**UNITED STATES DEPARTMENT OF AGRICULTURE ANIMAL AND PLANT HEALTH INSPECTION SERVICE** 

**VETERINARY SERVICES THE**  **CEN TER FOR VETERINARY B**  **IOLOGICS**



# Environmental Assessment **For Field Testing of a Can**  **ine Lymphoma Vaccine**  **, DNA** 

Febr uary 201 3 Prepared by: United States Department of Agriculture Animal and Plant Health Inspection Service

Veterinary Services The Center for Veterinary Biologics

____________________________ _______________________________________________________________________________________

Environmental Assessment Field Trial of an Experimental Canine Ly mphoma Vaccine , DNA**Environmental Assessment For**  **Fi eld Testing**  **Cani ne**  **Lymphoma Vaccine ,**  **DNA**
**I**
. **Proposed Action** APHI S is considering granting authorization to ship an unlicensed Canine L ymphoma Vaccine , DNA , for field testing in dogs diagnosed with B

- cell ly mphoma

. Merial, Inc., Athens, Georgia, has requested authorization to conduct the efficacy and safet y studies through veterinary

clinics that administer this vaccine. The dogs will c ontinue to reside with their owners during the course of the studies. The efficacy trial will measure the sparing effect of the vaccine on dogs diagnosed with ly mphoma. The safety

of the vaccine will be evaluated in animals participating in the studies. Under the provisions of the Virus

- Serum
- Toxin Act of 1913, as amended in 1985, the USDA must ensure that veterinary biologics are pure, safe, potent, and efficacious and not worthless, contaminated, dangerous, or h armful. Accordingl y, APHIS has conduct ed a risk anal ys is and has concluded that the safet y risks to animals, public health, and the environment are low. A copy of the firm’s risk analy sis with confidential business information re dact ed is available online and b y request, as indicated in the F ederal Register Notice announcing the proposed field testing

. The APHI S risk analy sis cover s th e firm’s document , but also in clude s review of other studies and tests conducted by the firm , the confirmat ory

testing completed by

APHIS personnel on the Maste r Seed to confirm the vaccine’s purity , identity, and expression of therapeutic protein , results of literary and regulatory

review, and comparison with similar p reviously tested or licensed products. **II. Background**  Canine ly mphoma is considered a progres sive, fatal disease arising from the malignant clo nal expansion of ly

mphoid cells , most commonly from bone marrow, thy

mus, ly mph nodes, and spleen. Itis estimated that 0.1% of older dogs are affected. Chemotherapy using a combination of agents aggressiv el y ha s been rewarding to some extent. But expected survival time is said to be only about 9

- 12 months. Surgical resection and radiation may

also be used with combination chemotherap y, but with mixed results (Kahn, 2008)

. The manufacturer, Merial, Inc., has developed a vaccine intended for use concurrent with or following chemotherap y to extend remission time and prolong life following vaccination. The experimental vaccine to be used in the proposed field tests is a r eplication

incompetent plasmid which has been genetically modified to contain a therapeutic gene. The route of administration for this vaccine is trans dermal, given by a trained veterin arian

.The proposed field safet y test will be conducted in at least three different geographical

location s and the product will be used according to instructions on the product circular. The potential for escape and dispersal of the experimental vaccine from the proposed release sites is low. The personnel to conduct the study are experienced in canine heal th management. Plasmid DNA ha s been used previousl y to express foreign genes for vaccine development. Examples of USDA

- licensed plasmid DNA vaccine s include West Nile Virus Vaccine licensed in 2005, and Canine Melanoma Vaccine licensed in 2006

. No safet y issues have arisen from the use of these plasmid DNA vaccines. The Canine Lymphoma vaccine has a similar safet y profile compared to the already - licensed Canine Melanoma V accine , first conditionally licensed in 2007 and given a full license in 2009. It has been used safely in thousands of dogs in the US. **III. Need for the Proposed Action**  There are no C anine Lymphoma vaccines approved for use in dogs. A replication

incompetent DNA vaccine can potentiall y be both safe and efficacious, inducing both antibodies and cy

totoxic T

- cells in a broad immune response. A vaccine found to be successful against canine ly mphoma may

have wider application in treating ly mpho ma in other animal species and in humans. It is expected that the data from these monitored f ield trials will confirm the safet y of this l ympho ma DNA vaccine for use in dogs and within the environment of the United States. **IV. Areas of Concerns**  The three areas of concern to APHI S are: 1) animal safet y, 2) public health, and 3) environmental saf

ety . APHI S has conducted its own risk anal ys is to assess whether risks are associated with the proposal to field test this experimental vaccine. The safety characteristics of this vaccine have been thoroughly evaluated. The conclusions derived from the risk anal ys is for each of the areas of concern are summarized below. **A. Animal Safety**  The risk to animals is low.
 Nine dogs participated in a study using a similar plasmid vaccine containing a therapeutic transgene against canine ly mphoma. Three dogs in each of three groups received four biweekl y intramuscular injections of either 50 μg, 100 μg, or 500 μg , all without adverse effects (Bergman, et. al, 2008).
 Merial is the manufacturer of the licensed canine melanoma vaccine which has been given to man y dogs with melanoma during prelicensing trials and following licensure

. No indication of t oxicity has been reported. In the prelicens ing trials, dogs were monitored for anti

- DNA antibody , tolerance, and allergic reaction. No negative effects were ident ified. It has been estimated that the probability of a DNA molecule to integrate into the chromosome of cells after intramuscular injection is at least 3000x less than the natural mutation rate ( Martin, et al.; Ledwith, et al. 2000).
 The vaccine is not a risk for non

- target species since it cannot replicate in eukaryotic cells, is not infectious, and is highl y purified. Reversion to virulence and shedding are not safety concerns applicable to this DNA vaccine. Should ingestion occur, it is known that pla smid DNA is readil y degraded in the stomach by gastric acid and in the duodenum by pancreatic and bile secretions, without negative effect to the host (Maturin & Curtiss, 1977)

. **B. Public Health**  The risk to public health is likewise low.
 The plasmid h as no virulence

- associated genes, and no known mobility or integration motifs which would allow integration into the human genome.
 Only qualified personnel administering the recombinant vaccine could potentially be exposed to it. In this case, qualified p ersonnel will be participating veterinarians and veterinary technical personnel at veterinary clinics

.  Although the safet y of this specific vaccine has not been evaluated in humans and is therefore unknown, accidental human injection is not expected to cau se adverse effects. Man y doses of various DNA vaccines using a plasmid vector have been administered to animals, including dogs, mice, guinea pigs, and horses (Grosenbaugh, et al. 2011; Martin, et al.1999; Ledwith, et al. 2000; Manam, et al. 2000; Davis, et al. 2001). No significant adverse events have been reported for those vaccines.
 Numerous DNA vaccines have entered human clinical trials (L iu & Ulmer, 2005). There has been no evidence of autoimmunity , immunological tolerance, or integration in these various trials. **C. Environmental Safety**  The risks to the environment are low.
 The vaccine is based on highl y purified and well

- characterized plasmid DNA that does not replicate in vaccinated animals.
 The vaccine will be stored in sealed labeled vials kept in participating clinics, and will be used only

by veterinarians and professional staff.
 In the unlikel y event the vaccine is released in the environment, the plasmid will not be maintained without selection pressure, using a toxic antibiotic not use d in human or agricultural applications. The plasmid construct cannot readil y i nfect bacteria without prior lab

- based chemical or ph ys ical treatment of the bacteria to generate cells competent to take up plasmids.
 The plasmid is unstable in the environm ent and exposure to nucleases, pH variations, oxidation, and/or other denaturing conditions will result in degradation of the DNA.The information above provides support to the claims made regarding the safet y of the

canine l ymphoma vaccine to animals, public health, and the environment. There are no apparent substantial issues with adverse environmental impacts concerning use of this vaccine

. **V. Alternatives**  Two alternatives were considered. T he onl y alternative considered other than the preferred act ion alternative is not to approve the proposed field tests, the “no action” alternative. We have considered the applicants’ goals in light of the agency ’s public interest and responsibilities and an y potential environmental impact. Based upon the results of our risk analy sis and the potential applications for this vaccine in disease control, APHI S adopts the alternative that the proposed field tests be approved. **VI. Conclusion**  Based upon the risk analysis documented in this EA, APHI S has determined that implementation of the proposal would not significantly affect the quality

of the human environment and that the preparation of an Environmental Impact Statement is not required (Finding of No Significant Impact).

**References** 1. Canine Malignant Ly

mphom a: Introduction. In: The Merck Veterinary Manual, Ninth Edition, 2008. C.M. Kahn (Ed), Merck & Co., Inc., Whitehouse Station, N.J. Available online: http://www/merckvetmanual.com 2. Bergman, P.J. Leibman, N.F., Wo lchok, J.D., et al. 2008. Phase Iclinical trial of murine CD20 DNA vaccination in dogs with B

- cell ly mphoma. Proc Veterinary Cancer Societ y, Seattle, WA. 3. Martin, T., Parker, S.E., Hedstrom, R., et al. 1999. Plasmid DNA malaria vaccine: the potential for g enomic integration after intramuscular injection. Hum Gene Therap y 10:759
- 768. 4. Ledwith, B.J., Manam, S., Troilo, P.J., et al. 2000. Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice. Intervi rol 43:258

- 272. 5. Maturin, L . & Curtiss, R. 1977. Degradation of DNA by nucleases in intestinal tract of rats. Science 196(4286):216

- 8. 6. Grosenbaugh, D.A., Leard, A.T., Bergman, P.J., et al. 2011. Safety and efficacy

of a xenogeneic DNA vaccine encoding for human t yr osinase as adjunctive treatment for oral malignant melanoma in dogs following surgical excision of the primary tumor. Am J Vet Res 72:1631
- 8.7. Manam, S., Ledwith, B.J., Barnum, A.B., et al . 2000. Plasmid DNA vaccines: tissue

distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA. Intervirol 43:273

- 81. 8. Davis, B.S., Chang, G.J., Cropp, B., et al . 2001. West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzy me - linked immunosorbent assay s. J Vir 75:4040

- 4047. 9. Liu, M.A. & Ulmer, J.B. 2005. Human clinical trials of plasmid DNA vaccines. Adv Genetics 55:25

- 40.