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Risk Assessment

For Field 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 

Adenovirus Vector, for field testing in wildlife. Artemis Technologies Inc., Guelph, 

Ontario, Canada, in collaboration with the USDA-APHIS-Wildlife Services (WS), has 

requested authorization to conduct field tests in the United States (US) under sylvatic 

conditions that are typical of the terrestrial rabies vector species 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 harmful. Accordingly, the 

CVB has conducted a risk analysis 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 analysis with confidential business information removed is available 

upon request. The CVB risk analysis includes that document, but also incorporates 

review of all the firm’s studies and tests related to the vaccine, the confirmatory testing 

completed by CVB personnel on the Master Seed Virus to confirm purity, identity, and 

stability, consultation with subject matter experts as needed, results of literary and 

regulatory review, and evaluation in light of similar previously tested or licensed 

products.

Based on the CVB risk analysis 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 Notice 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 encephalomyelitis found throughout the world and is fatal (in all 

but a few human cases) once clinical signs appear. Following human exposure to rabies 

virus and prior to development of signs, preventive vaccination can be successful in 

halting infection but is expensive and has supply limitations. This can result in 
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inadequate public health resources that endanger the survival of persons exposed to 

rabies. 

In the US, there are numerous rabies virus variants, generally each associated with a 

certain species but spillover infection into other species can occur.

1,2

 Although canine 

rabies has been largely eliminated from domestic dogs in North America and Europe due 

to successful parenteral vaccination programs, other rabies variants 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

reduces 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 

reduced. This has been demonstrated by the past success of the oral baited rabies 

vaccine, “V-RG”, which is a live recombinant vaccinia virus expressing the rabies 

glycoprotein. 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 serotype 5 adenovirus (hAd5) containing the rabies glycoprotein

gene inserted into a deletion in the adenoviral genome. The recombinant is replicationcompetent but host-restricted. Human adenoviruses can cause cold-like symptoms in 

people but are not considered to be pathogenic for other animal species.

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 Regarding 

hAd5, most persons are exposed early in life and develop immunity. The hAd5 vector is 

considered nonpathogenic in healthy adult humans and has a history of safe use as a 

rabies vaccine vector in over 7 million baits distributed in Canada since 2006.

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The 

product is intended for oral vaccination of raccoons, skunks, and foxes, as an aid in the 

prevention of rabies.

The proposed field safety 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 products for use in animals.

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 The potential for escape and dispersal of the 

experimental vaccine from the proposed release sites is low. The APHIS-WS personnel 

to conduct the study are experienced in wildlife health management.

III. Need for the Proposed Action

Inactivated rabies vaccines do not generate an adequate immune response when given 

orally. 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 

rabies that protects skunks, raccoons, and foxes using oral vaccination. 
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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 safety. The CVB 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. Adenoviruses are considered to be largely 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

young cotton rats, hamsters, and rabbits. In the case of this adenovirus vector, an E3

genetic deletion may have attenuated the virus even further. The E3 proteins not 

produced as a result of the deletion are believed to help prevent destruction of 

infected cells by the host’s immune system. In their absence, there may be less virus 

production and persistence in vivo.

2. Regarding the rabies virus, only its glycoprotein 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 conventionally prepared vaccines.

3. A dose titration study was conducted by the firm in 30 skunks with no adverse 

reactions, morbidity or mortality observed.

4. In 6 Canadian field trials 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 by 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 relatively higher doses (~5x) to 

red fox, raccoon, skunk, meadow vole, deer mouse, grey squirrel, rabbit, groundhog, 

horses, sheep, cows, pigs, chickens, dogs, cats, and immunocompromised mice.6

Live virus was recovered only from SCID mice, not even from nude mice which lack 

mature T cell function yet were still able to limit viral replication. These results in 
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SCID and nude mice would not be considered significant under the conditions of a 

field trial. Previous field trials conducted in Canada would be significant and, during 

those trials, adverse effects on species exposed to the vaccine baits were not detected.

7. Results of PCR, sequencing, and other testing on the Master Seed (MS) and on the 

MS following its 5th passage in cell culture (the highest pass to be used in vaccine 

production), were consistent with expected genetic identity, with genomic stability of 

the recombinant vector and the inserted rabies glycoprotein gene, and with sterility

and purity of the MS and MS+5. 

B. Public Health

The risk to public health is low. 

1. Special safety 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 distributing the vaccine and handling 

the animals that are captured for examination. In the Canadian field trials thus far, 

there has been an average of one report of finding 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 safely in previous field trials. In general, 

adenovirus vectors have been tested and used extensively in experimental human 

vaccines. As of July 2006, more gene therapy clinical trials had used adenoviral 

vectors (26%) than any other vector; most of those were derived from type 2 and type 

5 adenovirus.3 They are considered fairly innocuous, although one clinical trial death 

has been documented, following arterial infusion of a high dose of viral vector, 

resulting in a hypercytokinemic 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 It has 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 daily for 9 days post exposure yielded no virus from cats and only very low 

levels of virus in a few dog swabs taken within 2 days 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 safety of this experimental oral vaccine for use in wildlife has not been 

specifically evaluated in humans and is therefore unknown, no safety hazards to the 

public health are expected, and the public will not be exposed to the virus under 

hazardous conditions.
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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 oral swabs taken from rabies vector species in 

studies in Ontario at day 7 and beyond. Fecal swabs contained virus only 3-4 days 

for most species tested; skunks excreted virus in feces for 3 days only.

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3. A vaccination shedding study was conducted by the firm in 30 skunks, using oral and 

fecal swabs and a real time PCR assay. All oral swabs, taken at days 3, 7, 15, 21, and 

29, were negative. Only one fecal sample from each of only 6 animals was positive 

for virus within 3 days of ingestion and only at low levels. This would be residual 

virus from the oral baited vaccine, not newly replicating virus. 

4. The CVB conducted testing of the Master Seed (MS) of the vaccine virus, including a 

panviral microarray analysis for extraneous agents, and found the MS to be sterile and 

free of contaminating agents. In addition, CVB sequencing confirmed the identity

and stability of the rabies insert and flanking sequences, through the highest passage 

to be used in vaccine production (MS+5).

5. To determine phenotypic and genetic stability, the firm conducted several 

experiments.8 A collection of murine monoclonal antibodies against six distinct 

antigenic sites on the rabies glycoprotein determined the preservation of all the main 

immunogenic epitopes after viral growth in vitro, and the consistent co-expression of 

glycoprotein and hAd5 proteins in a double immunostain assay, indicating no loss of 

the glycoprotein 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 the virus through cotton rats five times was done. 

Sequence analysis of 105 virus clones obtained after the fifth passage did not identify

any mutation. Lastly, regarding the possibility of recombination with wild-type virus 

when placed in the field, a series 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. It is unlikely that an exchange of genetic material 

with wildtype 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-type

viruses.

There are no apparent substantial issues with adverse environmental impacts concerning 

this agent. The several successful field trials in Canada have reinforced the estimation of 

its safety. As there are no apparent significant adverse effects on animal, human, or 
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environmental safety associated with the use of the AdRG1.3 vaccine, this supports a

finding of no significant impact for conducting the proposed field trials.

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 risk assessment, 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. Rupprecht, 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 therapy. 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 

non-target species. Vaccine 27(47):6619-26.

7. Raper, S.E., Chirmule, N., Lee, F.S., et al. 2003. Fatal systemic inflammatory

response syndrome in a ornithine transcarbamylase deficient patient following 

adenoviral gene transfer. Mol Gen Metabol 80:148-58.

8. Knowles, M.K., Roberts, D., Craig, S., et al. 2009. In vitro and in vivo genetic 

stability studies of a human adenovirus type 5 recombinant rabies glycoprotein 

vaccine (ONRAB). Vaccine 27:2662-2668.