distance ⁸¹ and facilitate virus penetration of the surrounding cells (efficient cell-to-cell spread). EEVs represent only a fraction of a percent of total VV infectivity, but in a released form, they mediate long-range virus dissemination. Mutants with defects in EEV production are attenuated in long-range spread of the virus ^150,151.

VV–host interactions

The host response to VV infection is multifactorial. Immediately after VV invasion, nonspecific mechanisms involving apoptosis induction, complement, interferons, cytokines, and natural killer cells serve as the first-line host defense. Subsequently, adaptive immune responses that are mediated by cytotoxic and helper T cells assume importance in defense ²². Although neutralizing antibodies are also involved in host protection during VV infection (especially in preventing subsequent VV infections), the cell-mediated immune responses are known to be particularly potent and may be most critical for viral clearance ^3,32,58,189. Numerous studies have demonstrated that the T helper 1 (Th1) immune response, which is mediated by antiviral cytokines such as interleukin (IL)-12, IL-18, and interferon (IFN)-, plays a critical role in host defense against VV infection ^205,225. In contrast, the Th2 immune response (mediated by IL-4 and IL-10) may actually suppress the host response to vaccinia. It was demonstrated that IL-4 mediated down-regulation of expression of IL-2, IL-12, and IFN-; suppressed anti-vaccinia CTL responses; and delayed VV clearance in vivo ¹⁸². IL-4 and IL-10 knockout mice displayed enhanced vaccinia clearance ²⁰⁵. It is therefore not surprising that VV encodes multiple factors that actively suppress innate immunity and the Th1 immune response.

VV has developed a wide range of immune evasion strategies. Soon after entry into the cell, VV completely shuts off DNA, RNA, and protein synthesis of cellular origin ^19,44,57. An important consequence of this host cell shut-off is the abrogation of class I and class II major histocompatibility complex (MHC) molecule production and presentation, thereby leading to poor recognition of the virus by T cells. In addition, VV encodes multiple factors that suppress directly innate immunity and the Th1 immune response. For example, VV blocks the function of many immune defense molecules by secreting truncated, soluble receptors for these molecules (for examples, IFN-, - ^30,196, and - ⁷; tumor necrosis factor (TNF)- ⁵; IL-1 ^6,194; and IL-18 ^24,191), interfering with binding of these molecules to their natural cell surface receptors. VV also encodes a chemokine-binding protein (B29R), which binds to and antagonizes CC chemokines ⁸.

VV has evolved a number of other mechanisms to maximize its replication and spread in vivo. Apoptosis is a major mechanism by which the host organisms eliminate infected cells and terminate further replication and spread of the virus. VV encodes at least three proteins to inhibit premature cell death caused by apoptosis: SPI-2 ¹⁰¹, the E3L gene product ^102,110, and the F1L gene product ^216,217. Complement is another means by which the host organisms inactivate and clear viruses. At least two virus-encoded factors were shown to inhibit the complement cascade: C3L ^106,107 and B5R ⁵³. Furthermore, VV incorporates host complement control proteins in the outer envelope of EEV ²⁰⁹. These strategies allow VV to evade the consequences of complement activation. VV also encodes a double-stranded RNA binding protein (E3L) that prevents activation of protein kinase PKR ²⁷, and an eIF-2a homolog (K3L) that blocks phosphorylation of eIF2a ¹⁵. Taken together, these viral-encoded proteins allow VV to infect and replicate with remarkable efficiency in its host.

The interaction between VV and the host immune reaction is one of the most important issues in cancer therapies mediated by vaccinia vectors, especially when it comes to cancer immunotherapies and virotherapies (oncolytic virus). A quick and strong immune reaction may clear the virus prematurely, thus reducing the effectiveness of the therapy. On the other hand, a vigorous local immune response raised against viral-infected tumor cells may prime the immune system to recognize the tumor cells and/or tumor-associated antigens. This immunity can contribute to the clearance of both the local tumor and systemic metastasis and provide protection against future challenge. Thus, a strong immune response could serve either as a foe or as an ally to VV-mediated cancer therapies. The extensive panel of immunomodulating genes encoded by VV, along with the large cloning capacity, provides a unique opportunity to adjust precisely and safely the immune response to maximize overall antitumor efficacy.

Preclinical research and clinical experience with VV

VV exhibits a broad host range, allowing infection of many laboratory animals. This makes VV easy to study in the laboratory in animal models, and preclinical results can be more readily translated into clinical trials. This is in contrast to, for example, human adenovirus, for which a lack of good animal models has remained a major obstacle. VV particles are stable and can be stored as dry powder for prolonged periods of time without significant loss of infectivity, thus permitting easy transport and clinical application. Development in recombinant DNA technology has made efficient manipulation of the VV genome a reality ¹³². VV genes are largely nonoverlapping, which makes it relatively easy to manipulate the VV genome. Four approaches have been developed to create recombinant VVs ^75,132. The most widely used method is homologous recombination in permissive cells. It should be noted that, whereas DNA molecules of many other DNA viruses (for example, adenovirus) are infectious, i.e., a complete round of replication occurs after transfection of the naked viral DNA into a cell, VV genomic DNA is not infectious because the viral genome is not transcribed by cellular enzymes. Therefore, parental viral DNA must be introduced into the cells by infection. Other approaches that have been successfully used to create recombinant VVs include in vitro ligation, bacterial recombination, and Shope fibroma virus-mediated recombination ⁷⁵.

A wealth of clinical experience with VV is available, initially from its role in the worldwide smallpox eradication program and more recently from a number of clinical trials in which wild-type or recombinant VVs were used for the treatment of human cancers, as well as treatment of infectious diseases such as rabies and HIV. As a smallpox vaccine, VV induced rare adverse events (about 1000 per million vaccinations), including vaccinia necrosum, encephalitis, and eczema vaccinatum ⁵⁹. Aggressive dermal replication occurred almost exclusively in patients with severe T cell immunodeficiency. Despite worldwide use of this live virus vaccine, mutation of the virus to a more aggressive derivative has never been observed and virus-induced tumor has not been reported. The application of VV in the treatment of cancers and infectious diseases also demonstrated that the virus is well tolerated. VV has been delivered into patients in the form of subcutaneous ¹⁸⁵, intramuscular ¹⁶⁴, intratumoral ¹²⁰, and intravesical (bladder) injections ⁶⁹ without significant vector-related toxicity. Doses of up to 10⁹ plaque-forming units (pfu) have been delivered safely. Mild vector-related toxicities were observed only at high doses. Overall, VV is remarkably safe for use in humans.

VV as a delivery vehicle for cancer therapeutic genes

Several unique features of VV make it an excellent choice as a gene delivery vehicle in vivo. First, VV has a wide host range, capable of infecting almost all human cell types with high efficiency. Many other viruses have a more restricted host cell range. For example, infection by adenovirus is dependent on the abundance of the cell surface receptor coxsackievirus and adenovirus receptor (CAR), so cells that lack CAR are usually poorly infected by adenovirus ¹². Second, VV infection and gene expression occur extremely efficiently. A number of viral promoters can be chosen from to control the timing and level of transgene expression. Third, the VV genome can accommodate at least 25 kb of foreign DNA sequence ^128,188. This quantity could be further expanded by deleting viral DNA that is not required for replication in cultured cells. In comparison, other commonly used vector systems, such as adenovirus, adeno-associated virus, and retrovirus, can accommodate considerably less foreign DNA. Last, VV replication occurs exclusively in the cytoplasm, eliminating the possibility of chromosomal integration, in contrast to the retrovirus delivery system.

Despite these benefits, VV was initially not widely used as a gene therapy vector (except for delivery of tumor antigens and immunoregulatory molecules) because of the presumed high immunogenicity of the virus, which would likely make repeated injections impossible. However, new studies on animals immunized with vaccinia ¹¹¹ showed that VV-specific protein expression in tumors was as effective in vaccinated as in nonvaccinated animals. This important finding was confirmed in a clinical study showing that VV can infect and replicate in cancer despite the presence of systemic neutralizing antibodies ¹³⁵.

Several highly attenuated VV strains were developed during the smallpox era, and these have been employed as the vectors for cancer gene delivery to enhance the safety of VV vectors. For example, modified vaccinia virus Ankara (MVA) was obtained from serial passages in cultures of chicken embryo fibroblasts, resulting in the loss of substantial genomic information, including many genes regulating virus–host interactions. The virus has lost the ability to replicate in mammalian cells and became apathogenic even for immunodeficient animals. Importantly, the ability of MVA to infect and synthesize viral proteins (and transgene products) is not impaired ^16,132. Similarly, NYVAC is a derivative of the Copenhagen strain with multiple deletions whose replication in human cells is markedly impaired ^16,95,184. Attenuated VV strain Lister and its derivatives were evaluated in a number of studies as well ^28,72,142. In this section, we focus on gene delivery studies using attenuated VV strains or VVs that were rendered nonreplicative by physical–chemical treatments.

A recombinant VV that expressed the tumor suppressor p53 gene was created ²⁰⁰. The virus (rVV-p53) was built on the attenuated Lister strain backbone. Efficient expression of p53 was demonstrated in cells infected with this virus. Infected cells exhibited growth inhibition and underwent apoptosis as a result of p53 overexpression ²⁰⁰. In an in vivo experiment using a glioma tumor model, this virus was safely administered without serious toxicity and induced effective inhibition of tumor growth ²⁰¹. The antitumor activity was further enhanced when this approach was combined with radiation treatment ⁷². Mild treatment with psoralen and ultraviolet (UV) irradiation rendered this virus completely replication incompetent, further reducing the possibility of a toxic side effect due to viral replication ¹⁹⁹. The virus retained its ability to infect cells and demonstrated a prolonged, high level expression of p53, resulting in efficient induction of apoptosis ¹⁹⁹.

Cytotoxic T cells (CTLs) can recognize and kill tumor cells that present fragments of tumor-associated antigens (TAAs) in association with MHC class I molecules on their surface. However, such immune responses against cancer cells are often suppressed by a tumor-induced state of immune anergy, allowing tumor cells to grow unchecked ^76,149. Much work has attempted to overcome tumor-induced T cell anergy by the direct injection of vectors carrying one or a few cytokine genes (discussed below). Paul et al. proposed and tested an alternative strategy ¹⁴⁷. These researchers hypothesized that the polyclonal stimulation of T cells, preferably through the T cell receptor (TCR) complex, would result in a cascade of cytokines associated with T cell activation and would be best able to overcome T cell anergy. Toward this aim, they constructed MVA vectors expressing membrane-bound monoclonal antibodies specific for TCR complex. Tumor cells infected with these vectors expressed membrane-embedded antibodies and were able to induce T cell proliferation and cytokine production. When injected into growing tumors, these recombinant viral vectors induced the activation of both CD4⁺ and CD8⁺ T cells and resulted in tumor rejection in a murine tumor model. This approach represents a novel strategy to overcome T cell anergy in tumors and allow the stimulation of tumor-specific T cells.

Tumor cells (and their specific tumor antigens) are often ignored by the body's immune system, either because MHC I expression on tumor cells is down-regulated or the TAAs are mistaken for "self" molecules. A strategy to overcome this type of immune anergy by "targeting" cytolytic effector cells toward TAA-expressing cancer cells was designed and tested ^146,148. Recombinant vaccinia vectors that express membrane-anchored monoclonal antibodies for tumor antigens (for example, MUC1 and GA733-2/epithelial cell adhesion molecule) were constructed. These vectors were then used to infect immune effector cells such as activated macrophages and CTLs. Antibodies were expressed by the infected immune effector cells in a membrane-embedded form and directed the lytic function of these cells toward cancer cells that express the target TAAs. It is conceivable that this approach can be used for ex vivo cancer therapeutics: macrophages and T cells transduced and expanded in vitro can be injected into the patient to lyse the TAA-expressing tumor cells.

VV as a vector for cancer immunotherapy

The goal of cancer immunotherapy is to stimulate immune responses that not only target and eliminate existing tumor cells, but also establish a long-term antitumor memory. In general, cancer immunotherapy consists of two major strategies: nonspecific adjuvant immunotherapies and antigen-specific immunotherapies. Nonspecific adjuvant immunotherapies stimulate immune responses by modulating systemic or local concentration of cytokines or costimulatory molecules. Antigen-specific immunotherapies include delivery of TAAs or cells that express specific TAAs, adoptive transfer of T cells that are activated by specific tumor antigens, and therapies using dendritic cells (DCs) pulsed with TAAs. VV is highly immunogenic and is capable of inducing strong humoral as well as cell-mediated immune responses. More importantly, infection by VV provides "danger signals" to the host that help to prime T cell responses effectively ^60,122. Thus, VV represents a unique opportunity as a delivery vector for cancer immunotherapy. Indeed, a number of cancer vaccines based on VV vectors have shown promising results in preclinical animal models and numerous clinical trials ^75,109. VV has been engineered to delivery TAAs to elicit antigen-specific immune responses. It has also been used to deliver immune modulating genes, such as cytokines and costimulatory molecules, directly into established tumors to change the local microenvironment. In this review, we will focus our discussion on some of the most recent developments.

VV expressing tumor-associated antigens

Carcinoembryonic antigen (CEA) is a glycoprotein that is commonly expressed at low levels in normal tissues but overexpressed in most carcinomas of the colon, rectum, breast, lung, pancreas, and gastrointestinal tract. Recombinant VV vectors carrying CEA were constructed and examined in numerous preclinical studies. In general, vaccination with VV–CEA in animal models generated anti-CEA antibodies and CEA-specific T cells and induced antitumor responses against tumor cells overexpressing CEA ^{1,17,71,82,85,108,116,177}. Clinical trials have been conducted with these vectors to examine the toxicity, immune activities, and tumor responses in patients with advanced or metastatic CEA-expressing adenocarcinomas. In general, the vaccines have been well tolerated and effective at inducing CEA-specific cytotoxic T cell responses ^{10,31,116,123,203} and anti-CEA antibodies ³³. However, so far these studies failed to demonstrate objective antitumor responses. New vaccines and vaccination strategies were adopted to enhance antitumor efficacy. One strategy was to use a diversified prime-and-boost protocol ^85,118 combining VV-CEA and a replication-deficient avipox virus vector expressing CEA (ALVAC–CEA ^117,226). Another strategy was to combine CEA with T cell costimulatory molecules (for example, B7.1) ^{1,73, 85,90,131}. A third strategy is the incorporation of a modification in the CEA protein that resulted in higher affinity for the T cell receptor ²²⁴. All three strategies have significantly enhanced production of CEA-specific cytotoxic T cells ^116,118,124, and a clinical trial using avipox virus vector ALVAC expressing CEA and B7.1 has demonstrated disease stability in a subset of patients ⁹⁰. Other clinical trials are ongoing to test these new strategies ¹³¹.

Prostate-specific antigen (PSA) is a glycoprotein that is expressed at a relatively low level by normal prostate epithelial cells. It is a good target for immunotherapy because it is commonly overexpressed by most primary and metastatic prostate cancers. VV vectors encoding PSA (VV–PSA) have demonstrated an ability to induce PSA-specific T cell responses and therapeutic activity in animal prostate cancer models, especially when cytokines and costimulatory molecules were coexpressed ^87,89,91,198. Clinical trials in patients with advanced or recurrent prostate cancer demonstrated that vaccination with VV–PSA was well tolerated, stimulated PSA-specific T cell production, and resulted in stabilization of PSA levels ^52,74,174. A phase II clinical trial was conducted to evaluate a diversified prime-and-boost vaccine protocol involving VV–PSA and a fowlpox virus vector expressing PSA in patients with recurrent prostate cancer ⁹⁹. Of the eligible patients, 46% demonstrated an increase in PSA-reactive T cells, 45.3% maintained stable PSA level at 19.1 months, and 78.1% demonstrated clinical progression-free survival.

Many melanoma-associated tumor antigens have been identified ^129,176. Among them, melanocyte differentiation antigens, such as melan-A/MART-1, tyrosinase, and gp100, are most attractive as candidate targets for cancer immunotherapy as they are expressed in nearly all melanoma tumor cells ¹⁵³. In numerous preclinical studies ^{51,143,156,180,220,222}, VV vectors expressing single melanoma TAAs or different combinations have demonstrated powerful capabilities to stimulate specific CTL generation and induce cytotoxicity against tumor cells that express the relevant antigens. Several clinical studies are being conducted to evaluate the safety and immunogenicity of these vaccines ^46,170,193. In general, these vaccines were well tolerated and effective in generating cellular immune responses ^170,193. A recent report ²²¹ on a phase I/II clinical trial in metastatic melanoma patients with an UV-inactivated nonreplicating recombinant VV expressing an ER-targeted melan-A/MART-1 minigene, peptides from gp100 and tyrosinase, and CD80 and CD86 costimulatory proteins showed that 15 of 18 evaluable patients had specific T cell responses against the TAAs. Regression of individual metastases was observed in 3 patients, stable disease in 7 patients, and progressive disease in 7 patients. Other melanoma-associated antigens are also being explored as candidate targets for immunotherapy, such as CD63 ¹¹².

Human papillomavirus (HPV) is the etiologic agent associated with cervical carcinoma. The E6 and E7 oncoproteins of HPV are normally required for the maintenance of the malignant phenotype, whereas the HPV E2 protein is able to regulate the expression of E6 and E7 proteins negatively. The E2 protein can also promote cell arrest and apoptosis. Thus, the HPV E2 protein has the potential to inhibit the malignant phenotype. In addition, the E2 protein is required early in HPV infection and therefore may serve as a useful immune target for a vaccine aimed at prevention or therapy of premalignant lesions. Recombinant MVA expressing the HPV E2 (MVA–E2) was created and tested in animal models. In nude mice, MVA–E2 efficiently inhibited growth of HPV-associated human tumors and increased life expectancy of the animals three- to fourfold ²⁰⁴. In a papilloma cancer model in immunocompetent rabbits, treatment with MVA–E2 resulted in remarkable tumor regression. A humoral immune response against tumor cells was observed in these animals. These antitumor antibodies were capable of activating macrophages to destroy tumor cells efficiently, which is responsible, at least in part, for the antitumor activity of this vector ¹⁶⁷. MVA-E2 is currently being evaluated in a phase II clinical trial, in patients with HPV-related cervical intraepithelial neoplasia, a precursor lesion to invasive cervical carcinoma ³⁴. In a recent report, 36 women were treated with MVA–E2 at a total of 10⁷ virus particles injected directly into the uterus once every week over a 6-week period. The vaccine did not produce any apparent side effects in any of the patients treated. Thirty-four of 36 patients showed complete elimination of precancerous lesions. All patients developed antibodies against E2, showed a remarkable reduction of HPV viral load, and generated a specific cytotoxic response against HPV-transformed cells ³⁵. In addition to E2, the HPV E6 and E7 proteins are also potential immune targets for a cancer vaccine. A live recombinant VV expressing modified forms of the HPV-16 and -18 E6 and E7 proteins (TA–HPV) was examined in patients with early stage cervical cancer. Vaccination with TA–HPV was shown to be well tolerated and result in enhanced HPV-specific cytotoxic T cell responses ^4,100. A recent report ³⁸ confirmed these findings and further demonstrated that the HPV viral load was significantly reduced and that 67% (12/18) of the treated patients had significant reduction in lesion size or relief in symptoms.

Many other TAAs are being evaluated in preclinical and clinical settings for their potential as vaccines for cancer immunotherapy. MUC1 is a glycoprotein overexpressed and aberrantly glycosylated in most breast tumors and many other human cancers ⁴⁹. Recombinant VV vectors expressing the human MUC1 and IL-2 genes are currently under investigation and have been shown to stimulate MUC1-specific T cell responses both in mice ² and in humans in phase I and II clinical trials ¹⁷⁹. A recent study showed that 40% of the patients vaccinated with MUC1 had antigen-specific T cell response and 30% of the patients had stable disease ¹⁶⁴. Other tumor antigens that are currently being evaluated using VV expression platform include 5T4 ¹³⁶, GA733 ²²³, tumor suppressor p53 ^55,207, and the tumor antigens of Epstein–Barr virus (EBNA1, LMP1, and LMP2) ^70,197.

Cancer immunotherapy mediated by dendritic cells

DCs are professional antigen-presenting cells and have been explored widely as a presentation platform for TAAs in immunotherapies for cancer ¹⁹⁵. Moreover, pieces of dead DCs can be taken up by other DCs, resulting in antigen cross-presentation to amplify further the immune reaction ¹³⁴. Recombinant VVs are used to infect DCs and express TAAs. DCs loaded with TAAs then efficiently induce a strong T cell response against the TAA-expressing tumor cells while protecting the virus from the neutralizing antibody responses that may potentially limit future boosting ⁹². Numerous preclinical studies using recombinant VV vectors have demonstrated efficient delivery and expression of various TAAs (for example, gp100 ^137,156, MUC1 ²⁰², EBV-derived tumor antigens ^70,197) in human DCs and robust stimulation of antigen-specific CTL responses. A phase I clinical trial is ongoing to evaluate this technology in patients ⁴⁶.

VV-lysed tumor cell vaccine (Oncolysates)

Another form of VV-based cancer immunotherapy is to use VV-infected tumor cell lysates as vaccines. This strategy, also known as the oncolysate approach, exploits vaccinia's high level of immunogenicity. This approach showed promise in animal models, and earlier clinical trials involving several hundred patients have demonstrated safety and antitumor potential ^211,212. When used as an adjuvant therapy in patients with stage 3 malignant melanoma, this approach conferred a small survival advantage in a subset of patients ²¹³. However, there was no difference in survival when all patients were considered together. Other reports ^80,103 also showed no significant difference in disease-free interval or overall survival between vaccine patients and the control group. It should be noted, however, that the control group received live VV without tumor oncolysate, which could potentially have affected patient response ¹⁰³. Further studies are needed if the oncolysate approach is to become a viable treatment form for human cancers.

VV expressing immunoregulatory molecules

Cytokines and costimulatory molecules are often delivered to tumors using recombinant VV vectors, either as single agents or in various combinations. The overall hypothesis is that by modulating the immune milieu at the local tumor site, and thus recruiting antigen-presenting cells and effector cells, it will be possible to engender a systemic tumor-specific immune response. The important roles of cytokines and costimulatory molecules in promoting antitumor immune responses have been well documented ^{25,43,130,168,169}. Recombinant VV vectors have been employed to deliver local release of cytokines (such as IL-2, IL-12, granulocyte–macrophage colony-stimulating factor (GM-CSF), TNF-) and costimulatory molecules (such as B7.1, ICAM-1, and LFA-3). Encouraging results in augmenting antitumor immune responses have been documented in animal models and human clinical trials (reviewed in ¹⁰⁹). The most significant antitumor responses were seen, however, when TAAs, cytokines, and costimulatory molecules were simultaneously expressed in various combinations. The capacity of VV to accommodate large pieces of DNA and express multiple genes in the same vector allows for flexibility in engineering, such that multiple immune enhancing genes and TAA genes can be recombined together into the genome. One group has shown that cells infected with a recombinant VV expressing three costimulatory molecules (B7.1, ICAM-1, and LFA-3, designated TRICOM) were much more efficient at inducing antigen-specific CD4 and CD8 T cell activation than cells infected with vectors expressing any one or two costimulatory molecules or no costimulatory molecules at all ^83,84,86,160. As an extension of this research, recombinant VV encoding human CEA and the TRICOM was generated and tested in animal studies. Strong activation of T cells directed against CEA and CEA-bearing tumor cells was observed, which led to potent therapeutic antitumor immunity ^82,85. Various clinical trials combining costimulatory molecules with TAAs are ongoing ^90,97,98,131.

Numerous other studies have demonstrated the synergistic effect when cytokines and TAAs were coexpressed from recombinant VV ^{118,144,178,179,219} or when cytokines were combined with costimulatory molecules and TAAs ^{1,26,71,73,108,162}. Local administration of GM-CSF, IL-2, or IL-12 in patients receiving tumor vaccines was found to be safe and contributed significantly to the induction of TAA-specific T cell responses, leading to remarkable antitumor activity in animal tumor models and in cancer patients in early clinical trials. In conclusion, induction of a strong antitumor immunity requires both the presentation of TAAs and augmenting effects mediated by costimulatory molecules and cytokines. In most studies, there was a positive correlation between the number of costimulatory and immune-enhancing cytokine molecules being expressed and the overall antitumor immune response. The size and the cloning flexibility of VV make it an attractive vector for delivery of multiple genes to activate optimally antitumor immune responses. As more and more immunostimulatory molecules are coexpressed from the VV vectors, it will be important to determine the toxicity and the efficacy of the vaccines.

Prime-and-boost strategy

As mentioned above, a diversified prime-and-boost using two different vectors has proved to be highly effective in inducing antitumor immunity. There are at least two reasons why this strategy is beneficial. First, the host immune responses against the viral vector may prevent the effectiveness of subsequent vaccination using the same vector. Second, the danger signals induced by two vectors may be likely to prime the immune system to mount a stronger reaction. Although the optimal choice of vectors for maximizing antitumor immunity is not known, this strategy has been used extensively with two different poxviruses ^84,116. VV-based cancer vaccines were frequently used in prime-and-boost immunization, in combination with vaccines based on fowlpox virus ^73,82,85,108, Sindbis virus ¹¹³, plasmid DNA ^126,181, and peptides ^143,206. Improved antitumor effects were observed in animal models when a diversified prime-and-boost strategy was used ^84,116. In a phase II clinical trial with the advanced prostate cancer patients, this strategy has demonstrated improved PSA stabilization and progress-free survival ⁹⁹. In another clinical trial involving 18 patients with advanced tumors expressing CEA, this strategy increased the precursor frequency of CEA-specific T cells and produced limited clinical efficacy in a subset of patients ^116,118.

Conditionally replicating VV as oncolytic agent

The earliest report of using viruses to treat cancer was published 100 years ago ⁴⁸. However, it is only recently that advances in molecular biology, virology, and cancer biology have provided us with the tools necessary to develop novel oncolytic viruses for cancer therapy. The first tumor-selective oncolytic virus to demonstrate antitumor activity in a clinical setting was ONYX-015, a recombinant adenovirus with a functional deletion of the E1B-55K gene ^{18,127,140,163}. Since then, numerous viruses have been explored as tumor-selective replicating vectors, including adenovirus, herpes simplex virus (HSV), reovirus, Newcastle disease virus, vesicular stomatitis virus, measles virus, poliovirus, West Nile virus, and VV (reviewed in ^29,104,139).

Efficient replication, cell lysis, and spread of VV, its broad host range and natural tropism for tumor tissues (see below), along with its remarkable safety record in human use, make VV a very attractive vector for developing oncolytic viruses. The entire life cycle of VV is usually completed within 24 h, releasing as many as 10,000 IMV particles upon cell lysis. Infectious CEV and EEV may be released from cells as soon as 6 h postinfection, allowing rapid long-range spread. This is in sharp contrast to adenovirus, whose replication cycle typically lasts 48–72 h ¹⁸³. Many of the limitations found with ONYX-015 in clinical trials, including insufficient antitumor potency as a single agent, inability to infect efficiently tumor metastases following intravenous delivery, and insufficient virus spread within solid tumors, are, at least in part, due to slow replication of adenovirus. It is conceivable that these limitations can be relieved by viruses that are more potent in replication/spread.

Nonengineered VV

Wild-type VV has been noted to have a natural tropism for tumors. After intravenous injection into tumor-bearing animals, the highest amount of virus was recovered from the tumor, followed by the ovary, with little virus detected in other organs ^157,218,225. It has been suggested that the leaky vasculature in most tumors and in ovarian follicles may be responsible for this tropism ^125,225, because the large particle size of VV (350 nm in diameter) may require the leaky vasculature to exit from circulation and enter tissues. This natural tropism for tumors gives VV an advantage to be the base for oncolytic viruses.

Historically, wild-type VV has been used, whether deliberately or not, in the treatment of human cancers. In 1974, Roenigk et al. explored the antitumor activity of the Wyeth stain of VV (the vaccine strain used in the United States for smallpox immunization) by direct intralesional injection in 20 patients with metastatic malignant melanoma ¹⁶⁶. The trial demonstrated antitumor efficacy against injected lesions (objective response rate was approximately 50%) with only mild, transient side effects such as flu-like symptoms. In a 1978 report, a patient with untreated chronic lymphocytic leukemia was revaccinated for smallpox and was found to be in complete remission ⁷⁷. More recently, a clinical trial was conducted in five patients with recurrent melanoma using the wild-type Wyeth strain ¹¹⁹. Patients were treated twice weekly with increasing doses of intralesional injected virus. Minimal systemic side effects were observed in the trial. Three of five patients had partial but brief tumor regression, and one patient sustained clinically complete remission of the treated lesion and an untreated lesion in close proximity. Evidence of viral infection was demonstrated in the injected tumor throughout the treatment course even in the face of systemic antivirus immunity.

Genetically modified replication-selective VV

Given the remarkable efficiency of VV in inducing tissue destruction, genetic modifications of the virus have been designed to create oncolytic vectors that specifically infect and replicate in tumor cells. The common laboratory strain WR was selected by many researchers as the basis for tumor-selective oncolytic VVs because it is more efficient at producing tissue damage than other strains used in vaccination trials ⁷⁵. The WR strain produces a large amount of CEV compared to other VV strains ¹⁹⁰ and forms large plaques in cell cultures, reflecting its ability to spread efficiently and cause large areas of cell lysis. This feature is extremely useful for an oncolytic virus, as it allows for efficient viral penetration within solid tumors with minimal systemic spread.

Many strategies have been used to create tumor-selective oncolytic viruses (reviewed in ^105,139). These strategies can be grouped into three general approaches. The first approach is to delete gene functions that are critical for efficient viral replication in normal cells but dispensable in tumor cells, exemplified by the creation of the replication-selective adenovirus mutant ONYX-015 ^18,47. The second approach is to limit the expression of a critical viral gene to tumor tissues through the use of tumor- and/or tissue-specific promoters, represented by the adenovirus mutant CV706 ^45,165. The third general approach is to alter viral tropism through modification of surface proteins ^36,68. Thus far, only the first general approach has been successfully used to engineer tumor-specific VVs.

A recombinant VV with the thymidine kinase (TK) gene deleted was examined. TK is involved in the synthesis of deoxyribonucleotides to facilitate DNA replication in cells with suboptimal precursor pools. While the TK gene is necessary for replication in normal cells, where intracellular nucleotide concentration tends to be low, it is not necessary in cancer cells, which have relatively high concentrations of intracellular nucleotides ²³. Thus replication of the TK-deleted virus is dependent on the growth status of the host cells. This virus demonstrated tumor selectivity over normal tissues in a number of animal tumor models, including murine colon cancer and melanoma, human colon cancer and melanoma in nude mice, rat sarcoma, and rabbit kidney cancer ^65,157. Upon systemic administration into mice (such as intravenous or intraperitoneal injection), this virus specifically targeted its replication in subcutaneous tumors and led to an antitumor response ¹²⁴. The antitumor effect was shown to be related directly to viral replication within the tumor and was not the result of a bystander inflammatory response, as the effect was enhanced in athymic/nude mice, which cannot mount an effective immune response against the virus ⁶⁶.

Another example of generating tumor specificity of VV using the gene deletion approach is the deletion of the vaccinia growth factor (VGF) gene. VGF is expressed early during the VV infection cycle and is secreted from infected cells. It then binds growth factor receptors on surrounding resting cells and stimulates cell proliferation, thus preparing them for subsequent VV infection. This function is important for VV replication in normal tissues, but dispensable in tumors because tumor cells are naturally proliferating. Deletion of this gene would create a virus that preferentially replicates in tumors. A double-deletion VV was constructed ¹²⁵ with both the TK gene and the VGF gene eliminated. It was hypothesized ¹²⁵ that double deletion should further diminish virus replication in normal cells. Indeed, the TK^–/VGF^– virus was found to have markedly enhanced tumor specificity in vivo: it can be injected intravenously at doses of 10⁸ pfu into nude mice without pathogenicity, while preserving intratumoral virus replication and producing regression of established subcutaneous tumors ¹²⁵. The same dose of the wild-type WR strain killed all the mice with a medium survival of 5 days. The excellent safety profile of this double-deleted virus was also demonstrated in rhesus macaques at doses up to 10⁹ pfu ⁷⁵. This virus will soon be evaluated in phase I clinical trials.

Additional strategies are being explored using the gene deletion approach to create tumor-selective oncolytic VVs. VV encodes a number of host-range genes, such as the antiapoptotic SPI-1 and SPI-2 genes. It has been speculated that products from these host-range genes may enable VV to replicate in nontransformed cells, but their functions may be redundant in tumor cells ²²⁵. Therefore, deletion of such host-range genes may confer tumor-selectivity to the virus.

The second general approach, engineering tumor selectivity through the use of tumor- and/or tissue-specific promoters, is unlikely to work for VV, because transcription occurs exclusively in cytoplasm and gene expression is mostly regulated by viral transcription factors.

The third approach, improving tissue or tumor specificity of a viral vector by genetically modifying the surface proteins of the virus, has been successfully applied to adenovirus ^36,173 and HSV ⁶⁸ and other viral systems. However, this approach may be of limited usefulness for VV at the current stage. First, the precise molecular mechanisms that regulate VV entry process are not understood. The cellular receptor(s) for VV has not been defined. Second, VV produces three different forms of viral particles, IMV, CEV, and EEV, whose entry mechanisms are likely to be different ^152,208. Third, VV naturally infects all cell types, possibly by utilizing multiple cellular receptors. Although some attempts have been made to alter the EEV coat proteins ^61,225, specific binding or altered tropism toward target cell lines has not been demonstrated. Future investigation of this approach awaits a better understanding of the precise molecular mechanisms that regulate uptake of VV.

One interesting strategy that has been used to augment VV delivery to tumors following systemic administration is local hyperthermia ²²⁵. Hyperthermia increases the permeability of the endothelial vasculature and so may facilitate extravasation of VV particles into tissues. It was found that hyperthermia increased the permeability of an in vitro model of the endothelial cell monolayer to VV and the phenomenon was completely reversible. Importantly, infection, gene expression, and cytotoxicity of VV were not influenced by hyperthermia. Using an in vivo model (immunocompetent mice bearing subcutaneous MC-38 tumors) receiving systemic VV treatment, it was demonstrated that regional hyperthermia (41.5°C for 30 min) improved vaccinia targeting to tumors and increased marker gene expression by over 100-fold. This effect was tumor specific and correlated with significant antitumor responses. Presumably, tissues with a baseline leaky vasculature (such as tumors) may have increased susceptibility to hyperthermia, allowing virus particles to access these cells more efficiently. This strategy is currently being tested in nonhuman primate models.

Armed oncolytic VVs

Because infection and lysis of 100% of the tumor cells is hard to achieve in vivo using oncolytic viruses alone, oncolytic viruses are often "armed" with genes that can augment their cytolytic capacities. Meanwhile, tumor-directed gene therapy requires high levels of gene expression in a high percentage of tumor cells in vivo to be effective. Oncolytic viruses that specifically replicate in tumor tissues and amplify template copy numbers provide an ideal platform for achieving this goal ⁶⁵. Three groups of genes are commonly used in armed oncolytic virus therapy. The first group includes genes whose products can kill tumor cells directly or indirectly. Genes that have "bystander effects" are most attractive as they can lead to the death of uninfected surrounding tumor cells. The commonly used genes within this group are those that encode diffusible toxins, prodrug converting enzymes, or immunostimulatory molecules. The second group of genes includes those whose products may facilitate virus infection and/or replication, for example, suppressors of antiviral immune responses such as IL-4 or IL-10. The third group includes the so-called "safety valve" genes. They function to shut off virus replication/spread in case the virus becomes uncontrolled or the patient suffers serious adverse reactions.

Thus far, two prodrug converting enzyme/prodrug systems have been analyzed using replication-selective VV platform: the cytosine deaminase/5-fluorocytosine (CD/5-FC) system ^66,124 and the purine nucleoside phosphorylase/6-methylpurine deoxyriboside (PNP/6-MPDR) system ¹⁵⁸. In both cases, the prodrug converting enzymes were expressed from a TK-deleted, replication-competent VV vector. Complex interaction between the virus-mediated oncolysis and CD/5-FC cytotoxic effect was examined ^124,225. In vitro infection of cancer cells at high m.o.i. (>0.1) with a TK-deleted VV expressing CD (VV-CD) led to cell death due to viral cytopathic effect. No added effect was observed with addition of 5-FC. At low m.o.i., virus alone did not produce a cytopathic effect, but addition of 5-FC mediated significant cell death. Importantly, addition of 5-FC also inhibited virus replication by about 300-fold. Thus, 5-FU produced in this system appeared to have an inhibitory effect on viral replication, either by direct interference with viral DNA synthesis or by killing surrounding cells prior to their infection. Similar results were observed in nude mice bearing subcutaneous MC38 tumors ¹²⁴. Although VV-CD alone demonstrated an antitumor effect, this effect was augmented by the addition of 5-FC, especially at low dose of virus. 5-FC also retarded virus replication, prolonging animal survival from virus-mediated death. The PNP/6-MPDR system was found to have a similar interplay with replication of the viral vector ¹⁵⁸. Therefore, incorporating a prodrug converting enzyme/prodrug system to a replicating VV can have dual effects. On one side, it can enhance the antitumor response, especially when low levels of the virus reach the tumor. In the clinical setting this may be of particular importance because the amount of virus that can reach the patient's tumor is likely to be very low. On the other side, it can decrease viral pathogenicity by inhibiting viral replication. In this aspect the prodrug converting enzymes function as safety valves, capable of shutting off VV replication and spread whenever it is necessary. Extensive studies are needed to find the right balance and maximize the antitumor efficacy. One strategy for taking advantage of both the viral oncolysis and the enzyme/prodrug cytotoxicity is to place the prodrug converting enzyme under the control of an inducible expression system that would allow the transgene to be turned on after significant viral replication has occurred.

Expression of immunostimulatory molecules may be another way to enhance the overall antitumor efficacy of the oncolytic virus. A systemic antitumor immune response induced by the immunostimulatory molecules can target and kill uninfected tumor cells, thereby providing the bystander effect. An oncolytic VV alone may drive an antitumor immune response via the viral-induced oncolysis. However, this response is generally rather weak, as VV has evolved efficient mechanisms to retard the development of a Th1 immune response. It is likely that expression of an immunostimulatory molecule (such as a cytokine capable of driving a Th1/Tc1 immune response) from the vaccinia may significantly augment any antitumor immune response. By the same token, a strong immune response may clear the virus prematurely, reducing the efficiency of viral replication in vivo. Expression of cytokines from a VV vector would be expected to exacerbate this problem, as demonstrated with IL-2 ^96,154, IL-15 ^114,154, and TNF- ¹⁷². Although it is clear that expression of immunostimulatory molecules diminishes in vivo replication and oncolytic properties of the virus, it appears that the immunostimulatory capabilities are not reduced ^63,64,161. Therefore, the overall antitumor efficacy of oncolytic VVs expressing immunostimulatory genes is determined by the gain in antitumor immune response and the loss in direct viral oncolysis. Only empirical testing will tell us whether the enhanced bystander capabilities of a particular cytokine recombinant VV will outweigh the diminution of its direct oncolytic effects. Clinical studies with one such virus have generated promising results ^120,121. A TK-deleted, replication-competent VV expressing GM-CSF was shown to infect selectively melanoma cells and induce an antitumor immune response. This virus was administrated intralesionally in a phase I clinical trial involving seven immunocompetent patients with refractory and/or recurrent melanoma. Injected lesions contained an active inflammatory response and demonstrable viral replication. Of seven patients studied, one had a complete response and four had a mixed/partial response ¹²¹. These results clearly support further study of this virus and similar oncolytic viruses that express other cytokines.

Other therapeutic genes have been proposed to arm oncolytic viruses ⁷⁹. These include genes whose products may facilitate virus infection and/or replication and genes that inhibit angiogenesis. The interplay between the virus and the transgene products is very complicated, and thus far the beneficial effect of arming viruses with these transgenes has not been clearly demonstrated. For example, IL-4 and IL-10 have been inserted into VV to circumvent premature viral clearance and increase in vivo virus replication ^182,205. Although this approach is expected to enhance direct viral-mediated oncolysis, the safety concerns created by a virus that is not recognized by the immune system make it unlikely to be considered for use in virotherapy.

Older patients who received VV for smallpox vaccination have existing immunity against VV. This will limit the replication and spread of VV in these patients, reducing the effectiveness of VV-mediated virotherapies. This will not affect the younger generations who have never been immunized with this virus. Previous work with the oncolytic adenovirus ONYX-015 indicated that the overall tumor response was not significantly different in patients who were seropositive for adenovirus compared to those who were naïve for this virus ^47,138. The effect of a preexisting immunity against VV on virotherapy is unclear. New strategies need to be developed to circumvent this issue. For example, transient immunosuppression may temporarily tune down the immune reaction and allow the virus to replicate in and lyse cancer cells efficiently.

Safety considerations for using VV in cancer treatment

Even with the excellent safety record of VV in human clinical use, therapies based on VV vectors (especially when live viruses are used for virotherapies) still raise biosafety and risk management issues. The properties that make VV a useful tool for tumor therapy also make it potentially dangerous to the treated patients and their contacts. For example, efficient replication of VV in a wide spectrum of human cells raises the possibility of serious damage to untargeted, normal tissues. This is particularly important when replication-competent VVs are employed in systemic administration. Although intradermal delivery has proved to be safe for the vaccine strains, more virulent VV strains, such as WR, may be pathogenic following systemic administration or even local delivery as VV may replicate and be shed into the bloodstream. This would affect not only the treated patients, but also their relatives and contacts. An increasing population with immunodeficiency also adds to the possibility of an epidemic spread of the virus. Because many recombinant VVs carry transgenes, effects of the transgene products must be considered in addition to the pathogenicity of the viral vector. Extensive clinical studies are necessary to address these safety concerns.

Should unwanted replications occur, methods for treating or controlling VV replication do exist. Vaccinia immunoglobulin is approved for treating complications of vaccinia infection ⁵⁴. However, the effectiveness of this product still awaits further demonstration in randomized trials. In addition, many antiviral drugs have provided effective treatment in animal models of orthopoxvirus infections ⁴². Of the approved antiviral drugs, cidofovir has the greatest potential for protection against VV replications ^{41,141,159,186}. This drug is licensed to treat cytomegalovirus-induced complications in AIDS patients, but is not currently licensed to treat vaccinia. It will be an important task in the coming years to define the safety profile and efficacy of cidofovir and other antiviral agents in humans. Finally, as mentioned earlier, suicide genes engineered into the virus may convert nontoxic prodrugs into active drugs that can inhibit viral replication. Anti-vaccinia activity has been demonstrated experimentally using this approach both in vitro and in vivo ^124,158.

Conclusion

VV possesses many unique properties that make it a good tool in cancer treatment. Its large genome capacity, ability to infect a broad range of host cells, and powerful gene expression machinery make it an excellent choice to deliver multiple therapeutic genes at very high levels. When VV is used as a delivery vehicle for cancer immunotherapy, its high immunogenicity may contribute to eliciting a strong antitumor immune response. VV has also become an important platform on which to build oncolytic viral agents because it replicates and spreads efficiently, it traffics to tumors effectively in vivo, and it can carry therapeutic genes to enhance antitumor efficacy further. The huge database on safety and activity of vaccinia in humans will lend valuable information regarding its proper use in the clinics. Further understanding of the biology of this virus will improve our ability to manipulate it to our advantage. It can be anticipated that the potentials of this virus may become more apparent and its applicability may be more significant over time.

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Acknowledgements

We extend special thanks to Dr. Sean Tucker for his critical reading of the manuscript.