**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**  **Yersinia Pestis Vaccine, Live Raccoon Poxvirus Vector**

January 201 3 Prepared by: The Center for Veterinary Biologics National Center for Animal Health 1920 Dayton Avenue Ames, IA 50010

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_________________ Environmental Assessment Field Trial of an Experimental Yersinia Pestis Vaccine, Li ve Raccoon Poxvirus Vector**USDA -**  **APHIS -**  **VS -**  **Center for Veterinary Biologics Environmental Assessment**  **For Fi**  **eld Tes ting**  **Yersinia Pestis Vaccine, Live**  **Raccoon Pox virus Vector** 

**I**
. **Proposed Action** The United States Department of Agriculture (USDA)
- Animal and Plant Health Inspection Service (APHIS)
- Veterinary Services (VS)
- Center for Veterinary Biologics (CVB) is considering granting authorization to ship an unlicensed Yersinia Pestis Vaccine, L ive Raccoon Pox virus Vector, for field testing in wildlife
. The U.S. Geological Survey

(USGS)
-National Wildlife Health Center (NWHC ), has requested authorization to conduct a field safety trial in the United States (US) under sylvatic conditions that are typical of the sp e cies of interest, i.e. free
- ranging prairie dogs
. Under the provisions of the Virus
- Serum
- Toxin Act of 1913, as amended in 1985, the USDA
- APHIS
- VS
- CVB must ensure that veterinary biologics are pure, safe, potent, and efficacious and not worthless, contaminated, dangerous, or h armful. Accordingl y, the CVB has conducted a risk anal ys is and has concluded that , regarding use of this vaccine, the safety ris ks to animals, public health, and the environment are low. A copy of the firm’s risk anal ys is with confidential business information removed is available upon request. The CVB risk analy sis includes th at document, but also incorporates review of all the firm’s studies and tests related to the vaccine , the confirmat ory testing completed by CVB personnel on the Master Seed Virus to confirm purity , identit y, expression, and stability , consultation with subject matter e xperts, results of literary and regulato ry review, and evaluation in light of similar previously tested or licensed products. Based on the CVB risk analy sis and other relevant data, we have reached a preliminary determination that field testing this veterinary vaccine will not have a significant impact on the quality

of the human environment, and that an environmental impact statement need not be prepared. We intend to authorize shipment of this vaccine for field testing following the close of the comment period of the Federal Register N otice announcing the pending trial unless new substantial issues bearing on the effects of this action are brought to our attention. **II. Background**  The USGS has developed a live recombinant Yersinia pestis vaccine for oral use in wild, free
- ranging prairie dogs, as an aid in the prevention of plague caused by Y. pestis infection. The recombinant raccoon pox virus (RCN strain) vector expresses certain coding sequences of Y. pestis . The vaccine will be inco rporated into a bait matrix and distributed by

plane or vehicle to selected test sites t o combat plague in prairie dogs. Prairie dogs are very

susceptible to plague. Prairie dog colonies can experience mortality rates of 90 per cent or more due to plague , with large overall reductions in population andlocalized extinctions. Control of plague is necessary for prairie dog conservation as well as for ferret recovery

programs. Prairie dogs are the primary food source of the highl y endangered black
- footed f erret, an animal also susceptible to plague. In order to manage the disease in ferrets, it will be necessary to control the disease in prairie dogs. “RCN
- F1
- V307” , the experimental vaccine to be used in the proposed field trial, consists of the RCN vecto r derived from the attenuated Herman strain, the full coding sequence of the F1 capsular antigen from Y. pestis, and a truncated form of the low calcium response V (lcrV) gene from Y. pestis
. The Herman strain has been used in several experimental vaccines in numerous species with no apparent harmful effects. The gene insert s were plac ed in the RCN genome at the site of the non
- essential thy midine kinase (TK) gene
. The RCN strain is a member of the Poxviridae family containing a linear and nearl y 200 kb d ouble
- stranded DNA genome with a hairpin loop at each end. Like other pox viruses, RCN replicates in the cy

toplasm and uses its own transcription sy stems. 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 virus, and canary

pox virus. RCN has been used previously as a live vector to express foreign genes in a recombinant vaccine, although those constructs have not received USDA licensure. For this current vaccine construct, no genetic manipulation was made to the wild ty

pe RCN parent strain prior to insertion of the Y. pestis genes . RCN is considered to be nonpathogenic for raccoons. It was first isolated from the upper respiratory

tract of a raccoon with no obvious clinical sy mptoms by

Y.F. Herman in Aberdeen, Maryland, in 1961
- 1962. The F1 gene and truncated lcrV gene (V 307) were inserted into the RCN TK locus, further attenuating the wild type parent strain. The CVB conducted confirmatory

testing on the Master Seed for this vaccine with satisfactory results for identity , purity, extraneous agents, expression, stability , and genetic anal ys is. The proposed field studies to assess the safety of the sylvatic plague vaccine in prairie dogs and non
- target animals will potentially be conducted at numerous sites in the US , under State and Federal approval and oversight , in accordance with standard requirements published in Title 9, Code of Federal Regulations (CFR), Part 103.3, concerning shipment of experimental biological product s for use in animals
. 1 The potential for escape and dispersal of the experimental vaccine from the proposed release sites is low. The USGS
-NWHC personnel to conduct the study are experienced in wildlife health management. **III. Need for the Proposed Action**  The prairie dog’s range has been reduced b y habitat loss and poisoning to 1 to 2 percent of its former range, leaving colonies smaller and less centralized, thus more susceptible to elimination by

plague. The prairie dog and its burrows are highl y significant in the grassland ecos ys tem, b eing a major source of food and shelter for other species of wildlife.In activated vaccines do not generate an adequate immune resp onse when given orall y.

Live baited vaccines may have pathogenicity in some species or fail to provide sufficient protection to others. F errets were protected b y inoculation of the RCN
- vectored Y. pestis vaccine both in the laboratory and in the field. But, inoculation of free
- ranging animals is not practical for field use. This experimental vaccine represents an attempt by the USGS to produce an efficacious and safe baited vaccine against plague that protects p rairie dogs using o ral vaccination , and in so doing help preserve the endangered black
- footed ferret , as well
. **IV. Areas of Concerns**  The three areas of concern to the CVB regarding use of this live recombinant vaccine are: 1) animal safety, 2) public health, and 3) environmental safet y. The CVB has conducted its own risk analy sis to assess whether risks are associated with the proposal to field test this experimental vaccine. The safety characteristics of this vacci ne have been thoroughl y 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. In three experimental studies using oral RCN
- vectored plague vaccin es in prairie dogs, the target species, no adverse effects were observed. The data were published in peer
- reviewed journals. 2. In two field safety

studies of the oral baited RCN
- F1
- V307 at two remote, enclosed sites, a high percentage of sampled prairie d ogs ingested baits but no morbidity or mortality was detected. B iomarker anal ys is of hair and whisker samples indicated that certain rodent species consume d the baits but without any associated adverse effects. 3. The RCN virus has been reported to be avirulent for various domestic and wild animals. Safety studies have been conducted in raccoons, dogs, bobcats, cotton rats, striped skunks, gray

foxes, rabbits, domestic cats, American mongooses, sheep, swine, patas (African) and cy nomolgus (Asian) monke ys . 2, 3 However, rabbits inoculated intradermally

with RCN and a recombinant RCN developed skin lesions but no edema or hemorrhage. 4. One report indicated poor antibody response to a recombinant RCN expressing a rabies virus gl ycoprotein (rRCNV
- G) , when g iven orally to sheep . This implied that if ingested b y sheep the vaccine would not be harmful. Inaddition, transmission of the v irus between vaccinated and in
- contact controls was not observed or detected b y serology
. 4 Intradermal inoculation of sheep did result in mild ery thematous skin lesions with edema, but not sy stemic disease. 5. In studies comparing the pathogenicity of RCN, a rRCNV
- G construct, the Copenhagen strain of vaccinia virus (vv), and a vv
- rabies virus gl ycoprotein (vv
- G) construct, the rR CNV
- G construct was 100
- fold less pathogenic than RCN, 100
- to 1,000
- fold less pathogenic than vv
- G, and 1,000
- to 10,000
- fold less pathogenic thanvv. This support s the claim of viral attenuat ion by foreign gene insertion and the

relative avirulence of RCN when compared to the Copenhagen strain of vaccinia
. The results were determined using rabbit skin histopathology , suckling mouse lethality , and oral infection of monkeys and mice. 5 6. One case was reported of natural infection with RCN in a domestic Cana dian cat. 6 The cat presented with an ulcerative lesion on the forepaw that was determined to be caused b y a raccoon poxvirus infection using electron microscop y, PCR, and sequencing. The cat was not sy stemicall y affected and recovered ful ly from the infection. 7. USGS studies indicate th at the vaccine is avirulent in various species of prairie dogs and in all non
- target animals tested thus far . Infection via the oral route in prairie dogs and deer mice results in localized infection that does not become sy stemic. 8. The anticipated field dose is 5 x 10 7 pfu. Vaccine doses as high as 10 8 pfu have been injected or fed to animals then comparing results with wild ty pe and TK
- deleted RCN virus, with no adverse effects in mice or prairie dogs. 7, 8 9. Backpassage stu dies in prairie dogs indicated no reversion to virulenc e of the vaccine with passage. **B. Public Health**  The risk to public health is low. 1. Special safety measures will be taken to conduct the field trials including isolated or closed test sites and project signage providing safety information placed along trails and roads near the sites. 2. Human exposure will be limited to the qualified perso nnel handling the vaccine during distribution, and they will be using personal protective equipment (latex gloves)
. 3. There is one reported case of human exposure, caused by a needle prick to the finger of a scientist administering a recombinant raccoon p oxvirus expressing Y. pestis F1 capsular antigen during an experiment. A very small blister developed within 9 day s and was full y healed within four weeks. 9 4. Although the safety of this experimental vaccine in humans has not been fully evaluated and is therefore unknown, no significant safety hazards to the public health are expected following oral baited vaccination of the intended target species. It is reported that the baits disappear rapidl y when deposited in a prairie dog colony, within 3
- 4 day s. There are few baits available for people to encounter and the vaccine has low virulence in humans; thus, the risk to public health is expected to be low. Accidental human exposure is not expected to cause adverse effects. **C. Environmental Safety**  The risk to the environment is low. 1. Bait ingredients are food grade, FDA
- approved when used within the regulatory limits, and flavored with peanut butter. USGS has found that the baits are notattractive to wild pronghorn, mule deer, elk, fox, coyote, badger, s kunk, and not

highl y palatable for captive hoofed stock (cattle, horses), and domestic dogs. 2. The potential for dispersal of this recombinant vaccine is low. Shed/spread studies of the oral vaccine in prairie dogs indicated most (9/10) shed virus orall y, but fecal shedding was not detected. No horizontal transmissio n of the virus from co
- housed v accinated prairie dogs to in
- contact unvaccinated prairie dogs was detected. 3. The survivability

of the recombinant in the field has not yet been tested. However, USGS reports that the experimental baited vaccine was tested for virus stability at various temperatures in the laboratory , with no loss of RCN titer for at least 28 day s at 4 o C (39 o F), 14 day s at 20 o C (68 o F), and 7 day s at 28 o C (82 o F). For proposed field trial distribution during the summer and fall months, what remains of the vaccine after day three will be largel y degraded within seven day s. 4. There are no expected adverse ecological effects associated with the proposal to c onduct field testing with this experimental vaccine, which is derived from a strain that already exists in nature, in raccoon populations that are not clinicall y affected b y its presence. One earlier report from Maryland, in the area where RCN was isolate d, indicated that 23.8% (22/92) of raccoons had antibodies to RCN, suggesting the virus was present in nature in that region. 10 The vaccine candidate is more attenuated than the parental strain, suggesting its risk to the environment is minimal. 5. The potent ial for horizontal gene transfer is lo w and the risk of recombination between the RCN
- F1
- V307 construct and wild t yp e (wt) poxvirus es is low. There are no apparent substantial issues with adverse environmental impacts concerning this agent. T he two compl eted penned field trials have reinforced the estimation of its safety . As there are no apparent significant adverse effects o n animal, human, or environmental safety

associated with the use of th e RCN
- F1
- V307 vaccine, this supports a finding of no signifi cant impact for conducting the proposed field trials. **V. Alternatives**  Two alternatives were considered. T he onl y alternative considered other than the preferred action alternative is not to approve the proposed field test , 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 alternati ve that the proposed field test 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 env ironment and that the preparation of an Environmental Impact Statement is not required (Finding of No Significant Impact).**References**

1. Code of Federal Regulations, Washington, DC. Available online: http://www.gpoaccess.gov/cfr/index.html 2. Esposito JJ, Chandler FW, and Baer GM. Oral immunization of animals with raccoon poxvirus expressing rabies virus gl yc oprotein. In: Vaccines 89. (ed. R Lerner, H Ginsberg, RM Chanock, F Brown) Cold Spring Harbor Laboratory Press, NY, p 403
- 408, 1989. 3. Esposito J J, Sumner JW, Brown DR, Ebert JW, Shaddock JH, He BX, Dobbins JG, and Fekadu M. Raccoon poxvirus rabies
- gl ycoprotein recombinant oral vaccine for wildlife: further efficacy

and safety studies and serosurvey for raccoon poxvirus. In: Vaccines 92. (ed. F Bro wn, RM Chanock, HS Ginsberg, RA Lerner) Cold Spring Harbour Laboratory

Press, NY, p 321
- 329, 1992. 4. DeMartini JC, Bickle HM, He BX, and Esposito JJ. Raccoon poxvirus rabies virus gl ycoprotein recombinant vaccine in sheep. Arch Virol 133:211
- 222, 1993. 5. Esposito JJ. L ive poxvirus
- vectored vaccines in wildlife immunization programmes: the rabies paradigm. Res Virol 140:480
- 482. 1989. 6. Yager JA, Hutchison L, Barrett JW. Raccoonpox in a Canadian cat. Vet Dermatol 17:443
- 8, 2006. 7. Rocke TE, Iams KP, Dawe S, Smith SR, Williamson JL , Heisey DM, and Osorio JE. Further development of raccoon poxvirus
- vectored vaccines against plague ( Yersinia pestis ): Vaccine 28:338
- 344, 2010. 8. Rocke TE, Pussini N, Smith SR, Williamson J, Powell B, and Osorio JE. Consumption of baits containing raccoon pox
- based plague vaccines protects black
tailed prairie dogs ( Cynomys ludovicianus) . Vector
- borne & Zoonotic Dis 10:53
- 58, 2010. 9 . Rocke TE, Dein FJ, Fuchsberger M, Fox BC, Stinchcomb DT, and Osorio JE. L imited infection upon human exposure to a recombinant raccoon pox vaccine vector. Vaccine 22:2757
- 2760, 2004. 10 .Alexander AD, Flyger V, Herman YF, McConnell SJ, Rothstein N, and Yager RH. Survey of wild mamm als in a Chesapeake Bay area for selected zoonoses. Jour Wildlife Dis 8:119
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