Environmental Assessment

For Field Testing

Avian Influenza

- Marek’s Disease Vaccine, H5 Subtype, Serotype 3, Live Marek’s Disease Vector

I

. Proposed Action

APHIS is considering granting authorization to ship an unlicensed Avian Influenza

Marek’s Disea se Vaccine, H5 Subtype, Serotype 3, Live Marek’s Disease Vector , for

field sa fety testing. Biomune Company

, Lenexa

, Kansas

, has requeste d authorization to

conduct field test s under conditions of husbandry that are typical of the commercial poultry industr y 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 h armful. Accordin gly, 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 upon request. The APHI S risk analysis includes this document, but also incorporates review of all the firm’s studies and tests, the

confirmat ory testing completed by APHIS personnel on the Master Seed Virus to confirm purity, identity, and expression of immunogenic protein, con sultation with subject matter

e xperts as needed , results of literary and regulatory review, and comparison with similar previously tested or licensed products.

II. Background

Marek’s disease virus (MDV) can be foun d in chicken flocks worldwide. It is sa id that

e very flock, except those kept under stringent pathogen

- free conditions, can be presumed to be infected with this herpesvirus (1) . This infection may manifest itself as clinical disease consisting of depression, ataxia, enlarged nerves, and death, or as a subclinical decrease in egg production and growth rate. Early exposure to very virulent virus strains may be an important cause of significant losses due to Marek’s disease (MD).

Avian influenza virus ( AI V) infects domestic poultry and wild bi rds, and may cause mild respiratory disease or drops in egg production (2); these are known as low pathogenicity avian influenza (LPAI) viruses. Occasionally, AIV can cause severe systemic disease that results in high mortality; these are described as hig h pathogenicity viruses , or HPAI. The HPAI viruses can arise through mutations in LPAI viruses of H5 or H7 subtype and quickly cause severe and widespread disease. Quarantine and depopulation are used to control their spread.

The experimental vaccin e to be use d in the proposed field t rial s is a live rec ombinant virus consisting of a turkey herpesvirus (HVT) backbone that expresses a gene o f AI V.The product is intended for in ovo vaccination of 18 day old chick embryos or for

subcutaneous vaccinatio n of healthy day

- of

- age chicks, as an aid in th e prevention of MD and avian influenza (AI)

.

HVT is considered to be nonpathogenic for turkeys, chickens, and other animals. The HVT parent strain has been used commercially for at least two decades in chic kens as a vaccine against the closely

- related MDV without adverse effects on human health. The

current HVT vector is the same as for two USDA

- licensed vaccines : Bursal Disease

Marek’s Disease Vaccine, Serotype 3, Live Marek’s Disease Vector and Marek’s

Di sease

- Newcastle Disease Vaccine, Serotype 3, Live Marek’s Disease Vector.

The proposed field safety test will be conducted in at least t wo different geographical

locations and the product will be used according to instructions on the product circular.

Th e 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 poultry health

management.

III. Need for the Proposed Action

MDV is a ubiquitous and economica lly important cause of neoplasm in poultry. The advent of vaccination has dramatically reduced losses due to MDV in broiler and layer

flocks. HVT

- vectored vaccines have been shown to be effective against virulent MDV.

For AI V, H5 and H7 subtype vaccinat ion is restrict ed in the U.S. and requires specific approval granted by state authorities and the USDA

. Commercial poultry are under surveillance for AIV and exposure is reportable to the USDA; most exposed flocks are depopulated. In several countries ar ound the world, vaccination is used to control H5

AIV.

This experimental vaccine represents an attempt by Biomune Company to produce an efficacious and safe vaccine against MDV and AI V that protects chicks early in life with

one vaccination.

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 vaccin e. 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. 18 day

- old embryos or d ay

- of

- age chicks were inoculated in ovo or subcutaneously, respectively, with a 10X dose and observed for 120 days. No clinical signs of MD orAI were seen and average body weights were not statistically different from the negative control group.

2. Three ef ficacy studies using vaccinated chicks of varying age that were subsequently challenged by either HPAI or the MDV GA strain indicated no adverse effects during the observation periods.

3. Safety of the rHVT/A1

- H5 virus in chickens was tested in a backpassage study. Five passes in chickens followed by testing for reversion to virulence and genetic stability revealed no adverse reactions or clinical signs of MD or AI, out to 45 days after the fifth backpassage.

4. Purity testing of the master seed virus (MSV) i ndicated the absence of extraneous agents, including chicken anemia virus and reticuloendotheliosis virus.

5. The MSV was passed five times in vitro to generate MSV+5, the highest level to be used in production. Molecular testing by Southern blot, PCR/seque ncing, Western blot, and black plaque assay indicated the genetic and phenotypic stability of the recombinant during cell culture. These same tests were used to indicate the in vivo stability of the recombinant in the backpassage study, following five pas ses chickens.

6. Chickens inoculated with either a 10X dose of the vaccine or the HVT parent strain were evaluated for possible changes in tissue tropism. There were no apparent clinical signs, reactions, or lesions. The vaccine and HVT strain demonstrated the same tropism in the vaccinated chickens; both were isolated from purified white blood cells from the blood, spleen, thymus, and bursa.

7. Evaluation of vaccine safety in non

- target avian species consisted of inoculation of turkeys, quail, pheasants, and pigeons with either the vaccine or the HVT parent, and a comparison made of clinical signs, gross lesions, adverse vaccine reactions, and

virus isolation. These non

- target avian species had no signs, lesions, or reactions. The recombinant and parent disp layed a similar nonpathogenicity in these other avian species and a similar host range.

8. To evaluate the safety of the vaccine in non

- avian species, three mammalian cell lines (mouse , porcine , and canine) were inoculated with either the recombinant or the HVT parent but, after five passes, the viruses had not induced cytopathic effect. Previous

attempts by others to replicate the HV T parent in mammalian cell culture ( 3, 4) and

mammalian species such as newborn hamsters, rhesus monkeys

, and marmoset monkeys (5, 6) were reported to be unsuccessful, as well.

B. Public Health

The risk to public health is low.1. There are no indications that special safety measures should be taken to conduct this study. Human exposure will be limited to qualified personnel administering the vaccine and handling the poultry.

2. Accidental human injection is not expected to cause adverse effects. The HVT parent strain from which the recombinant is derived has been used safely for decades as a

vaccine in chickens.

3. The National Institutes of Health (NIH) Guidelines indicate that MDV is not associated with disease in healthy adult humans and classify MDV in Risk Group 1 (7 ). HVT is not specifically menti oned in the Guidelines but is also not associated

with human disease.

4. Alt hough the safety of this experimental vaccine in humans has not been specifically evaluated and is therefore unknown, no safety hazards to the public health are expected.

C. Environmental Safety

The risks to the environment are low.

1. The host range of the HVT parent is limited to certain avian species. The potential for escape and dispersal of this experimental vaccine from poultry houses is low. The transmission capabilities of the vaccine used in ovo or subcutaneously in chickens under normal husbandry conditions are limited.

2. In a shed/spread study conducted by the firm, chickens were vaccinated in ovo with a 10X dose of the vaccine or an equivalent amount of the HVT parent. Upon hatching, for each virus group, chicks were placed with a group of non

- v accinated chicks to evaluate shed/spread. Virus isolation was attempted at 10, 14, and 21 days of age, from the blood of all treatment groups. Virus was reisolated from the vaccinated groups but not from any control animal, suggesting that the vaccine an d the parent

were not transmitted to the in

- contact controls.

3. Regarding the potential for horizontal gene transmission, recombination between HVT and AIV has not been reported. Recombination between rHVT/AI

- H5 and field MDV is considered unlikely. Vacci nes commonly mix serotypes without apparent recombination among these or field MDV strains.

4. The firm conducted environmental stability studies on rHVT/AI

- H5 and the HVT parent in sterile wood shavings and cotton swabs at 37±3 o C or 25±3 o C. No significant difference between the two was noted.

There are no apparent substantial issues with adverse environmental impacts concerning

this vaccine

.V. Alternatives

Two alternatives were considered. T he only alternative considered other than t he 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 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. Marek’s Disease. In: The Me rck Veterinary Manual

, Te nth Edition, 20 1 0. C.M. Kahn, (Ed), Merck & Co., Inc. , Whitehouse Station, N.J. Available online: http://www.merckvetmanual.com/mvm/index.jsp

2. Avian Influenza : Introductio n. In: The Merck Veterinary Manual, Te nth Edition,

20 10 . C.M. Kahn, (Ed), Merck & Co., Inc., Whitehouse Station, N.J. Available

online: http://www.merckvetmanual.com/mvm/index.jsp

3. Muelemans, G. e t al . Susceptibility of mammalian and avian cell cultures to infection

with cell

- free turkey herpes virus. J. Comp. Pathol. 83:605

- 608. 1973.

4. Witter, R. L. and Sharma, J. M. Transient infectivity and heterokaryon formation in

hamster cell cultures inocula ted with cell

- associated stocks of Marek’s disease virus and herpesvirus of turkeys. J. Natl. Canc. Inst. 53:1731

- 1742. 1974.

5. Witter, R. L. and Schat K. A. Marek’s disease. In: Diseases of Poultry, Eleventh Edition, 2003. Y.M. Saif et al (Eds), Iowa State University Press, Ames, IA. pp. 407

- 465.

6. Sharma, J. M. et al . Lack of pathogenicity of Marek’s disease virus and herpesvirus of turkeys in marmoset monkeys. J. Natl. Canc. Inst. 49:1191

- 1197. 1972.

7. NIH Guidelines for Research Involving Recombinant DNA M olecules. Appendix B

V. NIH Recombinant DNA Advisory Committee, September 200 9 , p 43. Available

online: http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Gdlines_2002prn.pdf