UNITED STATES DEPARTMENT OF AGRICULTURE

ANIMAL AND PLANT HEALTH INSPECTION SERVICE

VETERINARY SERVICES

THE CENTER FOR VETERINARY BIOLOGICS

Environmental Assessment

For Field Testing of a

Canine Lymphoma Vaccine, DNA

February 2013

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 Lymphoma Vaccine, DNA
Environmental Assessment

For Field Testing 

Canine Lymphoma Vaccine, DNA

I. Proposed Action

APHIS is considering granting authorization to ship an unlicensed Canine Lymphoma 

Vaccine, DNA, for field testing in dogs diagnosed with B-cell lymphoma. Merial, Inc., 

Athens, Georgia, has requested authorization to conduct the efficacy and safety studies

through veterinary clinics that administer this vaccine. The dogs will continue 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 lymphoma. 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 harmful. Accordingly, APHIS has conducted 

a risk analysis and has concluded that the safety risks to animals, public health, and the 

environment are low. A copy of the firm’s risk analysis with confidential business 

information redacted is available online and by request, as indicated in the Federal 

Register Notice announcing the proposed field testing. The APHIS risk analysis covers 

the firm’s document, but also includes review of other studies and tests conducted by the 

firm, the confirmatory testing completed by APHIS personnel on the Master Seed to 

confirm the vaccine’s purity, identity, and expression of therapeutic protein, results of 

literary and regulatory review, and comparison with similar previously tested or licensed 

products.

II. Background

Canine lymphoma is considered a progressive, fatal disease arising from the malignant 

clonal expansion of lymphoid cells, most commonly from bone marrow, thymus, lymph 

nodes, and spleen. It is estimated that 0.1% of older dogs are affected. Chemotherapy

using a combination of agents aggressively has 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 chemotherapy, but with mixed results

(Kahn, 2008). The manufacturer, Merial, Inc., has developed a vaccine intended for use 

concurrent with or following chemotherapy to extend remission time and prolong life 

following vaccination.

The experimental vaccine to be used in the proposed field tests is a replicationincompetent plasmid which has been genetically modified to contain a therapeutic gene. 

The route of administration for this vaccine is transdermal, given by a trained 

veterinarian.
The proposed field safety test will be conducted in at least three different geographical

locations 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 health 

management.

Plasmid DNA has been used previously to express foreign genes for vaccine 

development. Examples of USDA-licensed plasmid DNA vaccines include West Nile 

Virus Vaccine licensed in 2005, and Canine Melanoma Vaccine licensed in 2006. No 

safety issues have arisen from the use of these plasmid DNA vaccines. The Canine 

Lymphoma vaccine has a similar safety profile compared to the already-licensed Canine 

Melanoma Vaccine, 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 Canine Lymphoma vaccines approved for use in dogs. A replicationincompetent DNA vaccine can potentially be both safe and efficacious, inducing both 

antibodies and cytotoxic T-cells in a broad immune response. A vaccine found to be 

successful against canine lymphoma may have wider application in treating lymphoma in 

other animal species and in humans. It is expected that the data from these monitored 

field trials will confirm the safety of this lymphoma DNA vaccine for use in dogs and 

within the environment of the United States.

IV. Areas of Concerns

The three areas of concern to APHIS are: 1) animal safety, 2) public health, and 3) 

environmental safety. APHIS has conducted its own risk analysis 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 analysis 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 lymphoma. Three dogs in each of three 

groups received four biweekly 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 many dogs with melanoma during prelicensing trials and following 

licensure. No indication of toxicity has been reported. In the prelicensing trials, 

dogs were monitored for anti-DNA antibody, tolerance, and allergic reaction. No 

negative effects were identified.
 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 highly purified. Reversion to virulence 

and shedding are not safety concerns applicable to this DNA vaccine. Should 

ingestion occur, it is known that plasmid DNA is readily 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 has 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 personnel will be participating 

veterinarians and veterinary technical personnel at veterinary clinics.

 Although the safety of this specific vaccine has not been evaluated in humans and 

is therefore unknown, accidental human injection is not expected to cause adverse 

effects. Many 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 (Liu & 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 highly 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 unlikely event the vaccine is released in the environment, the plasmid will 

not be maintained without selection pressure, using a toxic antibiotic not used in 

human or agricultural applications. The plasmid construct cannot readily infect 

bacteria without prior lab-based chemical or physical treatment of the bacteria to 

generate cells competent to take up plasmids. 

 The plasmid is unstable in the environment 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 safety of the 

canine lymphoma 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. The only alternative considered other than the 

preferred action 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 any potential environmental impact. Based upon the 

results of our risk analysis and the potential applications for this vaccine in disease 

control, APHIS adopts the alternative that the proposed field tests be approved.

VI. Conclusion

Based upon the risk analysis documented in this EA, APHIS 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 Lymphoma: 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., Wolchok, J.D., et al. 2008. Phase I clinical trial of 

murine CD20 DNA vaccination in dogs with B-cell lymphoma. Proc Veterinary

Cancer Society, Seattle, WA.

3. Martin, T., Parker, S.E., Hedstrom, R., et al. 1999. Plasmid DNA malaria vaccine: the 

potential for genomic integration after intramuscular injection. Hum Gene Therapy 

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. Intervirol 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 tyrosinase 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 enzyme-linked immunosorbent 

assays. 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.