Canine Melanoma Vaccine, DNA
- Environmental Assessment

For Public Release

November 3, 2011

Prepared and revised by:

Canadian Centre for Veterinary Biologics
Terrestrial Animal Health Division
Canadian Food Inspection Agency

The information in this environmental assessment was current at the time of its preparation. It is possible that the situation may have changed since that time. Please consult the Canadian Centre for Veterinary Biologics, if you have any questions.


Table of Contents

  • Summary
  • 1. Introduction
    • 1.1 Proposed Action
    • 1.2 Background
  • 2. Purpose and Need for Proposed Action
    • 2.1 Significance
    • 2.2 Rationale
  • 3. Alternatives
  • 4. Molecular and Biological Characteristics of Parental and Recombinant Organisms
    • 4.1 Identification and Description of Vaccine Organism
    • 4.2 Source, Description and Function of Foreign Genetic Material
    • 4.3 Method of Accomplishing Genetic Modification
    • 4.4 Genetic and Phenotypic Stability of the Vaccine Organism
    • 4.5 Horizontal Gene Transfer and Potential for Recombination
    • 4.6 Host Range/Specificity, Tissue Tropism and Shed/Spread Capabilities
    • 4.7 Route of Administration
  • 5. Human Safety
    • 5.1 Previous Safe Use
    • 5.2 Probability of Human Exposure
    • 5.3 Possible Outcomes of Human Exposure
    • 5.4 Pathogenicity of Parent Micro-organisms in Humans
    • 5.5 Effect of Gene Manipulation on Pathogenicity in Humans
    • 5.6 Risk Associated with Widespread Use of the Vaccine
  • 6. Animal Safety
    • 6.1 Previous Safe Use
    • 6.2 Fate of the Vaccine in Target and Non-Target Species
    • 6.3 Potential of Shed and/or Spread from Vaccinate to Contact Target and Non-Target Animals
    • 6.4 Reversion to Virulence Resulting from Back Passage in Animals
    • 6.5 Effect of Overdose in Target and Potential Non-Target Species
    • 6.6 The Extent of the Host Range and the Degree of Mobility of the Vector
  • 7. Affected Environment
    • 7.1 Extent of Release into the Environment
    • 7.2 Persistence of the Vector in the Environment and Cumulative Impacts
  • 8. Environmental Consequences
    • 8.1 Risks and Benefits
    • 8.2 Relative Safety Compared to Other Vaccines
  • 9. Mitigative Measures
    • 9.1 Worker Safety
    • 9.2 Handling Vaccinated or Exposed Animals
  • 10. Monitoring
    • 10.1 General
    • 10.2 Human
    • 10.3 Animal
  • 11. Consultations and Contacts
  • 12. Conclusions and Actions
  • 13. References

Summary

Canine Melanoma Vaccine, DNA (Trade name: Oncept) consists of highly purified plasmid DNA capable of expressing the human tyrosinase protein in transfected canine cells. The product is a therapeutic vaccine to be administered to dogs with stage II or stage III oral melanoma to aid in extending survival times. The vaccine was evaluated by the Canadian Centre for Veterinary Biologics of the Canadian Food Inspection Agency for licensing in Canada. As part of the requirements for licensing this product in Canada, an "Environmental Assessment" was conducted, and a public document containing information on the molecular and biological characteristics of the genetically modified organism, target animal and non-target animal safety, human safety, environmental considerations, and risk-mitigating measures was prepared.

1. Introduction

1.1 Proposed Action

The Canadian Centre for Veterinary Biologics (CCVB) of the Canadian Food Inspection Agency (CFIA) is responsible for licensing veterinary biologics for use in Canada. The legal authority for the regulation of veterinary biologics in Canada is provided under the Health of Animals Act and the Health of Animals Regulations. Any veterinary biologic manufactured, sold, or represented for use in Canada must comply with the requirements specified by the CFIA regarding the safety, purity, potency, and efficacy of the product. Merial Inc. (Athens, Georgia, USA) through Merial Canada Inc. (Baie d'Urfé, Quebec) has submitted the following vaccine for licensing in Canada:

  • Canine Melanoma Vaccine, DNA. (Trade Name: Oncept), United States Department of Agriculture (USDA) Product Code 9240.D0, CCVB File 820MS/C1.0/R2.1.

This Environmental Assessment was prepared by the CCVB as part of the overall assessment for licensing the above vaccine in Canada.

1.2 Background

Melanomas are neoplasms of melanin-producing cells (melanocytes or melanoblasts), which can occur in many domestic species and in humans. These neoplasms may occur in various areas of the body, such as the skin, eye, and oral cavity, and in dogs, they represent approximately 4% of all malignant neoplasms (Stell, 2011). Melanoma is the most common tumour of the oral cavity in dogs, and a high percentage of canine oral melanomas are malignant with a high rate of metastasis. Older dogs (aged 10-12 years) are most commonly affected, and the condition may be more common in breeds with pigmented mucous membranes. Clinical signs may include the presence of a mass, dysphagia, ptyalism, halitosis, oral bleeding, and loss of teeth or facial distortion. The most common site of canine oral melanoma is the gingiva, followed by the labial and buccal mucosa, and palate.

The World Health Organization (WHO) staging scheme for dogs with oral melanoma is based on size, with stage I involving a tumour of less than 2 cm in diameter, stage II involving a tumour of 2 to less than 4 cm in diameter, stage III involving a tumour equal to or more than 4 cm in diameter and/or lymph node metastasis, and stage IV involving distant metastasis (Bergman, 2007). Factors that may adversely affect prognosis include stage of disease, size of oral neoplasm, evidence of metastasis, and various histological criteria (Bergman, 2007). Treatments such as surgery, coarse-fractionation radiation therapy, and chemotherapy have provided stage-dependent clinical benefits to a minimal-to-modest degree, with most patients eventually succumbing to systemic metastasis (Bergman, 2007). When treated with surgery, median survival times for dogs with oral melanoma are approximately 17 to 18 months for stage I disease, five to six months with stage II disease, and three months with stage III disease (Bergman, 2007).

Tyrosinase is an enzyme involved in the synthesis of the pigment melanin by melanocytes present in the skin, hair, and eyes. The protein has been found to be expressed in most melanomas in both humans and dogs, and is recognized as a marker protein of this cancer cell type (Ramos-Vara & Miller, 2011).

Canine Melanoma Vaccine, DNA, is an unadjuvanted vaccine consisting of highly purified plasmid DNA. Following uptake by canine cells near the injection site, a gene encoding the human tyrosinase protein is expressed from the plasmid, using host cellular machinery. This vaccine-derived xenogeneic immunogen is then detected by the vaccinee's immune system, leading to both humoral and cell-mediated immune responses. Vaccination with human tyrosinase appears to break tolerance for the related self canine tyrosinase protein, and an immune response against the endogenous canine tyrosinase expressed by the melanoma cells is mounted. The plasmid is non-replicating in eukaryotic cells.

The Canine Melanoma Vaccine, DNA, has been developed for the therapeutic immunization of dogs diagnosed with melanoma. The vaccine can aid in extending survival times of dogs with stage II (tumours 2-4 cm diameter) or stage III (tumours ≥ 4 cm and/or lymph node metastasis) oral melanoma for which local disease control has been achieved.

2. Purpose and Need for Proposed Action

2.1 Significance

The labelling for Oncept indicates that the product is intended to aid in extending survival times of dogs with stage II or stage III oral melanoma for which local disease control has been achieved (negative local lymph nodes or positive lymph nodes that were surgically removed or irradiated).

2.2 Rationale

The CCVB evaluates veterinary biologic product submissions for licensure under the Health of Animals Act and the Health of Animals Regulations. The general criteria for licensing are as follows: a) the product must be pure, safe, potent, and efficacious; b) vaccine components must be relevant to Canadian disease conditions; c) foreign products must be licensed in the country of origin; and d) the product must be produced and tested in accordance with generally accepted "good manufacturing practices." This U.S.-origin vaccine meets these general criteria, and presents no unacceptable importation risk, and thus was evaluated for licensing by the CCVB.

3. Alternatives

The two alternative options for consideration are as follows: a) to issue a Permit to Import Veterinary Biologics to Merial Canada Inc. for the importation of Canine Melanoma Vaccine, DNA, if all licensing requirements are satisfactory, or b) not to issue a Permit to Import Veterinary Biologics, if licensing requirements are not met.

4. Molecular and Biological Characteristics of Parental and Recombinant Organisms

4.1 Identification and Description of Vaccine Organism

The vaccine is not an organism but a double-stranded, covalently closed, circular DNA molecule known as a plasmid. The plasmid is propagated for vaccine production in an E. coli host strain commonly used in laboratories. The vaccine plasmid is unable to replicate autonomously in a eukaryotic host cell.

4.2 Source, Description and Function of Foreign Genetic Material

The vaccine plasmid contains an origin of replication, a gene-conferring antibiotic resistance, and complementary DNA (cDNA), encoding human tyrosinase under the control of a viral immediate-early promoter/enhancer and a transcription termination sequence.

The origin of replication sequence allows the vaccine plasmid to be replicated within the E. coli cells cultured for vaccine production. The antibiotic resistance gene facilitates the selection of bacteria carrying the plasmid and the propagation of only those E. coli containing the vaccine plasmid during vaccine manufacturing. The antibiotic resistance gene is under the control of prokaryotic regulatory sequences, and is oriented in the opposite direction to the promoter-controlling tyrosinase expression, so it should not be expressed in eukaryotic cells. The human tyrosinase cDNA encodes the vaccine antigen, and the viral promoter plus the terminator sequences drive the expression of the antigen in dog cells following vaccination.

The cDNA encoding human tyrosinase sequence (approximately two kilo-base pairs in length) was obtained from a cDNA library prepared from a human melanoma cell line. The other functional elements of the vaccine plasmid correspond to sequences present in numerous commercially available plasmids.

4.3 Method of Accomplishing Genetic Modification

The vaccine plasmid was assembled using standard molecular biology techniques. Details of the methods used to create the vaccine plasmid are on file at the CCVB.

4.4 Genetic and Phenotypic Stability of the Vaccine Organism

Each batch (serial) of Canine Melanoma Vaccine, DNA, is tested for its continued ability to express the human tyrosinase antigen in a mammalian cell line. Restriction enzyme (RE) digests are also performed on each batch of the final vaccine to ensure that the RE profile is as expected and that no gross rearrangements (e.g. deletions, duplications) occurred to alter the predominant plasmid population. After establishing a master cell bank (MCB), which serves as the starting material for each batch of Canine Melanoma Vaccine, DNA, the manufacturer sequenced the entire plasmid isolated from a first passage culture and found that the sequence was 100% as expected.

The serial employed in the efficacy studies used to support product licensing was produced by isolating plasmid DNA from a culture at the maximum number of passages from the MCB permitted during the production of a vaccine serial.

4.5 Horizontal Gene Transfer and Potential for Recombination

A commonly raised concern associated with plasmid DNA vaccines is their potential for integrating into the genomic DNA of the host. The propensity of the present DNA vaccine to integrate into the genomes of vaccinated dogs has not been investigated. However, Canine Melanoma Vaccine, DNA, is intended for use only in dogs that already have cancer (melanoma), and the presumed negative repercussion of DNA vaccine chromosomal integration is insertional mutagenesis, which, in rare cases, could contribute to neoplastic transformation and the development of a tumour. Studies involving the intramuscular injection of other DNA vaccines in mice suggest that, while the vaccine DNA can persist for many months in the muscle surrounding the injection site, the vast majority of this DNA is extra-chromosomal. In these experiments, ≤ 8 copies of the plasmid DNA were typically detected per µg high molecular weight DNA or approximately 150,000 cells (Ledwidth et al., 2000; Manam et al., 2000). It has been estimated that 1 integrated plasmid per µg DNA (equivalent to approximately 150,000 diploid genomes) would correspond to a gene mutation frequency about 1,000 times lower than that of the spontaneous rate of gene-inactivating mutations (Nichols et al., 1995; Martin et al., 1999; Ledwidth et al., 2000). Again, considering that the intended recipients of Canine Melanoma Vaccine, DNA, are dogs with cancer and with an expected survival time of less than one year without treatment, we do not consider the potential risk of plasmid DNA integrating into host chromosomes a safety concern for these dogs.

The presence of an antibiotic resistance gene on the vaccine plasmid leads to the question of whether the plasmid is likely to participate in horizontal gene transfer (HGT). The vaccine will be delivered into the dermal and/or muscle tissues of dogs, where there should be no bacteria capable of propagating the plasmid. For intact plasmid DNA to reach bacteria making up the natural gut flora of the vaccinee, it will have to evade degradation by nucleases present in the extracellular space and blood, cross the intestinal barrier, and then survive degradation by nucleases present in the intestines. Accomplishing this feat, a bacterium then needs to take up the plasmid by DNA transformation, which is an inefficient process and a rare event outside of optimized laboratory conditions. It could be envisioned that a small amount of plasmid DNA could be exuded from the site of vaccine injection and licked by the dog as a minor source of oral exposure. Here, the likelihood of HGT to bacteria in the mouth is also minimal, due to the inefficiency of bacterial transformation of naked DNA and the presence of nucleases in saliva. In both the aforementioned cases, the chances of stable HGT are further reduced by the fact that bacteria transformed with the vaccine plasmid are unlikely to retain the plasmid in the absence of the appropriate antibiotic selection pressure. Taken together, there are many barriers that should restrict the chances of the vaccine plasmid participating in a HGT event. Since the antibiotic against which the vaccine plasmid's antibiotic resistance gene confers resistance is currently of limited therapeutic value in veterinary and human medicine, the impact of a rare HGT event would also be relatively minor.

It should be noted that antibiotic resistance genes, similar or identical to that present on the vaccine plasmid, are frequently found on plasmids used in research, in soil bacteria, and in transgenic plants approved for use in Canada as food and/or feed crops (Miki and McHugh, 2004). Use of Canine Melanoma Vaccine, DNA, is not expected to significantly contribute to the dissemination of genetic material encoding antibiotic resistance.

4.6 Host Range/Specificity, Tissue Tropism and Shed/Spread Capabilities

Plasmids containing the vaccine plasmid's type of origin of replication can only replicate in a limited range of bacterial hosts, which include E. coli and other related members of the Enterobacteriaceae family, and possibly members of the Legionellaceae family (Kües and Stahl, 1989). Although the tyrosinase gene present on the vaccine plasmid can be expressed in canine cells, the plasmid itself cannot replicate autonomously within a eukaryotic cell.

Following intramuscular administration of a plasmid DNA vaccine into an animal, some of the plasmid is taken up by myocytes surrounding the injection site, and possibly by some resident immune cells. Much of the remaining DNA is localized in the extracellular space, where it is subject to degradation by nuclease enzymes. Soon after inoculation, some plasmid DNA can be detected in the blood, although it is unclear how much of this DNA is intact plasmid. Nucleases are also present within blood and likely contribute to plasmid DNA degradation prior to clearance. Several minutes later, small quantities of plasmid DNA can be detected in the liver, spleen, draining lymph nodes, kidney, lungs, distant muscles, gonads and brain, presumably as a result of circulation within the bloodstream, and possibly via lymph and/or immune cells. Distribution to these vascularized organs is transient, and the only site where the intramuscularly injected plasmid appears to persist is in the muscle near the site of administration. Scavenger cells associated with the liver and kidney appear capable of clearing plasmid DNA from the circulation, and small DNA fragments may be eliminated by glomerular filtration. Plasmid DNA seems to be excreted in the urine and, to a lesser extent, in the feces (Parker et al., 1999; Dupuis et al., 2000; Kawase et al., 2003; Zang et al., 2005; Tonheim et al., 2008; Faurez et al., 2010).

The manufacturer of Canine Melanoma Vaccine, DNA, did not conduct experiments to determine the precise biodistribution properties of the vaccine plasmid, or to investigate whether the vaccine plasmid can be shed intact from vaccinated animals or spread to in-contact animals.

4.7 Route of Administration

Canine Melanoma Vaccine, DNA, is recommended for transdermal administration, targeting intramuscular deposition using the VET JET needle-free transdermal vaccination system (a high-pressure device placed on the skin which deposits injectate transdermally into the muscular tissue). Doses of 0.4 mL volume are administered into the muscle of the media thigh just caudal to the femur using the device. Initial treatment consists of four doses of vaccine given at two-week intervals, with a booster dose administered every six months for the life of the patient.

5. Human Safety

5.1 Previous Safe Use

Canine Melanoma Vaccine, DNA, has been used in the U.S. since 2007. Its use has also been authorized, on a case-by-case basis, by veterinarians in Canada through the Emergency Drug Release (EDR) program of Health Canada's Veterinary Drugs Directorate (HC-VDD) since 2007. To date, there have been no adverse event reports received by HC-VDD related to accidental human injection.

5.2 Probability of Human Exposure

Human exposure to the vaccine is likely to be limited, because the vaccine is to be administered by veterinary health professionals, and the vaccine plasmid should be largely degraded prior to clearance from the vaccinated animal.

5.3 Possible Outcomes of Human Exposure

The CCVB was provided a summary of the results of a clinical trial involving a small group of human melanoma patients who were administered a human tyrosinase DNA vaccine that was very similar to the plasmid contained in the present canine vaccine. The vaccine was well tolerated in patients, and the researchers did not detect any persistent elevation of anti-DNA antibodies, even following six doses at 1.5 mg/dose (more than 10 times the expected canine dose). Based on these results, we do not anticipate significant adverse consequences following human exposure to Canine Melanoma Vaccine, DNA.

Other vaccines expressing human tyrosinase have been administered to humans in clinical trials with no reported toxicity (Meyer et al., 2005; Lindsey et al., 2006).

This vaccine is produced using no ingredients of animal origin, which virtually eliminates the risk of the vaccine being contaminated with a zoonotic agent in the case of accidental human exposure.

5.4 Pathogenicity of Parent Micro-organisms in Humans

The vaccine is a double-stranded, covalently closed piece of DNA, and not a living organism or virus. The plasmid backbone has been administered to humans without serious complications, and the viral promoter/enhancer, terminator, and antibiotic resistance gene sequences have each been part of different constructs, previously administered to humans in various clinical trials. (References withheld to protect confidential business information.)

5.5 Effect of Gene Manipulation on Pathogenicity in Humans

The addition of the human tyrosinase gene to the parental plasmid vector is not expected to greatly increase the toxicity of the plasmid, based on the results of multiple clinical trials involving the administration of vaccines expressing human tyrosinase to humans (Meyer et al., 2005; Lindsey et al., 2006). The vaccine plasmid is not a pathogen capable of infecting humans or animals, but a double-stranded, covalently closed piece of DNA.

5.6 Risk Associated with Widespread use of the Vaccine

No risks to human safety associated with the widespread use of the vaccine have been identified.

6. Animal Safety

6.1 Previous Safe Use

The manufacturer conducted laboratory and field safety trials involving 145 dogs. Trials involved the administration of two to four doses of the vaccine given at two-week intervals, and observation periods of 18 days to 10 weeks. The most significant adverse event seen was transient fever, though all adverse events were transient, with none resulting in serious consequences. Efficacy trials involving 53 additional dogs yielded similar safety results. The local adverse event rate for the product was relatively high, and might normally be unacceptable for a veterinary biologic, but given that the product is to be used in terminally ill animals, these transient side effects were deemed to be acceptable and relatively safe in the target population of dogs. The Canadian labels for Canine Melanoma Vaccine, DNA, advise users that a transient low-grade fever may be observed in some dogs.

With respect to DNA vaccines in general, the potential induction of anti-DNA antibodies has been discussed as a theoretical risk. This outcome has not been observed in most studies conducted in laboratory animals, except in a single publication describing a study wherein lupus-prone mice received multiple injections of plasmid DNA (Gilkeson et al., 1995). In a preliminary feasibility trial for this vaccine, dogs were evaluated for the presence of anti-DNA antibody, and no increases or clear seroconversion were observed.

Another theoretical risk pertaining to DNA vaccines involves the persistent expression of plasmid protein(s) causing autoimmune disease. For this particular vaccine, auto-immunity is actually desired, as the therapeutic effect involves stimulation of the immune system to attack cancer cells expressing tyrosinase. A theoretical side effect of this treatment would be an attack on normal melanocytic cells expressing tyrosinase, potentially resulting in depigmentation of pigmented tissues. This side effect was not seen to a significant degree in either the manufacturer's studies or in the published literature.

As previously mentioned, the use of Canine Melanoma Vaccine, DNA, has been authorized on a case-by-case basis to veterinarians in Canada through the EDR program of HC-VDD since 2007. Although the Canadian experience with the product has been limited, to date, no serious adverse events have been reported from using this product in dogs.

6.2 Fate of the Vaccine in Target and Non-Target Species

Following vaccination of a dog, it is presumed that some of the plasmid is taken up by myocytes surrounding the injection site, and possibly some antigen-presenting immune cells. After reaching a transfected cell's nucleus, gene expression occurs using the host cellular machinery, resulting in production of the vaccine antigen, human tyrosinase. This foreign protein becomes the subject of host immune system surveillance, leading to cell-mediated and humoral immune responses against the antigenic protein. Following vaccination with Canine Melanoma Vaccine, DNA, not only is an immune response mounted against the vaccine-derived human tyrosinase protein, but the canine tyrosinase protein expressed by the melanoma cells also becomes targeted.

Plasmid DNA has been shown to persist at the administration site for many months following vaccination in various animal models. It appears that the vast majority of this plasmid DNA remains extra-chromosomally within differentiated myocytes (Ledwith et al., 2000; Manam et al., 2000). In at least some models, gene expression from the plasmid can continue for as long as the plasmid persists (Wolff et al., 1992).

Plasmid DNA not transfected into cells following injection is subject to degradation by ubiquitous extracellular nucleases. Immune cells may also play a role in eliminating free plasmid DNA or transfected cells or their products. A fraction of the plasmid DNA injected into a muscle may be distributed systemically via the blood and/or lymph, and reach various organs prior to vaccine clearance. It is unclear how much of this circulating DNA is intact plasmid versus degraded fragments.

Studies have shown that plasmid DNA can be detected in gonadal tissue samples for a short period of time post-vaccination (Parker et al., 1999). However, its presence in the reproductive organs is transient, being undetectable after a few days, and it does not appear to integrate into the chromosomal DNA of germ cells to be vertically transmitted to offspring (Manam et al., 2000). Nonetheless, it is not recommended to breed dogs undergoing treatment with Canine Melanoma Vaccine, DNA, because biodistribution and reproductive toxicity studies have not been done.

6.3 Potential of Shed and/or Spread from Vaccinate to Contact Target and Non-Target Animals

The manufacturer has not investigated the potential for Canine Melanoma Vaccine, DNA, to be shed from vaccinated animals or spread to other animals. Since the vaccine plasmid is unable to replicate in vaccinated dogs, the maximum amount of plasmid DNA that can be shed from an animal is equivalent to the amount injected (approximately 100 µg/dose). Based on tissue distribution and clearance studies performed using other plasmid DNA vaccines, it is anticipated that much of the vaccine plasmid DNA shed in the urine and feces will be at least partially degraded. If only a fraction of the plasmid DNA excreted in the urine and feces of a vaccinated dog is intact, and plasmid DNA is diluted over time and volume of excrement, the potential amount of exposure will be limited.

The likely route of exposure for another animal to any shed plasmid is oral, meaning this plasmid would be subject to degradation by stomach acid and digestive enzymes, and will also constitute only a minute quantity of all the DNA consumed by the animal. Unlike bacteria and viruses, plasmid DNA is non-infectious; it does not have efficient means of gaining entry into mammalian cells. Only a small portion of the plasmid DNA injected into an animal is capable of transfecting canine cells, and muscle cells appear to be more capable of taking up plasmids than other mammalian cells. Taken together, we do not expect that the vaccine plasmid will spread from a vaccinated dog to other animals.

6.4 Reversion to Virulence Resulting from Back Passage in Animals

The vaccine is a double-stranded, covalently closed piece of DNA, and not a living organism or virus. The foreign gene is not a pathogen or even a piece of a pathogen. Reversion to virulence is not a potential safety concern for this plasmid DNA vaccine.

6.5 Effect of Overdose in Target and Potential Non-Target Species

A high-dose safety study involving 25 healthy dogs was conducted wherein vaccinates received four transdermal doses, two weeks apart, of a serial of the product formulated to contain the "worst case scenario" levels of endotoxin, protein, and supercolied DNA. Animals were observed three times weekly for 10 weeks. All adverse events were transient, and none resulted in serious consequences.

6.6 The Extent of the Host Range and the Degree of Mobility of the Vector

Plasmids containing the vaccine plasmid's type of origin of replication can only replicate in a limited range of bacterial hosts, which include E. coli and other related members of the Enterobacteriaceae family, and possibly members of the Legionellaceae family (Kües and Stahl, 1989). Although the tyrosinase gene present on the vaccine plasmid can be expressed in canine cells, the plasmid itself cannot replicate autonomously within a eukaryotic cell.

7. Affected Environment

7.1 Extent of Release into the Environment

It is expected that the majority of vaccine plasmid DNA released into the environment will consist of degraded fragments, due to its metabolism/cleavage by nucleases in the vaccinated animal.

7.2 Persistence of the Vector in the Environment and Cumulative Impacts

Free DNA in the environment is subject to degradation by nucleases and other factors present in the soil (Dale et al., 2002). The amount of vaccine plasmid DNA released into the environment is expected to be minimal, compared with the vast amount of DNA contributed from microbes, plants, and animals.

8. Environmental Consequences

8.1 Risks and Benefits

For any vaccine, the risks of vaccination can be attributed to potential adverse reactions. In numerous laboratory and field studies, no significant risk has been posed by the vaccine molecule, and the safety of this vaccine in dogs has been demonstrated. The benefit of the vaccine is its ability to prolong the survival time of dogs diagnosed with stage II or stage III melanoma.

8.2 Relative Safety Compared to other Vaccines

One of the major benefits of plasmid DNA vaccines is their expected safety. Unlike live attenuated vaccines, no opportunity exists for the plasmid DNA vaccine to revert to virulence. Compared with an inactivated vaccine, there is no risk that the inactivation treatment was incomplete and that a few pathogenic particles remain in the vaccine. Canine Melanoma Vaccine, DNA, does not contain an adjuvant, so the vaccine will not be associated with the type of tissue damage often seen with oil-adjuvanted vaccines.

This particular vaccine is unique in that, unlike most vaccines, it is intended for use as a therapeutic agent in animals with cancer, rather than as prophylaxis against infectious disease. Therefore, it may be useful to consider its relative safety, compared with other cancer treatment modalities. This vaccine has not necessarily been designed to replace some degree of conventional treatment, since the intended recipients of this vaccine (and the animals involved in the manufacturer's efficacy study) have, or likely will have, undergone surgical treatment and possibly radiation treatment to achieve local disease control. Compared with other possible adjunctive treatments, such as chemotherapy and radiation, this vaccine is associated with far fewer possible serious adverse effects. Chemotherapy and radiation, for instance, can be associated with gastrointestinal upset and immunosuppression, leading to increased risk of serious infection, but these undesirable results were not associated with this vaccine in any studies conducted by the manufacturer or published in the literature.

9. Mitigative Measures

9.1 Worker Safety

Veterinarians and animal technicians could be exposed to the vaccine plasmid when administering Canine Melanoma Vaccine, DNA, to dogs. As was discussed in section 5, above, on human safety, such exposure is not expected to be a safety concern. Since the vaccine plasmid is not a pathogen capable of infecting humans or animals, but a double-stranded, covalently closed piece of DNA, human exposure is not expected to be a human health concern. Moreover, a small group of human melanoma patients was repeatedly administered a human tyrosinase DNA vaccine very similar to the present canine DNA vaccine, and this high level of exposure was not associated with any toxicity. The Canine Melanoma Vaccine, DNA, does not contain any adjuvant, and thus the risk of clinical problems due to accidental self-injection of oil adjuvant is removed.

9.2 Handling Vaccinated or Exposed Animals

Following administration of the vaccine using the recommended needle-free device, a small amount of fluid is occasionally seen at the injection site. It is possible that this fluid could contain some vaccine plasmid molecules and potentially lead to human exposure. Considering the inefficiency with which plasmid DNA is taken up by cells and the evidence suggesting that the vaccine plasmid is safe when intramuscularly administered to humans, it is unlikely that the limited amount of human exposure through the handling of vaccinated animals is a safety concern.

10. Monitoring

10.1 General

The vaccine licensing regulations in Canada require manufacturers to report all significant suspected adverse reactions to the CFIA within 15 days of receiving notice from an owner or a veterinarian. Veterinarians may also report suspected adverse reactions directly to the CFIA. If an adverse reaction complaint is received by the CCVB, the manufacturer is asked to investigate and prepare a report for the owner's veterinarian and the CFIA. If the problem is resolved to the satisfaction of the veterinarian or client, usually, no further action is requested by the CCVB. However, if the outcome of the investigation is unsatisfactory, the CCVB may, depending on the case, initiate regulatory action, which may include further safety testing, temporary stoppage of product sales, or product withdrawal from the market.

10.2 Human

No special monitoring of the human safety of the product will be carried out.

10.3 Animal

Veterinarians, vaccinators, and animal owners should report any suspected adverse reactions to the CCVB as indicated above. Suspected adverse reactions should be reported using Form CFIA/ACIA 2205 - Notification of Suspected Adverse Events to Veterinary Biologics.

11. Consultations and Contacts

Manufacturer

Merial, Inc.
115 Transtech Drive
Athens, Georgia, USA 30601

Importer

Merial Canada, Inc.
20000 Clark Graham
Baie d'Urfé, Quebec H9X 4B6

12. Conclusions and Actions

Based on our assessment of the available information, the CCVB has concluded that the importation and use of Canine Melanoma Vaccine, DNA, in Canada would not be expected to have any significant adverse effect on the environment, when manufactured and tested as described in the approved Outline of Production, and used according to label directions.

Following this assessment, and the completion of the Canadian veterinary biologics licensing process, the Permit to Import Veterinary Biologics of Merial Canada Inc. may be amended to allow the importation and distribution of the following product in Canada:

  • Canine Melanoma Vaccine, DNA. (Trade Name: Oncept), USDA Product Code 9240.D0, CCVB File 820MS/C1.0/R2.1.

All serials of this product must be released by the USDA prior to importation into Canada. All conditions described in the Permit to Import Veterinary Biologics must be followed with respect to the importation and sale of this product.

13. References

Bergman PJ. Canine oral melanoma. Clinical Techniques in Small Animal Practice 2007;22(2):55-60.

Dupuis M, Denis-Mize K, Woo C, et al. Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice. Journal of Immunology 2000;165(5):2850-8.

Faurez F, Dory D, Le Moigne V, et al. Biosafety of DNA vaccines: New generation of DNA vectors and current knowledge on the fate of plasmids after injection. Vaccine 2010;28(23):3888-95.

Gilkeson GS, Pippen AM, Pisetsky DS. Journal of Clinical Investigation 1998;95(3);1398-1402.

Kawase A, Nomura T, Yasuda K, et al. Disposition and gene expression characteristics in solid tumors and skeletal muscle after direct injection of naked plasmid DNA in mice. Journal of Pharmaceutical Sciences 2003;92(6):1295-304.

Kües U, Stahl U. Replication of plasmids in gram-negative bacteria. Microbiological Reviews 1989;53(4):491-516.

Ledwith BJ, Manam S, Troilo PJ, et al. Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice. Intervirology 2000;43(4-6):258-72.

Lindsey KR, Gritz L, Sherry R, et al. Evaluation of prime/boost regimens using recombinant poxvirus/tyrosinase vaccines for the treatment of patients with metastatic melanoma. Clinical Cancer Research 2006;12(8):2526-37.

Manam S, Ledwith BJ, Barnum AB, Troilo PJ, et al. Plasmid DNA vaccines: tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA. Intervirology 2000;43(4-6):273-81.

Martin T, Parker SE, Hedstrom R, et al. Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection. Human Gene Therapy 1999;10(5):759-68.

Meyer RG, Britten CM, Siepmann U, et al. A phase I vaccination study with tyrosinase in patients with stage II melanoma using recombinant modified vaccinia virus Ankara (MVA-hTyr). Cancer Immunology, Immunotherapy 2005;54(5):453-67.

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