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Neutralizing Ab to JEV

McClain (132)

Vaccinia-Hantaan virus

Neutralizing Ab to Hantaan virus

McAneny (133)

Vaccinia-CEA

Safe

Borysiewicz (134)

Vaccinia-HPV

HPV specific immunity

Picard (135)

Vaccinia-gp 160

HIV immunity in healthy controls

of this vector in more novel delivery systems in terminal cancer patients. As the field of tumor-directed cancer gene therapy has developed, the study of tumor-selective replicating viruses has become an important endeavor. Because of the known ability of vaccinia to destroy tissue (a complication of smallpox vaccination known as vaccinia necrosom), vaccinia has also been developed as a direct oncolytic virus for cancer therapy. The concept of using replication-competent viruses to selectively destroy tumors is quite appealing. Numerous viruses have been explored as tumor-selective replicating vectors, including adenovirus, herpes simplex virus, reovirus, newcastle disease virus, autonomous parvovirus, and vaccinia virus (111,112). Advantages and disadvantages exist for each of these vectors, and some limitations are common to all vectors.

Vectors are most limited by inefficient replication in vivo, inefficient tumor targeting, and safety concerns. Vaccinia has many characteristics that overcome these limitations:

1. It has a quick, efficient life cycle, forming mature virions in just 6 h after infection;

2. it spreads efficiently cell to cell thus increasing the efficacy of in vivo infection;

3. it has a large genome that can accept over 25 kb of inserted DNA without deletions;

4. vaccinia virus carries its own strong promoters capable of achieving very high levels of transgene expression;

5. it can infect a wide range of human tissues but does not cause any known human disease;

6. there is a large body of knowledge about its biology and extensive experience with it clinically as part of the smallpox vaccination program.

Preclinical development has focused on mutating the WR strain of vaccinia virus to make it replication selective for tumor cells (100,113,114). The WR strain of vaccinia virus appears to be more efficient in vivo than other strains used in vaccination trials. An intradermal injection of 106 pfu of a wild-type WR strain of vaccinia in rhesus macaques leads to a necrotic ulcer of 108 cm2 in diameter in only 8 days, without systemic spread of the virus. This compares with < 1 cm for NYCBH and Wyeth strains (115). This ability to quickly spread, express genes, and destroy tissue to this extent is unique among current vectors in clinical and preclinical development.

We and others have studied the wild-type virus and found that after intravenous injection, the highest amount of virus can be recovered from the tumor, the second highest from the ovary, and minimal to no virus is recovered from other organs (58,100,116,117) (Fig. 4). The natural tropism of this virus to tumor is surprising, and the mechanism of this is not established. This tumor tropism was demonstrated in numerous tumor models, including murine colon cancer and melanoma, rat sarcoma, human colon cancer in nude mice, and rabbit kidney cancer. Historically, smallpox virus was noted to have

Figure 4 GFP fluorescence is specific for this subcutaneous tumor after intravenous injection of vaccinia-GFP. See the color insert for a color version of this figure.

tropism for injured and irritated skin. This is believed to be secondary to histamine release, leading to leaky vasculature and allowing for transfer of the virus out of the circulation. Vaccinia is a large virus particle (350 nm in diameter) and would require leaky vasculature for extravasation into tissues. Notably, the tumor and ovarian follicles are both known to be sites of vascular endothelial growth factor production and leaky vasculature. As demonstrated by immunohistochemis-try, vaccinia tropism to the ovary is specific for ovarian follicles (100).

To develop a tumor-selective vaccinia, the WR strain of vaccinia was mutated such that the VGF and TK genes were deleted (100). The TK gene is important for nucleotide synthesis and DNA replication, and is near essential for replication in nondividing cells where the host nucleotide pool is low. Vaccinia growth factor is a protein that is expressed early by vaccinia virus and is secreted by infected cells. It binds growth factor receptors on surrounding resting cells and stimulates them to proliferate. This increases the available nucleotides in these resting cells, priming them for vaccinia infection. Deleting both the TK and VGF genes leads to near complete abrogation of replication in resting cells, without decreasing the ability of the virus to replicate in the tumor environment. This mutant virus was made and tested, and found to have markedly enhanced tumor specificity (Fig. 5). This virus was tested in rhesus macaques and was found to be completely nonpathogenic when delivered intravenously at doses up to 109 pfu (115). Intradermal inoculation at 106 pfu demonstrated no viral replication. Nevertheless, 4 days after intravenous virus delivery, equal titers of wild-type and double-deleted virus could be recovered from subcutaneous tumor in mice. This virus can be given systemically at doses of 109 pfu to a nude mouse without pathogenicity, and led to marked responses in established subcutaneous tumors (Fig. 6).

One limitation to mutating a virus for selective replication in dividing cells is potential toxicity to other dividing cells in

Figure 5 Differential viral recovery (median viral titers) from brain and tumor after systemic injection of 107 pfu wild-type, VGF-deleted, TK-deleted, and TK/VGF-deleted vaccinia virus in MC-38 tumor-bearing mice.

vivo. Vaccinia can efficiently infect almost all cell types in vitro, and it is hard to imagine delivering this systemically to animals or humans without significant pathogenicity. Other sites of dividing cells known to suffer toxicity from chemotherapy agents such as bone marrow-derived cells and gastrointestinal mucosa are not affected by systemic vaccinia in murine, rat, rabbit, or primate studies (10). The large virus seems to require a leaky vasculature for extravasation into tissues in addition to proliferating cells. This leaky vasculature is lacking in tissues such as the gastrointestinal mucosa. Despite bone marrow-derived cells having ready access to circulating vaccinia, no bone marrow toxicity is encountered, even

Figure 6 Antitumor response after systemic injection of 109 pfu of the TK/VGF-deleted virus in nude mice bearing 5-mm MC-38 tumors. (From Ref. 100.)

in animals succumbing to viral pathogenicity (unpublished observations, 1999). Bone marrow-derived cells are known not to infect well by vaccinia for unknown reasons. The one place that replicating virus is recovered is ovarian follicles. The developing follicle is much like a tumor with developing, leaky vasculature, high levels of VEGF, and replicating cells (118). Immunohistochemistry demonstrates that the double-deleted vaccinia replicates efficiently throughout developing ovarian follicles, without infection or spread through normal ovarian parenchyma.

Although no clinical trials to date have focused directly on the ''oncolytic'' activity of vaccinia virus, intratumoral injections have been explored. In 1974, Roenigk et al. described direct injection of melanoma with vaccinia from standard vialed smallpox vaccine (Wyeth strain) at unknown concentrations in 20 patients at 2-week intervals (119). Numerous interesting antitumor responses of injected lesions were described. Even older studies from the 1960s describe the treatment of warts with direct intralesional vaccinia injection with success (120).

Mastrangelo et al. reported their results of intratumoral injection of a NYCBH vaccinia strain expressing GM-CSF into cutaneous melanoma (60,121). This was a phase I trial of escalating doses up to 2 X 107 pfu per lesion and 8 X 107 pfu per session (multiple lesions injected). Patients were administered twice weekly intratumoral injections over 6 weeks. Systemic toxicity was limited to mild flulike symptoms that resolved within 24 h and local inflammation at the injection site with doses of > 107 pfu per lesion. All patients were vaccinated against vaccinia within weeks prior to receiving the vaccinia-GM-CSF. Interesting responses were seen in 5 of the 7 patients treated. Three patients had mixed responses with complete regression of treated and untreated dermal metastases, 1 patient had a partial response with regression of injected and uninjected regional dermal metastases, and 1 patient with multiple dermal metastases confined to the scalp achieved a complete remission. This group plans to extend their observations with continued clinical trials using this vector.

D. Treatment of Poxvirus Infection

The treatment of orthopoxvirus infections has become of widespread interest recently due to the terroristic threat of biological warfare with smallpox virus. Clinical trials using vaccinia as a gene delivery vector would benefit greatly from a drug or compound that could turn off viral replication. Vaccinia immunoglobulin (VIG) is the only approved product available for treating complications of vaccinia infection. VIG has been owned by the Department of Defense, with a small amount available through the Centers for Disease Control (55). No randomized controlled clinical trials have been performed to evaluate therapeutic efficacy; and prevention therefore, there is doubt as to its effectiveness in established complications from vaccinia. A randomized trial examining comcomitant treatment with vaccinia and VIG demonstrated a significantly lower rate of postvaccinal encephalitis.

Numerous antiviral drugs have been tested in animal models of orthopoxvirus infections (122) (Table 4). Of the

Table 4 Antiviral Compounds Effective In Vivo Against Vaccinia

Animal model

Compound

First author

Vaccinia keratitis in rabbits

Vaccinia tail lesion formation in mice following IV injection Vaccinia tail lesion formation in mice following IV injection Vaccinia tail lesion formation in mice following IV injection

Vaccinia tail lesion formation in mice following IV injection Vaccinia tail lesion formation in mice following IV injection Vaccinia tail lesion formation in mice following IV injection Vaccinia related death in SCID mice following IV injection Vaccinia related death in SCID mice following IV injection

Ribavarin Sidwell (136)

Interferon de Clercq (137)

Polyacrylic acid de Clercq (137)

Ara-C, ribavirin, 5-iodo-dUrd, 5-ethyl-dUrd, de Clercq (138)

5-thiocyano-dUrd

C-c3 Ado de Clercq (139)

3-Deazaneplanocin A, Ara-A Tseng (140)

H961 (diacetate ester prodrug of S2242) Neyts (143)

licensed antiviral compounds, cidofovir has the greatest potential for protection against and treatment of vaccinia infections (123,124). Numerous other agents have also been identified that demonstrate efficacy against different poxviruses. The pharmacokinetics and safety profile of these agents have not been defined in humans, so their true utility will have to be determined. Suicide genes engineered into the virus may function to decrease viral replication upon addition of the prodrug. This has been demonstrated experimentally both in vitro and in vivo using a vaccinia expressing CD followed by the addition of 5-FC (64). Prolonged survival after inoculation with a lethal dose of vaccinia in a murine model was achieved with addition of prodrug compared with controls. Future studies with more potent enzyme/prodrug systems may enhance this effect. Combinations of antiviral drugs and prodrugs may improve outcome.

The development of tightly regulated inducible gene expression systems would allow for in vivo induction of genes, which would be toxic to the virus itself and inhibit viral replication. This has been difficult in vaccinia virus, because of its unique transcription system. Nevertheless, Traktman et al. reported on a tetracycline-inducible expression system in vaccinia (125). This system should be explored further to demonstrate the potential for in vivo induction of genes that are self-toxic.

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