**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 Feline Interleukin**  **- 2 Immunomodulator, Live** 

**Canarypox Vector**

January 201 2 Prepar ed 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 Felin e Interleukin
- 2 Immunomodulator , Live Canary pox Vector**Environmental Assessment For**  **Fi eld Testing**  **Feline Interleukin**  **- 2 Immunomodulator**  **, Live Canarypox Vector** 

**I**
. **Proposed Action** APHI S is considering granting authorization to ship an unlicensed Feline Interleukin
- 2 Immunomodulator , Live Canary pox Vector , for field sa fety testing. Merial, Inc. , Athens, Georgia, has requeste d authorization to conduct field test s under conditions of ty pical 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 worthle ss, contaminated, dangerous, or h armful. Accordingl y, APHIS has conducted 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 inform ation removed is available online and b y request, as indicated in the Federal Register Notice announcing the proposed field testing
. The APHI S risk analy sis includes 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 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 p reviously

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 ha s been reported to occur in more than 70% of cases w ithin one year of the initial surgery (1). Chemotherap y 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 cy tokine previously reported to provide cancer immunotherapy without adverse toxicity to feline and human patients , when delivered b y intratumoral administration using recombinant canary pox viruses (2 , 3 )
. In one study of cats with spontaneous fibrosarcoma , tumor rec urrence 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 canary pox virus
- vectored FeIL
- 2 (2). The vaccine proposed for clinical field testing is a live recombinant virus consisting of a canary pox vector expressing the FeIL
- 2 immunomodulator gene . The route of administration for this vaccine is subcutaneous, at the tumor site. One mLof sterile diluent is used per dose, with one dose given pre
- surge ry and five doses administered over a 6
- week period, post
- surgery .Poxviruses have been used man y times as live vectors to express foreign genes for

vaccine development. Examples of USDA
- licensed poxvirus vaccine vectors include vaccinia virus, fowlpox v irus, and canary pox virus. Current licensed vaccines using a canary pox 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 ha s a similar safety profile compared to the already- licensed canary pox vectored 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 produc t 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 fel ine health management. **III. Need for the Proposed Action**  The cy tokine IL
- 2 is produced by T
- helper l ymphocy

tes 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 ai d in delaying post
- surgical recurrence following excision of the tumor. IL
- 2 has been used as an immunomodulating agent for cancer therap y in humans, but its use has been limited by the toxic effects of the large doses required for effective sy stemic treatment. It is suggested that adverse effects may be overcome b y local (e.g. , subcutaneous) delivery using a viral vector delivery s ys tem such as describ ed here. This is the first vaccine to be proposed as immunotherapeutic treatment f

or feline fibro sarcom a in field trials
. **IV. Areas of Concerns**  The three areas of concern to APHI S are: 1) animal safety, 2) public health, and 3) environmental safety . APHI S has conducted its own risk anal ys is to assess whether risks are associated with the proposal to fie ld 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. 1. The host range of the canary pox virus vector is highly restricted (4)
. Even i n 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 m orbidity 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 usi ng ~40 times the proposed field dose intravenously (IV) or subcutaneousl y (SC) had no significant adverse reactions, although all cats experienced a transient hy perthermia, some with apathy , and some with mild local reactions. These reactions have been ob served previously with high doses of canary pox virus. 4. The firm’s efficacy

trial was conducted in cats with fibrosarcoma with satisfactory results. C ats given the additional vaccine regimen SC as intratumoral therapy had less than half the tumor recurrenc e 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 unlikel y to replicate, revert to virulence, or be shed after injection in cats. 6. CVB results from agarose gel anal ys is, quantitative Western blotting, real time PCR amplification, and sequence analy sis 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 anal ys is supports accurate recombination of the IL
- 2 gene into the cloning site and 99
- 100% identity

to the sequence s supplied by the firm for MSV and MSV+10, respectivel y. 7. Recombination between vaccine and wild
- ty pe poxvirus co
- infecting one cell is very unlikely in the cat and most non
- target species because those species are rarely infected b y poxviruses. Cowpox vi rus infection in cats has occasionall y been detected but there is no report in the literature regarding recombination between any avipox and orthopox viruses. Canary pox virus constructs do not repl icate 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 b y the NIH Recombinant DNA Advisory Committee ( 5 ) . BL1 containment is suitable for work invol ving 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 c anary pox virus strain attenuated by more than 200 p assages on chicken embryo fibroblast cells (CEFs). The firm previouslyconducted 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 canary pox - vectored recombi nants in animals, several such constructs have been tested in humans without adverse effect, including vaccines for rabies, measles, c yt omegalovirus, HIV, and Japanese encephalitis virus. A similar IL
- 2 c onstruct 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 personn el, 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 canary pox - 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 canary pox - vectored vaccines licensed and sold in the US and Europe have not caused environmental problem s . T hey have been shown to be replication
deficient in mammals and have a relativel y long history of safe use. 2. Shed
- spread studies with other recombinants prepared from the same virus ve ctor in mammals and avian species other than canaries indicate that the se recombinant viruses are not shed, thus not spread into the environment. 3. No genes cause virulen ce , 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 mechanicall y transmitted primarily b y mosquitoes, but there is no evidence of replication in the insect vector or transmission to non
- avian species. Studies by

the firm on the canary

pox - ve ctored West Nile virus vaccine indicate d no viral replication in the four mosquito species studied. The information gi ven 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. Altern**  **atives**

Two alternatives were considered. T he onl y 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 analy sis and the potential applications for this vaccine in disease control, APHI S adopts the alternative that the proposed field tests be a pproved. **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 S tatement 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. L ocal immunotherapy of spontaneous feline fibrosarcomas using recombinant poxviruses expressing interleukin 2 (IL2). Gene Therap y 10:2126
- 2132. 3. Tarta glia, 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. NI H Recombinant DNA Advisory Committee, 7
- 8 June 1993; Federal Register , Sep 13, 1993 ; 58 ( 175 ): 47910
.