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A Recombinant Human Adenovirus Vaccine against Rabies Ludvik Prevec, James B. Campbell, Brian S. Christie, Larry Belbeck, and Frank L. Graham

From the Departments of Biology and Pathology, McMaster University, Hamilton, and the Department of Microbiology, University of Toronto, Ontario, Canada

It is estimated that >20,000 people die annually from rabies virus infection, mainly in southeast Asia [1]. Even in areas where the incidence of human deaths is negligible, there is considerable economic loss from the deaths of domestic animals and distress to the hundreds of thousands of individuals vaccinated [1]. Infected wildlife constitute a major reservoir of rabies [2], and while currently available vaccines for humans and domestic animals have proven safe and effective, economic and practical limitations preclude the use of these vaccines for large-scale disease control programs [2]. As an alternative, recombinant virus vaccines, in which the relevant rabies antigen is carried and expressed in an infectious vector virus, hold considerable potential. Vaccinia virus vectors expressing the rabies .glycoprotein antigen are effective in inducing an immune response and in protecting animals of several species against lethal rabies challenge [2-6]. Human adenoviruses also merit consideration as vectors since they possess properties, such as stability and the ability to infect by the oral route, that make them particularly useful vectors for development of vaccines for wildlife rabies [7]. Adenovirus vectors have been constructed that express various foreign glycoproteins including those of herpes virus [8], hepatitis B virus [9], human immunodeficiency virus type 1 (HIV-l) [10], and vesicular stomatitis virus (VSV) [11]. The VSV-carrying adenovirus recombinants are effective in inducing neutralizing antibodies to VSV glycoprotein in mice, dogs, pigs, and cows [12] and a vector expressing

Received 8 April 1989; revised 10 August 1989. Supported by a Strategic Grant from the Natural Science and Engineering Research Council of Canada (to F. L. G. and L. P.) and by the Ontario Ministry of Natural Resources (1. B. C.). F. L. G. is a Terry Fox Research Scientist of the National Cancer Institute of Canada. Reprints and correspondence: Dr. L. Prevec, Department of Biology, LSB429, McMaster University, Hamilton, Ontario, Canada L8S 4K1. The Journal of Infectious Diseases 1990;161:27-30 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6101-0006$01.00

a herpes virus glycoprotein has induced neutralizing antibodies and protected against herpes in mice [13].

Materials and Methods Cells and virus. Monolayer cultures of cells were maintained in F-ll medium while suspension cultures were grown in Joklik's modified medium. Media were supplemented with 10% newborn calf serum for cell growth and 5 % newborn calf serum for virusinfected cultures. Ad5 wild-type virus and recombinants were titrated on 293 cells [14] and recombinant viruses were grown in KB cells in suspension culture. All cell culture was carried out in incubators at 37°C, 100% humidity, and an atmosphere of 5 % CO 2 • Adenoviruses were purified by CsCI banding as described by Graham and VanDer Eb [15]with minor modifications. For twice banding of recombinants, the material removed from the first gradient was readjusted to the appropriate volume ofCsCI solution at density 1.34 and the banding repeated. All purified virus was then exhaustively dialyzed against 10 mM Tris-HCI (pH 8.0). Rescue of rabies gene into adenovirus type 5. DNA from wildtype adenovirus type 5 was extracted from gradient-purified virus as described elsewhere [16]. After complete digestion with EcoRI, this DNA was transfected into 293 cells using the calcium phosphate precipitation method of Graham and Van Der Eb [15] along with plasmid pBCRGI (figure 1). The resultant plaques were picked and the viral DNA in each screened by restriction analysis. One recombinant with the expected HindIII restriction pattern and designated AdRGI was selected for further study. The virus was plaque-purified by two successive plaque titrations on 293 cells and then grown in HeLa or KB cells in culture. Immunization ofanimals with the adenovirus-rabies recombinant. Dogs received vaccine in 0.5-ml doses. Primary doses were given by the intranasal or subcutaneous route; in two instances a second subcutaneous dose was given 2 weeks later. Serum antibody titers were measured at 2 and 4 weeks after primary vaccination. Mice (3- to 5-week-old female Swiss; Charles River) received 0.1 ml of inocula by either the intraperitoneal or oral route. Oral administration was performed under light flurothane anesthesia using microtubing fitted over a 23-gauge needle attached to a tuberculin syringe. Tubing was inserted f'\Jl cm past the pharynx. Mice were bled via the tail, and serum samples were assayed individually. Antibody titers

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Rabies continues to be a serious problem in both developed and developing nations due to the reservoir of rabies virus in wildlife vectors. The control and worldwide eradication of rabies depends on the development ofsafe, effective, and economical vaccines that might be used in preexposure vaccination programs for humans and animals. To this end an infectious human adenovirus type 5 recombinant virus that contains the rabies glycoprotein gene, and which may serve as the prototype for a new class of vaccines against rabies, was constructed and tested. This recombinant, when administered by either the parenteral or oronasal route, was highly effective in eliciting good levels of rabies-neutralizing antibodies in the sera of dogs and mice. Mice immunized by the recombinant virus were protected from lethal intracerebral challenge with rabies virus.

Prevec et ale

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A.

were expressed in fluorescent focus inhibition microtiter units (l unit was the highest twofold dilution of serum inhibiting replication of ERA strain rabies challenge virus by at least 50 %, as measured by fluorescent focus formation [20]). All sera were heat-inactivated at 56°C for 30 min before assay.

8

~ E

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1. Nde I site converted to Xbal. 2. Bam HI to EcoRI replaced by Xbal. 3. Hind III to Hpa I replaced with polyllnker containing Hind III, Bam HI, EcoRI, 8mal, Xhol.

Results E

EcoRI 8IImHI

~y_'

pFG dX1 (AdS· 59.5% to 100%) 6E3 78.5% to 84.3%

E

pBCRG

Xl

B.

X

raZZ22M220m -

......--

AdRG1 59.5% 0%

Ad5 EcoRI A FRAGMENT

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100%

.r

IE I 75.9%

Figure 1. Construction of AdRGl. A, In preparation for insertion of viral genes between an SV40 early promoter and the SV40 poly A addition signal, the plasmid pSV2neo [17] was modified through conversion of its single NdeI (N) site to an XbaI (X) site, replacement of the BamHI (B) to EcoRI (E) fragment by an XbaI linker and finally deletion of all the neo gene (stippled) and some SV40 sequences by replacing the HindllI to HpaI fragment with a synthetic polylinker that contained the sites for HindIII (H), BamHI, EcoRI, SmaI (S), and XhoI (Xh) enzymes. The resultant plasmid (pSV2X3) has the SV40 promoter sequence (solid) and polyA addition sequence (open) separated by the polylinker sequence that can be used to insert appropriate genes. This cassette is bounded by XbaI sites. The rabies (ERA strain) glycoprotein gene (modified gene described in [18]) was removed from pBR322 by digestion with EcoR! and BamHI and after filling in blunt ends with Klenow polymerase, was ligated into the SmaI site of pSV2X3. One resultant plasmid, pSV2X3RG, which had the rabies gene in the orientation that would allow transcription from the SV40 promoter, was used as the source of the XbaI fragment that was isolated from agarose and inserted into the XbaI site of plasmid pFGdXl [19], which contains the right end sequences of human adenovirus type 5 from the BamHI site at 59.5%-100%, except for theXbaI D fragment from 78.5%-84.3%. One resultant plasmid, pBCRG, with the rabies gene oriented parallel to the direction of transcription of the adenovirus E3 promoter, was selected for growth and purification. Infectious recombinant vi-

rus was rescued by cotransfection of this plasmid and EcoRI digested adenovirus DNA as described in Methods and a recombinant (B) with the desired insert of the rabies gene was identified and purified by two further plaque isolations. This virus, AdRG1, was used in subsequent studies. A PvuII site in pSV2neo is denoted by P.

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x

We inserted the structural gene of the glycoprotein from the ERA strain of rabies virus between the promoter and polyA addition site of the early region of SV40 and then placed this cassette into the deleted E3 region of human adenovirus type 5 as shown in figure 1. The final recombinant adenovirus (AdRG1) contained the rabies glycoprotein gene in the same orientation as the E3 genes it replaced. We and others have previously shown that, while foreign control elements may be active, inserts of this type are expressed principally from transcripts that originate at either the E3 promoter or the major late promoter of adenovirus [8-11]. Although the role, if any, of the SV40 promoter and poly A addition sequences in AdRGl remains to be determined, we have consistently found that genes inserted into the Ad5 genome in this format express the insert, frequently at high levels. Recombinant AdRGl virus, which was first purified by twice banding on CsCI to eliminate any carryover of antigen from the infected cell culture, was administered to mice and dogs. .As indicated in table 1, mice were inoculated orally or intraperitoneally while dogs were given virus by either the subcutaneous or intranasal route. Pre- and postinoculation serum samples were taken and coded before analysis, and rabies neutralizing antibody titers were determined by the fluorescence inhibition microtest [20]. Both species responded well to the parenterally administered vaccine. In mice responding to the oral vaccine route, seroconversion may be effected by either the buccal or intestinal routes, since some of the inoculum in individual animals may have been regurgitated or may have been deposited in the buccal cavity during withdrawal of the tubing. Particularly significant is the observation that virus given to dogs by the intranasal route was highly effective in inducing neutralizing antibodies. This is in keeping with our previous observations using an adenovirus vector expressing the VSV glycoprotein [12]. It is noteworthy that Ad5 replicates relatively poorly in mouse cells and cannot produce infectious virus in canine cells [12]. Nonetheless the AdRGl vector was clearly capable of infecting dogs and mice and of expressing the rabies glycoprotein to an extent sufficient to induce a good immune response. Since we previously showed that an adenovirus-VSV

Recombinant Adenovirus Rabies Vaccine

lID 1990;161 (January)

Table 1. Rabies neutralizing antibodies after administration of

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HUMAN ADENOVIRUS-RABIES RECOMBINANT (lP IN MICE)

AdRG1 vaccine. 100

Dose (pfu)

Animal

Vaccination route

80

Antibody titer by week

10 7 PFU

Survivors/total (Mean Ab titelrl\

60

1st

2nd

0

2

4*

'I'-

12/12 (3490)

40

Dog 1 Dog 2 Dog 3 Dog 4 Mice Mice Mice Mice

5 5 5 5 1 1 1 1

X X X X

x x x x

105 105 105 105 107 106 107 106

SC SC IN IN Oral Oral IP IP

SC NI SC NI NI NI NI NI

Neg Neg Neg Neg Neg Neg Neg Neg

256 1024 256 256

512 1024 512 4096 1124 424 2925 1312

20 0

(4/10) (4/10) (l0/10) (8/10)

10 6 PFU

12/16 (1432)

40 20 0

40

10 5

'I'20 0

recombinant was also effective in inducing VSV antibodies in calves and pigs, these results suggest that the AdRGl virus and similar adenovirus-based vectors may be effective immunogens in a wide range of animal species. After this initial success, we conducted a more extensive investigation of the efficacy of the AdRGl recombinant vaccine in mice. In these studies, mice in groups were given a single intraperitoneal dose of the recombinant, with titers of lQ4-107 plaque-forming units (pfu), and were later challenged intracerebrally with the ERA strain of rabies (Connaught Laboratories, Willowdale, Ontario). The results (figure 2) show that injection of AdRGl at 107 pfu/mouse induced high titers of neutralizing antibody in ~ll animals. Lower doses, in 10-folddilutions, produced responses in decreasing proportions of animals and also induced correspondingly reduced titers of antibodies in the responding animals. Although 100% of control (unimmunized) mice died after the rabies challenge, any animals that had developed even a minimal detectable antibody response after vaccine administration showed no signs of rabies infection during the 21-day observation period. Three mice that had been given the lower doses of AdRGl and in which no neutralizing antibodies were detected also survived rabies challenge, suggesting that subdetectable levels of antibody provided protection or that protection had been effected by stimulation of some other defense mechanism(s). This protective stimulation of the immune system by AdRGl was rabiesspecific since a group of 15 mice inoculated with adenovirus type 5 alone remained susceptible to challenge with rabies 2 weeks later (data not shown).

Discussion These experiments clearly demonstrate the efficacy of AdRGl in inducing rabies antibody response in mice and in protecting the animals against subsequent rabies infection. From these results and other work [9, 12, 13], it is clear that

100

10 4

80 60

'I'40 20 0 100 80

D

SURVIVORS

60



DEAD

0/13

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40

following IC challenge with rabies (ERA; 20TCID so)

'I'20

o

Neg. 16-32 64-128 256-512

~1024

RABIES NEUTRALIZING ANTIBODY rrrs RS (FIMT units; 3 weeks post-vacclnatlon)

Figure 2. AdRG1 protection of mice against rabies challenge. In each of two experiments, groups of 5 to 10 mice received intraperitoneal (IP) inoculations (0.1 ml) of CsCI gradient purified AdRG1, ranging in titer from 107 to 1Q4 plaque-forming units/OJ mI. Control groups received 0.1 mI of diluent alone (PBS + 2 % fetal calf serum). At 3 weeks, animals were bled via tail vein (0.2-0.3 mI/animal), serum was collected, heat-inactivated, and assayed for rabies-neutralizing antibodies by fluorescent focus inhibition microtiter (FIMT) [20]. Three days after collection of serum, the animals were challenged intracerebrally with 0.03 mI ERA rabies (20 TCIDso/dose) and observed for 21 days. Animals dying within 3 days of challenge (Le., due to inoculation trauma) were excluded from study. Death due to rabies (or euthanization) occurred mainly between days 10 and 14. All animals scored as developing rabies exhibited typical pathologic signs (ruffled hair, huddled posture, weight loss, spasms or paralysis of extremities, and death).

human adenovirus vectors will work effectivelyin certain nonhuman hosts. The fact that the adenovirus recombinant can be effectively administered by the oral route suggests that adenovirus-based vaccines may be easier to deliver than conventional rabies vaccines and thus should be more suited to

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NOTE. IN, intranasal; IP, intraperitoneal; Neg, negative; NI, no inoculation; pfu, plaque-forming units; SC, subcutaneous. * Parentheses show number of seropositive individual animals/total animals. Indicated antibody titers are the averages for the seropositive animals.

60

'I'-

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Prevec et at.

Acknowledgment We thank 1. Rudy and L. Barton for technical assistance. A DNA copy of the complete glycoprotein gene of rabies virus ERA strain in plasmid pBR322 was obtained from Dr. Shi-Hsiang Shen, Connaught Research Institute, Willowdale, Ontario.

References 1. Fernandes MV, Arambulo PV III. Rabies as an international problem. In: Koprowski H, Plotkin SA, eds. World's debt to Pasteur. New York: Alan R. Liss, 1985:187-218 2. Campbell JB, Charlton KM, eds. Rabies. Hingham MA: Kluwer Academic Publishers, 1988 3. Blancou J, Kieny MP, Lathe R, Lecoq 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 4. Rupprecht CE, Wiktor TJ, Johnston DH, Haimer AN, Dietzschold B, Wunner WH, Glickman LT, Koprowski H. Oral immunization and protection of raccoons (Procyon lotor) with a vaccinia-rabies glycoprotein recombinant virus vaccine. Proc Natl Acad Sci USA 1986; 83:7947-7950 5. 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 6. Tolson ND, Charlton KM, Casey GA, Knowles MK, Rupprecht CE, Lawson KF, Campbell JB. Immunization of foxes against rabies with

a vaccinia recombinant virus expressing the rabies glycoprotein. Arch Virol 1988;102:297-301 7. Ginsberg HS, Dingle JH. The adenovirus group. In: Horsfall FL, Tamm I, eds. Viral and rickettsial infections of man. Philadelphia: JB Lippincott, 1965;860-891 8. Johnson DC, Ghosh-Choudhury G, Smiley JR, Fallis L, Graham FL. Abundant expression of herpes simplex virus glycoprotein gB using an adenovirus vector. Virology 1988;164:1-14 9. 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 USA 1987; 84 :4626-4630 10. Dewar RL, Natarajan V, Vasudevachari MB, Salzman NP. Synthesis and processing of human immunodeficiency virus type 1 envelope proteins encoded by a recombinant human adenovirus. J Virol 1989; 63:129-136 11. Schneider M, Graham FL, Prevec L. Expression of the glycoprotein of vesicular stomatitis virus by infectious adenovirus vectors. J Gen Virol 1989;70:417-427 12. 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 13. McDermott MR, Graham FL, Hanke T, Johnson DC. Protection of mice against lethal challenge with herpes simplex virus by vaccination with an adenovirus vector expressing HSV glycoprotein B. Virology 1989;169:244-247 14. Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 1977;36:59-72 15. Graham FL, Van Der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973;52:456-467 16. Graham FL, Prevec L. Manipulation of adenovirus vectors. In: Murray EJ, ed. Methods in molecular biology. Vol 7. Gene expression in vivo. Clifton NJ: Humana Press, 1989 (in press) 17. Southern PJ, Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet 1982;1:327-334 18. Malek LT, Soostmeyer G, Garvin RT, James E. The rabies glycoprotein gene is expressed in Escherichia coli as a denatured polypeptide. In: Chanock 'R, Lerner R, eds. Modern approaches to vaccines. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1984:203-208 19. Haj-Ahmad Y, Graham FL. Development of a helper-independent human adenovirus vector and its use in the transfer of the herpes simplex virus thymidine kinase gene. J Virol 1986;57:267-274 20. Zalan E, Wilson C, Pukitis D. A microtest for the quantitation of rabies virus neutralizing antibodies. J BioI Stand 1979;7:213-220

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large-scale vaccination programs designed for protection of wildlife or domestic animals. The effectiveness of the AdRGl recombinant in humans or other animals that may already possess some immunity to human adenovirus type 5 remains to be investigated. Our results suggest that continued development of both human and nonhuman adenovirus could play an important role in the control and possible elimination of rabies. The recombinant adenovirus vectors, along with existing vaccinia virus recombinants [3], will also provide a useful tool to probe the relative roles of humoral and cell-mediated immune mechanisms to protect against rabies infection and to determine the relevant epitopes involved in these responses in a number of animal species.

JID 1990;161 (January)

A recombinant human adenovirus vaccine against rabies.

Rabies continues to be a serious problem in both developed and developing nations due to the reservoir of rabies virus in wildlife vectors. The contro...
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