Studies
on
the Stability of a Human Adenovirus-Rabies Recombinant Vaccine
Kishna K. Kalicharran, V. Susan Springthorpe and Syed A. Sattar
ABSTRACT Human adenovirus type 5 containing the rabies virus glycoprotein gene (rHAd-RG1) has potential for the oral vaccination of animals. The stability of this recombinant was tested indoors and outdoors by measuring the loss in virus infectivity. Under indoor conditions the stability of the recombinant virus was studied in an egg yolkcontaining commercial stabilizer and a simple buffered salt solution (EBSS; Earle's balanced salt solution) at 40C and room temperature (24-25°C). Over 16 days, there was a more rapid loss in virus titer at room temperature than at 40C in both suspending media; however, these differences were slight and may be insignificant when the overall stability of the vaccine is considered. When the virus was mixed with either 10% (w/v) fox or skunk feces or EBSS, placed on stainless steel disks and the disks kept under ambient conditions (air temperature 24-25°C; relative humidity 45-50%o), there was a more rapid decline in virus titer in the fecal suspensions (37o remained after 72 h) than in EBSS (26%o remained after 72 h). When bait-coated blister packs of the vaccine were placed in an outdoor location in the fall (October) season, there was a larger drop in the virus titer for vaccines placed in the sun (547o over 32 days) than for those in the shade (40%o over 32 days). Incorporating proteinaceous stabilizers in the vaccine samples for outdoor study showed virus stability was not enhanced in their presence. This study shows rHAd5-RG1 in EBSS has halflives of 66 days at 4°C and 51 days at room temperature and that it is sufficiently stable outdoors for use as a vaccine in baits for wildlife.
RESUME
eprouves au-dehors n'a pas
augmente
la stabilite du virus. Cette etude montre L'adenovirus humain type 5 conte- que le recombinant rHAd5-RG1 dans nant le gene de la glycoproteine du le EBSS possede des demi-vies de virus de la rage (rHAd5-RG1) possede 66 jours a 4 IC et de 51 jours a la un potentiel pour la vaccination orale temperature de la piece et qu'il est sufdes animaux. La stabilite de ce recom- fisamment stable 'a l'exterieur pour etre binant a ete testee a l'interieur et utilise comme vaccin dans des appats au-dehors en mesurant la perte du pour la faune. (Traduit par Dr Ronald pouvoir infectieux du virus. A l'inte- Magar) rieur, la stabilite du virus recombinant melange, d'une part a un stabilisateur commercial contenant du jaune d'oeuf INTRODUCTION et d'autre part a une simple solution saline tamponnee (solution saline Rabies continues to be a problem in equilibree de Earle EBSS), a ete etudiee Canada, with foxes (Vulpes vulpes) tant a 4 IC qu'a la temperature de la and skunks (Mephitis mephitis) being piece (24-25 IC). Sur une peiode de the major wildlife reservoirs (1-3). The 16 jours, il y avait une perte de titre use of an oral attenuated rabies virus viral plus rapide a la temperature de la vaccine was introduced in Canada piece qu'a 4 IC dans les deux milieux based on its success in fox rabies conde suspension; toutefois, ces dif- trol in certain European countries (4). ferences etaient legeres et pourraient However, the attenuated vaccine is etre non-significatives lorsque la ineffective in skunks (3), shows stabilite generale du vaccin est con- residual pathogenicity for rodents (5) sideree. Lorsque le virus a ete melange and is relatively unstable (2,3). a une suspension (10 No p/v) de feces Research into recombinant vaccines de renard ou de mouffette ainsi qu'au led to the development of a vaccinia EBSS puis a ete place sur des disques virus containing the rabies glycoprod'acier inoxydable, qui ont ete gardes tein gene (6). This vaccine proved ensuite en conditions ambiantes effective in foxes, but failed to (temperature 24-25 IC; humidite immunize skunks when given orally relative (45-50 %), une chute plus (7). Prevec et al (8) were successful in rapide du titre du virus a ete notee dans inserting the rabies glycoprotein gene les suspensions fecales (3 0o restant in the deleted E3 region of human apres 72 h) que dans le EBSS (26 % adenovirus type 5. Adenoviruses have restant apres 72 h). Quand des pla- proved to be relatively innocuous and quettes de vaccin enrobees d'appat ont stable to gastric acid. Adenovirus types ete placees sur une site a l'exterieur en 4 and 7 are administered orally to automne (octobre), il y avait une plus immunize army recruits against acute grande chute du titre viral pour les vac- respiratory disease (9-12); these viruses cins places au soleil (54 %7o sur l'espace can induce high levels of heterologous de 32 jours) que pour ceux places a gene expression in mammalian cells l'ombre (40 sur l'espace de 32 jours). (13-17). L'incorporation de stabilisateurs proAmong the major concerns on the teiques dans les echantillons de vaccins release of recombinant viruses is their
Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K 1 H 8M5. Reprint requests to Dr. S.A. Sattar. Support for this work was provided by a grant from the Ontario Ministry of Natural Resources. Submitted April 2, 1991.
28
Can J Vet Res 1992; 56: 28-33
ability to remain viable in the environment for prolonged periods. On the other hand a recombinant vaccine virus must have a suitable shelf-life and retain its infectivity long enough in the field to successfully immunize the target species. Knowledge of virus survival is, therefore, a prerequisite to planning vaccine production and field trials. In view of this, we studied the stability of the adeno-rabies recombinant under both indoor and field conditions. MATERIALS AND METHODS VIRUS
The adeno-rabies recombinant (rHAd5-RGl) pool used throughout this study was in its third passage in MRC-5 cells and was prepared in Dr. J.B. Campbell's Laboratory at the University of Toronto, Toronto, Ontario. The virus was received through the courtesy of Dr. A. Wandeler, Animal Diseases Research Institue (ADRI), Agriculture Canada, Nepean, Ontario. CELLS
The Vero line of African green monkey kidney cells was used throughout this study and a seed culture of these cells was provided by Dr. Campbell. The cells were grown in Eagle's minimal essential medium (EMEM; Gibco Laboratories, Grand Island, New York) supplemented with 507o fetal bovine serum (Cell Culture Laboratories, Cleveland, Ohio), 50 jtg/mL gentamicin sulfate (Cidomycin; Roussel, Montreal, Quebec), 200 mM glutamine (Gibco) and 0.22507o (w/v) sodium bicarbonate (BDH Chemicals, Toronto, Ontario). The cells were maintained in the same medium with only 207o serum.
made in Earle's balanced salt solution (EBSS; Gibco). For each dilution, three wells were inoculated with 0.1 mL of inoculum each. The cell control wells were exposed to an equivalent amount of EBSS. The plates were rotated gently to redistribute the inoculum over the monolayers before incubating them at 37°C for 60 min in a 57o CO2 atmosphere. Redistribution of the inoculum was carried out every 10 min during the virus adsorption period. Each well then received 2.0 mL of an overlay containing EMEM with 200 mM glutamine (Gibco), 10 mM magnesium chloride (Sigma Chemical, St Louis, Missouri), 50 /tg/mL gentamicin, 2 ,ug/mL amphotericin B (Fungizone; Gibco), 2% serum, 0.225Wo (w/v) sodium bicarbonate and 0.75% (w/v) agarose type II (Sigma). The plates were kept at room temperature to allow the medium to solidify, sealed individually in laminated plastic bags (Dazey Corp., Industrial Airport, Kansas) and incubated at 37°C for five to six days. Once plaques were readily visible under an inverted microscope, 2.0 mL of a 3.7% (v/v) formaldehyde (Fisher Scientific, Ottawa, Ontario) solution in normal saline were added to each well to fix the monolayers and to inactivate the virus. After holding the plates overnight at room temperature, the overlay was removed with running tap water. Each monolayer was then stained for 5 min with 0.5 mL of a 1 07o aqueous solution of crystal violet. The stain was washed off in tap water, the plates dried and the plaques counted. The virus infectivity titer is expressed as plaque forming units (PFU)/mL. The data are plotted with the means and standard deviations. The parameters were calculated and graphed by the SigmaPlotTM software (Jandel Scientific, Corte Madera, California).
VIRUS TITRATION
For virus plaque assay, monolayers were prepared by seeding approximately 1 x 105 cells into each well of a twelve-well cell culture plate (Costar, Cambridge, Massachusetts) in 2.0 mL of the growth medium. The seeded plates were incubated at 37°C overnight in a 5Wo CO2 atmosphere. Serial tenfold dilutions (10-3, 10-4, 10- 5) of the virus samples to be titrated were
VACCINE
Polystyrene blister packs and a bait mixture were provided by Dr. K. Lawson (Connaught Laboratories, Willowdale, Ontario) and both of these are currently being used in the field trials of the attenuated (ERA) rabies virus vaccine (18). One part of the stock virus was mixed with one part of either EMEM without serum or the
chemical stabilizer under test and 2.0 mL of the mixture was injected into each blister pack using a 2.0 mL syringe with a 26 gauge needle (Becton, Dickinson & Co., Mississauga, Ontario); the needle puncture was sealed with a plastic glue. For embedding the blister packs in the bait, the bait mixture, contained in a 250 mL glass conical flask, was melted in a water bath at 70°C. Approximately 18 g was then poured into each one of the clear plastic moulds (4.0 x 4.0 x 2.5 cm). While the bait mixture was still soft, the blister pack with the virus was inserted into it with an applicator stick, making sure that the entire surface of the blister pack was completely and evenly covered with the bait. To monitor the vaccine temperature, 16-gauge hypodermic probes (Product No. Hyp3-16-1 1/2-K-V-48RP; Omega Corp., Stamford, Connecticut) containing type K thermocouples were inserted into two of the blister packs before embedding them in the bait. The bait temperature was recorded by plugging the thermocouple probes into a portable thermometer (Omega). INDOOR SURVIVAL EXPERIMENTS
The stock virus was mixed in a 1:1 ratio with an egg yolk-containing commercial stabilizer provided by Dr. Lawson. This stabilizer is used with the attenuated (ERA) rabies virus vaccine (19). The virus-stabilizer mixture was vortexed for 10 s and 200 AiL quantities were aliquoted into 2.0 mL sterile plastic vials (Sarstedt, St. Laurent, Quebec). As virus control, the stock virus was mixed with an equal volume of EBSS. Immediately, 100 ML samples of the virus-EBSS and the virus-stabilizer mixtures were removed for titration (zero min control). Five vials of each of the test mixtures were placed at room temperature (24-25°C) in a plastic box; a corresponding set was placed at 4°C. One vial of each mixture was removed at the end of 2, 4, 8, 12 and 16 days. The samples were stored at - 80°C and all of them were titrated at the end of the experiment. This experiment was repeated three times. To determine the survival of the recombinant virus on nonporous inanimate surfaces under indoor conditions, it was suspended in fecal 29
samples from an unvaccinated fox and an unvaccinated skunk kept at the ADRI. The fecal samples were prepared as a 1007o (w/v) suspension in normal saline, clarified by centrifugation at 1,000 x g for 5 min and passed through a 47 mm diameter membrane filter (Nalge Co, Rochester, New York) with a pore diameter of 0.22 zm to remove bacterial and fungal contamination. The filtrates were found to be noncytotoxic to Vero cells and free from any indigenous viruses that could produce cytopathology in these cells. A 1:10 dilution of the virus was prepared in either EBSS or the fecal filtrates and, using a positive displacement pipette (Gilson, Villiers le Bel, France), 10 ,L of virus suspension were placed separately at the center of each stainless steel disk (1 cm in diameter). The disks were punched out of locally purchased sheets (0.75 mm thick) of #4 polished stainless steel. A 12-well plastic cell culture plate, with one virus-inoculated disk per well, was held uncovered in a laminar flow hood. The air temperature and relative humidity were monitored throughout the experiment using a recording hygrothermograph (Cole-Parmer, Chicago, Illinois). One disk each of the two different virus suspensions was removed at the end of 1, 2, 4, 6, 12, 24, 48 and 72 h and placed in a tube containing 1.0 mL EBSS. The tubes with the disks were sonicated for 10 min in an ultrasonic bath (Bransonic Ultrasonic Corp, Danbury, Connecticut) to assist in the elution of the virus from the disks. The eluates were then titrated. This experiment was performed twice with fox feces and once with skunk feces. OUTDOOR SURVIVAL EXPERIMENTS
Baits with the attenuated rabies virus vaccine are currently dropped in the fall season in Eastern Ontario (18). Therefore, the survival of rHAd5-RGI in baits was also tested at this time (October) of the year under outdoor conditions. Each blister pack containing 2.0 mL of the recombinant virus (grown in MRC-5 cells) was embedded in the bait. The baits were placed outdoors, with one set of samples exposed to sunlight and another placed in the shade; a thermocouple-fitted vaccine pack
30
was
included
in
each
set
to mea-
8 bait temperature. Outdoor air temperature and relative humidity were also monitored with a recording hygrothermograph throughout the sampling period. Samples were withdrawn at 1, 2, 4, 8, 16 and 32 days and titrated. 6 A set of samples containing the following organic materials was also prepared in order to determine their virus 5stabilizing potential: peptone (Oxoid Ltd., Basingstoke, Hampshire, England), lactalbumin hydrolysate 4 0 2 4 6 8 10 12 14 16 18 (Gibco), yeast extract (Difco LaboraTIME (days) tories, Detroit, Michigan) and tryptose phosphate broth (Difco). All the above Fig. 1. Comparison of the stability of were prepared as 10% (w/v) solutions HAd5-RG1 virus in the commercial stabilizer EBSS at room temperature expressed as in phosphate buffered saline (pH 7.2) and log10 PFU/mL. The mean virus starting titers and each was mixed separately with the were 4.5 x 106 and 4.6 x 106 PFU/mL in virus in a 1:1 ratio. The mixtures were EBSS and stabilizer respectively. placed in blister packs and inserted into 8, baits as described above. After 32 days * VIRUS/STABILIZER v VIRUS/EBSS of exposure to the sun outdoors, the contents were titrated.
sure
U)
0
7
E 7 :D
RESULTS c.
SURVIVAL OF rHAd5-RGl UNDER INDOOR CONDITIONS
These experiments were designed to give an indication of the shelf-life of the rHAd5-RG1 vaccine when it is stored either at room temperature (Fig. 1) or under refrigeration (Fig. 2). As can be seen from Table I, the estimated half-lives of the stabilizercontaining virus held at 4°C and at room temperature were 88 and 64 days, respectively. The rate of loss of the EBSS-containing virus was somewhat greater, with the estimated half-lives at 4°C and room temperature being 66 and 51 days, respectively. The results thus indicate that the virus may be sufficiently stable in EBSS, and for the remainder of the indoor studies, EBSS was used as the suspending medium.
6
U)
0
5
4
0
4
2
6
8
14
16
Excretion of the virus in the feces of experimentally-vaccinated animals could contaminate animal holding
on
storage in liquid
coefficientb 0.90 0.83
Half-life (days) 88 64
EBSS at 40C 0.0104 0.86 66 EBSS at 24-25°C 0.84 0.0135 51 aKi Loss in logl0 virus PFU/day bCorrelation coefficient refers to the measure of the linear association between two variables =
18
SURVIVAL OF rHAd5-RGl ON ENVIRONMENTAL SURFACES
Correlation Kia 0.0078 0.0106
12
Fig. 2. Comparison of the stability of rHAd5-RG1 virus in the commercial stabilizer and EBSS at 4°C expressed as logl0 PFU/mL. The mean virus starting titers were 7.6 x 106 and 8.3 x 106 PFU/mL in EBSS and stabilizer respectively.
TABLE I. Estimated half-lives and Ki values of rHAd-5RG1
Experiment Stabilizer at 4°C Stabilizer at 24-25°C
10
TIME (days)
VIRUS/FECES VIRUS/EBSS
O *
_:"
- ATIVE HUMIDITY 1 REL Al H1} '\ EM,' 'P . 8AI TMEP
6
TIME (DAY S
0
8
24
16
32
40
TIME
(hours)
56
48
64
72
80
Fig. 3. Survival of rHAd5-RG1 virus on stainless steel disks at room temperature expressed in loglo PFU/mL. The virus was suspended in either EBSS or 10%o fox feces. The relative humidity ranged from 45-50%M and the air temperature remained between 24-25°C. The mean
starting titers of infectious virus on the disks were 1.0 x 105 and 3.3 x 105 PFU/mL in feces and EBSS respectively. 8
V *
E
7
11 -
A-
F--
VACCINE/SHADE VACCINE/ SUN
Il
-
8
12
X
6
x
c) 0
Fig. 5. Fluctuations in relative humidity, air temperature and bait temperature during the outdoor survival experiment on rHAd5-RG1.
temperature (Table I and Fig. 1). After 72 h, 26Wo and 3Wo of the initial virus remained in EBSS and feces, respectively. The most rapid loss of virus infectivity occurred within the first 8 h after virus deposition. The mechanisms of virus inactivation are not clear but in the case of EBSS may include inactivation of the virus population more sensitive to drying and the effects of increased salt concentration as the inoculum dries. In fecal suspension, enzymatic activity or interaction with other organic or inorganic constituents of the fecal suspension may be superimposed on the drying effects. Tests with one sample of skunk feces deposited on disks have confirmed the results obtained with the fox feces.
4r+ 0
4
'6
20
24
28
32
36
TIME (days)
Fig. 4. Survival of rHAd5-RG1 virus under outdoor conditions expressed as log10 PFU/mL. The virus-containing blister packs were placed in the baits and kept outdoors in October. The mean virus starting titers were 5.7 x 106 and 6.3 x 106 PFU/mL in the shade and sun respectively.
facilities with the possibility of virus spread to cage mates, neighboring animals or human attendants. These experiments therefore examined the stability of the vaccine virus when it was dried onto metal disks. Figure 3 summarizes the results of these experiments; the RH ranged from 45 to 50% and the air temperature remained between 24 and 25°C. The results indicated that the virus survived better in EBSS than in feces, but the rate of virus inactivation was much greater than when the virus was held in liquid suspension at the same Can J Vet Res 1992; 56: 34-40
SURVIVAL OF rHAd5-RGl UNDER OUTDOOR CONDITIONS
These experiments were designed to mimic the conditions to which the vaccine may be exposed in the field before consumption by target animals. The results (Fig. 4) showed that the virus was more stable in the shade than in the sun; with virus PFU after 32 days of 60% and 46%o respectively. The air temperature ranged from 4°C to 16°C during the sampling period (Fig. 5) and the bait temperature was generally about 3°C higher than the air temperature. The relative humidity ranged between 4007o and 1000/o (Fig. 5). The results indicate that the presence of stabilizers did not increase the stability of the virus compared to EMEM (Fig. 6). Except for lactalbumin hydrolysate, the virus titers were lower, compared to the EMEM control. This further confirms the results reported above with the com-
0 32
0 32
0 32 0 32 TIME (days)
0 32
Fig. 6. The effect of peptone, tryptose phosphate broth (Trp P. B), yeast extract (Yeast Ext) and lactalbumin hydrolysate (Lact/Hy) in stabilizing the infectivity of rHAd5-RGI under outdoor conditions.
mercial stabilizer used for the ERA vaccine. DISCUSSION Adenoviruses have been used as vectors for genes from hepatitis B virus
(15), cytomegalovirus (16), vesicular stomatitis virus (14) and the human immunodeficiency virus (17), but none of the above recombinants is intended for direct release into the environment. On the other hand, adeno-rabies recombinants may be distributed outdoors in targeted areas. It was, therefore, considered essential to determine how long the recombinant virus could retain its infectivity under outdoor conditions. MRC-5-grown vaccine seed stock of rHAd5-RGl was used in all the experiments. The stability of the rHAd5-RG1 virus in liquid suspension at 4°C and room temperature, with or without a stabilizer, suggests that this recombinant virus would be a suitable vaccine virus from this point of view (18). Refrigeration would be adequate for vaccine storage over a period of a few months, and at ambient temperatures up to 24-25°C, the half-life of the vaccine would be several weeks. However, further work is needed to determine the minimum level of infectious virus necessary for efficient immunization of the wildlife targets. The recombinant tested in this study also proved to be more stable under outdoor conditions than the stabilizercontaining attenuated rabies virus currently being used as a vaccine for
31
wildlife (19). Addition of various organic compounds to the recombinant virus suspension failed to increase its stability. This suggests that rHAd5RG 1 vaccine could be prepared without any stabilizer, thereby making it simpler and perhaps cheaper to produce. The commercial stabilizer used with the attenuated rabies vaccine contains yolk from chicken eggs; such yolk requires screening to ensure freedom from virus inhibitors and undesirable microorganisms. Mixing the adenovirus with the egg yolk actually resulted in a drop in infectivity. The recombinant virus is excreted in the feces of orally vaccinated animals. Several samples of feces from experimentally-vaccinated foxes and skunks were screened but none had a reasonably high titer of infectious virus. In view of this, cell culture-grown recombinant virus was added to a 1007o suspension of clarified fecal material from a fox and a skunk; higher concentrations of feces tended to be cytotoxic and failure to remove particulates gave erratic results. The rate of loss in virus infectivity was higher in the fecal suspension than the control (Fig. 3). Additional tests with fecal samples kept at a range of RH levels and air temperatures will be required to assess the potential for spread of excreted virus to other animals. Virus naturally shed in feces may be better protected than virus added to fecal matter in suspension. On the other hand, bacteria and fungi were removed from the samples by filtration whereas whole feces would contain large numbers of viable microorganisms which could inactivate viruses contained therein. It was considered important to determine how long the fecallyexcreted virus could remain viable on ponporous inanimate surfaces as a measure of the duration of contamination of animal holding facilities. Stainless steel disks were selected in this study, because an earlier investigation showed no apparent differences in virus survival on these and disks made out of two different types of plastic and glass (20). It is not possible to determine the absolute risk of virus transmission to animal handlers, but the data suggest a relatively rapid loss in virus infectivity upon drying on a surface under the conditions of the 32
test. Many nonenveloped viruses are relatively resistant to a variety of chemical disinfectants (21) and viruses in feces are afforded an even greater degree of protection against disinfection by the organic matter present. It would be useful to determine the efficacy of commonly used disinfectants in decontamination of recombinant virus-contaminated environmental surfaces. Establishment of proper decontamination procedures using environmentally safe chemicals should also assist in satisfying the regulatory concerns on the handling and disposal of rHAd5-RG1 in vaccine production and testing facilities. The outdoor virus survival experiments could be done only once because of the need to coincide such testing with the present vaccine drop season (19). However, it is planned to repeat these experiments in the fall season again and also test the survival of the virus in the spring when a second vaccine drop is contemplated. Has the incorporation of the rabies glycoprotein gene in the adenovirus in some way enhanced or diminished its capacity to survive in the environment? Experiments are now underway to answer this crucial question. If the recombinant virus is found to retain its infectivity longer than the parent adenovirus under outdoor conditions, the release of the recombinant into the environment will require a much more careful assessment from a regulatory view point.
ACKNOWLEDGMENTS We wish to thank Drs. A. Wandeler, K. Charlton, J. Campbell, K. Lawson and L. Prevec for advice and help in this investigation. REFERENCES 1. BLACK JG, LAWSON KF. Sylvatic rabies studies in the silver fox (Vulpes vulpes). Susceptibility and immune response. Can J Comp Med 1970; 34: 309-311. 2. LAWSON KF, JOHNSTON DH, PATTERSON JM, BLACK JG, RHODES AJ, ZALAN E. Immunization of foxes
Vulpes vulpes by the oral and intramuscular routes with inactivated rabies vaccines. Can J Comp Med 1982; 46: 382-385. 3. TOLSON ND, CHARLTON KM, LAWSON KF, CAMPBELL JB, STEWART
RB. Studies of ERA/BHK-21 rabies vaccine in skunks and mice. Can J Vet Res 1988; 52: 58-62. 4. WANDELER Al, CAPT S, KAPPLER A, HAUSER R. Oral immunization of wildlife against rabies: concepts and first field experiment. Rev Infect Dis 1988; 10: S649-S653. 5. NICHOLSON KG, BAUER SP. Enteric inoculation with ERA rabies virus: evaluation of a candidate wildlife vaccine in laboratory rodents. Arch Virol 1981; 67: 51-56. 6. BLANCOU J, KIENY MP, LATHE R, LECOCQ JP, PASTORET PP, SOULEBOT JP, DESMETTRE P. Oral vaccination of the fox against rabies using a live recombinant vaccinia virus. Nature 1986; 322: 373-375. 7. TOLSON ND, CHARLTON KM, STEWART RB, CAMPBELL JB, WIKTOR TJ. Immune response in skunks to a vaccinia virus recombinant expressing the rabies virus glycoprotein. Can J Vet Res 1987; 51: 363-366. 8. PREVEC L, CAMPBELL JB, CHRISTIE BS, BULBECK L, GRAHAM FL. A recombinant human adenovirus against rabies. J Infect Dis 1990; 161: 27-30. 9. CHANOCK RM, LUDWIG W, HEUBNER RJ, CATE TR, CHIU LW. Immunization by selective infection with type 4 adenovirus grown in human diploid tissue culture. J Am Med Assoc 1966; 195: 151-158. 10. GRIFFIN JP, BURTON H, GREENBERG MD, GREAT LAKES III. Live and inactivated adenovirus vaccines: clinical evaluation of efficacy in prevention of acute respiratory disease. Arch Intern Med 1970; 125: 981-986. 11. CHALONER-LARSSON G, CONTRERAS G, FURESZ J, BOUCHER DW, KREPPS D, HUMPHREYS GR, MOHANNA SM. Immunization of Canadian armed forces personnel with live types 4 and 7 adenovirus vaccine. Can J Public Health 1986; 77: 367-370. 12. TOP FH Jr, GROSSMAN RA, BARTELLONI PJ, SEGAL HE, DUDDING BA, RUSSELL PK, BUESCHER EL. Immunization with live types 7 and 4 adenovirus vaccines. 1. Safety, infectivity, antigenicity, and potency of adenovirus type 7 vaccine in humans. J Infect Dis 1971; 124: 148-154. 13. PREVEC L, SCHNEIDER M, ROSENTHAL KL, BELBECK LW, DERBYSHIRE JB, GRAHAM FL. Use of human adenovirus-based vectors for antigen expression in animals. J Gen Virol 1989; 70: 429-434. 14. SCHNEIDER M, GRAHAM FL, PREVEC L. Expression of vesicular stomatitis virus by infectious adenovirus vectors. J Gen Virol 1989; 70: 417-427. 15. MORIN JE, LUBECK MD, BARTON JE, CONLEY AJ, DAVIS AR, HUNG PP. Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters. Proc Natl Acad Sci 1987; 84: 4626-4630. 16. MARSHALL GS, RICCIARDI RP, RANDO RF, PUCK J, GE R, PLOTKIN S, GONCZOL E. An adenovirus recombinant that expresses the human cytomegalovirus major envelope glycoprotein
and induces neutralizing antibodies. J Infect Dis 1990; 162: 1177-1181. 17. DEWAR RL, NATARAJAN V, VASUDEVACHARI MB, SALZMAN NP. Synthesis and processing of human immunodeficiency virus type 1 envelope protein encoded by a recombinant human adenovirus. J Virol 1989; 63: 129-136.
18. BACHMANN P, BRAMWELL RN, FRASER SJ, GILMORE DA, JOHNSTON DH, LAWSON KF, MacINNES CD, MATEJKA FO, MILES HE, PEDDE MA, VOIGT DR. Wild carnivore acceptance of baits for delivery of liquid rabies vaccine. J Wildl Dis 1990; 26: 486-501. 19. LAWSON KF, HERTLER R, CHARLTON KM, CAMPBELL JB, RHODES AJ. Safety and immunogenicity of the ERA strain of rabies virus propagated in a BHK-21 cell line. Can J Vet Res 1989; 53: 438-444.
20. SATTAR SA, LLOYD-EVANS N, SPRINGTHORPE VS. Institutional outbreaks of rotavirus diarrhoea: Potential role of fomites and environmental surfaces. J Hyg (Lond) 1986; 96: 277-289. 21. SPRINGTHORPE VS, SATTAR SA. Chemical disinfection of virus-contaminated surfaces. Critical Rev Env Control 1990; 20: 169-229.
33
on
the Stability of a Human Adenovirus-Rabies Recombinant Vaccine
Kishna K. Kalicharran, V. Susan Springthorpe and Syed A. Sattar
ABSTRACT Human adenovirus type 5 containing the rabies virus glycoprotein gene (rHAd-RG1) has potential for the oral vaccination of animals. The stability of this recombinant was tested indoors and outdoors by measuring the loss in virus infectivity. Under indoor conditions the stability of the recombinant virus was studied in an egg yolkcontaining commercial stabilizer and a simple buffered salt solution (EBSS; Earle's balanced salt solution) at 40C and room temperature (24-25°C). Over 16 days, there was a more rapid loss in virus titer at room temperature than at 40C in both suspending media; however, these differences were slight and may be insignificant when the overall stability of the vaccine is considered. When the virus was mixed with either 10% (w/v) fox or skunk feces or EBSS, placed on stainless steel disks and the disks kept under ambient conditions (air temperature 24-25°C; relative humidity 45-50%o), there was a more rapid decline in virus titer in the fecal suspensions (37o remained after 72 h) than in EBSS (26%o remained after 72 h). When bait-coated blister packs of the vaccine were placed in an outdoor location in the fall (October) season, there was a larger drop in the virus titer for vaccines placed in the sun (547o over 32 days) than for those in the shade (40%o over 32 days). Incorporating proteinaceous stabilizers in the vaccine samples for outdoor study showed virus stability was not enhanced in their presence. This study shows rHAd5-RG1 in EBSS has halflives of 66 days at 4°C and 51 days at room temperature and that it is sufficiently stable outdoors for use as a vaccine in baits for wildlife.
RESUME
eprouves au-dehors n'a pas
augmente
la stabilite du virus. Cette etude montre L'adenovirus humain type 5 conte- que le recombinant rHAd5-RG1 dans nant le gene de la glycoproteine du le EBSS possede des demi-vies de virus de la rage (rHAd5-RG1) possede 66 jours a 4 IC et de 51 jours a la un potentiel pour la vaccination orale temperature de la piece et qu'il est sufdes animaux. La stabilite de ce recom- fisamment stable 'a l'exterieur pour etre binant a ete testee a l'interieur et utilise comme vaccin dans des appats au-dehors en mesurant la perte du pour la faune. (Traduit par Dr Ronald pouvoir infectieux du virus. A l'inte- Magar) rieur, la stabilite du virus recombinant melange, d'une part a un stabilisateur commercial contenant du jaune d'oeuf INTRODUCTION et d'autre part a une simple solution saline tamponnee (solution saline Rabies continues to be a problem in equilibree de Earle EBSS), a ete etudiee Canada, with foxes (Vulpes vulpes) tant a 4 IC qu'a la temperature de la and skunks (Mephitis mephitis) being piece (24-25 IC). Sur une peiode de the major wildlife reservoirs (1-3). The 16 jours, il y avait une perte de titre use of an oral attenuated rabies virus viral plus rapide a la temperature de la vaccine was introduced in Canada piece qu'a 4 IC dans les deux milieux based on its success in fox rabies conde suspension; toutefois, ces dif- trol in certain European countries (4). ferences etaient legeres et pourraient However, the attenuated vaccine is etre non-significatives lorsque la ineffective in skunks (3), shows stabilite generale du vaccin est con- residual pathogenicity for rodents (5) sideree. Lorsque le virus a ete melange and is relatively unstable (2,3). a une suspension (10 No p/v) de feces Research into recombinant vaccines de renard ou de mouffette ainsi qu'au led to the development of a vaccinia EBSS puis a ete place sur des disques virus containing the rabies glycoprod'acier inoxydable, qui ont ete gardes tein gene (6). This vaccine proved ensuite en conditions ambiantes effective in foxes, but failed to (temperature 24-25 IC; humidite immunize skunks when given orally relative (45-50 %), une chute plus (7). Prevec et al (8) were successful in rapide du titre du virus a ete notee dans inserting the rabies glycoprotein gene les suspensions fecales (3 0o restant in the deleted E3 region of human apres 72 h) que dans le EBSS (26 % adenovirus type 5. Adenoviruses have restant apres 72 h). Quand des pla- proved to be relatively innocuous and quettes de vaccin enrobees d'appat ont stable to gastric acid. Adenovirus types ete placees sur une site a l'exterieur en 4 and 7 are administered orally to automne (octobre), il y avait une plus immunize army recruits against acute grande chute du titre viral pour les vac- respiratory disease (9-12); these viruses cins places au soleil (54 %7o sur l'espace can induce high levels of heterologous de 32 jours) que pour ceux places a gene expression in mammalian cells l'ombre (40 sur l'espace de 32 jours). (13-17). L'incorporation de stabilisateurs proAmong the major concerns on the teiques dans les echantillons de vaccins release of recombinant viruses is their
Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K 1 H 8M5. Reprint requests to Dr. S.A. Sattar. Support for this work was provided by a grant from the Ontario Ministry of Natural Resources. Submitted April 2, 1991.
28
Can J Vet Res 1992; 56: 28-33
ability to remain viable in the environment for prolonged periods. On the other hand a recombinant vaccine virus must have a suitable shelf-life and retain its infectivity long enough in the field to successfully immunize the target species. Knowledge of virus survival is, therefore, a prerequisite to planning vaccine production and field trials. In view of this, we studied the stability of the adeno-rabies recombinant under both indoor and field conditions. MATERIALS AND METHODS VIRUS
The adeno-rabies recombinant (rHAd5-RGl) pool used throughout this study was in its third passage in MRC-5 cells and was prepared in Dr. J.B. Campbell's Laboratory at the University of Toronto, Toronto, Ontario. The virus was received through the courtesy of Dr. A. Wandeler, Animal Diseases Research Institue (ADRI), Agriculture Canada, Nepean, Ontario. CELLS
The Vero line of African green monkey kidney cells was used throughout this study and a seed culture of these cells was provided by Dr. Campbell. The cells were grown in Eagle's minimal essential medium (EMEM; Gibco Laboratories, Grand Island, New York) supplemented with 507o fetal bovine serum (Cell Culture Laboratories, Cleveland, Ohio), 50 jtg/mL gentamicin sulfate (Cidomycin; Roussel, Montreal, Quebec), 200 mM glutamine (Gibco) and 0.22507o (w/v) sodium bicarbonate (BDH Chemicals, Toronto, Ontario). The cells were maintained in the same medium with only 207o serum.
made in Earle's balanced salt solution (EBSS; Gibco). For each dilution, three wells were inoculated with 0.1 mL of inoculum each. The cell control wells were exposed to an equivalent amount of EBSS. The plates were rotated gently to redistribute the inoculum over the monolayers before incubating them at 37°C for 60 min in a 57o CO2 atmosphere. Redistribution of the inoculum was carried out every 10 min during the virus adsorption period. Each well then received 2.0 mL of an overlay containing EMEM with 200 mM glutamine (Gibco), 10 mM magnesium chloride (Sigma Chemical, St Louis, Missouri), 50 /tg/mL gentamicin, 2 ,ug/mL amphotericin B (Fungizone; Gibco), 2% serum, 0.225Wo (w/v) sodium bicarbonate and 0.75% (w/v) agarose type II (Sigma). The plates were kept at room temperature to allow the medium to solidify, sealed individually in laminated plastic bags (Dazey Corp., Industrial Airport, Kansas) and incubated at 37°C for five to six days. Once plaques were readily visible under an inverted microscope, 2.0 mL of a 3.7% (v/v) formaldehyde (Fisher Scientific, Ottawa, Ontario) solution in normal saline were added to each well to fix the monolayers and to inactivate the virus. After holding the plates overnight at room temperature, the overlay was removed with running tap water. Each monolayer was then stained for 5 min with 0.5 mL of a 1 07o aqueous solution of crystal violet. The stain was washed off in tap water, the plates dried and the plaques counted. The virus infectivity titer is expressed as plaque forming units (PFU)/mL. The data are plotted with the means and standard deviations. The parameters were calculated and graphed by the SigmaPlotTM software (Jandel Scientific, Corte Madera, California).
VIRUS TITRATION
For virus plaque assay, monolayers were prepared by seeding approximately 1 x 105 cells into each well of a twelve-well cell culture plate (Costar, Cambridge, Massachusetts) in 2.0 mL of the growth medium. The seeded plates were incubated at 37°C overnight in a 5Wo CO2 atmosphere. Serial tenfold dilutions (10-3, 10-4, 10- 5) of the virus samples to be titrated were
VACCINE
Polystyrene blister packs and a bait mixture were provided by Dr. K. Lawson (Connaught Laboratories, Willowdale, Ontario) and both of these are currently being used in the field trials of the attenuated (ERA) rabies virus vaccine (18). One part of the stock virus was mixed with one part of either EMEM without serum or the
chemical stabilizer under test and 2.0 mL of the mixture was injected into each blister pack using a 2.0 mL syringe with a 26 gauge needle (Becton, Dickinson & Co., Mississauga, Ontario); the needle puncture was sealed with a plastic glue. For embedding the blister packs in the bait, the bait mixture, contained in a 250 mL glass conical flask, was melted in a water bath at 70°C. Approximately 18 g was then poured into each one of the clear plastic moulds (4.0 x 4.0 x 2.5 cm). While the bait mixture was still soft, the blister pack with the virus was inserted into it with an applicator stick, making sure that the entire surface of the blister pack was completely and evenly covered with the bait. To monitor the vaccine temperature, 16-gauge hypodermic probes (Product No. Hyp3-16-1 1/2-K-V-48RP; Omega Corp., Stamford, Connecticut) containing type K thermocouples were inserted into two of the blister packs before embedding them in the bait. The bait temperature was recorded by plugging the thermocouple probes into a portable thermometer (Omega). INDOOR SURVIVAL EXPERIMENTS
The stock virus was mixed in a 1:1 ratio with an egg yolk-containing commercial stabilizer provided by Dr. Lawson. This stabilizer is used with the attenuated (ERA) rabies virus vaccine (19). The virus-stabilizer mixture was vortexed for 10 s and 200 AiL quantities were aliquoted into 2.0 mL sterile plastic vials (Sarstedt, St. Laurent, Quebec). As virus control, the stock virus was mixed with an equal volume of EBSS. Immediately, 100 ML samples of the virus-EBSS and the virus-stabilizer mixtures were removed for titration (zero min control). Five vials of each of the test mixtures were placed at room temperature (24-25°C) in a plastic box; a corresponding set was placed at 4°C. One vial of each mixture was removed at the end of 2, 4, 8, 12 and 16 days. The samples were stored at - 80°C and all of them were titrated at the end of the experiment. This experiment was repeated three times. To determine the survival of the recombinant virus on nonporous inanimate surfaces under indoor conditions, it was suspended in fecal 29
samples from an unvaccinated fox and an unvaccinated skunk kept at the ADRI. The fecal samples were prepared as a 1007o (w/v) suspension in normal saline, clarified by centrifugation at 1,000 x g for 5 min and passed through a 47 mm diameter membrane filter (Nalge Co, Rochester, New York) with a pore diameter of 0.22 zm to remove bacterial and fungal contamination. The filtrates were found to be noncytotoxic to Vero cells and free from any indigenous viruses that could produce cytopathology in these cells. A 1:10 dilution of the virus was prepared in either EBSS or the fecal filtrates and, using a positive displacement pipette (Gilson, Villiers le Bel, France), 10 ,L of virus suspension were placed separately at the center of each stainless steel disk (1 cm in diameter). The disks were punched out of locally purchased sheets (0.75 mm thick) of #4 polished stainless steel. A 12-well plastic cell culture plate, with one virus-inoculated disk per well, was held uncovered in a laminar flow hood. The air temperature and relative humidity were monitored throughout the experiment using a recording hygrothermograph (Cole-Parmer, Chicago, Illinois). One disk each of the two different virus suspensions was removed at the end of 1, 2, 4, 6, 12, 24, 48 and 72 h and placed in a tube containing 1.0 mL EBSS. The tubes with the disks were sonicated for 10 min in an ultrasonic bath (Bransonic Ultrasonic Corp, Danbury, Connecticut) to assist in the elution of the virus from the disks. The eluates were then titrated. This experiment was performed twice with fox feces and once with skunk feces. OUTDOOR SURVIVAL EXPERIMENTS
Baits with the attenuated rabies virus vaccine are currently dropped in the fall season in Eastern Ontario (18). Therefore, the survival of rHAd5-RGI in baits was also tested at this time (October) of the year under outdoor conditions. Each blister pack containing 2.0 mL of the recombinant virus (grown in MRC-5 cells) was embedded in the bait. The baits were placed outdoors, with one set of samples exposed to sunlight and another placed in the shade; a thermocouple-fitted vaccine pack
30
was
included
in
each
set
to mea-
8 bait temperature. Outdoor air temperature and relative humidity were also monitored with a recording hygrothermograph throughout the sampling period. Samples were withdrawn at 1, 2, 4, 8, 16 and 32 days and titrated. 6 A set of samples containing the following organic materials was also prepared in order to determine their virus 5stabilizing potential: peptone (Oxoid Ltd., Basingstoke, Hampshire, England), lactalbumin hydrolysate 4 0 2 4 6 8 10 12 14 16 18 (Gibco), yeast extract (Difco LaboraTIME (days) tories, Detroit, Michigan) and tryptose phosphate broth (Difco). All the above Fig. 1. Comparison of the stability of were prepared as 10% (w/v) solutions HAd5-RG1 virus in the commercial stabilizer EBSS at room temperature expressed as in phosphate buffered saline (pH 7.2) and log10 PFU/mL. The mean virus starting titers and each was mixed separately with the were 4.5 x 106 and 4.6 x 106 PFU/mL in virus in a 1:1 ratio. The mixtures were EBSS and stabilizer respectively. placed in blister packs and inserted into 8, baits as described above. After 32 days * VIRUS/STABILIZER v VIRUS/EBSS of exposure to the sun outdoors, the contents were titrated.
sure
U)
0
7
E 7 :D
RESULTS c.
SURVIVAL OF rHAd5-RGl UNDER INDOOR CONDITIONS
These experiments were designed to give an indication of the shelf-life of the rHAd5-RG1 vaccine when it is stored either at room temperature (Fig. 1) or under refrigeration (Fig. 2). As can be seen from Table I, the estimated half-lives of the stabilizercontaining virus held at 4°C and at room temperature were 88 and 64 days, respectively. The rate of loss of the EBSS-containing virus was somewhat greater, with the estimated half-lives at 4°C and room temperature being 66 and 51 days, respectively. The results thus indicate that the virus may be sufficiently stable in EBSS, and for the remainder of the indoor studies, EBSS was used as the suspending medium.
6
U)
0
5
4
0
4
2
6
8
14
16
Excretion of the virus in the feces of experimentally-vaccinated animals could contaminate animal holding
on
storage in liquid
coefficientb 0.90 0.83
Half-life (days) 88 64
EBSS at 40C 0.0104 0.86 66 EBSS at 24-25°C 0.84 0.0135 51 aKi Loss in logl0 virus PFU/day bCorrelation coefficient refers to the measure of the linear association between two variables =
18
SURVIVAL OF rHAd5-RGl ON ENVIRONMENTAL SURFACES
Correlation Kia 0.0078 0.0106
12
Fig. 2. Comparison of the stability of rHAd5-RG1 virus in the commercial stabilizer and EBSS at 4°C expressed as logl0 PFU/mL. The mean virus starting titers were 7.6 x 106 and 8.3 x 106 PFU/mL in EBSS and stabilizer respectively.
TABLE I. Estimated half-lives and Ki values of rHAd-5RG1
Experiment Stabilizer at 4°C Stabilizer at 24-25°C
10
TIME (days)
VIRUS/FECES VIRUS/EBSS
O *
_:"
- ATIVE HUMIDITY 1 REL Al H1} '\ EM,' 'P . 8AI TMEP
6
TIME (DAY S
0
8
24
16
32
40
TIME
(hours)
56
48
64
72
80
Fig. 3. Survival of rHAd5-RG1 virus on stainless steel disks at room temperature expressed in loglo PFU/mL. The virus was suspended in either EBSS or 10%o fox feces. The relative humidity ranged from 45-50%M and the air temperature remained between 24-25°C. The mean
starting titers of infectious virus on the disks were 1.0 x 105 and 3.3 x 105 PFU/mL in feces and EBSS respectively. 8
V *
E
7
11 -
A-
F--
VACCINE/SHADE VACCINE/ SUN
Il
-
8
12
X
6
x
c) 0
Fig. 5. Fluctuations in relative humidity, air temperature and bait temperature during the outdoor survival experiment on rHAd5-RG1.
temperature (Table I and Fig. 1). After 72 h, 26Wo and 3Wo of the initial virus remained in EBSS and feces, respectively. The most rapid loss of virus infectivity occurred within the first 8 h after virus deposition. The mechanisms of virus inactivation are not clear but in the case of EBSS may include inactivation of the virus population more sensitive to drying and the effects of increased salt concentration as the inoculum dries. In fecal suspension, enzymatic activity or interaction with other organic or inorganic constituents of the fecal suspension may be superimposed on the drying effects. Tests with one sample of skunk feces deposited on disks have confirmed the results obtained with the fox feces.
4r+ 0
4
'6
20
24
28
32
36
TIME (days)
Fig. 4. Survival of rHAd5-RG1 virus under outdoor conditions expressed as log10 PFU/mL. The virus-containing blister packs were placed in the baits and kept outdoors in October. The mean virus starting titers were 5.7 x 106 and 6.3 x 106 PFU/mL in the shade and sun respectively.
facilities with the possibility of virus spread to cage mates, neighboring animals or human attendants. These experiments therefore examined the stability of the vaccine virus when it was dried onto metal disks. Figure 3 summarizes the results of these experiments; the RH ranged from 45 to 50% and the air temperature remained between 24 and 25°C. The results indicated that the virus survived better in EBSS than in feces, but the rate of virus inactivation was much greater than when the virus was held in liquid suspension at the same Can J Vet Res 1992; 56: 34-40
SURVIVAL OF rHAd5-RGl UNDER OUTDOOR CONDITIONS
These experiments were designed to mimic the conditions to which the vaccine may be exposed in the field before consumption by target animals. The results (Fig. 4) showed that the virus was more stable in the shade than in the sun; with virus PFU after 32 days of 60% and 46%o respectively. The air temperature ranged from 4°C to 16°C during the sampling period (Fig. 5) and the bait temperature was generally about 3°C higher than the air temperature. The relative humidity ranged between 4007o and 1000/o (Fig. 5). The results indicate that the presence of stabilizers did not increase the stability of the virus compared to EMEM (Fig. 6). Except for lactalbumin hydrolysate, the virus titers were lower, compared to the EMEM control. This further confirms the results reported above with the com-
0 32
0 32
0 32 0 32 TIME (days)
0 32
Fig. 6. The effect of peptone, tryptose phosphate broth (Trp P. B), yeast extract (Yeast Ext) and lactalbumin hydrolysate (Lact/Hy) in stabilizing the infectivity of rHAd5-RGI under outdoor conditions.
mercial stabilizer used for the ERA vaccine. DISCUSSION Adenoviruses have been used as vectors for genes from hepatitis B virus
(15), cytomegalovirus (16), vesicular stomatitis virus (14) and the human immunodeficiency virus (17), but none of the above recombinants is intended for direct release into the environment. On the other hand, adeno-rabies recombinants may be distributed outdoors in targeted areas. It was, therefore, considered essential to determine how long the recombinant virus could retain its infectivity under outdoor conditions. MRC-5-grown vaccine seed stock of rHAd5-RGl was used in all the experiments. The stability of the rHAd5-RG1 virus in liquid suspension at 4°C and room temperature, with or without a stabilizer, suggests that this recombinant virus would be a suitable vaccine virus from this point of view (18). Refrigeration would be adequate for vaccine storage over a period of a few months, and at ambient temperatures up to 24-25°C, the half-life of the vaccine would be several weeks. However, further work is needed to determine the minimum level of infectious virus necessary for efficient immunization of the wildlife targets. The recombinant tested in this study also proved to be more stable under outdoor conditions than the stabilizercontaining attenuated rabies virus currently being used as a vaccine for
31
wildlife (19). Addition of various organic compounds to the recombinant virus suspension failed to increase its stability. This suggests that rHAd5RG 1 vaccine could be prepared without any stabilizer, thereby making it simpler and perhaps cheaper to produce. The commercial stabilizer used with the attenuated rabies vaccine contains yolk from chicken eggs; such yolk requires screening to ensure freedom from virus inhibitors and undesirable microorganisms. Mixing the adenovirus with the egg yolk actually resulted in a drop in infectivity. The recombinant virus is excreted in the feces of orally vaccinated animals. Several samples of feces from experimentally-vaccinated foxes and skunks were screened but none had a reasonably high titer of infectious virus. In view of this, cell culture-grown recombinant virus was added to a 1007o suspension of clarified fecal material from a fox and a skunk; higher concentrations of feces tended to be cytotoxic and failure to remove particulates gave erratic results. The rate of loss in virus infectivity was higher in the fecal suspension than the control (Fig. 3). Additional tests with fecal samples kept at a range of RH levels and air temperatures will be required to assess the potential for spread of excreted virus to other animals. Virus naturally shed in feces may be better protected than virus added to fecal matter in suspension. On the other hand, bacteria and fungi were removed from the samples by filtration whereas whole feces would contain large numbers of viable microorganisms which could inactivate viruses contained therein. It was considered important to determine how long the fecallyexcreted virus could remain viable on ponporous inanimate surfaces as a measure of the duration of contamination of animal holding facilities. Stainless steel disks were selected in this study, because an earlier investigation showed no apparent differences in virus survival on these and disks made out of two different types of plastic and glass (20). It is not possible to determine the absolute risk of virus transmission to animal handlers, but the data suggest a relatively rapid loss in virus infectivity upon drying on a surface under the conditions of the 32
test. Many nonenveloped viruses are relatively resistant to a variety of chemical disinfectants (21) and viruses in feces are afforded an even greater degree of protection against disinfection by the organic matter present. It would be useful to determine the efficacy of commonly used disinfectants in decontamination of recombinant virus-contaminated environmental surfaces. Establishment of proper decontamination procedures using environmentally safe chemicals should also assist in satisfying the regulatory concerns on the handling and disposal of rHAd5-RG1 in vaccine production and testing facilities. The outdoor virus survival experiments could be done only once because of the need to coincide such testing with the present vaccine drop season (19). However, it is planned to repeat these experiments in the fall season again and also test the survival of the virus in the spring when a second vaccine drop is contemplated. Has the incorporation of the rabies glycoprotein gene in the adenovirus in some way enhanced or diminished its capacity to survive in the environment? Experiments are now underway to answer this crucial question. If the recombinant virus is found to retain its infectivity longer than the parent adenovirus under outdoor conditions, the release of the recombinant into the environment will require a much more careful assessment from a regulatory view point.
ACKNOWLEDGMENTS We wish to thank Drs. A. Wandeler, K. Charlton, J. Campbell, K. Lawson and L. Prevec for advice and help in this investigation. REFERENCES 1. BLACK JG, LAWSON KF. Sylvatic rabies studies in the silver fox (Vulpes vulpes). Susceptibility and immune response. Can J Comp Med 1970; 34: 309-311. 2. LAWSON KF, JOHNSTON DH, PATTERSON JM, BLACK JG, RHODES AJ, ZALAN E. Immunization of foxes
Vulpes vulpes by the oral and intramuscular routes with inactivated rabies vaccines. Can J Comp Med 1982; 46: 382-385. 3. TOLSON ND, CHARLTON KM, LAWSON KF, CAMPBELL JB, STEWART
RB. Studies of ERA/BHK-21 rabies vaccine in skunks and mice. Can J Vet Res 1988; 52: 58-62. 4. WANDELER Al, CAPT S, KAPPLER A, HAUSER R. Oral immunization of wildlife against rabies: concepts and first field experiment. Rev Infect Dis 1988; 10: S649-S653. 5. NICHOLSON KG, BAUER SP. Enteric inoculation with ERA rabies virus: evaluation of a candidate wildlife vaccine in laboratory rodents. Arch Virol 1981; 67: 51-56. 6. BLANCOU J, KIENY MP, LATHE R, LECOCQ JP, PASTORET PP, SOULEBOT JP, DESMETTRE P. Oral vaccination of the fox against rabies using a live recombinant vaccinia virus. Nature 1986; 322: 373-375. 7. TOLSON ND, CHARLTON KM, STEWART RB, CAMPBELL JB, WIKTOR TJ. Immune response in skunks to a vaccinia virus recombinant expressing the rabies virus glycoprotein. Can J Vet Res 1987; 51: 363-366. 8. PREVEC L, CAMPBELL JB, CHRISTIE BS, BULBECK L, GRAHAM FL. A recombinant human adenovirus against rabies. J Infect Dis 1990; 161: 27-30. 9. CHANOCK RM, LUDWIG W, HEUBNER RJ, CATE TR, CHIU LW. Immunization by selective infection with type 4 adenovirus grown in human diploid tissue culture. J Am Med Assoc 1966; 195: 151-158. 10. GRIFFIN JP, BURTON H, GREENBERG MD, GREAT LAKES III. Live and inactivated adenovirus vaccines: clinical evaluation of efficacy in prevention of acute respiratory disease. Arch Intern Med 1970; 125: 981-986. 11. CHALONER-LARSSON G, CONTRERAS G, FURESZ J, BOUCHER DW, KREPPS D, HUMPHREYS GR, MOHANNA SM. Immunization of Canadian armed forces personnel with live types 4 and 7 adenovirus vaccine. Can J Public Health 1986; 77: 367-370. 12. TOP FH Jr, GROSSMAN RA, BARTELLONI PJ, SEGAL HE, DUDDING BA, RUSSELL PK, BUESCHER EL. Immunization with live types 7 and 4 adenovirus vaccines. 1. Safety, infectivity, antigenicity, and potency of adenovirus type 7 vaccine in humans. J Infect Dis 1971; 124: 148-154. 13. PREVEC L, SCHNEIDER M, ROSENTHAL KL, BELBECK LW, DERBYSHIRE JB, GRAHAM FL. Use of human adenovirus-based vectors for antigen expression in animals. J Gen Virol 1989; 70: 429-434. 14. SCHNEIDER M, GRAHAM FL, PREVEC L. Expression of vesicular stomatitis virus by infectious adenovirus vectors. J Gen Virol 1989; 70: 417-427. 15. MORIN JE, LUBECK MD, BARTON JE, CONLEY AJ, DAVIS AR, HUNG PP. Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters. Proc Natl Acad Sci 1987; 84: 4626-4630. 16. MARSHALL GS, RICCIARDI RP, RANDO RF, PUCK J, GE R, PLOTKIN S, GONCZOL E. An adenovirus recombinant that expresses the human cytomegalovirus major envelope glycoprotein
and induces neutralizing antibodies. J Infect Dis 1990; 162: 1177-1181. 17. DEWAR RL, NATARAJAN V, VASUDEVACHARI MB, SALZMAN NP. Synthesis and processing of human immunodeficiency virus type 1 envelope protein encoded by a recombinant human adenovirus. J Virol 1989; 63: 129-136.
18. BACHMANN P, BRAMWELL RN, FRASER SJ, GILMORE DA, JOHNSTON DH, LAWSON KF, MacINNES CD, MATEJKA FO, MILES HE, PEDDE MA, VOIGT DR. Wild carnivore acceptance of baits for delivery of liquid rabies vaccine. J Wildl Dis 1990; 26: 486-501. 19. LAWSON KF, HERTLER R, CHARLTON KM, CAMPBELL JB, RHODES AJ. Safety and immunogenicity of the ERA strain of rabies virus propagated in a BHK-21 cell line. Can J Vet Res 1989; 53: 438-444.
20. SATTAR SA, LLOYD-EVANS N, SPRINGTHORPE VS. Institutional outbreaks of rotavirus diarrhoea: Potential role of fomites and environmental surfaces. J Hyg (Lond) 1986; 96: 277-289. 21. SPRINGTHORPE VS, SATTAR SA. Chemical disinfection of virus-contaminated surfaces. Critical Rev Env Control 1990; 20: 169-229.
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