|H|||||||||||| USOO5225336A United States Patent (19) 11 Patent Number: 5,225,336 Paoletti 45) Date of Patent: Jul

|H|||||||||||| USOO5225336A United States Patent (19) 11 Patent Number: 5,225,336 Paoletti 45) Date of Patent: Jul. 6, 1993

54 RECOMBINANT POXVIRUS HOST RANGE Drillien, R., Spehner, D., and A. Kirn, J. Virol., 28, SELECTION SYSTEM 843-850 (1978). 75) Enventor: Enzo Paoletti, Albany, N.Y. Falkner, F. G. and B. Moss, J. Virol., 62, 1849-1854 (1988). 73 Assignee: Health Research Incorporated, Fathi, Z., Sridhar, P., Pacha, R. F., and R. C. Condit, Albany, N.Y. Virology, 155,97-105 (1986). 21 Appl. No.: 902,428 Fenner, F. and J. F. Sambrook, Virology, 28, 600-609 (1966). 22 Filed: Jun. 23, 1992 (List continued on next page.) Related U.S. Application Data Primary Examiner-Richard A. Schwartz 63 Continuation of Ser. No. 478,179, Feb. 14, 1990, aban Assistant Examiner-David Guzo doned, which is a continuation-in-part of Ser. No. Attorney, Agent, or Firm-Curtis, Morris & Safford 320,471, Mar. 8, 1989, Pat. No. 5,155,020. 57 ABSTRACT 51) Int. Cl...... C12P 21/00; C12N 7/01; A61K 39/00; A61K 39/12 What is described is a modified recombinant virus for 52 U.S. C...... 435/69.1; 435/235.1; expressing a gene product in a host. The modified re 435/320.1; 424/89; 424/88 combinant virus has host range genes deleted therefrom 58 Field of Search ...... 435/69.1, 320.1, 235. 1, so that the virus has restricted replication in the host. The modified recombinant virus also contains DNA 435/172.3; 424/88, 89; 935/32, 57 which codes for and expresses the gene product in the 56 References Cited host even with restricted replication of the virus in the FOREIGN PATENT DOCUMENTS host. The modified recombinant virus is used in a method for expressing a gene product in a host or in a 78906/87 SA1988 Australia . cell cultured in vitro, and in a vaccine for inducing an 0262043 3/1988 European Pat. Off. . immunological response in a host inoculated with the WO89/12103 12/1989 PCT Int'l Appl. . vaccine. What is also described is a selection system for OTHER PUBLICATIONS the cloning and expression of open reading frames in Perkins et al., Virology, vol. 179, No. 1, pp. 276-286 poxviruses, particularly vaccinia virus. The selection (1990). system is based on a conditional lethal mutant (host Lai et al., Microbiol. Pathogenesis, vol. 6, No. 3, pp. range) of poxviruses. A deletion/recombinant mutant of 219-226 (1989). the vaccinia virus was generated which is capable of Goebel et al., Virology, vol. 179, No. 1, pp. 247-266 plaquing on primary chick embryo fibroblasts and two (1990). monkey cell lines (BSC-40 or VERO) but is defective in Beck, E., Ludwig, G., Auerswald, E. A., Reiss, B., and replication in the human cell line MRC-5. Insertion of H. Schaller, Gene, 19, 327-336 (1982). the host range gene into the deletion/recombinant re Boyle, D. B. and B. E. H. Coupar, Gene, 65, 123-128 stores the ability for growth on MRC-5 cells. A series of (1988). plasmids were constructed which allow for the rapid Chakrabarti, S., Brechling, K., and B. Moss, Mol. Cell. single-step cloning and expression of any open reading Biol. 5,3403-3409 (1985). frame when recombined with the deletion/recombinant Clewell, D. B., J. Bacteriol., 110, 667-676 (1972). and scored for growth on MRC-5 cells. Clewell, D. B. and D. R. Helinski, Proc. Natl. Acad. Sci. USA, 62, 1159-1166 (1969). Drillien, R., Koehren, F., and A. Kirn, Virology, 111, 488-499 (1981). 6 Claims, 60 Drawing Sheets 5,225,336 Page 2

OTHER PUBLICATIONS Rosel, J. L., Earl, P. L., Weir, J. P., and B. Moss, J. Franke, C. A., Rice, C. M., Strauss, J. H., and D. E. Viol., 60, 436-449 (1986). Hruby, Mol. Cell. Biol. 5, 1918-1924 (1985). Shapira, S. K., Chou, J., Richaud, F. V., and M. J. Gangemi, J. D. and D. G. Sharp, Virology, 85,262-270 Casadaban, Gene, 25, 71-82 (1983). (1978). Southern, P. H. and P. Berg, J. Mol. Appl. Genet., 1, Gemmell, A. and F. Fenner, Virology, 11, 219-235 327-341 (1982). (1960). Tagaya, I., Kitamura, T., and Y. Sano, Nature, (Lon Gillard, S., Spehner, D., and R. Drillien, J. Virol., 53, don), 192,381-382 (1961). 316-318 (1985). Wachsman, M., Aurelian, L., Smith, C. C., Lipinskas, B. Gillard, S., Spehner, D., Drillien, R., and A. Kirn, Proc. R., Perkus, M. E., and E. Paoletti, J. Inf. Dis., 155, Natl. Acad. Sci. USA, 83, 5573-5577 (1986). 1188-1197 (1987). Graham, F. L. and A. J. Van der Eb, Virology, 54, Wilson, E.M., Hodges, W. M., and D. E. Hruby, Gene, 536-539 (1973). 49, 207-213 (1986). Hruby, D. E., Lynn, D. L., Condit, R., and J. R. Kates, Yuen, L. and B. Moss, Proc. Natl. Acad. Sci. USA, 84, J. Gen. Virol, 47, 485-488 (1980). 6417-6421 (1987). Kieny, M. P., Lathe, R., Drillien, R., Spehner, D., Kotwal, G. J. and B. Moss, Virology, 167, 524-537 Skory, S., Schmitt, D., Wictor, T., Koprowski, H., and (1988). J. P. Lecocq, Nature, (London), 312, 163-166 (1984). Taylor, J. Weinberg, R., Kawaoda, Y., Webster, R. G., Lake, J. R. and P. D. Cooper, J. Gen. Virol., 48, and E. Paoletti, Vaccine, 6, 504-508 (1988). 135-147 (1980). Taylor, J. Weinberg, R., Languet, B., Desmettre, P., Mackett, M., Smith, G. L. and B. Moss, Proc. Natl. and E. Paoletti, Vaccine, 6, 497-503 (1988). Acad. Sci. USA, 79,7415-7419 (1982). Pickup, D.J., Ink, B. S., Hu, W., Ray, C. A., and W. K. Mackett, M. and J. R. Arrand, EMBO, 4, 3229-3235 Joklik, Proc. Natl. Acad. Sci. USA, 83, 7698-7702 (1985). (1986). Mayr, A., Hochstein-Mintzel, V., and H. Stickl, Infec Southern, E. M., J. Mol. Biol., 98, 503-517 (1975). tion, 3, 6-14 (1975). Kotwal, G.J. and B. Moss, J. Virol., 63, 600-606 (1989). McClain, M. E., Aust. J. Exp. Biol. Med. Sci., 43, 31-44 Tabor, S. and C. C. Richardson, Proc. Natl. Acad. Sci. (1965). USA, 84,4767-4771 (1987). Moyer, R. W. and C. T. Rothe, Virology, 102, 119-132 Patel, D. D. and D. J. Pickup, EMBO, 6, 3787-3794 (1980). (1987). Nakano, E., Panicali, D., and E. Paoletti, Proc. Natl. Patel, D. D., Ray, C. A., Drucker, R. P., and D. J. Acad. Sci. USA, 79, 1593-1596 (1982). Pickup, Proc. Natl. Acad. Sci. USA, 85,9431-9435 Panicali, D., Davis, S. W., Mercer, S. R., and E. Pao (1988). letti, J. Virol., 37, 1000-1010 (1981). Bertholet, C., Drillien, R., and R. Wittek, Proc. Natl. Panicali, D. and E. Paoletti, Proc. Natl. Acad. Sci. Acad. Sci. USA, 82, 2096-2100 (1985). USA, 79, 4927-4931 (1982). Guo, P., Goebel, S., Davis, S. Perkus, M. E., Languet, Panicali, D., Grzelecki, A., and C. Huang, Gene, 47, B., Desmettre, P., Allen, G., and E. Paoletti, J. Virol., 193-199 (1986). 63, 4189-4198 (1989). Perkus, M. E., Panicali, D., Mercer, S., and E. Paoletti, Tamin, A., Villarreal, E. C., Weinrich, S. L., and D. E. Virol., 152, 285-297 (1986). Hruby, Virology, 165, 141-150 (1988). Perkus, M. E., Piccini, A., Lipinskas, B. R., and E. Boursnell, M. E. G., Foulds, I.J., Campbell, J. I., and Paoletti, Science, 229,981-984 (1985). M. M. Binns, J. Gen. Virol. 69,2995-3003 (1988). Piccini, A., Perkus, M. E., and E. Paoletti, In: Methods Mandecki, W., Proc. Natl. Acad. Sci. USA, 83, . in Enzymology, vol. 53, ed. Wu, R. and L. Grossman, 7177-7181 (1986). (Academic Press), pp. 545-563 (1987). (List continued on next page.) 5,225,336 Page 3

OTHER PUBLICATIONS Vos, J. C. and H. G. Stunnenberg, EMBO, 7,3487-3492 Spehner, D., Gillard, S., Drillien, R., and A. Kirn, J. (1988). Virol., 62, 1297-1304 (1988). Bucher, D., Popple, S., Baer, M., Mikhail, A., Gong, Altenburger, W., Suter, C.-P., and J. Altenburger, Y-F., Whitaker, C., Paoletti, E., and A. Judd, J. Virol., Arch. Virol., 105, 15-27 (1989). 63,3622-3633 (1989). Hruby, D. E., Maki, R. A., Miller, D. B., and L. A. Ball, Saiki, R. K., Gelfand, D. H., Stoffel, S. Scharf, S. J. Proc. Natl. Acad. Sci. USA, 80, 341-3415 (1983). Higuchi, R., Horn, G. T., Mullis, K. B., and H. A. Er Robbins, A. K., Dorney, D. J., Wathen, M. W., lich, Science, 239, 487-491 (1988). Whealy, M. E., Gold, C., Watson, R.J., Holland, L. E., Kaplan, J. M., Mardon, G., Bishop, J. M., and H. E. Weed, S. D., Levine, M., Giorioso, J. C., and L. W. Varmus, Mol. Cell. Biol, 8, 2435-2441 (1988). Enquist, J. Virol., 61,2691-2701 (1987). Baroudy, B. M., Venkatesan, S., and B. Moss, Cell, 28, Robbins, A. K., Watson, R. J., Whealy, M. E., Hays, W. 315-324 (1982). W., and L. W. Enquist, J. Virol., 58,339-347 (1986). Wathen, M. W. and L. M. K. Wathen, J. Virol., 51, Wittek, R. and B. Moss, Cell, 21, 277-284 (1980). 57-62 (1984). Wittek, R., Muller, H. K., Menna, A., and R. Wyler, Mettenleiter, T. C., Lukacs, N., Thiel, H.-J., Schreurs, FEBS Letters, 90, 41-46 (1978). C., and H. J. Rziha, Virology, 152, 66-75 (1986). Slabaugh, M., Roseman, N., Davis, R., and C. Mathews, Petrovskis, E. A., Timmins, J. G., Armentrout, M. A., J. Virol., 62,519-527 (1988). Marchioli, C. C., Yancey, Jr., R. J., and L. E. Post, J. Mulligan, R. C. and P. Berg, Science, 209, 1422-1427 Virol., 59, 216-223 (1986). (1980). Schmitt, J. F. C. and H. G. Stunnenberg, J. Virol., 62, Pratt, D. and S. Subramani, Nuc. Acids Res., 11, 1889-1897 (1988). 8817-8823 (1983). U.S. Patent July 6, 1993 Sheet 1 of 60 5,225,336

ECOR ECOR Smal BamHI HincI PSt. Smal HindII

ECOR pUC8HR pMP528E HindIII v.A Pst EcoR Smal Smal HindII Smal Small FIG. 2A Fragment isolation KenOW

Ligate

ECORI Transfection MRC-5 pMP528HR VP457-- a. Small infection vF293 2/ PSt. FIG 2B Hindi

U.S. Patent July 6, 1993 Sheet 3 of 60 5,225,336

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U.S. Patent July 6, 1993 Sheet 9 of 60 5,225,336

IoeSK

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ECOR ECOR Sal

Sail

Ligate ECOR ra SSt. ECOR Hind Sall HindII Smal Pst BamH Sa Xbal Sall HindII PSt. HindII

HindII ECOR pMP528 FIG. A A sk EcoRI Hind Sal Sai, KenOW SmaI inkerS Ligate Small HindII ECOR Sma M. Small Sma Transfection CEF pipe VP293-- ligate infection VK79

HindII FIG. B Sall HindIII." a HindII VTK79 EZ38. - Q1 FIG. C HindII VP293 U.S. Patent July 6, 1993 Sheet 11 of 60 5,225,336

Sall M TCGACTGACGACAATAACAAAATCACAACATCGTTTTTGATATTATTATTTTTCTTGGTA {X W S S L L L I W W D N. K I N N N K K T ACGTATGCCTTTAATGGAGTTTCACCATCATACTCATATAATGGATTTGCACCACTTTCT W Y A K L P T E G D Y E Y P N A G S E 26 12 ATCAATGATTGTGCACTGCTGGCATCGATGTTAAATGTTTTACAACTATCATAGAGTATC CI L S Q A S S A D I N F T K C S D Y L. I TTATCGTTAACCATGATTGGTTGTTGATGCTATCGCATTTTTTGGTTTCTTTCATTTCAG K D N W M CC9L 74.7kDa (fragment) 24 TTATGTATGGATTTAGCACGTTTGGGAAGCATGAGCTCATATGATTTCAGTACTGTAGTG CN H J S K A R K P L M L E Y S K L W T T TCAGTACTATTAGTTTCGATCAGATCAATGTCTAGATCTATAGAATCAAAACACGATAGG D T S N T E I L D D L D S D F C S L 1145 361 TCAGAAGATAATGAATATCTGTACGCTCTTTTTGTACTGTAACTTCTCGTTTTGTTAGA CD S S L S Y R Y A E K Q W T W E R K T L TGTTTGCATCGTGCTTTAACATCAATGGTACAAATTTTATCCTCGCTTTGTGTATCATAT H K C R A K W D I T C I K D E S Q T D Y 105 481 TCGTCCCTACTATAAAATTGTATATTCAGATTATCATGAGATGTGTATACGCTAACGGTA CE D R S Y F Q I N N D H S T Y V S W T TCAATAAACGGAGCACACCATTTAGTCATAACCGTAATCCAAAAATTTTTAAAGTATATC D F P A C W K T M W T W F N K F Y I 65 601 TTAACGAAAGAAGTTGTATCATCGTTAGGATTTGGTAAATCATTACTACAGTGTATGGT CK V F S T T D D N P N P D N D W T Y P ACTAGATCCTCATAAGTGTATATATCTAGAGTAATGTTTAATTTATCAAATGGTTGATAA W L D E Y T Y D L T N. L. K D F P Q Y 25 721 TATGGATCCTCATGACAATTTCCGAAGATGGAAATGAGATATAGACATGCAATAAATCTA CY P D E H C N G F I S I Li Y L. C A F R ATCGAAGACATGGTTACTCCTTAAAAAAATACGAATAATCACCTTGGCTATTTAGTAAGT I S S M CC8L 21, 6 kDa 84 GTCATTTAACACTATACTCATATTAATCCATGGACTCATAATCTCTATACGGGATTAACG CD M S E Y D R Y P I L. P GATGTTCTATATACGGGGATGAGTAGTTCTCTTCTTTAACTTTATACTTTTTACTAATCA H E Y P S S Y N E E . K V K Y K K S I M 119 96 TATTTAGACTGATGTATGGGTAATAGTGTTTGAAGAGCTCGTCTCATCATCAGAATAAA CN L S Y P Y Y H K F L E N E D D S Y I TCAATATCTCTGTTTTTTTGTTATACAGATGTATTACAGCCTCATATATTACGTAATAGA L E T K K N Y L H W A E Y I V Y Y F FIG.8A U.S. Patent July 6, 1993 Sheet 12 of 60 5,225,336

08 ACGTGTCATCTACCTTATTAACTTCACCGCATAGTTGTTTGCAAATACGGTTAATCCTT CT D D V K N V K W A Y N N A F W T L G K TGACCTCGTCGATTTCCGACCAATCTGGGCGTATAATGAATCTAAACTTTAATTTCTTGT W E D E S W D P R F R F K L K K Y 39 120 AATCATTCGAAATAATTTTTAGTTTGCATCCGTAGTTATCCCCTTTATGTAACTGTAAAT {D N S I K K C G Y N D G K H L Q N TTCTCAA CGCGATATCTCCATTAATAATGATGTCGAATCGTGCTGTATACCCATACTGA R L A I D G N I I I D F E H Q G M C7L 18, OKDa 32 ATGGATGAACGAATACCGACGGCGTTAATAGTAATTACTTTTTCATCTTTACAATTGG GTACTAGTTTTACTATCATAAGTTTATAAATTCCACAAGCTACTATGGAATAAGCCAACC lily ATCTTAGTATAACACACATGTCTTAAAGTTTATTAATTAATTACATGGTTTTATATAT

CGCTACGAATTTAAACAGAGAAATCAGTTAGGAAAAAAAATTATCTATCTACATCATCA CR D W D D 147 156 CGTCTCTGTATTCTACGATAGAGTGCTACTTTAAGATGAGA CATATCCGTGTCATCAAAA {R R Q I R R Y L A V K L H S M D T D D F ATAACICCATTAAAATGATTATTCCGGCAGCGAACTTGATATTGGATATATCACAACCT Y E M L I G A A. F. K I N S I D C G 107 681 TTGTTAATATCTACGACAATAGACAGCAGTCCCATGGTTCCATAAACAGTGAGTTTACT CK N I D W W I S L L G M T G Y W T K D TTCTTTGAAGAGATATTTTGTAGAGATCTTATAAAACTGTCGAATGACATCGCATTATA K K S S I N Q. L S R F S D F S M A N 67 1801. TCTTTAGCTAAATCGTATATGTTACCATCGTAATATCTAACCGCGTCTATCTTAAACGTT {D K A L. D Y N G D Y Y R W A D K F TCCATCGCTTTAAAGACGTTTCCGATAGATGGTCTCATTTCATCAGTCATACTGAGCCAA E M A K F W N G S P R M E D T M S L W 27 1921 CAAATATAATCGTGTATAACATCTTGATAGAATCAGA CTCTAAAGAAAACGAATCGGC CC Y D H I W D K I S D S E L S F S D A TTATTAACGCATCATGATAAACTTAATGAAAAATGTTTTTCGTTGTTTAAGTTGGATG K. N. Y. A N M CC6L 17, likDa 2041 AATAGTATGTCTTAATAATTGTTATTATTTCATTAATTAATATTTAGTAACGAGTACACT CTATAAAAACGAGAATGACATAACTAGTATCAAAGTGTCTAGGACGCGTAATTTTCATA CN D F H R P R T I K M 194 U.S. Patent July 6, 1993 Sheet 13 of 60 5,225,336

26 TGGTATAGATCCTGTAAGCATTGTCTGTATTCTGGAGCTATTTTCCTATCGCATAGTG KH Y L. D. Q C Q R Y E P A K E I A N AGTTCAGAATATGTTATAAATTTAAATCGAATAACGAACATAACTTTAGTAAAGCGTCT L E S Y T F K F R V F M V K T F D D, 154 228 ATATTAACTCTTTTATTTTCTAGCCATCGTAATACCATGTTTAAGATAGTATATTCTCTA {I N W R K N E L W R L. W. M. N. L. I. T. Y E R GTTACTACGATCTCATCGTGTCTAGAATATCACATACTGAACTACATCCAATTTTAGA T W W I E D N D L I D C W S D W D L K L 114 240 AATTGGTCTGGTTACATATCTCTCTATATTATTGTTGATGTATTGTCGTAGAAAACTA CF Q D T N C I E E I N N N Y Q R L. F S TTACGTAGACCATTTTCTTTATAAAACGAATATATAGTACTCCAATTATCTTTACCGATA N R L G N E K Y F S Y I T S W N D K G 74 2521. TATTTGCACACAAATCCATTCTCTCAATCACTACACTTAAGATTTTCGTTGTTAAGA CY K C W Y D M R E I W W D K L N E N N TATTTGGCTAAACTATATAATTCTATTAGATCATCAACAGAATCAGTATATATTTTTCTA Y K A S Y E I D D W S D Y I K R 34 264 GATCCAAAGACGAA CTCTTGGCGTCCCTATAATATCCCAGAAAAGATATTTTCGTGT CS G F W F E K A D E I I N G S F N E H TTTAGTTTATCGAGATCTGACTGTTCATATACGCCATGATTGTACGGTACGTTATGATA K L. K D D S R N M Y A M CC5L 24,5kDa 276 AccGCTAATAAATCCATTCATTACAATACTACAATsAGIG TGTAATACTTTGTTACTTTGAACGTAAAGACAGTACACGGATCCGTATCTCCAACAAGCA Y Y K T V K F T F W T C P D T D G W L V 291 2881 CGTAGTAATCAAATTTGGTGTTGTTAAACTTCGCAAATTCATCAATTTAGATAGAAACT CY Y D F K T N N F K A I N M L K S L F K TATACT CATCATCTGTTTTAGGAATCCATGTATTATTACCACTTTCCAACTTATCATTAT Y E D D T K P I W T N N G S E L K. D N D 25 500 CCCAGGCTATGTTTCGTCCATCATCGTTGCGCAGAGTGAATAATTCTTTTGTATTCGGTA CW A N R G D D N R L T F L E K T N P L. GTTCAAATATATGACCATGCATAGATCGGCAAAGCTATTGTAGATGTGATTTTTCCTAA E F I H D M C L D A F S N Y H. N. K R F 21 3121 ATCTAAATAAAA CTCGTTTACTAGCAAACACTTTCCTGATTTACGACCAAGACACATA CR I Y F E N W L. C K G S K D W W C I TGGTTTCTAAATCTATCAAGGGTGGGGATCCATAGTTATGACGCAGTAACATATATTAT T E L D L H H P D M T I V C Y C N N 171 FIG.8C U.S. Patent July 6, 1993 Sheet 14 of 60 5,225,336

32ll TACATTCTTGACGTCGCTAATATCTAAATATTTATTGTTATCGTATTGGATTCTGCATA CC E Q S D S I D L Y K N N D Y Q I R C I TAGATGGCTTGTATGT CAAAGATATAGAACACATAACCAATTTATAGTCGCGCTTTACAT S P K Y T L S I S C M W L K Y D R K V N 13 3361 TCTCGAATCTAAAGTTAAGAGATTTAGAAAACATTATATCCTCGGATGATGTTATCACTG {E F R F N L S K S F M I D E S S T L V T TTTCTGGAGTAGGATATATTAAAGTCTTTACAGATTTCGTCCGATTCAAATAAATCACTA E P T P Y I L T K V S K T R N Li Y I W L 9. 3.48 AATAATATCCCACATTATCATCTGTTAGAGTAGTATCATTAAATCTATTATATTTTATGA CY Y G V N D D T L T T D N F R N Y K F AAGATATATCACTGCTCACCTCTATATTTCGTACATTTTTAAACTGTTTGTATAATATCT S I D S S W E N R V N K F Q K Y L. E 51 36Ol CTCTGATACAATCAGATATATCTATTGTGTCGGTAGACGATACCGTTACATTTGAATTAA {R C D S I D I T D T S S W T V N S N I TGGTGTTCCATTTTACAACTTTTAACAAGTTGACCAATTCATTTCTAATAGTATCAAACT T N W K V V K L L N W L E N R I D F E 1. 372 CTCCATGATTAAATATTTTAATAGTATCCATTTTATATCACTACGGACACAAAGTAGCTG KG H N F I K I D M {CAL 57,2kDa ACATAAACCATTGTATAATTTTTATGTTTTAGTTTATTAGCGTACA CATTTTGGAAGTT CR W C K P L E 257 38l. CCGGCTTCCATGTATTTCCTGGAGAGCAAGTAGATGATGAGGAACCAGATAGTTTATATCCP K W T N G P S C T S S S S G S L K Y G CGTACTTGCACTTAAAGTCTACATTGTCGTTGTATGAGTATGATCTTTTAAACCCGCTAG Y K C K F D V N D N Y S Y S R K F G S S 27 3961 ACAAGTATCCGTTTGATATTGTAGGATGTGGACATTTAACAATCTGACACGTGGGTGGAT CL Y G N S I T P H P C K W I Q C T P P D CGGACCATTCTCCTCCTGAACACAGGACACCAGAGTTACCAATCAA CGAATATCCACTAT S W E G G S C L W G S N G I L S Y G S N 177 408 TGCAACTATAAGTTACAA CGCTTCCATCGGTATAAAAATCCTCGTATCCGTTATGTCTTC CC S Y T V V S G D T Y F D E Y G N H R G CGTTGGATATAGATGGAGGGGATTGGCATTTAACAGATTCACAAATAGGTGCCTCGGGAT N S I S P P S Q C K V S E C P A E P N 137 420 TCCATACCATAGATCCAGTAGATCCTAATTCACAATACGATTTAGATTCACCGATCAAAT CW W M S G T S G L E C Y S K S E G H GATATCCGCTATTACAAGAGTACGTTATACTAGAGCCAAAGTCTACTCCACCAATATCAA Y G S N C S Y T I S S G F D W G G D 97 FIG8D

U.S. Patent July 6, 1993 Sheet 16 of 60 5,225,336

540 TATTGTTAATCATGCCGCCAATAATGATAGACAATTATGAAAACATTGCATTAAGA {N N I M G G I Y L. C N H L V N A N Y F

ATTGTCTATCTGTATTACCGACTATCGTCCAATATTCTGTCCTAGGAGAGTAATGGGTTAG R D T N G W T W Y E T R P S Y H T 232 552. TTGTGGATATATAATCAGAGTTTTTAATGACTACTATATTATGTTTTATACCATTTCGTG CT S I Y D S N K I V V N H K I. G. N. R. T. TCACTGGCTTTGTAGATTTGGATATAGTTAATCCCAACAATGATATAGCATTGCGCATAG V P K T S K S I T L G L L S I A N R M T 192 564 TATAGTCATAAACTTGGGATGTAAAATGTTGATGATATCTACATCGTTTGGATTTTTAT CN T M F K P H N I D W D N P N K H GTATCCACTTTAATAATATCATAGCTGTAACATCCTCATGATTTACGTTAACGTCTTCGT I W K L L M A T W D E H N V N W D E H 152 576 GGGATAAGATAGTTGTCAGTTCATCCTTGATAATTTTCCAAATTCTGGATCGGATGTCA CS L T T L E D K S L K G F E P D S T W CCGCAGTAATATTGTTGATTATTTCTGACATCGACGCATTATATAGTTTTTTAATTCCAT A T I N N I E S M S A N Y L. K K I G Y 112 588 ATCTTTTAGAAAAGTTAAACATCCTTATACAATTTGTGGAATTAATATTATGAATCATAGCR K S F N F M R C N T S N N H J M T TTTTTACACATAGATCTACTACAGGCGGAACATCAATTATTACGGCAGCAACTAGTATCA K W C L D V V P P W D I W A A W L I M 72 6001 TTTCTACATTGTTTATGGTGATGTTTATCTTCTTCCAGCGCATATAGTCTAATAGCGATT KE V N N I T. N I K K W R M Y D L L S E CAAACGCGTGATAGTTTATACCATTCAATATAATCGCTTCATCCTTAGATGGTGATCCT F A H Y N I. G. N. L. I. I. A E D K L H H D Q 52 621. GAATGCGTTTAAAAAAATTATACGGAGACGCCGTAATAATTTCCTTATTCACTTGTATAA {I R K F F N Y P S A T I I E K N W Q I I TTTCCCCATTGATAGAAAATATCACGCTTTCCATTCTTGAAGTACTATAAGTAATTATAG E G N I S F I W S E M CC2. 59, KDa TATATGTAGGTTATATATCATATITITIATAAAATCATeg. TA CCTTTTTAAATTTGCGTCTATCATCTATAGAAACATATTCTATGAATTTATAAAATGCTT K K F K R R D D S W Y E F K Y F A K 200 636 TTACGTGTCCTATCGTAGGCGATAGAACCGCTAAAAAGCCTATCGAATTTCTACAAAAGACW H G L T P S L V A. L. F G S N R C F F ATCTGTATATGGTAAGGGAGAGATAAAACATTAAATGTCCGTACTTATTAAAGTATT R N Y P P L T Y F M L H G Y K N F Y E 160 FIG.8F U.S. Patent July 6, 1993 Sheet 17 of 60 5,225,336

648 CAGTAGCCAATCCTAACTCTTTCGAATACTTATTAATGGCTCTTGTTCGTACGAATCTA CT A L G L E K S Y K N I. A R T R Y S D TTTTTTTGAACAACGGACCTAGTGGTATATCTTGTTCTATGTATCTAAAATAATGTCTGA K K F L P G L P I D Q E Y R F Y H R V 20 660 CTAGATCCGTAGTTTAATATCCTCAGTCATCTTGTCTAGAATGGCAAATCTAACTGCGG KL D T K I D E M K D L I A F R W A P GTTTAGGCTTAGTTTAGTTTCTATATCACATCTATGTCTTTATCTAACACCAAAAATA K P K L K E I D W D I D K D L V F I 80 672 TAATAGCTAATATTTTATTACAATCATCCGGATATTCTTCTACGATCTCACTAACTAATG CI A L I K N C D D P Y E E W E S W L T TTTCTTTGGTTATACTAGTATAGTCACTATCGGACAAATAAAGAAAATCAGATGATCGAT E K T I S Y D S D S L Y L. F D S S R H 40 6841 GAATAAACATTTAAATTCACATCTGTAAGATTTTTGAGATGTCTCATTAGAATATTAT { I C K F E D D T L N K L H R M L E N N TAGGGTTAGTACTCATTATCATTCGGCAGCTATTACTATTTTATTATTTTTCACCATAT P N T S M I M R C S N S I K N k *E 's "Cl25.4kDa 696 AGATCAATCATTAGACACAAAATATGTTTCAATCATCCTAAAGAGTATGGGAATGAC { D M L D D F Y E I M R F L T F S TCTTCCCATCTAATTTCTGAACGTCACCAATGTCTCTAGCCA CTTTGGCACTAATAGCG E E W R E S R E G I D R A V K A S I A 74 7081 ATCATTCGCTTAGCGTCTCTATATATTAACTGGTTGATTCAATCTATCTAGCAATGGA K M R K A D E I N N W P Q N L R D L L P CCGTCGGACAGCGTCATTCTCATGTTCTAATCAATGTACATACATCGCCGTCATCTACC G D S L T M R M N K I L T C W D G D D W 34 720 AATTCATCCAA CAA CATA AG TTTTTAAAATCATCATTATAATAGGTTTGATCGTTGTCA K E D L L M K F D D N Y Y T Q D N D TTCTCCAAAGAATATATCTAATAAGTAGAGTCCTCATGATTAGTTAACAACTATTTTTT N R W L Y R L. L T R M. CNL 14 OkDa 732 ATGAAATCAATTAGTACACCGCTATGTTTAATACTTATTCATATTTTAGTTTTTAGGA

TTGAGAATCAATACAAAAATAATGCATCATTAATTTAGAAATACTTAGTTTCCACGTA CF Y K T E W Y 169 7441 GTTAATGAAACATTTGAACTCATCGTACAGGACGTTCTCGTACAGGACGTAACTATAAAC CN F C K F E D Y L V N E Y L. V Y S Y V CGGTTTATATTTGTTCAAGATAGATACAAATCCGATAACTTTTTTTACGAATICTACGGG P K Y K. N. L. I. S. V. F G | V K K V F E W P 129 FIG.8G U.S. Patent July 6, 1993 Sheet 18 of 60 5,225,336

7561 ATCCACTTTAAAAGTGTCATACGGGGTTCTTTTTATTTTTTTAAACAGATCAAGGTGTG CD V K F T D Y P T R K I K K F L D I T H ATGTTGATTAGGTCTTTTACGAATTGATATAGAATAGCGTTTACATATTCTCCATAATG H Q N P R K R Q Y L I A N W Y E G Y H 89 768 GTCAATCGCCATTTGTTCGTATGTCATAAATTCTTTAATTATATGACACTGTGTATTGTT CD I A M Q E Y T M F E K I I H C Q T N N TAGTTCATCCTTGTTCATTGTTAGGAATCTATTCAAAATGGCAATTATACTAGAACTATA L E D K N M T. L. F R N L. I. A I I S S S Y 49 780 GGTGCGTTGTATACACATATTGATGTGTCTGTTATACAATCCATGATATTTGGATCCAT CT R Q C M N | H R N J C D M I N P D M GCTACTACCTTCGGGTAAAATTGTAGCATCATATACCATTTCTAGTACTTTAGGTTCATT S S G E P L I T A D Y W M E L V K P E N 9 792 GTTATCCATTGCAGAGGACGTCATGATCGAATCATAAAAAAATATATTATTTTTATGTTA CN D M A S S T M CN2L 20,8kDa TTTTGTTAAAAAAATCATCGAATACTTCGTAAGATACTCCTTCATGAACATAATCAGTT KF Y D D F W E Y S W G E H W Y D T 456 8041 ACAAAACGTTTATATGAAGTAAAGTATCTACGATTTTTACAAAAGTCCGGATGCATAAGT CW F R K Y S T F Y R R N K C F D P H M A CAAAGTACGCGATAAACGGAATAATAATAGATTTATCTAGTTTATCTTTTTCTATAGCT W. F. Y. A F P I I I S K D L. K D K E I A ll6 86 TTCATAGTTAGATA CATGGTCTCAGAAGTAGGATTATGTAACATCAGCTTCGATAAAATG CK M T L Y M T E S T P N H L M L K S L I ACTGGGTTATTTAGTCTTACACATTCGCTCATACATGTATGACCGTTAACTACAGAGTCT W P N N L R W C E S M C T H G N V V S D 376 828. ACACTAAAATGATTGAACAATAGATAGTCTACCATTGTTTCGTATTCAGATAGTACAGCG CW S F H N F L L Y D W M T E Y E S L W A TAGTACATAGCATCTTCACAAATTATATCATTGTCTAATAGATATTTGACGCATCTTATG Y Y M A D E C D N D L L Y K W C R 336 8401 GATCCCACTTCA ACAGCCATCTTAAAATCGGTAGAATCATATTGCTTTCCTTTATCATTA CS G W E W A M K F D T S D Y Q K G K D N

ATAATTTCTAGAACATCATCTCTATCATAAAAGATACAAATATTAACTGTTTGATCCGTAI I E L W D D R D Y F I C J N W T Q D T 296 852. ATAACATTGCTAGTCGATAGCAATTGAATAAGAGCGCTGGGCTCAATGTCTTAATA {I V N S T S L K N I L H A P S L T K I AGAAGTGTAAGAGGACTATCTCCGAATTTGTTTTGTTTATTAACATCCGTTGATGGAAGT L. L T L P S D G F K N Q. K N V D T S P L 256 FIG.8H U.S. Patent July 6, 1993 Sheet 19 of 60 5,225,336

864 AAAAGATCTATAATGTCTACATTCTTGACTGTTTTAGAGCATACAATATGGAGAGGTGTA CL - D I D V N K W T K S C W I H L P T TTTCCATCATGATCTGGTTTTGAGGGACTAATTCCTAGTTTCATCATCCATGAGATTGTA N G D H D P K S P S I G L K M M W S I T 216 8761 GAAGCTTTTGGATTGTCTGACATAAGATGTCTATGAATATGATTTTTGCCAAATTTATCC CS A K P N D S M L H R H I H.N. K. G. F. K D ACTATCCTGGCTTCGAATCCGATGGACATTATTTTTTTAAACACTCTTTCTGAAGGATCT W I R A E, F G 1 S M I K K F W R E S P D 76 888 GTACACGCCAACAACGGACCACATCCTTCTTCATCAACCGAGTTGTTAATCTTGGCTCCA {T C A P G C G E E D W S N N I K A G TACTGTACCAATAAATTTATTCTCTCTATGACTTCATCATCTGTTCCCGAGAGATAATAT Y Q W L N I R E I W E D D T G S L Y Y 36 900 AGAGGTGTTTATATGTTTATCACACGCGTTTGGATCTGCGCCGTGCGTCAGCAGCATC CL P T K N H K D C A N P D A G H T L L M GCGACTATTCTATATTATTAATTTTAGAAGCTATATGCAATGGATAATTTCCATCATCA A W H R N N N I K S A I H L P Y N G D D 96 912. TCCGTCTCATTTGGAGAGTATCCTCTATGAAGAAGTTCTTCGACAAATCGTTCATCTAGT KD T E N P S Y G R H L L E E W F R E D L CCTTTAATTCCACAATACGCATGTAGAATGTGATAATTATTTCCAGAAGGTTCGATAGCT G K G C. Y. A H L I H Y N N G S P E I A 56 924l TGTAGCATATTCCTAAATACATCTAAATTTTTACTATTATATTTGGCATAAAGAGATAGA KQ L M N R F W D N K S N Y K A Y L S TAATA CTCGGCCGACATAATGTTGTCCATTGTAGTATAAAAATAATATTTCTATTTCTA Y Y E A S M N D M T T Y F N I N R N R 6 9361 TTTCTGTATATTTGCAACAATTTACTCTCTATAACAAATATCATAACTTAGTTCTTTTAT KN R Y Q. L. L. K S E W F M {Mill 542 kDa CE R Y C I D Y S L E K I GTCAAGAAGGCACTGGTTTAGTTCATCTATAAATGTCACGCCATAACTACCACGCATGCC D L L C Q N E D F T W G Y S G R M G 89 9481 ATACTCAGAATTATGATAAAGATATTATCCTTGGGGTGAGGTAATGGGGATTAATCTT CY E S N H Y L. Y. K D K P H L Y H P N I K TGTTGGATCAGTCTCTAAGTTAACACATGTCACACATGATCCATTTATAGTTATATCACA T P D T E L N V C T V C S G N I T I D C 149 96.Oil CGAGATGATTTATGAATTGATTCCGGAAGATCGCATCGTATTTTGTGGTTCCACAATT CS S S K H I S E P L D S D Y K T G C N CATTTCCATACATGTTATTGTCACACTAATATTATGATGAACTTTATCAGCCGCTGAGT M E M C T | T W S N H H V K D L R Q T 109 FIG.8 U.S. Patent July 6, 1993 Sheet 20 of 60 5,225,336

972). GGTAAACAACAGAACAGATAGTTTATATCTTACCAA CACCCTCAGCCGCTGCCACAAA {T F L L V S L K N D K G W G E A A A W F TCTCTGATCCGTATCCATGATGGTCATGTTTATTTCTAGTCCGTATCCAGTCAACACTAT R Q D T D M I T M N E L G Y G T L V 69 984 GTAGCATTTCTGTCGATATAGCTTTCACTCATATGACACTCACCAATAATAGTAGAATT KN A N R D I. Y S E S M H C E G I T S N AATGTCGTAATTTACACCAATAGGAGTTCGGCGGCAAAGTACCAATACCGGTAATCTTG I D Y N W G I. T L E A A F Y W Y R Y D Q 29 996 TCGAGGAGGACATATAGATTCTTGTATTCTACCGAATACCCGAGAGATGCGATACAAAA {R P P C T N K Y E W S Y G L S A I C F GAGCAAGACTAATTTGTAAA CCATCTTACT CAAAATATGTAACAATAGTACGATGCAATG L W L K Y W M CM2L 25, 1 kDa 10081 AGTAAGACAATAGGAAATCTATCTTATATACACATAATTATTCTATCAATTTTACCAATT AGTTAGTGTAATGTTAACAAAAATGTGGGAGAATCTAATTAGTTTTTCTTTACACAATTG CN K K W C N 278 1020 ACGTACATGAGTCTGAGTTCCTTGTTTTTGCTAATTATTTCATCCAATTTATTATTCTTG CW Y M L R L E K N K S I I E D L K N N K ACGATATCGAGATCTTTTGTATAGGAGTCAGACTTGATTCAA CATGCTTTTCTATAATC W I D L. D. K. T. Y S D S K Y E W H K E I 238 10321 ATCTTAGTTATTTCGGCATCATCCAATAGTACATTTTCCAGATTAACAGAGTAGATATTA gM K T | E A D D L L V N E L N V S Y I N ATGTCGTATTTGAACAGAGCCTGTAACATCTCAATGTCTTTATTATCTATAGCCAATTTA I D Y K F L A Q L. M. E I D K N K I A L K 198 1044 ATGTCCGGAATGAAGAGAAGGGAATTATTGGTGTTTGTCGACGTCATATAGTCGAGCAAG { D P I F L L S N N T N T S T M Y D L. L. AGAATCATCATATCCACGTGTCCATTTTTTATAGTGGTGTGAATACAACTAAGGAGAATA L I M M D W H G N K I T T H I C S L L I 158 10561 GCCAGAT CAAAAGTAGATGGTATTTCTGAAAGAAAGTATGAACAATACTTACATCATTA CA. L. D F T S P I E S L. F Y S W I S W D N

AGCATGACGGCATGATAAAATGAAGTTTTCCATCCAGTTTTCCCATAGAACATCAGTCTCL M W A H Y F S T K W G T K G Y F M L R 18 1068). CAATTTTTCTTAAACAGTTTCACCGTTTGCATGTTACCACTATCAACCGCATAAACAAT CW N K K F L K W T Q M N G S D W A Y Y L. GCGGGTTTCCTTGTCATCAAATTGTGAATCATCCATTCCACTGAATAGCAAAATCTTT A N G K D D F Q S D D M G S F L I K 78 FIG.8J U.S. Patent July 6, 1993 Sheet 21 of 60 5,225,336

1080 ACTATTTTGGTATCTTCTAATGTGGCTGCCTGATGTAATGGAAATTCATTCCTAGAAGA {W I K T D E L T A A Q H L P F E N E L L TTTTTCAATGCTCCAGCGTTCAACAACGTACATACTAGACGCACGTTATTATCAGCTATT . N K L A G A N L. L T C W L R V N N D A 38 092 GCATAATACAAGGCACTATGTCCATGGACATCCGCCTTAAATGTATCTTTACTAGAGAGA CA Y Y L. A S H G H W D A K F T D K S S L AAGCTTTTCAGCTGCTTAGACTTCCAAGTATTAATTCGTGACAGATCCATGTCTGAAA CG K L Q K S K W T N I R S L D M CKll L 325kDa 104 AGA CGCTAATTAGTGTATATTTTTTCATTTTTTATAATTTTGTCATATTGCACCAGAATT AATAATATCTCTAAT AGATCTGATTAGTAGATACAGGCTATCGCAAAACAACAATACA l61 CATTTAATAAAAATAATATTTATTAAGAAAATTCAGATTCACGTACCCATCAATATAAA

TAAAATAATGATTCCTTCCACCGTATCCATAAACAATATTAAGGAGATICTACCTTACCCCP S E V K G 364 28 ATAAACAATATAAATCCAGTAATATCATGTCTAATGATGAACACAAATGGTGTATTAAAT CM F L J F G T | D H R F W F P T N F TCCAGTTTTTCAGGAGATGATCTCGCCGTAGCTACCATGATAGTAGATGCCTCTGCTACA E L K E P S S R A A W M I T S A E A W 324 1140l GTTCCTTGTTCGTCGACATCTATCTTTGCATTCTGAAACATTTTATAAATATATAATGGG {T G Q E D W D I K A N Q F M K Y I Y L. P TCCCAGTCATATGTTTAAA CGACGCATTATCTGGATTAAACATACTAGGAGCCATCATT D R T M H K F S A N D P N F M S P A M M 284 1521. TCGGCTATCGACTTAATATCCCTCTTATTTTCGATAGAAAATTTAGGGAGTTTAAGATTG KE A S K I D R K N E I S F K P L K L N TACA CTTTATTCCCTAATTGAAA CGACCAATAGTCTAATTTTGCAGCCGTAATAGAATCT Y V K N G L Q F S W Y D L. K. A A T I S D 244 11641 GTGAAATGGGTCATATTATCACCTATTGCCAGGTACATACTAATATTAGCATCCTTATAC CT F H T M N D G A Li Y M S I N A D K Y GGAAGGCGCACCATATCATATTCTTCGTCATCGATTGTGATTGTATTTCCTTGCAATTTA P L R W M D Y E E D D T T N G Q L. K. 204 1761 GTAACTACGTTCATCATGGGAACCGTTTCGTACCGTACTTATTAGTAAAACAGCATTG CT V V N M M P W T K T G Y K N T F S A N CGTGTTTTAGTGATATCAAACGGATATTGCCATGTACCTTTAAAATATATAGTATTAATG R T K T D F P Y Q W T G K F Y I T N 164 FIG.8K U.S. Patent July 6, 1993 Sheet 22 of 60 5,225,336

1881 ATGCCCATAGAGTATTATTGTCGAGCAATTAGAATCTACA CATTAGACATACCGGAT CI A W L T N N D M N S D V V N S M G S CTACGTCTACTATAGAATTAATTTTATTAACCGCATCTCGTCTAAAGTTTAATCTATAT R R E V S N K N V A D R R F N L R Y 124 1200l AGGCCGAATCTATGATATTGTTGATAATACAACGGTTTAATGCACACAGTATTATCTACG CL G F R H Y Q Q Y Y L. P K I C W T N D V AAACTTGATAAGTTAGATCAGTGACGTATATTTAGATGTTTCAGCTTAGCTAATCCT F S Q Y T L D T Y T Y K S T K L K A L G 84 l2121 GATATTAATTCTGTAAATGCTGGACCCAGATCTCTTTTTCTCAAATCCATAGTCTTCAAT CS I L E T F A P G L D R K R L D M T K L. AATTCTATTCTAGTATTACCTGATGCAGGCAATAGCGACATAAA CAT AGAAAACGAATAA E I R T N G S A P L L S M F M S F S Y ll 122ll CCAAACGGTGAGAAGACAAATTATCATCTTGAATATTTTTATA CGCTACTATACCGGCA gG F P S F W I N D D Q I N K Y A W H G A TTGGTAAATCCTTGCAGACGATAGGTAGACACTGAACACGTTAACGATAGTATCAATAAC N T F G Q L R Y T S W S C T L S L I L. L. l 12361 GCAATCATGATTTTATGGTATTAATAATTAACCTTATTTTTATGTTCGGTATAAAAATTA {A M {K2L 42.3 kDa - TTGATGTCTACACATCCTTTTGTAATTGACATCTATATATCCTTTTGTATAATCAA CTCT CQ H R C M R K Y N W D Y G K T Y D W R 69 1248l AATCACTTTAACTTTTACAGTTTTCCCTACCAGTTTATCCCTATATTCAA CATATCTATC CI V K V K W T K G W L K D R Y E W Y R D CATATGCATCTTAACA CTCTCTGCCAAGATAGCTTCAGAGTGAGGATAGTCAAAAAGATA M H M K W S E A L I. A E S H P Y D F L Y 29 1260 AATGTATAGAGCATAATCCTTCTCGTATACTCTGCCCTTTATTACATCGCCCGCATTGGG {I Y L. A Y D K E Y V R G K I W D G A N P CAACGAATAACAAAATGCAAGCATCTTGTTAACGGGCTCGAAATTGGGATAAAAATTAT L S Y C F A L. M CK3L 10,5kDa l272 GTTTTTATATCTATTTTATTCAAGAGAATATTCAGGAATTTCTTTTTCCGGTTGTATCTC CE L S Y E P I E K E P Q E ATCGCAGTATATATCATTTGTACATTGTTTCATATTTTTTAATAGTTTACACCTTTTAGT D C Y D N T C Q K M N K L L K C R K T 391 284 AGGACTAGTATCGTACAATTCATAGCTGTATTTTGAATTCCAATCACGCAAAAAATATC CP S T D Y L E Y S Y K S N W D R M F I D TTCCAATTGTTGACGAAGACCTAATCCATCATCCGGTGTAAATTAATAGATGCTCCACA E L Q Q R L G L G D D P T I N I S A G C 551 FIG.8L

U.S. Patent July 6, 1993 Sheet 26 of 60 5,225,336

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(QudX)OAGOEyQSd

U.S. Patent July 6, 1993 Sheet 29 of 60 5,225,336

U.S. Patent July 6, 1993 Sheet 31 of 60 5,225,336

39 + O

U.S. Patent July 6, 1993 Sheet 32 of 60 5,225,336

pSD42O pUC8 pSD454 Xibal HindIII Bgll Bgll O ECORV

B al 55ral Ban Na4. EcoR Ef in vivo recombination VP664 VP458 WP668 (deletion C6 through KL (deletion C7L through K1 plaques on MRC-5) no plaques on MRC-5) FIG 4 U.S. Patent July 6, 1993 Sheet 33 of 60 5,225,336

FIG 5 Dra MPSYN238 pMPCTK1 -> --> p"PCS-1 partial MPSYN239

P.St. MPSYN249 pMPCS-1 -> --> pCOPCS-4

HindIII H6 pMPCS-1 -> --> pCOPCS-3H Asp718 promoter

PSt. MPSYN249 pCOPCS-3H -> --> pCOPCS-5H

Nru MPSYN250 pCOPCS-5H --> -> pCOPCS-6H Bg II MPSYN251

Nru MPSYN252 pCOPCS-5H --> -> pCOPCS-7H Bg II MPSYN253

Niru MPSYN254 pCOPCS-5H --> -> pCOPCS-8H Bgl II MPSYN255

Niru MPSYN271 pCOPCS-6H --> -> pCOPCS-9H Asp718 MPSYN272

Nru MPSYN273 pCOPCS-6H --> -> pCOPCS-10H Asp718 MPSYN274

U.S. Patent July 6, 1993 Sheet 39 of 60 5,225,336

FIG. 8

ARM FROM VP452 ARM FROM HindIII F Xhol Hind B isolate termini pUCS/eOW pSD478VC Smal EC Ncol "Bgll -/ ind Cal ECOR Smal Clal KlenOW Ball Hindl Bgll isolate MPSYN26 M isolate fragment 262 fragment

D indl

Clai Bg KlenOW Bgll pMPRENDA Cama-n-Ho Ncolxy 2 BamH Bglica LEFT DELETION RIGHT DELETION PLASMD PLASMD U.S. Patent July 6, 1993 Sheet 40 of 60 5,225,336

K K

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U.S. Patent July 6, 1993 Sheet 42 of 60 5,225,336

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19IO 0+NÅ1X|M|__H_2

U.S. Patent July 6, 1993 Sheet 52 of 60 5,225,336

S.W+1SH|I||'SÅT:N3-KSK3 480

1010—>(yz)y— 3HH00NSIÒIld_M_3'N}+1(1S(I]|?}{T+N34×HA8G 0D.C1

U.S. Patent July 6, 1993 Sheet 55 of 60 5,225,336

K0+80

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U.S. Patent July 6, 1993 Sheet 57 of 60 5,225,336

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5,225,336 1. 2 gives a poxvirus modified by the presence, in a nones RECOMBINANT POXVIRUS HOST RANGE sential region of its genome, of foreign DNA sequences. SELECTION SYSTEM The term “foreign' DNA designates exogenous DNA, particularly DNA from a non-pox source, that codes for This invention was made with Government support 5 gene products not ordinarily produced by the genome under contract DAMD17-85-C-5232 awarded by the into which the exogenous DNA is placed. Department of the Army. The Government has certain Genetic recombination is in general the exchange of rights in this invention. homologous sections of DNA between two strands of CROSS REFERENCE TO RELATED DNA. In certain viruses RNA may replace DNA. Ho 10 mologous sections of nucleic acid are sections of nucleic APPLICATIONS acid (DNA or RNA) which have the same sequence of This application is a continuation of application Ser. nucleotide bases. No. 07/478,179, filed Feb. 14, 1990, now abandoned Genetic recombination may take place naturally dur which is a continuation-in-part of application Ser. No. ing the replication or manufacture of new viral genomes 07/320,471, filed Mar. 8, 1989, now U.S. Pat. No. 15 within the infected host cell. Thus, genetic recombina 5,155,020. tion between viral genes may occur during the viral replication cycle that takes place in a host cell which is FIELD OF THE INVENTION co-infected with two or more different viruses or other The present invention relates to modified recombi genetic constructs. A section of DNA from a first ge nant viruses, methods for expressing gene products in a 20 none is used interchangeably in constructing the sec host using such modified recombinant viruses, and to tion of the genome of a second co-infecting virus in vaccines comprising such modified recombinant vi which the DNA is homologous with that of the first SeS. viral genome. The present invention also relates to modified poxvi However, recombination can also take place between rus, particularly modified vaccinia virus, and to meth 25 sections of DNA in different genomes that are not per ods of making and selecting for the same. More in par fectly homologous. If one such section is from a first ticular, the invention relates to a selection system for genome honologous with a section of another genome the cloning and expression of an open reading frame in except for the presence within the first section of, for recombinant poxvirus, particularly recombinant vac example, a genetic marker or a gene coding for an anti cinia virus. 30 genic determinant inserted into a portion of the homolo Several publications are referenced in this application gous DNA, recombination can still take place and the by arabic numerals within parentheses. Full citation to products of that recombination are then detectable by these references is found at the end of the specification the presence of that genetic marker or gene in the re immediately preceding the claims. These references combinant viral genome. describe the state-of-the-art to which this invention 35 Successful expression of the inserted DNA genetic pertains. sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessen BACKGROUND OF THE INVENTION tial region of the virus in order that the modified virus Vaccinia virus and more recently other poxviruses remain viable. The second condition for expression of have been used for the insertion and expression of for inserted DNA is the presence of a promoter in the eign genes. The basic technique of inserting foreign proper relationship to the inserted DNA. The promoter genes into live infectious poxvirus involves recombina must be placed so that it is located upstream from the tion between pox DNA sequences flanking a foreign DNA sequence to be expressed. genetic element in a donor plasmid and homologous Unperturbed, successful recombination occurs at a sequences present in the rescuing poxvirus (32). 45 frequency of approximately 0.1%. Specifically, the recombinant poxviruses are con A basic screening strategy for recovering those vi structed in two steps known in the art and analogous to ruses modified by a successful recombination involves the methods for creating synthetic recombinants of the in situ hybridization of recombinants on replica filters vaccinia virus described in U.S. Pat. No. 4,603, 112, the with a radiolabeled probe homologous to the inserted disclosure of which patent is incorporated herein by 50 sequences (26,28). A number of modifications have been reference. reported to increase the efficiency of recombination First, the DNA gene sequence to be inserted into the itself o to facilitate the identification of recombinants. virus, particularly an open reading frame from a non Among these modifications are included: using single pox source, is placed into an E. coli plasmid construct stranded donor DNA (38); identification of recombi into which DNA homologous to a section of nonessen 55 nants expressing unique enzymatic functions such as tial DNA of the poxvirus has been inserted. Separately, 12.Iododeoxycytidine incorporation into DNA via ex the DNA gene sequence to be inserted is ligated to a pression of the Herpes simplex virus thymidine kinase promoter. The promoter-gene linkage is positioned in (28); chromogenic substrates for (co)expression of for the plasmid construct so that the promoter-gene linkage eign genes along with B galactosidase (3,29); selection is flanked on both ends by DNA homologous to a DNA for thymidine kinase expression (20,28); antibody based sequence flanking a nonessential region of pox DNA. reactions to visualize recombinant plaques (21); use of The resulting plasmid construct is then amplified by conditional lethal ts or drug mutants (9,18); selection of growth within E. coli bacteria (4) and isolated (5,22). recombinants using the neomycin resistance gene from Second, the isolated plasmid containing the DNA Tn5 and the antibiotic G418 (11); or selection pressures gene sequence to be inserted is transfected into a cell 65 with mycophenolic acid and the E. coli gpt gene (2,8). culture, e.g. chick embryo fibroblasts, along with the Disadvantageously, these known methods for identi poxvirus. Recombination between homologous pox fying or selecting recombinant poxvirus all involve DNA in the plasmid and the viral genome respectively tedious multi-step identification of the recombinants, 5,225,336 3 4. the introduction of radiochemicals, chromogenic sub portions of the organism responsible for the replication strates, biochemicals useful for selection such as myco of the pathogen. phenolic acid and bromodeoxyuridine which may be Thus, it can be appreciated that a method which detrimental (mutagenic) to the viral genome itself, use confers on the art the advantages of live virus inocula of serological reagents that may introduce contami tion but which reduces or eliminates the previously nants, and typically the presence of an exogenous gene discussed problems would be a highly desirable ad in the final recombinant in addition to the foreign ge vance over the current state of technology. This is even netic element of interest. more important today with the advent of the disease It can thus be appreciated that provision of a method known as acquired immune deficiency syndrome of making and selecting for poxvirus recombinants, 10 (AIDS). Victims of this disease suffer from severe im particularly vaccinia recombinants, which method munological dysfunction and could easily be harmed by avoids the previously discussed problems, would be a an otherwise safe live virus preparation if they came in highly desirable advance over the current state of tech contact with such virus either directly or via contact nology. with a person recently immunized with a vaccine com Methods have been developed in the prior art that 15 prising such a live virus. permit the creation of recombinant vaccinia viruses and OBJECTS OF THE INVENTION avipox viruses by the insertion of DNA from any source (e.g. viral, prokaryotic, eukaryotic, synthetic) into a It is therefore an object of the present invention to nonessential region of the vaccinia or avipox genome, provide a vaccine for inducing an immunological re 20 sponse in a host which has the advantages of a live virus including DNA sequences coding for the antigenic vaccine, and which has few or none of the disadvan determinants of a pathogenic organism. Recombinant tages of either a live virus vaccine or a killed virus vaccinia viruses created by these methods have been vaccine as enumerated above. used to induce specific immunity in mammals to a vari It is a second object of this invention to provide mod ety of mammalian pathogens, all as described in U.S. 25 ified recombinant viruses for use in such vaccines. Pat. 4,603,112, incorporated herein by reference. Re It is an additional object of this invention to provide combinant avipox viruses created by these methods a method for expressing a gene product in a host by have been used to induce specific immunity in avian inoculating the host with a modified recombinant virus species (41) and in non-avian species (42). which codes for and expresses the gene product in the Unmodified vaccinia virus has a long history of rela 30 host with restricted replication of the virus in the host. tively safe and effective use for inoculation against It is also an object of the invention to provide meth smallpox. However, before the eradication of smallpox, ods for expressing a gene product in a cell cultured in when unmodified vaccinia was widely administered, vitro, which method comprises introducing into the cell there was a modest but real risk of complications in the a modified recombinant virus containing DNA which form of generalized vaccinia infection, especially by 35 codes for and expresses the gene product with restricted those suffering from eczema or immunosuppression. replication of the virus in the cell. Another rare but possible complication that can result It is a further object of this invention to provide mod from vaccinia inoculation is post vaccination encephali ified recombinant viruses, which modified recombinant tis. Most of these reactions resulted from inoculating viruses express gene products in a host with restricted individuals with skin diseases such as eczema or with replication of the viruses in the host, and to provide a impaired immune systems, or individuals in households method of making such modified recombinant viruses. with others who had eczema or impaired immunologi It is a further object of this invention to provide rapid cal responses. Vaccinia is a live virus, and is normally one-step identification of recombinant viruses and rapid harmless to a healthy individual. However, it can be screening for expression of the foreign open reading transmitted between individuals for several weeks after 45 frames in the recombinants. inoculation. If an individual with an impairment of the It is a further object of this invention to provide a normal immune response is infected either by inocula method of making and selecting for a recombinant pox tion or by contagious transmission from a recently inoc virus, particularly recombinant vaccinia virus, and to ulated individual, the consequences can be serious. provide DNA sequences, produced or involved as in Suitably modified virus mutants carrying exogenous 50 termediates in the method. genes which are expressed in a host as an antigenic It is a still further object of this invention to provide determinant eliciting the production by the host of anti a selection system for the cloning and expression of an bodies to a host pathogen with restricted replication of open reading frame in recombinant poxvirus, particu the virus in the host represent novel vaccines which larly recombinant vaccinia virus, wherein the recombi avoid the drawbacks of conventional vaccines employ 55 nant virus contains no foreign gene other than the open ing killed or attenuated live organisms. Thus, for in reading frame of interest. stance, the production of vaccines from killed organ These and other objects and advantages of the pres isms requires the growth of large quantities of the or ent invention will become more readily apparent after ganisms followed by a treatment which will selectively consideration of the following. destroy their infectivity without affecting their antige nicity. On the other hand, vaccines containing attenu STATEMENT OF THE INVENTION ated live organisms present the possibility of a reversion In one aspect, the present invention relates to a modi of the attenuated organism to a pathogenic state. In fied recombinant virus having host range genes deleted contrast, when a recombinant poxvirus suitably modi therefrom so that the virus has restricted replication in fied is used as a vaccine, the possibility of reversion to a 65 a host, wherein the modified recombinant viruscontains pathogenic organism is avoided since the poxvirus con DNA which codes for and expresses a gene product in tains only the gene coding for the antigenic determinant the host with restricted replication of the virus in the of the disease-producing organism and not those genetic host. The virus according to the present invention is 5,225,336 5 6 advantageously a poxvirus, particularly a vaccinia vi FIG. 2A schematically shows a method for cloning S. of the host range gene K1L into the plasmid pMP528L In another aspect, the present invention relates to a and its insertion into vF293 to generate vaccinia virus method for expressing a gene product in a host by inoc vP457; ulating the host with a modified recombinant virus FIG. 2B is a map of the left end of vP293 through having host range genes deleted therefrom so that the HindIII. K. virus has restricted replication in the host. The modified FIG. 2C is a map of the left end of vP457 through recombinant virus contains DNA which codes for and HindIII K; expresses the gene product in the host even with re FIG. 3A schematically shows a method for the con stricted replication of the virus in the host. The virus 10 struction of plasmids pMP528HRH and pHES1-4; used in the method according to the present invention is FIG. 3B shows the DNA sequence of the synthetic advantageously a poxvirus, particularly a vaccinia vi H6 promoter and downstream restriction sites present rus. The gene product expressed in the host is advanta in pMP528HRH; geously an antigen. More in particular, the host is a FIG. 3C shows the DNA sequence (with restriction vertebrate and the antigen induces an immunological 15 sites, stop codons and early transcriptional termination response in the vertebrate. signal) which replaces the bracketed sequence of FIG. In yet another aspect, the present invention relates to 3B in plasmid pHES1; a vaccine for inducing an immunological response in a FIG. 3D shows the DNA sequence (with restriction host inoculated with the vaccine, said vaccine including sites, stop codons and early transcriptional termination a carrier and a modified recombinant virus having host 20 signal) which replaces the bracketed sequence of FIG. range genes deleted therefrom so that the virus has 3B in plasmid pHES2; restricted replication in the host. The modified recombi FIG. 3E shows the DNA sequence (with restriction nant virus contains DNA which codes for and expresses sites, stop codons and early transcriptional termination a gene product in the host even with restricted replica signal) which replaces the bracketed sequence of FIG. tion of the virus in the host. The Virus used in the vac 25 3B in plasmid pHES3; cine according to the present invention is advanta FIG. 3F shows the DNA sequence (with restriction geously a poxvirus, particularly a vaccinia virus. sites, stop codons and early transcriptional termination In a further aspect, the invention relates to a method signal) which replaces the bracketed sequence of FIG. for selecting for a recombinant poxvirus in a host by 3B in plasmid pHES4; combining donor DNA and a modified poxvirus to 30 FIG. 4A schematically shows a method for the con form a recombinant poxvirus and identifying the recom struction of plasmids pHES31-34; binant poxvirus by its ability to replicate in the host. In FIG. 4B shows the DNA sequences of the synthetic a still further aspect, the invention relates to a method oligonucleotides HRL15-22; for cloning and expressing an open reading frame in a FIG. 5 shows the DNA sequence of the vaccinia u recombinant poxvirus in a host by combining donor 35 promoter present in plasmids pHES31-34. Additionally, DNA and a modified poxvirus to form a recombinant FIG. 5 shows in bracketed sequence the restriction poxvirus, replicating the recombinant poxvirus in the sites, stop codons and early transcriptional termination host and expressing the open reading frame. According signals present in pHES31-34 and the initiation codons to the present invention, the modified poxvirus has a present in pHES31-33; host range gene deleted therefrom so that the modified FIG. 6A schematically shows a method for the con poxvirus does not replicate in the host and the donor struction of plasmids pHES61-64; DNA contains an open reading frame from a non-pox FIG. 6B shows the DNA sequences of the synthetic source and the host range gene for permitting the re oligonucleotides HRL33-40; combinant poxvirus to replicate in the host. FIG. 7 shows the DNA sequence of the synthetic In still another aspect, the invention relates to a donor 45 ATI promoter present in plasmids pHES61-64. Addi plasmid for making the recombinant poxvirus of the tionally, FIG. 7 shows in bracketed sequence the re selection system. The donor plasmid contains an open striction sites, stop codons and early transcriptional reading frame from a non-pox source and a host range termination signals present in pHES61-64 and the initia gene for permitting the recombinant poxvirus to repli tion codons present in pHES61-63; cate in the host. Advantageously, the donor plasmid 50 FIG. 8, PARTS A-O, shows the DNA sequence may also contain a promoter upstream from the poxvi (with restriction sites) of 15,537 bp located near the left rus host range gene, a translation initiation codon down end of the Copenhagen strain of vaccinia; stream from the promoter followed by unique multiple FIGS. 9-1 and 9-2 schematically show a method for restriction sites, translational termination signal sequen the construction of recombinants vP548 and vP661; ces and an early transcription termination signal se 55 FIG. 10 is a map of the left end of the vaccinia virus quence. genome; FIG. 11 schematically shows a method for the testing BRIEF DESCRIPTION OF THE DRAWINGS of potential vaccinia host range genes in the vP293 A better understanding of the present invention will system; be had by referring to the accompanying drawings, in FIG. 12 schematically shows a method for the con which: struction of recombinants vF665, vP683, vp706 and FIG. 1A schematically shows a method for the con vP716; struction of the vaccinia virus deletion/recombinant FIG. 13 schematically shows a method for the con vP293; struction of plasmids pCP3 and pCP5 and for the testing FIG. 1B is a map of the left end of the rescuing vac 65 of a potential cowpox host range gene in the vaccinia cinia virus VTK79 through HindIII K; system; FIG. 1C is a map of the left end of the vaccinia virus FIG. 14 schematically shows a method for the con deletion/recombinant vP293 through HindIII K; struction of recombinants vP66 and vp668; 5,225,336 7 8 FG. 15 schematically shows a method for the con resistance gene from transposon Tn5 and selection with struction of a series of plasmids derived from the antibiotic G418. This deletion/recombinant, VF293, pMPCTK1A; lacks approximately 21.7 Kb of DNA beginning 3.8Kb FIG. 16 shows the DNA sequences of synthetic oli from the left end of the genome. vP293 is capable of gonucleotides MPSYN238, MPSYN239, MPSYN250 plaquing on primary chick embryo fibroblasts (CEF), 255 and MPSYN271-274; two monkey cell lines (BSC-40 or VERO) but is defec FIGS. 17-1, 17-2, and 17-3 show the synthetic DNA tive in replication in the human cell line MRC-5. sequence containing restriction sites, stop codons and Insertion of the host range gene, K1L, into vP293 early transcriptional termination signals present in plas restores the ability for growth on MRC-5 cells. mids pMPCS-1 and pMPCS-4. Additionally, FIG. 17 10 A series of plasmids were constructed which in addi shows the synthetic H6 promoter region present in tion to the K1 L host range gene contain a vaccinia pCOPCS-3H and pCOPCS-5H through pCOPCS-10H. early/late promoter, H6, preferably followed by unique Additionally, FIG. 17 shows in bracketed sequence the polylinker sequence multicloning restriction sites, trans restriction sites, stop codons and early transcriptional lational initiation and termination codons, and an early termination signals present in pCOPCS-3H and 15 transcription termination signal. These plasmids, pCOPCS-5H through pCOPCS-10H and the initiation pMP528HRH and pHESI-4, allow for the rapid single codons present in pCOPCS-6H through pCOPCS-10H; step, cloning and expression of any open reading frame FIG. 18 schematically shows a method for the con when recombined with vp293 and scored for growth on struction of plasmids pMPLENDA and pMPRENDA; MRC-5 cells. FIG. 19, PARTS A-P, shows the DNA 13,978 bp 20 Insertion of a foreign open reading frame into these sequence from HindIII C of the vaccinia virus Copen plasmids followed by recombination with vP293 will hagen genome, including coding sequences located to simultaneously restore the host range function (K1L the left of the sequence presented in FIG. 8; gene) and introduce the foreign open reading frame into FIG. 20, PARTS A-R, shows the complete DNA the rescuing virus, vP293. The recombinant viruses are sequence for HindIII Flocated immediately to the right 25 identified by their ability to plaque on MRC-5 cells. of HindIII K in FIG. 8; and Advantages of this system include the absence of any FIG. 21, PARTS A-U, shows the DNA sequence non-vaccinia exogenous gene in the final recombinant contained in HindIII B near the right terminus of the other than the genetic element of interest, no genetic vaccinia virus genome. reversion of the virus since vF293 is a deletion mutant 30 of K1 L, and the rapid one step identification of recom DETAILED DESCRIPTION OF THE binants. This single step can also be used for rapid INVENTION screening of expression of the foreign gene, for exam The invention is directed to modified recombinant ple, for epitope mapping. viruses having host range genes deleted therefrom so Additional plasmids containing the KL host range that the virus has restricted replication in the host and 35 gene have been constructed where the H6 early/late containing DNA which codes for and expresses a gene promoter has been replaced with either a strictly early product in the host with restricted replication of a virus or a strictly late vaccinia promoter. With such addi in the host. The invention is also directed to a selection tional plasmids the subtleties of temporal regulation of system for poxvirus recombinants, particularly vaccinia expression of foreign genetic elements can be studied. recombinants, and for the cloning and expression of an The host range restricted systems of the present in open reading frame in poxvirus, particularly vaccinia vention advantageously ar used in vaccines for inducing virus, using a conditional lethal host range mutant of the an immunological response in a host inoculated with the poxvirus. vaccine. In this respect, the vaccine comprises a carrier Host range mutants of rabbitpox virus (24,13) and and a modified recombinant virus. The modified recom vaccinia virus (6,7,12,17,23,36) are known. 45 binant virus has host range genes deleted therefrom so Host range mutants of rabbitpox virus are believed to that the virus has restricted replication in the host. In be defective in some control function required for virus addition, the modified recombinant virus contains DNA replication (10). Subsequent genomic analysis of these which codes for and expresses a gene product in the rabbitpox virus mutants demonstrated extensive termi host with restricted replication of a virus in the host. nal deletions (up to 30 Kb) of DNA (19,25). 50 Modified recombinant viruses have been constructed Nitrous acid mutagenesis of the Copenhagen strain of which express gene products, particularly antigens, vaccinia virus allowed Drillien et al. (6) to isolate a host with restricted replication of the virus due to the dele range mutant defective in replication in most human tion of the host range genes in the virus. In one embodi cells. Genomic analysis of this mutant revealed an ex ment, the host is a vertebrate and the antigen induces an tensive deletion of approximately 18Kb toward the left 55 immunological response in the vertebrate. In another terminus (6). Additional analysis by marker transfer embodiment, the host is a cell cultured in vitro. studies mapped the genetic function responsible for host One can readily appreciate that additional viruses and range to a 5.2 Kb EcoRI fragment (14) and finally to an species beyond those cited in this application can be 855 bp open reading frame overlapping the HindIII scored for host range restriction. Moreover, one can M/K fragments (15). readily appreciate that additional "host range genes' The host range gene of the WR strain of vaccinia exist in poxvirus. Furthermore, one can readily appreci virus (27,30) is located between 24 and 25.2 Kb from the ate that a variety of foreign genes can be utilized in left end of the vaccinia genome. This host range gene, these host range mutants. transcribed leftward from HindIII Kinto HindIIIM, is A better understanding of the present invention and described herein as the K1 L gene following the nomen 65 of its many advantages will be had from the following clature recommended by Rosel et al. (33). examples, given by way of illustration. A host range gene deletion mutant of the vaccinia In some of these examples, the WR strain of vaccinia WR strain was generated by insertion of the neomycin virus was utilized. Its origin and conditions of cultiva 5,225,336 9 10 tion have been previously described (27). In some of tained the foreign gene encoding neo. The restriction these examples, the Copenhagen strain of vaccinia virus map of the left terminus of the rescuing virus VTK-79 was utilized. Its origin and conditions of cultivation and of the recombinant virus vP293 expressing the neor have been previously described (50). Primary chick gene and selected on primary CEF in the presence of embryo fibroblasts (CEF), monkey cell lines (VERO 5 G418 are indicated in FIGS. 1B and 1C. ATCC# CCL81) and BSC40), and the human cell line In the absence of the antibiotic G418, vP293 pro MRC-5 (ATCC# CCL171) were cultivated in Eagle's duced large plaques on primary CEF and plaqued well minimal essential medium (MEM) containing 5% on BSC40 or VERO cells although vP293 plaques were (VERO and BSC40) or 10% (MRC-5, CEF) fetal bo detectably smaller than the parent VTK-79 on VERO vine serum (FBS). 10 cells. Significantly, vP293 gave no measurable replica Plasmids were constructed, screened, and grown by tion and failed to form plaques on the human cell line standard procedures (22,31,32). MRC-5. EXAMPLE EXAMPLE 2 CONSTRUCTION OF PLASMID pMP528PiN AND 15 GENERATION OF VP293 RECONSTRUCTION OF vF293 WITH THE HOST RANGE GENE, K1L Referring to FIG. 1A, an EcoRI/Sali fragment com prising the left terminal 3.8 Kb of vaccinia DNA was To demonstrate that the host range gene, K1L, when isolated from paC5 (30) and inserted into puC13 previ reconstituted into the deletion mutant vP293 of the WR ously cut with EcoRI and SalI. The resulting plasmid, 20 strain of vaccinia would allow the virus to replicate on pMP5, was digested with HindIII and SalI and ligated human cells, the host range gene, K1L, was cloned into with a 3.8 HindIII/Sal fragment containing vaccinia the plasmid pMP528L and inserted into vP293. sequences corresponding to the right end of the vac Referring now to FIG. 2A, the vaccinia DNA se cinia HindIII fragment K. The resulting plasmid quence composing the right arm of pMP528L (FIGS. pMP528 thus contains the 3.8 Kb of the left terminus of 25 1A and 2A) was shortened to eliminate unwanted re the vaccinia genome and 3.8 Kb from the right end of striction sites and to facilitate future cloning steps. the HindIII K fragment deleting the intervening 21.7 pMP528L was cut by EcoRV/HindIII, blunt ended Kb between the Sall sites at 3.8 and 25.5 Kb from the with the Klenow fragment of the E. coli polymerase and left end of the genome. The unique Sal site in pMP528 self ligated. In this manner, the right arm of the result was changed to a SmaI site by addition of synthetic 30 ing plasmid pMP528E was reduced in length to 0.4. Kb linkers (commercially available from Collaborative of DNA. Research, Inc., Bedford, Mass.) producing pMP528L. An 891 bp vaccinia BglII (partial)/HpaI fragment A 1.4 Kb SmaI fragment containing the gene for neo containing the entire coding sequence and promoter mycin phosphotransferase from transposon Tn5(1) was from the K1L host range gene (15) was prepared from isolated from pSV2-neo (35, ATCC# 37149) and put 35 pSD452VC, a subclone of Copenhagen strain vaccinia under the control of an early vaccinia promoter (desig containing sequences from HindIIIM and K. The K1L nated here as Pi). The Pi promoter, from the Aval H region of vac containing fragment was cloned into the polylinker cinia, has been described (37). More in particular, this region of puC8 for the sole purpose of flanking the gene promoter is derived from the Aval H (XhoI G) frag- 40 with convenient restriction sites. The resulting plasmid ment of the L-variant WR vaccinia strain, in which the pUC8HR was digested with HindIII and SmaI to iso promoter directs transcription from right to left. The late the K1L containing fragment. The HindIII end was map location of the promoter is approximately 1.3 Kb filled in with the Klenow fragment of the E. coli DNA from the left end of Aval H, approximately 12.5 Kb polymerase and the fragment cloned in the SmaI site of from the left end of the vaccinia genome, and about 8.5 45 pMP528E. A plasmid pMP528HR with the orientation Kb left of the HindIII C/N junction. The promoter was of the K1L host range gene reading leftward as shown located by standard transcriptional mapping analysis. in FIG. 2A was isolated by standard procedures. The Pi DNA sequence corresponds to the region up Procedures for recombination and hybridization on stream from an open reading frame coding for a 5kDa nitrocellulose filters were as known in the art and as glycine-rich protein recently described (40). This pro- 50 previously described (28) with the following modifica moter element has been shown to express foreign genes tions. in vaccinia recombinants at early times after infection The donor plasmid pMP528HR was introduced by (37). electroporation into either VERO or MRC-5 cells each A SmaI ended Pi promoter/neo gene cassette was coinfected with vp293. Subconfluent monolayers of ligated into the SmaI site of pMP528L producing 55 VERO or MRC-5 cells were infected with rescuing pMP528PiN. pMP528PiN contains 0.4. Kb of vaccinia virus for 1 hour. The cells were harvested with trypsin, sequences derived from a Sau3A subclone of Aval H washed with Hepes buffered saline (HeBS) (16), and containing the Pi promoter region followed by 1 Kb of electroporated in the presence of 25 ug of plasmid DNA Tn5 sequences from the BglII through SmaI sites (I). in HeBS. Virus-infected cells were electroporated using pMP528PiN was transfected into primary CEF and 60 a Bio-Rad Gene Pulser equipped with a Bio-Rad Gene coinfected with the rescuing vaccinia virus, VTK79, Pulser Capacitance Extender. The cell suspension (0.8 by standard procedures (28). Recombinant virus was ml) was placed on ice for 10 minutes in a Bio-Rad gene selected and grown on primary CEF in the presence of pulser cuvette, then pulsed at 200 volts (capacitance 960 300 ug/ml G418 (1,11,35). uFD) and placed on ice for another 10 minutes. The The genomic configurations of recombinant vaccinia 65 cells were then diluted in 8ml MEM--5% FBS, plated vP293 were confirmed by Southern blot hybridization in 60 mm dishes containing corresponding VERO or analysis. The recombinant vaccinia vp293 had been MRC-5 cell monolayers (4 ml per dish), and incubated deleted of 21.7 Kb of vaccinia as predicted and con at 37 C. overnight. 5,225,336 11 12 Progeny was harvested and plated on either VERO oligonucleotides HRL1 5'(TCGACCATG or MRC-5 cells. The number of plaques obtained on GGATCCCCGGGTACCGAGCTCTCGAG VERO cells was 10 to 100 times greater than the num TAAATAAATAATTTTTAT)3' and HRL2 ber of plaques obtained on MRC-5 cells. Isolated 5'(CCGGATAAAAATTATTTATTTACT plaques (of uniform size) were picked from MRC-5 and CGAGAGCTCGGTACCCGGGGATC from VERO cell cultures (both large and small sized CCATGG)3' cloned into this site. pHES2, pHES3 and plaques). These plaque isolates were replated on VERO pHES4 were similarly constructed. pHES2 was con cells and after three days the resulting plaques were structed with the oligonucleotides HRL3 5'(TCGAC lifted onto nitrocellulose filter disks and prepared for in CATGGGGATCCCCGGGTACCGAGCTCT situ hybridization (26). All the plaques originating from 10 CGAGTAAATAAATAATTTTTAT)3 and HRL4 MRC-5 cells and all the larger plaques but not the 5'(CCGGATAAAAATTATTTATTTACT smaller sized plaques derived from VERO cells gave CGAGAGCTCGGTACCCGGGGATCC positive hybridization signals when probed with a 32P CCATGG)3', pHES3 was constructed with the oligo labeled probe to the K1L coding sequences. This data is nucleotides HRLS 5'(TCGACCATGGG consistent with restoration of host range functions con 5 tained in the K1L coding sequence. GGATCCCCGGGTACCGAGCTCTCGAG An isolate obtained from MRC-5 cells was further TAAATAAATAATTTTTAT)3' and purified and designated vP457. In vP457 the K1L gene 5'(CCGGATAAAAATTATTTATTTACT had been restored and was situated within the deletion CGAGAGCTCGGTACCCGGGGATCCC in its native orientation reading from right to left. The 20 CCATGG)3' and pHES4 was constructed with the K1L sequences had replaced the Pi promoter/neomy oligonucleotides HRL7 5'(TCGAGGATCCCGG cin phosphotransferase gene cassette present in vP293 GTACCGAGCTCTAAATAAATAATTTTTAT)3 as shown in FIGS. 2B and 2C. Compared to the genome and HRL8 5'(CCGGATAAAAATTATTTATT of the L variant vaccinia (30,27) vP457 contains a 291 TAGAGCTCGGTACCCGGGATCC)3'. bp deletion to the right of the K1 L gene and a 20.2 Kb 25 The pertinent DNA sequence elements, restriction deletion to the left of the K1 L gene. sites, and transcriptional and translational signals of pMP528HRH and pHES1-4 are as follows. EXAMPLE 3 The sequence of the synthetic H6 promoter (positions CONSTRUCTION OF PLASMIDS pMP528HRH - 124 through -1, with the altered base at position AND pHE81-4 - 102 underlined) and downstream restriction sites To demonstrate that the conditional lethal mutation present in pMP528HRH are shown in FIG. 3B. in vP293 could be exploited for constructing donor The bracketed sequence is replaced in plasmids plasmids into which additional open reading frames pHES1-4, with restriction sites, stop codons, and early could be cloned, a series of plasmids, pMP528HRH and transcriptional termination signal as indicated, as shown pHES1-4, were constructed. Recombination of exoge 35 in FIG. 3C for pHES1, in FIG. 3D for pHES2, in FIG. nous open reading frames present in a plasmid contain 3E for pHES3, and in FIG. 3F for pHES4. ing the K1L host range gene into vP293 would yield a In addition to the elements contained in simple method for generating vaccinia recombinants by pMP528HRH, each plasmid, pHES1-3, contains a trans virtue of host range restriction. lation initiation codon downstream from the H6 pro A vaccinia promoter, H6, was added upstream from moter followed by unique multiple restriction sites, the K1L gene in pMP528HR. This early/late promoter translational termination signal sequences, and a specific was previously identified by standard transcriptional vaccinia early transcription termination signal sequence mapping and DNA sequence analysis. It has the se (39). Each plasmid, pHES1-3, contains a translation quence (positions - 125 to +3) ATTCTTTATT initiation codon in one of the three reading frames. CTATACTTAAAAAAT 45 Therefore any DNA sequence which contains an open GAAAATAAATACAAAGGTTCTT reading frame can be expressed when cloned into one of GAGGGTTGTGTTAAATTG AAAGC these plasmids and recombined into vaccinia virus. GAGAAATAATCATAAATTATTTCATTATCG The fourth plasmid, pHES4, does not contain a trans CGATATCCGTTAAGTTTGTATCGTAATG. The lation initiation codon but does contain unique multiple sequence is that described as being upstream of open SO reading frame H6 by Rosei et al. (33). restriction sites, translational termination sequences, Referring now to FIG. 3, DNA corresponding to and an early transcription termination signal sequence. positions - 124 to -1 (with position -102 changed A DNA sequence which contains an open reading from A to G in order to prevent the introduction of any frame and an initiation codon can be expressed when potential initiation codons) and followed by XhoI, 55 cloned into pHES4 and recombined into vaccinia virus. Kipni, and SmaI restriction sites was synthesized chemi EXAMPLE 4 cally (FIG. 3B) and cloned into the SmaI site of INCORPORATION OF THE BACTERAL LACZ pMP528HR producing pMP528HRH (FIG. 3A). Thus, GENE INTO VACCNA VIRUS AND pMP528HRH contains the H6 promoter upstream from SELECTION OF THE RECOMBINANT VIRUSES the Kll gene which is expressed under the control of 60 the Kll endogenous promoter. Both are in a right to ON THE BASIS OF HOST RANGE left orientation with respect to vaccinia arms (genome). RESTRICTION The H6 promoter in pMP528HRH is immediately up To demonstrate the utility of the pHES1-4/VP293 stream of unique XhoI, Kipni, and SmaI restriction sites. host range selection system, a recombinant vaccinia To increase further the utility of the system a series of 65 virus containing the E. coli lacZ gene encoding B galac plasmids pHES1-4 were derived from pMP528HRH. tosidase was constructed. pHES1 was constructed by the following procedure: A BamHI fragment containing codons 8 through the pMP525HRH was cut with XhoI and XmaI, and the end of the lacz gene was obtained from pMC1871 (34). 5,225,336 13 14 This lacZ BamHI fragment was cloned into the unique toic membrane (CAM) has been reported (43). This BamHI site of the plasmids pHES1-4. gene, u, is highly expressed at early times post infection. Recombination between the resulting plasmids The u gene maps to a 1465 bp NcoI-HaeIII fragment. pHESLZ1-4 transfected individually into VERO cells The region of the Copenhagen strain vaccinia virus coinfected with the host range mutant vP293 was per genome containing the equivalent of the cowpox u gene formed. was determined by Southern blot analysis (44) using After 24 hours post infection, progeny virus was cloned cowpox DNA as a probe. The Copenhagen harvested by three freeze/thaw cycles and plated on equivalent of the cowpox u gene maps to HindIII B, and either VERO (Table 1A) or MRC-5 (Table 1B and 1C) corresponds to the location of the u gene in WR strain cells. 10 vaccinia virus recently reported (45). VERO and MRC-5 monolayers (Table 1A and 1B), In cowpox, the u promoter region is located down stained with neutral red, were lifted after 3 days onto stream from an Nicosite at -294 (43). DNA containing nitrocellulose filters and prepared for in situ hybridiza the Copenhagen u gene equivalent and promoter was tion (26) using a 32P labeled lacz gene probe. VERO sequenced (46). The Copenhagen u gene equivalent is (data not shown) and MRC-5 monolayers (Table 1C) 5 nonfunctional, resulting in white pock formation for were exposed to X-gal (5-bromo-4-chloro-3-indolyl-B- Copenhagen vaccinia virus grown on CAM due to D-galactopyranocide, Boehringer Mannheim) and blue frameshift mutations within the coding region. The color development scored after 8 hours. upstream region is highly homologous to the cowpox When progeny was plated on VERO cells and ex promoter region and functional. Recombinant vaccinia pression of B galactosidase assayed in the presence of 20 containing. E. coli beta-galactosidase expressed under X-gal no blue plaques were observed in cells transfected the control of the Copenhagen vaccinia u promoter with pHESLZ1, 2 or 4. Significantly, approximately form blue plaques in the presence of the chromogenic 20% of the plaques generated with plasmid pHESLZ3 substrate X-gal. As in cowpox, the Copenhagen genome gave blue plaques in the presence of X-gal (data not contains the Ncol site upstream from the u promoter shown). 25 region. When progeny was plated on VERO cells and re To move the Copenhagen u promoter region to the combinant viruses screened by in situ hybridization, 12 pHES system, a HindIII site was added to the Nco site to 22% of the plaques gave positive hybridization sig upstream from the u promoter by ligation with self nals to lacz (Table 1A). When analyzed by in situ DNA annealed oligonucleotide HRL14 (5' CATG hybridization (26) every plaque on MRC-5 demon 30 GAAGCTTC 3'; HindIII site underlined). A 299 bp strated the presence of the lacz gene (Table 1B). B HindIII-Clal fragment containing the u promoter re galactosidase activity, however, was seen only in those gion from the NcoI site through the Clal site at -6 was plaques on MRC-5 which were derived from isolated. The H6 promoter was removed from pHES1 pHESLZ3 (Table 1C). Only the plasmid construct (Example 3) by partial HindIII digestion, followed by pHESLZ3 had the lacZ gene in frame with the transla 35 digestion with KonI. Referring now to FIG. 4, the 7.8 tional initiation codon. kb HindIII-KpnI vector fragment was isolated from an TABLE 1 Analysis of recombinant lacZ/vaccinia virus generated with plasmids pHESLZ-4 and VP293 vaccinia virus Donor Plasmid pHESLZ) pHESLZ2 pHESLZ3 pHESLZ4 A. Total Plaques 056 637 793 1344 Hybridization 153 41 95 269 Positive Percent Positive 4.5 22 12 20 B. Total Plaques 60 56 ND 71 Hybridization 60 56 ND 71 Positive Percent Positive 100 00 100 C. Total Plaques 60 55 59 70 X-gal Positive O O 59 O Percent Positive 0 O 100 O

EXAMPLE 5 agarose gel (FIG. 4A). To replace promoter sequences CONSTRUCTION OF PLASMIDS pHES31-34 55 downstream from the Clal site and polylinker sequences AND pHES61-64 through the Kpnl site, eight oligonucleotides, HRL15 To demonstrate that the conditional lethal mutation through HRL22, were synthesized (FIG. 4B). Pairs of of vP293 could be exploited for constructing further oligonucleotides were annealed and ligated with the 7.8 donor plasmids, and to extend the vaccinia WR vP293 kb HindIII-Kpnl vector fragment from pHES1 and the based host range selection system, the pHES plasmid HindIII-Clal u promoter fragment generating plasmids series (Example 3) was expanded by replacing the H6 pHES31-34 (FIG. 4A). early/late vaccinia promoter with other temporally Referring now to FIG. 5, the resulting plasmids, regulated promoters. Plasmids pHES31-34 and plasmid pHES31 through pHES34, contain polylinker regions series pHES61-64 were generated to regulate expres downstream from the Copenhagen u promoter region sion of foreign genes early or late, respectively. (FIG. 5). The 0.3 kb DNA sequence specifying the u The localization and sequence of the gene for a 38 promoter is indicated in FIG. 5 for plasmid pHES31 kDa protein in cowpox virus required for (hemor only. The identical sequence is present in plasmids rhagic) red pock formation on the chicken chorioallan pHES32 through pHES34. The bracketed sequence 5,225,336 15 16 following the promoter region in pHES31 is replaced Plasmid pHES64 does not contain an ATG initiation by the bracketed sequences indicated for pHES32 codon. through pHES34. Restriction sites are indicated. In As in the pHES plasmid series containing other pro pHES31 through pHES33, the polylinker region is moters, all members of the pHES61 through pHES64 located downstream from the initiating ATG in the plasmid series contain polylinker regions followed by three different reading frames. Plasmid pHES34 does translational (underlined) and transcriptional termina not contain an initiating ATG. In all members of the tion signals (overlined). Since derivatives of the pHES31 through pHES34 series, the polylinker region pHES61 through 64 series contain the vaccinia KlL is followed by translational stop codons in all three human host range gene, recombinant vaccinia progeny reading frames, underlined, followed by the sequence 10 virus generated by recombination of these plasmids TTTTTAT, overlined, which has been shown to spec with vP293 are selected by their ability to grow on ify transcriptional termination for early genes in vac human cells. cinia (39). As with the pHES1 through pHES4 series of plas EXAMPLE 6 mids (Example 3) the pHES31 through pHES34 series 15 CONSTRUCTION OF RECOMBINANTS vP548 allows expression of foreign coding sequences inserted AND VP661 into the polylinker region. Foreign coding sequences The sequence of 15,537 bp of DNA located near the containing an initiation codon are expressed under the left end of the Copenhagen genome is shown from left control of the vaccinia u promoter by insertion into to right in FIG.8. The sequence includes 7218 bp be pHES34. Foreign coding sequences devoid of an initia 20 tween the rightmost Sall site in HindIII C and the Hin tion codon are expressed in the appropriate reading dIII C/N junction, and extends through the entire se frame by insertion into pHES31, pHES32 or pHES33. quences for HindIII N (1544 bp; positions 7219-8762), As with the original pHES series, pHES31 through HindIIIM (2219 bp; positions 8763-10981) and HinDIII pHES34 contain the K1L human host range gene (15). K (4551 bp; positions 10982-15532). For clarity, coding Recombination between vaccinia deletion mutant 25 sequences and restriction sites are designated by base vP293 (Example 1) and all plasmid derivatives of the positions as indicated in FIG. 8. By conventional no pHES series generate recombinant vaccinia virus which menclature, vaccinia open reading frames (ORFs) are are selected by their ability to grow on human cells. designated by numbering from left to right within each To further adapt the pHES plasmid system to allow 30 HindIII fragment (33). Since different vaccinia strains expression of foreign genes in recombinant vaccinia at contain significant differences toward the left end of late times post infection, the promoter for the 160 kDa - HindIII C. (the left terminus of the vaccinia genome), ATI gene of cowpox was chosen (47). The 533 bp re ORFs located within the vaccinia HindIII C fragment gion immediately upstream from the cowpox ATI gene, are designated herein by numbering from right to left when inserted into vaccinia virus, has been shown to 35 starting at the HindIII C/N junction. By this nomencla direct high levels of expression of foreign genes at late ture, ORF ClL is the rightmost ORF beginning in the times after infection (48). The 63 bp cowpox DNA HindIII C fragment of Copenhagen vaccinia DNA (see region extending from the upstream BglII site to the FIG. 8). initiation codon is sufficient to act as a promoter for the Referring now to FIG. 9, plasmids were constructed expression of foreign genes in vaccinia. DNA specify to delete the K1L human host range gene (15) from ing this promoter region was synthesized and inserted Copenhagen virus in the expectation that removal of into the pHES system as detailed below. the K1L gene would result in loss of the ability of the The H6 promoter was removed from pHES1 (Exam resultant virus to replicate on human cells. Copenhagen ple 3) by partial HindIII digestion, followed by diges KonI fragment D, which includes approximately 2.5 kb tion with BamHI. Referring now to FIG. 6, the 7.8 kb 45 of DNA to the left of the sequence presented in FIG. 8 HindIII-BamHI vector fragment was isolated from an and extends rightward through position 12998, was agarose gel (FIG. 6A). To replace H6 promoter sequen cloned into the KonI site of pUC18, resulting in ces with cowpox ATI promoter sequences and BamHI pSD435 (FIG.9). (Note: in FIG.9 plasmids in the pSD linkage to polylinker sequences, eight oligonucleotides, series containing vaccinia Copenhagen inserts appear HRL33 through HRL40, were synthesized (FIG. 6B). 50 with the optional "VC" designation. Thus, pSD435 is Pairs of oligonucleotides were annealed and ligated equivalent to pSD435VC. The "VC" designation is with the 7.8 kb HindIII-BamHI vector fragment from omitted in FIG. 10.) The Kpn D fragment contains the pHES1 generating plasmids pHES61-64. Each annealed K1L gene (pos. 11030-10179). For ease of manipulation pair of oligonucleotides contains the 63 bp synthetic of the K1L gene and its flanking region, pSD452, a cowpox ATI promoter region flanked by HindIII and 55 subclone of pSD435 which includes sequences between BamHI restriction sites as indicated. the Sohl site (pos. 9478) in HindIII and the Clal site in Referring now to FIG. 7, the resulting plasmids, HindIII K (pos. 11731) was constructed (FIG. 9). The pHES61 through pHES64, contain polylinker regions K1 L gene is indicated by a striped block, direction of downstream from the cowpox ATI late promoter re transcription indicated by an arrow. pSD452, which gion (FIG. 7). The identical sequence for the cowpox contains two HpaI sites (pos. 9561, 101.55) was linear ATI promoter, which is present in pHES61 through ized by partial digestion with HpaI and Bgll linkers pHES64, is indicated here for pHES61 only. The brack were ligated into the HpaI site (pos. 10155) immediately eted sequence following the promoter region in downstream from the K1L gene. The resulting plasmid pHES61 is replaced by the bracketed sequences indi was cut with BglII and self-ligated, generating pSD453. cated for pHES62 through pHES64. Restriction sites 65 In pSD453, the K1L gene and its promoter are deleted. are indicated. In pHES61 through pHES63, the poly The site of deletion is indicated by a triangle (FIG. 9). linker region is located downstream from the ATG A fragment containing the coding sequences of beta initiation codon in the three different reading frames. galactosidase (stippled block, direction of gene indi 5,225,336 17 18 cated by an arrow) under the control of the vaccinia 11 or the EcoRI junction (pos. 1295) between EcoRI C kDa late promoter (dark arrow) (49) was inserted into and J would have been missed. the BglII site of pSD453, generating pSD453BG (FIG. Analysis of the amino acid translation of the 15.5 kb 9). pSD453BG was used as donor plasmid for recombi sequence described herein reveals four potential genes nation with vp410, a thymidine kinase minus derivative which would not have been tested earlier (14,15). All of Copenhagen strain vaccinia virus (50). Progeny virus four open reading frames are oriented right to left. The were assayed in the presence of X-gal. Blue plaques positions of these four ORFs, C7L (pos. 1314-863), were picked and purified by growth on VERO cells. As N2L (pos. 7943-7416), M1L (pos. 9329-7985) and K2L expected, the resulting recombinant, vp548, was shown (pos, 12367-11258) are indicated in FIG. O. The first to be missing the K1L gene when probed with 32P 10 ATG in the M1L ORF is located at position 9403. Since labelled K1L sequences. Surprisingly, vP548 plaqued this location is upstream from (to the right of) the pub on MRC-S cells. lished locations for transcriptional start sites for M1L To test whether the presence of the gene for B-galac (51) and within coding sequences for the M2L gene, one tosidase in vP548 was instrumental in its ability to can interpret the ATG at position 9329 as the true trans plaque on MRC-5 cells, the 11 kDa/B-galactosidase 15 cassette was removed from vp548 by recombination lational start of the M1L gene. with donor plasmid pSD453. The resulting vaccinia Of the four potential genes, M1L initially seemed the deletion recombinant, vP661, also plaqued on MRC-5 most likely candidate as a host range gene. 32P-labelled cells. DNA probe for K1 L cross reacts weakly with M1L sequences on a Southern blot (44) (data not shown). EXAMPLE 7 The M1L gene sequence in Copenhagen strain of DENTIFICATION OF THE C7L HOST RANGE vaccinia described herein differs at the amino terminus GENE FROM COPENHAGEN STRAIN OF from the published ML sequence in WR strain of vac VACCINIA VIRUS cinia (51). Insertion of two bases (pos. 9307,9312) in the present sequence relative to the published sequence The results described in Example 6 suggest that K1L 25 is not the only vaccinia host range gene capable of result in frame shift mutations. In the sequence de conferring growth on human cells. The possibility was scribed herein, translation begins at the ATG at pos. investigated that the deleted regions of vaccinia virus 9329. In the reported sequence (51) potential translation vP293 (Example 3) and the host range 18kb deletion from this ATG would be terminated by an in frame stop mutant vaccinia virus (14) were deleted for another 30 codon at pos. 9278 and translation of M1L would begin gene which like K1L confers the ability to grow on at pos. 9247. In the sequence described herein, transla human cells. FIG. 10 presents the restriction map of the tion from pos. 9329 is continuous to the stop codon of left end of the vaccinia virus genome showing locations M1L previously reported (pos. 7987). The net result is of potential host range genes. The HindIII and EcoRI. that the M1L gene described herein contains 27 extra maps of the left end of the vaccinia virus genome are 35 amino acids at the amino terminus as well as two amino shown at the top. Only the relevant EcoRI sites are acid substitutions relative to the reported M1L gene indicated. The extent of the host range deletions in (51). The sequence for the WR N2L gene has also been vaccinia virus deletion mutants vP293 (Example 3) and published (51). The Copenhagen N2L gene described the 18 kb host range deletion (14), as well as the deleted herein has four amino acid substitutions relative to the region common to both deletion mutants are shown by published sequence. heavy lines. The 15537 bp sequenced region (FIG. 8) Computer analysis of the protein encoded by the from the rightmost Sal site through the HindIII K Copenhagen vaccinia virus M1L ORF described herein fragment is expanded. Only the relevant restriction sites reveals a higher level of similarity to the Copenhagen are indicated. The locations of genes discussed here are vaccinia virus KlL protein (15) than to any other pro indicated within open boxes. The locations of fragments 45 tein in the PIR or Swiss-Prot data bases (data not used to test genes for host range capability are indicated shown). This data, coupled to the data derived by above troughs. The locations of vaccinia inserts in plas Southern blot analysis, suggests that the M1L gene mids described herein, along with relevant restriction product might serve a similar host range function as the sites, are also indicated. Code: S = Sall; E = EcoRI; K1L gene product. H=HindIII; Bg=BglII; Kp=KpnI; B=BamHI; SO Therefore of the four potential human host range Sp=Sphl; C=Cla; Hip=HpaI. genes, the M1L gene was tested first in the vaccinia As indicated in FIG. 10, the deletion region common virus vp293 host range selection system to assay for its to the 18 kb host range deletion and the deletion in ability to allow growth of vaccinia virus on human vP293 extends from an undetermined point in EcoRIC cells. To recombine the gene for M1L and subsequent to the Sal site in HindIII K (pos. 11412). It has been 55 genes into the vP293 selection system, pMP528 (Exam determined that a host range function mapped to the ple 1) and its derivative pMP528E (Example 2) were EcoRI K (pos. 7550-12875) fragment (14) and a host utilized as vector plasmids. pMP528 is the original vac range gene, K1L (positions 11030-10179), has been cinia deletion plasmid from which vaccinia recombi identified in a 846 bp BglII D fragment (pos. nant vP293 was derived. pMP528 contains a Sal site at 10271-11116) from within EcoRI K (15). The BglII A the deletion junction between vaccinia flanking arms fragment (pos. 8645-10270) from EcoRI K did not re derived from vaccinia DNA regions HindIII C/HindIII store growth on human cells. However, in these analy K. In pMP528E the right flanking arm derived from ses possible host range genes in the EcoRI K fragment vaccinia HindIII K DNA has been shortened, and a which are situated between the EcoRI (pos. 7749) and Small site has been substituted for the original Sal site at BglII (pos. 8644) sites, or between the BglII (pos. 65 the HindIII C/HindIII K deletion junction. 11116) and Clal (pos. 11731) sites, would have been Testing of potential vaccinia host range genes in the missed. Also, genes which cross the EcoRI (pos. 7749), vP293 system is presented schematically in FIG. 11. BglII (pos. 8644) or Clai (pos. 11731) sites of EcoRI K, The HindIII map of the left end of the vaccinia virus 5,225,336 19 20 genome is given on the top line. Locations of genes are nant vaccinia viral progeny were unable to plaque on indicated within boxes, direction of transcription indi MRC-5 cells, indicating that K2L was not a human host cated by arrows. Location of vaccinia fragments used to range gene. test host range activity of genes are indicated by The Copenhagen vaccinia C7L gene described herein troughs. corresponds exactly on the amino acid level with the A DNA fragment extending from the 3' end of M2L WR 18 kDa gene previously reported (40). To test the (pos. 9384, 55 bases upstream from the M1L initiation C7L gene (pos. 1314-863) for its host range ability, codon) through the Scal site (pos. 7906) downstream plasmid pSD420 was cut with BamHI and BglII and a from M1L was ligated into pMP528E cut with SmaI 1040 bp fragment extending from the BamHI site at (FIG. 11). The resulting plasmid, pMP528m, was used 10 position 724 to the BgllI site at position 1764 was iso as donor plasmid for recombination with vaccinia virus lated. This BglII/BamHI fragment, which contains the vP293. Although analysis with 32P-labelled DNA probe entire gene for C7L, was ligated into pMP528K2 which for the M1L gene revealed that M1L sequences were had been cut with BamHI (FIG. 11). When the resulting inserted into vaccinia, progeny virus does not plaque on plasmid, pMP528C7L, was used as donor plasmid for MRC-5 cells. 15 recombination with vaccinia virus vp293, viral progeny Since the size of the promoter region necessary for were produced which plaque on MRC-5 cells. This initiation of transcription of the M1L gene is unknown, indicated that C7L, like K1L, was a host range gene it is possible that the 55 bases upstream from M1L cod capable of specifying growth on human cells. Since the ing sequences in plasmid pMP528M were not sufficient C7L gene spans the EcoRIC/Jjunction, it had not been to specify transcription of the M1L gene. Therefore, a 20 tested previously (14). larger fragment of Copenhagen strain vaccinia virus DNA containing the entire genes for M2L, M1L and EXAMPLE 8 N2L was tested. A 2849 bp HpaI fragment (pos. DELETION OF THE C7L GENE FROM 7307-10155) was obtained by partial HpaI digestion of COPENHAGEN STRAIN OF VACCINIA VIRUS pSD420, a SalI clone of Copenhagen vaccinia virus 25 DNA (pos. 1-10477). This HpaI fragment contains the Since, like K1 L, the vaccinia virus C7L gene was entire genes for M2L (pos. 10043-9381), M1L (pos. capable of restoring the ability of the WR strain vac 9329-7985) and N2L (pos. 7943-7416). The HpaI frag cinia vP293 deletion mutant to plaque on human cells, ment was cloned into pMP528E cut with SmaI (FIG. the effect of deleting the C7L gene from the Copenha 11). The resulting plasmid, designated pMPm 12n2 in 30 gen strain of vaccinia, both as a single deletion and as a FIG. 3, was used as donor plasmid for recombination double deletion with the other host range gene, K1L, with vP293. Although analysis with 32P-labelled DNA was investigated. probes indicated that the three genes were inserted into The construction of plasmids for the deletion of the vaccinia, recombinant viral progeny did not plaque on gene for C7L and the generation of vaccinia recombi MRC-5 cells. This indicated that M1L and N2L were 35 nants deleted for C7L are presented schematically in not the presumptive host range gene(s). M2L was not FIG. 12. A HindIII map of the left end-of the vaccinia expected to be the host range gene, since the gene for genome is presented on the top line. The C7L gene is M2L is wholly contained in the BglII A fragment of indicated by a striped box, direction of transcription EcoRI K previously tested (15). indicated by an arrow. The BamHI-Bgll DNA frag The remaining possible vaccinia virus human host 40 ment (pos 725-1764) derived from plasmid pSD420 was range genes were K2L, which is missing in the 18 kb blunt ended with Klenow fragment of E. coli polymer host range mutant and truncated in vP293, and C7L, an ase and ligated into puC18 which had been cut with ORF in HindIII C which spans the EcoRI C/J junction PvuII, generating plasmid pMP420BB (FIG. 12). and has the coding capacity for an 18 kDa protein. pMP420BB was linearized with EcoRV, which cuts The K2L gene described herein corresponds to the 45 within coding sequences for C7, and a 3.2 kb Smal "ORF K1L" previously reported (52) for WR strain ended DNA fragment consisting of the vaccinia 11 kDa vaccinia, differing by two amino acid substitutions. promoter (dark arrow)/B galactosidase (dark stippled Vaccinia virus deletion mutant vP293 contains the bulk box, direction of gene indicated by an arrow) cassette of the coding sequences for K2L immediately to the was inserted. The resulting plasmid, pMPC7LKBG, right of the deletion junction in HindIII K (equivalent 50 contains the 11 kDa promoter/B-galactosidase cassette to the Sall site at position 11412). To test the K2L gene in a left to right orientation relative to vaccinia sequen (pos. 12367-11258) for its ability to permit vaccinia viral ces. Recombination was performed using donor plasmid growth on human cells, the 3' end of the vaccinia K2L pMPC7LKBG and rescuing Copenhagen vaccinia vi gene was restored to plasmid pMP528. Synthetic poly rus, vP410, resulting in vaccinia recombinant, vP665, linkers MPSYN52 (5' ATTATTTTTATAAGCTT 55 which was identified as a blue plaque in the presence of GGATCCCTCGAGGGTACCCCCGGGGAGCT X-gal. CGAATTCT 3) and MPSYN53 (5' AGAATT To delete the C7L gene from pMP420BB, the plas CGAGCTCCCCGGGGGTACCCTCGAGGGATC mid was linearized by cutting at the unique SacI (pos. CAAGCTTATAAAAATAAT 3") were annealed and 999) site in the C7L gene, followed by digestion with ligated into the Sso site (pos. 11177) downstream from Bal 31 exonuclease. Mutagenesis (53) was performed on the K2L gene in a plasmid subclone of Copenhagen the double stranded template using a synthetic 46-mer HindIII K, and a XhoI/Sal fragment containing the 3' oligonucleotide MPSYN234 (5' TGTCATT end of the K2L gene was isolated. Plasmid pMP528 was TAACACTATACTCATATACTGAATGGAT cut with Sall, and the XhoI/Sall fragment containing GAACGAATACC 3"). In the resulting plasmid, the 3' end of the K2L gene was inserted in the correct 65 pMPC7A, the C7L gene is deleted (site of deletion indi orientation (FIG. 11). The resulting plasmid, cated by a triangle, FIG. 12), leaving flanking vaccinia pMP528K2, was used as donor plasmid for recombina arms of 140 bp to the left and 400 bp to the right. tion with vaccinia virus vP293. Once again, recombi pMPC7A was used as donor plasmid to remove the 5,225,336 21 22 interrupted C7L gene/11 kDa promoter/B galactosi contrast to WR, the Copenhagen strain of vaccinia dase sequences from vP665, generating vP706, which virus contains a 4.1 kb deletion encompassing the 5' end was identified as a colorless plaque in the presence of of the ATI equivalent gene and the 3' end of the gene X-gal. Both vP665 and vP706 grow on MRC-5 cells. immediately preceding it. The remnants of the two This is expected, since these recombinants still contain 5 ORFs are joined in frame to produce a hybrid ORF of the KlL host range gene. 966 bp. Copenhagen vector plasmid pSD494VC is an To create a virus devoid of the genes for both K1L Xbal/BplI plasmid subclon of Copenhagen HindIII A and C7L, pMPC7LKBG was used as donor plasmid for in which the hybrid ORF formed by the fusion of the recombination with the KL-deleted vaccinia virus cowpox ATI counterpart gene in Copenhagen and its recombinant vP661. The resulting virus, vP683, was 10 upstream neighbor are replaced by a polylinker region. selected as a blue plaque in the presence of X-gal. The The polylinker region consists of the sequence 5'A- C7L gene was deleted from vaccinia recombinant virus GATCTCCCGGGAAGCTTGGATC vP683 by recombination with donor plasmid pMPC7A. CGAGCTCCTCGAGGAATTCGTTAAC 3' speci The resulting double deletion recombinant vaccinia fying restriction sites BglII, SmaI, HindIII, BamHI, virus, VP716, was selected as a colorless plaque in the 15 presence of X-gal. Both vP683 and vP716 fail to plaque SstI, XhoI, EcoRI and HpaI. pSD494VC contains 0.7 on MRC-5 cells, indicating that the deletion of the two kb of flanking vaccinia DNA to the left of the poly genes, K1L and C7L, is sufficient to prevent growth of linker region and 1.3 kb of flanking vaccinia DNA to vaccinia virus on human cells. the right of the polylinker region. Table 2 compares the ability of vaccinia virus genes 20 A 2.3 kb EcoRI-BamHI fragment containing the to restore host range functions to vaccinia virus deletion cowpox. 77 kDa gene and its promoter was isolated mutant, vP293. What is compared is the relative ability from pCP3. This fragment was ligated into the poly to replicate on human or monkey cells after the noted linker region of pSD494VC cut with EcoRI and genes have been reintroduced into vaccinia virus vP293 BamHI, generating plasmid pCP5 (FIG. 13). As ex by recombination. pected, recombination between pCP5 containing the 77 25 kDa cowpox gene and Copenhagen vaccinia virus TABLE 2 vP410 produced a recombinant virus, vp695, which Titer (pfu/ml) was able to plaque on CHO cells. Virus Genes inserted WERO MRC-5 To test whether the 77 kDa cowpox CHO host range vP293 M1L 1.7 x 105 O wP293 K2 2.5 x 105 O 30 gene was also capable of specifying growth of vaccinia wP293 M2, M1, N2L 1.5 x 105 O virus on human cells, recombination was performed wp293 KL 1.7 x 105 4.7 x 103 between plasmid pCP5 containing the cowpox. 77 kDa wP293 CTL 1.4 x 10. 5.9 × 103 gene and vP293, the WR vaccinia host range deletion mutant which does not plaque on human cells. Recom binant progeny virus, vP698, plaqued on MRC-5 cells. EXAMPLE 9 35 This indicates that in addition to being a CHO host COWPOX GENE ENCODING A 77 kDa range gene, the 77 kDa cowpox gene, like the vaccinia PRODUCT genes Kll and C7L, is also a human host range gene Unlike vaccinia virus, cowpox virus is capable of (FIG. 13). growth on Chinese Hamster ovary (CHO) cells. A re- 40 In light of the observation that the cowpox virus 77 gion of the cowpox genome that permits vaccinia virus kDa gene is capable of specifying the growth of vac replication on CHO cells has been identified (54). The cinia virus on both CHO and human MRC-5 cells, it cowpox gene and promoter map to a 2.3 kb Hpa I frag was of interest to determine the roles of C7L and K1L, ment. The gene encodes a predicted translation product the two vaccinia human host range genes, on the ability of 77 kDa. The cowpox gene has no significant homol 45 of vaccinia virus to replicate in vitro on cells derived ogy at the DNA or protein level to either of the two from other species. Also, it was of interest to determine vaccinia virus human host range genes; K1L (54) or whether other vaccinia-encoded genes were specifi C7L described herein. cally required for growth of vaccinia virus on cells from Referring now to FIG. 13, as a preliminary to ex other species. Initially, the series of Copenhagen vac pressing the 77 kDa cowpox gene in the present vac 50 cinia virus C7L and K1L deletion mutants were tested cinia systems, cowpox DNA was digested with HpaI for their ability to plaque on LLC-PK1 cells, a cell line and the 2.3 kb fragment containing the gene and its derived from pig kidney. promoter were isolated from an agarose gel. To flank Confluent monolayers of VERO, MRC-5 and LLC the gene with polylinkers, the cowpox HpaI fragment PK1 cells in 60 mm dishes were infected with 10-fold was ligated into SmaI digested pIBI25 (International 55 serial dilutions of virus in 200 ul volume Eagles Biotechnologies, Inc., New Haven, Conn.), generating MEM+2% newborn calf serum. After a 1 h adsorption pCP3 (FIG. 13). For insertion into vaccinia, the cow period the inoculum was removed and the monolayers pox gene was cloned into the ATI deletion region of the were overlayed with 5 ml Eagles MEM containing Copenhagen vector plasmid pSD494VC as described 0.7% Seaken Le Agarose and 10% newborn calf below, serum. Dishes were incubated at 37 C. At 4 d post The vaccinia equivalent of the cowpox ATI gene infection, the monolayers were stained by adding an region in vaccinia WR strain was initially located by additional layer consisting of 5 ml 0.6% agarose con sequencing appropriate vaccinia WR clones using prim taining 0.04% neutral red. Plaques were observed 6 h ers synthesized in accordance with the published DNA after staining. sequence at the 5' end of the cowpox ATI coding se As shown in Table 3A, Copenhagen deletion mutants quence (47). In contrast to cowpox, whose ATI gene show identical plaquing abilities on pig kidney LLC encodes a 160 kDa protein, the WR vaccinia counter PK1 cells compared to human MRC-5 cells. Recombi part gene encodes a 94 kDa protein (see also 48). In nant viruses which are deleted for either K1L (vP661) 5,225,336 23 24 or C7L (vP706), while retaining the other human host vP293 and pHES-based plasmids containing the K1L range gene, plaqued both on MRC-5 and LLC-PKl gene (Example 3) form large plaques on an MRC-5 cells. Recombinant virus deleted for both Kll and C7L monolayer which are clearly distinguishable from the (vP716) did not plaque on either MRC-5 or LLC-PK1 background of vP293 small plaques. Therefore, the cells. Thus, based on the criterion of in vitro plaquing ability of vP293 and the Copenhagen set of human host ability on the LLC-PK1 cell line, both vaccinia human range deletion mutants to mount a restricted infection in host range genes Kll and C7L are also porcine host MRC-5 and VERO cells under a liquid overlay was range genes. As was observed with the human cell line investigated. MRC-5, the presence of either K1L or C7L in the vac cinia genome is sufficient to allow plaquing of Copenha 10 Duplicate T-75 flasks were seeded with 5X106 gen vaccinia virus on pig kidney LLC-PK1 cells. As in MRC-5 or VERO cells as indicated. After two days the case of vaccinia human host range genes, KL and confluent monolayers were infected at an moi of 0.01 C7L are the only vaccinia porcine host range genes pfu per cell (input titer 10 pfu per flask) of vaccinia encoded in the Copenhagen strain of vaccinia virus viruses as indicated in Table 3 in a volume of 0.5 ml since recombinant vaccinia virus vP716 (KL; C7L) 15 MEM--newborn calf serum (NCS). After a 1 h adsorp did not plaque on LLC-PK1 cells. tion period 10 ml of medium was added to each flask. These results were confirmed using vaccinia virus One flask of each set was frozen immediately (1 hpi recombinants containing the host range genes inserted sample). The remaining flasks were incubated at 37 C. into the WR vaccinia deletion mutant vP293 (Table until 96 hpi and then frozen. Virus from all samples 3B). As expected, vP293, which contains a large dele 20 were harvested by 3 cycles of freezing and thawing and tion spanning the C7L through KlL region, lacks the titered on VERO cells. ability to plaque on LLC-PK1 cells. Insertion of the MRC-5 and VERO cells were infected at an moi of gene for K1L into vP293 is sufficient to permit growth 0.01 pfu per cell. After 96 h incubation (96 hpi), virus of the resulting vaccinia recombinant (vP457) on LLC was harvested and titered on VERO cells. Copenhagen PK1 cells. However, as was seen with human MRC-5 25 mutants containing deletions of either human host range cells, insertion of the M1L gene into the WR deletion gene vP661 (C7L+, KIL-) and vP706 (C7L-, K1Lt) mutant, vP293, is not sufficient to permit plaquing of the displayed approximately equal multiplication (3 to 4 resultant virus (vP596) on LLC-PK1 cells (Table 3B). log10) on both MRC-5 and VERO cells, equivalent to When either the vaccinia virus C7L or K1L gene or the vP410 (C7L, K1 Lit) control. Copenhagen mu the cowpox virus 77 kDa gene is inserted into the WR 30 tants deleted for both human host range genes vP716 deletion mutant vP293, the ability to plaque on human (C7L, K1L) and vP668 (C7L through KlL)) as MRC-5 cells is restored (Table 3B). Similarly, the well as WR deletion mutant vP293 (21.7 kb deletion) vP293-based vaccinia virus recombinants containing showed multiplication on VERO cells approximately either C7L (vP638) or the cowpox 77 kDa gene (vP698) plaque on LLC-PK1 cells. Thus the cowpox 77 kDa equivalent to vP410, vP661 and vP706. Multiplication gene, in addition to being a host range gene for Chinese 35 of deletion mutant viruses vP716, vP668 and vP293 was hamster ovary (54) and human cells, is also a host range definitely positive on human MRC-5 cells, though dras gene for porcine cells. tically reduced compared to multiplication of these viruses on VERO cells. Under the conditions used here, TABLE 3 all three vaccinia viruses, vF716, vP668 and vp293, A. Copenhagen based deletion mutants which are deleted for both vaccinia human host range Virus Deletion VERO MRC.S LLC-PKI CHO genes K1L and C7L are capable of productive but wF410 ------greatly restricted infection of human MRC-5 cells (ap vP66 KL ------proximately tenfold multiplication during 96 h infec vP706 C7L ------vP716 Kl, C7L -- -- - 45 tion). These results, shown in Table 4, are in agreement vP668 C7L-K1 L) ------with earlier reports of a 2.3 fold multiplication during B. WR v P293 based deletion nutans 36 h infection (6). Virus Insert WERO MRC-5 LLC-PK CHO TABLE 4 wP293 -- - - Growth of vaccinia deletion mutants on VERO and MRC-5 cells vP457 KL ------SO VPS96 M1L -- - - Multiplication of wP638 CL ------Titer at 96 hpi' Virus? vP698 cowpox 77kDa ------Virus Deletions VERO MRC-5 VERO MRC-5 vP40 1.9 x 107 1.0 x 106 5588 6250 vP661 KL 3.8 x 107 1.1 x 107 9268 3235 EXAMPLE 10 55 vP706 C7L 2.3 x 107 8.6 x 106 10000 3440 vP716 C7L, K1L 1.3 x 107 3.4 x 10' 4375 1. GROWTH OF COPENHAGEN DELETION vP668 (C7-K1L 8.0 x 106 6.4 x 10' 2857 21 MUTANTS ON HUMAN CELL LINES vP293 WR 21.7kb) 6.9 x 106 6.4 x 10° 3833 7 input titer equals 10 Under customary conditions of growth (3 days, Ratio of titer at 96 hpi (hours post infection) to titer at 1 hpi (end of adsorption Noble Difco agar overlay), WR deletion mutant vP293 60 period) did not form plaques on human MRC-5 cell monolay ers. However, with increased length of incubation or To determine whether vaccinia virus deleted for the modification of the agar overlay vF293 can form small human host range genes C7L and K1 L were capable of plaques on MRC-5 cell monolayers. Specifically, use of limited multiplication on human cell lines other than 0.6% to 1% Seaken agarose or low melting point aga 65 MRC-5, the multiplication of Copenhagen vaccinia rose for the overlay instead of agar favors small plaque virus mutant vP668 C7L through KIL deletion on formation of vF293 virus on MRC-5 cells. Recombinant three additional human cell lines compared to MRC-5 vaccinia progeny generated by recombination between and VERO cells was assayed (Table 5). 5,225,336 25 26 Cells were seeded in 60 mm dishes at 1.5x 106 cells readily appreciate that a variety of foreign genes can be per dish 2 days prior to infection. Vaccinia virus vP410 utilized in these host range mutants. Furthermore, one or vp668 at a moi of 0.01 pfu per cell in a volume of 0.5 can readily appreciate that additional species beyond m) of MEM-5% newborn calf serum (NCS) was those cited in this application can be scored for host added to duplicate dishes containing monolayers of 5 range restriction of these vaccinia mutants by the pres each cell line. After an adsorption period of 1 h, 4 ml of ent methods described herein. medium was added to each dish and half of the dishes were frozen (1 hpi samples). The remaining dishes were Furthermore, one can readily appreciate that addi incubated at 37° C. until 96 hpi, then frozen (96 hpi tional host range genes exist in poxvirus. For example, samples). All samples were harvested by 3 cycles of 10 the vaccinia MVA vaccine strain is reported to be atten freezing and thawing, and virus titered on VERO cells. uated, particularly in immune suppressed animals. Re Two dishes were infected for each time point and the cently it was reported that the K1L human host range titers were averaged. Multiplication of each virus on gene is partially deleted in MVA (55). The present each cell line is expressed as the ratio of titer obtained at analysis of the MVA genome confirms the reported 96 hpi over the titer at 1 hpi. deletion in the K1 L gene, but indicates that the second 15 human host range gene, C7L, is present in MVA, even TABLE 5 though the MVA vaccinia virus does not plaque on Multiplication of Copenhagen vaccinia virus vP40 and VP668 on monkey and human cell lines human cells. The promoter region upstream from the virus % yield C7L gene in MVA is identical to the upstream region in Celine wP40 wF668 wP668AvP40 20 Copenhagen presented here. The amino acid sequence Monkey of the putative C7L translation product for MVA is VERO 70,212 3,103 18.6 identical with that of Copenhagen. This indicates that Hunan the C7L human host range gene, which in both WR and MRC-5 7,333 O 0.14 Copenhagen appears to be functionally equivalent to WISH 19,480 0.4 0.002 25 the KL human host range gene, is incapable, by itself, HeLa 50,000 0.4 00008 Detroit 9,660 2.8 0.03 of specifying growth of MVA vaccinia virus on human Cell lines used: VERO. Monkey kidney ATCC CCL 81; MRC-5: Human embry. cells. Further, replacement of the defective K1L gene onal lung ATCC CC. 171: Hella: Human cervix. epithelioid carcinoma ACC in MVA with the intact K1L gene from Copenhagen CCL 2: WISH: Human amnion (Hella markers) ATCC CCL 25; Detroit: Human does not confer to the hybrid vaccinia virus recombi foreskin ATCC CC 54. % yield vp668/vP410 for each cell line is the ratio of the multiplication of vp668 30 nant the ability to grow on human cells. (96 hpi/l hip) divided by multiplication of vP410 (96 hpi/1 hpi) x 100. MVA vaccinia virus is also impaired in its ability to grow on monkey cells, suggesting the existence of vP668 virus shows a one log multiplication on other, as yet unidentified, host range gene(s). Utilizing MRC-5 cells during a 96 h incubation period. Yield of approaches similar to those used here it should be possi vP668 virus 96 h post infection (hpi) of Detroit (human 35 ble to define the genes necessary for these restrictions. foreskin) cell line is 2.8 times the titer following adsorp Furthermore, it is well appreciated that other poxvi tion of the virus (1 hpi). For WISH (human amnion) and ruses such as avipox and swinepox are host restricted in HeLa (human cervix epithelioid carcinoma) cell lines, regards replication to avian and swine species, respec yield of vp668 virus 96 hpi was less than that observed tively. These host restrictions clearly suggest the exis following adsorption at 1 hpi, indicating no viral repli tence of a number of host range genes in the poxviruses. cation of the Copenhagen vaccinia host range deletion Definition of these genes by approaches defined in this mutant vF668 on these cell lines. All cell lines were specification can increase the repertoire of host range permissive for vaccinia virus, as shown by multiplica tion of control virus vp410. Others have also found constructed poxvirus vectors. differences in the ability of various human cell lines to 45 EXAMPLE 2 support growth of their host range mutant (6). INSERTION OF RABIES GLYCOPROTEIN EXAMPLE 11 GENE INTO THE TK DELETION LOCUS OF VARIOUS COPENHAGEN VACCINIA HOST RANGE MUTANTS OF VACCINIAVIRUS DELETION MUTANTS AS VACCINE VECTORS 50 Host range mutants of vaccinia virus would provide The rabies glycoprotein was chosen as a model for advantages as recombinant vaccine vectors. Reduction eign antigen for insertion into various Copenhagen vac or absence of replication should increase the perception cinia deletion mutants to allow comparative analysis of of safety since the viral vector is replication defective in the relative effects of these deletions. The gene for the the subject species, for example man or swine as de 55 rabies glycoprotein (18,42) was placed under the con scribed above. This would advantageously reduce the trol of the synthetic vaccinia H6 promoter. This expres opportunity of a runaway infection due to vaccination sion cassette was inserted into the Copenhagen TK in the vaccinated individual and also diminish transmis deletion vector plasmid pSD513VC. pSD513VC is a sion from vaccinated to unvaccinated individuals or subclone of Copenhagen vaccinia HindIIIJ fragment in contamination of the environment. which the coding sequences for the thymidine kinase To this end, these host range mutants are useful vac (TK) gene (56) are replaced by a polylinker region. The cine vectors. The vP293 deletion mutant (Example 3) polylinker region consists of the sequence 5' harbors a foreign genetic element. Further to this end, CCCGGGAGATCTCTCGAGCTGCAGGGCGCC recombinants containing pseudorabies virus genes (a GGATCC 3' specifying restriction sites SmaI, BglII, pertinent swine vaccine) and recombinants expressing 65 XhoI, Pst, Nari and BamHI. The resulting plasmid rabies virus glycoprotein (which has relevance for not containing the rabies glycoprotein gene was designated only veterinary applications but also humans) also have pRW842. In pRW842, coding sequences for the vac been constructed and are described herein. One can cinia TK gene are replaced by the H6 promoter/rabies 5,225,336 27 28 glycoprotein gene cassette which is oriented in a left to phage. The nucleotide sequence for the gll gene was right orientation relative to vaccinia flanking arms. determined (46). Recombination between pRW842 and any vaccinia The coding sequences for the PRV gI gene were virus results in a TK minus virus which contains the inserted into the Copenhagen vaccinia vector plasmid rabies glycoprotein gene under the control of the H6 pTP15 (50). In the resulting plasmid, pPR18, the g|I promoter. gene is located in the Copenhagen vaccinia hemaggluti Recombination was performed between pRW842 and nin (HA) deletion locus under the control of the H6 the set of Copenhagen vaccinia viruses containing dele vaccinia promoter. Recombination between plasmid tions of one or both of the human host range genes. The pPR18 and Copenhagen vaccinia deletion mutant resulting set of vaccinia recombinants containing the vP668 resulted in vaccinia recombinant vp726. In rabies glycoprotein gene are listed in Table 6. Monkey vP726 the PRV gII gene is inserted in the HA deletion (VERO) cells were infected with the set of vaccinia locus under the control of the vaccinia H6 early/iate recombinants containing the rabies gene. Immune pre promoter. All extraneous PRV DNA 5' and 3' to the cipitations were performed using a monoclonal anti gene has been removed. A sequence specifying termina body specific for the rabies glycoprotein (42). All re 15 tion of early vaccinia transcription (39) has been in combinants express the gene. serted downstream from PRV gll coding sequences. TABLE 6 B. Insertion of the PRV gp50 gene into the ATI Copenhagen deletion mutants containing rabies deletion locus of Copenhagen vaccinia virus glycoprotein gene DNA encoding the gene for the PRV glycoprotein Rabies Parental Plasmid Recombinant Glycoprotein gp50 is located on the BamHI fragment 7 of the PRV Virus Donor Virus Deletions Expression genome (61). Plasmid pPR7.1 contains the PRV BamHI vP410 pRW842 vP744 TK -- fragment 7 cloned into the BamHI site of pBR322. A vP661 pRW842 vP745 TK, Kl -- StuI/NdeI subfragment of pPR7.1 containing the entire vP706 pRW842 vP746 TK, C7L -- 25 gene for PRV gp50 was subcloned into pIBI25 generat vP716 pRW842 vP750 TK, C7L, -- ing plasmid pPR22. The nucleotide sequence for the KL gp50 gene was determined (46). vP668 pRW842 vP752 TK, -- The coding sequences for PRV gp50 were placed C7L-K1L) under the control of the early/intermediate vaccinia 30 promoter equivalent to the immediate upstream sequen Vaccinia recombinant vP750 contains the rabies gly ces of I3L (62,63). This promoter element has been used coprotein gene in a C7L, K1L background. vP752 previously to express foreign genes in vaccinia virus contains the rabies gene in a C7L through K1L) dele recombinants (31,64). DNA corresponding to promoter tion background. Since both of the human host range sequences upstream from the I3L open reading frame genes are missing in both of these vaccinia recombi 35 (62) was synthesized by a polymerase chain reaction nants, productive infection of human cells by these (65) using synthetic oligonucleotide primers recombinants would not be expected. To test whether P5OPPBAM (5' ATCATC the rabies gene can be expressed in human cells in the GGATCCCGGTGGTTTGCGATTCCG 3') and absence of the human host range genes, MRC-5 cells P5OPPATG (5' GATTAAACCTAAATAATTG 3') were infected with the entire set of vaccinia rabies re 40 and pMP1VC, a subclone of Copenhagen HindIII I, as combinants, including vP750 and vP752. In all members template. The resulting fragment was digested with of the set, immunofluorescence was detected on the BamHI to generate a BamHI cohesive end at the 5' end surface of infected cells. of the promoter sequence. The 3' end remained blunt ended. EXAMPLE 13 45 The PRV gp50 coding sequences were excised from CLONING AND EXPRESSION OF plasmid pPR22. Plasmid pPR22 was digested with Nsil, PSEUDORABIES (PRV) GENES IN A VP668 which cuts 7 bp upstream from the ATG and results in BACKGROUND a 3' overhang. The 3' overhang was blunt ended with T4 DNA polymerase in the presence of 2 mM dNTPs. vP668, the Copenhagen vaccinia deletion mutant SO The resulting DNA was partially digested with BglI, which contains a deletion spanning the region encom and a 1.3 kb blunt/BglII fragment containing the PRV passing the human and porcine host range genes (C7L gp50 gene was isolated. through KiL) was chosen as the basic vector. vp668 The 126 bp I3L promoter fragment (BamHI/blunt) does not plaque on human MRC-5 cells or pig kidney and the 1.3 kb gp50 gene containing fragment (blunt/B- LLC-PK1 cells (see Table 3). Pseudorabies genes g|I, 55 gliI) were ligated into a pBS-SK plasmid (Stratagene, gIII and gp50, which contain homology to herpes sim La Jolla, Calif.) vector digested with BamHI. The re plex virus (HSV) genes gB (57), gC (58) and gld (59), sulting plasmid was designated pbSPRV503. The ex respectively, were inserted into the vF668 vector as pression cassette containing the I3L promoter linked to detailed below. the PRV gp50 gene was removed by BamHI digestion followed by partial SmaI digestion. A 1.4 kbp fragment A. Insertion of the PRV glycoprotein g|I gene into the containing the I3L promoter/PRVgp50 gene was iso HA deletion locus of Copenhagen vaccinia virus lated and blunt ended using Klenow fragment of E. coli PRV DNA was digested with BamHI and the result polymerase. ing fragments were cloned into pbR322 cut with pSD541 is a Copenhagen deletion plasmid in which BamHI. Plasmid pPR9.25, containing PRV BamHI 65 flanking arms for the ATI deletion region (see fragment 1 (60) contains the entire gene for PRV glyco pSD494VC) were generated by polymerase chain reac protein glI. Portions of pPR9.25 containing the gene for tion (PCR) (65) using subclones of Copenhagen HindIII gII (57) were subcloned into pbR322, puC18 and M13 A as template. Synthetic oligonucleotides MPSYN267 5,225,336 29 30 (5' GGGCTGAAGCTTGCGGCCGCTCAT have been removed. Recombination between plasmid TAGACAAGCGAATGAGGGAC 3') and pPRVIIIVCTK and Copenhagen vaccinia deletion MPSYN268 (5 AGATCTCCCGGGCTCGAG mutant vP668 was performed. TAATTAATTAATTTTTATTACAC CAGAAAAGACGGCTTGAGATC3") were used as 5 D. Expression of PRV gI in vaccinia recombinant primers to make the 420 bp vaccinia arm to the right of vP726 the deletion. Synthetic oligonucleotides MPSYN269 (5 Expression of PRV gII in Copenhagen vaccinia re TAATTACTCGAGCCCGGGAGATCTAATT combinant vF726 was tested in VERO, LLC-PK1 and TAATTTAATTTATATAACTCATTTTTT. MRC-5 cells. vP726 contains PRV gII in a vP668 back GAATATAC T 3) and MPSYN270 (5' TATCT 10 ground C7L through KlL deletion), and thus would CGAATTCCCGCGGCTTTAAATGGACG not be expected to mount a productive infection in pig GAACTCTTTTCCCC 3') were used as primers to kidney LLC-PK1 and human MRC-5 cells. Neverthe make the 420 bp vaccinia ar to the left of the deletion. less, immunofluorescence analysis using a monoclonal The left and right vaccinia arms generated above were antibody specific to PRV gpII surprisingly demon mixed together and extended by a further polymerase 15 strated the expression of the PRV gpII gene product in chain reaction to generate a DNA fragment consisting pig, monkey and human cells. of both left and right flanking vaccinia arms separated by a polylinker region specifying restriction sites BglII, E. Construction of double and triple PRV recombinants SmaI and XhoI. The PCR-generated fragment was cut in the vP668 Copenhagen vaccinia virus background with HindIII and EcoRI to yield sticky ends, and li 20 Recombination was performed to construct vaccinia gated into puC8 cut with HindIII and EcoRI. The recombinants containing multiple PRV genes. Recom resulting plasmid is pSD541. binations have been performed using donor plasmids The 1.4 kb blunt ended fragment containing the I3L pATIp50 (PRV gp50, ATI deletion locus) and promoter/PRVgp50 gene was inserted into Copenha pPRVIIIVCTK (PRV gIII, TK deletion locus) and gen vector plasmid pSD541 digested with SmaI. In the 25 rescuing virus vP726, the Copenhagen vaccinia recom resulting plasmid, paTIp50, the PRV gp50 gene is binant which contains PRV g|I in the HA deletion located in the Copenhagen vaccinia ATI deletion locus locus in a C7L through Kll deletion background. under the control of a 126 bp vaccinia I3L promoter These recombinations generate vaccinia recombinants element. In paTIp50 all extraneous PRV DNA 3’ to containing double insertions of PRV genes gI+ gp50 the gene has been removed. 7 bp of extraneous PRV 30 sequences remain immediately upstream of the PRV and g|I--g|II, respectively. One of these vaccinia dou gp50 ATG. An early vaccinia transcriptional termina ble PRV recombinants is used as rescuing virus for tion sequence (39) is located downstream from PRV recombination with the appropriate plasmid to generate gp50 coding sequences. Recombination between plas the triple recombinant containing PRV genes mid paTIp50 and Copenhagen vaccinia deletion mu 35 tant vP668 was performed. EXAMPLE 1. C. Insertion of the PRV glycoprotein g|II gene into the CONSTRUCTION OF A COPENHAGEN STRAIN TK deletion locus of Copenhagen vaccinia virus VACCINA VIRUS BASED HOST RANGE The coding sequences for PRV glycoprotein g|II SELECTION SYSTEM map to BamHI fragments 2 and 9 of the PRV genome To construct a Copenhagen vaccinia virus based host (58). Plasmids pPR9.9 and pPR7.35 contain PRV range selection system (COPCS), plasmids were con BamHI fragments 2 and 9, respectively, cloned into the structed to delete DNA encompassing the region en BamHI site of pbR322. An Sphl/BamHI fragment coding the genes from C7L on the left through K1L on containing the 5' end of the PRV gIII gene was isolated 45 the right (FIG. 8). Vaccinia viruses containing this from pPR9.9. An NcoI/BamHI fragment containing deletion would not be expected to grow on human cells the remainder of the g|II gene was isolated from since both host range genes C7L (Example 7) and K1L pPR7.35. The entire PRV gIII gene was assembled by (15) were deleted. Plasmids were also constructed to ligating the two fragments into pIBI25, resulting in delete the region extending from C6L through K1L. plasmid pPR17. The nucleotide sequence for the gIII 50 Since the C6L through K1L deletion does not remove gene was determined (46). the human host range gene, C7L, vaccinia viruses con The PRV g|II gene was placed under the control of taining this deletion would be expected to grow on a Copenhagen vaccinia u promoter element resulting in human cells. plasmid pPR24 (vaccinia u promoter sequence is de Referring now to FIG. 14, a plasmid pSD420 (FIG. scribed in Example 5, FIG. 5). An expression cassette 55 14), containing a Sall clone of Copenhagen vaccinia containing a 120 bp vaccinia u promoter element and virus DNA (FIG. 8,10) was prepared. A fragment from the entire PRV gpIII gene was excised from plasmid the HindIII C region of Copenhagen strain vaccinia pPR24 by digestion with SnaBI (at position - 120 up virus was derived from pSD420 by cleavage with Xba stream from the initiation codon and with Dra down (pos. 685) followed by blunt ending with Klenow frag stream from the PRV gIII gene. The 1.5 kb bluntended ment of E. coli polymerase and cleavage with BglII fragment containing the u promoter/PRV gpIII gene (pos. 1764). The resulting 1079 bp fragment was isolated was isolated and ligated into SmaI digested Copenha from an agarose gel. pSD451 (FIG. 9,14) is a plasmid gen vector plasmid pSD513VC to yield containing Copenhagen vaccinia DNA between the pPRVIIIVCTK. In pPRVIIIVCTK, vaccinia TK cod Sph site in HindIII M (pos. 9478) and the Kpni site in ing sequences are replaced by the PRV gIII gene in 65 HindIII K (pos. 12998). A fragment from the HindIII K serted in a right to left orientation under the control of region of Copenhagen strain vaccinia virus was derived the 120 bp Copenhagen vaccinia u promoter element. from pSD451 by cleavage with BglII (pos. 11116) and All extraneous PRV sequences 5' and 3' to the gIII gene EcoRV (pos. 11834). The 718 bp restriction fragment 5,225,336 31 32 was isolated from an agarose gel. Both fragments were reading frames entering or leaving the polylinker re ligated into pljC8 which had been cleaved with Hin glon. dIII, blunt ended with Klenow fragment of E. coli poly To add the vaccinia H6 promoter to the polylinker merase, and cleaved with SmaI (FIG. 14). The resulting region, pMPCS-1 was cut with HindIII and Asp718. A plasmid was designated pMP581CK. pMP581CK (FIG. 5 synthetic HindIII/Asp718 DNA fragment consisting of 14) contains the C7L gene (solid block, direction of the modified H6 promoter (Example 3) was inserted, transcription indicated by arrow). pMP581 CK contains resulting in plasmid pCOPCS-3H (promoter sequence a unique Bgll site flanked by a left vaccinia arm (pos. given in FIG. 17). All subsequent plasmids, pCOPCS 685-1764) derived from Hind III C and by a right vac 5H through pCOPCS-10H, derived from pCOPCS-3H cinia arm (pos. 11116-11834) derived from Hind III K. 10 contain the H6 promoter region which is indicated in The left vaccinia arm contains the entire gene for C7L FIG. 17 for pCOPCS-3H. The bracketed sequence fol (coding sequences pos. 1314-863). Relative to the Co lowing the promoter region in pCOPCS-3H is replaced penhagen vaccinia genome, the two arms are separated by the bracketed sequences indicated for pCOPCS-5H by a 9351 bp deletion (pos. 1315-11 115). The site of through pCOPCS-10H. The ATG initiation codons for deletion between HindIII C sequences and HindIII K 15 plasmids pCOPCS-6H through pCOPCS-10H are un sequences is indicated by a triangle in FIG. 14. derlined. Note that pCOPCS-3H and pCOPCS-5H do To remove excess DNA at the deletion junction, not contain ATG initiation codons upstream from the pMP581CK was cut with BglII, followed by digestion polylinker region. Translational frame beginning from with Bal 31 exonuclease. Mutagenesis (53) was per the ATG in plasmids pCOPCS-6H through pCOPCS formed on the double stranded template using a syn 20 10H is indicated. To add a stop codon to the small open thetic 49mer oligonucleotide MPSYN228. (5 reading frame from pMPCS-1 referred to above, the TTTCTTAATAAATATTATTTTTATT. equivalent mutagenesis using MPSYN249 was per TAAATTCGTAGCGATATATAAAAC 3') The re formed on pCOPCS-3H, resulting in plasmid pCOPCS sulting plasmid, pMPCTK1A retains the vaccinia 5H. human host range gene, C7L. It is deleted between 25 To add an ATG initiation codon to plasmid positions 1513-1165, and is deleted for eleven genes pCOPCS-5H downstream from the H6 promoter in all C6L through K1L (FIG. 8). Recombination between reading frames relative to the polylinker restriction plasmid pMPCTK1A and vP458, a recombinant Copen sites, pCOPCS-5H was cut at the Nirul site in the H6 hagen vaccinia virus containing the E. coli lacz gene in promoter and at the BglII site in the polylinker region. the M2L deletion locus, generated vaccinia recombi 30 Vector fragment was isolated from an agarose gel. Syn nant vP664. As expected, vP664 is able to plaque on thetic oligonucleotides MPSYN250/MPSYN251 were human cells since it retains an intact C7L gene. annealed and inserted into the pCOPCS-5H vector, To remove the coding sequences pos. 1314-863) for resulting in plasmid pCOPCS-6H C7L and excess DNA at the deletion junction, Synthetic oligonucleotides MPSYN252/MPSYN253 pMP581CK was cut with Nicol, followed by digestion 35 were annealed and inserted into the pCOPCS-5H vec with Bal31 exonuclease. Mutagenesis (53) was per tor, resulting in plasmid pCOPCS-7H. formed on the double stranded template using a syn Synthetic oligonucleotides MPSYN254/MPSYN255 thetic 44mer oligonucleotide MPSYN233. (5 were annealed and inserted into the pCOPCS-5H vec TGTCATTTAACACTATACTCATAT tor, resulting in plasmid pCOPCS-8H. TAATAAAAATAATATTTATT 3"). The resulting pCOPCS-6H, pCOPCS-7H and pCOPCS-8H contain plasmid, pMPCSK1A, is deleted between positions the H6 promoter with ATG initiation codon followed 862-11163 and is deleted for twelve genes C7L through by restriction sites in the three different reading frames. K1L. Recombination between plasmid pMPCSK1A The first and second amino acids encoded in these plas and vP458 generated vaccinia recombinant vP668. As mids are as follows: pCOPCS-6H met/val; pCOPCS expected, vP668 is unable to plaque on human cells 45 7H met/gly and pCOPCS-8H met/gly. Since the since both host range genes K1L and C7L have been met/gly motif in some contexts (66) can specify myris deleted. tylation of the translated polypeptide, plasmid A series of plasmids were derived from pMPCTK1A pCOPCS-6H was modified to generate plasmids con by addition of synthetic polylinker DNA at the deletion taining ATG initiation codons in the other two reading junction. Construction of plasmids in the COPCS series 50 frames which, like pCOPCS-6H, do not begin transla is summarized in FIGS. 15-7. DNA sequence for all tion with the met/gly motif. pCOPCS-6H was cut with synthetic oligonucleotides used in the construction of Nrul and Asp718 and vector fragment was isolated these plasmids are presented in FIGS. 15-17. from an agarose gel. Synthetic oligonucleotides Plasmid pMPCTK1A (FIG. 14) was subjected to MPSYN271/MPSYN272 were annealed and inserted partial Dral digestion and linear DNA was isolated 55 into the pCOPCS-6H vector, resulting in plasmid from an agarose gel. Synthetic oligonucleotides pCOPCS-9H. MPSYN238/MPSYN239 were annealed and ligated Synthetic oligonucleotides MPSYN273/MPSYN274 into pMPCTK1A in a right to left orientation at the were annealed and inserted into the pCOPCS-6H vec deletion junction, resulting in plasmid pMPCS-1. tor, resulting in plasmid pCOPCS-10H. The first two To add a stop codon to a small open reading frame amino acids encoded in these plasmids is as follows: entering the polylinker region from the left (ATG pos. pCOPCS-9H met/ser and pCOPCS-10H met/thr. 1485), pMPCS-1 was cut with PstI. Mutagenesis was In the final COPCS series, DNA consisting of coding performed (53) using a synthetic 72 mer oligonucleotide sequence with a promoter are inserted for expression MPSYN249. (5' GTTTGTTTTATATATCGCTAC into pCOPCS-4; coding sequences containing an ATG GAATTTAAATAAAAATTATTTATT 65 are inserted for expression into pCOPCS-5H; and cod TATAGATCTAGAGTCGAC CCGGGTACC 3'). ing sequences without an ATG initiation codon are The resulting plasmid, pCOPCS-4 (referred to in FIG. inserted for expression in the appropriate reading frame 17 by its alternate designation, pMPCS-4), has no open into one or more of the pCOPCS-6H through pCOPCS 5,225,336 33 34 10H series. The resulting plasmids are recombined into an agarose gel, the terminal fragments exhibit heteroge Copenhagen vaccinia virus deletion mutant vP668, re neity. Rather than running as a single band, terminal storing the ability of vaccinia virus to plaque on human fragments appear as a ladder, the rungs of which are cells. separated in size by about 1 kb. About 80% of the vac EXAMPLE 15 cinia virus recombinants derived as plaque isolates from VC-2 or its derivatives which themselves contain heter UTILITY OF THE COPCS SYSTEM FOR ogeneous termini are found by restriction analysis to ANALYZING PROMOTER STRENGTH contain heterogeneous termini. In the remaining 20% of The ability of recombinant vaccinia progeny gener vaccinia recombinants, heterogeneity of termini has ated by recombination using the Copenhagen vaccinia 10 been lost, and the terminal DNA restriction fragments virus vp668/COPCS plasmid host range selection sys appear as discrete bands. When new recombinants are tem to plaque on human MRC-5 cells permits rapid derived from virus with discrete termini, these recombi identification of recombinants. The vP668/COPCS nants are always observed to contain discrete termini. system can be used to generate vaccinia recombinants Since the termini of stock virus VC-2 were heteroge for a variety of purposes. 15 neous, we chose to clone into a plasmid the terminal Plasmid pCOPCS-4, a member of the COPCS series fragment from recombinant virus vp452, a VC-2 deriv which does not contain a promoter upstream from its polylinker region, was cut with BglII. A BglII fragment ative which contains discrete termini. vp452 is deleted containing the complete coding sequence for the rabies for vaccinia genes TK (thymidine kinase) and HA glycoprotein gene (18,42) was inserted into pCOPCS-4 20 (hemagglutinin) (50). DNA was extracted from vP452 in a right to left orientation, resulting in plasmid and digested with XhoI, and the 2 molar terminal band pCOPCS-RAB. In pCOPCS-RAB the polylinker re of approximately 7 kb was isolated from an agarose gel. gion is located upstream from the rabies gene. A variety Isolated fragment was subjected to limited digestion of synthetic promoter regions and promoters derived with BAL-31 exonuclease, followed by blunt ending from vaccinia virus or other poxviruses have been in- 25 with Klenow fragment of E. coli polymerase. The blunt serted into the polylinker region of pCOPCS-RAB, ended fragment was cloned into the SmaI site of puC8, upstream from the rabies glycoprotein gene. The result producing pSD522VC (FIG. 18). ing plasmids are used in recombination with vaccinia DNA sequencing of pSD522VC reveals that, as in virus Copenhagen deletion mutant vP668. Recombinant the case of WR vaccinia, the termini of Copenhagen progeny are selected by their ability to plaque on 30 vaccinia recombinant vP452 contain tandem repeat MRC-5 cells. Relative promoter strength can be as units. In addition to the blocks of 70 bp tandem repeat sayed by quantitating expression of the rabies glycopro units reported for the plaque cloned WR isolate, the tein gene in the recombinant progeny virus using mono termini of vF452, unlike the WR isolate, contain tandem clonal antibody. Additional utilities are comparable to repeat units composed of 54 bp located internal to the the vp293 host range selection system. 35 70 bp tandem repeat units and proximal to coding se quences. FIG. 19 lists the sequence of a portion of the EXAMPLE 6 Copenhagen genome, beginning with the most internal DELETION OF THE INVERTED TERMINAL copy of the 54 bp tandem repeat unit (pos. 1-54). The REPEATS OF VACCINIA VIRUS 13978 bp sequence presented in FIG. 19 was derived Large amounts of DNA can be deleted from vaccinia 0 from pSD522VC and various clones of VC-2 Copenha virus without destroying its ability to grow in tissue gen DNA in puC-based plasmids. It includes coding culture. To increase stability of the vaccinia genome sequences in HindIII C rightward of the final block of and remove nonessential genes which may be associated tandem repeats. The sequence presented in FIG. 19 with virulence, a deletion within a single vaccinia virus ends at the Sall site which is the beginning of the se recombinant of 32.7 kb of DNA from the left terminus 45 quence of Copenhagen DNA presented in FIG. 8. and 14.9 kb of DNA from the right terminus was engi To generate a plasmid containing the vaccinia repeti neered. tive DNA derived from the terminus of vp452 but de The genome of vaccinia virus is composed of double leted for vaccinia coding sequences, pSD522VC was stranded DNA. At each terminus, the DNA of comple digested with ClaI and HindIII, and a 7 kb fragment mentary strands is crosslinked by a DNA strand which 50 isolated. Synthetic oligonucleotides MPSYN261 (5' forms an incompletely base-paired terminal loop (67). CGATTCAGACACACGCTTTGAGTTTTGTT Immediately internal to the terminal loop the genome GAATCGAGATCTA 3') and MpSYN262 (5 AGCT contains sets of tandem repeats. A cloned version of the TAGATCTCGATTCAACAAAACT WR genome has been reported to contain 13 tandem CAAAGCGTGTGTCTGAAT 3) were annealed and copies of a 70 bp repeat unit near each end of the ge- 55 ligated into the pSD522VC vector fragment, generating nome, separated by 435bp of non-repetitive DNA from pMPVCEND. pMPVCEND contains vaccinia DNA an additional block of 17 tandem copies of the 70 bp from the end of pSD522VC (approximately 50 bp from repeat unit (68). The terminal loop and repetitive DNA the end of the genome) through all blocks of tandem form the distal portions of the vaccinia inverted termi repeats, ending at the Clal site at position 338 of FIG. nal repetition. The inverted terminal repetition, Which 60 19. A small ORF (positions 292-336) which crossed the has been estimated at 10 kb for the cloned version of Cial site at position 305 was reconstructed in the syn WR (69), contains a number of genes which, since they thetic oligonucleotides MPSYN261/MPSYN262, are contained in both the left and right copies of the which also introduce a BglII site for ease of future inverted terminal repetition, are present in two copies in cloning steps. pMPVCEND, which contains no ORFs the vaccinia genome. 65 proceeding from internal vaccinia DNA toward the When DNA extracted from the plaque-cloned stock terminus, was used as the plasmid vector and external of Copenhagen vaccinia virus (VC-2) utilized here is arm in the creation of plasmids designed to delete genes digested with restriction endonucleases and analyzed on from both the left and right termini of vaccinia. 5,225,336 35 36 Near the left terminus, all genes through the gene To delete genes from the right end of the genome, encoding the small subunit of ribonucleotide reductase, plasmid pmPRENDA was constructed to provide which resides in HindIII F (70), were deleted. The flanking vaccinia arms for the deletion of the vaccinia sequence for Copenhagen HindIII F was determined, hemorrhagic (u) region (Example 5) and all genes to the and is presented in FIG. 20. Vaccinia HindIII F is lo right of this region. The sequence of HindIII B, the cated immediately to the right of HindIII K. The DNA rightmost HindIII fragment in the genome was deter sequence presented in FIG. 20 is contiguous with the mined by sequencing various puC-based clones of this sequence presented in FIG. 8, which includes the entire region (FIG. 21). Comparison of the sequences derived sequence for HindIII. K. The small subunit for ribonu from the left and right regions of the genome reveals cleotide reductase is encoded by ORF F4 (positions 10 that the terminal repetition extends to position 8104 of 3506-2547, FIG. 20). FIG. 19. Thus the inverted terminal repetition of the To test whether the 10 genes (K2L through F4L) Copenhagen strain of vaccinia virus analyzed here is immediately to the right of the vP668 deletion (C7L composed of 8.1 kb of coding region in addition to the through Kl) were nonessential, a plasmid, blocks of tandem repeats. The leftmost 9 ORFs in Hin pMLPCTFRA, was constructed as follows. pSD521VC 15 dIII C, ORFs C23L through C15L, correspond to the is a subclone of Copenhagen HindIII F, containing rightmost 9 ORFs in HindIII B, ORFs B29R through sequences from the HindIII K/Fjunction (junction of B21R. FIG. 21 contains the sequence for Copenhagen FIG. 8/FIG. 20) Appendices A/C through the unique HindIII B beginning at the HindIII A/B junction and BamHI site of HindIII F (FIG. 20, position 5663). To continuing rightward through the rightmost ORF obtain a flanking arm to the right of F4, pSD521VC was 20 which begins in unique DNA sequences (B20R). The cut with clal at position 3576, upstream from F4 coding right copy of the terminal repetition begins at position sequences, and with BgllI at position 2841, within F4L 17,132 of FIG. 21, 14 bp before the end of the B20R coding sequences. Synthetic oligonucleotides ORF. MPSYN256 (5' CGATGTACAAAAATCCAAG pSD477VC is a puC-based Nco Nrul subclone of TACAGGCATATAGATAACTGA 3') and 25 Copenhagen vaccinia HindIII B (FIG. 21, positions MPSYN257 (5' GATCTCAGTTATCTATATGCCT 9713-11299) which contains the hemorrhagic (u) region GTACTTGGATTTTTTGTACAT 3") were annealed (ORFs B13R and B14R). pSD478VC (FIG. 18) is a and ligated into the vector plasmid pSD521VC between derivative of pSD477VC in which the entire u region the ClaI and BglII sites. In the resulting plasmid, (positions 10,024-11,014, FIG. 21, is replaced by a mul pMP256/257, the promoter region upstream from the 30 tiple cloning region including a Bgll site. The pair of F4 ORF is recreated, linked to a BglII site. To obtain a synthetic oligonucleotides which were annealed for this right vaccinia flanking arm, pMP256/257 was cut with purpose were SD41mer (5' CGATTACTAGATCT BglII nd EcoRI, and a 2.3 kb fragment containing vac GAGCTCCCCGGGCTCGAGGGATCCGTT 3) cinia sequences upstream from the F4 gene was isolated. and SD39mer (5' AACGGATCCCTCGAGCCCGG The left vaccinia flanking arm from HindIII C was 35 GGAGCTCAGATCTAGTAAT 3"). To obtain a obtained from plasmid pCOPCS-4 (Example 14), which flanking vaccinia arm to the left of the u region, contains the gene for C7L and a further 140 bp of vac pSD478VC was cut with EcoRI at the junction of cinia DNA to the left. pCOPCS-4 was cut with BglII pUC/vaccinia sequences, blunt ended by Klenow frag and EcoRI, and the 3.5 kb vector fragment ligated with ment of E. coli polymerase, and cut with BglII. A 0.3 kb the 2.3 kb fragment containing the right arm from Hin fragment containing the vaccinia u promoter region and dIII F. The resulting plasmid, pMPCTFRA, contains a flanking sequences to the left of the u region was iso left vaccinia arm from HindIII C and a right arm from lated. This fragment was ligated with a vector fragment HindIII F flanking a deletion of 20 genes C6L-F4L). obtained by cutting pMPVCEND with HindIII, blunt pMPCTFRA was used as donor plasmid for recombina ending with Klenow fragment of E. coli polymerase, tion with vP668 (Example 9), and recombinant virus 45 and cutting with BglI. The resulting plasmid, selected by growth on MRC-5 cells. Viable vaccinia pMPRENDA, contains a left vaccinia arm derived from progeny vP749 (C6L-F4L deletion) was recovered, HindIII B DNA upstream from the B13R ORF, and proving that all genes in the deleted region are nones including the B13R (u) promoter region. The right sential. vaccinia arm in pMPRENDA consists of blocks of tan To delete all genes from the left end of vaccinia up to 50 dem repeats, and is identical to the left vaccinia arm and including F4L, plasmid pMPLENDA (FIG. 18) present in the left end deletion plasmid, pMPLENDA. was constructed as follows. A right flanking arm from The two arms of pMPRENDA flank a deletion of 17 HindIII F was obtained by digestion of pMPCTFRA ORFs B13R-B29R). The total size of the deletion be with SmaI and BglII, followed by isolation of the 2.3 kb tween the flanking vaccinia arms in the right end dele fragment. pMPVCEND (FIG. 18), which contains 55 tion plasmid, pMPRENDA is 14,873 bp, all from Hin DNA tandem repeats from the terminus of vP452, was dIII B (sequence presented in FIG. 21 positions 10,024 digested with HindIII followed by blunt ending with through 17,145; continuing in the inverted terminal Klenow fragment E. coli polymerase and cutting with repetition, with deleted sequence equivalent to that BglII. The two fragments were ligated together, gener presented in FIG. 19, positions 8090 through 340). The ating pMPLENDA. In pMPLENDA the left vaccinia strategy for the construction of deletion plasmids arm is composed of tandem repeat units and the right pMPLENDA and pMPRENDA is presented schemati vaccinia arm is composed of DNA derived from Hin cally in FIG. 18. Filled blocs indicate Copenhagen dIII F. In plasmid pMPLENDA, the leftmost 38 genes vaccinia DNA consisting of the tanden repeats derived C23L-F4L) of the Copenhagen genome are deleted, from the terminus of vP452; open blocs indicate other totalling 32,681 bp (from HindIII C: FIG. 19, position Copenhagen vaccinia DNA. The location of the dele 340 through end (13,638 bp deleted); from HindIII C, tions in plasmids pMPCTFRA, pSD478VC, M, N and K: all of FIG. 8 (15,537 bp) and from HindIII pMPLENDA and pMPRENDA is indicated by trian F: FIG. 20 positions 1-3506). gles. 5,225,336 37 38 To take advantage of selective pressure in generating TABLE 7-continued recombinant vaccinia virus deleted for large amounts of ATI, HA) Exogpt) DNA at both ends of the genome, two selectable mark vP617 (TK-, pMPLgpt (C23L-F4L, vP791 ers were used. The first is the vaccinia C7L human host ATI, HA) Ecogpth) range gene (Example 7) with selection of recombinant vP723 (TK-, pMPLgpt (C23L-F4L), vP796 virus progeny on human MRC-5 cells. The second is ATI, HAT, u) Ecogpth) vP796 (TK, pMPRAC7 ((B 13R-B29R) -- wP8 the E. coli gene encoding the gene for guanine phos ATI, HA-, C7L) phoribosyl transferase (Ecogpt gene) with selection of (C23L-F4L), recombinant vaccinia virus progeny using mycophe Ecogpt) nolic acid (2,8). To create a moveable fragment containing only the vaccinia C7L gene and its promoter, pCOPCS-4 was Recombinant vaccinia virus deletion mutant, vP796, cut with Nicol near the 3' end of the C7L gene (position was generated by recombination between the left end 870, FIG. 8) and with BamHI 48 bp downstream from deletion plasmid carrying the selectable Ecogpt marker, the C7L coding sequences. The end of the C7L gene 15 pMPLgpt, and rescuing virus vP723, which is addition was reconstructed using synthetic oligonucleotides, ally deleted for the TK and HA genes, as well as the MPSYN258 (5' CATGGATTAATTAATTTTTTTG ATI and u equivalent regions. By DNA restriction 3') and MPSYN259 (5' GATCCAAAAAAATTAAT analysis, vP796 is deleted for the C23L through F4L TAATC3"), which were annealed and ligated with the region, as well as the TK, HA, ATI and u regions. Since vector fragment, producing plasmid pMP258/259. 20 the 38 gene deletion near the left end of vP796 encom pMP258/259 was cut with BglII and BamHI, and a 660 passes both C7L and K1 IL, vP796 was used as rescuing bp fragment containing the C7L gene and its promoter virus for recombination with pMPRAC7, the right end was isolated for insertion into the left and right end deletion plasmid containing C7L. The resulting vac deletion plasmids, pMPLENDA and pMPRENDA, cinia recombinant containing deletions near both ter respectively. 25 mini, vP811, was selected by growth on MRC-5 cells. A 670 bp BglII/BamHI fragment containing the REFERENCES Ecogpt gene was derived from plasmid pSV2gpt (ATCC #37145) (71) by the addition of a BamHI linker 1. Beck, E., Ludwig, G., Auerswald, E. 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Patel, D. D. and D. J. Pickup, EMBO 6,3787-3794 replication in the cell and said modified recombinant (1987). virus comprising DNA which codes for and expresses 48. Patel, D. D., Ray, C. A., Drucker, R. P., and D. J. the gene product in the cell with restricted replication Pickup, Proc. Natl. Acad. Sci. U.S.A. 85,9431-9435 of the virus in the cell. (1988). 2. A modified recombinant vaccinia virus for express 49. Bertholet, C., Drillien, R., and R. Wittek, Proc. ing a gene product in a host, said modified recombinant Natl. Acad. Sci. U.S.A. 82, 2096-2100 (1985). vaccinia virus having host range genes deleted there 50. Guo, P., Goebel, S., Davis, S., Perkus, M. E., Lan from so that the virus has restricted replication in the guet, B., Desmettre, P., Allen, G., and E. Paoletti, J. 65 host and said modified recombinant vaccinia virus com Virol. 63, 4189-4198 (1989). prising DNA which codes for and expresses the gene 51. Tamin, A., Villarreal, E. C., Weinrich, S. L., and D. product in the host with restricted replication of the E. Hruby, Virology 165, 141-150 (1988). virus in the host. 5,225,336 41 42 3. A virus as claimed in claim 2, wherein the gene is selected from the group consisting of rabies glycopro product is an antigen. teino antigen and pseudorabies glycoprotein antigen. 4. A virus as claimed in claim 3, wherein the host is a g ps - glycop 3. - vertebrate and the antigen induces an immunological 6. A virus as claimed in claim 2, wherein the host is a response in the vertebrate. 5 cell cultured in vitro. 5. A virus as claimed in claim 4, wherein the antigen k . . . .