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

Feline Interleukin-2 Immunomodulator, Live 

Canarypox Vector

January 2012

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 Feline Interleukin-2 Immunomodulator, Live Canarypox Vector
Environmental Assessment

For Field Testing 

Feline Interleukin-2 Immunomodulator, Live Canarypox Vector

I. Proposed Action

APHIS is considering granting authorization to ship an unlicensed Feline Interleukin-2 

Immunomodulator, Live Canarypox Vector, for field safety testing. Merial, Inc., Athens, 

Georgia, has requested authorization to conduct field tests under conditions of typical

feline clinical practice in the United States.

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

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

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 Virus 

to confirm purity, identity, and expression of feline interleukin-2 (FeIL-2), consultation 

with subject matter experts, results of literary and regulatory review, and comparison 

with similar previously tested or licensed products.

II. Background

Fibrosarcomas are the most common soft-tissue tumors in cats. Surgical excision is the 

treatment of choice, but recurrence has been reported to occur in more than 70% of cases 

within one year of the initial surgery (1). Chemotherapy and radiation therapy have also 

been used to treat fibrosarcomas, with a goal of increasing tumor-free intervals for as 

long as possible. 

Interleukin-2 is an immunostimulatory cytokine previously reported to provide cancer 

immunotherapy without adverse toxicity to feline and human patients, when delivered by

intratumoral administration using recombinant canarypox viruses (2, 3). In one study of 

cats with spontaneous fibrosarcoma, tumor recurrence was observed in 61% of animals 

within 12 months after surgery and radiotherapy, but in only 28% of animals (n=18 per 

group) also receiving treatment with canarypox virus-vectored FeIL-2 (2). 

 

The vaccine proposed for clinical field testing is a live recombinant virus consisting of a 

canarypox vector expressing the FeIL-2 immunomodulator gene. The route of 

administration for this vaccine is subcutaneous, at the tumor site. One mL of sterile 

diluent is used per dose, with one dose given pre-surgery and five doses administered 

over a 6-week period, post-surgery.
Poxviruses have been used many times as live vectors to express foreign genes for 

vaccine development. Examples of USDA-licensed poxvirus vaccine vectors include 

vaccinia virus, fowlpox virus, and canarypox virus. Current licensed vaccines using a 

canarypox vector that is host-restricted in mammalian tissues include vaccines for canine 

distemper, equine influenza, feline rabies, feline leukemia, and West Nile virus. The 

FeIL-2 vaccine has a similar safety profile compared to the already-licensed canarypoxvectored vaccines used in the US and in Europe. 

The proposed field safety test will be conducted in clinical settings 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 feline health management.

III. Need for the Proposed Action

The cytokine IL-2 is produced by T-helper lymphocytes and is a growth and activation 

factor for T-cells and natural killer (NK) cells. The recombinant vaccine is for use in 

adult cats diagnosed with fibrosarcoma as an aid in delaying post-surgical recurrence 

following excision of the tumor. IL-2 has been used as an immunomodulating agent for 

cancer therapy in humans, but its use has been limited by the toxic effects of the large 

doses required for effective systemic treatment. It is suggested that adverse effects may

be overcome by local (e.g., subcutaneous) delivery using a viral vector delivery system

such as described here. This is the first vaccine to be proposed as immunotherapeutic 

treatment for feline fibrosarcoma in field trials.

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. 

1. The host range of the canarypox virus vector is highly restricted (4). Even in 

canaries, studies by the firm show that, following scarification of skin and 

administration of high-dose titer material, the virus can be re-isolated but there is no 

morbidity or mortality in the canaries.
2. The firm has conducted animal safety studies in cats (the intended target species), 

dogs, chickens, and mice using the vaccine containing the FeIL-2 insert with 

satisfactory results. 

3. Overdose studies in kittens using ~40 times the proposed field dose intravenously

(IV) or subcutaneously (SC) had no significant adverse reactions, although all cats 

experienced a transient hyperthermia, some with apathy, and some with mild local 

reactions. These reactions have been observed previously with high doses of 

canarypox virus.

4. The firm’s efficacy trial was conducted in cats with fibrosarcoma with satisfactory

results. Cats given the additional vaccine regimen SC as intratumoral therapy had 

less than half the tumor recurrence within 12 months, when compared to the rate of 

recurrence in cats treated only with surgery and radiotherapy.

5. Back passage and shed-spread studies using the recombinant FeIL-2 vaccine 

demonstrated that the vaccine virus would be extremely unlikely to replicate, revert to 

virulence, or be shed after injection in cats.

6. CVB results from agarose gel analysis, quantitative Western blotting, real time PCR 

amplification, and sequence analysis of the master seed virus (MSV) and MSV+10, 

the highest passage to be used in production, were consistent with genetic stability of 

the vaccine virus. Both MSV and MSV+10 express the target protein. Genetic 

analysis supports accurate recombination of the IL-2 gene into the cloning site and 

99-100% identity to the sequences supplied by the firm for MSV and MSV+10, 

respectively.

7. Recombination between vaccine and wild-type poxvirus co-infecting one cell is very

unlikely in the cat and most non-target species because those species are rarely

infected by poxviruses. Cowpox virus infection in cats has occasionally been 

detected but there is no report in the literature regarding recombination between any 

avipox and orthopox viruses. Canarypox virus constructs do not replicate in most 

species, except avian species which will not be exposed to the vaccine.

B. Public Health

The risk to public health is likewise low. 

1. The vector virus was classified as a Biosafety Level 1 (BL1) agent by the NIH 

Recombinant DNA Advisory Committee (5). BL1 containment is suitable for work 

involving well-characterized agents not associated with disease in healthy adult 

humans, and of minimal potential hazard to laboratory personnel and the 

environment. 

2. The vaccine vector was generated from a canarypox virus strain attenuated by more 

than 200 passages on chicken embryo fibroblast cells (CEFs). The firm previously
conducted in vitro replication studies that indicated the vector grows in CEFs and in 

the quail fibroblast cell line, but not in human cell lines nor in other mammalian cell 

lines.

3. In addition to safe use of canarypox-vectored recombinants in animals, several such 

constructs have been tested in humans without adverse effect, including vaccines for 

rabies, measles, cytomegalovirus, HIV, and Japanese encephalitis virus. A similar 

IL-2 construct with a human IL-2 insert was tested in humans and found to be safe 

(3).

 

4. Only qualified personnel administering the recombinant vaccine will be exposed to it. 

In this case, qualified personnel will be participating veterinarians, Merial personnel, 

and veterinary nurses at veterinary hospitals and veterinary oncology centers.

5. 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. After millions of doses of various canarypox-vectored vaccines administered 

to animals, no adverse effect to human health has been reported.

C. Environmental Safety

The risks to the environment are low. 

1. The numerous canarypox-vectored vaccines licensed and sold in the US and Europe 

have not caused environmental problems. They have been shown to be replicationdeficient in mammals and have a relatively long history of safe use.

2. Shed-spread studies with other recombinants prepared from the same virus vector in 

mammals and avian species other than canaries indicate that these recombinant 

viruses are not shed, thus not spread into the environment. 

3. No genes cause virulence, no toxins are produced, and no adjuvant is present in the 

vaccine. 

4. The vaccine is for parenteral use in cats and, thus, administration of the vaccine will 

not result in environmental release, given the lack of replication and shed/spread of 

the virus in cats.

5. Avipoxviruses can be mechanically transmitted primarily by mosquitoes, but there is 

no evidence of replication in the insect vector or transmission to non-avian species. 

Studies by the firm on the canarypox-vectored West Nile virus vaccine indicated no 

viral replication in the four mosquito species studied.

The information given above provides support to the claims made regarding the safety of 

the FeIL-2 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. Soft-Tissue Sarcomas. 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. Jourdier, T-M., Moste, C., Bonnet M-C., et al. 2003. Local immunotherapy of 

spontaneous feline fibrosarcomas using recombinant poxviruses expressing 

interleukin 2 (IL2). Gene Therapy 10:2126-2132.

3. Tartaglia, J., Bonnet, M-C., Berinstein, N., et al. 2001. Therapeutic vaccines against 

melanoma and colorectal cancer. Vaccine 19:2571-2575.

4. Paoletti, E., Tartaglia, J., Taylor, J. Safe and Effective Poxvirus Vectors – NYVAC 

and ALVAC. In: Recombinant Vectors in Vaccine Development. Dev Biol Stand. 

1994. F. Brown (Ed), Karger, Basel, SW. 82:65-69. 

5. NIH Recombinant DNA Advisory Committee, 7-8 June 1993; Federal Register, Sep

13, 1993;58(175):47910.