1 **Risk Assessment**  **For Fi**  **eld Testing Rabies Vaccine, Live Adenovirus Vector**  **Center for Veterinary Biologics**

**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 Rabies Vaccine, Live Ade novirus Vector, for field testing in wildlife . Artemis Technologies Inc., Guelph, Ontario, Canada, in collaboration with the USDA
- APH IS
- Wildlife Services (WS), has requested authorization to conduct field tests in the United States (US) under sylvatic conditions that are t ypical of the terrestrial rabies vector spe cies of interest, i.e. raccoons and skunks
. 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 rabies vaccine, the safety

risks to animals, public health, and the environment are low. A copy of the firm’s risk analy sis with confidential business information removed is available upon request. The CVB risk anal ys is 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 b y CVB personnel on the Master Seed Virus to confirm purity , identity , and stability , consult ation with subject matter e xperts as needed , results of literary and regulatory

review, and evaluation in light of similar previousl y tested or licensed products. Based on the CVB risk analy sis and other relevant data, we have reached a preliminary determ ination 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 testi ng 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**  Rabies is an acute viral encephalo myelitis found throughout the world and is fatal (in all but a few human case s ) once clinical signs appear. Following human exposure to rabies virus and prior to development of signs, pr eventive vaccination can be successful in halting infection but is expensive and has suppl y limitations
. This can result in2

inadequate public health resources that endanger the survival of persons exposed to rabies. In the US, there are numerous rabies virus variants, generall y each associated with a certain species but spill over infection into other species can occur . 1,2 Although canine rabies has been largel y el iminated from domestic dogs in North America and Europe due to successful parenteral vaccination programs, other rabies vari ants are maintained in wildlife. About 90% of all reported cases in the US occur in wildlife. This ongoing source of exposure necessitates continued vaccination of domestic animals, which in turn reduce s the risk of human infection . Although no feline rabies virus variant is known, cats are the most frequently reported rabid domestic animal in the US, and reported cases in US domestic cats have outnumbered cases in dogs since 1988. 1 By providing effective oral vaccines to wildlife, the risk of infection in domestic animals and humans can be reduc ed. This has been demonstrated b y the past success of the oral baited rabies vaccine, “ V
- RG ” , which is a live recombinant vaccinia virus expressing the rabies gl ycoprotein. It has been effective in reducing the occurrence of rabies in raccoons, coyotes, and gray

foxes in the US
. However, no single vaccine has proven effective in all the vector species. 2 The experimental vaccine to be used in the proposed field trial , “AdRG1.3”, is a live recombinant human seroty pe 5 adenovirus (hAd5) containin g the rabies gl ycoprotein gene inserted into a deletion in the adenoviral genome. The recombinant is replication

competent but host

- restricted
. Human adenoviruses can cause cold

- like s ymptoms in people but are not considered t o be pathogenic for other an imal species
. 3 Regarding hAd 5, most persons are exposed early in life and develop immunity
. The h Ad5 vector is considered nonpathogenic in health y adult humans and has a history of safe use as a rabies vaccine vector in over 7 million baits distributed in Canada since 2006
. 4 The product is intended for oral vaccination of raccoons , skunks , and foxes , as an aid in the prevention of rabies
. The proposed field safet y test will be conducted in West Virginia 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
. 5 The potential for escape and dispersal of the experimental vaccin e from the proposed release sites is low. The APHI S
- WS personnel to conduct the study

are experienced in wildlife health management. **III. Need for the Proposed Action**  In activated rabies vaccines do not generate an adequate immune response when given ora lly
. Live baited vaccines may have pathogenicity

in some species or fail to provide sufficient protection to others. This experimental vaccine represents an attempt by Artemis Technologies Inc. to produce an efficacious and safe baited vaccine against ra bies that protects skunks, raccoons, and foxes using o ral vaccination.3

**IV. Areas of Concerns**  The three areas of concern to the CVB regarding use of this live recombinant vaccine are: 1) animal safet y, 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 vaccine have been thoroughl y evaluated. The conclusions derived from the ris k anal ys is for each of the areas of concern are summarized below. **A. Animal Safety**  The risk to animals is low. 1. Adenoviruses are considered to be largel y host

- specific and a human adenovirus has little to no infectivity

for mammals other than humans , including the target species (raccoons, skunks) to be exposed to the vaccine. There are reports in the literature of experimentally

- induced viral replication and sometimes pathogenicity only in very yo ung cotton rats , hamsters, and rabbits
. In the case o f this adenovirus vector , an E3 genetic dele tion may

have attenuated the v i r

us even further
. The E3 proteins no t produced as a result of the deletion are believed to help prevent destruction of infected cells by

the host’s immune sy stem. In their absence , there may be less virus production and persi stence in vivo
. 2. Regarding the rabies virus, o nly its gl ycoprotein gene is incorporated into the hAd5 vector. The recombinant virus has been shown to cause no adverse clinical signs or lesions and can elicit an immune response that protects a number of species from virulent rabies virus challenge. Antibody to the remaining rabies viral structural proteins is not generated and this can be used to differentiate between rabies

- exposed animals or those vaccinated with conventionall y prepared vaccines. 3. A dose titration study was conducted b y the firm in 30 skunks with no adverse reactions, morbidity

or mortality

observed. 4. In 6 Canadian field tri als conducted between 2006 and 2009, a total of 3877 raccoons and 1613 skunks were live

- trapped, samples collected, and released. During these trials, no adverse reactions, morbidity , or mortality were observed in the captured animals or in the wildlife population at large. 5. In vaccination/challenge studies conducted b y the firm, 58 skunks and 20 raccoons in the vaccinated groups have shown no adverse reactions attributable to the vaccine. 6. In overdosing studies, the vaccine was given orally at relativel y higher doses (~5x) to red fox, raccoon, skunk, meadow vole, deer mou se, grey squirrel, rabbit, groundhog, horses, sheep, cows, pigs, chickens, dogs, cats, and immunocompromised mice. 6 Live virus was rec overed only from SCID mice, not even from nude mice which lack mature T cell function yet were still able to limit viral replication . T hese results in4

SCID and nude mice would not be considered significant under the conditions of a field trial
. P revious field trials conducted in Canada would be significant and, during those trials , adverse effects on species exposed to th e vaccine baits were not detected
. 7. Results of PCR , sequencing, and other testing on the M aster S eed (MS) and on the MS following its 5 th passage in cell culture (the highest pass to be used in vaccine production), were consistent with expected genetic ide ntity , with genomic stability of the recombinant vector and the inserted rabies glycoprotein g ene, and with sterility and purity

of the MS and MS+5
. **B. Public Health**  The risk to public health is low. 1. S pecial safet y measures will be taken to conduct this study
. Each bait packet will contain an appropriate label warning. There will be a communication network the public can use to report any bait or vaccine contact. It is intended that human exposure will be limited to qualified personnel distribut i ng the vaccine and handling the animals that are captured for examination
. Inthe Canadian field trials thus far, there h as been an average of one report of f inding or having contact with a bait for every 83,500 baits distributed, with no adverse effects
. 2. Accidental human exposure is not expected to cause adverse effects. The hAd5

- vectored rabies vaccine has been used safel y in previous field trials
. In general, adenovirus vectors have been tested and used extensively in experimental human vaccines. A s of July

2006, more gene therap y clinical trials had used adenoviral vectors (26%) than an y other vector; most of those were derived from type 2 and type 5 adenovirus. 3 They

are considered fairl y innocuous, although one clinical trial death has been documented, following arterial infusion of a high dose of viral vector, resulting in a h yp ercy

tokinemic immune reaction. 7 3. The vaccine is not virulent for domestic livestock species that could potentially encounter baits and expose humans, based on testing in horses, sheep, cows, pigs, and chickens. 6 Ithas also been shown to not be virulent in companion animals in experimental testing at higher doses
. The close interaction of dogs and cats with their owners provides a potential source of exposure; oral and fecal swabs taken from dogs and cats dail y for 9 days post exposure yielded no virus from cats and onl y very low levels of virus in a few dog swabs taken within 2 day s of exposure. In the Canadian trials run thus far, there are only a very low number of human contacts with the vaccine baits via dogs and there have been no adverse reactions associated with them. 4 4. Although the safet y of this experimental oral vaccine for use in wildlife has not been specificall y evaluated in humans and is therefore unkn own, no safet y hazards to the public health are expected , and the public will not be exposed to the virus under hazardous conditions
.5

**C. Environmental Safety**  The risk to the environment is low. 1. The host range of the hAd5 vector is limited to humans
. The potential for escape and dispersal of this experimental vaccine from a targeted wildlife population that cannot support viral replication is low. 2. Virus could not be recovered from o ral swabs taken from rabies vector species in studies in Ontario at day 7 and beyond . Fecal swabs contained virus only 3
- 4 day s for most species tested; skunks excreted virus in feces for 3 day s onl y. 6 3. A vaccination shedding study was conducted b y the firm in 30 skunks, using oral and fecal swabs and a real time PCR assay
. All oral swabs, taken at day s 3, 7, 15, 21, and 29, were negative. Onl y one fecal sample from each of onl y 6 animals was positive for virus within 3 day s of ingestion and onl y at low level s. This would be residual virus from the oral baited vaccine, not newl y replicating virus
. 4. The CVB conducted testing of the Master Seed (MS) of the vaccine virus, including a panviral microarray

analy sis for extraneous agents, and found the MS to be sterile and free of contaminating agents. In addition, CVB sequencin g confirmed the ide ntity and stability

of the rabies insert and flanking sequences, through the highest passage to be used in vaccine production (MS+5)
. 5. To determine phenot ypic and genetic stability, the firm conducted several experiments. 8 A collection of murine monoclonal antibodies against six distinct antigenic sites on the rabies gl ycoprotein determine d the preservation of all the main immunogenic epitopes after viral growth in vitro , and the consistent co

- expression of gl ycoprotein and hAd5 protein s in a double immunostain assay , indicating no loss of the gl ycoprotein gene insert. Also, 67 independent clones were recovered after 20 passages in cell culture and sequenced; they showed no variation across the transgene region. In vivo passaging of th e virus through cotton rats five times was done. Sequence anal ys is of 105 virus clones obtained after the fifth passage did not identify any mutation. Lastl y, regarding the possibility of recombination with wild

- ty pe virus when place d in the field, a se ries of competitive in vitro passaging experiments were conducted to assess the stability of the recombinant in the presence of the parental hAd5. The parent and the recombinant maintained their relative proportions during the co

- infection in cell culture
. Itis unlikel y that an exchange of genetic material with wildty

pe virus would occur in the field
. Even if it did occur, the event would not be expected to generate a more virulent virus than the already present wild

- ty pe viruses
. There are no apparent substantial issues with adverse environmental impacts concerning this agent. T he several successful field trials in Canada have reinforced the estimation of its safety . As there are no apparent significant adverse effects o n animal, human, or6

environmental safety

associated with the use of th e AdRG1.3 vaccine, this supports a finding of no significant impact for conducting the proposed field trials. **V. Alternatives**  Two alternatives were considered. T he onl y alternative considered other than the prefer red 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 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 alternative that the proposed field tests be approved. **VI. Conclusion**  Based upon the ri sk analy sis documented in this risk assessment , 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 Statement is not required (Finding of No Significant Impact).

**References** 1. Rupprec ht, C.E. 2005. Rabies. pp. 1067

- 1071. In: The Merck Veterinary Manual, Ninth Edition. C.M. Kahn, (Ed), Merck & Co., Inc., Whitehouse Station, N.J. Available online: http://www.merckvetmanual.com/mvm/index.jsp 2. Slate, D., Algeo T.P., Nelson, K.M., et al. 2009. Oral rabies vaccination in North America: opportunities, complexities, and challenges. PLOS 3(12):e549. 3. Douglas, J.T. 2007. Adenoviral vectors for gene therap y. Mol Biotechnol 36:71

- 80. 4. Rosatte , R.C., Donovan, D., Davies, J.C., et al . 2007. Aerial distribution of ONRAB® baits as a tactic to control rabies in raccoons and striped skunks in Ontario, Canada. J Wildl Dis 45:363

- 74. 5. Code of Federal Regulations, Washington, DC. Available online: http://www.gpoaccess.gov/cfr/index.html 6. Knowles, M.K., Nadin

- Davis, S.A., Sheen, M., Rosatte, R., et al . 2009. Safety studies on an adenovirus recombinant vaccine for rabies (AdRG1.3

- ONRAB) in target and n on - target species. Vaccine 27(47):6619

- 26. 7. Raper, S.E., Chirmule, N., L ee, F.S., et al. 2003. Fatal sy stemic inflammatory response s yndrome in a ornithine transcarbam yl ase deficient patient following adenoviral gene transfer. Mol Gen Metabol 80:148

- 58. 8. Kno wles, M.K., Roberts, D., Craig, S., et al . 2009. In vitro and in vivo genetic stability studies of a human adenovirus t ype 5 recombinant rabies glycoprotein vaccine (ONRAB). Vaccine 27:2662

- 2668.