Proc. Natl. Acad. Sci. USAVol. 86, pp. 1287-1291, February 1989Genetics

Highly attenuated vaccinia virus mutants for the generation of saferecombinant viruses

(human immunodeficiency virus type 1 gag and env genes)

DOLORES RODRIGUEZ*, JUAN-RAMON RODRIGUEZ*, JOSE F. RODRIGUEZ*, DAVID TRAUBERt,AND MARIANO ESTEBAN*tDepartments of *Biochemistry, Microbiology, and Immunology, and tMedicine, State University of New York Health Science Center at Brooklyn,Brooklyn, NY 11203

Communicated by Chandler McCuskey Brooks, October 20, 1988

ABSTRACT An attenuated vaccinia virus mutant withspecific genetic lesions has been used to develop a vehicle forsafer live recombinant virus vaccines. The mutant virus 48-7has an 8-MDa deletion starting 2.2 MDa from the left end of theviral genome and point mutations in the gene encoding the14-kDa fusion protein that determines the plaque-size pheno-type of the virus. Using the highly sensitive reporter geneluciferase, we have shown that this mutant can generaterecombinant viruses that infect cultured cells and animals withnormal vaccinia virus tropism. Insertion of the envelope andgag genes of human immunodeficiency virus type 1 into theattenuated vaccinia mutant resulted in their efficient expres-sion and precursor processing in infected cultured cells. Infec-tion of mice with human immunodeficiency virus-vacciniarecombinant viruses elicited human immunodeficiency virus-specific antibodies. Using mice pretreated with cyclophospha-mide as a model for immunosuppression, the reduced virulenceof the mutant recombinant virus was clearly evident. Thesefindings demonstrate that the highly attenuated vaccinia virusmutant 48-7 can be used to generate effective and safervaccines.

Genetically engineered recombinants of vaccinia virus arenow used as eukaryotic expression vectors and, because theyare potent immunogens, have a potential as vaccines againsta broad spectrum of infectious pathogens of animals andhuman significance (1-3). However, in humans, accidentalvaccination with live vaccinia virus of an immunocompro-mised host can lead to serious and lethal complications, suchas systemic vaccinia infection with encephalitis (4, 5). Thispoint was confirmed by the vaccination of a soldier whounknowingly was seropositive for human immunodeficiencyvirus (HIV) (6). To diminish the chances of producing suchcomplications, an expert panel from the World Health Or-ganization has recommended the development of attenuatedstrains of vaccinia virus that carry identifiable genetic mark-ers upon which to base the construction of future recombi-nant vaccines (7). Attenuated variants of vaccinia virus havebeen obtained by several methods, including (i) inactivationof the viral thymidine kinase gene (8), (ii) generation ofdeletions at the left end of the viral genome (9, 10), (iii)mutation of the gene encoding a 14-kDa fusion protein thatinfluences viral plaque size (11), (iv) alterations in size ofvirion core proteins (12, 13), and (v) insertion of the inter-leukin 2 gene that greatly decreases virus virulence inathymic nude mice (14, 15).

In this report we describe the effectiveness of recombinantvaccines based on an attenuated vaccinia virus mutant thathas specific genetic lesions. Using luciferase as a reporter

gene and HIV gag and envelope genes, we have demon-strated the advantage of basing recombinant vaccines on thisattenuated variant.

MATERIALS AND METHODSConstruction of Vaccinia Recombinants Expressing Lu-

ciferase and HIV Envelope and gag Genes. A 1892-base-pair(bp) BamHI fragment encoding the luciferase gene (16) wasinserted into the vaccinia virus expression vector pSC11 andrecombinant viruses were selected by marker rescue byfollowing standard protocols (17). Luciferase activity wasmeasured with a spectrophotometer and results were quan-titated as described (18). For the construction of HIV-vaccinia recombinants, an Xba I-Xho I DNA fragmentcontaining the coding region of HIV env was created bystandard recombinant DNA techniques from a full-lengthHIV type 1 (HIV-1) DNA genome (19). This fragmentextends to the Ssp I site 66 bp upstream from the start site oftranslation of the env gene and 100 bp downstream from thetermination codon. The env DNA fragment was blunt-endedby treatment with the Klenow fragment of DNA polymeraseI and cloned into the Sma I site of the vaccinia virus insertionvector pSC11. As a result of this cloning strategy, we isolateda plasmid containing HIV env coding sequence under thecontrol of the 7.5-kDa vaccinia virus promoter. A similarstrategy was used to construct vaccinia virus insertionvectors containing the intact HIV gag gene. A BssHII-EcoRIDNA fragment containing the coding region of gag wasisolated from the full-length HIV-1 DNA. This fragmentcontains 79 bp upstream from the start site of translation ofthe gag gene and, from the termination site, it contains 2358bp of the pol gene. The luciferase, env, and gag genes wereinserted in the correct orientation relative to the vaccinia7.5-kDa promoter and into the thymidine kinase region ofwild-type and mutant viruses by DNA homologous recom-bination. P -Galactosidase-producing plaques (17) werepicked, cloned three times, and amplified in African greenmonkey kidney cells (BSC-40) grown in culture.

RESULTSExpression and Tissue Tropism of Attenuated Vaccinia

Recombinants. Since attenuated viruses might multiply lessefficiently in infected animals than wild-type virus, our firstinterest was to document the extent of viral gene expressionand tissue tropism of attenuated recombinants. This wasaccomplished by introducing the highly sensitive reportergene luciferase into vaccinia DNA. Two classes of recombi-nants were obtained, one derived from wild-type virus and

Abbreviations: HIV, human immunodeficiency virus; pfu, plaque-forming unit(s).tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Expression of the firefly luciferase gene by wild-type and mutant recombinants of vaccinia virus. The construction of vaccinialuciferase recombinants and assays of luciferase activity have been described (18). (A) Expression of luciferase in cultured cells. BSC-40 cellswere infected with wild-type vaccinia luciferase-recombinant viruses (WRLUC, open symbols) or with mutant 48-7 luciferase-recombinantviruses (MUT7LUC, solid symbols) at 5 plaque-forming units (pfu) per cell with (A, A) or without (o, *) cytosine arabinonucleoside (ARA-C)at 40,u g/ml. At various times after infection, cells were collected and cell extracts prepared in luciferase buffer (100mM potassium phosphate/1mM dithiothreitol) were tested for luciferase activity in the presence of luciferin and ATP (18). (B) Expression of luciferase in organs of infectedmice. BALB/c female mice, 6 weeks old (Charles River Breeding Laboratories), were inoculated i.p. with 5 x 105 pfu of recombinant vacciniaviruses and 3 days later tissues were washed in isotonic phosphate-buffered saline (PBS), weighed, homogenized in luciferase buffer containing1 mM phenylmethylsulfonyl fluoride and leupeptin at 10ttg/ml (5t 1/mg of tissue), and supernatants were assayed for luciferase activity (18).WRLUC, wild-type vaccinia luciferase-recombinant; MUT7LUC, mutant 48-7 luciferase-recombinant.

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the other derived from the attenuated vaccinia mutant 48-7.Mutant 48-7 has an 8-MDa deletion starting 2.2 MDa from theleft end of the viral genome and two point mutations on thegene encoding the 14-kDa fusion protein that result in highattenuation (refs. 10-12; S. C. Gong, C. F. Lai, and M. E.,unpublished results). Vaccinia recombinants containing theluciferase gene were then tested for luciferase activity both incultured cells and in experimental animals. As shown in Fig.1A, the levels of luciferase activity were equivalent inmonkey cells infected with the luciferase-wild-type recom-binant or with the luciferase-mutant 48-7 recombinant. Ininfected mice luciferase activity was observed in spleen andliver, the known target organs for wild-type vaccinia virusmultiplication (20). The levels of luciferase activity found inspleens of mice inoculated with the luciferase-mutant recom-binant virus were =30% lower than in mice inoculated withthe wild-type recombinant virus. Thus, the mutant recombi-nant virus efficiently expresses a foreign gene and shows thesame tissue tropism as the wild-type virus.

Expression of HIV Envelope and gag Genes by AttenuatedVaccinia Recombinants. To assess that the attenuated vac-cinia virus mutant has a vaccine potential, we constructedrecombinant viruses containing either the HIV-1 env or thegag gene and studied their expression and immunogenicity.Because mature retroviral gag and envelope proteins arederived from precursor molecules by processing (21-23), wefirst examined processing of env and gag by immunoprecip-itation analysis. The results are shown in Fig. 2 A and B. In

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FIG. 2. Expression of HIV env and gag by vaccinia (wild-typeand mutant) recombinant viruses. Immunoprecipitation of env (A)and gag (B) proteins from lysates of HIV-vaccinia virus-infectedcells, as examined by NaDodSO4/PAGE. Hela S3 cells infected (5pfu per cell) with vaccinia recombinants were labeled with [35S]_methionine (50,u Ci/ml; 1 Ci = 37 GBq) from 3 to 18 hr after infectionand cell extracts were immunoprecipitated with mouse monoclonalantibodies against gpl20 (env) and p24 (gag) (DuPont). Lanes: 1, 3,5, and 7, total cell extracts; 2, 4, 6, and 8, immunoprecipitates.Uninfected cells (lanes 1 and 2), cells infected with wild-type virus(lanes 3 and 4), and cells infected with HIV-vaccinia recombinants,from wild-type virus (lanes 5 and 6) and from mutant virus (lanes 7and 8), are shown. (C) Processing of env products in cells infectedwith HIV-vaccinia env mutant virus (lanes 1-5). Six hours afterinfection, Hela cells were pulse-labeled with [35S]methionine (l00.t Ci/ml) for 30 min and chased for various times with a 100-foldexcess of unlabeled methionine, and cell extracts were immunopre-cipitated with a monoclonal antibody to gpl20. Proteins were

resolved on a 10% NaDodSO4/polyacrylamide gel. Total cell lysate(lane 1) and immunoprecipitates after a 30-min pulse (lane 2) and afterchase periods of 30 min (lane 3), 60 min (lane 4), and 90 min (lane 5)are shown as well as purified HIV-1 (lane 6) and gpl20 released frominfected cells (lane 7). Released gpl20 was measured in culturesupernatants from mutant HIV-vaccinia env virus-infected Helacells at 24 hr after infection after a 50-fold concentration by using an

Amicon ultrafiltration cell. Lanes 6 and 7 show a blot reacted withrabbit anti-HIV gpl60 serum and developed by immune peroxidasestaining.

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FIG. 3. Humoral immune response elicited in mice by HIV-vaccinia recombinants. (A) Four BALB/c mice per group were

inoculated i.p. (5 x 107 pfu) with purified HIV-vaccinia env mutantrecombinant virus, and 21 days later mice were revaccinated i.v. andi.p. with 108 pfu of recombinant virus. After 21 days, mice were givena booster injection i.v. with 100tug of purified gpl60 isolated fromHIV-vaccinia-infected cells. Serum was collected from the tail vein6 days later and tested for specific antibodies on a Western blot byimmunoperoxidase staining. Proteins were separated by a minigel(10% polyacrylamide). Lanes: 1, lysates of mutant virus-infectedCV-1 cells 24 hr after infection; 2, lysates of HIV-vaccinia env

mutant-infected CV1 cells 24 hr after infection; 3, purified HIV-vaccinia env wild-type virions; 4, purified HIV-vaccinia env mutantvirions; 5, purified gpl60. (B) Mice were primed with purified HIV-vaccinia gag mutant recombinant virus and 21 days later revacci-nated as above. Serum was collected 2 weeks after secondaryvaccination and tested on a Western blot. Proteins were separated ona long gel (10% polyacrylamide). Lanes: 6, purified HIV-1 virus (areverse transcriptase mutant with unprocessed gag, isolated from the8E5 human T-cell line); 7, purified gpl60; 8, lysates of HIV-vacciniaenv wild-type virus-infected Hela cells 36 hr after infection; 9, lysatesof wild-type virus-infected Hela cells 36 hr after infection. Proteinmarkers are myosin,, -galactosidase, bovine serum albumin, oval-bumin, and carbonic anhydrase.

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lysates of Hela S3 cells infected with wild-type HIV-vacciniaenv virus, monoclonal antibodies against gp120 immunopre-cipitates both gp120 and its precursor gpl60 (Fig. 2A, lane 6).These polypeptides were also detected in lysates of cellsinfected with the mutant recombinant virus (lane 8). How-ever, these polypeptides were not detected in lysates ofuninfected cells (lane 2) or in cells infected with nonrecom-binant virus (lane 4). In lysates of cells infected with HIV-vaccinia gag recombinants (either wild-type or mutant virus),a monoclonal antibody against p24 has specific reactivitywith polypeptides p56, p42, p32, and p24 (Fig. 2B, lanes 6 and8). Next we establish a precursor-product relationship forenv by pulse-chase experiments (Fig. 2C). The polypeptidethat was most labeled during the pulse period was gpl60,which was chased into gpl20. The chased gpl20 was releasedextracellularly and was recovered in the cell culture super-natant (lane 7). Two other immunoreactive bands of 70 and50 kDa (lane 7) were also found and appear to result from asingle nick in gp120 (unpublished results). To determine ifmutant HIV-vaccinia recombinants elicit specific antibodiesagainst env and gag proteins, mice were vaccinated with therecombinants and serum was tested by Western blot andELISA. We found that mice vaccinated with mutant HIV-vaccinia env virus have weak antibody response to gpl60.However, induction of antibodies to gp160 was markedlyincreased if vaccinated mice were given a booster injectionwith purified gpl60 (Fig. 3A, lane 5). Mice only vaccinatedwith mutant HIV-vaccinia gag virus had a strong immuneresponse to gag (Fig. 3B, lane 6). Immune reactivity to env

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and gag proteins in serum samples was also confirmed byELISA with HIV-infected cell extracts (Pasteur Institute).Reduced Virulence of Attenuated HIV-Vaccinia Recombi-

nants in Immunosuppressed Animals. Since it is known thatinoculation of immunocompromised individuals with vac-cinia virus may lead to generalized vaccinia and encephalitis(4, 5) [and indeed, this has been documented in a vaccinatedmale seropositive for HIV (6)], it was important to assess thedegree of virulence of attenuated HIV-vaccinia recombi-nants in an immunosuppressed host. Thus, BALB/c micewere first treated with the immunosuppressor drug cyclo-phosphamide (24) and subsequently infected, and their sur-vival was evaluated daily (Fig. 4). As expected, in normalmice the mortality produced by the vaccinia mutant wasabout 100 times lower than that induced by wild-type virus(Fig. 4A). Significantly, the differences in mortality betweenthe two viruses were greater with immunosuppressed mice(Fig. 4B). This was also the case with mutant recombinantvirus. As shown in Fig. 4 C and D, mutant HIV-vaccina envvirus caused markedly decreased mortality in immunosup-pressed mice as compared with wild-type recombinant virus.

DISCUSSIONThese studies demonstrate that a vaccinia virus mutant, 48-7,containing two defined genetic lesions can be used to gener-ate safe recombinant live vaccines. During infection mutantrecombinant viruses express significant levels of foreigngenes, are good immunogens, and have greatly reduced

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FIG. 4. Reduced virulence of HIV-vaccinia recombinants in immunosuppressed mice. Four BALB/c mice per group were inoculated i.p.(0.2 ml) with cyclophosphamide (Sigma) at 300 mg/kg in PBS, and 5 days later mice were treated with a second dose of the drug but at 150 mg/kg.Twenty-four hours later, untreated and drug-treated mice were inoculated i.p. with various doses of purified parental and recombinant HIV-vaccinia env viruses. Survival was followed daily. (A and B) Results obtained after 1 month with parental viruses in normal (A) andimmunosuppressed (B) animals. o, MUT-7; *, WR. (C and D) Results obtained in imniunosuppressed animals with HIV-vaccinia env recom-binants. o, MUT-7 ENV;m, WR ENV. The surviving mouse (WR ENV) in D died on day 21. The survival pattern shown with mutant recombinantvirus was maintained for more than 2 months.

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virulence in an immunosuppressed host. Expression of thehighly sensitive reporter gene luciferase in spleen and liverbut not in other organs provided direct evidence that tissuetropism of attenuated recombinant virus is the same aswild-type virus. Cells infected with attenuated HIV-vacciniaviruses were efficient in their ability to process env and gagprecursors. Similar processing for HIV-1 env products hasbeen observed with other HIV-vaccinia recombinants de-rived from wild-type virus (25, 26). However, the processingof HIV-1 gag precursors that we have observed with HIV-vaccinia recombinant has not been observed, and only theunprocessed form of gag p56 has been reported with awild-type HIV-vaccinia gag recombinant (27). Processing ofgag precursors by our vaccinia recombinants is likely to bedue to the function of the protease encoded by the 5' end ofthe pol gene contained in the BssHII-EcoRI fragment used tointroduce the gag sequence as a result of ribosomal "frame-shifting" (28). Of significance in our study is that micevaccinated with attenuated HIV-vaccinia recombinants de-velop antibodies to env and gag proteins. The immuneresponse to the gag protein was stronger than to the envprotein. However, if after vaccination mice were given abooster injection i.v. with purified gpl60, we then observedgood antibody response to the env protein (Fig. 3A).

In this study we have established an immunosuppressedmouse model system to evaluate virulence of vaccinia virusrecombinants. In this animal-virus system, we observedclear differences in mortality between recombinant virusesthat correlated with the viral genetic markers (Fig. 4). In fact,inactivation of wild-type vaccinia thymidine kinase locus thatis known to reduce virulence in normal mice (8), has littleeffect in decreasing virulence in immunosuppressed animals.However, a vaccinia recombinant with a left-end deletion andalterations in the 14-kDa fusion protein encoding gene hasabout 100 times less virulence in immunosuppressed animalsthan a wild-type recombinant with a thymidine kinase-negative phenotype. Thus, a potential advantage in generat-ing highly attenuated vaccinia virus recombinants with de-letions and point mutations is that these types of recombi-nants will greatly decrease the risk of virus disseminationafter vaccination, particularly in an immunocompromisedhost.

Considering the characteristics of the highly attenuatedvaccinia recombinants that we have generated, these recom-binants and derivatives might be useful as immunogens instudies of recognition specificity of immune T cells and in thedesign of safer vaccines against pathogens of veterinary andhuman importance.

We thank J. A. Lewis and R. Bablanian for reading the manu-script, E. McGowan for luciferase assays, B. Moss for the vectorpSC 11, S. H. Howell for the luciferase plasmid pD0432, F. Ruscettifor the plasmid containing the full-length HIV-1 DNA strain (HTLV-IIIB), and J. J. Skehel for the HIV-1 reagents.

1. Panicali, D. & Paoletti, E. (1982) Proc. Natl. Acad. Sci. USA79, 4927-4931.

2. Mackett, M., Smith, G. L. & Moss, B. (1982) Proc. Natl. Acad.Sci. USA 79, 7415-7419.

3. Moss, B. & Flexner, C. (1987) Annu. Rev. Immunol. 5, 305-324.

4. Lane, J. M., Ruben, F. L., Neff, J. M. & Millar, J. D. (1969)N. Engl. J. Med. 281, 1201-1208.

5. Arita, I. & Fenner, F. (1985) in Vaccinia Viruses as Vectors forVaccine Antigens, ed. Quinnan, J. (Elsevier, New York), pp.49-60.

6. Redfield, R. R., Wright, D. C., James, W. D., Jones, T. S.,Brown, C. & Burke, D. S. (1987) N. Engl. J. Med. 316, 673-676.

7. Brown, F., Schild, G. C. & Ada, G. L. (1986) Nature (London)319, 549-550.

8. Buller, R. M., Smith, G. L., Cremer, K., Notkins, A. L. &Moss, B. (1985) Nature (London) 317, 813-815.

9. Paez, E., Dallo, S. & Esteban, M. (1985) Proc. Natl. Acad. Sci.USA 82, 3365-3369.

10. Dallo, S. & Esteban, M. (1987) Virology 159, 408-422.11. Dallo, S., Rodriguez, J. F. & Esteban, M. (1987) Virology 159,

423-432.12. Paez, E., Dallo, S. & Esteban, M. (1987) J. Virol. 61, 2642-

2647.13. Maa, J.-S. & Esteban, M. (1987) J. Virol. 61, 3910-3919.14. Ramshaw, I. A., Andrew, M. E., Phillips, S. M., Boyle, D. B.

& Coupar, B. E. H. (1987) Nature (London) 329, 545-546.15. Flexner, C., Hugin, A. & Moss, B. (1987) Nature (London) 330,

259-262.16. Ow, D. W., Wood, K. V., DeLuca, M., de Wet, J. R., Helin-

ski, D. R. & Howell, S. H. (1986) Science 234, 856-859.17. Chakrabarti, S., Brechling, K. & Moss, B. (1985) Mol. Cell.

Biol. 5, 3403-3409.18. Rodriguez, J. F., Rodriguez, D., Rodriguez, J.-R., McGowan,

E. & Esteban, M. (1988) Proc. Natl. Acad. Sci. USA 85, 1667-1671.

19. Fisher, A. G., Collalti, E., Ratner, L., Gallo, R. C. & Wong-Staal, F. (1985) Nature (London) 316, 262-265.

20. Dales, S. & Pogo, B. G. T. (1981) Biology of Poxviruses,Virology Monographs (Springer, New York), Vol. 18.

21. Muesing, M. A., Smith, D. H., Cabradilla, C. D., Benton,C. V., Lasky, L. A. & Capon, D. J. (1985) Nature (London)313, 450-458.

22. Sanchez-Pescador, R., Power, M. D., Barr, P. J., Steiner,K. S., Stempien, M. M., Brown-Shimer, S. L., Gee, W. W.,Renara, A., Randolph, A., Levy, J. A., Dima, D. & Luciw,P. A. (1985) Science 227, 484-492.

23. Wain-Hobson, S., Sonigo, P., Damos, O., Cole, S. & Alizon,M. (1985) Cell 40, 9-17.

24. Bach, J. F. (1975) Frontiers ofBiology (Elsevier Biomed., NewYork), Vol. 41, pp. 173-225.

25. Chakrabarti, S., Rober-Guroff, M., Wong-Staal, F., Gallo,R. C. & Moss, B. (1986) Nature (London) 320, 535-537.

26. Hu, S.-L., Kosowski, S. G. & Dalrymple, J. M. (1986) Nature(London) 320, 537-540.

27. Walker, B. D., Chakrabarti, S., Moss, B., Paradis, T. J.,Flynn, T., Durmo, A. G., Blumberg, R. S., Kaplan, J. C.,Hirsch, M. S. & Schooley, R. T. (1987) Nature (London) 328,345-348.

28. Jacks, T., Power, M. D., Masiarz, F. R., Luciw, P. A., Barr,P. J. & Varmus, H. E. (1987) Nature (London) 331, 280-283.

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