USOO5766598A United States Patent (19) 11 Patent Number: 5,766,598 Paoletti et al. 45) Date of Patent: Jun. 16, 1998

54 RECOMBINANTATTENUATED ALVAC Murphy. F., 1996. "Virus Taxonomy", in Fields Virology, CANARYPOXVIRUS EXPRESSION Third Edition, Fields et al., eds. Lippincott-Raven Publish WECTORS CONTAINING HETEROLOGOUS ers. Philadelphia, pp. 15-57. DNA SEGMENTS ENCODNG LENTWRAL Taylor et al., 1991. Vaccine 9(3):190-193. PRODUCTS Berman et al., 1990, Nature 345:622-625. Girard et al., 1991 Proc. Natl. Acad. Sci., USA 88:542-546. 75 Inventors: Enzo Paoletti, Delmar; James Tartaglia. Schenectady; William Irvin Hu et al., 1986. Nature 320:537-540. Cox. Troy, all of N.Y. Ho et al., 1990, Eur, J. Immunol. 20:477-483. Virology, vol. 173, No. 1, issued Nov. 1989, S. Dallo et al. 73) Assignee: Wirogenetics Corporation, Troy, N.Y. "Humoral Immune Response Elicited by Highly Attenuated Variants of Virus and by an Attenuated Recombi nant Expressing HIV-1 Envelope ". pp. 323-328 21 Appl. No.: 303,275 (entire document). (22 Filed: Sep. 7, 1994 Virology, vol. 152, issued 1986. M.E. Perkus et al., “Inser tion and Deletion Mutants of Vaccinia Virus". pp. 285-297 Related U.S. Application Data (entire document). Nature, vol. 317, issued 31 Oct. 1985, R.M.L. Buller et al., 63 Continuation of Ser. No. 897,382, Jun. 11, 1992, abandoned, which is a continuation-in-part of Ser. No. 715,921, Jun. 14, "Decreased Virulence of Recombinant Vaccinia Virus 1991, abandoned, and Ser. No. 847,951, Mar. 6, 1992, which Expression Vectors is Associated With a Thymidine is a continuation-in-part of Ser. No. 713,967, Jun. 11, 1991, Kinase-Negative Phenotype", pp. 813-815 (entire docu which is a continuation-in-part of Ser. No. 666,056, Mar. 7, ment). 199. Journal of Virology, vol. 62. No. 12, issued Dec. 1988, H. (51) Int. Cl...... A61K 39/12: A61K 39/295; Shida et al., “Effects and Virulences of Recombinant Vac A61K 39/21: A61K 39/275 cinia Viruses Derived From Attenuated Strains That Express 52 U.S. C...... 424/199.1; 424/208.1: the Human T-Cell Leukemia Virus Type 1 Envelope Gene". 424/232.1; 424/2011; 435/320.1; 435/236: pp. 4474-4480 (entire document). 435/1723 Critical Reviews in Immunology, vol. 10, No. 1 issued 58) Field of Search ...... 435/69.1, 172.3, 1990.J. Tartaglia et al., "Poxvirus-Based Vectors as Vaccine 435/235. 236, 237; 424/199.1, 202.1, 208.1. Candidates", pp. 13-30 (entire document). (Tartaglia 900). 209.1 Virology, vol. 188, No. 1, issued May 1992, J. Tartaglia et al., "NYVAC: A Highly Attenuated Strain of Vaccinia 56) References Cited Virus". pp. 217-232 (entire document). U.S. PATENT DOCUMENTS Hosmalin. A. Nara, P.L., Zweig, M., Lerche. N.W., Cease. K.B., Gard, E.A., Markham, P.D., Putney, S.D. Daniel, 5,093,258 3/1992 Cohen ...... 435/235.1 M.D.. Desrosiers, R.C. and Berzofsky, J.A. J. Immunol. 5,174,993 12/1992 Paoletti ...... 424/89 146. 1667-1673 (1991). 5,180,675 1/1993 Prillien ... 435/235. 5,185,146 2/1993 Attenburger ...... 424/89 (List continued on next page.) FOREIGN PATENT DOCUMENTS Primary Examiner-Laurie Scheiner WO 89/O3429 of 1989 WIPO Assistant Examiner-Jeffrey S. Parkin WOA Attorney, Agent, or Firm-Frommer Lawrence & Haug 89/O3429 4f1989 WIPO. LLP: William S. Frommer; Thomas J. Kowalski WOA 90/10693 9/1990 WIPO 57 ABSTRACT WOA 90/210. Of 1990 WIPO This invention is directed toward recombinant attenuated canarypox virus expression vectors containing exogenous OTHER PUBLICATIONS DNA segments encoding lentiviral gene products. A parental Turner, P.C. et al. (90) Current topics in Microbiol. Immu canarypox virus (Rentschler strain) was obtained and attenu no. 163: 125-151. ated through more than 200 serial passages on chick embryo Lerner. R.A. et al. (83) in: The Biology of Immunologic fibroblasts. A master viral seed was subjected to four suc Disease, eds. F.J. Dixon & D.W. Fisher, H.P. Publishing Co. cessive plaque purifications under agar and one plaque clone New York, NY, pp. 331-338. was amplified through five additional passages after which Wain-Hobson. S. et al. (85) Cell 40:9-17. the stock virus was used as the parental virus in in vitro Cooney. E.L. et al. (91) Lancet 337:567-572. recombination studies. This attenuated plaque purified Tartaglia, J. et al. (906) in: Immunochemistry of Viruses, canarypox isolate was designated ALVAC. A series of eds. M.H.V. van Regenwortel and A.R. Newrath, Elsevier ALVAC recombinants were generated that are capable of Science Publishers B.V. pp. 125-151. expressing different HTV and SIV gene products including Norley, S. et al. Immunobiology 184: 193-207 (1992). Gag, Pol, , and . These recombinants provide useful Cohen, J. et al. Science 262: 980-81 (1993). reagents for the generation of viral-specific immune Moss, B., 1996, "Poxviridae: The Viruses and Their Repli responses. cation" in Fields Virology, Third Edition, Fields et al., eds. Lippincott-Raven Publishers, Philadelphia, pp. 2637-2671. 14 Claims, 9 Drawing Sheets 5,766,598 Page 2

OTHER PUBLICATIONS Tartaglia.J. and Paoletti. E. In Immunochemistry of viruses, II, eds. van Regenmortel, M.H.V and Neurath, A.R., Letvin, N.L., and King. N.W..J. AIDS3. 1023-1040 (1990). McMichael, A.J., Gotch, F.M., Noble, G.R., and Beare, (Elsevier Science Publishers B.V., Amsterdam) pp. 125 P.A.S. New Engl. J. Med. 309, 13-17 (1983). (1990). Moss, D.J. Rickinson, A.B., and Pope, J.H., Int. J. Cancer Taylor, J. et al., Vaccine 6, 466-467 (1988). 22, 662-668 (1978). Taylor, J. et al., Vaccine 9, 190-193 (1991). Schmaljohn et al., Virology 155, 633–643 (1986). Taylor. J. Weinberg. R. Lanquet. B. Desmettre, P., and Schmitt et al., J. Virol. 62, 1889-1897 (1988). Paoletti, E. Vaccine 6, 497-503 (1988a). Tartaglia, J. Winslow, J. Goebel, S. Johnson, G.P. Taylor, Taylor, J. Weinberg, R. Kawaoka. Y. Webster. R.G. and J., and Paoletti, E. J. Gen. Virol. 71, 1517-1524 (1990). Paoletti, E. Vaccine 6, 504-508 (1988b). Tartaglia et al. Virology 188. 217-232 (1992). Walker. B.D., Flexner, C. 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LaRosa, G.J. Davide, J.P. Weinhold, K. Waterbury, J.A. Hu. S.-L. Travis, B.M., Garrigues. J. Zarling, J.M. Eich Profy, A.T., Lewis, J.A. Langlois, A.J., Dressman, G.R. berg, J.W. and Alpers, C.E.. In Vaccine 90, eds. Chanock. Boswell. R.N. Shadduck, P. Holley, L.H. Karpus. M., R.M. Lerner, R.A., Brown. F. and Ginsberg. H., (Cold Bolognesi, D.P. Matthews, T.J. Emini, E.A., and Putney, Spring Harbor Press. Cold Spring Harbor, New York) pp. S.D., Science 249, 932-935 (1990). 231-236 (1990). Mandiecki, W., Proc. Natl. Acad. Sci. USA 83, 777-7182 Hu. S.-L., Kosowski. S. and Dalrymple, J., Nature 320, (1986). 537-540, (1986). Maniatis, T., Fritsch, E.F., and Sambrook, J. In Molecular Hu, S.L. Zarling.J.M. Chinn, J. Travis, B.M. Moran, PA. cloning: a laboratory manual, (Cold Spring Harbor Labora Sias, J., Kuller, L. Morton, W.R., Heidecker, G., and Ben tory. Cold Spring Harbor, NY) (1982). veniste, R.E. Proc. Natl. Acad. Sci. USA 86, 7213-7217 Michel. F. Hoffenbach. A. Langlade-Demoyen. P. Guy, B. (1989). Lecocq, J.-P. Wain-Hobson, S. Kieny, M.-P. and Plata, F. Javeherian, K. Langlois, A.J. McDanal, C. Ross. K.L. Eur. J. Immunology 18, 1917-1927 (1988). Eckler, L.I. Jellib. C.L., Profy, A.T. Rusche. J.R., Bolog Murphy-Corb, M., Martin. L.N. Davison-Fairburn. B., Montellaro, R.C. Miller, M., West, M. Ohkawa, S., Baskin, nesi. D.P., Putney, S.D., and Matthews, T.J. Proc. Natl. G.B., Zhang.J.-Y. Putney, S.D. Allison, A.C. and Eppstein, Acad. Sci. USA 86, 6768-6772 (1989). D.A. Science 246, 1293-1297 (1989). Behbehani, A.M., Microbiological Reveiws 47, 455-509 Franchini. G. Fargnoli. K.A., Giomnini, F. Jagodzinski, L. (1983). DeRossi, A., Bosch, M., Biberfield, G. Fenyo, E.M., Albert, Bertholet, C., Drillien, R. and Wittek, R., Proc. Natl. Acad. J. Gallo, R.C. and Wong-Staal. F. Proc. Natl. Acad. Sci. Sci. 82, 2096-2100 (1985). USA 86, 2433-2437 (1989). Chakrabarti, S., Robert-Guroff. M., Wong-Staal, F. Gallo, Gillard, S. Spehner. D. Drillien, R., and Kirn, A., Proc. R.C., and Moss. B., Nature 320, 535-537 (1986). Natl. Acad. Sci. USA 83.5573-5577 (1986). Cianciolo, G.J. Copeland T.D., Oroszlan S., and Snyder Goebel, S.J.. Johnson, G.P., Perkus, M.E., Davis, S.W., man, R., Science 230. 453-456 (1985). Winslow, J.P. and Paoletti, E., Virology 179, 247–266 Clewell, D.B., and Helinski, D.R., Proc. Natl. Acad. Sci. (1990a). USA 62. 1159-1166 (1969). Goebe, S.J.. Johnson, G.P. Perkus, M.E., Davis, S.W., Clewell, D.B., J. Bacteriol 110, 667-676 (1972). Winslow, J.P. and Paoletti. E., Virology 179, 517-563 Colinas, R.J., Condit, R.C., and Paoletti, E., Virus Research (1990b). Guo, P. Goebel, S., Davis. S. Perkus, M.E., Taylor, M. 18, 49-70 (1990). Norton, E. Allen, G. Lanquet, B. Desmetre. P. and E. Desrosiers, R.C., M.S., Wyand, T., Kodama, T.J. Ringler. Paoletti, J. Virol. 64. 2399-2406 (1990). L.O. Arthur, P.K. Seghal. N.L. Letvin, N.W. King, and M.D. Guo. P. Goebel, S., Davis, S. Perkus, M.E., Lanquet, B., Daniel. Proc. Natl. Acad. Sci. USA 86,6353-6357 (1989). Desmettre, P., Allen, G., and Paoletti, E. J. Virol. 63, Dreyfuss. G., Adam, S.A., and Choi.Y.-D. Mol. Cell. Biol. 4.189-4198 (1989). 4. 415-423 (1984). Haffar, O., Garrigues. J., Travis, B. Moran, P. Zarling, J., Engelke. D.R., Hoener, P.A., and Collins. F.S. Proc. Natl. and Hu, S.-L.J. Virol. 64, 2653-2659 (1990). Acad. Sci. USA 85. 544-548 (1988). Hu.S.-L. Fultz, P. McClure. H. Eichberg.J. Thomas, E. Franchini, G., Gurgo, C., Guo, H.-G. Gallo, R.C. Collati, Zarling. J. Singhal, M. Kosowski, S., Swenson. R. Ander E., Fargnoli, K.A. Hall, L.F. Wong-Staal, F. and Reitz, Jr., son. D., and Todaro. G. Nature 328, 721-723 (1987). M.S., Nature (London) 328, 539-543 (1987). U.S. Patent Jun. 16, 1998 Sheet 1 of 9 5,766,598

ECOR Hind Hindl No Ssp Pvu Yi'Gi" Hind /PWu + puC8 Hind? /Smal

Hind AECOR SOLATE O.8 kb HindIII/Ssp No SOLATE O.5 kb SOLATE VECTOR -- MPSYN 43/44 -- MPSYN

+ puCl8 HindIII/Eco R 45/46

Hind EcoR

NOSmo EcoR Nld Sspi Simo Not Hindi/Smo Hind ASrnal g/ O.5 kb ISOLATE VECTOR

U.S. Patent Jun. 16, 1998 Sheet 2 of 9 5,766,598

-e WACCNA ------VACCNA So G i. So J Salfuci G Nicol Clo Hinc Sol Hpal Niru s J pSD49 +pUC8 Ncol/Smal pSD419

ECOR pSD476 Sal Nico AS Thal \ (Hinc) Hinc SOLATE O.3 kb

Hpal/Cla SOLATE VECTOR + SD42mer M4Orner N -- SO22ner/2Omer pSD478 EcoRI-Qe2 Clair Hpol Bom- EcoR/KENOW y Bom H. pSD478E +BETA GALACTOSEDASE -- HEM5/6 pSD479BG pSD486 In vivo in vivo RECOMBINATION RECOMBINATION WP4O vP533 wP533 wd553 BLUE PLAGUES CLEAR PLAGUES FIG. 2 U.S. Patent Jun. 16, 1998 Sheet 3 of 9 5,766,598

SoO I XbalO -, SoO I WACCNA A26L So B Solis B Hpal Ndel pSD414 Xbo/Hind KLENOW ECOR SO ECOR Cld ECOR EcoRI/Bgll? KLENOW Hai Bgll KLENOW EcoRV pSD484 pSD489

HoOO 2EcoRV Bgl Hpa 2.É. RW Nde Nde Ndel/Hpal SOLATE VECTOR CloyEcoRV - AT3/4 SOLATE VECTOR

COMECO RW pSD492 pSD485 ) -beso ATE oz. A

S-2 Bg| || Cld Ni?-(EcoRV Co EcoRV Hpa Nde

Hpof 7 YNdel EcoRI Bg || EcoRI Bg || + K/BetaBgll galactosidase Wutaceness pSD493KBG pMP494 a in vivo vP55 vP58 WP58 VP68 BLUE PLAQUES CLEAR PLAQUES FIG. 3 U.S. Patent Jun. 16, 1998 Sheet 4 of 9 5,766,598

So I -- Hind So WACCNA So G Sal G + puC8 pSD419 Hind

pSD456 ECOR Smo Born H So S-2 Hind" RSO Eag Rsal/Eag SOLATE VECTOR MPSYN59-62

Eco R/Born H KLENOW Hind? So Hind Rsa | 1 YEag| Rsa (1 YEag Bg || | Bg| || Bg K/Beto galactosidase FERENA pSD466KBG TION V P7O8 In vivo RECOMBINATION wd723 vP68 CLEAR PLAQUES VP7O8 BLUE PLAOUES FIG. 4 U.S. Patent Jun. 16, 1998 Sheet S of 9 5,766,598

C N M K Hind VACCINA- H C7L-KL So I pSD42O So

Xba I Bg Sph Kpni Xbo pSD45 KLENOW Bg || EcoRV SOLATE Bgll kb Bgll/EcoRV

SOLATE O/ kb

pUCS Sf9/9'Smd/Hind - pMP58CK KLENOW

(IK/BetagalactosidasepMP4O9DBG MESSSIsMPsyn233 N M2L DELETION) in vivo pMPCSKA FEEINATIONW S-2 WP784 In vivo RECOMBINATION BLUE PLAQUES VP784

VP8O4 CLEAR PLAGUES FIG. 5 U.S. Patent Jun. 16, 1998 Sheet 6 of 9 5,766,598

aq Hind Bg|| 4L EcoRV Hind WACCNA Hind RSO Rso UC8 Hind p Bom-/EcoRpUC8 pSD4O5+pUC8 ES5'--58B/B2 + 58A A2 EcoRV/Smal pSD53 pSD53. RSO/ECOR RSO/BOnH Hind? EcoR SE SOLATE Bg (SD RSO/ECOR VECTOR VECTOR RSO1V Rsal... SOLATE-- O6 kb Rsaf/Bg HpaI/BamH SOLATE KE, BOTH O.5 kb --galactosidose ef O. Bgll Smal pSD524 KBQ RECOMBINATION EcoRI/Bg || 'BPS3.in vivo N SEE VP855 BLUE PLAQUES EcoRI/Bg SOLATE VECTOR Pst S. RSC / YECOR Bg Bg Smal EcoRMPst SOLATE kb In vivo WP866CE RECOMBINATION EcoRV SD548 E°F/frcRAPS PLAGUES VP855 Psi PQ2 EcoRiso AE RSO 7 RSO VECTOR Bglli Smal FIG. 6 U.S. Patent Jun. 16, 1998 Sheet 7 of 9 5,766,598

OO

7

X-- YZvP9 | / 2. is8 75 V2 V1 O 5O W12 M2 He2 M 1 GEy 25 2.Y 12 O Y Y

PRIMARY MEMORY SECONDARY CTL RESPONSE FIG. 7

90 PRIMARY 8O YZMEMORY 7O X- SECONDARY H 6O X 5O

O 4O H 3O O C1 2O O

P85 B MN SF2 HIV V3 PEPTIDE F.G. 8 U.S. Patent Jun. 16, 1998 Sheet 8 of 9 5,766,598

O5 O ALMAC,VP3, NYWAC

0 VCPI 2

WEEKS FIG. 9

PRIMARY 7 MEMORY

8O SECONDARY

t L1. 1. 2.E.

NONE C Thy +C CD4-C Lyl2 +C SPLEEN CELL TREATMENT FIG. O U.S. Patent Jun. 16, 1998 Sheet 9 of 9 5,766,598

OO

TARGE CELLS NFECTED WITH: VACCIN-A

VACCIN A-env

4O

2O

NONE VPE8 NYWAC ALWAC -eV -er V in vitro STEMULATION OF PEMC FIG. 5,766.598 1 2 RECOMBINANTATTENUATED ALWAC bination between homologous pox DNA in the plasmid and CANARYPOXVIRUS EXPRESSION the viral respectively gives a poxvirus modified by WECTORS CONTAINING HETEROLOGOUS the presence, in a nonessential region of its genome, of DNA SEGMENTS ENCODNG LENTWRAL foreign DNA sequences. The term “foreign" DNA desig GENE PRODUCTS nates exogenous DNA, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced CROSS-REFERENCE TO RELATED by the genome into which the exogenous DNA is placed. APPLICATIONS Genetic recombination is in general the exchange of This application is a continuation of application Ser. No. homologous sections of DNA between two strands of DNA. 07/897,382, filed Jun. 1, 1992 now abandoned, which in In certain viruses RNA may replace DNA. Homologous turn is a continuation-in-part of application Ser. No. 07/715. sections of nucleic acid are sections of nucleic acid (DNA or 92 filed Jun. 14, 1991. now abandoned. Application Ser. RNA) which have the same sequence of nucleotide bases. No. 07/897.382 is also a continuation-in-part of copending Genetic recombination may take place naturally during application Ser. No. 07/847,951, filed Mar. 6. 1992, also the replication or manufacture of new viral within incorporated herein by reference application Ser. No. 5 the infected host cell. Thus, genetic recombination between 07/847,951 is a continuation-in-part of application Ser. No. viral may occur during the cycle that 07/713.967, filed Jun. 11, 1991, which in turn is a takes place in a host cell which is co-infected with two or continuation-in-part of application Ser. No. 07/666,056, more different viruses or other genetic constructs. A section filed Mar. 7, 1991. of DNA from a first genome is used interchangeably in constructing the section of the genome of a second FELD OF THE INVENTION co-infecting virus in which the DNA is homologous with that of the first viral genome. The present invention relates to a modified poxvirus and However, recombination can also take place between to methods of making and using the same. More in sections of DNA in different genomes that are not perfectly particular, the invention relates to recombinant poxvirus, 25 homologous. If one such section is from a first genome which virus expresses gene products of an immunodefi homologous with a section of another genome except for the ciency virus gene, and to immunogenic compositions which presence within the first section of, for example, a genetic induce an immunological response against immunodefi marker or a gene coding for an antigenic determinant ciency virus when administered to a host. inserted into a portion of the homologous DNA, recombi Several publications are referenced in this application. nation can still take place and the products of that recom Full citation to these references is found at the end of the bination are then detectable by the presence of that genetic specification preceding the claims. These references marker or gene in the recombinant viral genome. describe the state-of-the-art to which this invention pertains. Successful expression of the inserted DNA genetic BACKGROUND OF THE INVENTION 35 sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential Vaccinia virus and more recently other poxviruses have region of the virus in order that the modified virus remain been used for the insertion and expression of foreign genes. viable. The second condition for expression of inserted DNA The basic technique of inserting foreign genes into live is the presence of a promoter in the proper relationship to the infectious poxvirus involves recombination between pox inserted DNA. The promoter must be placed so that it is DNA sequences flanking a foreign genetic element in a located upstream from the DNA sequence to be expressed. donor plasmid and homologous sequences present in the In recent years much attention within the field of medical rescuing poxvirus (Piccini et al.. 1987). virology has been focused on the escalating incidence of Specifically, the recombinant poxviruses are constructed acquired immunodeficiency syndrome (AIDS) caused by in two steps known in the art and analogous to the methods 45 human immunodeficiency virus (HIV). HIV-1 is a member for creating synthetic recombinants of the vaccinia virus of the virus family. Retroviridae, and more specifically of the described in U.S. Pat. Nos. 5,110,587. 4,769,330, 4.722,848 Lentivirus subfamily. This viral system along with other and 4.603.112. the disclosures of which are incorporated related viruses such as HIV-2 and simian immunodeficiency herein by reference. In this regard, reference is also made to virus (SIV) have been scrutinized with respect to their copending U.S. applications Ser. Nos. 881,995 filed May 4, 50 molecular biology, immunology, and pathogenesis in an 1992 and 537,890, filed Jun. 14, 1990, also incorporated effort to develop safe and efficacious vaccines and antiviral herein by reference. therapies. Two major obstacles, however, have haunted the First, the DNA gene sequence to be inserted into the virus, design of HIV vaccine candidates. For one, there exists particularly an open reading frame from a non-pox source. considerable sequence heterogeneity among HIV isolates, is placed into an E. coli plasmid construct into which DNA 55 and secondly, there exists a lack of information pertaining to homologous to a section of DNA of the poxvirus has been protective immunity, Infected individuals develop antibody inserted. Separately, the DNA gene sequence to be inserted (Starcich et al., 1986; Weiss et al., 1985) and CD8*-T cell is ligated to a promoter. The promoter-gene linkage is (Walker et al., 1987: Plata et al., 1987) responses to HIV but positioned in the plasmid construct so that the promoter are not protected since they eventually develop AIDS. gene linkage is flanked on both ends by DNA homologous Vaccinia virus has been used extensively to express a to a DNA sequence flanking a region of pox DNA containing number of HIV gene products to investigate their a nonessential locus. The resulting plasmid construct is then biochemical functional and immunological properties, par amplified by growth within E. coli bacteria (Clewell. 1972) ticularly cell-mediated responses. Using these specific and isolated (Clewell et al., 1969; Maniatis et al., 1986). reagents, cell-mediated cytotoxic activities have been dem Second, the isolated plasmid containing the DNA gene 65 onstrated in seropositive individuals towards the envelope sequence to be inserted is transfected into a cell culture. e.g. (Riviere et al., 1989; Walker et al., 1987; Koup et al., 1989), chick embryo fibroblasts, along with the poxvirus. Recom core (gag) (Riviere et al., 1989: Koup et al., 1989, 5,766.598 3 4 Nixon et al., 1988) (Walker et al., 1989; Walker et al., like particles budding from the plasma membrane and 1988). and the nef (Riviere et al., 1989) gene products. extracellular forms which were indistinguishable from par Additionally, vaccinia virus recombinants containing HTV ticles observed in HIV-1 infected cells. Biochemically, these genes have been shown to elicit both cell-mediated and particles are also similar to native HIV-1 particles. humoral immune responses in small laboratory animals Moreover, rodents and chimpanzees inoculated with a vac (Chakrabarti et al., 1986; Hu et al., 1986; Rautman et al. cinia virus-HIV gag recombinant generated both humoral 1989; Michel et al., 1988). macaques (Zarling et al., 1986). and cell-mediated immune responses to the HIV-1 core chimpanzees (Hu et al., 1987) and significantly, humans (Hu et al., 1990). (Zagury et al., 1988; Koff et al., 1989). To date, limited The co-expression of the HIV-1 envelope vaccination/protection studies in primates have been 1 with the core proteins in mammalian cells by vaccinia virus reported with vaccinia virus recombinants expressing gene also resulted in the assembly and release of HIV-1 particles products from HIV and related viruses. (Haffar et al., 1990). These typical type C- par Vaccination of primates with a recombinant vaccinia virus ticles contained both the envelope (gp120 and expressing the envelope glycoprotein from AIDS-causing ) and the gag proteins (p24. p17, p55. and p39). These retroviruses have elicited humoral and cell-mediated 15 proteins were also described as being present in these immune responses and, more significantly, have protected recombinant-made particles in the same ratio as observed in against HIV (Zagury et al., 1988; Hu et al., 1989). HIV-1 virions. The production of these non-infectious HIV In the results described by Hu et al... a vaccinia virus particles either in purified form or synthesized in a vaccinee recombinant expressing the envelope glycoprotein from upon inoculation with a multivalent vaccinia virus recom simian AIDS retrovirus (SRV-2) was used to vaccinate binant may provide valuable "whole-virus" vaccinating pig-tailed macaques (Macaca nemestrina). All immunized agents against HIV. This is especially appealing since pro animals developed both SRV-specific cell-mediated and tection against the lentivirus. SIV. has been demonstrated humoral immune responses, including neutralizing antibod using whole inactivated virus preparations (Murphy-Corb et ies and antibodies which mediate ADCC towards SRV-2 al., 1989: Derosiers et al., 1989). infected cells. Animals were challenged intravenously with 25 The above reviewed examples demonstrate the potential 5x1OTCIDso of SRV-2/W. The challenged control animals use of vaccinia virus recombinants expressing HTV antigens (non-vaccinated) became virenic by two weeks post to induce pertinent immunological responses necessary for challenge and those that did not seroconvert died by seven protection. A major concern, however, about the use of live weeks post-challenge. Significantly, all of the challenged viral vaccines is the issue of safety. Rare complications from animals which were previously inoculated with the vaccinia 30 immunization with vaccinia virus have been documented, virus-env recombinant remained healthy, virus-free and particularly in immunocompromised individuals, and have seropositive exclusively against the envelope . raised some objections for their acceptance as vaccine In a pilot experiment in humans, HIV seronegative indi candidates (Behbehani, 1983; Lane et al., 1969). Recently, viduals were vaccinated with a vaccinia virus/HIV-1 enve however, significant strides have been made in understand lope recombinant. This primary inoculation resulted in weak 35 ing viral virulence factors with the hope of modifying strains immunological responses (Zagury et al.. 1988). These pri for use as immunization vehicles (Tartaglia et al., 1990). mary responses were subsequently boosted with various With more relevance to the development of vaccinia virus protocols. The use of an intravenous injection of based HIV vaccine candidates for the immunoprophylaxis paraformaldehyde-fixed autologous cells infected in vitro and immunotherapy of AIDS, the genes responsible for the with the vaccinia virus-HIV recombinant, however, pro productive replication of vaccinia virus in human cell sys vided the most significant booster effect. With this immu tems have been identified (Gillard et al., 1986; Perkus et al. nization protocol, anamnestic immune responses were 1990). Potential use of vaccinia virus vectors devoid of these achieved against the envelope antigen consisting of group genes provide a non-replicating vector vaccine candidate specific neutralizing antibodies, cytotoxic T-lymphocytes that may present appropriate HTV antigens in a fashion that and delayed-type hypersensitivity (Zagury et al.. 1988). In 45 elicits a protective immune response. clinical trials performed in the United States using a recom Another approach towards the generation of safe and binant vaccinia virus expressing the HTV envgene (HIVAV effective poxvirus based HIV vaccine candidates utilize le: Bristol-Meyers), individuals receiving the recombinant avipoxvirus vectors (i.e. canarypoxvirus and fowlpoxvirus) mounted T-cell proliferative responses to HIV (Koff et al., to express pertinent HIV antigens. These viruses are natu 1989). The intensity of these responses, however, was 50 rally host-restricted and only productively replicate in avian affected by prior exposure to vaccinia virus. Consequently, species. Therefore, there exists a built-in safety factor for individuals immune to vaccinia virus mounted a weak and their use in humans, particularly immunocompromised indi transient T-cell response whereas in individuals not immune viduals. In this regard, these viruses have been engineered to to vaccinia virus, a strong response was observed towards express the rabies G glycoprotein and have been demon the HIV envelope antigen (reviewed by Koff et al., 1989). 55 strated to protect various nonavian species from live rabies These results are encouraging and have provided evidence challenge (Taylor et al., 1988a; 1991). that an immune state can be obtained in man prior to HIV It can thus be appreciated that provision of an immuno exposure using a poxvirus-based immunization vehicle. deficiency virus recombinant poxvirus, and of an immuno An intriguing potential in terms of HTV-1 vaccinology is genic composition which induces an immunological provided by expression of the HIV-1 gag products by response against immunodeficiency virus infections when vaccinia virus either alone or in combination With the administered to a host, particularly a composition having envelope glycoprotein. Several laboratories have demon enhanced safety, would be a highly desirable advance over strated that expression of gag or gag?pol sequences by the current state of technology. vaccinia virus results in the production of non-infectious particles (Hu et al., 1990; Shioda et al., 1990; Karacostaset 65 OBJECTS OF THE INVENTION al., 1989). Analysis of these vaccinia virus recombinant It is therefore an object of this invention to provide infected cells by electron microscopy revealed retrovirus recombinant poxviruses, which viruses express gene prod 5,766,598 5 6 ucts of an immunodeficiency virus, and to provide a method FIG. 8 shows the specificity of cytotoxic T lymphocyte of making such recombinant poxviruses. antigen receptor for the HIV II B hypervariable V3 loop of It is an additional object of this invention to provide for gp120, but not for the V3 loop of HTV MN or SF2: the cloning and expression of immunodeficiency virus cod FIG.9 shows the antibody responses to HIV DII B gp120 ing sequences, particularly human immunodeficiency virus of mice immunized with vectors (NYVAC. ALVAC) or with (HIV) and simian immunodeficiency virus (STV) coding vaccinia virus recombinant v911 or canarypox recombi sequences in a poxvirus vector. nant vCP112 expressing HIV-1 env (inverted triangle indi It is another object of this invention to provide an immu cates time of administration of second inoculation); nological composition having enhanced safety and which is FIG. 10 shows the sensitivity of the cytotoxic effector capable of inducing an immunological response against O cells from the spleens of mice immunized with vCP112 to immunodeficiency virus infections when administered to a antibodies against cytotoxic T lymphocyte cell surface anti host. gens Thy 1.2 and Lyt 2.2; and These and other objects and advantages of the present FIG. 11 shows percent cytotoxity versus in vitro stimu invention will become more readily apparent after consid iation of PBMC. eration of the following. 15 DETALED DESCRIPTION OF THE NVENTION STATEMENT OF THE INVENTION A better understanding of the present invention and of its In one aspect, the present invention relates to a recombi many advantages will be had from the following examples, nant poxvirus containing therein a DNA sequence from an given by way of illustration. immunodeficiency virus, particularly HIV or SIV. in a In the Examples, the following methods and materials are nonessential region of the poxvirus genome. The poxvirus is employed. advantageously a vaccinia virus or an avipox virus, such as DNA Cloning and Synthesis fowlpox virus or canarypox virus. Plasmids were constructed, screened and grown by stan According to the present invention, the recombinant pox 25 dard procedures (Maniatis et al., 1982; Perkus et al., 1985; virus expresses gene products of the foreign immunodefi Piccini et al., 1987). Restriction endonucleases were ciency virus gene, particularly an HIV or SIV gene. obtained from GIBCO/BRL. Gaithersburg, Md.; New In another aspect, the present invention relates to a England Biolabs, Beverly, Mass.; and Boehringer Man vaccine for inducing an immunological response in a host nheim Biochemicals. Indianapolis, Ind. Klenow fragment of animal inoculated with the vaccine, said vaccine including a E. coli polymerase was obtained from Boehringer Man carrier and a recombinant poxvirus containing, in a nones nheim Biochemicals. BAL-31 exonuclease and phage T4 sential region thereof. DNA from an immunodeficiency DNA ligase were obtained from New England Biolabs. The virus, particularly HIV or SIV. The poxvirus used in the reagents were used as specified by the various suppliers. vaccine according to the present invention is advantageously Synthetic oligodeoxyribonucleotides were prepared on a 35 Biosearch 8750 or Applied Biosystems 380B DNA synthe a vaccinia virus or an avipox virus, such as fowlpox virus or sizer as previously described (Perkus et al., 1989). DNA canarypox virus. sequencing was performed by the dideoxy-chain termination BRIEF DESCRIPTION OF THE DRAWINGS method (Sanger et al., 1977) using Sequenase (Tabor et al., 1987) as previously described (Guo et al., 1989). DNA A better understanding of the present invention will be amplification by polymerase chain reaction (PCR) for had by referring to the accompanying drawings, in which: sequence verification (Engelke et al., 1988) was performed FIG. 1 schematically shows a method for the construction using custom synthesized oligonucleotide primers and of plasmid pSD460 for deletion of thymidine kinase gene GeneAmp DNA amplification Reagent Kit (Perkin Elmer and generation of recombinant vaccinia virus vP410; Cetus. Norwalk, Conn.) in an automated PerkinElmer Cetus FIG. 2 schematically shows a method for the construction 45 DNA Thermal Cycler. Excess DNA sequences were deleted of plasmid pSD486 for deletion of hemorrhagic region and from plasmids by restriction endonuclease digestion fol generation of recombinant vaccinia virus v553; lowed by limited digestion by BAL-31 exonuclease and FIG.3 schematically shows a method for the construction mutagenesis (Mandecki, 1986) using synthetic oligonucle of plasmid pMP494A for deletion of ATI region and gen otides. SO Cells, Virus, and Transfection eration of recombinant vaccinia virus v018; The origins and conditions of cultivation of the Copen FIG. 4 schematically shows a method for the construction hagen strain of vaccinia virus has been previously described of plasmid pSD467 for deletion of hemagglutinin gene and (Guo et al.. 1989). Generation of recombinant virus by generation of recombinant vaccinia virus vp723; recombination, in situ hybridization of nitrocellulose filters FIG. 5 schematically shows a method for the construction 55 and screening for B-galactosidase activity are as previously of plasmid pMPCSK1A for deletion of gene cluster described (Panicali et al., 1982: Perkus et al.. 1989). C7L-K1L and generation of recombinant vaccinia virus The parental canarypox virus (Rentschler strain) is a vP804; vaccinal strain for canaries. The vaccine strain was obtained FIG. 6 schematically shows a method for the construction from a wild type isolate and attenuated through more than of plasmid pSD548 for deletion of large subunit, ribonucle 200 serial passages on chick embryo fibroblasts. A master otide reductase and generation of recombinant vaccinia virus viral seed was subjected to four successive plaque purifica wP866 (NYVAC); tions under agar and one plaque clone was amplified through FIG. 7 shows the cytotoxic response of spleen cells of five additional passages after which the stock virus was used mice and immunized with vaccinia virus or canarypox virus as the parental virus in in vitro recombination tests. The vectors (NYVAC, ALVAC) or with vaccinia virus or canary 65 plaque purified canarypox isolate is designated ALVAC. pox virus recombinants expressing HIV III B env (vP911, ALVAC was deposited under the terms of the Budapest vCP112); Treaty with the ATCC. accession number VR-2547. 5,766,598 7 8 The strain of fowlpox virus (FPV) designated FP-1 has spleens or pooled spleens from a single experimental group been described previously (Taylor et al., 1988a). It is an were processed to single cell suspensions by a 1 minute attenuated vaccine strain useful in vaccination of day old cycle in a Stomacher blender. The spleen cell suspensions chickens. The parental virus strain Duvette was obtained in were washed several times in either Assay Medium or Stim France as a fowlpox scale from a chicken. The virus was 5 Medium, as appropriate. The spleen cells were enumerated attenuated by approximately 50 serial passages in chicken by the use of a Coulter Counter or by trypan blue dye embryonated eggs followed by 25 passages on chicken exclusion using a hemacytometer and microscope. embryo fibroblast cells. The virus was subjected to four Sera successive plaque purifications. One plaque isolate was Mice were lightly anesthetized with ether and blood was further amplified in primary CEF cells and a stock virus, 10 collected from the retroorbital plexus. Blood from mice designated as TROVAC. established. comprising an experimental group was pooled and allowed NYVAC. ALVAC and TROWAC viral vectors and their to clot. The serum was collected and stored at -70° C. until derivatives were propagated as described previously (Piccini Sc. et al., 1987; Taylor et al., 1988a,b). Vero cells and chick In Vitro Stimulation for the Generation of Secondary Cyto embryo fibroblasts (CEF) were propagated as described 15 toxic T Lymphocytes (CTL) previously (Taylor et al., 1988a,b). P815 murine mastocy The pooled spleen cells from the various experimental toma cells (H-2) were obtained from ATCC (#TIB64) and groups (responders) were diluted to 5x10/ml in Stim maintained in Eagles MEM supplemented with 10% fetal Medium. The spleen cells from syngeneic, naive mice bovine serum CFBS and 100 IU/ml penicillin and 100 g (stimulators) were diluted to 1x10 cells per ml and infected streptomycin per ml. 20 for 1 hour in tissue culture medium containing 2% FBS at Mice 37 C. with the appropriate poxvirus at an m.o. i. of 25 Female BALB?c) (H-2) mice were purchased from The PFU/cell. Following infection, the stimulator cells were Jackson Laboratories (Bar Harbor. Me.) and maintained on washed several times in Stim Medium and diluted to 1x10 mouse chow and water ad libitum. All mice were used cells per ml with Stim Medium. Five mls of stimulator cells between the ages of 6 and 15 weeks of age. 25 and 5 mls of responder cells were added to a 25 cm tissue Media culture flask and incubated upright at 37° C., in 5% CO2 for Assay Medium for immunological assays was comprised 5 days. On the day of the assay, the spleen cells were washed of RPMI 1640 medium supplemented with 10% FBS. 4 mM several times in Assay Medium and counted on a hemacy L-glutamine, 20 mM HEPES (N-2-hydroxyethylpiperazine tometer in trypan blue with the use of a microscope. N-2-ethanesulfonate), 5x10M 2-mercaptoethanol. 100 IU 30 Target Cell Preparation penicillin per ml, and 100 pg/ml streptomycin. Stim Medium For poxvirus specific CTL activity, tissue culture cells was comprised of Eagle's Minimum Essential Medium were infected overnight by incubation at 1x10 cells per ml supplemented with 10% FBS. 4 mM L-glutamine, 10 M in tissue culture medium containing 2% FBS at an m.o. i. of 2-mercaptoethanol. 100 IU penicillin per ml, and 100 ug 25 PFU/cell for 1 hour at 37° C. Following incubation, the streptomycin per ml. 35 cells were diluted to between 1-2x10 cells per ml with Radioimmunoprecipitation Analysis tissue culture medium containing 10% FBS and further Cell monolayers were infected at 10 PFU/cell in modified incubated at 37° C. in 5% CO until use. For HIV specific Eagle's methionine-free medium (MEM met-). At 2 hours CTL activity, tissue culture cells were incubated overnight post-infection, 20 uCi/ml of (S)-methionine were added in with 20 pg/ml of peptide HBX2 (American Biotechnologies. MEM (-met) containing 2% dialysed fetal bovine serum Cambridge, Mass.). SF2 (American Biotechnologies, (Flow). Cells were harvested at 15 hrs post-infection by Cambridge. Mass.) or MN. (American Biotechnologies, resuspending them in lysis buffer (150 mM. NaCl. 1 mM Cambridge, Mass.) corresponding to the V3 loop region of EDTA pH 8, 10 mM Tris-HCl (pH 7.4), 0.2 mg/ml PMSF, gp120 of HIV-1 isolates III SF2. and MN. respectively. On 1% NP40, 0.01% Na Azide) and 50 ulaprotinin, scraped into the day of the assay, the targets were washed several times eppendorf tubes and the lysate was clarified by spinning 20 45 by centrifugation in Assay Medium. After the final wash, the minutes at 4°C. One third of the supernatant of a 60 mm cells were resuspended in approximately 100 uCi of diameter Petri dish was incubated with 1 pil normal human Na"CrO (Cr). Following incubation at 37° C. for 1 hr. serum and 100 pil of protein A-Seraphose CL-4B (SPA) the cells were washed at least 3 times in Assay Medium by (Pharmacia) for 2 hours at room temperature. After spinning centrifugation, counted on a hemacytometer, and diluted to for 1 minute, the supernatant was incubated for 90 min at 4 50 1x10/ml in Assay Medium. C. with immune sera specifically recognizing HIV-1. HIV-2, Cytotoxicity Assays or SIV proteins and 100 pil SPA. For primary CTL assays, freshly prepared spleen cells The pellet was washed four times with lysis buffer and were diluted with Assay Medium to 1x10 cells per ml. For two times with lithium chloride/urea buffer (0.2M LiCl, 2M secondary CTL assays (following either in vivo inoculation urea, 10 mM Tris-HCl pH 8) and the precipitated proteins 55 or in vitro stimulation), the spleen cells were diluted to were dissolved in 60 pil Laemmli buffer (Laemmli, 1970). 2x10/ml in Assay Medium. One tenth ml of spleen cell After heating for 5 minutes at 100° C. and spinning for 1 suspension was added to 'Cr labelled target cells in the minute, proteins were resolved on a SDS 10% polyacryla wells of a 96 well, round-bottom microtiter plate (EXP). In mide gel and fluorographed. most cases, the spleen cells being assayed for primary CTL Inoculations activity were further 2-fold diluted in the wells of the Mice were intravenously inoculated with 5x10' plaque microtiter plate prior to the addition of the target cells. As a forming units (PFU) in 0.1 ml of phosphate-buffered saline measure of spontaneous release of 'Cr (SR), target cells via the lateral tail vein. were incubated in only Assay Medium. To determine the Spleen Cell Preparations maximum release of "Cr (MAX), target cells were delib Following euthanasia by cervical dislocation, the spleens 65 erately lysed at the beginning of the assay by adding 0.1 ml of mice were aseptically transferred to a sterile plastic bag of 10% sodium dodecyl sulfate to the appropriate wells. To containing Hank's Balanced Salt Solution. Individual initiate the assay, the microtiter plates were centrifuged at 5,766,598 9 10 200xg for 2 min and incubated for 4 or 5 hrs at 37° C., in was modified by the deletion of six nonessential regions of 5% CO. Following incubation, the culture supernatants of the genome encoding known or potential virulence factors. each well were collected using the Skatron Supernatant The sequential deletions are detailed below. All designations Collection System. Released 'Cr was determined by a of vaccinia restriction fragments, open reading frames and Beckman 5500B gamma counter. The percent specific cyto nucleotide positions are based on the terminology reported toxicity was determined from the counts by the following formula: in Goebel et al. (1990a,b). The deletion loci were also engineered as recipient loci % CYTOTOXICTTY-(EXP-MAX(MAX-SR)x100 for the insertion of foreign genes. O Depletion of T Helper Cells and Cytotoxic T Lymphocytes The regions sequentially deleted in NYVAC are listed Using Monoclonal Anti-CD4 and Monoclonal Anti-CD8 below. Also listed are the abbreviations and open reading Spleen cell suspensions were diluted to a density of 10'/ml in cytotoxicity medium (RPMI 1640 containing frame designations for the deleted regions (Goebel et al., 0.2% BSA and 5 mM HEPES) containing a 1:5 dilution of 1990a,b) and the designation of the vaccinia recombinant anti-CD4 (monoclonal antibody 1724) or a 1:200 dilution of 15 (vP) containing all deletions through the deletion specified: anti-CD8 (monoclonal antibody anti-Lyt 2.2) and a 1:16 (1) thymidine kinase gene (TK; J2R) vP410; dilution of Cedar Lane Low-Tox rabbit complement. Appro priate controls for the single components (complement. (2) hemorrhagic region (u; B13R+B14R) w?553; anti-CD4. anti-CD8) were included. (3) A type inclusion body region (ATI; A26L) vP618: Anti-HIV-1 gp160 ELISA (4) hemagglutinin gene (HA; A56R) wP723; The wells of ELISA plates (Immulon II) were coated overnight at 4° C. with 100 ng of purified HIV-1 gp160 (5) host range gene region (C7L-K1L) vp804; and (Immuno) in carbonate buffer, pH 9.6. The plates were then (6) large subunit, ribonucleotide reductase (I4L) vP866 washed with phosphate-buffered saline containing 0.05% (NYVAC). Tween 20 (PBST). The plates were then blocked for 2 hrs at 25 As described in following Examples, any or any combi 37° C. with PBST containing 1% bovine serum albumin nation of these regions can be a site either alone or in (BSA). After washing with PBST. sera were initially diluted combination with other sites for inserting exogenous DNA 1:20 with PBST containing 0.1% BSA (dilution buffer). The from an immunodeficiency virus, immunodeficiency viruses sera were further 2-fold serially diluted in the wells of the 30 or from such virus or viruses and other exogenous DNA to ELISA plate. The plates were incubated at 37° C. for 2 hrs obtain a useful recombinant. and washed with PBST. Horseradish peroxidase conjugated (1) Construction of Plasmid pSD460 for Deletion of Thy rabbit anti-mouse immunoglobulins (DAKO) was diluted midine Kinase Gene (J2R) 1:2000 in dilution buffer and added to the wells of the ELISA plated and incubated at 37° C. for 1 hour. After Referring now to FIG. 1. plasmid pSD406 contains vac washing with PBST, OPD (o-phenylenediamine 35 cinia HindIII J (pos. 83359–88377) cloned into puC8. dihydrochloride) in substrate buffer was added and the color pSD406 was cut with HindIII and Pvu, and the 1.7 kb was allowed to develop at ambient temperature for about 20 fragment from the left side of HindIIIJ cloned into puC8 cut minutes. The reaction was extinguished by the addition of with HindIII/SmaI, forming pSD447. pSD447 contains the 2.5M HSO. The absorbance at 490 nm was determined on entire gene for J2R (pos. 83855-84385). The initiation a Bio-Tek EL-309 ELISA reader. The serum endpoint was codon is contained within an Nla site and the termination defined as the reciprocal of the dilution giving an absorbance codon is contained within an SspI site. Direction of tran value of 1.0. scription is indicated by an arrow in FIG. 1. Lymphocyte Proliferation Assays To obtain a left flanking arm, a 0.8 kb HindIII/EcoRI Single cell suspensions of the spleen cells of individual 45 fragment was isolated from pSD447, then digested with mice were diluted to 2x10/ml in Assay Medium and 0.1 ml NaII and a 0.5 kb HindIII/NIla fragment isolated. were added to the wells of 96 well, flat-bottom microtiter Annealed synthetic oligonucleotides MPSYN43/MPSYN44 plates containing Assay Medium alone, 1, 5, or 10 ug of HIV-1 peptide T1, 1, 5, or 10 ug of HIV-1 peptide T2. and (SEQID NO:1/SEQ ID NO:2) 1 or 10 g of purified HTV-1 gp160 (Immuno). The cells 50 MPSYN435' TAATTAACTAGCTACCCGGG 3' were incubated for 5 days at 37°C., in 5% CO. To each well MPSYN443' GTACATTAATTGATCGATGGGCCCTTAA 5' was added 1.0 uCi of H-thymidine for the final 6 hrs of NaIII EcoRI incubation and then harvested onto Beckman Ready Filters using a Cambridge PHD cell harvester. The filter disks were were ligated with the 0.5 kb HindIII/NlaIII fragment into dry-counted in a liquid scintillation counter. 55 pUC18 vector plasmid cut with HindIII/EcoRI. generating SIMULATION INDEX=CPMs/CPMs plasmid pSD449. To obtain a restriction fragment containing a vaccinia EXAMPLE 1. right flanking arm and pljC vector sequences. pSD447 was cut with Ssp (partial) within vaccinia sequences and HindIII ATTENUATED WACCNAVACCNE STRAN at the plJC/vaccinia junction, and a 2.9 kb vector fragment NYWAC isolated. This vector fragment was ligated with annealed To develop a new vaccinia vaccine strain. NYWAC synthetic oligonucleotides MPSYN45/MPSYN46 (SEQ ID (vP866), the Copenhagen vaccine strain of vaccinia virus NO:3/SEQID NO:4) 5,766,598 11 12

Hind Smal MPSYN455' AGCTTCCCGGGTAAGTAATACGTCAAGGAGAAAACGAA MPSYN463" AGGGCCCATTCATTATGCAGTTCCTCTTTTGCT Not Ssp ACGATCTGTAGTTAGCGGCCGCCTAATAACTAAT 3MPSYN45 TGCTAGACATCAATCGCCGGCGGATTAATTGATTA SMPSYN46 generating pSD459. 10 pSD479BG. pSD479BG was used as donor plasmid for To combine the left and right flanking arms into one recombination with vaccinia virus WP410. Recombinant plasmid, a 0.5 kb HindIII/SmaI fragment was isolated from vaccinia virus vP533 was isolated as a blue plaque in the - - presence of chromogenic substrate X-gal. In vP533 the ESACA SPS ES5i"SE with galactosidase.B13R-B14R region is deleted and is replaced by Beta used as donor plasmid for recombination with wild type is To remove Beta-galactosidase sequences from vp533. parental vaccinia virus Copenhagen strain VC-2. 3-p plasmid pSD486, a derivative of pSD477 containing a labelled probe was synthesized by primer extension using polylinker region but no initiation codon at the u deletion MPSYN45 (SEQID NO3) as template and the comple- junction, was utilized. First the Cla/HpaI vector fragment mentary 20mer oligonucleotide MPSYN47 (SEQID NO:5) from pSD477 referred to above was ligated with annealed (5' TTAGTTAATTAGGCGGCCGC 3') as primer. Recom- 20 synthetic oligonucleotides SD42mer/SD40mer (SEQ ID binant virus vP410 was identified by plaque hybridization. NO:8/SEQ ID NO:9)

Cla Sac XhoI HpaI SD42mer 5'CGATTACTAGATCTGAGCTCCCCGGGCTCGAGGGATCCGTT 3 SD40er 3" TAATGATCTAGACTCGAGGGGCCCGAGCTCCCTAGGCAA 5 Bg Smal BamHI

(2) Construction of Plasmid pSD486 for Deletion of Hem- generating plasmid pSD478. Next the EcoRI site at the orrhagic Region (B13R+B14R) ' pUC/vaccinia junction was destroyed by digestion of Referring now to FIG2 plasmid pSD419 contains yac pSD478 with EcoRI followed by bluntending with Klenow cinia Sali G (pos. 1607-173351) cloned into PUC8. fragment of E. coli polymerase and ligation, generating pSD422 contains the contiguous vaccinia Sal fragment to plasmid pSD478E. pSD478E was digested with BamHI Tothe constructright, Sal a Jplasmid (pos. 173351-182,746) deleted for the hemorrhagic cloned into region,puC8. s HpaI s ligated with Alled synthetica- - - Ele B13RBros. 172549-1735.52).sp415 wassed otides HEM5/HEM6 (SEQID NO:10/SEQID NO:11) as the source for the left flanking arm and pSD422 was used Ban EcoRI Hpal as the source of the right flanking arm. The direction of HEM55 GATCCGAATTCTAGCT 3 for the u region is indicated by an arrow in FIG. HEM63 GCTTAAGATCGA 5' 2. 40 To remove unwanted sequences from pSD419, sequences to the left of the Neo site (pos. 72,253) Were removed by generating plasmid pSD486. pSD486 was used as donor digestion of PSD19 with NcoSmall followed by blunt plasmid for recombination with recombinant vaccinia virus ligatiending withgenerating Klenow AirpSD476.fragment of E. AVAght coli polymerase flank- and 45 vP533, generating vP553, which was isolated- as a clear ing arm was obtained by digestion of pSD422 with HpaI at plaque in the presence of X-gal. s origin? E.E (3) Construction of Plasmid plMP494A for Deletion of ATI ligated with a 3.4 kb HincII vector fragment isolated from Region (A26L) pSD476, generating plasmid pSD477. The location of the O Referring now to FIG. 3. pSD414 contains Sal B cloned Early its,A. (i. into puC8. To remove unwanted DNA sequences to the left sequences in psD47 were removed by digestion with of the A26L region, pSD1 was cut with Xbal within Cla/HpaI, and the resulting vector fragment was ligated vaccinia sequences (pos. 137,079) and with HindIII at the with annealed synthetic oligonucleotides SD22mer? 55 puC/vaccinia junction, then blunt ended with Klenow frag SD2Omer (SEQID NO:6/SEQ ID NO:7) ment of E. coli polymerase and ligated, resulting in plasmid pSD483. To remove unwanted vaccinia DNA sequences to Sa Briga the right of the A26L region, pSD483 was cut with EcoRI SEC coSSSSSSS 60 (pos. 140,665 and at the pJC/vaccinia junction) and ligated, forming plasmid pSD484. To remove the A26L coding generating pSD479. pSD479 contains an initiation codon region, pSD484 was cut with Ndel (partial) slightly underlined followed by a BamHI site. To place E. coli upstream from the A26L ORF (pos. 139.004) and with HpaI Beta-galactosidase in the B13-B14 (u) deletion locus under (pos. 137.889) slightly downstream from the A26L ORF the control of the u promoter a 3.2 kb BamHI fragment 65 The 5.2 kb vector fragment was isolated and ligated with containing the Beta-galactosidase gene (Shapira et al., 1983) annealed synthetic oligonucleotides ATI3/ATI4 (SEQ ID was inserted into the BamHI site of pSD479, generating NO:12/SEQ ID NO:13) 5,766,598 13 14

Ndle ATI35'TATGAGTAACTTAACTCTTTTGTTAATTAAAAGTATATTCAAAAAATAAGT A43 ACTCATTGAATTGAGAAAACAATTAATTTTCATATAAGTTTTTTATTCA Bgll EcoRI HpaI TATATAAATAGACTGAATTCGT3"ATI3 ATATATTTATCTAGACTTAAGCAA 5AT4

reconstructing the region upstream from A26L and replacing 10 4. Vaccinia sequences derived from HindIII B were removed the A26L ORF with a short polylinker region containing the by digestion of pSD419 with HindIII within vaccinia restriction sites Bgll, EcoRI and HpaI, as indicated above. sequences and at the plJC/vaccinia junction followed by The resulting plasmid was designated pSD485. Since the ligation. The resulting plasmid, pSD456, contains the HA Bgland EcoRI sites in the polylinker region of pSD485 are gene, A56R, flanked by 0.4 kb of vaccinia sequences to the not unique, unwanted BglLI and EcoRI sites were removed 15 left and 0.4 kb of vaccinia sequences to the right. A56R from plasmid pSD483 (described above) by digestion with coding sequences were removed by cutting pSD456 with Bgl (pos. 140,136) and with EcoRI at the puC/vaccinia RsaI (partial; pos. 161,090) upstream from A56R coding junction, followed by bluntending with Klenow fragment of sequences, and with Eag (pos. 162,054) near the end of the E. coli polymerase and ligation. The resulting plasmid was gene. The 3.6 kb Rsa/Eagl vector fragment from pSD456 designated pSD489. The 1.8 kb Clai (pos. 137,198)/EcoRV was isolated and ligated with annealed synthetic oligonucle (pos. 139,048) fragment from pSD489 containing the A26L otides MPSYN59 (SEQ ID NO:15), MPSY62 (SEQ ID ORF was replaced with the corresponding 0.7 kb polylinker NO:16). MPSYN60 (SEQ ID NO:17), and MPSYN 61 containing Cla/EcoRV fragment from pSD485, generating (SEQ ID NO:18)

RsaI MPSYN595'TACACGAATGATTTTCTAAAGTATTTGGAAAGTTTTATAGGTAGTTGATAGA MPSYN623" TGTGCTTACTAAAAGATTTCATAAACCTTTCAAAATATCCATCAACTATCT 5 MPSYN59 -ACAAAAACATAATTT 3' Bgll MPSYN605 TGTAAAAAAAATCACTTTTTATACTAAGATCT MPSYN613'TGTTTTATGTATTAAAACATTTTTATTTAGTGAAAAATATGATTCTAGA Smal Ps Eag MPSYN6OCCCGGGCIGCAGC 3. MPSYN61-GGGCCCGACGTCGCCGG5 pSD492. The BglI and EcoRI sites in the polylinker region reconstructing the DNA sequences upstream from the A56R of pSD492 are unique. ORF and replacing the A56R ORF with a polylinker region A 3.3 kb Bg II cassette containing the E. coli Beta as indicated above. The resulting plasmid is pSD466. The galactosidase gene (Shapira et al., 1983) under the control of 40 vaccinia deletion in pSD466 encompasses positions 161, the vaccinia 11 kDa promoter (Bertholet et al.. 1985; Perkus 185-162,053). The site of the deletion in pSD466 is indi et al., 1990) was inserted into the BglII site of pSD492. cated by a triangle in FIG. 4. forming pSD493KBG. Plasmid pSD493KBG was used in A 3.2 kb Bg/BamHI (partial) cassette containing the E. recombination with rescuing virus wP553. Recombinant coli Beta-galactosidase gene (Shapira et al., 1983) under the vaccinia virus, vp581, containing Beta-galactosidase in the 45 control of the vaccinia 11 kDa promoter (Bertholet et al. A26L deletion region, was isolated as a blue plaque in the 1985; Guo et al., 1989) was inserted into the Bgll site of presence of X-gal. pSD466, forming pSD466KBG. Plasmid pSD466KBG was To generate a plasmid for the removal of Beta used in recombination with rescuing virus vP618. Recom galactosidase sequences from vaccinia recombinant virus binant vaccinia virus, wi08, containing Beta-galactosidase vP581, the polylinker region of plasmid pSD492 was 50 in the A56R deletion, was isolated as a blue plaque in the deleted by mutagenesis (Mandecki, 1986) using synthetic presence of X-gal. oligonucleotide MPSYN177 (SEQ ID NO:14) (5" Beta-galactosidase sequences were deleted from vp708 AAA A T G G G C G T G GATT G T TA A CT T using donor plasmid pSD467. pSD467 is identical to TATATAACTTATTTTTTGAATATAC 3'). In the resulting pSD466, except that EcoRI, SmaI and BamHI sites were plasmid, pMP494A, vaccinia DNA encompassing positions 55 removed from the pUC/vaccinia junction by digestion of 137,889-138.937, including the entire A26L ORF is pSD466 with EcoRI/BamHI followed by blunt ending with deleted. Recombination between the pMP494A and the Klenow fragment of E. coli polymerase and ligation. Beta-galactosidase containing vaccinia recombinant, vp581. Recombination between vP708 and pSD467 resulted in resulted in vaccinia deletion mutant v618, which was recombinant vaccinia deletion mutant, wi23, which was isolated as a clear plaque in the presence of X-gal. isolated as a clear plaque in the presence of X-gal. (4) Construction of Plasmid pSD467 for Deletion of Hemag (5) Construction of Plasmid pMPCSK1A for Deletion of glutinin Gene (A56R) Open Reading Frames C7L-K1L) Referring now to FIG. 4, vaccinia Sal G restriction Referring now to FIG. 5, the following vaccinia clones fragment (pos. 160,744-173.351) crosses the HindIII A/B were utilized in the construction of pMPCSK1A. pSD420 is junction (pos. 162.539). pSD419 contains vaccinia Sal G 65 Sal H cloned into puC8. pSD435 is Kpn F cloned into cloned into pUC8. The direction of transcription for the pUC18. pSD435 was cut with Sph and religated, forming hemagglutinin (HA) gene is indicated by an arrow in FIG. pSD451. In pSD451, DNA sequences to the left of the Sph 5,766.598 15 16 site (pos. 27.416) in HindIIIM are removed (Perkus et al. and the Beta-galactosidase containing vaccinia recombinant, 1990). pSD409 is HindIIIM cloned into puC8. vP784, resulted in vaccinia deletion mutant, w804, which To provide a substrate for the deletion of the C7L-K1L was isolated as a clear plaque in the presence of X-gal. gene cluster from vaccinia. E. coli Beta-galactosidase was (6) Construction of Plasmid pSD548 for Deletion of Large first inserted into the vaccinia M2L deletion locus (Guo et 5 Subunit. Ribonucleotide Reductase (4L) al., 1990) as follows. To eliminate the Bgll site in pSD409, Referring now to FIG. 6, plasmid pSD405 contains vac the plasmid was cut with Bg II in vaccinia sequences (pos. cinia HindIII I (pos. 63.875-70.367) cloned in puC8. 28.212) and with BamHI at the puC/vaccinia junction, then pSD405 was digested with EcoRV within vaccinia ligated to form plasmid pMP409B. pMP409B was cut at the sequences (pos. 67.933) and with SmaI at the puC/vaccinia unique SpHI site (pos. 27.416). M2L coding sequences were 10 junction, and ligated, forming plasmid pSD518. pSD518 removed by mutagenesis (Guo et al., 1990: Mandecki, 1986) was used as the source of all the vaccinia restriction frag using synthetic oligonucleotide. ments used in the construction of pSD548.

Bg MPSYN82 (SEQDD NO:19).5'TTTCTGTATATTTGCACCAATTTAGATCTTACTC AAAATATGTAACAATA3

The resulting plasmid, pMP409D. contains a unique Bgll The vaccinia I4L gene extends from position 67,371-65. site inserted into the M2L deletion locus as indicated above. 20 059. Direction of transcription for I4L is indicated by an A 32 kb BamHI (partial)/Bg I cassette containing the E arrow in FIG. 6. To obtain a vector plasmidfragment deleted coli Beta-galactosidase gene (Shapira et al., 1983) under the for a portion of the I4L coding sequences. pSD518 was control of the 11 kDa promoter (Bertholet et al.. 1985) was digested with BamHI (pos. 65.381) and HpaI(pos. 67.001 inserted into pMP409D cut with BglII. The resulting t blunt ended E. Anot f E. plasmid, pMP409DBG (Guo et al., 1990), was used as donor 25 plasmid for recombination with rescuing vaccinia virus merase. This 4.8 kb vector fragment was ligated with a 3.2 vP723. Recombinant vaccinia virus, vp784, containing kb SmaI cassette containing the E. coli Beta-galactosidase Beta-galactosidase inserted into the M2L deletion locus, was gene (Shapira et al., 1983) under the control of the vaccinia isolated as a blue plaque in the presence of X-gal. 11 kDa promoter (Bertholet et al.. 1985; Perkus et al., 1990). A plasmid deleted for vaccinia genes (C7L-K1L was resulting in plasmid pSD524KBG. pSD524KBG was used assembled in pUC8 cut with Smal, HindIII and bluntended as donor plasmid for recombination with vaccinia virus with Klenow fragment of E. coli polymerase. The left wP804. Recombinant vaccinia virus, vp855, containing flanking arm consisting of vaccinia HindIIIC sequences was Beta-galactosidase in a partial deletion of the 4L gene, was obtained by digestion of pSD420 with Xba (pos. 18.628) followed by blunt ending with Klenow fragment of E. coli isolated as a blue plaque in the presence of X-gal. polymerase and digestion with BglTI (pos. 19.706). The right 35 To delete Beta-galactosidase and the remainder of the I4L flanking arm consisting of vaccinia HindIIIK sequences was ORF from VP855. deletion plasmid PSP548 was con obtained by digestion of pSD451 with BglII (pos. 29,062) structed. The left and right Yaccinia flanking arms were and EcoRV pos. 29.778). The resulting plasmid, assembled separately in pC8 as detailed below and pre pMP581CK is deleted for vaccinia sequences between the sented schematically in FIG. 6. Bgll site (pos. 19.706) in HindIII Cand the BglII site (pos. ' To construct a vector plasmid to accept the left vaccinia 29,062) in HindIII K. The site of the deletion of vaccinia flanking arm, pluC8 was cut with BamHIEcoRI and ligated sequences in plasmid pMP581CK is indicated by a triangle with annealed synthetic oligonucleotides 518A1/518A2 in FIG. 5. (SEQ ID NO:21/SEQ ID NO:22)

BamHI Risa 518A15 GAICCTGAGTACTITGTAATATAATGATATATATTTTCACTTTATCTCAT 518A2 3' GACTCATGAAACATTATATTACTATATATAAAAGTGAAATAGAGTA BglI EcoRI TTGAGAATAAAAAGATCTTAGG 3518A AACTCTTATTTTTCTAGAATCCTTAA5518A2

To remove excess DNA at the vaccinia deletion junction, forming plasmid pSD531. pSD531 was cut with RsaI plasmid pMP581CK, was cut at the NcoI sites within 55 (partial) and BamHI and a 2.7 kb vector fragment isolated. vaccinia sequences (pos. 18,811; 19.655), treated with Bal- pSD518 was cut with BglII (pos. 64.459)/RsaI(pos. 64.994) 31 exonuclease and subjected to mutagenesis (Mandecki, and a 0.5 kb fragment isolated. The two fragments were 1986) using synthetic oligonucleotide MPSYN233 (SEQID ligated together, forming pSD537, which contains the com NO:20) 5' TGTCATTTAACACTATACT CATAT- plete vaccinia flanking arm left of the 4L coding sequences. TAATAAAAATAATATTTATT 3'. The resulting plasmid, so To construct a vector plasmid to accept the right vaccinia pMPCSK1A, is deleted for vaccinia sequences positions flanking arm, puC8 was cut with BamHI/EcoRI and ligated 18.805-29,108, encompassing 12 vaccinia open reading with annealed synthetic oligonucleotides 518B11518B2 frames C7L-K1L). Recombination between pMPCSK1A (SEQ ID NO:23/SEQID NO:24)

BanhI Bg Smal 58B 5' GACCAGATCTCCCGGGAAAAAAATIATTTAACTTTCATTAATAGGGATTT 5,766,598 17 18 -continued S18B2 3' GTCTAGAGGGCCCTTTTTTTAAAAATTGAAAAGTAATTATCCCTAAA Rsa EcoRI GACGTATGTAGCGTACTAGG 3'518B1 CTGCATACTACGCATGATCCTTAA5'58B2

forming plasmid pSD532. pSD532 was cut with RsaI (provided by Dr. R. C. Gallo, NCI-NIH. Bethesda, Md.). (partial)/EcoRI and a 2.7 kb vector fragment isolated. Oligonucleotides HIV88A (SEQ ID NO:27) (5'- pSD518 was cut with Rsal within vaccinia sequences (pos. ATGAATGTGACAGAAAATTTTAACAT GTGG 67.436) and EcoRI at the vaccinia/pUC junction, and a 0.6 AAAAATGTAGAAATTAATTGTACAAGACCC-3") and kb fragment isolated. The two fragments were ligated HIV 88B (SEQ D NO:28) (5'- together, forming pSD538, which contains the complete GGGTCTTGTACAATTAATTTCTACATTTTTCCACAT. vaccinia flanking arm to the right of I4L coding sequences. 15 GTTAAAATTTTCTGTCACATTCAT-3") were annealed The right vaccinia flanking arm was isolated as a 0.6 kb together to produce a double-stranded fragment containing EcoRI/Bgll fragment from pSD538 and ligated into the HIV-1 epitope 88 (amino acids 95-105. Shaffermann et pSD537 vector plasmid cut with EcoRI/BglI. In the result al., 1989). The 150 bp V3-containing PCR fragment con ing plasmid, pSD539, the I4L ORF (pos. 65.047-67,386) is taining the epitope and the 42 bp fragment containing the 88 replaced by a polylinker region, which is flanked by 0.6 kb epitope sequences were fused together by PCR by virtue of vaccinia DNA to the left and 0.6 kb vaccinia DNA to the the existence of complementary sequences. The reactions right, all in a puC background. The site of deletion within were performed using oligonucleotides HIV88C (SEQ ID vaccinia sequences is indicated by a triangle in FIG. 6. To NO:29) (5'-AGTAATGTGACAGAAAATTTTAAC-3') and avoid possible recombination of Beta-galactosidase HTV3BL3. The 192 bp PCR-derived fragment contains the sequences in the puC-derived portion of pSD539 with 25 epitope 88 sequences fused upstream to the V3 loop Beta-galactosidase sequences in recombinant vaccinia virus sequences. A termination codon (TAA) was incorporated wP855, the vaccinia I4L deletion cassette was moved from into oligonucleotide HTV3BL3P to terminate translation of pSD539 into pRC11, a puC derivative from which all the open reading frame and an initiation codon was incor Beta-galactosidase sequences have been removed and porated into oligonucleotide HIV88C to serve as the start of replaced with a polylinker region (Colinas et al., 1990). 30 translation to express the epitope 88/V3 loop . pSD539 was cut with EcoRI/PstI and the 1.2 kb fragment Additionally, oligonucleotide HTV3BL3 was synthesized so isolated. This fragment was ligated into prC11 cut with that an EcoRI site existed at the 3'-end of the 192 bp PCR EcoRI/PstI (2.35 kb) forming pSD548. Recombination fragment. between pSD548 and the Beta-galactosidase containing vac The entomopoxvirus promoter, 42 kDa (early) was gen cinia recombinant, vp855, resulted in vaccinia deletion 35 erated by PCR using oligonucleotides RG273 (SEQ ID mutant vp866, which was isolated as a clear plaque in the NO:30) (5'- presence of X-gal. AGGCAAGCTTTCAAAAAAATATAAATGATTC-3') and DNA from recombinant vaccinia virus w866 was ana RG 274 (SEQ D NO:31) (5'- lyzed by restriction digests followed by electrophoresis on TTTATATTGTAATTATATATTTTC-3) with plasmid, an agarose gel. The restriction patterns were as expected. pAM12, as template. The 108 bp fragment containing the 42 Polymerase chain reactions (PCR) (Engelke et al., 1988) kDa promoter was synthesized to contain a HindIII site at using vP866 as template and primers flanking the six dele the 5'-end. The 42 kDa promoter containing segment was tion loci detailed above produced DNA fragments of the kinased and digested with HindIII prior to ligation to the expected sizes. Sequence analysis of the PCR generated epitope 88/V3 fragment digested with EcoRI and pRW831 fragments around the areas of the deletion junctions con 45 digested with HindIII and EcoRI. The resultant plasmid was firmed that the junctions were as expected. Recombinant designated as pC5HIVL88. This plasmid was used in in vitro vaccinia virus vP866, containing the six engineered dele recombination assays with CPpp as rescue virus to generate tions as described above, was designated vaccinia vaccine vOP95. ALVAC recombinant, vCP95. contains the epitope Strain 'NYWAC.' 88/V3 loop in the de-ORFed C5 locus of CPpp. 50 The plasmid pC5HIVL88 was digested with HindIII and EXAMPLE 2 EcoRI to liberate a 300 bp fragment containing the epitope 88/V3 expression cassette described above. This fragment EXPRESSION OF HIV GENE PRODUCTS BY was excised from a LMP-agarose gel and isolated by phenol HOSTRESTRICTED POXVIRUSES extraction (2X) and ether extraction (1X). The isolated This Example describes the generation of host-restricted 55 fragment was blunt-ended using the Klenow fragment of the poxviruses that express HIV gene products. The vectors E. coli DNA polymerase in the presence of 2 mM dNTPs. employed are NYVAC, ALVAC and TROVAC. The blunted fragment was ligated to pSD550VC digested ALVAC and NYWAC Recombinants Containing the HIV-1 with SmaI to yield plasmid pHIVL88VC. This plasmid was (IIIB) V3 Loop and Epitope 88 used with vP866 as the rescue virus to generate wip878. A 150 bp fragment encompassing the V3 loop (amino vP878 contains the epitope 88/V3 loop cassette in the acids 299-344; Javeherian et al., 1989; La Rosa et al., 1990) de-ORFed 4L locus of VP866. of HIV-1 (DIB) was derived by PCR using oligonucleotides ALVAC- and NYVAC-Based Recombinants Expressing the HIV 3BL5 (SEQ ID NO:25) (5'- HIV-1 (IIIB) Envelope Glycoproteins ATGGTAGAAATTAATTGTAC-3') and HIV3BL3 (SEQID An expression cassette composed of the HIV-1 (ITB) env NO:26) (5'- 65 gene juxtaposed 3' to the vaccinia virus H6 promoter (Guo ATCATCGAATTCAAGCTTATTATTTT GCTC et al., 1989; Taylor et al., 1988a,b) was engineered for TACTAATGTTAC-3') with pHXB.2D (III) as template expression of gp160 from HIV-1 by the ALVAC and 5,766,598 19 20 NYVAC vectors. A 1.4 kb fragment was amplified from the 5" portion (150 bp) of the HIV-1 env gene. This KpnI pHXB.2D (III) (provided by Dr. R. C. Gallo, NCI-NIH. fragment was ligated into pBSHTV3B3P2768 digested with Bethesda, Md.) using oligonucleotides HIV3B1 (SEQ ID KpnI to yield plasmid pBSHTV3BEII. A 2.8 kb fragment NO:32) (5'- GTTTTAA was derived from pBSHTV3BEII by digestion with Xbal TTGTGGAGGGGAATTCTTCTACTGTAATTC-3) and followed by a partial KpnI digestion. This fragment was HIV3B5 (SEQ ID NO:33) (5'-ATCA blunt-ended and inserted into SmaI digested pSD550. The TCTCTAGAATAAAAATTATAGCAAAATCCTTTC-3'). plasmid pa H6HIV3B was generated and used in recom This fragment contains the 3' portion of the env gene. PCR bination experiments with vP866 as the rescue virus. This amplification with these primers placed a vaccinia virus generated vP911 which contains the HIV-1 envgene in the early transcription termination T5NT sequence motif foll O I4L locus of the NYVAC genome. lowing the coding sequence and removed the T5NT motif To insert the HIV-1 env gene into an ALVAC vector, situated at position 6146 to 6152 (Ratner et al., 1985) pBSHIV3BEAI was digested with NruI and Xba. The without altering the amino acid sequence. This change (T to derived 2.7 kb fragment was blunt-ended with the Klenow C) creates an EcoRI site (GAATTC) at this position. This 1.4 fragment of the E. coli DNA polymerase in the presence of kb fragment was digested with EcoRI (5'-end) and Xbal (3'- 5 2 mM dNTPs. This fragment contains the entire HIV-1 env end) and inserted into EcoRI and Xbal digested pBS-SK gene juxtaposed 3' to the 3'-most 21 bp (to Niru site) of the (Stratagene. La Jolla, Calif.). The resultant plasmid was vaccinia H6 promoter. This fragment was ligated to a 3.1 kb designated as pBSHIVENV1.5. Nucleotide sequence analy fragment derived by digestion of pRW838 with NruI and sis of this fragment demonstrated that the sequence was EcoRI with subsequent blunt-ending with Klenow. The entirely correct except for a T to C transition at position pRW838 derived fragment contains the homologous arms 7048. This transition was corrected as follows: A 250 bp derived from canarypox to direct the foreign gene to the C5 fragment was derived by PCR using oligonucleotides locus. It also contains the 5'-most 100 bp of the H6 promoter. HIV3B 1 (SEQ ID NO:32) (5'-GTTTT Therefore, ligation of these fragments resulted in an inser AATTGTGGAGGGGAATTCTTCTACTGTAATTC-3) tion plasmid containing an expression cassette for the HIV-1 and HIV3B 17 (SEQ ID NO:34) (5'- 25 env gene and was designated pC5HTV3BE. This plasmid TGCTACTCCTAATGGTTC-3') with pHXB.2D (III) as was used in in vitro recombination experiments with template. This fragment was digested with Bg|II and EcoRI. ALVAC as the rescue virus to generate vCP112. The fragment was inserted into p3SHIV3B15. digested NYVAC-Based Recombinants Expressing the HIV-1 (IIEB) with Bg|II and EcoRI and thus substituted for the region gp120 with the incorrect nucleotide to yield plasmidpbSHIV3B3P. 30 The plasmid pBSHIV3BEAII was digested with EcoRI PCR was utilized to derive a 150 bp fragment containing and Xbal to liberate a 4.3 kb fragment. This fragment the 5' portion of the envgene with oligonucleotides HIV3B9 contains the vaccinia virus H6 promoter linked to the HTV-1 (SEQ ID NO:35) (5'-CATATGCTTTAGCATCTGATG-3') envgene to nucleotide 6946 (Ratner et al., 1985). The 4.3 kb and HIV3B 10 (SEQ ID NO:36) (5'- fragment was ligated to 300 bp EcoRIXba digested PCR ATGAAAGAGCAGAAGACAGTG-3") with pHXB.2D 35 derived fragment corresponding to the 3' portion of the (III) as template. PCR was also used to generate a 128 bp gp120 coding sequence. The 300 bp PCR fragment was fragment containing the vaccinia virus H6 promoter from derived using oligonucleotides HIV1-120A (SEQ ID pC3FGAG using oligonucleotides VV6KSP (SEQ ID NO:43) (5'-ATCATCTCTAGAATAAAAATTATGGTTC NO:37) (5'-ATCATCGGTACCGATTCTTTATTCTATAC AATTTTTACTACTTTTATATTATATATTTC-3') and 3') and VV H63P (SEQ ID NO:38) (5'- 40 HIV 1 - 12 OB (SEQ D NO:44) (5'- TACGATACAAACTTAACGG-3). Both fragments were CAATAATCTTTAAGCAAATCCTC-3') with pHXB.2D digested with KpnI and the 150 bp fragment was kinased (III) as template. The ligation of the 4.3 kb Xba/EcoRI prior to co-insertion of these fragments into pBS-SK fragment and the 300 bp Xba/EcoRI fragment yielded digested with Kpn. The resultant plasmid was designated as plasmid pBSHIVB120. pBSH6HIV3B5P 45 A 1.6 kb KpnI/Xbal fragment was derived from PCR was used to generate a 600 bp fragment from pBSHTVB120 by initially linearizing the plasmid with Xbal pHXB.2D (III) with oligonucleotides HIV3B2 (SEQ ID followed by a partial Kpndigestion. The 1.6 kb fragment NO:39) (5'- GAATTACA GTA GAA was blunt-ended by treatment with the Klenow fragment of GAATTCCCCTCCACAATTAAAAC-3') and HIV3B7 the E. coli DNA polymerase in the presence of 2 mM (SEQ ID NO:40) (5'-CAATAGATAATGATACTAC-3'). 50 dNTPs. This fragment was inserted into pSD541 digested This fragment was digested with EcoRI and kinased. PCR with SmaI to yield pATIHIVB120. This plasmid was used in was also used to derive a 500 bp fragment with the same in vitro recombination experiments to generate vP921. This template but with oligonucleotides HIV3B6 (SEQ ID recombinant contains the portion of the HIV-1 env gene NO:41) (5'- encoding gp120 in the ATI locus of NYVAC. GTATTATATCAAGTTTATATAATAATGCATATTC-3) 55 To determine the authenticity of the HIV-1 gene products and HIV3B 8 (SEQ ID NO:42) (5'- expressed by vP911, vp921 and vCP112, immunoprecipita GTTGATGATCTGTAGTGC-3'). This fragment was tion analyses were performed. digested with KpnI. These fragments together correspond to Lysates derived from the infected cells were analyzed for nucleotide 5878 to 6368 (Ratner et al., 1985). The engineer HIV-1 envgene expression using pooled serum from HIV-1 ing of these fragments with these primers also removes a seropositive individuals (obtained from Dr. Genoveffa T5NT sequence positioned at nucleotide 6322 to 6328 Franchini, NCI-NIH. Bethesda, Md.). The sera was pread without altering the amino acid sequence. These two frag sorbed with vP866-infected Vero cells. The preadsorbed ments were inserted into pbSHTV3B3P digested with KpnI human sera was bound to protein A-sepharose in an over and EcoRI. This plasmid was designated as night inoculation at 4 C. In some cases a monoclonal pBSHIV3BP2768. 65 antiserum specific to gp120 (Dupont) was used as the Plasmid pbSH6HIV3B5P was digested with KpnI to primary serum and a rat anti-mouse as the second antibody. liberate a 360 bp fragment containing the H6 promoter and Following this incubation period, the material was washed 4 5,766,598 21 22 times with 1X Buffer A. Lysates precleared with normal to poor immunization since vaccinia-specific memory CTL human sera and protein A-Sepharose were then incubated activity was apparent after in vitro stimulation with spleen overnight at 4° C. with the human sera from seropositive cells infected with any of the vaccinia viruses used. individuals bound to protein A-Sepharose. Following the In a similar study, the ability of a canarypox recombinant overnight incubation period, the samples were washed four expressing the V3 loop region fused to the 88 epitope (vOP95) to generate HIV-specific memory CTLs was exam times with 1XBuffer A and 2xwith LiClfurea buffer. Pre ined (Table 3). Three weeks following a single inoculation cipitated proteins were dissociated from the immune com of 10 PFUs of vCP95 or the canarypox vector. CPpp. plexes by the addition of 2xLaemmli's buffer (125 mM Tris HIV-specific memory CTL responses were compared to that (pH6.8), 4% SDS, 20% glycerol: 10% 2-mercaptoethanol) elicited by the recombinant vaccinia virus analog, w878. and boiling for 5 minutes. Proteins were fractionated on a O Vaccinia and canarypox CTL responses were included as 10% Dreyfuss gel system (Dreyfuss et al., 1984) fixed and controls for proper immunization. Only spleen cells from treated with 1M - Na - salicylate for fluorography. vP878 and vCP95 immunized mice produced HIV-specific The results of immunoprecipitation using sera pooled memory CTL activity which could be stimulated by vP878. from HIV-1 seropositive individuals showed specific pre The inability of vCP95 to stimulate existing memory CTLs cipitation of the gp120 and gp41 mature forms of the gp160 15 to functional cytolytic CTLs may have been related to the in envelope glycoprotein from vP911 infected cell lysates. No vitro conditions employed which were maximized based such specific gene products can be detected in the parental upon the use of vaccinia virus recombinants. Nonetheless, virus (NYWAC; we866) infected cell lysates, Specific pre vCP95 was fully capable of generating significant HTV cipitation of gp120 was also found in vp921 infected cell specific memory CTLs in the spleens of immunized mice. lysates. Characterization of the In Vitro Stimulated Cytotoxic Cells Immunofluorescence analysis with the same Sera illus It is conceivable that the cells mediating cytotoxicity trated that the gp160 and gp120 species expressed by vP911 against the HIV peptide-pulsed target cells represent a and vP921, respectively, were present on the surface of population of nonspecific effector cells unrelated to CTLs. infected cells. such as natural killer cells. To test this, the spleen cells of Immunoprecipitation was also performed with vCP112 25 mice immunized with vp21 and restimulated in vitro with infected CEF cells. No HIV-specific polypeptides were vP921 infected spleen cells were depleted of T-lymphocytes precipitated with a monoclonal antibody directed against the bearing surface antigens characteristic of T helper lympho gp120 extracellular moiety from cells infected with the cytes (CD4) or of cytotoxic T lymphocytes (CD8) and ALVAC parental virus and uninfected CEF cells. Two HIV assayed against V3 loop peptide pulsed target cells (Table 4). specific polypeptides species were, however, precipitated 30 As before, only v21 immunized mice generated memory from vCP112 infected cells. These species migrated with HIV-specific CTL activity which could be stimulated in vitro apparent mobilities of 160 kDa and 120 kDa, corresponding with wP921 infected syngeneic spleen cells. Although the to the precursor env gene product and the mature extracel complement preparation (C) and the monoclonal anti-CD4 lular form, respectively. and anti-CD8 produced some toxic effects, only the cultures A Recombinant Vaccinia Virus Expressing HIV gp120 Elic 35 depleted of CD8-bearing cells (anti-CD8+C) were also its Primary HIV-specific Cytotoxic T Lymphocyte Activity depleted of HIV-specific cytotoxic effector cells. Thus, the Following iv administration with 5x10' PFUs of vaccinia cells mediating cytolytic activity against the HTV peptide virus recombinants vp878, vp-11, or vP921, or, as a control, pulsed target cells possessed CD8 antigens on their cell with NYVAC, the vector. splenic CTL activity of BALB?c membranes, a characteristic of MHC class I restricted CTLS. mice was assessed against syngeneic P815 cells which had Specificity of CTL Antigen Receptor Recognition of the V3 been incubated overnight with peptide HBX2 (Table 1). Loop Region of HIV gp120 Modest, but significant (P<0.05) primary CTL activity was T lymphocyte antigen receptors are exquisitely sensitive generated in the spleens of mice administered vP921. to small alterations in the primary amino acid sequence of expressing HTV gp120. No other recombinant vaccinia virus the epitope fragment. The V3 loop region of HIV gp120 is nor the vector was able to elicit primary HIV-specific CTL 45 hypervariable and differs immunologically among HIV iso activity. This was not due to inadequate infection as each lates. The hypervariability resides in substitutions and addi group of mice administered a vaccinia virus responded with tions of only a few amino acids. To examine the specificity primary vaccinia-specific CTL activity. Control, unimmu of cytotoxic cells generated by HIV vaccinia virus nized mice responded to neither target. recombinants, susceptibility to CTL activity was compared Recombinant Poxviruses Expressing HTV env Peptides Gen among P815 target cells pulsed with peptides corresponding erate HIV-Specific Memory Cytotoxic T Lymphocytes the V3 loop region of HIV isolates III SF2, and MN. Only At least one month following a single inoculation with immunization with w811 and vP921 induced HIV specific one of the recombinant vaccinia viruses, mouse spleen cells primary CTL activity (Table 5). Furthermore, HTV specific were stimulated in vitro with syngeneic, naive spleen cells CTL activity was confined only to P815 target cells pulsed previously infected with NYVAC or with each of the HIV 55 with peptide corresponding to the V3 loop of HIV isolate recombinant vaccinia viruses (Table 2). Strong HIV-specific III. Similar results were obtained with in vitro stimulated, CTL activity was detected only in the spleen cell cultures of HIV specific secondary CTL activity induced by immuni mice immunized with w878, w1, and vp921 which were zation with the vaccinia virus recombinants wF878, voll, restimulated in vitro by cells infected with one of the same and wP921 (Table 6). Thus, HTV specific CTLs elicited by vaccinia virus HIV recombinants (vP878, wP911, or vpg21). recombinant vaccinia viruses expressing various portions of The vaccinia virus recombinants expressing HIV gp120 or the env gene of HIV isolate III recognize only target gp160 were better able to generate memory CTLs than the epitopes derived from the same antigenic isolate. vaccinia virus recombinant expressing only the V3 loop Lymphocyte Proliferation Responses to HIV Epitopes Fol fused to the 88 epitope. HIV-specific memory CTL activity lowing Immunization with Vaccinia Virus HIV Recombi could not be elicited from unimmunized control or NYWAC nants immunized spleen cells. The absence of HIV-specific CTL Lymphocyte proliferation to antigens is an in vitro cor activity from vector immunized mice could not be attributed relate of cell-mediated immunity. Presentation of the appro 5,766,598 23 24 priate antigen induces cellular proliferation in the immune population of cells expressing receptors for the antigen. The TABLE 2 initiation and continuation of proliferation requires the Secondary CTL activity of spleen cells involvement of Thelper lymphocytes via soluble mediators. following in vitro stimulation with vaccinia To evaluate cell-mediated immunity to HIV antigens in mice virus recombinants. immunized with recombinant vaccinia viruses expressing PERCENT CYTOTOXCTY HIV antigens, spleen cells from mice immunized 27 days MMUNIZATION TARGET earlier were incubated for 5 days with peptides correlating to in vivo in vitro P85 WAC HTV W3 T helper lymphocyte epitopes designated T and T as well 10 as with purified HIV gp160 (Table 7). No proliferative NONE NONE -0.1 19 0.5 NYWAC 3. 8.9 38 responses to the T helper cell epitopes T and T. were wP878 4.6 9.0 5.5 observed in any of the spleen cell cultures. However, the yP91 -1.7 2.9 4.8 spleen cells of mice previously immunized with vP921 vP921 2.9 29 .5 15 NYWAC NONE O.O 4.4 1. vigorously responded to HIV gp160 as determined by the NYWAC 3.5 478 9.2 incorporation of 13 H-thymidine. A stimulation index (SI) of w878 6.3 44.1% 14.4 vP91 7.9 48.68 10.6 greater than 2.0 is considered indicative of immunity. Thus, wP921 6.8 50.8. 7.9 inoculation of mice with v21 elicited cell-mediated wP878 NONE 0.1 17 1.3 immunity to HIV gp160. NYWAC 10.2 58.5: 130 w878 1.6 57.94 59.9 wP911 7.8 56.2 40.8 wpS2 4.9 42.0 14.8 Antibody Responses of Mice Inoculated with Vaccinia Virus wpg1 NONE O.3 2.9 40 HIV Recombinants NYWAC 6.2 50.7 x 8.5 wP878 5.9 50.9k 774 25 wp31. 5.0 54.2 82.6 To evaluate humoral responses to HIV, mice were immu v92 10.9 55.04: 878: nized at day 0 with one of the vaccinia virus HIV recom wP921 NONE 2.9 5. 9.4 NYWAC 8.3 544 227 binants and received a secondary immunization at week 5. wP878 10.4 56.23 85.6 The mice were bled at various intervals through 9 weeks 30 wP911 8.7 58.2 86.5 after the initial immunization. Pooled sera from each treat wP921 7.8 55.2 81.0 ment group were assayed for antibodies to HIV by ELISA BALB?c) spleen cells from mice immunized approximately 1 month earlier employing purified gp160 as antigen (Table 8). Primary with the indicated vaccinia virus recombinants and were incubated with antibody responses were generally modest, but detectable infected syngeneic spleen cells for 5 days and assayed for cytotoxicity at an effector to target cell ratio of 20.1. with the highest levels induced by vP911. Following the 35 * PC 0.05 compared to controls, Student's t-test. secondary immunization, the antibody titers of mice immu nized with vP911 and vP921 increased and peaked at week 7 with titers of over 4,600 and 3.200, respectively, before TABLE 3 declining slightly by week 9. Thus, two vaccinia virus HTV 40 Anamnestic CTL responses of the spleen cells recombinants. VP911 and vP921, were capable of inducing of mice administered a single inoculation of recombinant vaccinia or canarypox virus a significant antibody response. expressing the V3 loop of HIV gp120. TABLE 1. MMUNIZATION PERCENT CYTOTOXCTY Primary CTL activity of spleen cells from 45 PRIMARY BOOSTER TARGET mice immunized with vaccinia virus recombinants against vaccinia virus infected in vivo in vitro P815 Wac CP HIV W3 targets and targets pulsed with peptide NONE NONE 0.4 -2.5 -2.3 -1.5 corresponding to the W3 loop region of HTV-1 w804 0.5 88 O.7 0.8 gp120. 50 vP878 18 6.1 0.4 1.6 CP 5.8 4.2 4.9 0.4 PERCENT CYTOTOXCITY WCP95 4.4 2.6 6. 0.1 TARGET SB13S -0.2 -O. -0.4 O.5 wP804 NONE O. 1. O. 1.3 MMUNIZATION P815 WAC HTV W3 wP804 5.5 435 5.8 3.5 55 vP878 3.6 42.5 1.6 -0.3 NONE -3.5 -0.6 -4.8 CP 8.5 70 5.6 3.9 20 5 1.6 yCP95 5.8 5.3 44 4.0 NYWAC -44 95. -5.9 SB135 1.2 -0.9 -0.5 -0.2 19 3.2 7 wP878 NONE 0.2 -29 -0.8 -0.2 wp878 -49 7.1: -4.0 yP804 5.3 56.4 7.5 4. 1.8 2.2 .2 wps78 6.7 6O.2 77 417 wS11 -40 46 1.4 CP 8.7 134 9.4 4. t 2.5 2.0 5.1 wcpg5 7.1 10.5 8.7 19.0 SB135 9 -O. -0.2 -1.4 wP92 -3.4 10.72 15.5-k cP NONE 46 -0.6 2.3 -O.0 t 09 15 2.8 vP804 1.0 177 it 5.7 6.1 wP878 71. 146 12.3 5.5 ET = 00:1 65 CP T4 59 19.3 3. * P K 0.05 vs appropriate controls, Student's t-test wCP95 6.8 54 20.4 2.8 5,766,598 26

TABLE 3-continued TABLE 5-continued Anamnestic CTL responses of the spleen cells of mice administered a single inoculation of Specificity of primary CTL activity for the V3 loop of recombinant vaccinia or canarypox virus HIV-1 isolate II following a single inoculation with expressing the V3 loop of HIV gp120. HTV recombinant vaccinia viruses. MMUNIZATION PERCENTCYTOTOXCTY PERCENTCYOTOXCTY O PRiMAR BOOSTER TARGET TARGET

in vivo in vitro P85 Wac CP HW W3 W3 PEPIDE SB135 1.4 -0.4 0.8 -14 wCP95 NONE -0.8 -2.2 -1.3 0.3 15 MMUNIZATION P85 B SF2 MN wP804 9.4 26.4 9.3 6.6 vP878 0.4 22.54 16.9 32.1: CP 8.8 7.2 2O.O. 3.2 wP921 -2.5 12.5- -0. -1.2 WCP95 5. 4.2 19.6 78 O. 3.6 0.5 0.5 SB135 1.9 -1.5 -0.3 -1.2 Mice were administered a single iv inoculation with the indicated vaccinia Twenty-three days after immunization, the spleen cells were stimulated in virus recombinant and assayed for CTL activity 7 days later against P815 vitro for 5 days with virus infected or peptide-pulsed syngeneic spleen cells targets and P815 targets pulsed with one of three peptides corresponding to and then assayed for specific cytotoxicity against virus infected or peptide the W3 loop region of HIV-1 isolates III, SF2, and MN. Although assayed pulsed P815 target cells at an effector to target cell ratio of 20:1. at effector to target cell ratios of 100:1, 50:1, and 25:1, only the 100:1 data * P K 0.05 compared to appropriate controls, Student's t-test. 25 are shown. * P K 0.05 vs appropriate controls, Student's t-test TABLE 4 Depletion of cytotoxic activity with monoclonal TABLE 6 antibodies to CD8 plus complement. 30 Specificity of secondary CTL activity for the PERCENT CYTOTOXCIY V loop of HIV-1 isolate III following a single MMUNIZATION ARGETS inoculation with HIW recombinant vaccinia viruses.

in vivo in witro TREATMENT P815 WAC HV W3 35 PERCENT CYTOTOXCTY TARGET NONE NONE NONE l. 5 -0.3 NONE NYWAC NONE -74 0.4 -0.4 NONE wP921. NONE -0.2 .1 -O.7 MMUNIZATION W3 PEPTOE NYWAC NONE NONE -3. -0.3 -1.4 NWAC NYWAC NONE -2.6 40.5 -0.3 in vivo in vitro P815 B SF2 MN NWAC wP92 NONE 3.3 31.4 -29 wP92 NONE NONE 30 -1.3 -0. NONE NONE O 1. 0.5 -00 wP921 NYWAC NONE -49 25.9 2.2 NYWAC 0.4 O.S -0.6 -0.3 wP92 wP92 NONE -0.2 2.3 30.5 wP92 wP92 C. 4.6 2O. 22.9 w878 O.2 O.2 -0.5 -10 wP92 wP92 anti-C4 4.2 22.6 23.2 wP911 ---.S O3 -0.5 O.2 wps2 wP92 anti-CD8 -50 22.5 26.9 45 vP921 -0.6 14 O1 -0.5 wP921 wP92 anti-CD4+C" 1OO 26.6 3O. NYWAC NONE -2.2 O2 0.5 -10 wP921 wP92 anti-CD8 -- C 9.2 7. 2.3 NYWAC 3.2 2.2 3.9 2.5 vP878 44 59 S.O. 6.1 v911 58 11.1 5.0 5.3 TABLE 5 50 wP921 50 6.5 2.9 2.9 wP878 NONE 0.1 -0.2 -0.9 -10 Specificity of primary CTL activity for the W3 loop of NWAC 30 48 4.4 4.5 HIV-1 isolate DI following a single inoculation with HIV recombinant vaccinia viruses. vP878 79 O.2 8 8.6 wP911 48 7.8 4.5 4.7 PERCENT CYTOTOXCITY 55 wP921 2. 6.9 2.8 3.0 TARGET wp1 NONE O9 8 1.4 0.5 V3 EPTDE NWAC 8.8 8.3 8. 6.6 wP878 6.6 57.2 6.8 8.2 IMMUNIZATION P815 B SF2 MN wP91 4.6 63.7 2.9 4.2 wP921 7.2 63.6 4. 4.9 NONE -2.7 -9 -0.9 -1.2 -0.5 O.5 0.5 O.5 wP921 NONE 0.5 0.8 1.2 O.6 NYWAC -1.6 -0.3 -0.6 -0.3 NYWAC 4.4 7.9 7.5 6.0 -0.5 O.8 0.7 0.2 wP878 8.1 59. 7. 7.5 vP878 -2.8 0.5 -0.5 -i.2 wP91 6.4 71.4 79 66 0.8 1.0 0.6 0.5 wP91 -2.6 7.5 -0.5 -1.1 65 wpS21 9.3 63.4 9.0 8.1 0.2 3.2 O.5 0.4 5,766,598 27 28

TABLE 7 Lymphocyte proliferative responses to HTV gp160 epitopes 27 days after a single immunization with HTV recombinant vaccinia viruses. COUNTS PERMNTUE HTWANGEN gp160 (Lig) TI (ug) T2. (ug) MMUNIZATION RPM 1 O 5 O 5 10 NONE MEAN 5,185 6,397 7,808 7,682 8,64 11,541 6,141 8.835 6,774 SD 102O 274 2,596 1,274 2,033 2,036 2,103 1883 2,806 SI 10 1.2 1.5 O .1 15 O 1.4 1.1 NYWAC MEAN 10,327 13,589 15,969 11360 2,654 15,369 10,339 9,834 8,868 SD 1,543 3,323 4,583 1,352 2,272 1821 762 1,731 5O2 SI 10 3 1.5 1.0 .1 1.4 O 10 O.9 wP878 MEAN 10,126 13,150 18,329 11, 114 11.956 13,754 10,415 11.442 9,147 SD 1.269 103 4,245 1,217 1,106 1,568 335 1.288 1,033 SE 10 1.3 18 1.0 1.1 1.2 O 1. 0.9 wP911 MEAN 12,155 15,564 26,083 12,417 15,380 17,007 10,681 1412 0.702 tSD 1,307 9,707 16,327 873 1847 6,266 2428 3,201 468 SI 10 1.3 2.1 O 1.2 1.4 O 1.1 1.0 wpS21 MEAN 9,701 49,256 * 61,036 10,550 15,367 15,816 8,818 9,232 8,803 SD 2,601 23,673 25,866 3,447 3,481 7, 176 954 2,265 2,860 S 10 5.1 6.3 O 15 1.5 O 10 10

SI - stimulation index. * P C 0.05 compared to unstimulated control cultures Student's t-test esis with this oligonucleotide generates a T to C transition TABLE 8 and disrupts the T5CT motif at nucleotide positions 6929-6935 of the ARV-2 genome (Sanchez-Pescador et al. HIV gp160 ELISA titers of mice immunized with HIV 1985). This mutation does not alter the amino acid sequence recombinant vaccinia viruses. of the env gene and creates an EcoRI site, which was used WEEKSAFEREMMMUNIZATION to screen for mutagenized plasmid clones. Sequence confir mation was done by the dideoxynucleotide chain termina MMUNIZATION O 1 2 4. 7 9 plasmidtion method was (Sanger designated et al.. as 1977). pIBI25mutenv11. The resultant mutagenized cNES : . : s A 1.45 kb BglII fragment was derived from wP878 2O 43 2 46 65 63 pIBI25mutenv11. This fragment contained the mutated env wP9 O O 90 453 4,614 3,263 sequences. It was used to substitute for the corresponding wP92 O 26 25 77 2,614 1689 unmutated fragment in pIRI25env. The resultant plasmid 40 was designated as p3.25mutenv8. Further modifications were made to pIBI25mutenv8. In vitro mutagenesis was EXAMPLE 3 performed to remove the sequence coding for the rex protein and the LTR sequence (LTR region) from the 3'-end of the EXPRESSION OF THE HIV-1 (ARV-2 OR SF-2 gene and to delete the putative immuno-suppressive (IS) STRAIN) env GENE IN ALVAC, TROVAC AND 45 region amino acids 583 through 599 (SEQID NO:46:Leu NYWAC VECTORS Gln-Ala-Arg-Val-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Arg-Asp Plasmid Constructions Gln-Gln-Leu) (Klasse et al., 1988). These reactions were The lambda clone containing the entire HIV-1 (ARV-2 or done with the single-stranded template derived from SF-2 strain) genome was provided by J. Levy and was pIBI25mutenv8 with oligonucleotides LTR2 (SEQ ID described previously (Sanchez-Pescador et al., 1985). The 50 NO:47) (5'-TTGGAAAGGCTTTT GGCAT env sequences were subcloned into plJC13, creating plasmid GCCACGCGTC-3) and MUENSVISR (SEQ DD NO:48) pMP7MX373, which contains the sequences from -1 rela (5'-ACAGTCTGGGGCATCAAGCAGCTAGGGATTT tive to the initiation codon (ATG) of the env gene product to GGGGTTGCTCT-3"). Mutagenized clones were identified 715 bp downstream of the termination codon (TAA) of the by hybridization and restriction analysis. A clone env gene. These env sequences were excised from 55 mutagenized such that it was deleted both of the IS and the pMP7MX373 by digestion with EcoRI and HindIII and LTR region and another deleted of the LTR was confirmed inserted into the plasmid vector, pIBI25 (International by nucleotide sequence analysis and designated Biotechnologies, Inc.. New Haven, Conn.) generating plas pIBI25mut3env40 and p3.25mut2env22, respectively. mid pIBI25env. A3.4 kb SmaI/HindIII fragment containing the entire env Recombinant plasmid pLEBI25env was used to transform gene was derived from pIBI25mut3env40 and from competent E. coli CJ236 (dut- ung-) cells. Single-stranded pIBI25mut2env22 and inserted into pCPCV1 and pFPCV2. DNA was isolated from phage derived by infection of the digested with SmaI/HindIII. The plasmid pCPCV1 is an transformed E. coli CJ236 cells with the helper phage. insertion plasmid which enables the generation of ALVAC MG408. This single-stranded template was used in in vitro recombinants with insertion occurring in the C3 locus. The mutagenesis reactions (Kunkel et al., 1985) with oligonucle 65 plasmid, pFPCV2, is an insertion plasmid which enables the otide MUENVT 12 (SEQ ID NO:45) (5'- generation of TROVAC recombiants with insertion occur AGAGGGGAATTCTTCTACTGCAATACA-3). Mutagen ring in the F7 locus. Plasmids pCPCV1 and pFPCV2 have 5,766,598 29 30 been described previously in PCT International Publication Expression analyses with all six recombinant viruses were No. WO 89/03429 published Apr. 20, 1989. performed in CEF, Vero, and MRC-5 cell monolayers. Oligonucleotide PROVECNS (SEQ ID NO:49) (5'- radioimmunoprecipitation experiments using pooled Sera CCGTTAAGTTTGTATCGTAATGAAAGTGAAGGGGA from HIV seropositive individuals were performed as CCAGG-3") was used for in vitro mutagenesis reactions via described above. All six recombinants directed the synthesis the method of Mandecki (1986) to make a precise ATG:ATG of the HIV-1 gp160 envelope precursor. The efficiency of construction with the VVH6 promoter and the env processing of gp160 to gp120 and gp41. however, varied sequences. Potential mutants were screened for the loss of between cell types and was also affected by deletion of the the SmaI site. Plasmid clones devoid of a SmaI site Were immunosuppressive region. Recognition of gp41 by the identified and confirmed by nucleotide sequence analysis. pooled sera from HIV seropositive individuals also varied Properly mutagenized plasmid clones were identified and O between the virus background and the cell type. designated as pCPenvS+ or pCPenvS- and pFPenvS+ or pFPenviS. EXAMPLE 4 The HIV-1 env genes were excised from pCPenviS- by EXPRESSION OF THE HIV-2 (ISSYSTRAIN) digestion with NruI and HindIII. The two env fragments of env GENE IN NYWAC 2.5 kb (envS+) and 2.4 kb (envS-), respectively, were 15 Expression of gp160 isolated and blunt-ended by reaction with the Klenow frag Oligonucleotides HIV25PA (SEQ ID NO:50) (5'- ment of the E. coli DNA polymerase in the presence of 2 ATGAGTGGTAAAATTCAGCTGCTTGTTGCCTTTCT mM dNTPs. These fragments were ligated with the 3.5 kb GCTAACTAGTGCTTGCTTA-3") and HIV25PB (SEQ ID fragment derived by digestion of pSTVenvVV with NruI and NO:51) (5'-TAAGCAAGCACTAGTTAGCAGAAAGGC Pst with a subsequent blunting step with the Klenow 20 AACAAGCAGCTGAATTTTACCACTCAT-3") were fragment of the E. coli DNA polymerase in the presence of annealed to constitute the initial 54 bp of the HIV-2 ISSY 2 mM dNTPs. The plasmid pSIVenvVV contains the SIV strain (Franchini et al.. 1989) env coding sequence. This envgene expression cassette regulated by the vaccinia virus fragment was fused 3' to a 129bp fragment derived by PCR H6 promoter in the ATI insertion locus. Digestion of with oligonucleotides H65PH (SEQ ID NO:52) (5'- pSIVenvVV with NruI and Pst excises the entire SIV env 25 ATCATCAAGCTTGATTCTTTATTCTATAC-3") and coding sequences and the 3'-most 20 bp of the promoter H63PHIV 2 (SEQ ID NO:53) (5'- element. Ligation to the env IS- and env IS+ fragments CAGCTGAATTTTACCACCATTACGATACA AACTT restores the 20 bp of the H6 promoter and inserts the HIV-1 AACG-3") using pTP15 (Guo et al., 1989) as template. The env gene into the ATI insertion plasmid. The resultant fusion of these two fragments was done by PCR using plasmids were designated as pARSVV+ and pAR6VV- for 30 oligonucleotides HIV25PC (SEQ ID NO:54) (5'- env IS- and env IS-, respectively. TAAGCAAGCACTAGTTAG-3") and H65PH (SEQ ID In Vitro Recombination and Purification of Recombinants NO:52). The 174 bp PCR derived fragment was digested Recombination was performed introducing plasmid DNA with HindIII and SacI and inserted intopBS-Sk(Stratagene, into infected cells by calcium phosphate precipitation both La Jolla, Calif.) digested with HindIII and SacI. The result for ALVAC and for TROVAC recombinants, as previously 35 ant fragment was designated p3SH6HTV2. The insert was described (Piccini et al., 1987). Plasmids pCPenvS+ and confirmed by nucleotide sequence analysis. pCPenvS- were used to make recombinants vCP61 and The 3' portion of the HIV-2 env gene was also derived by vCP60, respectively. Plasmids pFPenvS+ and pFPenvS PCR. In this reaction a 270 bp fragment was amplified with for recombinants wFP63 and vFP62, respectively. The plas oligonucleotides HIV2B1 (SEQ ID NO:55) (5'- mids pAR5VV+ and paR6VV- were used in in vitro CCGCCTCTTGACCAGAC-3') and HIV2B2 (SEQ ID recombination experiments with wP866 as rescue to yield NO:56) (5'-ATCATCTCTAGAATAAAAATTACAGGA vP939 and vP940, respectively. Recombinant plaques were GGGCAATTTCTG-3") using pISSY-KPN (provided by Dr. selected by autoradiography after hybridization with a p Genoveffa Franchini, NCI-NIH, Bethesda, Md.) as template. labeled env specific probe and passaged serially three times This fragment fragment was digested with BamHI and Xba. to assure purity, as previously described (Piccini et al., 45 The 150 bp fragment derived from this digestion contained 1987). a 5' BamHI and a 3’ Xbal cohesive end. The fragment was Expression of the HIV-1 env Gene engineered to contain a T5NT sequence motif known to be Six different recombinant viruses were prepared where recognized as vaccinia virus early transcription termination the HIV env gene of the ARV-2 or SF-2 strain was inserted signal (Yuen et al., 1987), following the termination codon downstream from a vaccinia early-late promoter. H6. For 50 (TAA). simplicity, the two ALVAC-based recombinant viruses, The majority of the HIV-2 env gene was obtained from wCP61 and vCP60, will be referred to as CPS-- and CPS pISSY-KPN by digestion with SacI and BamI. The 2.7 kb the two TROVAC-based recombinants, w83 and vFP62, fragment generated by this digestion was coinserted into as FPS- and FPIS-, and the two NYWAC-based pBS-SK digested with SacI and Xbal with the 150 bp recombinants, vp39 and wP940, as VV- and VV+, respec BamHI/Xbal fragment corresponding to the 3' end of the tively. gene. The resultant plasmid was designated pBSHIV2ENV. All the constructs were precise, in that, the ATG initiation The 174 bp Spe/HindIII fragment from pBSH6HTV2 and codon of the HIV-1 env gene was superimposed on the ATG the 2.5 kb Spe/Xbal fragment from pBSHIV2ENV were of the vaccinia H6 promoter. Moreover, all extraneous ligated into pBS-SK digested with HindIII and Xbal to yield genetic information 3' to the termination codon was elimi pBSH6HIV2ENV. The 2.7 kb HindIII/Xbal insert from nated. CPIS-FPIS-, and VV- were all obtained by deletion pBSH6HIV2ENV was isolated and blunt-ended with the of a 51 bp region, corresponding to amino acids 583-599. Klenow fragment of the E. coli DNA polymerase in the located near the 5' portion of the gp41 gene product. This presence of 2 mM dNTP. The blunt-ended fragment was region shares homology with putative immunosuppressive inserted into a SmaI digested pSD541VC insertion vector. regions (Klasse et al., 1988: Ruegget al., 1989a,b) occurring 65 The resultant plasmid was designated as paTHIV2ENV. in the transmembrane polypeptide of other retrovirus gly This plasmid was used in vitro recombination experiments coproteins (Cianciolo et al., 1985: Ruegg et al., 1989a,b). with vP866 (NYVAC) as the rescuing virus to yield wP920. 5,766,598 31 32 Immunoprecipitation analysis was performed to deter of the entire HIV-2 env gene. No HTV-specific species were mine whether vP920 expresses authentic HIV-2 gp160. precipitated from mock infected or wF866 infected Vero Lysates from the infected cells were analyzed for HIV-2 cells. A protein species of 120 kDa was, however, precipi env gene expression using pooled serum from HTV-2 serop tated from lysates derived from cells infected with vP922. ositive individuals (obtained from Dr. Genoveffa Franchini, NCI-NIH. Bethesda, Md.). The sera was preadsorbed with EXAMPLE 5 vP866 infected Vero cells. The preadsorbed human sera was EXPRESSION OF STV GENES IN NYVAC bound to Protein A-sepharose in an overnight incubation at Generation of NYVAC/SIV gp140 Recombinant 4° C. Following this incubation period, the material was A plasmid pSS11E containing the STV () env gene washed 4xwith 1xbuffer A. Lysates precleared with normal O was obtained from Dr. Genoveffa Franchini (NCI-NTH. human sera and protein A-sepharose were then incubated Bethesda, Md.). This plasmid was digested with HindIII and overnight at 4 C. with the human sera from seropositive Pst to liberate a 2.2 kbp fragment containing from nucle individuals bound to protein A-sepharose. After the over otide 220 of the STV env gene to a region 160 bp down night incubation period, the samples were washed 4xwith stream from the translation termination codon. It should be 1xbuffer A and 2xwith a LiClfurea buffer. Precipitated 15 noted that an expression cassette containing this fragment proteins were dissociated from the immune complexes by will result in the expression of a gp140 protein species rather the addition of 2xLaemmli's buffer (125 mM Tris(pH6.8), that a gp160 species. This 40% deletion of the transmem 4% SDS. 20% glycerol. 10% 2-mercaptoethanol) and boil brane region results from a premature termination at nucle ing for 5 min. Proteins were fractionated on a 10% Dreyfuss otide 7.934 of the genome (Franchini et al.. 1987). Such gel system (Dreyfuss et al., 1984), fixed and treated with 1M premature terminations of the env gene product are noted Na-salicylate for fluorography. after propagation of SIV in culture (Kodama et al., 1989). Human sera from HTV-2 seropositive individuals specifi The amino portion of the gene was derived by PCR using cally precipitated the HIV-2 gp160 envelope glycoprotein pSS11E as template and oligonucleotides SIVENV1 (SEQ from v920 infected cells. Furthermore, the authenticity of ID NO:59) (5'-CGATATCCGTTAAGTTTGTATCGTAA the expressed HIV-2 env gene product was confirmed, since 25 TGGGATGTCTTGGGAATC-3") and SIVENV2 (SEQ ID the gp160 polyprotein is processed to the mature gp120 and NO:60) (5'-CAAGGCTTTATTGAGGTCTC-3). The result gp41 protein species. No HIV-specific protein species were ant 250 bp fragment contains the 5'-most 230 bp of the SIV precipitated from mock-infected cells or cells infected with envgene juxtaposed downstream from the 3'-most 20 bp of the parental virus, w866. Also, supportive of the proper the vaccinia virus H6 promoter (3'-end of NruI site). A 170 expression of the HIV-2 env by vP920 was the observation 30 bp fragment was obtained by digestion of the fragment with by an immunofluorescence assay that the gene product is HindIII, which removes 80 bp of SIV env sequences. expressed on the surface of vP920 infected cells. The sequences containing the remainder of the SIV env Expression of gp120 gene following the premature termination signal were The plasmid pBSH6HIV2 containing the vaccinia virus derived by PCR from pSS35E (obtained from Dr. Genoveffa H6 promoter fused to the 5'-end of the HIV-2 env gene was 35 Franchini). This plasmid contains sequences containing the digested with Spe and HindIII to liberate the 180 bp C-terminal portion of the SIV env gene into the LTR region fragment containing these sequences. This fragment was downstream from the envgene. The oligonucleotides used to ligated into pbS-SK digested with HindIII and Xba along derive the 360 bp fragment were SIVENV3 (SEQ ID with the 1.4 kb Spe/Xbal fragment of p3SHIV2120A to NO:61) (5'-CCTGGCCTTGGCAGATAG-3') and yield pBSHIV2120B. SIVENV 4A (SEQ ID NO:62) (5'- The plasmid pBSHIV2120A was derived by initially ATCATCGAATTCAAAAATATTACAAAGAGCGTGA deriving the 3' portion of the gp120 coding sequence by GCTCAAGTCCTTGCCTAATCCTCC-3"). This fragment PCR. The PCR was performed using oligonucleotides was digested with Pst and EcoRI to generate a 260 bp HIV 22 OA (SEQ D NO:57) (5'- fragment having a 5' Pst cohesive end and a 3'- EcoRI ATCATCTCTAGAATAAAAATTATCTCTTATGTCT 45 cohesive end. CCCTGG-3") and HIV2120B (SEQ ID NO:58) (5'- The 2.2 kb HindIII/Pst fragment from pSS11E, the 170 AATTAACTTTACAGCACC-3") with pISSY-KPN as tem bp Nru/HindIII fragment containing the 5' end of the gene, plate. The PCR-derived fragment was digested with EcoRI and the 260 bp Pst/EcoRI containing the 3' end of the gene and Xbal to yield a 300 bp fragment which contained a were ligated with a 3.1 kb NruI/EcoRI fragment derived 5'-EcoRI cohesive end and a 3'-Xbal cohesive end. The SO from pRW838. pRW838 contains the vaccinia virus H6 fragment was engineered with a translation termination promoter linked to the rabies G gene flanked by canarypox sequence (TAA) and a T5NT sequence motif just 5' to the virus sequences which enable the insertion of genes into the Xba site. The 300 bp Xbal/EcoRI PCR fragment was C5 locus. Digestion with NruI and EcoRIliberates the rabies ligated into pBS-SK digested with SacIFXbal along with a G gene and removes the 3'-most 20 bp of the H6 promoter, 1.4 kb SacI/EcoRI fragment derived from pISSY-KPN to 55 The resultant C5 insertion plasmid containing the SIV env generate pBSHIV2120A. gene linked to the vaccinia H6 promoter was designated as The plasmid p3SHTV2120B was digested with HindIII pC5SIVENV. and Xbaltogenerate a 1.8 kb fragment containing the HIV-2 The plasmid, pC5SIVENV. was digested with HindIII and gp120 coding sequence juxtaposed 3' to the vaccinia virus EcoRI to liberate a 2.2 kb fragment, containing from nucle H6 promoter. This fragment was blunted with the Klenow otide 150 of the SIV env gene to the end of the entire gene. fragment of the E. coli DNA polymerase in the presence of PCR was used to derive the vaccinia H6 promoter/SIV env 2 mM dNTPs. The blunt-ended fragment was ligated to linkage from pC5SIVENV with oligonucleotides SmaI digested pSD541VC to generate paTHIV2120. This MP SYN286 (SEQ D NO:63) (5'- plasmid was used in in vitro recombination experiments to CCCCCCAAGCTTTTTTATTCTATACTT-3') and yield vP922. 65 SIVENV2 (SEQ D NO:64) (5'- Immunoprecipitation experiments with vP922 infected CAAGGCTTTATTGAGGTCTC-3). The 320 bp fragment cells were performed as described above for the expression was digested with HindIII to derive a 240 bp fragment. The 5,766,598 33 34 2.2 kb HindIII/EcoRI and the 240 bp HindIII fragment were used in in vitro recombination experiments with vp873 as coligated into pC3I digested with HindIII and EcoRI. The rescue virus to yield vP948. resultant plasmid containing the HindIII fragment in the The plasmid to insert both gag and pol into NYVAC proper orientation relative to the STV env coding sequence based vectors was engineered in the following manner. was designated pC3SIVEM. The plasmid pC3I was derived pSIVG1, described above. contains extraneous 3'-noncoding as follows. The nucleotide sequence analysis of an 2.5 kb sequences which were eliminated using a 1 kb PCR frag BglII canarypoxvirus genomic fragment revealed the entire ment. This fragment was generated from plasmid C3 open reading frame and the 5' and 3' flanking regions. In pSIVGAGSS11G with the oligonucleotides SIVP5 and order to construct a donor plasmid for insertion of foreign SIVP6. This PCR derived fragment containing the 3' end of genes into the C3 locus with the complete excision of the C3 the pol gene was digested with BamHI and HpaI. The 1 kb open reading frame. PCR primers were used to amplify the BamHI/HpaI fragment was ligated to the 7.400 bp partial 5' and 3' sequences relative to C3. Primers for the 5' BamHI/HpaI fragment of pSIVG1 to yield pSIVG4. sequences were RG277 (SEQ ID NO: 65) (5'- Sequence analysis of pSIVG4 revealed a single base pair CAGTTGGTACCACTGGTATTTTATTTCAG-3") and deletion within the pol gene. To correct this error the 2.300 RG278 (SEQ ID NO:66) (5'- 15 bp BglTI/Stu fragment from pSIVG1 was inserted into the TATCTGAATTCCTGCAGCCCGGGTTTTTATAGCTAA 6,100 bp partial BglII/Stu fragment of pSIVG4 to yield TTAGTCAAATGTGAGTTAATATTAG-3"). pSIVG5. The plasmid, pSIVG5, was used in in vitro recom Primers for the 3' sequences were RG279 (SEQ ID bination experiments with vP873 as rescue to generate NO: 67) (5'- VP943. T C G C T G AAT T C GATAT C A A G CTTATC Generation of NYVAC/SIV p16 and p28 Recombinants GATTTTTATGACTAGTTAATCAAATAAAAAGCATA The pol gene and the portion of the gag gene encoding CAAGC-3") and RG280 (SEQ ID NO:68) (5'- p28, p.2.p8, p1, and p6 were eliminated from pSIVG1. This TTATCGAGCTCTGTAACATCAGTATCTAAC-3'). The was accomplished by cloning the oligonucleotides STVL10 primers were designed to include a multiple cloning site (SEQ ID NO:73) (5'-AG ACC AACAGC flanked by vaccinia transcriptional and translational termi 25 ACCATCTAGCGGCAGAGGAGGAAATTACTAA nation signals. Also included at the 5'-end and 3'-end of the TTTTTATTCTAGAG-3') and SIVL11 (SEQ ID NO:74) left arm and right arm were appropriate restriction sites (5'-GATCCTCTAGAATAAAAATTAGTAATTTCCTC (Asp718 and EcoRI for left arm and EcoRI and SacI for right CTCTGCCGCTAGATGGTGCTGTTGGT-3') into the arm) which enabled the two arms to ligate into Asp718/SacI 4.430 bp AccI/BamHI fragment of pSTVG1 to generate digested pBS-SK plasmid vector. The resultant plasmid was 30 pSIVG3. This plasmid contains an expression cassette for designated as pC3I. the SIV p16 gene product expressed by the vaccinia I3L The plasmid pC3SIVEM was linearized by digestion with promoter, EcoRI. Subsequent partial digestion with HindIIIliberated a The entomopoxvirus 42 kDa-promoted SIV p28 gene (5' 2.7 kb HindIII/EcoRI fragment. This fragment was blunt end only) was inserted downstream from the I3L-promoted ended by treatment with Klenow fragment of the E. coli 35 p16 gene. This was accomplished by cloning the 360 bp DNA polymerase in the presence of 2 mM dNTPs. The BspMI/BamHI fragment of pSTVG1, containing the 5' end fragment was ligated into pSD550VC digested with SmaI. of the p28 gene, the oligonucleotides pSIVL14 (SEQ ID The resultant plasmid was designated as pSIVEMVC. This NO:75) (5'-TAGACAAAATT GAAAATATATAATTA plasmid was used in in vitro recombination experiments CAATATAAAATG CCA GTA CAA CAAATAG with vP866 as rescue virus to generate vP873. vP873 GTGGTAACTATGTCCACCTGCC ATT-3") and SIVL15 contains the SIV env gene in the 4L locus. (SEQ D NO:76) (5'- Generation of a NYVAC/gag?pol and gag Recombinants G CTTA AT G G CAG G T G GA CATA GT TA A plasmid, pSIVAGSS11G, containing the SIV cDNA CCACCTATTTGTTGTACTGGCATTTTATATTGTAATT sequence encompassing the gag and pol genes was obtained ATATATTTTCAATTTTGT-3"), containing the entomopox from Dr. Genoveffa Franchini (NCI-NIH, Bethesda, Md.). 45 42 kDa promoter into the 4,470 bp partial Xbal/BamHI The gag and pol genes from this plasmid were juxtaposed 3' fragment of pSTVG3. The resultant plasmid was designated to the vaccinia I3L promoter between vaccinia tk flanking as pSIVG6. arms. This was accomplished by cloning the 4,800 bp The 3' portion of the p28 gene was then inserted into CfoI/Tagfragment of pSIVGAGSS11G, containing the gag pSTVG6. A 290 bp PCR fragment, containing the 3' end of and the oligonucleotides SIVL1 (SEQ ID NO:69) (5'- 50 the SIV p28 gene, was derived from pSIVGI using oligo TCGAGTGAGATAAAGTGAAAATATATATCATTATA nucleotides SIVP12 (SEQ ID NO:77) (5'- TTACAAGTACAATTATTTAGGTTTAATCATGGGCG TGGATGTACAGACAAC-3') and SIVP13 (SEQ ID 3') and SIVL 2 (SEQ ID NO:70) (5'- NO: 78) (5'- CCCATGATTAAACCTAAATAATGTACTTTGTAATA AAGGATCCGAATTCTTACATTAATCTAGCCTTC-3). TAATGCTATATATTTTCACTTTATCT CAC-3) corre 55 This fragment was digested with BamHI and ligated to the sponding to the I3L promoter into the 4,070 bp XhoI/AccI 4,830 bp BamHI fragment of pSIVG6. The resultant fragment of pSD542, a derivative of pSD460 (FIG. 1). The plasmid, pSIVG7, was used in in vitro recombination plasmid generated by this manipulation was designated experiments with vP866 and vP873 as rescue experiments to pSTVG1. generate wP942 and wP952. respectively. To eliminate the pol gene, a 215 bp PCR fragment was Expression Analyses derived from pSIVGAGSS11G using oligonucleotides The SIV gp140 envgene product is a typical glycoprotein SIVP5 (SEQID NO:71) (5'-AATCAGAGAGCAGGCT-3') associated with the plasma membrane of infected cells. It is and SIVP6 (SEQ ID NO:72) (5'- expressed as a polyprotein of 140 kDa that is proteolytically TTGGATCCCTATGCCACCTCTCT-3"). The PCR-derived cleaved to an extracellular species of 112 kDa and a trans fragment was digested with BamHI and Stu and ligated 65 membrane region of 28 kDa (Franchini et al., 1987). Immu with the 5370 bp partial BamHI/StuI fragment of SIVG1. nofluorescence analysis using sera from rhesus macaques This resulted in the generation of pSIVG2. pSIVG2 was seropositive for SIV followed by fluorescein conjugated 5,766.598 35 36 rabbit anti-monkey IgG demonstrated expression of the env Six week old female BALB/c mice were inoculated intra gene product on the surface of recombinant infected Vero venously with 5x10 pfu of vaccinia virus (NYVAC). cells. Surface expression was not detectable on the surface recombinant vaccinia virus expressing HIV-1 (IDB) env of mock infected cells or cells infected with the NYWAC (vP911), canarypoxvirus (ALVAC), or with recombinant (vP866) parent virus. Furthermore, cells infected with canarypoxvirus expressing HIV-1 (IIIB) env (vOP112). recombinants containing only gag genes were not shown to Seven days later, the spleen cells were assayed for primary express any SIV components on the surface. Surface expres CTL activity against unmodified P815 cells or P815 cells Sion in cells infected with w873, WA3, w948 and v52 that had been incubated overnight with a peptide corre all demonstrated surface expression and significantly, all sponding to the hypervariable V3 loop region of HIV-1 contain the SIV env gene. (IIIB) gp120. Twenty-two days after the initial The authenticity of the expressed SIV gene products (env immunization, the spleen cells of the experimental mice and gag) in Vero cells infected with the NYVAC/HIV were incubated with poxvirus infected stimulator spleen recombinants was analyzed by immunoprecipitation as cells and assayed for memory CTL activity against peptide described above, except all samples were harvested at 17 pulsed targets as before. To determine secondary CTL hours post infection by the addition of 1 ml of 3xBuffer A. 15 activity. 29 days after the primary immunization mice Lysates from the infected cells were analyzed with pooled received a second inoculation of identical dosage and con sera from STV seropositive rhesus macaques or a mono tent as the first. Five days later, the spleen cells were assayed clonal antibody specific for gag p24 gene product (both for cytolytic activity against peptide pulsed targets. For obtained from Dr. Genoveffa Franchini, NCI-NIH, Bethesda cytotoxicity assays, H-2 P815 murine mastocytoma cells Md.). were incubated overnight in medium (Minimum Essential Immunoprecipitation with the SIV seropositive macaques Medium containing Earle's salts and supplemented with sera was performed in the following manner. The macaque 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml sera was incubated with a protein A-sepharose at 4 C. for penicillin, and 100 pg/ml streptomycin) with or without 20 16 hours. After washing with buffer A, the sera bound to ug/ml V3 peptide (CNTRKRIRIQRGPGRAFVTGK. protein A sepharose was added to lysates precleared with 25 American Bio-Technologies, Inc.) (SEQ D NO:79). The normal monkey sera and protein Asepharose. Following an following morning, the P815 cells were washed by centrifu overnight incubation at 4° C. the precipitates were washed gation and labeled for 1 hr at 37°C. in 100 uCi of NaCrO. 4xwith buffer A and 2xwith LiCl/urea buffer. To dissociate per 2x10 cells. Intact spleens were aseptically removed the precipitated protein from the antibody, the samples were from euthanized mice, bathed in ice cold Hank's Balanced boiled in 80 ul2xLaemmlibuffer for 5 minutes. The samples 30 Salt Solution, and disrupted into single cell suspensions were fractionated on a 12.5% gel using the Dreyfuss gel using a Stomacher blender. The spleen cell suspensions were system (Dreyfuss et al.. 1984). The gel was fixed and treated washed several times by low speed centrifugation and with 1M Na-salicylate for fluorography. All the recombi resuspended in Assay Medium (RPMI 1640 containing 10% nants containing SV genes were expressing the pertinent fetal bovine serum, 20 mM HEPES, 2 mM L-glutamine. gene products. The NYVAC recombinants vP873, vp43. 35 5x10M 2-mercaptoethanol. 100 U/ml penicillin, and 100 vP948 and v952 which contain the SIV env gene all ug/ml streptomycin). For memory CTL activity, the spleen expressed the authentic gp140. However, it is difficult to cells from immunized mice were resuspended in Stimulation assess the processing of the gp140 protein to the 112 kDa Medium (Minimum Essential Medium with Earle's salts and 28 kDa mature forms. No species with an apparent containing 10% fetal bovine serum, 2 mM L-glutamine. molecular weight of 140 kDa was precipitated by macaque 40 10M 2-mercaptoethanol, 100 U/ml penicillin, and 100 anti-SIV sera from mockinfected Vero cells, w866 infected ug/ml streptomycin) and stimulated in vitro in upright 25 Vero cells and Vero cells infected with NYWAC/SIV recom cm tissue culture flasks with naive syngeneic stimulator binants not containing the STV env gene. Expression of the spleen cells that had been infected with one of the poxvi SIV gag encoded gene products by vP942, vp943, vp48, ruses orpoxvirus recombinants. After five days at 37° C., the and vP952 was demonstrated using the pooled sera from 45 cells were washed, counted, and resuspended in Assay macaques infected with SIV and the monoclonal antibody Medium. 'Chromium labelled target cells were added to specific to the p28 gag component. Expression of the entire titrated effector cells in 96-well microtiter plates for a 4 hr p55 gag protein without the pol region, which contains the 'Cr release assay. Effector to target cell ratios (E:T) shown function, by NYVAC (vP948) in Vero cells is for the three assays were 100:1 (primary). 20:1 (memory), evident. These results demonstrate that lack of SIV protease 50 and 50:1 (secondary). Percent cytotoxicity was calculated as expression prevents the complete proteolysis of p55 into its described above, i.e., (experimental 'Cr release mature form. This is demonstrated much more clearly when spontaneous 'Cr release)/(maximum 'Cr release a monoclonal antibody specific to p28 was used to precipi spontaneous 'Cr release)x100. Maximum release was tate gag specific gene products from v948 infected Vero determined by the addition of 5% sodium dodecyl sulfate to cells. Contrary to this result, expression of STV gag with the 55 target cells while spontaneous release was determined by pol gene (includes protease in vp43 infected Vero cells) incubating target cells in the absence of effector cells. In enabled the expressed p55 gag precursor polypeptide to be none of the experiments presented did spontaneous release proteolytically cleaved to its mature forms. of Cr from target cells exceed 20% of maximum 'Cr Expression of both the p16 and p28 SIV gene products in release. Error bars in FIG.7 represent 1 standard deviation wP942 and vpg52 infected Vero cells was demonstrated from the mean. PC0.05, Student's t-test compared to appro using the pooled sera from macaques infected with SIV. priate vaccinia or canarypoxvirus immunized mice. Using the monoclonal antibody specific to p24 obviously Cell Surface Phenotype of Cytotoxic Effector Cells. Mice only recognized the p28 expressed component. spleen cells were immunized with vaccinia virus or canary Certain Materials and Methods Which Relate to Examples pox virus vectors (NYVAC. ALVAC) or with vaccinia virus 6 to 8. 65 or canarypox virus recombinants expressing HTV IIIB env Assay for Cytotoxic T Lymphocytes and In Vitro Stimu (vP911, vCP112). A second inoculation was administered 30 lation of Memory Precursors of Cytotoxic T Lymphocytes. days after the first. Prior to addition to V3 peptide pulsed 5,766,598 37 38 targets, the spleen cells were treated with monoclonal anti vitro stimulation or a second inoculation, the levels of bodies or alloantiserum to murine T-lymphocyte surface cytotoxicity of the spleen cells of mice given the canary antigens in a two-stage protocol. Briefly, the spleen cells poxvirus recombinant increased and were comparable to were resuspended at 10" viable cells per ml of Cytotoxicity spleen cells from mice similarly administered the NYVAC Medium (RPMI 1640 containing 0.2% BSA and 5 mM recombinant. No such cytotoxic responses were detected HEPES) to which was added alloanti-Thy 1.2 (Cedarlane). from spleen cells of mice inoculated with the non monoclonal anti-CD4 (172.4, K. J. Weinhold, Duke Univer recombinant NYWAC or ALVAC vectors confirming the sity Medical Center). or monoclonal anti-Lyt 2.2 requirement for immunization with a poxvirus recombinant (Cedarlane). After 30 min at 5° C., the cells were washed and expressing the HIV env gene. Furthermore, no cytotoxic resuspended in the original volume of Cytotoxicity Medium, reactivity was detected against unmodified P815 cells from divided into two equal portions with or without complement the spleen cells of any of the mice regardless of the inocu (Rabbit Lo-Tox M. Cedarlane) and incubated at 37° C. for lation regimen. Thus, only mice inoculated with recombi 45 min. The cells were then washed in Assay Medium and, nant NYVAC or, more significantly, recombinant ALVAC based on the pre-treatment cell densities, resuspended in expressing the env coding sequence from HIV-1 demon volumes of Assay Medium approximating effector to target 5 strated V3-specific cytotoxic responses. cell ratios of 100:1 (primary), 10:1 (memory), or 8.0:1 (secondary) before addition to a 5 hr 'Cr release assay. EXAMPLE 7 Error bars in FIG. 10 represent 1 standard deviation from the CHARACTERIZATION OF CYTOTOXC CS, Specificity of CTL Antigen Receptor Recognition of the EFFECTOR CELLS V3 Loop Region of HIV IIIB gp120. Cytotoxic T lympho To determine the identity of the spleen cells associated cytes and memory precursors of cytotoxic T lymphocytes with the lysis of HIV-1 V3 peptide pulsed target cells, mice were generated by inoculation of mice with vCP112 as were immunized with vCP12. After each immunization, or described above. Assays for cytotoxic T lymphocytes were in vitro stimulation 21 days after the first inoculation a performed as described above except that P815 target cells 25 two-step depletion procedure was performed, and the spleen were pulsed overnight with V3 peptide from HIV-1 IIIB (CN cells were assessed for cytotoxicity against V3 peptide TRKRRIORGPGRAFVTGK) (SEQ ID NO:79). MN (CN pulsed P815 cells. Mice inoculated with the canarypox KRKRHIGPGRAFYTTKN) (SEQID NO:80), or SF2 (CN vector ALVAC did not generate spleen cells capable of TRKSIYIGPGRAFHTTGR) (SEQ ID NO:81). Effector to killing peptide pulsed targets. Following a single target cell ratios were 100:1 (primary), 20:1 (memory), and 30 immunization, vCP112 induced spleen cells able to kill V3 50:1 (secondary). peptide pulsed targets. The lytic effector cells were sensitive Antibody Responses to HIV-1 (IIIB) gp120. The wells of to treatment with anti-murine Thy 1.2 or Lyt 2.2 plus ELISA plates (Immulon II) were coated overnight at 4° C. complement and were resistant to anti-CD4. FIG. 10 shows with 0.5g of partially purified HIV-1 (IDB) gp120 (Dr. G. the sensitivity of the cytotoxic effector cells from spleen Franchini, NCI-NIH. Bethesda. Md.) in carbonate buffer, pH 35 cells of mice immunized with vCP112 to antibodies against 9.6. The plates were then washed with phosphate-buffered cytotoxic T lymphocyte cell surface antigens Thy 1.2 and saline containing 0.05% Tween 20 (PBST). The plates were Lyt 2.2. Neither complement nor any of the monoclonal then blocked for 2 hr at 37° C. with PBST containing 1% antibodies or alloantisera alone affected the cytolytic action bovine serum albumin (BSA). After washing with PBST, of these cells. Similar results were obtained five days after sera were initially diluted 1:20 with PBST containing 0.1% a second immunization administered on day 30. Twenty-one BSA (dilution buffer). The sera were further 2-fold serially days after a single inoculation, in vitro stimulation with diluted in the wells of the ELISA plate. The plates were vCP112 infected syngeneic spleen cells gave rise to lytic incubated at 37 C. for 2 hr and washed with PBST. effector cells only partially sensitive to anti-Thy 1.2 Horseradish peroxidase conjugated rabbit anti-mouse immu although completely sensitive to anti-Lyt 2.2 and resistant to noglobulins (DAKO) was diluted 1:2000 in dilution buffer 45 anti-CD4. These Thy 1.2-, CD4-, Lyt 2.2+effector cells are and added to the wells of the ELISA plated and incubated at not seen following in vitro stimulation with vP911 of spleen 37° C. for 1 hour. After washing with PBST, OPD cells from vCP112 inoculated mice. Nonetheless, it is clear (o-phenylenediamine dihydrochloride) in substrate buffer that HIV V3 loop specific cytotoxicity was mediated by a was added and the color was allowed to develop at ambient population of T lymphocytes expressing Thy 1.2 and Lyt 2.2, temperature for about 20 min. The reaction was extinguished 50 but not CD4. This cell surface phenotype is characteristic of by the addition of 2.5M HSO. The absorbance at 490 nm classical cytotoxic T lymphocytes. was determined on a Bio-Tek EL-309 ELISA reader. The serum endpoint was defined as the reciprocal of the dilution EXAMPLE 8 giving an absorbance value of 0.4. SPECIFICITY OF CTLANTIGEN RECEPTOR 55 EXAMPLE 6 RECOGNITION OF THE V3 LOOP REGION OF HIV gp120 A RECOMBINANT CANARYPOXVIRUS To examine the specificity of cytotoxic cells generated by EXPRESSING HTV env ELICTTS HIV-SPECIFIC the HTV canarypox virus (ALVAC) recombinant, vCP112 CYTOTOXCT LYMPHOCYTE ACTTVTTY susceptibility to CTL activity was compared among P815 Seven days after the initial inoculation with the HTV target cells pulsed with peptides corresponding the V3 loop canarypoxvirus recombinant (v0P112; defined in Example region of gp120 of HIV isolates IIIB, MN, or SF2. HIV 2), cytotoxic responses of spleen cells against HIV V3 specific primary CTL activity was confined only to P815 peptide pulsed target cells were roughly equivalent to the target cells pulsed with peptide corresponding to the V3 loop cytotoxic responses elicited by the same dose.5x10 pfu, of 65 of HIV isolate IIIB. but not target cells pulsed with peptides the NYVAC recombinant, vP911 (Example 2) expressing corresponding to the V3 loop region of gp120of HIV isolates the same HIV env gene (FIG. 7). Following appropriate in MN or SF2, as shown in FIG. 8 which illustrates the 5,766,598 39 40 specificity of cytotoxic T lymphocyte antigen receptor for ID NO:83) (5'-ATCATCGAGCTCTGTTCCTTGGG the HIVIIIB hypervariable V3 loop of gp120, but not for the TTCTTAG-3") and HIVMN3P (SEQ ID NO:84) (5'- V3 loop of HIV MN or SF2. Similar results were obtained ATCATCTCTAGAATAAAAATTATAGCAAAGCCCTTT with in vitro stimulated, HTV specific memory CTL activity CCAAGCC-3") followed by digestion with SacI and Xba. and secondary CTL activity induced by immunization with This fragment was co-ligated into pbS-SK digested with the ALVAC recombinant vCP112. Thus, HIV specific CTLs EcoRI and Xbal with a 404 bp EcoRI/SacI fragment. The elicited by a recombinant canarypox virus expressing the 404 bp fragment was derived by PCR with pMN1.8-9 as env gene of HIV isolate IIIB recognize only target epitopes template and oligonucleotides HTV3B1 (SEQ ID NO:32) derived from the same antigenic isolate. These results and HIVMN 4 (SEQ ID NO:85) (5'- clearly indicate the exquisite specificity of the lymphocyte 10 ATCATCGAGCTCCTATCGCTGCTC-3). The resultant effector cells generated by immunization with the HIV plasmid was designated as pBS3MN. canarypox virus recombinant and eliminate such nonspecific The 1,026 bp EcoRI/KpnI fragment from p3SMIDMN was inserted into the 4.315 bp pbS3MN digested with effector mechanisms as natural killer (NK) cell activity. EcoRI/KpnI to generate pbSMID3MN. This plasmid con EXAMPLE 9 15 tains most of the env gene except the 5'-most region. The vaccinia virus H6 promoter (Goebel et al., 1990a,b) and the ANTBODY RESPONSES OF MICE 5'-most region of the env gene were obtained by isolating a NOCULATED WITH NY WAC- and ALVAC 318 bp Kpn fragment from pBSH6HTV3B5P (defined in BASED HIV RECOMBINANTS Example 2). This fragment was ligated into Kpn/Xbal digested PBS-SK along with the 2.9 bp Kpn/Xba fragment To evaluate humoral responses to HIV, mice were immu 20 from pBSMID3MN. The resultant plasmid was designated nized at day 0 with a NYVAC HIV recombinant or canary as pH6HMNE. poxvirus (ALVAC) recombinant and received a secondary The 2.7 kb NruI/Xbal fragment from pH6HMNE, con immunization at week four. The mice were bled at various taining the entire HIV-1 (MN) env gene juxtaposed 3' to the intervals through 20 weeks after the initial immunization. 25 3'-most 26 bp of the H6 promoter, was blunt-ended and Pooled sera from each treatment group were assayed for inserted into NruI/SmaI digested pSPHAH6. This generated antibodies to HTV by ELISA employing purified gp120 as plasmid pHAHIVMNE. Plasmid pSPHAH6 was derived as antigen; the results are shown in FIG. 9 which provides the follows. Plasmid pMP2VCL (containing a polylinker region antibody responses to HTV IIIB gp120 of mice immunized within vaccinia sequences upstream of the K1L host range with vectors (NYVAC. ALWAC) or with NYVAC recombi 30 gene) was digested within the polylinker with HindIII and nant vF911 or ALVAC recombinant (vicP112) expressing XhoI and ligated to annealed oligonucleotides SPHPRHAA HTV-1 env, wherein the inverted triangle indicates the time through D (SPHPRHA A (SEQ ID NO: 86) of administration of the second inoculation. Primary anti 5'-AGCTTCTTTATTCTATACTTAAAAAGTGAAAAT body responses were generally modest, but detectable. Fol AAATACAAAGGTTCTTGAGGGT - 3'SPHPRHA, B lowing the secondary immunization, the antibody titers of 35 (SEQ D NO:87) (5'- mice immunized with both vp-11 and vCP112 increased and T G T G T TAAATTGAA A G C G A GAAATAAT peaked at week six with titers of over 10,000. These anti CATA AAT TATTT CAT TAT C GC GATATC body titers remained at approximately the same levels CGTTAAGTTTG TATCGTAC-3). SPHPRHAC (SEQ ID throughout the duration of the study. Thus, an ALWAC HIV NO:88) (3'-TTATTAGTATTTAATAAAGTAATAGCG recombinant, vCP112, was capable of inducing a significant CTATAGGCAATTCAAACATAGCATGAGCT-5') SPH antibody response. PRHA D (SEQ D NO:89) (3'- Inoculation of mice with ALWAC expressing the env gene AGAAATAAGATATGAATTTTTCACTTTTATTTATG of HIV-1 elicits spleen cell reactivity with characteristics of TTTCCAAGAACTCCCAACACAATTTAA- CTT cytotoxic T lymphocytes: the requirement for immunization. TCGCTCT-5') generating pSP126 containing a HindIII site, cell surface phenotype, memory, and elegant epitope speci 45 H6 promoter-124 through -1 (Perkus et al., 1989) and XhoI. ficity. Furthermore, antibody responses to HIV-1 gp120 are KpnI, SmaI, SacI and EcoRI sites. induced by inoculation with this ALVAC recombinant. Plasmid pSD544 (containing vaccinia sequences sur rounding the site of the HA gene replaced with a polylinker EXAMPLE 10 region and translation termination codons in six reading 50 frames) was digested with XhoI within the polylinker, filled DERVATION OF NYWAC- AND ALVAC-BASED in with the Klenow fragment of DNA polymerase I and HIV-1 RECOMBINANTS AND EXPRESSION OF treated with alkaline phosphatase. SP126 was digested with HIV-1(MN) env BY ALVAC AND NYVAC HindIII, treated with Klenow and the H6 promoter isolated HIV-1 (MN) env sequences were derived from plasmid by digestion with SmaI. Ligation of the H6 promoter frag pMN1.8-9 and pMN1.8-10 which contain a 1774 bp and 55 ment to pSD544 generated pSPHAH6 which contained the 1803 bp subfragment from a genomic cDNA clone of H6 promoter in the polylinker region (in the direction of HA HIV-1 (MN), respectively. These plasmids were provided by transcription). This insertion plasmid enables the replace the laboratory of Dr. R. C. Gallo (NCI-NIH). A 1,026 bp ment of the vaccinia HA gene (A56; Goebel et al., 1990a,b) Kpn/EcoRI fragment was derived by amplifying these with foreign genetic material. sequences from pMN1.8-9 by PCR using oligonucleotides The C5L insertion plasmid was derived as follows. Using HIVMN 6 (SEQ ID NO:82) (5'- the cosmid vector pVK102 (Knauf and Nester, 1982), a GGGTTATTAATGATCTGTAG-3") and HIV3B2 (SEQ ID genomic library for vCP65 (ALVAC-based rabies G recom NO:39) followed by digestion with Kipni/EcoRI. This frag binant with rabies in C5 locus) was constructed. This library ment was inserted into pBS-SK digested with KpnI and was probed with the 0.9 kb PvuII canarypoxvirus genomic EcoRI to yield pBSMIDMN. 65 fragment contained within pRW764.5 (C5 locus). These A 1,028 bp Sal/Xba fragment was derived from canarypox DNA sequences contain the original insertion pMN1.8-10 by PCR using oligonucleotides HIVMN5 (SEQ locus. A clone containing a 29 kb insert was grown up and 5,766,598 41 42 designated pHCOS 1. From this cosmid containing C5 contains a poxvirus expression cassette for HIV-1 (MN) sequences, a 3.3 kb Cla fragment was subcloned. Sequence gp120 in pbS-SK and was designated pBSHIVMN120. analysis from this Clafragment was used to extend the map A 1.7 kb Xbal/partial KpnI fragment was isolated and of the C5 locus from -1372. inserted into pC5L digested with KpnI/Xbal. The resultant The C5 insertion vector, pC5L, was constructed in two plasmid was designated as pC5HIVMN120. The insertion steps. The 1535 bp left arm was generated by PCR ampli plasmid for integrating the HIV-1 (MN) gp120 gene into fication using oligonucleotides C5A (SEQ D NO:90) (5'- NYVAC was obtained by first isolating the 1.6 kb NruI/SmaI ATCATCGAATTCTGAATGTTAAATGTTATACTTTG-3") fragment from p3SHTVMN120. This fragment was inserted and C5B (SEQ ID NO:91) (5'- into pSPHAH6 digested with NruI and SmaI to provide GGGGGTACCTTTGAGAGTACCACTTCAG-3"). The O pHAHIVMN120. template DNA was canarypoxvirus genomic DNA. This Insertion plasmids. p C5HIVMN 120 and fragment was cloned into EcoRISmaI digested puC8. The pHAHIVMN120. were used in recombination experiments sequence was confirmed by standard sequencing protocols. with ALVAC (CPpp) and NYVAC (vP866) as the rescuing The 404 bp right arm was generated by PCR amplification virus. These assays and plaque identification and purifica using oligonucleotides C5C (SEQ ID NO:92) (5'- 15 tion were performed by standard procedures (Piccini et al. ATCATCCTGCAGGTATTCTAAACTAGGAATAGATG 1987). Hybridization analyses were performed with a radio 3') and C5DA (SEQ ID NO: 93) (5'- labeled HIV-1 (MN) gp120-specific probe. Purified recom ATCATCCTGCAGGTATTCTAAACTAGGAATAGATG binants were amplified. The ALVAC-based HIV-1(MN) 3). This fragment was then cloned into the vector previously gp120 recombinant was designated as vCP124 and the generated containing the left arm digested with SmaIPst. NYVAC-based HIV-1 (MN) gp120 recombinant as vP1004. The entire construct was confirmed by standard sequence Cells infected with vCP124 and wP1004 were analyzed analysis and designated pC5L. This insertion plasmid for the presence of the recombinant expressed HIV-1 (MN) enables the insertion of foreign genes into the C5 locus. gp120 by immunofluorescence and immunoprecipitation. The 2.8 kb Xbal/partial KpnIfragment from pH6HMNE These assays were performed as previously described was isolated and inserted into pC5L digested with Xbaland 25 (Taylor et al., 1990) using a pooled human sera from KpnI. The resultant plasmid was designated as HTV-seropositive individuals (obtained from K. Steimer, pC5HIVMNE. Chiron Corporation, Emeryville, Calif.). Results from these Plasmids pHAHIVMNE and pC5HIVMNE were used in studies clearly indicated that cells infected with either vitro recombination experiments with NYVAC (vP866) and vOP124 and vP1004 contained HIV-1(MN) gp120, whereas ALVAC (CPpp), respectively, as the rescue virus. These 30 gp120 was not observed in uninfected cells and cells were done by standard procedures (Piccini et al., 1987). infected with parental viruses, ALVAC and NYVAC. Plaques derived from recombinant virus were identified by plaque hybridization using a radiolabeled env-specific DNA EXAMPLE 12 probe (Piccini et al., 1987). After three rounds of plaque purification, the recombinant viruses were amplified. The 35 EXPRESSION OF ANON-CLEAVABLE FORM NYVAC-based HIV-1(MN) env recombinant was desig OF HIV-1 gp160 BY ALVAC AND NYVAC nated vR1008 and the ALVAC-based recombinant v(P125. In order to express a non-cleavable form of the HIV-1 Recombinant viruses, vCP125 and vp1008, were ana (ITB) gp160 an arginine to threonine mutation was engi lyzed for expression of the HIV-1(MN) env gene by immu neered at amino acid 511 (Ratner et al., 1985) as was nofluorescence and immunoprecipitation using previously demonstrated by Guo et al. (1990). These modifications reported procedures (Taylor et al., 1990). Pooled human sera were made to decrease the shedding of gp120 from the from HIV-seropositive individuals (obtained from Dr. K. surface of infected cells. These manipulations were per Steimer. Chiron Corp., Emeryville, Calif.) was used in these formed as follows. A 376 bp Pst/Xbal fragment was assays. Results from immunofluorescence revealed that cells 45 obtained by first amplifying the sequences from infected with either vCP125 or vp1008 express the HIV-1 pBSHTV3BEII (described in Example 2) using oligonucle (MN) gene product on their surface. Immunoprecipitation otides HIV3 B2A (SEQ ID NO:96.) (5'- from lysates prepared from vP1008 and vCP125 infected GAAATAATAAAACAATAATC-3") and HIVECB (SEQID cells demonstrated the presence of three predominant HIV NO:97) (5'- 1-specific proteins with apparent molecular masses of 160 50 GCTCCTATTCCCACTGCAGTTTTTTCTCTCTGCAC kDa, 120 kDa, and 41 kDa, respectively. These are consis 3') followed by digestion with Pst and Xbal. This fragment tent with expression of the precursor envelope glycoprotein was ligated with a 1.061 bp PstIXbafragment and a 4.5 kb (160 kDa) and the proteolytically derived mature forms (120 EcoRIVXbal fragment from psSHIV3BEII to yield kDa and 41 kDa). pBSHIV3BEEC. 55 The central region of the Hantaan virus S segment was EXAMPLE 11 generated by PCR using oligonucleotides T5HT3PPS (SEQ D NO: 98) EXPRESSION OF THE HIV-1(MN) gp120 BY (5'GTCCTGCAGGATGGAAAAGAATGCCCCAAGC-3') NYVAC AND ALVAC and HTS55PN (SEQ ID NO: 99) (5'- A 391 bp EcoRI/Xbal fragment was amplified from GGGGGAGGCAAACTACCAAGG-3") and the S specific pBS3MN using oligonucleotides T7 (SEQ ID NO:94) (5'- cDNA clone as template. The 581 bp fragment contains a AATACGACTCACTATAG-3") and HIVMN120 (SEQ ID Pst site at its 3' end and the 5' end includes the Nici site of NO: 95) (5'- ATCAT CTCTAGAAT position 499 of the S segment (Schmaljohn et al., 1986). AAAAATTATCTTTTTTCTCTCTGCACCACTC-3") fol Furthermore, using the oligonucleotide T5HT3PPS (SEQID lowed by digestion with EcoRI and Xbal. This fragment was 65 NO:98) eliminates the TNT element at position 1029 to ligated to the 4.2 kb EcoRIIXbal fragment derived from 1035 without altering the amino acid sequence. This frag pH6HMNE (defined in Example 10). The resultant plasmid ment was then digested with NciI and Pst. The PCR 5,766,598 43 44 fragment containing the 5' end of the coding sequence fused ATCATCTCTAGAATAAAAATTATAGGAAAGCCC to the H6 promoter (HindIII/Ncil digested above) was TTTCCAAGCC-3') and HIVECA (SEQ ID NO:103) (5'- ligated into pBS-SK digested with HindIII and PstI along GTGCAGAGAAAAAACTGCAGTGGGAATAGGAGC with the 581 bp Nci/Pst fragment containing the central 3") followed by digestion with Pst and Xbal. This 1061 bp region of the S segment. The resultant plasmid was desig fragment was ligated with the 391 bp EcoRI/PstI from nated pHSHTSH65P pBSHIVMNT (described in Example 13) and the 4.2 kb The 3' most 438 bp of the S segment was derived by PCR EcoRIXbal fragment from pH6HMNE (defined in Example using oligonucleotides HTS3PXBA (SEQID NO:100) (5'- 10). The resultant plasmid was designated as pBSHTVM ATCATCTCTAGAATAAAAATTAGAGTTTCAAAGGC NEEC. Sequence analysis of the HIV env insert demon 3') and T5HT5 PSP (SEQ ID NO: 101) (5'- 10 strated that a single nucleotide was missing (between the CGCCAGCATGCAGAAGCAGC-3') and the S-specific SacI and Xbal sites). To correct this, the following manipu cDNA clone as template. The 5' end of this fragment lations were performed. The 1,028 kb SacI/Xba from contains the Pst site situated at position 1039 of the S pH6HMNE was used to substitute for the corresponding segment coding sequence (Schmaljohn et al., 1986) and the fragment from pBSHIVMNEEC resulting in the formation 3' end contains a TNT sequence motif and a unique Xbal 15 of pBSHIVMNEEC. prior to insertion into Pst/Xbal digested p3S-SK to yield The 2.6 NruI/Xbal fragment from pBSHIVMNEEC was pBSHTS3P. isolated, blunt-ended with Kenow, and inserted into Niruf To generate the entire S segment expression cassette, a SmaI digested pSPHAH6 (defined in Example 10). The 1122 bp Pst/partial HindIII fragment was derived from resultant plasmid was designated pHAHIVMNEEC. The 2.6 pBSHTSH56P. This fragment was co-inserted into HindIII/ kb NruI/Xba fragment from PBSHIVMNEEC was also Xbal digested pBS-SK with a 290 bp Pst/Xbal fragment inserted into NruI/Xba digested pVQH6C5LSP6 (below) to from pBSHTS3P. The resultant plasmid was designated yield pC5HIVMNEEC. pBSHVS by linearization with Xbal followed by a partial Insertion plasmids. pH AHIVMNEEC and HindIII digestion. pC5HIVMNEEC, were used in standard recombination The 2.6 kb NruI/Xbal fragment from peSHIV3BEEC, 25 experiments (Piccini et al., 1987) with NYVAC (vP866) and containing the 3'-most 26 bp of the H6 promoter linked to ALVAC (CPpp), respectively, as rescue virus. Recombinant the gp160 cassette, was isolated and ligated to a 3.0 kb virus was identified and plaque purified by standard plaque NruI/Xbafragment of pBSHVS to yield pPSHTV3BEECM. hybridization (Piccini et al., 1987) using a radiolabeled HIV Digestion with NruI and Xbal excises the 3'-most 26 bp of env-specific DNA probe. Purified recombinant virus were the H6 promoter and the Hantaan virus S sequence. The 3.0 30 then amplified. The NYWAC-based recombinant containing kb NruIXbal fragment contains the 5'-most 100 bp of the the HIV-1 (MN) non-cleavable gp160 was designated as H6 promoter in a PBS-SK plasmid. vP1078 and the ALVAC equivalent vCP144. The 2.8 kb XbalApartial Kpn fragment from Expression analysis of vCP126 and vp1078 was per pBSHTV3BEECM was ligated to Xbai/KpnIdigested pC5L formed as described above. These results demonstrated that to yield pC5HIV3BEEC. A 2.7 kb NruI/Xba fragment from 35 expression was qualitatively equivalent to the HIV-1 (IIIB) pBSHIV3BEECM was blunt-ended with the Klenow frag counterparts, vp1020 and vCP126. ment of the E. coli DNA polymerase and inserted into NruI/SmaI digested pSPHAH6 to yield pHAHIV3BEEC. EXAMPLE 13 The insertion plasmids, pC5HIV3BEEC and pHAHIV3BEEC, were used in in vitro recombination EXPRESSION OF ANON-C EAVABLE, experiments by standard procedures (Piccini et al., 1987) SECRETED FORM OF HIV-1 env BY ALWAC using ALVAC (CPpp) and NYVAC (vP866), respectively, as AND NYWAC rescue virus. Recombinant plaques were identified by stan ALVAC- and NYWAC-based recombinant viruses were dard plaque hybridization analysis (Piccini et al., 1987) 45 generated which express an HIV-1 (MN) env that is not using a radiolabeled probe specific for the HIV-1 env gene. proteolytically cleaved and is secreted by virtue of the Recombinant viruses were amplified following three rounds elimination of the transmembrane sequence near the carboxy of purification. The recombinant ALVAC-based HIV-1 (IDB) terminus of the gene product. A502 bp PstLIXba fragment gp160 (non-cleavable) was designated as vCP126 and the was obtained by first amplifying these sequences from NYVAC-based equivalent as wP1020. 50 pH6HMNE (defined in Example 10) using oligonucleotides Immunofluorescence and immunoprecipitation analyses HIVECA (SEQ ID NO:103) and HTVMNT1 (SEQ ID were performed by previously described procedures on NO: 104) (5'- ATCAT CTCTAGAATAAAAA vP1020 and vCP126 infected cells using pooled human TTACAAACTTGCCCATTTATCCAATTCC-3") followed serum from HIV-seropositive individuals (obtained from K. by digestion with Pst (5'-end) and Xbal (3'-end). This Steimer. Chiron Corp., Emeryville, Calif.). Immunofluores 55 fragment corresponds to nucleotides 7219 to 7808 (Ratner et cence results clearly demonstrated the surface expression of al., 1985). This fragment will serve as the 3'-end of the env the HIV-1 (IIB) gp160 (non-cleavable form) on the surface expression cassette. As such, the env gene product will lack of cells infected with either vCP126 or wF1020. the transmembrane region, will be terminated by a termina Furthermore, immunoprecipitation results demonstrated the tion codon provided by oligonucleotide HIVMNT1 (SEQID presence of a HIV-1 (IIIB) gp160 in these infected cells that NO:104). and will not be cleaved due to an amino change at was not proteolytically cleaved into the mature gp120 and 511 (defined in Example 12) provided using oligonucleotide gp41 frames. HIVECA (SEQ ID NO:103). This 502 bp fragment was A non-cleavable form of the HIV-1(MN) gp160 was also ligated to the 391 bp EcoRIVPst fragment derived by PCR pursued and the recombinant viruses obtained as follows. from pH6HMNE using oligonucleotides HTV3B1 (SEQ ID APst/Xbal fragment was obtained by PCR amplification 65. NO:32) and HIVECB (SEQ ID NO:97), and the 4.2 kb from pH6HMNE (described in Example 10) using oligo EcoRIIXbal fragment to pH6MNE. The resultant plasmid nucleotides HIVMN3P (SEQ ID NO: 102) (5'- was designated pBSHIVMNT. 5,766,598 45 46 The 2.2 kb Xbal/partial KpnI fragment from following manipulations were performed. A 200 bp frag pBSHIVMNT was isolated and inserted into pC5L digested ment corresponding to the 3'-most region of the gp120 with Xbal and Kipni. The resultant plasmid was designated coding sequence was derived by PCR from pH6HMNE as pC5HIVMNT. The NYVAC insertion plasmid was (defined in Example 10) using oligonucleotides HIV3B1 derived by isolating the 2.1 kb NruI/Xbal fragment from 5. (SEQ ID NO:32) and HIVMN18 (SEQ ID NO:106) (5'- pBSHIVMNT. This fragment was then blunt-ended with the GCCTCCTACTATCATTATGAATAATCTTTTTCTCTC Klenow fragment of the E. coli DNA polymerase in the TG-3'). This fragment was fused by PCR to annealed presence of 2 mM dNTPs and inserted into pSPHAH6 oligonucleotides HIVTM1 (SEQ ID NO:107) (5'- digested with NruI and SmaI to yield pHAHIVMNT. TTATTCATAATGATAG TAGGAG G CTTG G The insertion plasmids, pC5HIVMNT and O TAG G T T TAAG AATA GTTTTT G C T G - pHAHIVMNT, were used in standard recombination experi TACTCTCTGTAGT GAATAGAGTTAGGCAGGGATAA ments (Piccini et al., 1987) with ALVAC (CPpp) and 3') and HIVTM2 (SEQ ID NO: 108) (5'- NYVAC (vP866). respectively, as the rescue virus. Recom TTATCCCTGCCTAACTCTATTCACTACAGAGAGTA binant virus was identified by standard plaque hybridization CAGC AAAAACTATTCTTAAA CCTACCAAGC assays (Piccini et al., 1987) using a radiolabeled HIV 5 CTCCTACTATCATTATGAATAA-3") using oligonucle env-specific probe. Recombinant virus was subjected to otides HIV3B1 (SEQ ID NO:32) and HIVTM3 (SEQ ID three rounds of purification prior to amplification. The NO:109) (5'-ATCATCTCTAGAATAAAAATTATCCC ALVAC-based HIV-1 (MN) env (non-cleavable; secreted) TGCCTAACTCTATTCAC-3). Oligonucleotides HIVTM1 was designated as vCP120 and the NYVAC equivalent as (SEQID NO:107) and HTVTM2 (SEQ ID NO:108) corre VP994, spond to nucleotides 7850 to 7934 (Ratner et al., 1985) and Immunoprecipitation analyses were performed as previ represent the region encoding the HIV env hydrophobic ously described (above) for vCP120 and vP994 infected anchor sequence. Fusion with HIVTM3 (SEQID NO:109) cells using pooled human sera from HTV-seropositive indi engineers the 3'-end of the eventual cassette with a termi viduals. Both vCP120 and vP994 expressed an HIV-1(MN) nation codon and a 3Xbal site. The derived fragment was env-specific gene product with a molecular weight consis 25 digested with EcoRIIXbal and ligated to pH6HMNE tent with a non-cleavable, truncated gene product. digested with EcoRI and Xbal to yield p3SHIVMN120T. Furthermore, immunoprecipitation of the cell-free medium The 1.7 bk NruI/Xba fragment from pBSHTVMN120T, from vCP 120 and v994 infected cell cultures indicated the containing the 3'-most 26 bp of the H6 promoter and the secretion of this env gene product. entire HTV-1 cassette, was isolated and inserted into the 5.1 A similar construction was engineered for the HIV-1 (IB) 30 kb Nru/Xba fragment from pVQH6C5LSP6 to derive env. The following manipulations were performed to accom pC5HIVMN120T. The plasmid pVQH6C5LSP6 was plish this. A 487 bp Pst/Xbal fragment was obtained by first derived as follows. amplifying these sequences from peSH6HIV3B5P (defined pCSL (defined in Example 10) was digested within the in Example 2) using oligonucleotides HIVECA (SEQ ID polylinker with Asp718 and Not, treated with alkaline NO:103) and HIV3BT (SEQ ID NO: 105) (5'- 35 phosphatase and ligated to kinased and annealed oligonucle ATCATCTCTAGAATAAAAATTACAAACTTGCCCA otides CP26 (SEQ ID NO: 110) (5'- TTTATCTAATTCC-3') followed by digestion with Pst and GTACGTGACTAATTAGCTATAAAAAGGATCCGGTA Xbal. A 397 bp EcoRIVPst fragment was isolated from CCCTCGAGTCTAGAATCGATCCC- GGGTTTT pBSHTV3BEEC and a 4.2 kb EcoRIIXba fragment was TATGACTAGTTAATCAC-3') and CP27 (SEQID NO:111) isolated from pH6HIBEM. These three fragments were (5'-GGCCGTGATTAACTAGTCAT. AAAAACCCGG ligated together to yield pBSHIV3BT1. Plasmid GATCGATTCTAGACTCGAGGGTACCGGATCCTTTT pH6HIBEM was derived from pBSHTV3BEII (defined in TATAGCTAATTAGTCAC-3") (containing a disabled Example 2) by digestion with Kpn to liberate a second copy Asp718 site, translation stop codons in six reading frames, of the H6 promoter linked to the 5' portion of the HIV-1 vaccinia early transcription termination signal (Yuen and (IB) envgene. The 5.4 kp KpnI was then religated to form 45 Moss, 1987), BamHI, Kipni. XhoI, Xbal, Clal, and SmaI pBSHIV3BEII. restriction sites, vaccinia early transcription termination The 2.1 kb and 2.9 kb fragments derived by HindIII/Xbal signal, translation stop codons in six reading frames, and a digestion of p8SHIV3BEECM were ligated to the 105 bp disabled NotI site) generating plasmid pC5LSP. HindIII/Xba fragment from pH SHIV3BT1 to yield pC5LSP was digested with BamHI and ligated to pBSHIV3BT. This plasmid was digested with NruI and Xba 50 annealed oligonucleotides CP32 (SEQ ID NO:112) (5'- to excise a 2.1 kb fragment. This fragment was blunt-ended G AT C T TA AT TA AT TA GT C A T C A G - and inserted into pSPHAH6 digested with Nru and SmaI to G C A G G G C G A GAA C G A GA CTAT C T - generate pHAHIV3BT. GCTCGTTAATTAATTAGGT CGACG-3") and CP33 (SEQ The plasmid pHAHTV3BT was used in recombination 55 ID NO: 113) (5'- experiments, as above, with NYVAC (vP866) as the rescue GAT C C GT CGAC CTA ATTAATTAA CGA G virus. Recombinant virus was identified and purified as CA CATA GT C T C GTT C T C GC C C T G CCT above and the resultant recombinant was designated as GATGACTAATTAATTAA-3') to generate pVQCSLSP6. vP1036. This recombinant had all the expression character The 1.7 kb NruI/Xbal fragment from peSHIVMN120T istics noted above for vCP120 and vP994. was also blunt-ended and inserted into pSPHAH6 digested with Niru and SmaI. The resultant plasmid was designated EXAMPLE 14 as pHAHIVMN120T. EXPRESSION OF HIV-1 (MN) gp120 Insertion plasmids, pC5HIVMN 120T and ANCHORED WITH A TRANSMEMBRANE pHAHIVMN120T. were used in standard recombination SEQUENCE BY NYVAC AND ALVAC 65 experiments (Piccini et al., 1987) with ALVAC and NYVAC. To fuse the env region encoding the gp120 to the region respectively, as the rescue virus. Recombinant virus was encoding the hydrophobic transmembrane sequence, the identified and purified by standard plaque hybridization 5,766.598 47 48 (Piccini et al., 1987) using a radiolabeled HIV-1 gp120 of pHTVG3. The plasmid generated by this manipulation is specific DNA probe. The pure populations were amplified called pHIVG4. and the ALVAC-based anchored HIV-1 (MN) gp120 recom pHIVG4 was used in recombination experiments with binant was designated vCP138. The NYVAC-based equiva vP866 (NYWAC) as the rescuing virus to yield vP969. lent was designated vP1035. Immunoprecipitation analysis was performed to deter Immunofluorescence and immunoprecipitation analyses mine whether wP969 expresses authentic HIV-1 gag precur were performed by standard procedures (above) to evaluate sor protein. expression of the HTV-1 (MN) anchored gp120 in vP138 and Lysates from the infected cells were analyzed for HIV-1 wP1035 infected cells. The assays were performed using gag precursor expression using pooled serum from HIV-1 pooled human sera from HIV-seropositive individuals O seropositive individuals (obtained from Chiron, Emeryville. (obtained from Dr. K. Steimer, Chiron Corp., Emeryville, Calif.). The sera was preadsorbed with vP866 infected Vero Calif.). Investigation of surface immunofluorescence indi cells. The preadsorbed sera was bound to Protein cated that vCP138 and w?1035 infected cells contained A-sepharose in an overnight incubation at 4° C. Following HIV-1(MN) gp120 in the plasma membrane. Significantly. this incubation period, the material was washed 4xwith the surface staining of vCP138 and vP1035 infected cells 15 1xbuffer A. Lysates precleared with normal human sera and was enhanced compared to cells infected with recombinant protein A-sepharose were then incubated overnight at 4 C. viruses (i.e. vCP125, vCP124, vp1004, and vP1008) with the HTV-1 seropositive human sera bound to protein expressing gp160 or a non-anchored gp120. Results from A-sepharose. After the overnight incubation period, the immunoprecipitation analyses confirmed the expression of samples were washed 4xwith 1xbuffer A and 2xwith a gp120 in vCP138 and vP1035 infected cells and that the O LiClfurea buffer, Precipitated proteins were dissociated expressed product was of the expected molecular mass. from the immune complexes by the addition of 2xLaemmli's buffer (125 mM Tris (pH6.8), 4% SDS. 20% EXAMPLE 1.5 glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. GENERATION OF NYVAC/HIV-1 GAG Proteins were fractionated on a 10% Dreyfuss gel system 25 (Dreyfuss et al., 1984), fixed and treated with 1M (PROTEASE) RECOMBINANT Na-salicylate for fluorography. Plasmid pSD542 (a NYVACTK locus donor plasmid; see Human sera from HIV-1 seropositive individuals specifi Example 5) was derived from plasmid pSD460 (Tartaglia et cally precipitated the HIV-1 gag precursor protein from al., 1992) by forming vector plasmids pSD513 as described 30 vP969 infected cells, but did not precipitate HIV-1-specific above in Example 7. The polylinker region in pSD513 was proteins from mock infected or NYVAC infected cells. modified by cutting with Pst/BamHI and ligating to annealed synthetic oligonucleotides MPSYN288 (SEQ ID EXAMPLE 16 NO:114) (5'GGTCGACGGATCCT 3') and MPSYN289 (SEQ ID NO:115) (5'GATCAGGATCCGTCGACCTGCA GENERATION OF NYVAC/HIV-1 gag?pol 3') resulting in plasmid pSD542. 35 RECOMBINANT A plasmid, pHXB2D. containing human immunodefi The sequence encoding the 5'-end of the gag gene was ciency virus type 1 (HIV-1) cDNA sequence was obtained cloned between vaccinia virus tik flanking arms. This was from Dr. R. C. Gallo (NCI-NIH). The sequence encoding the accomplished by cloning the 1,625 bp Bg|II fragment of 5'-end of the gag gene was cloned between vaccinia virus tk pHXB2D, containing the 5'-end of the gag gene, into the flanking arms. This was accomplished by cloning the 1,625 40 4,075 bp BglII fragment of pSD542 (defined in Example bp BglII fragment of pHXB2D, containing the 5'-end of the 15). The plasmid generated by this manipulation is called gag gene, into the 4,075 bp Bgll fragment of pSD542. The pHTVG2. plasmid generated by this manipulation is called pHTVG2. The 3'-end of the gag gene was then cloned into pHTVG2. The 3'-end of the gag gene was then cloned downstream 45 This was accomplished by cloning a 280 bp Apal-BamHI from the rest of the gag gene. This was accomplished by PCR fragment, containing the 3'-end of the gag gene, into cloning a 280 bp Apa-BamHIPCR fragment, containing the the 5,620 bp Apal-BamHI fragment of pHIVG2. This PCR 3'-end of the gag gene, into the 5,620 bp Apa-BamHI fragment was generated from the plasmid, pHXB2D, with fragment of pHTVG2. This PCR fragment was generated the oligonucleotides. HIVP5 (SEQED NO:116) and HIVP6 from the plasmid, pHXB2D, with the oligonucleotides, 50 (SEQID NO:117). The plasmid generated by this manipu HIVP5 (SEQID NO:116) (5'-TGTGGCAAAGAAGGGC lation is called pHTVG3. 3') and HIVP6 (SEQ ID NO: 117) (5'- The I3L promoter was then cloned upstream of the gag TTGGATCCTTATTGTGACGAGGGGTC-3). The plasmid gene. This was accomplished by cloning the generated by this manipulation is called pHIVG3. oligonucleotides. HIVL17 (SEQ ID NO:118) and HIVL18 The I3L promoter was then cloned upstream of the gag 55 (SEQID NO:119), encoding the vaccinia virus I3L promoter gene. This was accomplished by cloning the and the 5'-end of the gag gene, into the 5.540 bp partial oligonucleotides, HIVL17 (SEQ ID NO:118) (5'- Bgll-Clal fragment of pHTVG3. The plasmid generated by GAT CTTGA, GATAAA GT GAA AATATATAT this manipulation is called pHTVG4. CATTATATTACAAAGTACAATTATITAGGTTTAATCA The portion of the gag gene encoding p24, p.2, p7 and p6 TGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAG was then eliminated. This was accomplished by cloning the AATTAGAT-3") and HIVL18 (SEQ ID NO:119) (5'- oligonucleotides, HIVL19 (SEQ ID NO:120) (5'- CGAT CTAATT C T C C C C C G CTTA, ATA CT C T G ACA CAG GA C A C A G CAAT CAG GT GA C G C T C T C GCA C C CAT GATTA AA C CAGCCAA AATTACTA ATTTTTAT CT C CTA AATAATT GTA CTTT GT GAGGTCGACAGGAC CCG-3) and HTVL20 (SEQ ID AATATAATGATATATATTTTCACTTTATCTCAA-3"), 65 NO: 121) (5'- encoding the vaccinia virus I3L promoter and the 5'-end of G AT C C G G G T C C T G T C G A C C T C - the gag gene, into the 5,540 bp partial BglII-Clal fragment GAGATAAAAATTA GTA ATTTT GGCTGAC 5,766.598 49 50 CTGATTGCTGTGTCCT GTGTCAG-3'), into the 4450 bp EXAMPLE 19 partial Pvu-BamHI fragment of pHIVG4. The plasmid GENERATION OF NYVAC/HIV-1 p17, p24 generated by this manipulation is called pHTVG5. RECOMBINANT The remainder of the gag gene, as well as the pol gene, A plasmid, pHXB2D. containing HIV-1 cDNA sequence, was then cloned downstream of the p17 "gene". This was 5 was obtained from Dr. R. C. Gallo (NCI-NIH). The sequence accomplished by cloning the 4.955 bp Cla-Safragment of encoding the 5'-end of the gag gene was cloned between pHXB2D, containing most of the gag gene and all of the pol vaccinia virus th: flanking arms. This was accomplished by gene, into the 4,150 bp Cla-SalIfragment of pHIVG5. The initially preparing pHIVG5, as described above (see plasmid generated by this manipulation is called pHTVG6. Example 16), Extraneous 3'-noncoding sequence was then eliminated. 10 The 3'-end of the p24 “gene" was then cloned into This was accomplished by cloning a 360 bp AftDI-BamHI pHTVG5. This was accomplished by cloning a 660 bp PCR fragment, containing the 3'-end of the pol gene.into the Sal-BamHIPCR fragment, containing the 3'-end of the p24 8,030 bp Afl-BamHI fragment of pHIVG6. This PCR “gene", into the 4450 bp Sall-BamHIfragment of pHIVG5. fragment was generated from the plasmid, pHXB2D, with This PCR fragment was generated from the plasmid, the oligonucleotides. HIVP7 (SEQ ID NO: 122) (5'- 15 pHXB2D, with the oligonucleotides. HTVP25 (SEQ ID NO:124) (5'-AAAGTCGACCCATATCACCTAGAAC-3') AAGAAAATTATAGGAC-3") and HIVP8 (SEQ ID and HIVP26 (SEQ ID NO:125) (5'- NO:123) (5'-TTGGATCCCTAATCCTCATCCTGT-3"). The TTTGGATCCTTACAAAACTCTTGCCTTAT-3"). The plasmid generated by this manipulation is called pHIVG7. plasmid generated by this manipulation is called pHIVG8. pHIVG7 was used in recombination experiments with 20. The entomopox 42 kid promoter was then cloned upstream vP866 (NYVAC) as the rescuing virus to yield vP989. of the p24 “gene". This was accomplished by cloning the Immunoprecipitation experiments with vP989 infected oligonucleotides, HIVL21 (SEQ ID NO:126) (5'- cells were performed as described above for the expression T C G A G CAA AATTGAA AATATATA ATTA of the HTV-1 gag precursor protein. No HIV-1-specific CAATATAAA AT G. C CTATA GT GC A GAA - species were precipitated from mock infected or NYVAC CATCCAGGGGC A AATG GTA CAT infected Vero cells. Protein species corresponding to the gag CAGGCCATATCACCTAGAACTTTAAATGCA-3") and precursor protein, as well as various intermediate and mature HVL22 (SEQ ID NO: 127) (5'- gag cleavage products, were precipitated, however, from TTTAAA GTT CTA G G T GATAT G GC CT GAT lysates of vP989 infected cells. GT ACC ATTT G CCCCTG GAT GTTCT GCAC T A T A G G C A T EXAMPLE 17 30 TTTATATTGTAATTATATATTTTCAATTTTGC-3'). encoding the entomopox 42 kid promoter and the 5'-end of GENERATION OF NYVAC/HIV-1 gag?pol AND the p24 “gene”. into the 5,070 bp XhoI-Nsi fragment of env (gp120) RECOMBINANT pHTVG8. The plasmid generated by this manipulation is The sequence encoding the 5'-end of the gag gene was called pHTVG9. cloned between vaccinia virus tik flanking arms. This was pHTVG9 was used in recombination experiments with accomplished by initially preparing plasmid pHIVG7, as vP866 (NYVAC) as the rescuing virus to yield vP970. Immunoprecipitation experiments with vP970 infected described above (see Example 16). cells were performed as described above for the expression pHIVGT was used in recombination experiments with of the HIV-1 gag precursor protein. No HIV-1-specific vP921 as the rescuing virus to yield vo91. 40 species were precipitated from mock infected or NYVAC Immunoprecipitation experiments with vP991 infected infected Vero cells. A protein species corresponding to p24 cells were performed as described above for the expression was precipitated, however, from lysates of v370 infected of the HIV gag precursor protein. No HIV-specific species cells. were precipitated from mock infected Vero cells. Protein EXAMPLE 2.0 species corresponding to the env and gag precursor proteins, 45 as well as various intermediate and mature gag cleavage GENERATION OF NYVAC/HIV-1 p17, p24 AND products, were precipitated, however, from lysates of vP991 env (gp120) RECOMBINANT infected cells. A plasmid, pHXB2D. containing HIV-1 cDNA sequence, was obtained from Dr. R. C. Gallo (NC-NIH). The sequence EXAMPLE 18 50 encoding the 5'-end of the gag gene was cloned between vaccinia virus tk flanking arms. This was accomplished by GENERATION OF NYVAC/HIV-1 gag?pol AND initially preparing plasmid pHTVG9 as described above (see env (gp160) RECOMBINANT Example 19). The sequence encoding the 5'-end of the gag gene was pHIVG9 was used in recombination experiments with cloned between vaccinia virus tk flanking arms. This was ss vP921 as the rescuing virus to yield vP973. accomplished by initially preparing plasmid pHTVG7, as Immunoprecipitation experiments with vpg73 infected described above (see Example 16). cells were performed as described above for the expression pHTVG7 was used in recombination experiments with of the HIV-1 gag precursor protein. No HIV-1-specific vP911 (above) as the rescuing virus to yield wP990. species were precipitated from mock infected Vero cells. Immunoprecipitation experiments with vP990 infected 6 Protein species corresponding to env and p24 were cells were performed as described above for the expression precipitated, however, from lysates of vP973 infected cells. of the HIV-1 gag precursor protein. No HIV-1-specific EXAMPLE 21 species were precipitated from mock infected Vero cells. Protein species corresponding to the env and gag precursor GENERATION OF NYVAC/HIV-1 p17, p24 AND proteins. as well as various intermediate and mature gag 65 env (gp160) RECOMBINANT cleavage products, were precipitated, however, from lysates A plasmid, pHXB2D, containing HIV-1 cDNA sequence, of VP990 infected cells. was obtained from Dr. R. C. Gallo (NCI-NIH). The sequence 5,766,598 51 S2 encoding the 5'-end of the gag gene was cloned between EXAMPLE 23 vaccinia virus tk flanking arms. This was accomplished by initially preparing plasmid pHIVG9, as described above (see GENERATION OF NYVAC/HIV-1 gag?pol AND Example 19). env (TRUNCATED) RECOMBINANT pHTVG9 was used in recombination experiments with The sequence encoding the 5'-end of the gag gene was vP911 as the rescuing virus to yield vP971. cloned between vaccinia virus tik flanking arms. This was Immunoprecipitation experiments with vP971 infected accomplished by initially preparing plasmid pHIVG7 as cells were performed as described above for the expression described above (see Example 16). of the HIV-1 gag precursor protein. No HIV-1-specific An H6-promoted truncated HIV-1 envelope gene was species were precipitated from mock infected Vero cells. then inserted into pHIVG7. This was accomplished by Protein species corresponding to env and p24 were cloning the E. coli DNA polymerase I (Klenow fragment) precipitated, however, from lysates of vpg71 infected cells. filled-in 1,600 bp XhoI-Not fragment of pHIVE10 (defined EXAMPLE 22 in Example 22), containing an H6-promoted truncated 5 HIV-1 envelope gene, into the filled-in BamHI site of GENERATION OF NYVAC/HIV-1 gag pHIVG7. The plasmid generated by this manipulation is (PROTEASE) AND env (TRUNCATED) called pHTVGE12. RECOMBINANT pHIVGE12 was used in recombination experiments with A plasmid, pHXB2D, containing HTV-1 cDNA sequence vP866 (NYVAC) as the rescuing virus to yield vP978. was obtained from Dr. R. C. Gallo (NCI-NIH). The sequence Immunoprecipitation experiments with vP978 infected encoding the 5'-end of the gag gene was cloned between cells were performed as described above for the expression vaccinia virus the flanking arms. This was accomplished by of the HIV-1 gag precursor protein. No HIV-1-specific initially preparing plasmid pHTVG9 as described above (see species were precipitated from mock infected or NYVAC Example 19). infected Vero cells. Protein species corresponding to the env An H6-promoted truncated HIV-1 envelope gene was 25 and gag precursor proteins, as well as various intermediate then inserted into pHIVG4. This was accomplished by and mature gag cleavage products, were precipitated, cloning the E. coli DNA polymerase I (Klenow fragment) however, from lysates of vP978 infected cells. filled-in 1,600 bp XhoI-Not fragment of pHIVE10, con taining an H6-promoted truncated HIV-1 envelope gene, EXAMPLE 24 into the filled-in BamHI site of pHTVG4. The plasmid 30 GENERATION OF NYVAC/HIV-1 gag?pol AND generated by this manipulation is called pHTVGE11. env (gp120) RECOMBINANT The plasmid pHTVE10 was derived by inserting a SacI/ partial KpnI fragment from pBSHIV3BCDT1 into the mul The sequence encoding the gag gene was cloned between tiple cloning region of pIBI25 (IBI, New Haven, Conn.). The vaccinia virus th flanking arms. This was accomplished by plasmid p3SHIV3BCDT1 contains an H6 promoted cassette 35 initially preparing plasmid pHTVG7, as described above (see to express a severely truncated form of the HIV-1 (IIIB) Example 16). envelope (amino acid 1 to 447; Ratner et al., 1985). Expres The I3L-promoted gag and poll genes were then inserted sion of this cassette was evaluated to eliminate CD4 binding into a canary pox insertion vector. This was accomplished by while retaining the V3 loop region and the T1 epitope. cloning the 4,360 bp partial BglII-BamHI fragment of To construct pBSHIV3BCDT1 the following manipula pHTVG7, containing the 3L-promoted gag and pol genes, tions were performed, A PCR-derived fragment of 200 bp into the BamHI site of pVQH6CP3L. The plasmid generated was amplified from peSH6HIV3B5P (defined in Example by this manipulation is called pHTVGE14. 2) using oligonucleotides HTV3B2A (SEQ ID NO:96) and The H6-promoted HIV-1(MN) envelope (gp120) gene HIV CD4A (SEQ ID NO: 128) (5'- 45 was then inserted into pHTVGE14. This was accomplished GCCTCCTACTATCATTATGAATAAACTGATGGGA by cloning the oligonucleotides, HTVL29 (SEQID NO:129) GGGGCATAC-3). This fragment was fused by PCR to (5'-GGCCGCAAC-3') and HIVL30 (SEQ ID NO:130) (5'- annealed oligonucleotides HIVTM1 (SEQID NO:107) and TCGAGTTGC-3"), and the 1,600 bp NruI-Not fragment of HIVTM2 (SEQ ID NO:108) using oligonucleotides pBSHIVMN 120, containing the H6-promoted gp120 gene, HIV3B2A (SEQ ID NO:96) and HIVTM3 (SEQ ID 50 into the 11,500 bp NruI-XhoI fragment of pHTVGE14. The NO:109). These manipulations create the 3'-end of the plasmid generated by this manipulation is called truncated env cassette by placing sequences encoding the pHIVGE15. HIV-1 env transmembrane anchor (amino acids 691 to 718; The H6-promoted envelope (gp120) gene and the I3L Ratner et al., 1985), a translation termination codon (TAA), promoted gag and pol genes were then inserted into a and a 3’ Xbal site. This PCR-fusion product was digested 55 vaccinia virus insertion vector. This was accomplished by with EcoRI and Xbal to yield a 243 bp fragment. The cloning the 6.400 bp Not-BamHI fragment of pHTVGE15. fragment was ligated to the 4.5bp EcoRIIXbal fragment of containing the H6-promoted gp120 gene and the 3L pH6HIIIBE to generate pBSHIV3BCDT1. promoted gag and pol genes, into the 4,000 bp Not-BglDI pHTVGE11 was used in recombination experiments with fragment of pSD542VCVQ. The plasmid generated by this vP866 (NYVAC) as the rescuing virus to yield vP979. manipulation is called pHIVGE16. Immunoprecipitation experiments with vP979 infected pHIVGE16 was used in in vitro recombination experi cells were performed as described above for the expression ments with vP866 (NYWAC) as the rescuing virus to yield of the HIV-1 gag precursor protein. No HIV-1-specific WP988. species were precipitated from mock infected or NYVAC Immunoprecipitation experiments with vP988 infected infected Vero cells. Protein species corresponding to env and 65 cells were performed as described above for the expression the gag precursor proteins were precipitated, however, from of the HIV-1 gag precursor protein. No HIV-1-specific lysates of vP979 infected cells. species were precipitated from mock infected or NYVAC 5,766,598 53 S4 infected Vero cells. Protein species corresponding to the env vCP117 infected cells, but did not precipitate HIV-1-specific and gag precursor proteins, as well as various intermediate proteins from mock infected or ALVAC infected cells. and mature gag cleavage products, were precipitated, however, from lysates of w888 infected cells. EXAMPLE 27 EXAMPLE 25 GENERATION OF ALVAC/HIV-1 gag?pol AND env (gp160) RECOMBINANT GENERATION OF NYVAC/HIV-1 gag?pol AND env (gp160) RECOMBINANT The sequence encoding the 5'-end of the gag gene was cloned between vaccinia virus the flanking arms. This was The sequence encoding the 5'-end of the gag gene was accomplished by initially preparing plasmid pHIVGE15 as cloned between vaccinia virus tik flanking arms. This was accomplished by initially preparing plasmid pHIVGE16 as described above (see Example 24). The gp120 gene was then replaced by the gp160 gene. described above (see Example 24). This was accomplished by cloning the 2.600 bp NruI-Not The gp120 gene was then replaced by the gp160 gene. fragment of pH6HMNE. containing the entire HIV-1(MN) This was accomplished by cloning the 2,600 bp NruI-Not 15 fragment of pH6HMNE. containing the entire HIV-1(MN) envelope (gp160) gene, into the 9,800 bp NruI-Not frag envelope (gp160) gene. into the 8,000 bp partial Niru-Not ment of pHTVGE15. The plasmid generated by this manipu fragment of pHIVGE16. The plasmid generated by this lation is called pHIVGE18. manipulation is called pHIVGE19. The canary pox flanking arm deleted in the previous step was then cloned into pHIVGE18. This was accomplished by pHTVGE19 was used in in vitro recombination experi cloning the 1,500 bp Not fragment of pHTVGE15. contain ments with vp866 (NYVAC) as the rescuing virus to yield ing the C3 flanking arm, into the 12.400 bp Not fragment WP1.009. of pHIVGE18. The plasmid generated by this manipulation Immunoprecipitation experiments with vP1009 infected is called pHIVGE20. cells were performed as described above for the expression of the HIV-1 gag precursor protein. No HIV-1-specific 25 pHTVGE20 was used in recombination experiments with species were precipitated from mock infected or NYVAC ALVAC (CPpp) as the rescuing virus to yield vCP130. infected Vero cells. Protein species corresponding to the env Immunoprecipitation analysis was performed with CEF and gag precursor proteins, as well as various intermediate cell monolayers as described above to determine whether and mature gag cleavage products, were precipitated. vCP130 expresses authentic HIV-1 gag and env gene prod however, from lysates of vP1009 infected cells. 3. luctS. Lysates from the infected cells were analyzed for HIV-1 EXAMPLE 26 gag and env gene expression using pooled serum from HIV-1 seropositive individuals (obtained from Chiron. GENERATION OF ALVAC/HIV-1 gag?pol AND Emeryville, Calif.). The sera was preadsorbed with ALVAC env (GP120) RECOMBINANT 35 infected CEF cells. The preadsorbed sera was bound to The sequence encoding the 5'-end of the gag gene was Protein A-sepharose in an overnight incubation at 4 C. cloned between vaccinia virus th: flanking arms. This was Following this incubation period, the material was washed accomplished by initially preparing plasmid pHTVGE15, as 4xwith 1xbuffer A. Lysates precleared with normal human described above (see Example 24). sera and protein A-sepharose were then incubated overnight pHTVGE15 was used in recombination experiments with at 4°C. with the HTV-1 seropositive human sera bound to ALVAC (CPpp) as the rescuing virus to yield vCP117. protein A-sepharose. After the overnight incubation period, Immunoprecipitation analysis was performed as the samples were washed 4xwith 1xbuffer A and 2xwith a described above but with CEF cell monolayers to determine LiClurea buffer. Precipitated proteins were dissociated whether vCP117 expresses authentic HIV-1 gag and env from the immune complexes by the addition of gene products. 45 2xLaemmli's buffer (125 mM Tris (pH6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. Lysates from the infected cells were analyzed for HIV gag Proteins were fractionated on a 10% Dreyfuss gel system and env gene expression using serum from HIV-1 seroposi (Dreyfuss et al., 1984), fixed and treated with 1M tive individuals (obtained from New York State Department Na-salicylate for fluorography. Human sera from HIV-1 of Health). The sera was preadsorbed with ALVAC infected 50 CEF cells. The preadsorbed sera was bound to Protein seropositive individuals specifically precipitated the HIV-1 A-sepharose in an overnight incubation at 4° C. Following gag and env proteins from vCP130 infected cells, but did not this incubation period, the material was washed 4xwith precipitate HIV-1-specific proteins from mock infected or 1xbuffer A. Lysates precleared with normal human sera and ALWAC infected cells. protein A-sepharose were then incubated overnight at 4°C. 55 with the HIV-1 seropositive human sera bound to protein EXAMPLE 28 A-sepharose. After the overnight incubation period, the GENERATION OF ALVAC/HIV-1 gag?pol samples were washed 4xwith 1xbuffer A and 2xwith a RECOMBINANT LiCl/urea buffer. Precipitated proteins were dissociated from the immune complexes by the addition of A plasmid, pHXB2D, containing HIV-1 cDNA sequence 2xLaemmli's buffer (125 mM Tris (pH6.8), 4% SDS. 20% was obtained from Dr. R. C. Gallo (NCI-NIH). The sequence glycerol. 10% 2-mercaptoethanol) and boiling for 5 min. encoding the 5'-end of the gag gene was cloned between Proteins were fractionated on a 10% Dreyfuss gel system vaccinia virus the flanking arms. This was accomplished by (Dreyfuss et al., 1984), fixed and treated with 1M initially preparing plasmid pHTVG7 as described above (see Na-salicylate for fluorography. 65 Example 16). Human sera from HIV-1 seropositive individuals specifi The gag and pol genes were then cloned between canary cally precipitated the HIV-1 gag and env proteins from pox flanking arms. This was accomplished by cloning the 5,766,598 SS S6 4,400 bp SmaI-Not fragment of pHIVG7, containing the env genes would also produce such particles. Furthermore, I3L-promoted gag and pol genes, and the oligonucleotides. if these ALWAC-based recombinants were used to infect HIV2L6 (SEQ ID NO:131) (5'-GGCCAAAC-3") and non-avian cells (i.e. Vero, MRC-5, etc.) then HIV-1 virus HTV2L7 (SEQ ID NO:132) (5'-TCGAGTTT-3"), into the like particles could be purified without any poxvirus virion SmaI-XhoI site of pSPCP3L. The plasmid generated by this contaminants. manipulation is called pHTVG24. To evaluate particle formation using Vero cells infected pHTVG24 was used in recombination experiments with with vCP156, the following experiment was performed. ALVAC (CPpp) as the rescuing virus to yield vCP152. Vero cells were infected at an m.o. i. of approximately 5 Immunoprecipitation experiments with vCP152 infected pfu?cell. After a 24 hr infection period, the supernatant was cells were performed as described above for the expression 10 harvested and clarified by centrifugation at 2000 rpm for 10 of the HIV-1 env and gag proteins. No HIV-1-specific min. The supernatant was then spun through filters which species were precipitated from mock infected or ALVAC have a molecular weight cutoff of 30,000 kDa (Centricell 60. infected cells. Protein species corresponding to the gag Polysciences. Inc.. Warrington, Pa.). Thus, any smaller mol precursor protein, as well as various intermediate and mature ecules would pass through these filters. The material gag cleavage products, were precipitated, however, from 15 retained by the filters was then analyzed by standard Western lysates of vCP152 infected cells. blot analysis (Maniatis et al., 1990) using pooled human serum from HIV-seropositive individuals (obtained from Dr. EXAMPLE 29 J. Conroy, New York State Department of Health). The results from the Western blot analysis demonstrated the GENERATION OF ALVAC/HIV-1 gag?pol AND 20 presence of the major core protein p24 and the HTV-1(MN) env (TRUNCATED) RECOMBINANT anchored gp120 in the material retained by the filters. With the size exclusion noted above, the p24 would have passed A plasmid, pHXB2D, containing HIV-1 cDNA sequence through unless it was in a higher structural configuration (i.e. was obtained from Dr. R. C. Gallo (NCI-NTH). The sequence virus-like particles). Therefore, these results strongly sug encoding the 5'-end of the gag gene was cloned between 25 gest that HIV-1 virus-like particles containing the gp120 vaccinia virus th flanking arms. This was accomplished by initially preparing plasmid pHTVG24 as described above envelope component are produced in vCP156 infected cells. (see Example 28). EXAMPLE 31 pHTVG24 was used in recombination experiments with vOP120 as the rescuing virus to yield vCP155. EXPRESSION OF THE T1, T2, AND TH4.1 Immunoprecipitation experiments with vCP155 infected EPTOPES OF THE HIV-1 env GENE N ALWAC cells were performed as described above for the expression AND NY WAC of the HIV-1 env and gag proteins. No HIV-1-specific Recombinant poxviruses wP1062 and vCP146 were gen species were precipitated from mock infected cells. Protein erated to express the T1, T2, and TH4.1 epitopes of HIV-1 species corresponding to the env and gag precursor proteins, 35 env (Hosmalin et al., 1991) as individual peptides. as well as various intermediate and mature gag cleavage Construction of plasmid p731T1. Plasmid pMPI3H con products, were precipitated, however, from lysates of tains the vaccinia I3L early/intermediate promoter element vCP155 infected cells. (Schmitt and Stunnenberg, 1988; Vos and Stunnenberg. EXAMPLE 30 1988) in a pUC8 background. The promoter element was synthesized by polymerase chain reaction (PCR) using GENERATION OF ALVAC/HIV-1 gag?pol AND pMPVC1, a subclone of vaccinia HindIII, as template and env (gp120 WITH TRANSMEMBRANE synthetic oligonucleotides MPSYN283 (SEQ ID NO:133) ANCHOR) RECOMBINANT (5'- CCCCCCAAGCTTACATCATGCAGTGGTTAAAC 45 -3") and MPSYN287 (SEQID NO:134) (5'- GATTAAAC A plasmid, pHXB2D, containing HIV-1 cDNA sequence CTAAATAATTGT-3") as primers. DNA from this reaction was obtained from Dr. R. C. Gallo (NCI-NIH). The sequence was cut with HindIII and RsaI and a 0.1 kb fragment encoding the 5'-end of the gag gene was cloned between containing the promoter element was purified. A linker vaccinia virus the flanking arms. This was accomplished by region was assembled by annealing complementary syn initially preparing plasmid pHTVG24 as described above 50 thetic oligonucleotides MPSYN398 (SEQ ID NO:135) (5'- (see Example 28). ACAATTATTTAGGTTAACTGCA -3") and MPSYN399 pHTVG24 was used in recombination experiments with (SEQ ID NO:136) (5'- GTTAACCTAAATAATTGT -3'). vCP138 as the rescuing virus to yield vCP156. The PCR-derived promoter element and the polylinker Immunoprecipitation experiments with vCP156 infected region were ligated with vector plasmid PUC8 which had cells were performed as described above for the expression 55 been cut with HindIII and Pst. The resulting plasmid, of the HIV-1 env and gag proteins (CEF Cell monolayer). pMPI3H, contains the I3L promoter region from positions No HIV-1-specific species were precipitated from mock -100 through-6 relative to the initiation codon, followed by infected cells. Protein species corresponding to the env and a polylinker region containing HpaI, Pst, Sal, BamHI, gag precursor proteins, as well as various intermediate and SmaI and EcoRI sites. Cleavage with HpaI produces blunt mature cleavage products, were precipitated, however, from ended DNA linearized at position -6 in the promoter. lysates of vCP156 infected cells. A cassette containing the T1 peptide driven by the vac Expression of HIV-1 gag-specific gene products either cinia I3L promoter was generated by ligating complemen alone or in combination with env by vaccinia virus has been tary oligonucleotides T1C (SEQ ID NO:137) (5'- shown to lead to the production of non-infectious virus-like TAATCATGAAACAAATTATTAATATGTGGCAAGAA particles (Haffar et al., 1990; Hu et al., 1990). With this 65 GAGG- AAAAGCTATGTACGCTTGACT AGTTAAT background it was investigated whether cells infected with CACTCGAG-3") and T1N (SEQID NO:138) (5'- GATC ALVAC-based recombinant expressing HIV-1 gag-pol and C T C G A G T G AT TAA CTA GT CAA GC GTA 5,766,598 57 58 CATAG CTTTTCCTACTT CTTGCCA CATATT CAAATAGTTTCGTTTTCACCTTGTCTAATAACTAAT AATAATTTGTTTCATGATTA -3") to plasmid pMPI3H TAATTAAGGATCCTGATTAA-3") as template for a third digested with HpaI and BamHI. This ligation reconstitutes PCR using primers 42KTH41 (SEQ ID NO:146) and the last 5 base pairs of the promoter, provides the complete BAMVQ (SEQ ID NO:149) (5'- TTAATCAGGATCCT. TAATTAATTAGTTATTAGAC-3). Subsequent nucleotide coding sequence of the T1 peptide, and creates a XhoI site sequence analysis revealed an error in the sequence of between the stop codon and BamHI site. This is plasmid oligonucleotide 42KTH41 (SEQ ID NO:146) such that an p731T1. The sequence of the fragment was confirmed by extra base (an A) was inserted after position 24 as indicated nucleotide sequence analysis. by the underline in the above sequence for 42KTH41. This Construction of plasmid pH6T2. A cassette containing the was corrected with a final PCR employing the 272 bp T2 peptide driven by the vaccinia H6 promoter was gener O fragment derived from the third PCR as template with ated in two steps: The H6 promoter through the EcoRV site primers BAMVQ (SEQID NO:149) and 42KTH41A (SEQ was derived from a plasmid containing the synthetic H6 ID NO:150) (5'- ATCATCGGATCCTCGAGTGATTAAC promoter (Perkus et al., 1989), using PCR and primers TAGTCATCTAATAGCTC -3'). After the final PCR, the cassette was to contain BamHI. Pst, and SmaI sites 5' to H6PCR1 (SEQID NO:157) and H6PCR2 (SEQID NO:205) 42K-TH4.1 with XhoI and BamHI sites 3". This 271 bp (5'-TTAACGGATATCGCGATAATG-3") creating a 5' Hin 15 PCR-derived fragment was digested with BamHI and cloned dII site. This 122 bp PCR-derived fragment was digested into the BamHI site of pBS-SK+ (Stratagene, La Jolla, with HindIII and EcoRV followed by ligation to similarly Calif.) generating plasmid pVQ42KTH4.1. Nucleotide digested pBS-SK-- (Stratagene, La Jolla, Calif.), generating sequence analysis of this plasmid confirmed that the plasmid pBSH6. Complementary oligonucleotides T2C sequence of the promoter and coding region was correct. (SEQID NO:139) (5'- ATCCGTTAAGTTTGTATCGTAAT 20 However, a 3 bp deletion was revealed resulting in loss of GCACGAAGATATTATTTCTTTGTGGGATCAATCTTA the 3' BamHI site. AAATGACTAGTTAATCAG -3") and T2N (SEQ ID Construction of plasmid pt 1T2TH4.1. These three cas NO:140) (5'- GATCCTGATTAACTAGTCATTTTAAA settes were combined into a singular plasmid pt 1T2TH4.1 such that I3L-T1 is opposite in orientation to the other two GATTGATCCCACAAAGAAATAATATCTTCGTGCA genes in the following manner: A 170 bp HindIII/XhoI TTACGATACAAACTTAACGGAT-3") which complete the 25 fragment was isolated from p731T1 and ligated to similarly 3' end of the H6 promoter from the EcoRV site, encode the digested pH6T2 generating pr1T2. A 290 bp BamHI/SacI T2 peptide and create a BamHI site at the 3' end of the gene fragment from pVQ42KTH4.1 was ligated to similarly were annealed then ligated to pbSH6 that was digested with digested pT1T2, creating pt 1T2TH4.1. The sequence of the EcoRV and BamHI. This plasmid was designated pH6T2 insert was confirmed by nucleotide sequence analysis. following confirmation of the fragment by nucleotide 30 pC5LSP (defined in Example 14) was digested with sequence analysis. EcoRI, treated with alkaline phosphatase and ligated to Construction of plasmid pVQ42KTH4.1. A cassette con self-annealed oligonucleotide CP29 (SEQ ID NO:151) (5'- AATTGCGGCCGC-3"), digested with Not and linear puri taining the TH4.1 peptide driven by the AmEPV 42K fied followed by self-ligation. This procedure introduced a promoter was generated by sequential PCR reactions: the Not site to pCSLSP, generating pNC5LSP5. 107 bp 42Kpromoter with 5'Pstand SmaI sites was derived 35 Plasmid pSD550 was derived from pSD548 as follows. by PCR from plasmid pa2KRABI, a plasmid containing the Plasmid pSD548 (Tartaglia et al., 1992) is a vaccinia vector gene for the rabies glycoprotein under control of the 42K plasmid in which the I4L ORF (Goebel et al., 1990a,b) is promoter, using primers 42KVQ1 (SEQ ID NO:141) (5'- replaced by a cloning region consisting of BglDI and SmaI AATTA AT TA G C T G C A G C C C C G G G T - sites. To expand the multicloning region, pSD548 was cut CAAAAAAATATAAATG -3') and 42KVQ2 (SEQ ID with Bg II and SmaI and ligated with annealed complemen NO:142) (5'- CCTTGTACTACTTCAATTACTC tary synthetic oligonucleotides 539A (SEQ ID NO:152) TATCCATTTTATATTGTAATTATATATTTTC). The (5'-AGAAAAATCA GT TAG CTA AGAT CTC sequence of the 107 bp promoter region of this PCR-derived CCGG GCTC GAGGGTAC CG GAT C CTG AT T AGTTAATTTTTG T-3") AND 539B (SEQ ID NO:153) fragment is (SEQ D NO: 143) 5'- (5'-GATCACAAAAATTAACTAATCAGGATCCGG T CAAAAAAATATAAATGATT CAC CAT CT 45 TACCCTCGAGCCCGGGAGATCTTAGCTAACTGATT GATAGAAAAAAAATTTATTGG GAAGAATAT TTTCT-3"). In the resulting plasmid, pSD550, the multiclon G AT AAT ATTTT G G GATTT CAA AAT ing region contains BglII. SmaI, XhoI, KpnI and BamHI TGAAAATATATAATTACAATATAAA -3'. The 159 bp restriction sites. PCR-derived fragment was fused to the coding sequences of The 602 bp XhoI fragment from pT1T2TH4.1 containing TH4.1 with a second PCR using this fragment and synthetic 50 the genes for the epitopes driven by their respective pro oligonucleotides encoding the TH4.1 peptide TH41C (SEQ moters was cloned into donor plasmids pNC5LSP5 and DD NO:144) (5'- ATGGATAGAGTAATTGAAGTAGTA pSD550 in their XhoI sites. Nucleotide sequence analysis was used to confirm the sequence and the orientation of the CAAGGAGCTTATAGAGCTATAGATGACTAGTTAAT insert. The resulting plasmids pC5T1T2TH4.1 and CACTCGAGGATCC -3") and TH41N (SEQ ID NO:145) pI4T1T2TH4.1 were used in in vitro recombination experi (5'- GG ATC CTCGA GT GATTAACTAGT 55 ments with ALWAC and NYVAC to generate recombinant CATCTAATAGCTCTATAAGCTCCTTGTACTACTCAA viruses vCP146 and vP1062, respectively. These recombi TTACTCTATCCAT-3") as template and primers 42KTH41 nant viruses were demonstrated to contain the desired genes (SEQ ID NO:146) (5'- ATCATCGGATCCTCGAGTGAT. by hybridization of a specific DNA probe to viral plaques. TAAACTAGTCATCTAATAGCTC-3') and 42KVQ1 (SEQ ID NO:141). This 210 bp PCR-derived fragment was EXAMPLE 32 extended in the 5' direction, incorporating a BamHI site at EXPRESSION OF TWO FUSION PEPTDES the 5' end using the fragment and synthetic oligonucleotides CONTAINING THE T1, T2, AND TH4.1 VQC (SEQ ID NO:147) (5'- TTAATCAGGATCCTTAAT EPTOPES OF HIV-1 env WTTH AND WITHOUT TAATTA GT TATTAGA CAAG G T GAAAA C A TRANSMEMBRANE ANCHOR DOMAN GAAACTATTTGTAGC TTAATTAATTAGCTGCAGC 65 FROM HIV-1 enly CCGGG -3') and VQN (SEQ ID NO:148) (5'- Recombinant poxviruses vP1060, vp1061, vCP154 and CCC GGG CTG CAG CTAATTAATTAAG CTA vCP148 were created to express a fusion peptide consisting 5,766.598 59 60 of the signal sequences from HIV-1 env coupled to CAT TAT GAATAATTTT CTA AAAG G A G - sequences corresponding to the T1, T2, and TH4.1 epitopes GTCTAATAGCTCTATAAGCTCCTTGTAC TACT of HIV-1 env by cleavable linker regions. vp1060 and TCAATTACTC-3), altering the 3' end to accommodate the vCP154 differ from v1.061 and vCP148 in that the former transmembrane region. This 195bp PCR-derived fragment recombinant viruses express the fusion peptide along with was fused by PCR with oligonucleotides comprising the sequences corresponding to the transmembrane region of anchor, HIVTM1 (SEQID NO:107) and HIVTM2 (SEQID HIV-1 env, NO:108) using primers PCRT1T2 (SEQ ID NO:161) and Both fusion peptides include the 51 amino acid PCRT4END (SEQID NO:162). This 276 bp PCR-derived N-terminal portion of HIV-1 (IIB) env, residues 1-50 (plus fragment was fused with the 314 bp PCR-derived fragment initiating Met) based on Ratner et al. (1985). The amino acid containing the promoter and signal sequences by PCR with sequence of this signal region (SEQ ID NO:154) is primers H6PCR1 (SEQID NO:157) and PCRT4END (SEQ MKEQKTVAMRVKEKY QHLWRWGWRWGTM ID NO:162). Following digestion of this 572 bp PCR LLGMLMICSATEKLWVTVYYGVP. This is followed by derived fragment with HindIII and Xbala fragment of 554 the T1, T2, and TH4.1 epitopes (Hosmalin et al., 1991) bp was isolated from an agarose gel, ligated to similarly separated from the signal, each other, and anchor sequence 15 digested pBS, and the sequence of the insert verified by where present, by a cleavable linker region up to 5 amino nucleotide sequence analysis. The resulting plasmid was acids in length. The amino acid sequence of this region of designated p3ST1T2TH4.1A. the peptide (SEQ ID NO: 155) is signal-PFR pC5LSP (defined in Example 14) was digested with KQIINMWQEVGKAMYA-PPFRK-HEDIISLWDQSLK BamHI and ligated to annealed oligonucleotides CP32 (SEQ PPFRK-DRVIEVVQGAYRAIR-IPPFRK-anchor). The ID NO:112) and CP33 (SEQ ID NO:113) to generate anchor domain is a 28 amino acid transmembrane region of pVQC5LSP6. pVQC5LSP6 was digested with EcoRI, HIV-1 (IITB) env, residues 691-718. The amino acid treated with alkaline phosphatase and ligated to self sequence of this region (SEQ ID NO:156) is annealed oligonucleotide CP29 (SEQID NO:151), digested LFIMIVGGLVGLRIVFAVLSVVNRVRQG-stop). with Not and linear purified followed by self-ligation. This For both versions of the fusion peptide, the H6 promoter 25 procedure introduced a Not site to pVQC5LSP6, generating and HTV-1 env signal sequences were derived by PCR from pNVQC5LSP7. plasmid pbSH6HIV3B5P (defined in Example 2) using Both cassettes were placed individually between the XhoI primers H6PCR 1 (SEQ ID NO:157) and Xbal sites of insertion plasmid pNVQC5LSP7. These (5'ACTACTAAGCTTCTTTATTCTATACTTAAAAA plasmids pC5STIT1TH4.1 and pCSST1T2TH4.1A were GTG-3") and PCRSIGT1 (SEQ ID NO:158) (5'- CATAT 30 used to generate canarypoxvirus recombinants, vCP148 and TAATTT G T TTT CTAAAAG G A G G T A C vCP154 respectively. BamHI-SmaI fragments were excised CCCATAATAGACTGTG -3"). This 314 bp PCR-derived from pC5ST1TITH4.1 and pC5ST1T2TH4.1A and ligated fragment consists of a 5' HindIII site followed by the H6 to similarly digested pSD550 (defined in Example 31) promoter and coding sequences for the signal, linker, and the generating plasmids p14 ST 1 T2 TH4. 1 and first 6 amino acids of the T1 peptide. 35 pI4ST1TITH4.1A respectively. These plasmids were used The remainder of the coding region for the construct in recombination experiments with NYWAC as the rescuing without the transmembrane region was generated by PCR virus resulting in recombinants wP1061 and vp1060, respec amplification of oligonucleotides T2T4A (SEQDD NO:159) tively. These recombinant viruses were demonstrated to (5'- GCTCCTCCTTTTAGAAAACAC GAA contain the desired genes by hybridization of a specific DNA GATATTATTTCTTTGTGGGATCAATCTTTAAAACCT 40 CCTTTTAGAAAAGATAGAGTAATTGAAGTAGTAC probe to viral plaques. -3") and T2T4B (SEQID NO:160) (5'- GTACTACTTCAAT EXAMPLE 33 TACT CTAT CTTTT CTAAAAG GAG (GTTT TAAAGATTGATCCCACAAAG AAATAATATCTTCGT EXPRESSION OF THE HIV-1 nef GENE IN GTTTTCTAAAAGGAGGAGC -3") using primers 45 ALVAC, TROVAC, AND NYVAC PCRT1T2 (SEQ ID NO:161) (5'- AAACAAATTAT TAATAT G T G GCA A GAA GTAGGAAAA G C Recombinant poxviruses vF1084, wrP174, and vCP168 TATGTACGCTCCTCCTTTTAGA AAACACGAAG -3') were generated expressing HIV-1 nef (MN) as follows: and PCRT4END (SEQ ID NO:162) (5'- ACTACTTCTA The I3L promoter was derived by PCR from plasmid GATTAT CTA ATA G CT CTATA A G CTC CT 50 pMPI3H using primers I3PCR1 (SEQ ID NO:164) (5'- TGTACTACTTCAATTACTC -3"). This 177 bp PCR ATCATCGGATCCAAGCTTACATCATGCAGTGG -3') derived fragment encodes the T1 peptide, a linker region, the and PI3NEF2 (SEQ ID NO: 165) (5'- CGTTTTGAC T2 peptide, another linker region, and the TH4.1 peptide. C AT T T G C C A C C C A T G AT T A A A C - followed by a 3'Xbasite. This fragment was fused with the CTAAATAATTGTACTTTG-3"). The coding region of nef 314 bp PCR-derived fragment containing the promoter and 55 was generated by PCR amplification of pMN1.8-10 (a clone signal sequences by PCR with primers H6PCR1 (SEQ ID of that portion of the MN genome between Sst sites at 7792 NO:157) and PCRT4END (SEQ ID NO:162). Following and 9595) using primers PI3NEF1 (SEQ ID NO:166) (5'- digestion of this 473 bp PCR-derived fragment with HindIII A G T A C A AT TAT T T A G G T T TA AT and Xbala fragment of 455 bp was isolated from an agarose CATGGGTGGCAAATGGTCAAAACG -3") and PNEF gel. ligated to similarly digested PBS-SK. and the sequence BAM (SEQ ID NO:167) (5'- ATCATCGGATCCTAA of the insert verified by nucleotide sequence analysis. The CACTTCTCTCTCCGG-3"). Fusion of the coding region resulting plasmid was designated p3ST1T2TH4.1. with the promoter was accomplished by amplification of the The remainder of the coding region of the version with the previous PCR products using primers I3PCR1 (SEQ ID transmembrane anchor was generated by PCR amplification NO:164) and PNEFBAM (SEQ ID NO:167). Following of oligonucleotides, T2T4A (SEQID NO:159) and T2T4B 65 digestion of this product with BamHI a fragment of 0.7 kb (SEQ ID NO:160), using primers PCRT1T2 (SEQ ID was isolated from an agarose gel and ligated to similarly NO:161) and PCRT4TM (SEQ ID NO:163) (5'- TACTAT digested pBS-SK+ (Stratagene. LaJolla, Calif.), generating 5,766,598 61 62 plasmid p3NEF. It was at this point that sequence devia consisted of fowlpox sequences flanked by a 5" EcoRI site tions were first observed. The sequence of the cassette was and a 3'NdeI site. Central in the fragment is a polycloning determined to differ from the published sequence (Gurgo et region containing SmaI, BamHI. and HindIII sites, flanked al., 1988. GenBank Accession Number M17449) due to by Not sites and translation stop codons in six reading deviations in plasmid pMN1.8-10 (provided by Dr. R. C. frames. An early transcription termination signal (Yuen and Gallo, NCI-NIH). These differences are summarized below Moss, 1987) is adjacent to the 3'NotI site. This PCR-derived relative to the published sequence. fragment, digested with EcoRI and NdeI, was ligated to similarly digested pRW715 creating plasmid pRW864. An 11K promoted lac Z gene was excised from pAM1 by partial Base Gurgo et al. MN1.8-10 Result Cassette BamHI, total Pst digestion. This fragment was made blunt 88.34 T T silent C (aa = Arg) ended with Klenow polymerase and ligated to Smaldigested 8863 G A. Lys->Arg A (aa e Lys) pRW864. creating pRW867A. The Not fragment from 8890 T C Pro->Leu C (aa = Pro) 9028 A. G Arg->Lys G (aa = Arg) pRW867A was made blunt ended with Klenow polymerase 912.7 A. A silent G (aa = Gln) in the presence of dNTPs so that the Not sites would be 9330-9331 GG GGG frameshift GG 5 regenerated when ligated into an FspI site, and ligated to pRW866 which was partially digested with FspI such that The two silent mutations in the cassette (at positions 8834 the insertion was made corresponding to position 1955 and 9127) were apparently errors in PCR. Since there is no described by Tartaglia et al. (1990). The resulting plasmid, effect on the encoded protein, these were allowed to persist. pRW868, was then digested with Not to remove the lac Z The frameshift at 9930 results in a lengthened open reading cassette, and ligated to the 66 bp polylinker from pRW864 frame more closely resembling other HIV-1 isolates. In which was excised by Not digestion. The resulting plasmid keeping with the published size of nef from the MN isolate, was designated pRW673. An 81 bp SmaI fragment was this cassette required a fourth PCR to generate the truncated derived from pVQ42KTH4.1 (defined in Example 31) and 3' end of the coding region. inserted into SmaI digested pRW873 generating plasmid Removal of the extra base at position 9930 was accom 25 pVQ873. plished by PCR amplification of the insert in p13.NEF with The nef cassette was excised from pBSI3NEF as a 684 bp primers I3PCR1 (SEQID NO:164) and PNEFFIX1 (SEQID HindIII fragment for insertion into pVQ873 followed by NO:168) (5'- ATCATCGGATCCTAACACTTCTCTCTC recombination into the F16 locus of TROVAC to generate CGGGTCATCCATCCATGCTGGCTCATAG-3") Follow WFP74. ing digestion of this 678 bp PCR-derived fragment with 30 BamHI a fragment of 660 bp was isolated from an agarose EXAMPLE 34 gel and ligated to similarly digested pBS, generating plas mid pBSI3NEF. The insert was verified by nucleotide EXPRESSION OF HIV-2 GENES IN NYVAC sequence analysis. Generation of NYWAC/HIV2 gag?pol recombinant. A The 660 bp BamHI fragment from pBSI3NEF containing 35 plasmid, PISSYEGP containing the human immunodefi the nef gene was placed in the BamHI site of insertion ciency virus type 2 (HIV2) gag and pol genes was obtained plasmid PNVQC5LSP7 (defined in Example 32). The result from Dr. G. Franchini (NCI-NIH). The gag and pol genes ing plasmid pC5I3NEF was employed in a recombination from this plasmid were cloned downstream of the 3L into the C5 locus of ALVAC, generating the recombinant promoter and between vaccinia virus tik flanking arms. This vCP168. The same 660 bp BamHIfragment was also placed was accomplished by cloning the 4400 bp BstUI-BglII in the BamHI site of insertion plasmid pSD550 (defined in fragment of pISSYEGP, containing the HIV2 gag and pol Example 31), creating plasmid plaNEF. A recombination genes, and the oligonucleotides. SIVL1 (SEQ ID NO:69) with this plasmid with NYVAC generated recombinant and HIV2L 1 (SEQ ID NO:173) (5'- wP1084. C G C C C AT GATTA AAC CTA AATAATT G An insertion plasmid for the F16 locos of TROVAC was 45 T A C T T T G T A A T A T A A T - derived in the following manner. A 7.3 kb NaeI/Nde frag GATATATATTTTCACTTTATCTC AC-3"), containing the ment was isolated from a plasmid containing the 10.5 kb I3L promoter, into the 4,070 bp XhoI-Bgll fragment of HindIII fragment of fowlpox virus described by Tartaglia et pSD542 (defined in Example 15). The plasmid generated by al. (1990) and ligated to similarly digested pCC9 creating this manipulation is called pHTV21. plasmid pRW866. 50 Extraneous 3'-noncoding sequence was then eliminated. pUC9 was digested with Pvu and an EcoRI inker This was accomplished by cloning a 280 bp Bcl-SmaIPCR ligated between the Pyu sites creating plasmidpRW715. A fragment. containing the 3'-end of the pol gene, into the cloning site flanked by fowlpox sequences was generated by 8.100 bp Bcl-SmaI fragment of pHIV21. This PCR frag PCR amplification of a portion of the 10.5 kb fragment with ment was generated from the plasmid, pISSYEGP, with the primers RW264 (SEQ ID NO:169) (5'- AATTAACCCGG 55 GAT C C A A G CTT CTA G CTA G CTAATTTT oligonucleotides, HIV2P2 (SEQ ID NO:174) (5'- TATAGCGGCCGCTATAATCGTT AACTTATTAG-3') and ATGGCAGTTCATTGCAT-3') and HIV2P3 (SEQ ID RW267 (SEQ ID NO:170) (5'- GCTAGAAATCTCT NO:175) (5'- TAGTTTTTATAGTTG-3). An adjacent region was also TTCCCGGGAGATCTCTATGCCATTTCTCCAT-3). The amplified by PCR using primers RW266 (SEQID NO:171) plasmid generated by this manipulation is called pHIV22. (5'- GTTACATATGTACAGAATCTGATCATAG-3") and pHIV22 was used in recombination experiments with RW265 (SEQ ID NO:172) (5'- CTAGCTAGAAGCTTG NYVAC (vP866) as the rescuing virus to yield vF1045. G. A. T C C C G G G T T A A T T A A T - Immunoprecipitation analysis was performed to deter TAATAAAAAGCGGCCGCGTTAAAGTAG AAAAATG mine whether vF1045 expresses authentic HIV2 gag gene -3"). These PCR-derived fragments were fused by a third 65 products. PCR using primers RW266 (SEQID NO:171) and RW267 Lysates from the infected cells were analyzed for HIV2 (SEQ ID NO: 170). The resulting PCR-derived fragment gag expression using pooled serum from HIV2 seropositive 5,766,598 63 64 individuals (obtained from Dr. G. Franchini (NCI-NIH)). Example 4), contains the H6-promoted HIV2 env (gp160) The sera was preadsorbed with vP866 infected Vero cells. gene. The H6-promoted env gene from this plasmid was The preadsorbed sera was bound to Protein A-sepharose in cloned between canary pox flanking arms. This was accom an overnight incubation at 4° C. Following this incubation plished by cloning the 2,700 bp XhoI-SacII fragment of period, the material was washed 4xwith 1xbuffer A. Lysates pBSH6HIV2ENV, containing the H6-promoted env gene. precleared with normal human sera and protein A-sepharose and the oligonucleotides, HIV2LA (SEQ ID NO:176) (5'- were then incubated overnight at 4° C. with the HIV2 GGTTG-3') and HIV2L5 (SEQ ID NO:177) (5'- seropositive human sera bound to protein A-sepharose. After AATTCAACCGC-3"), into the XhoI-EcoRI site of pC6L the overnight incubation period, the samples were washed (defined in Example 50). The plasmid generated by this 4xwith 1xbuffer A and 2xwith a LiCl/urea buffer. Precipi tated proteins were dissociated from the immune complexes 10 manipulation is called pHIV23. by the addition of 2xLaemmli's buffer (125 mM Tris The HIV2 gag and pol genes were then cloned into (pH6.8), 4% SDS. 20% glycerol, 10% 2-mercaptoethanol) pHIV23. This was accomplished by cloning the 4450 bp and boiling for 5 min. Proteins were fractionated on a 10% Xma-Not fragment of pHIV22. containing the I3L Dreyfuss gel system (Dreyfuss et al., 1984), fixed and promoted HIV2 gag and poll genes, and the oligonucleotides. treated with 1M Na-salicylate for fluorography. 15 HIV2L6 (SEQID NO:131) and HIV2L7 (SEQID NO:132). Human sera from HIV2 seropositive individuals specifi into the 7,000 bp Xma-XhoI fragment of pHIV23. The cally precipitated the HIV2 gag precursor protein, as well as plasmid generated by this manipulation is called pHIV25. various intermediate and mature gag cleavage protein pHIV25 was used in recombination experiments with products, from vP1045 infected cells, but did not precipitate ALVAC (CPpp) as the rescuing virus to yield vCP153. HIV2-specific proteins from mock infected or NYVAC Immunoprecipitation analysis was performed as infected cells. described above, but with CEF cell monolayers to determine EXAMPLE 35 whether vCP153 expresses authentic HIV2 gag and env GENERATION OF NYVAC/HIV2 gag?pol AND gene products. env (gp160) RECOMBINANT 25 Lysates from the infected cells were analyzed for HTV2 A plasmid, pISSYEGP, containing the HIV2 gag and pol gag and envgene expression using pooled serum from HIV2 genes was obtained from Dr. G. Franchini (NCI-NIH). The seropositive individuals (obtained from Dr. G. Franchini gag and poll genes from this plasmid were cloned down (NCI-NIH)). The sera was preadsorbed with ALVAC stream of the I3L promoter and between vaccinia virustl. infected CEF cells. The preadsorbed sera was bound to flanking arms. This was accomplished by initially preparing 30 Protein A-sepharose in an overnight incubation at 4° C. plasmid pHTV22 as described above (see Example 34). Following this incubation period, the material was washed pHIV22 was used in recombination experiments with 4xwith 1xbuffer A. Lysates precleared with normal human vP920 as the rescuing virus to yield vP1047. sera and protein A-sepharose were then incubated overnight Immunoprecipitation experiments with vP1047 infected at 4° C. with the HTV2 seropositive human sera bound to 35 protein A-sepharose. After the overnight incubation period. cells were performed as described above for the expression the samples were washed 4xwith 1xbuffer A and 2xwith a of the HIV2 gag proteins. No HTV2-specific species were LiCl/urea buffer. Precipitated proteins were dissociated precipitated from mock infected cells. Protein species cor from the immune complexes by the addition of responding to the HIV2 env and gag precursor proteins, as 2xLaemmli's buffer (125 mM Tris (pH6.8), 4% SDS, 20% well as various intermediate and mature gag cleavage 40 glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. products. were precipitated, however, from lysates of Proteins were fractionated on a 10% Dreyfuss gel system wP1047 infected cells. (Dreyfuss et al., 1984), fixed and treated with 1M EXAMPLE 36 Na-salicylate for fluorography. GENERATION OF NYVAC/HIV2 gag?pol AND Human sera from HIV2 seropositive individuals specifi 45 cally precipitated the HIV2 env and gag precursor proteins, env (gp120) RECOMBINANT as well as various intermediate and mature gag cleavage A plasmid, pISSYEGP, containing the HTV2 gag and pol products, from vCP153 infected cells, but did not precipitate genes was obtained from Dr. G. Franchini (NCI-NIH). The HIV2-specific proteins from mock infected or ALVAC gag and poll genes from this plasmid were cloned down infected cells. stream of the I3L promoter and between vaccinia virus tk 50 flanking arms. This was accomplished by initially preparing EXAMPLE 38 plasmid pHIV22 as described above (see Example 34). pHIV22 was used in recombination experiments with EXPRESSION OF STV GENES IN NYVAC wP922 as the rescuing virus to yield vF1044. GENERATION OF NYVAC/SIV env (gp120-gp28) Immunoprecipitation experiments with vp1044 infected 55 AND gag (PROTEASE) RECOMBINANT cells were performed as described above for the expression Immunoprecipitation analysis was performed to deter of the HIV2 gag proteins. No HTV2-specific species were mine whether wP948 (defined in Example 5) expresses precipitated from mock infected cells. Protein species cor authentic SIV env and gag precursor proteins. responding to the HIV2 env and gag precursor proteins, as Lysates from the infected cells were analyzed for SIV env well as various intermediate and mature gag cleavage and gag precursor expression using serum from SIV serop products, were precipitated, however, from lysates of ositive macaques (obtained from Dr. G. Franchini (NCI wP1044 infected cells. NIH)). The sera was preadsorbed with NYVAC (vP866) EXAMPLE 37 infected Vero cells. The preadsorbed sera was bound to Protein A-sepharose in an overnight incubation at 4° C. EXPRESSION OF HIV2 GENES IN ALVAC 65 Following this incubation period, the material was washed Generation of ALVAC/HIV2 gag?pol and env (gp160) 4xwith 1xbuffer A. Lysates precleared with normal macaque recombinant. The plasmid, pFBSH6HIV2ENV (defined in sera and protein A-sepharose were then incubated overnight 5,766,598 65 66 at 4°C. with the SIV seropositive macaque sera bound to expression of the SIV env and gag precursor proteins. No protein A-sepharose. After the overnight incubation period, SIV-specific species were precipitated from mock infected the samples were washed 4xwith 1xbuffer A and 2xwith a Vero cells. Protein species corresponding to p16 and p28 LiCl/urea buffer. Precipitated proteins were dissociated were precipitated, however, from lysates of vp42 infected from the immune complexes by the addition of 5 cells. 2xLaemmli's buffer (125 mM Tris (pH6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. EXAMPLE 43 Proteins were fractionated on a 10% Dreyfuss gel system (Dreyfuss et al., 1984), fixed and treated with 1M GENERATION OF NYVAC/SIV p16, p28 AND Na-salicylate for fluorography. env (gp120-gp28) RECOMBINANT Macaque sera from SIV seropositive individuals specifi Immunoprecipitation experiments with vP952 (Example cally precipitated the SIV gag precursor protein and the 5) infected cells were performed as described above for the envelope glycoprotein from vP948 infected cells, but did not expression of the SIV env and gag precursor proteins. No precipitate SIV-specific proteins from mock infected cells. SIV-specific species were precipitated from mock infected 15 Vero cells. Protein species corresponding to env and p16 and EXAMPLE 39 p28 were precipitated, however, from lysates of vpg52 GENERATION OF NYVAC/SIV gag?pol infected cells. RECOMBINANT EXAMPLE 44 A plasmid, pSTVGAGSS11G, containing SIV cDNA sequence was obtained from Dr. G. Franchini (NCI GENERATION OF NYVAC/SIV env (gp120-gp41) NIH). The gag and pol genes from this plasmid were cloned RECOMBINANT downstream of the I3L promoter and between vaccinia virus tk flanking arms. This was accomplished by preparing The plasmid, pSIVEMVC, contains the H6-promoted 25 SIVc envelope gene (in vitro selected truncated plasmid pSIVG5 as described above (see Example 5). version). The region of the envelope gene containing the pSIVG5 was used in recombination experiments with premature termination codon was cloned into pBSK+. This NYVAC (vP866) as the rescuing virus to yield vP1042. was accomplished by cloning the 1,120 bp Clal-BamHI Immunoprecipitation experiments with vp1042 infected fragment of pSIVEMVC into the Cla-BamHI site of cells were performed as described above for the expression 30 pBSK+. The plasmid generated by this manipulation is of the SIV env and gag precursor proteins. No SIV-specific called pSIV10. species were precipitated from mock infected or NYVAC The upstream termination codon, TAG, was then changed infected Vero cells. Protein species corresponding to the gag to the original CAG codon. This was accomplished by precursor protein, as well as various intermediate and mature cloning the oligonucleotides, SIVL20 (SEQ ID NO:178) gag cleavage products, were precipitated, however, from 35 ( 5 w lysates of vP1042 infected cells. CTA GCTA A GT TAAG GCAGGGGTATAGGC CA G T G T T C T C T T C C C C A C C C T C T - EXAMPLE 40 TATTTCCAGCAGAC TCATACCCAACAG-3") and GENERATION OF NYVAC/SIV gag?pol AND env SVL 21 (SEQ D NO: 179) (5'- (gp120-gp41) RECOMBINANT GT C C T G T T G G G T A T G A GT C T G C T G - GAA ATA A G A G G G T G G G GAA GA GAA - pSIVG5 (Example 5) was used in recombination experi CACTGGCCTATACCCCT GCCTTAACTTAG-3"), into the ments with vP1050as the rescuing virus to yield vR1071. 4,000 bp NheI-PpuMI fragment of pSIV10. The plasmid Immunoprecipitation experiments with v1071 infected generated by this manipulation is called pSIV11. cells show expression of SIV genes. 45 The region containing the modified codon was then cloned back into pSIVEMVC. This was accomplished by EXAMPLE 41 cloning the 380 bp Bgll-Nhe fragment of pSIV11, con GENERATION OF NYVAC/SIV gag?pol AND env taining the modified codon, into the 5,600 bp partial Bgll (gp120-gp28) RECOMBINANT Nhe fragment of pSIVEMVC. The plasmid generated by SO this manipulation is called pSTV12. pSTVG5 (Example 5) was used in recombination experi pSIV12 was used in in vitro recombination experiments ments with vP874 as the rescuing virus to yield vP943. with NYVAC (vP866) as the rescuing virus to yield vP1050. Immunoprecipitation experiments with vP943 infected Immunoprecipitation experiments with vP1050 infected cells were performed as described above for the expression cells were performed as described above for the expression of the SIV env and gag precursor proteins. No SIV-specific 55 of the STV env and gag precursor proteins. No SIV-specific species were precipitated from mock infected Vero cells. species were precipitated from mock infected or NYVAC Protein species corresponding to the env and gag precursor infected Vero cells. A protein species corresponding to env proteins, as well as various intermediate and mature gag was precipitated, however, from lysates of vF1050 infected cleavage products. were precipitated, however, from lysates cells. of vpg43 infected cells. EXAMPLE 42 EXAMPLE 45 GENERATION OF NYVAC/SIV p16, p28 EXPRESSION OF SIV GENES IN ALWAC RECOMBINANT 65 Generation of ALVAC/SIV gag?pol recombinant. A Immunoprecipitation experiments with vp942 (Example plasmid, pSTVGAGSS11G. containing SIV.142 cDNA 5) infected cells were performed as described above for the sequence was obtained from Dr. G. Franchini (NCI-NIH). 5,766,598 67 68 The gag and pol genes from this plasmid were cloned Cla-BamHI fragment of pSIVEMVC into the Cla-BamHI downstream of the I3L promoter and between vaccinia virus site of pBSK+. The plasmid generated by this manipulation flanking arms. This was accomplished by initially preparing is called pSIV10. plasmid pSIVG5 as described above (see Example 5). The upstream termination codon, TAG, was then changed The gag?pol genes were then cloned between canary pox to the original CAG codon. This was accomplished by flanking arms. This was accomplished by cloning the 4,500 cloning the oligonucleotides, SIVL20 (SEQD NO:178) and bp SmaI-Not fragment of pSVG5, containing the 3L SIVL21 (SEQ ID NO:179), into the 4,000 bp NheI-PpuMI promoted gag?pol genes, into the SmaI-Not site of pC5L fragment of pSIV10. The plasmid generated by this manipu (defined in Example 10). The plasmid generated by this lation is called pSIV11. manipulation is called pSTVGC13. O The region containing the modified codon was then pSTVGC13 was used in recombination experiments with cloned back into pSIVEMVC. This was accomplished by ALVAC (CPpp) as the rescuing virus to yield vCP172. cloning the 380 bp Bgll-Nhe fragment of pSIV11 into the Immunoprecipitation experiments with vCP172 infected 5.600 bp partial BglII-Nhe fragment of pSIVEMVC. The cells show expression of SIV genes. plasmid generated by this manipulation is called pSIV12. 15 The env gene was then cloned between canary pox EXAMPLE 46 flanking arms. This was accomplished by cloning the 2.700 bp NruI-Asp718 fragment of pSIV12, containing the EXPRESSION OF STV GENES IN ALVAC H6-promoted env gene, into the 7400 bp NruI-Asp718 Generation of ALVAC/SIV env (gp120-gp41) and gag?pol O fragment of pNVQH6C5SLP18. The plasmid generated by Recombinant. A plasmid. pSIVGAGSS11G, containing sim this manipulation is called pSIVGC15. ian immunodeficiency virus (SIV) cDNA sequence pSTVGC15 is used in in vitro recombination experiments was obtained from Genoveffa Franchini (NCI-NIH). The with ALVAC as the rescuing virus. gag?pol genes from this plasmid were cloned downstream of the I3L promoter and between vaccinia virus flanking arms. 25 EXAMPLE 47 This was accomplished by cloning the 4,800 bp Cfo-Tag fragment of pSIVGAGSS11G, containing the gag?pol genes, GENERATION OF HIV1 GENES IN ALVAC and the oligonucleotides. SIVL1 (SEQ ID NO:69) and Generation of ALVAC/HIV1 gag (pro) (ITB) and gp120 SIVL2 (SEQ ID NO:70), encoding the vaccinia virus I3L (+transmembrane) (MN) Recombinant. A plasmid, promoter, into the 4,070 bp XhoI-AccI fragment of 30 pHXB2D. containing human immunodeficiency virus type 1 pSD542VCVO. The plasmid generated by this manipulation (HIV1) cDNA sequence (ITB) was obtained from Dr. R. C. is called pSIVG1. Gallo (NCI-NIH). The sequence encoding the 5'-end of the Extraneous 3'-noncoding sequence was then eliminated. gag gene was cloned between vaccinia virus tk flanking This was accomplished by cloning a 1,000 bp BamHI-HpaI arms. This was accomplished by cloning the 1.625 bp Bg I PCR fragment, containing the 3'-end of the poll gene, into the 35 fragment of pHXB2D, containing the 5'-end of the gag gene. 7.400 bp partial BamHI-HpaI fragment of pSIVG1. (This into the 4,075 bp Bgll fragment of pSD542VCVQ. The PCR fragment was generated from the plasmid, plasmid generated by this manipulation is called pHTVG2. pSTVGAGSS11G, with the oligonucleotides, SIVP5 (SEQ The 3'-end of the gag gene was then cloned into pHIVG2. ID NO:71) and SIVP6 (SEQ ID NO:72)). The plasmid This was accomplished by cloning a 280 bp Apa-BamHI generated by this manipulation is called pSTVG4. PCR fragment, containing the 3'-end of the gag gene, into Sequencing analysis revealed that pSIVG4 contains a the 5,620 bp Apal-BamHI fragment of pHIVG2. (This PCR single base pair deletion within the pol gene. To correct this fragment was generated from the plasmid. pHXB2D, with error the 2.320 bp Bgll-StuI fragment of pSIVG1 was the oligonucleotides. HTVP5 (SEQID NO:116) and HTVP6 cloned into the 6,100 bp partial Bg-Stu fragment of (SEQID NO:117)). The plasmid generated by this manipu pSIVG4. The plasmid generated by this manipulation is 45 lation is called pHIVG3. called pSIVG5. The I3L promoter was then cloned upstream of the gag The gag?pol genes were then cloned between canary pox gene. This was accomplished by cloning the flanking arms. This was accomplished by cloning the 4,500 oligonucleotides, HTVL17 (SEQ ID NO:118) and HIVL18 bp SmaI-Not fragment of pSIVG5, containing the I3L (SEQID NO:119), encoding the vaccinia virus I3L promoter promoted gag?pol genes, into the Smal-Not site of pC5L. SO and the 5'-end of the gag gene, into the 5.540 bp partial The plasmid generated by this manipulation is called BglII-Clafragment of pHTVG3. The plasmid generated by pSIVGC13. this manipulation is called pHTVG4. The SIV env gene was then cloned into pSIVGC13. This The portion of the gag gene encoding p24, p.2, p7 and p6 was accomplished by cloning the E. coli DNA polymerase 55 was then eliminated. This was accomplished by cloning the I (Klenow fragment) filled-in 2,750 bp partial Bgll-XhoI oligonucleotides. HIVL19 (SEQ ID NO:120) and HTVL20 fragment of pSIV12, containing the H6-promoted SIV env (SEQ ID NO:121). into the 4450 bp partial Pvul-BamHI gene, into the SmaI site of pSTVGC13. The plasmid gener fragment of pHIVG4. The plasmid generated by this ated by this manipulation is called pSIVGC14. manipulation is called pHIVG5. pSIVGC14 is used in in vitro recombination experiments The remainder of the gag gene, as well as the pol gene, with ALVAC as the rescuing virus. was then cloned downstream of the p17 "gene". This was Generation of ALVAC/SIV env (gp120-gp41) Recombi accomplished by cloning the 4,955bp Cla-Salfragment of nant. A plasmid, pSIVEMVC, contains the H6-promoted pHXB2D, containing most of the gag gene and all of the pol simian immunodeficiency virus envelope gene (in vitro gene, into the 4.150 bp Cla-SalI fragment of pHTVG5. The selected truncated version). The region of the envelope gene 65 plasmid generated by this manipulation is called pHIVG6. containing the premature termination codon was cloned into Extraneous 3'-noncoding sequence was then eliminated. pBSK+. This was accomplished by cloning the 1.120 bp This was accomplished by cloning a 360 bp AflII-BamHI 5,766,598 69 70 PCR fragment, containing the 3'-end of the poll gene, into the The remainder of the gag gene, as well as the pol gene. 8,030 bp AfII-BamHI fragment of pHIVG6. (This PCR was then cloned downstream of the p17 "gene". This was fragment was generated from the plasmid, pHXB2D, with accomplished by cloning the 4,955bp Clal-SalIfragment of the oligonucleotides, HIVP7 (SEQID NO:122) and HTVP8 pHXB2D, containing most of the gag gene and all of the pol (SEQID NO:123)). The plasmid generated by this manipu gene, into the 4.150 bp Cla-Sal fragment of pHIVG5. The lation is called pHIVG7. plasmid generated by this manipulation is called pHIVG6. Extraneous 3'-noncoding sequence was then eliminated. The I3L-promoted gag and poll genes were then inserted This was accomplished by cloning a 360 bp AflII-BamHI into a canary pox insertion vector. This was accomplished by PCR fragment, containing the 3'-end of the polgene, into the cloning the 4360 bp partial Bgll-BamHI fragment of 8,030 bp Afl-BamHI fragment of pHIVG6. (This PCR pHIVG7, containing the I3L-promoted gag and pol genes, O fragment was generated from the plasmid. pHXB2D. with into the BamHI site of pVQH6CP3L. The plasmid generated the oligonucleotides. HIVP7 (SEQID NO:122) and HIVP8 by this manipulation is called pHIVGE14. (SEQID NO:123)). The plasmid generated by this manipu The H6-promoted HIV1 gp120 (+transmembrane) gene lation is called pHIVG7. was then cloned into pHTVGE14. This was accomplished by The I3L-promoted gag and pol genes were then inserted cloning the 1,700 bp NruI-SmaI fragment of 15 into a canary pox insertion vector. This was accomplished by pC5HTVMN120T. containing the gp120 (+transmembrane) cloning the 4.360 bp partial BglII-BamHI fragment of gene. into the 11.400 bpNruI-SmaIfragment of pHIVGE14. pHTVG7, containing the I3L-promoted gag and pol genes, The resulting plasmid is called pHTVGE14T. into the BamHI site of pVQH6CP3L. The plasmid generated Most of the pol gene was then removed. This was by this manipulation is called pHTVGE14. accomplished by cloning a 540 bp Apa-BamHI PCR 20 The H6-promoted HIV1 envelope "gp120 " gene (MN) fragment, containing the 3'-end of the HTV1 protease was then inserted into pHIVGE14. This was accomplished “gene", into the 10,000 bp Apa-BamHI fragment of by cloning the 1.600 bp NruI-NotI fragment of pHTVGE14T. (This PCR fragment was generated from the pBSHTVMN 120, containing the H6-promoted envelope plasmid, pHIVG7, with the oligonucleotides. HIVP5 (SEQ "gp120" gene, and the oligonucleotides. HIVL29 (SEQID 25 NO:129) and HIVL30 (SEQID NO:130), into the 11,500 bp ID NO: 116) and HIVP37 (SEQ ID NO: 180; NruI-XhoI fragment of pHIVGE14. The plasmid generated 5'-AAAGGATCCCCCGGGTTAAAAATTTAAAGTGC by this manipulation is called pHIVGE15. AACC-3')). This manipulation removes most of the pol The H6-promoted HIV1 envelope "gp120" gene (MN) gene, but leaves the protease "gene" intact. The plasmid and the 3L-promoted gag and poll genes (IB) were then generated by this manipulation is called pHIV32. 30 inserted into a vaccinia virus insertion vector. This was pHIV32 is used in in vitro recombination experiments accomplished by cloning the 6.400 bp Not-BamHI frag with ALVAC as the rescuing virus. ment of pHIVGE15, containing the H6-promoted HTV1 envelope"gp120” gene (MN) and the I3L-promoted gag and EXAMPLE 48 pol genes (IB), into the 4,000 bp Not-BglII fragment of pSD542 (defined in Example 15). The plasmid generated by GENERATION OF HIV1 GENES IN NYWAC 35 this manipulation is called pHIVGE16. Generation of NYVAC/HIV1 gag (pro) and gp120 The “gp120" gene was then replaced by the "gp160" (+transmembrane) Recombinant. A plasmid, pHXB2D, con gene. This was accomplished by cloning the 2,600 bp taining human immunodeficiency virus type 1 (HIV1) NruI-Notifragment of pH6HMNE (defined in Example 10), cDNA sequence (IIIB) was obtained from Dr. R. C. Gallo containing the entire HIV envelope gene (MN), into the (NCI-NIH). The sequence encoding the 5'-end of the gag 8,000 bp partial NruI-Not fragment of pHIVGE16. The gene was cloned between vaccinia virus tk flanking arms. plasmid generated by this manipulation is called This was accomplished by cloning the 1.625 bp Bg pHTVGE19. fragment of pHXB2D, containing the 5'-end of the gag gene. The part of the env gene encoding gp41 was then replaced into the 4075 bp BglII fragment of pSD542VCVQ. The with the env transmembrane region. This was accomplished plasmid generated by this manipulation is called pHIVG2. 45 by cloning the 1,700 bp NruI-Not fragment of The 3'-end of the gag gene was then cloned into pHTVG2. pBSHIVMN120T. containing the H6-promoted gp120 This was accomplished by cloning a 280 bp Apal-BamHI (+transmembrane) gene, into the 8,500 bp partial NruI-Not PCR fragment, containing the 3'-end of the gag gene, into fragment of pHTVGE19. The resulting plasmid is called the 5,620 bp Apa-BamHI fragment of pHTVG2. (This PCR 50 pHIVGE19T. fragment was generated from the plasmid, pHXB2D, with Most of the pol gene was then removed. This was the oligonucleotides, HIVP5 (SEQID NO:116) and HIVP6 accomplished by cloning a 540 bp Apa-SmaI PCR (SEQID NO:117)). The plasmid generated by this manipu fragment, containing the 3'-end of the HTV1 protease lation is called pHIVG3. "gene". into the 7.000 bp ApaI-SmaI fragment of The 3L promoter was then cloned upstream of the gag 55 pHIVGE19T. (This PCR fragment was generated from the gene. This was accomplished by cloning the plasmid, pHIVG7, with the oligonucleotides, HTVP5 (SEQ oligonucleotides, HIVL17 (SEQ ID NO:118) and HIVL18 ID NO:116) and HIVP37 (SEQID NO:180)). This manipu (SEQID NO:119), encoding the vaccinia virus I3L promoter lation removes most of the pol gene, but leaves the protease and the 5'-end of the gag gene, into the the 5.540 bp partial "gene" intact. The plasmid generated by this manipulation is BglTI-Clafragment of pHIVG3. The plasmid generated by called pHIV33. this manipulation is called pHIVG4. pHIV33 is used in in vitro recombination experiments The portion of the gag gene encoding p24, p.2, p7 and p6 with NYWAC as the rescuing virus. was then eliminated. This was accomplished by cloning the EXAMPLE 49 oligonucleotides, HIVL19 (SEQ ID NO:120) and HIVL20 (SEQ ID NO:121), into the 4.450 bp partial Pvu-BamHI 65 GENERATION OF HTV2 GENES IN ALVAC fragment of pHIVG4. The plasmid generated by this Generation of ALVAC/HIV2 gag?pol Recombinant. A manipulation is called pHTVG5. plasmid, pBSH6HIV2ENV (defined in Example 4), contains 5,766,598 71 72 the H6-promoted human immunodeficiency virus type 2 The gag and poll genes were then removed. This was (HIV2) env gene. The H6-promoted env gene from this accomplished by cloning the oligonucleotides, HTV2L10 plasmid was cloned between canary pox flanking arms. This (SEQID NO:183) and HIV2L11 (SEQID NO:184), into the was accomplished by cloning the 2.700 bp XhoI-SacII 7,000 bp partial BamHI-SmaI fragment of pHIV25. The fragment of p3SH6HIV2ENV and the oligonucleotides. plasmid generated by this manipulation is called pHTV28. HIV2LA (SEQID NO:176) and HIV2L5 (SEQID NO:177). into the XhoI-EcoRI site of pC6L (defined in Example 50). The part of the env gene encoding gp41 was then The plasmid generated by this manipulation is called removed. This was accomplished by cloning the 360 bp pHIV23. PstI-Xbal fragment of p8SHTV2120B. containing the 3'-end The HIV2 gag and pol genes were then cloned into of the gp120 gene, and the oligonucleotides, HTV2L12 (SEQ pHIV23. This was accomplished by cloning the 4.450 bp O ID NO:185:5'-CTAGAAAACCGC-3') and HIV2L13 (SEQ XmaI-Not fragment of pHIV22, containing the I3L ID NO:186; 5'-GGTTTT-3"), into the 5,600 bp Pst-SstII promoted HTV2 gag and pol genes, and the oligonucleotides, fragment of pHIV28. The plasmid generated by this manipu HIV2L6 (SEQID NO:131) and HIV2L7 (SEQID NO:132). lation is called pHIV29. into the 7.000 bp XmaI-XhoI fragment of pHIV23. The pHIV29 is used in in vitro recombination experiments plasmid generated by this manipulation is called pHTV25. 15 with ALVAC as the rescuing virus. The env gene was then removed. This was accomplished Generation of ALVAC/HIV2 gag?pol and gp120 Recom by cloning the oligonucleotides, HIV2L8 (SEQID NO:181; binant. A plasmid, pBSH6HIV2ENV (defined in Example 5'-CGATAAACCGC-3") and HIV2L9 (SEQ ID NO:182; 4), contains the H6-promoted human immunodeficiency 5'-GGTTTAT-3"), into the 8,800 bp partial Cla-SstII frag virus type 2 (HTV2) env gene. The H6-promoted env gene ment of pHIV25. The plasmid generated by this manipula 20 from this plasmid was cloned between canary pox flanking tion is called pHTV27. arms. This was accomplished by cloning the 2,700 bp pHIV27 was used in in vitro recombination experiments XhoI-SacII fragment of pBSH6HIV2ENV and the with ALVAC as the rescuing virus to yield vCP190. oligonucleotides. HIV2LA (SEQ ID NO:176) and HIV2L5 Generation of ALVAC/HIV2 env (gp160) Recombinant. A (SEQ ID NO:177), into the XhoI-EcoRI site of pcGL plasmid, pFBSH6HIV2ENV (defined in Example 4), contains 25 (defined in Example 50). The plasmid generated by this the H6-promoted human immunodeficiency virus type 2 manipulation is called pHIV23. (HIV2) env gene. The H6-promoted env gene from this The HTV2 gag and pol genes were then cloned into plasmid was cloned between canary pox flanking arms. This pHTV23. This was accomplished by cloning the 4.450 bp was accomplished by cloning the 2,700 bp XhoI-SacII 30 Xma-Not fragment of pHTV22, containing the I3L fragment of p8SH6HIV2ENV and the oligonucleotides, promoted HTV2 gag and pol genes, and the oligonucleotides, HIV2LA (SEQID NO:176) and HTV2L5 (SEQLD NO:177). HIV2L6 (SEQID NO:131) and HIV2L7 (SEQID NO:132). into the XhoI-EcoRI site of pC6L (defined in Example 50). into the 7,000 bp Xma-XhoI fragment of pHTV23. The The plasmid generated by this manipulation is called plasmid generated by this manipulation is called pHTV25. pHIV23. 35 The gag and pol genes were then removed. This was The HIV2 gag and pol genes were then cloned into accomplished by cloning the oligonucleotides, HTV2L10 pHIV23. This was accomplished by cloning the 4.450 bp (SEQID NO:183) and HIV2L11 (SEQID NO:184), into the Xma-NotI fragment of pHIV22, containing the I3L 7,000 bp partial BamHI-SmaI fragment of pHTV25. The promoted HIV2 gag and pol genes, and the oligonucleotides, plasmid generated by this manipulation is called pHTV28. HIV2L6 (SEQID NO:131) and HIV2L7 (SEQID NO:132). 40 The part of the env gene encoding gp41 was then into the 7,000 bp Xma-XhoI fragment of pHIV23. The removed. This was accomplished by cloning the 360 bp plasmid generated by this manipulation is called pHTV25. Pst-Xbal fragment of pBSHTV2120B. containing the 3'-end The gag and poll genes were then removed. This was of the gp120 gene, and the oligonucleotides. HIV2L12 (SEQ accomplished by cloning the oligonucleotides, HTV2L10 ID NO:185) and HIV2L13 (SEQID NO:186), into the 5,600 (SEQ ID NO:183; 5'-GGGAAAG-3) and HIV2L11 (SEQ 45 bp Pst-SstII fragment of pHTV28. The plasmid generated by ID NO:184:5'-GATCCTTTCCC-3"), into the 7,000 bp par tial BamHI-SmaI fragment of pHIV25. The plasmid gener this manipulation is called pHTV29. ated by this manipulation is called pHIV28. The gp120 gene was then cloned into pHIV25. This was accomplished by cloning the 1.550 bp Nru-SacII fragment pHIV28 was used in in vitro recombination experiments of pHTV29, containing the H6-promoted gp120 gene. into with ALVAC as the rescuing virus to yield vCP188. 50 Generation of ALVAC/HIV2 gp120 Recombinant. A the 9,000 bpNirul-SacII fragment of pHIV25. The plasmid plasmid, pBSH6HTV2ENV. contains the H6-promoted generated by this manipulation is called pHIV30. human immunodeficiency virus type 2 (HIV2) env gene. pHTV30 is used in in vitro recombination experiments The H6-promoted env gene from this plasmid was cloned with ALVAC as the rescuing virus. between canary pox flanking arms. This was accomplished 55 Generation of ALVAC/HIV2 gp120 (+transmembrane) by cloning the 2,700 bp XhoI-SacII fragment of Recombinant. A plasmid, p3SH6HIV2ENV (defined in pBSH6HIV2ENV (defined in Example 4) and the Example 4), contains the H6-promoted human immunode oligonucleotides. HIV2LA (SEQID NO:176) and HIV2L5 ficiency virus type 2 (HTV2) env gene. The H6-promoted (SEQID NO:177), into the XhoI-EcoRI site of pC6L. The envgene from this plasmid was cloned between canary pox. plasmid generated by this manipulation is called pHIV23. flanking arms. This was accomplished by cloning the 2,700 The HIV2 gag and pol genes were then cloned into bp XhoI-SacII fragment of p8SH6HIV2ENV and the pHIV23. This was accomplished by cloning the 4.450 bp oligonucleotides. HTV2LA (SEQED NO:176) and HIV2L5 Xma-Not fragment of pHIV22, containing the I3L (SEQ ID NO:177), into the XhoI-EcoRI site of pC6L promoted HIV2 gag and pol genes, and the oligonucleotides, (defined in Example 50). The plasmid generated by this HTV2L6 (SEQID NO:131) and HIV2L7 (SEQID NO:132). 65 manipulation is called pHIV23. into the 7,000 bp XmaI-XhoI fragment of pHIV23. The The HIV2 gag and poll genes were then cloned into plasmid generated by this manipulation is called pHIV25. pHIV23. This was accomplished by cloning the 4450 bp 5,766,598 73 74 Xma-Not fragment of pHIV22, containing the 3L HIV2L13 (SEQ ID NO:186), into the 5,600 bp Pst-SstII promoted HIV2 gag and pol genes, and the oligonucleotides, fragment of pHIV28. The plasmid generated by this manipu HTV2L6 (SEQID NO:131) and HIV2L7 (SEQED NO:132), lation is called pHIV29. into the 7,000 bp Xma-XhoI fragment of pHTV23. The The HIV1 env transmembrane region was then cloned plasmid generated by this manipulation is called pHTV25. onto the end of the gp120 gene. This was accomplished by The gag and pol genes were then removed. This was cloning the 500 bp EcoRI fragment of pHIV31, containing accomplished by cloning the oligonucleotides, HIV2L10 the 3'-end of the gp120 gene and the env transmembrane (SEQID NO:183) and HTV2L11 (SEQID NO:184), into the region, into the 5,600 bp EcoRI fragment of pHIV29. 7,000 bp partial BamHI-SmaI fragment of pHIV25. The pHIV31 was derived by cloning a 500 bp PstI-Xbal PCR plasmid generated by this manipulation is called pHIV28. fragment, containing the 3'-end of the gp120 gene and the The part of the env gene encoding gp41 was then env transmembrane region, into the Pst-Xbal site of pIBI25 removed. This was accomplished by cloning the 360 bp (IBI. New Haven. Conn.). This PCR fragment was generated Pst-Xba fragment of p8SHIV2120B, containing the 3'-end from the PCR fragment. PCRTM1, and the oligonucleotides, of the gp120 gene, and the oligonucleotides. HIV2L12 (SEQ HIVTM1 (SEQ ID NO:107) and HIVTM2 (SEQ ID ID NO:185) and HIV2L13 (SEQID NO:186). into the 5,600 15 NO:108), with the oligonucleotides. HIV2P14 (SEQ ID bp Pst-Sst Ifragment of pHIV28. The plasmid generated by NO:187) and HIVTM3 (SEQ ID NO:109). PCRTM1 was this manipulation is called pHIV29. generated from the plasmid, pHIV28, with the The HIV1 env transmembrane region was then cloned oligonucleotides. HIV2P14 (SEQID NO:187) and HTV2P15 onto the end of the gp120 gene. This was accomplished by (SEQID NO:188). The plasmid generated by this manipu cloning the 500 bp EcoRI fragment of pHIV31, containing lation is called pHIV34. the 3'-end of the gp120 gene and the env transmembrane The gp120 (+ transmembrane) gene was then cloned into region, into the 5,600 bp EcoRI fragment of pHIV29. a plasmid containing the HTV2 gag?pol genes. This was (pHTV31 was derived by cloning a 500 bp PstI-Xbal PCR accomplished by cloning the 2,700 bp Clal fragment of fragment, containing the 3'-end of the gp120 gene and the pHIV34, containing the H6-promoted gp120 env transmembrane region, into the PstI-Xbal site of pBI25. 25 (+transmembrane) gene, into the 6,800 bp Clafragment of This PCR fragment was generated from the PCR fragment, pHIV30. (pHIV30 was derived by cloning the 1.550 bp PCRTM1, and the oligonucleotides, HIVTM1 (SEQ ID NruI-SacII fragment of pHIV29, containing the NO:107) and HIVTM2 (SEQ ID NO:108), with the H6-promoted gp120 gene, into the 9,000 bp NruI-SacII oligonucleotides. HIV2P14 (SEQ ID NO: 187; fragment of pHIV25.) The plasmid generated by this 5'-CAGAAGTAGCATATATGT-3') and HIVTM3 (SEQ ID 30 manipulation is called pHIV35. NO:109). PCRTM1 was generated from the plasmid, pHIV35 is used in in vitro recombination experiments pHTV28, with the oligonucleotides, HTV2P14 (SEQ ID with ALVAC as the rescuing virus. NO:187) and HIV 2P15 (SEQ ID NO: 188: 5'-GCCTCCTACTATCATTATGAATAATCTCTTATGTC EXAMPLE 50 TCCCTGGAGC-3')). The plasmid generated by this 35 manipulation is called pHTV34. EXPRESSION OF TWO FUSION PEPTDES pHIV34 is used in in vitro recombination experiments CONTAINING THE p24 EPITOPE OF HIV-1 gag with ALVAC as the rescuing virus. FUSED TO THE T AND V3 Generation of ALVAC/HIV2 gag?pol and gp120 (+ transmembrane) Recombinant. A plasmid. LOOPEPTOPES OF HIV-1 env WTTH AND pBSH6HIV2ENV (defined in Example 4), contains the WTTHOUT THE SIGNAL DOMAN FROM HIV-1 H6-promoted human immunodeficiency virus type 2 (HIV2) W envgene. The H6-promoted env gene from this plasmid was Two expression cassettes were generated by a series of cloned between canary pox flanking arms. This was accom 45 polymerase chain reactions described below. These cassettes plished by cloning the 2.700 bp XhoI-SacI fragment of differ in that one version encodes the signal sequences of pBSH6HIV2ENV and the oligonucleotides, HIV2LA (SEQ HIV-1 env fused to the epitopes whereas the other does not. ID NO:176) and HTV2L5 (SEQ ID NO:177), into the The version of the fusion peptide with the signal is XhoI-EcoRI site of pC6L (defined in Example 50). The preceded by the 51 amino acid N-terminal portion of HIV-1 plasmid generated by this manipulation is called pHTV23. 50 (IB) env, residues 1-50 (plus initiating Met) based on The HIV2 gag and pol genes were then cloned into Ratner et al. (1985) followed by a cleavable linker region. pHTV23. This was accomplished by cloning the 4.450 bp The amino acid sequence of this region is (SEQID NO:189) Xma-Not fragment of pHIV22, containing the I3L MKEQKTVAMRVKEKYQHLWRWGWRWGTMLLGM promoted HTV2 gag and pol genes, and the oligonucleotides. LMCSATEKLWWTVYYGVP-PFRK. Both versions of the HTV2L6 (SEQID NO:131) and HIV2L7 (SEQID NO:132), 55 fusion peptide, contain an amino acid sequence based on the into the 7.000 bp Xma-XhoI fragment of pHIV23. The defined T-cell epitopes of p24, the V3 loop (MN), and T1 of plasmid generated by this manipulation is called pHIV25. HIV-1 (MN) env. The peptide is designed such that the The gag and poll genes were then removed. This was epitopes are separated from each other and the signal where accomplished by cloning the oligonucleotides. HIV2L10 present by the sequence (SEQ ID NO:189) PPFRK. The (SEQID NO:183) and HIV2L11 (SEQID NO:184), into the sequence of this region of the peptide is (SEQD NO:190) 7.000 bp partial BamHI-SmaI fragment of pHIV25. The signal-PFRK-GPKEPFRDYVDRFYK-PPFRK plasmid generated by this manipulation is called pHIV28. VQINCTRPNYNKRKRIHIGPGRAFYTTKNIIGTIRQAH The part of the envgene encoding gp41 was then removed. CNISRAK-PPFR-KQINMWQEVEKAMYA. In the ver This was accomplished by cloning the 360 bp PstI-Xbal sion lacking the signal sequence, the region indicated by fragment of pBSHIV2120B (defined in Example 4), con 65 signal-PFRK is replaced by an initiating methionine only. taining the 3'-end of the gp120 gene, and the For the cassette with the signal, the H6 promoter and oligonucleotides, HIV2L12 (SEQ ID NO:185) and signal were derived by PCR from plasmid p3ST1T2TH4.1 5,766,598 75 76 (described above) using primers H6PCR1 (SEQID NO:157; 239 bp PCR-derived fragments using primers H6PCR1 5'- ACTACTAAGCTTCTTTATTCTATACTTAAAAAGTG (SEQ ID NO:157) and T1MNPCR (SEQ ID NO:200). -3") and SIGPCR 24 (SEQ ID NO: 191; Following digestion of this 418 bp PCR-derived fragment 5'-AGGTCCCTTCCTGAATGGAGGTACCCCATAATAG with HindIII, a fragment of 400 bp was isolated from an ACTG-3"). The p24 epitope was fused to the signal sequence agarose gel, ligated to similarly digested p3S (Stratagene, by PCR amplification of this 307 bp PCR-derived fragment La Jolla, Calif.) creating plasmid pMN24EV3T1NSA. The and oligonucleotides P24A (SEQ ID NO: 192; insert was verified by nucleotide sequence analysis. 5'-CCATTCAGGAAGGGACCTAAAGAACCTTTTAGA A C6 insertion vector containing 370 bp upstream of C6. GATTATGTAGATAGATTTTATAAACCACCTTT polylinker containing SmaI, Pst, XhoI, and EcoRI sites, and TAGAAAA-3") and P24B (SEQ ID NO: 193; O 5'-TTTTCTAAAAGGTGGTTTATAAAATCTATCTACA 1156 bp of downstream sequence was derived in the fol TAAT CTCTAAAAGGTTCTTTAGGTCCCTTCC lowing manner. The 0.4 kb upstream sequence was gener TGAATGG-3') using primers H6PCR1 and P24PCR (SEQ ated by PCR amplification of a cosmid clone derived from LD NO:194; 5'-GTACAATTAATTTGTACTTTTCTAAAA purified genomic canarypox DNA using oligonucleotides GGTGGTTTATAAAATC-3'). This 377 bp PCR-derived C6A1SG (SEQ ID NO:201; 5'- ATCATCGAGCTCGCG fragment consists of the H6 promoter coupled to coding 15 GCCGCCTATCAAAAGTCTTAATGAGTT -3") and C6B1SG (SEQ ID NO:202; 5'- GAATTCCTCGAGCTG sequences for the signal, the p24 epitope and the first 6 CA G C C C G GGTTTTTATA G CTA ATTA GT amino acids of the V3 loop. CATTTTTTCGTAAGTAAGTATTTTTATTTAA-3"). The For the cassette without the signal, the H6 promoter were 1.2 kb downstream arm was generated by PCR amplification derived by PCR from plasmid pH6T2 (described above) of the same template using oligonucleotides C6C1SG (SEQ using primers H6PCR1 (SEQID NO:157) and H6P24 (SEQ ID NO:203; 5'- CCCGGGCTGCAGCTCGAGGAAT ID NO: 195: 5'-GGTGGTTTATAAAATCTATCT ACAT. T CTTTTT ATT G AT TAA CTA GT CAAAT AATCT CTAAAAG GTTCTTTAG GT C C CAT GAGTATATATAATTGAAAAAGTAA -3") and C6D1SG TACGATACAAACTTAACGG-3"). The p24 epitope was (SEQ ID NO:204; 5'- GATGATGGTACCTTCATAAATA fused to the promoter by PCR amplification of this 187 bp 25 CAAGTTTGATTAAACTTAAGTTG-3'). These fragments PCR-derived fragment and oligonucleotides P24A (SEQID were fused by a third PCR employing gel purified 0.4 and NO:192) and P24B (SEQ ID NO:193) using primers 1.2 kb fragments as template for primers C6A1SG (SEQID H6PCR1 (SEQ ID NO:157) and P24PCR (SEQ ID NO:201) and C6D1SG (SEQID NO:204). The resulting 1.6 NO:194). This 214 bp PCR-derived fragment consists of the kb fragment was isolated from an agarose gel, digested with H6 promoter coupled to coding sequences for the p24 30 epitope and the first 6 amino acids of the V3 loop. SacI and Kpn and ligated to similarly digested pBS Coding sequences for the V3 loop region were derived by (Stratagene, La Jolla, Calif.) generating C6 insertion plas PCR amplification of the SmaI fragment from plasmid mid pCOL. Both expression cassettes were excised by Pst/XhoI pHIVGE16EV (described above) using primers V3PCR1 digestion of pMNT1P24 and pMN24EV3T1NSA. isolated (SEQ ID NO: 196: 35 5'-AAACCACCTTTTAGAAAAGTACAAATTAA from agarose gels and ligated separately to similarly TTGTAC-3') and V3PCR2 (SEQ ID NO: 197; digested pC6L creating plasmids pC6P24FS and 5'-CTGCTTACGGAACGGTGGTTTTGCTCTACTAAT pC6P24FNS respectively, for recombination into the C6 GTTACAATG-3'). The T1 epitope was joined to the coding locus of ALWAC. The resulting recombinants are designated region for the V3 loop by PCR amplification of this 171 bp vCP189 and vCP195, respectively. PCR-derived fragment and oligonucleotides MNT1A (SEQ BamHI/XhoI fragments from pMNT1P24 and ID NO: 198: pMN24EW3T1NSA were ligated to similarly digested 5'- CCACCGTTCCGTAAG CAGATAATAAA C pSD550VC (defined in Example 33) creating pI4P24FS and ATGTGGCAAGAAGTAGAAAAAGCTATGTATGCTT pI4P24FNS for recombination into the I4 locus of NYVAC AA-3") and MNT 1B (SEQ ID NO: 199; 45 generating vP1117 and vP1110, respectively. 5'-TTAAGCATACATAGCTTTTTCTACTTCTTGCCAC Expression of Tetanus Toxin Fragment C in Poxviruses ATGTTTATTATCTGCTTACGGAACGGTGG-3") as tem Expressing HTV-1 Proteins. It has been proposed that the plate using primers V3PCR1 (SEQ ID NO:196) and addition of various Th epitopes from homologous (Good et T 1 MNPCR (SEQ D NO:200; al., 1987) and heterologous proteins (Francis et al., 1987) 5'-TCATCAAAGCTTCTCGAGAAAAATTAAGCATAC 50 may be capable of recruiting T-cell help for specific B cell ATAGCTTTTTC-3). This 239 bp PCR-derived fragment responses to synthetic peptide vaccines. In an effort to elicit consists of the last codon of p24 epitope, the V3 loop, ant the enhanced immune responses to HIV-1 antigens, various T1 epitope followed 3' by an early transcription termination T-cell epitopes derived from HIV-1 have been incorporated signal (TTTTTNT) and XhoI and HindIII sites. into recombinant poxviruses. To further pursue this strategy, For the cassette with the signal, the promoter, signal and 55 tetanus toxin fragment C, which contains other known p24 epitope coding sequences were joined to the coding human T-helper cell epitopes (Ho et al., 1990), will be region for the V3 loop/T1 epitope by PCR amplification of co-expressed with HIV-1 antigens in an ALVAC recombi the 377 bp and 239bp PCR-derived fragments using primers nant. The presence of these epitopes from tetanus toxin may H6PCR1 (SEQ ID NO:157) and T1MNPCR (SEQ ID enhance the immune response against HTV-1 by providing NO:200). Following digestion of this 581 bp. PCR-derived 60 nonspecific T-cell help. fragment with HindIII, a fragment of 563 bp was isolated Pst/SmaI digested pCGP24FS (described above) was from an agarose gel, ligated to similarly digested pBS modified by ligating the Pst/SmaI fragment from plasmid (Stratagene, La Jolla. Calif.) creating plasmid pMNT1P24. pVQ42KTH4.1 (described above) creating pC6P24FSVQ. The insert was verified by nucleotide sequence analysis. This plasmid was digested with Niru within the H6 promoter For the cassette without the signal, the promoter and p24 65 and XhoI at the 3' end and ligated to a 1.4 kb fragment coding sequences were joined to the coding region for the isolated from similarly digested pH6TETC (described V3 loop/T1 epitope by PCR amplification of the 171 bp and above). The resulting plasmid, pC6VQTETC. was con 5,766,598 77 78 firmed by restriction digestion and nucleotide sequence lated with the fully replication competent vPE16 infected analysis of the regions surrounding the cloning sites. Fol PBMC. It is not known whether this apparent enhancement lowing confirmation pC6VQTETC was employed in recom in CTL stimulation by NYVAC/gp160 and ALVAC/gp160 bination experiments with vCP112, vCP125 and vCP156. was due to differences in expression by the recombinants as compared to vpE16 or to the attenuation characteristics of EXAMPLE 51 the NYWAC and ALWAC vectors. PERIPHERAL BLOOD MONONUCLEAR CELL Extensive flow cytometric analyses of the various stimu STUDIES WITH NY WAC/HIV-1 AND ALVAC lated cultures was also performed in order to more thor HIV-1 RECOMBINANTS oughly identify the effector cell phenotype which was elic O ited. When compared to either unstimulated or control While broad issues concerning the immunogenicity of vector stimulated cultures, generation of anti-gp160 NYVAC/HIV-1 and ALWAC/HIV-1 constructs in man are cytolytic reactivities generally resulted in a decline in CD3 best addressed within the context of clinical trials, a number fCD4" subpopulations and a compensatory rise in CD3"| of relevant insights have already been gleaned by in vitro CD8" cells. Significant increases in CD3/CD25". CD3"/ cellular response assays. A central question regarding the 15 HLA-DR, CD8/S6F1, CD-8/CD38, and CD3/CD69 use of these vaccine constructs in man is their capacity to cells were also noted. When the cytolytic activities of each impact on cellular anti-HIV-1 reactivities, especially cyto culture were plotted against increases in particular cell toxic T-lymphocytes (CTL) responses. Whether as a com subpopulations, a linear relationship was found between ponent of a preventative or a therapeutic vaccine strategy, cellular anti-gp160 reactivity and increases in the CD8"f there are ample precedents suggesting a beneficial role for S6F1" population. This cellular phenotype broadly defines CTL in a number of viral infections in man (McMichael et al., 1983; Moss et al., 1978; Borysiewicz et al., 1988). CTL (Morinoto et al., 1987). Although the precise contribution of anti-HIV-1 CTL in These early pre-clinical studies with cells from HIV-1 preventing or controlling viral infection remains to be infected patients clearly illustrate the capacity of NYWAC elucidated, studies by Letvin and co-workers using the 25 and ALVAC/HIV-1 vectors to elicit potent CTL activities SIV/macaque animal model suggest that anti-STV-1 CTL. from precursor populations which are contained within the especially gag-specific CTL, may represent a major deter PBMC pool of patients. Furthermore, these studies strongly minant controlling disease progression (Letwin and King, exemplify the potential clinical utility of these vectors in the 1990). This coupled with the observations of numerous context of a therapeutic vaccine strategy. This could take the investigators that anti-HIV-1 CTL activities can be measured 30 form of simple immunization with these vectors or could directly from fresh peripheral blood mononuclear cells involve an ex vivo component of either cellular targeting and (PBMC) in a relatively high proportion of asymptomatic re-infusion or ex vivo CTL generation followed by large scale adoptive transfer. In either case. the combined safety of patients (reviewed in Walker and Plata, 1990; Autran et al. the non-replicating vectors and their inherently strong cel 1991), support the contention that elicitation of anti-HIV-1 lular immunogenicity make them ideal candidates for CTL reactivities should be included as a major goal of both 35 preventative and therapeutic vaccines. immune-based therapy in man. To test the capacity of NYVAC/HIV-1 and ALVAC/HIV-1 Having thus described in detail preferred embodiments of constructs to impact on relevant CTL reactivities, a series of the present invention, it is to be understood that the inven experiments were conducted involving a cohort of HIV-1 tion defined by the appended claims is not to be limited to infected patients devoid of detectable direct anti-HIV-1 the particular details set forth in the above description as gp160 CTL reactivities. PBMCs were obtained from these many apparent variations thereof are possible without patients and a portion of the PBMC were infected with either departing from the spirit or scope of the present invention. the fully replication-competent vaccinia/gp 160 construct vPE16 (Walker et al., 1987), or the replication-attenuated REFERENCES NYVAC/gp160 or ALVAC/gp160 constructs. The acutely 45 1. Autran, B., Plata, F. and Debre, P., J. AIDS, 4, 361-367 infected PBMC were washed and used as stimulators for the (1991) remaining PBMC in a 10-day in vitro stimulation protocol. 2. Behbehani. A. M. Microbiological Reviews 47.455-509 Controls included both unstimulated and control vector (i.e. (1983). parental poxvirus minus HIV-1 genes) stimulated cultures. 3. Bertholet, C. Drillien, R. and Wittek, R. Proc. Natl. Following the 10-day incubation in the absence of exog Acad. Sci. 82. 2096-2100 (1985). enous IL-2, cells were washed and evaluated for CTL 4. Borysiewicz, I., Graham, S. 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SEQUENCE LISTING

( 1) GENERAL INFORMATION: ( i i i y NUMBER OF SEQUENCES: 205

( 2) INFORMATION FOR SEQED NO:1: ( i SEQUENCE CHARACTERISTICs: ( A ) LENGTH: 20 base pairs ( B TYPE: nucleic acid ( C ), STRANDEDNESS: single (D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID No:1:

TAATAA CA C C A C C C C G G 2

( 2) INFORMATION FOR SEQID No:2: ( i SEQUENCE CHARACTERISTICs: ( A LENGTH: 28 base pairs (By TYPE: nucleic acid ( C ), STRANDEDNESS: single D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQ D NO:2:

AA T C C C C G G T A CTATA A TA CAT 5,766,598 83 84 -continued ( 2) INFORMATION FOR SEQID NO:3: ( ; ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 73 base pairs (B) TYPE: mucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:3: A G CT T C C C G G G T A A GAATA C GT CAA. G. G. A. G AAAAC GAA. A. C. GAT C T G T A GT TAG C G G C C GC 6 0

CTA AT TAA CT AAT 7 3

( 2) INFORMATION FOR SEQID NO:4: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 69 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single { D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:4:

A TA GT TAAT AG G C G GCC G CTA A CTA CAG AT C G T T C GT TTT C T C C T T G A C G T AT TACT 6 0.

A C C C G G GA 69

( 2) INFORMATION FOR SEQID NO:5: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: mucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQ ID NO:5:

T TA GT TAATT AG G C G GCC GC 2 O

( 2) INFORMATION FOR SEQID NO:6: ( i ) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 22 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:6:

CGATT ACT A T GAA. G. GAT C C G TT 22

( 2) INFORMATION FOR SEQID No.:7; ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ). SEQUENCE DESCRIPTION: SEQID No.:7;

AA CG GA. T C C T T CAT AG TAAT 20

( 2) INFORMATION FOR SEQID NO:8: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 41 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ). SEQUENCE DESCRIPTION: SEQID NO:3:

CGATT ACT AG AT C T G A G C T C C C C G G G C T C G A G G GAT C C GT T 4 5,766,598 85 86 -continued

( 2) INFORMATION FOR SEQ ID NO:9: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 39 base pairs B ) TYPE: nucleic acid ( C ) STRANDEDNESS: single { D TOPOLOGY: inear ( x i ) SEQUENCE DESCRIPTION: SEQED NO:9:

AA CGG AT C C C T C GAGCC CGG G GA, GCT CAA CTA GTA A 39

( 2) INFORMATION FOR SEQID NO:10: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 16 base pairs B TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ). SEQUENCE DESCRIPTION: SEQID NO:10:

A T C CAA. T. C. A. GC

2 INFORMATION FOR SEQID NO:11: i y SEQUENCE CHARACTERISTICS: (A) LENGH: 12 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID No:11;

AGC A GAA. T C G

( 2) INFORMATION FOR SEQID NO:12: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 75 base pairs ( B. ) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQD NO:12:

TA T G A GT AAC TAA CTC TTT TTAA AA AA GTA A T C AAAAAAAAG TAA AAA 6

A GA CT GAA C GT 7 5

( 2) INFORMATION FOR SEQID No:13: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 73 base pairs B TYPE: nucleic acid ( C ) STRANDEDNESS: single : ( D TOPOLOGY: Einear ( x i SEQUENCE DESCRIPTION: SEQID NO:13:

AAC GAA. T C A GAT CATT A TATA Act TAT TTTTGAAA ACT TAAT AA CAAA. A. GA 60

GT TAAG TA C T CA 7 3

( 2) INFORMATION FOR SEQID NO:14: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 49 base pairs ( B TYPE: nucleic acid ( Cy STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQIO NO:14: 5,766,598 87 -continued

AAAATCG G C G T G A T G T A A C TT TATA A A CTTATTTT GAATAAC 49

( 2) INFORMATION FOR SEQID No:15: ( ; ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 67 base pairs (B) TYPE: nucleic acid C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:15: A CAC GAA T G A TTT CT AAA G T A TTT G GAAA TTT ATA G O A GT TGAT AG AACAA AA TAC 6

A TAATTT 6

( 2) INFORMATION FOR SEQID NO:16: i SEQUENCE CHARACTERISTICS: A LENGTH:51 base pairs B TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:16: CTAT CAA CT ACC AT AAAA CTTC CAAAT A CTTAGAAA, ATCAT I C G T G T 5 1

( 2) INFORMAION FOR SEQID NO:17: ( i SEQUENCE CHARACTERISTICS: A ) LENGTH: 46 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQDD NO:17:

GAAAAAA AA CA CT TT TA TA CTA AGA C T C C C C C C T G CA. GC 4 6

( 2 INFORMATION FOR SEQID NO:18: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 66 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear x i y SEQUENCE DESCRIPTION: SEQID NO:18: GGC CGCT is CA, G C C C C GA, GA. T C TA GTATA AAAAG AT A TACA. AAA A T G T A 6 O

TTT GT 6 6

( 2 INFORMATION FOR SEQID NO:19: ( i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 50 base pairs ( B, ) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:19: TT T. CT G TATA TT GCAC CAA TTTAGAT CTT A CT CAA AATA T G T AACAA A 5 O

( 2 INFORMATION FOR SEQID NO:20; ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 44 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D TOPOLOGY: linear 5.766,598 89 90 -continued

( x i ) SEQUENCE DESCRIPTION: SEQED NO:20:

GT CAT T TAA CA CTATA, C T C A TAT AATAA AAATAA TAT TA T 4 4

( 2) INFORMATION FOR SEQ ID NO:21: ( i SEQUENCE CHARACTERISTICs: ( A LENGTH: 72 base pairs { B TYPE: nucleic acid ( C STRANDEDNESS: single { D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:21:

GAT C CT GA GT ACT T G T AAT ATA A GAA. T A AT TT CAC T TA CT CA TTGACAATAA 6

AAA GA. T. CT TA GG 2

( 2) INFORMATION FOR SEQED NO:22: ( i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 72 base pairs ( B, TYPE: nucleic acid ( C STRANDEDNESS: single (D TOPOLOGY: inear ( x i SEQUENCE DESCRIPTION: SEQID NO:22:

AATT C CTA AG. A.T. CT TTA. T C T CAAATGA GATAAA GT CA. AAA. TATATA CAT TATA A 5 O

CAAA G A CTC AG 2

( 2) INFORMATION FOR SEQID NO:23: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 72 base pairs ( B ) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D ). TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:23:

GAT C CAGA C T C C C G G GAAA AA AA TATT. AACTTT CAT TAAT AG G GAT TTGAC GTATG 6 O

TAG CT ACT A G G 7

( 2 INFORMATION FOR SEQ to No:24: ( i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 72 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single { D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:24:

AATT C CTA GT A C GCA CATA CGT CAAAT C C CTAT TAATGA AAA GT AAA AA TT TTC 6

CCG G GAGAT C T G. T 2

( 2) INFORMATION FOR SEQID NO:25: i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:25:

A T G G TAGAAA. TAA TT GAC O

2 INFORMATION FOR SEQID NO:26: 5,766.598 91 92 -continued

SEQUENCE CHARACTERISTICS: ( A LENGTH: 42 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear x SEQUENCE DESCRIPTION: SEQID NO:26:

AT CAT CAAT TCA A G C T TAT TATTTT G CT C TA CTAA T G T T AC 4

( 2 INFORMATION FOR SEQID NO:27: SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 60 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: singie ( D TOPOLOGY: inear ( x i SEQUENCE DESCRIPTION: SEQED NO:27: A GAA T G GA, C A GAA AAT T TAA, CA T G T G G AAA AA T G T A G AAA TTA. A TT G T ACA A GA CCC 5 O

( 2) INFORMATION FOR SEQID NO:28: i SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 60 base pairs ( B, ) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:28:

GGG CT GTA CAAT AAT CA CAT IT C CA CAT G T A A. AATTTT C G T CA CAT CAT 6 O

( 2 INFORMATION FOR SEQID NO:29: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 24 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single (D) TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID NO:29:

Act AA T G T A CAGAAAAIT TAAC 2 4

( 2 INFORMATION FOR SEQID NO:30: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 31 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQED NO:30: A G. G. CAA G CT CAAAAAAAT ATA AA T G A TT 3

( 2 INFORMATION FOR SEQID NO:31: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 24 base pairs ( B TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:31:

TT ATA T G T AATTA ATA TTT C 2 4

( 2 INFORMATION FOR SEQID NO:32: ( i SEQUENCE CHARACTERISTICS: 5,766,598 93 94 -continued ( A LENGTH: 37 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:32:

GT AATT T G G A G GAA TT CTT C T A C T G T AAT C 37

2 INFORMATION FOR SEQID NO:33: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 37 base pairs B ) TYPE: nucleic acid CSTRANDEDNESS: single D ) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:33:

AT CAT C T C A GAATAAAAAT TATA G CAAAA. T C CTTC 3 7

( 2) INFORMATION FOR SEQID NO:34: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 18 base pairs ( B, ) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: Einear ( x i SEQUENCE DESCRIPTION: SEQID NO:34:

C C T A C T C C AAT G TT C 8

( 2) INFORMATION FOR SEQID NO:35: { i SEQUENCE CHARACTERISTICs: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:35:

CATA T G CT T A G CA CT A. G. 2

2 INFORMATION FOR SEQID NO:36: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 21 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:36:

A T GAA A GA C C A GAA GACA GT G 1

( 2 INFORMATION FOR SEQID NO:37: i SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: mucleic acid C ) STRANDEDNESS: single D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:37:

A CAT C C G T A C C GATT CTTT ATC ATAC 9

(2) INFORMATION FOR SEQID NO:38: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 19 base pairs ( B. TYPE: nucleic acid 5,766,598 95 96 -continued ( C ) STRANDEDNESS: single (D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:38:

TAC GAA CAA ACT A. A C G G 19

( 2) INFORMATION FOR SEQID NO:39: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: mucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:39:

GAAT ACA GT A GAA GAA. T C C C C C CA CAA TT AAA. A. C. 3

( 2 INFORMATION FOR SEQID NO:40: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 19 base pairs B ) TYPE: nucleic acid ( C ), SRANDEDNESS: single (D) TOPOOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:40:

CAA TAGA AA T GATA CAC 1 9

( 2) INFORMATION FORSEQ ID NO:41: ( i SEQUENCE CHARACTERISTICs: (A) LENGTH: 34 base pairs B ) TYPE: nucleic acid ( Cy STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ). SEQUENCE DESCRIPTION: SEQID NO:41:

GTATT ATA. T C AA GT TT ATA AATAA T G CA. T A C 3 4

( 2) INFORMATION FOR SEQID NO:42: ( i ) SEQUENCE CHARACTERISTICS: A LENGTH: 18 base pairs ( B. ) TYPE: nucleic acid (C) STRANDEDNESS: singie (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID No:42:

GT GAT GA, C T G T A G T G C 8

( 2) INFORMATION FOR SEQID NO:43: ( ; ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 58 base pairs ( 8 TYPE: nucleic acid ( Cy STRANDEDNESS: singie ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQDD NO:43:

AT CAT C C T A GAATAAAAAT TA T G TT CAA TT TA CAC TTT TAA TA ATA C 58

2 INFORMATION FOR SEQ ID NO:44: ( i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 23 base pairs ( B. TYPE: nucleic acid ( C STRANDEDNESS: single { D TOPOLOGY: linear 5,766,598 97 98 -continued

x i ) SEQUENCE DESCRIPTION: SEQED NO:44:

CAA AA T C T TAAG CAAA C C T C

( 2 INFORMATION FOR SEQID NO:45: SEQUENCE CHARACTERISTICS: ( A LENGTH: 27 base pairs (B) TYPE: nucleic acid C ) STRANDEDNESS: single O TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:45:

A GA. GGG GAA T T C T C T A CTG CAA TACA 7

2 INFORMATION FOR SEQID No:46: ( i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 17 amino acids (B) TYPE: amino acid ( C STRANDEDNESS: single (D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:46: e u G E in A la A rig Wa L. eu Al a Wa G 1 u Arg Ty r L. eu. A rig A s p G 1 in G 1 in 1. 5 1 0 5

u.

2 INFORMATION FOR SEQID NO:47: ( . ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 29 base pairs ( B TYPE: mucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:47:

TT G CAAA. G. G. C T T G G CAT CCA C C C GT C a 9

( 2) INFORMATION FOR SEQID NO:48: ( i SEQUENCE CHARACTERISTICS: ( A ENGH: 42 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D.) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:48:

A CA. G. C. G. G. G. GCA CAA G CA G CTA G G GATT T G GT T C C CT 4 2

( 2) INFORMATION FOR SEQ Id NO:49: { i SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid ( C ) STRANOEDNESS: single D ) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQI No:49:

CCG T AA GT T G T A T C GAA GAAAG GAA GCAC CA. GC 4

2 INFORMATION FOR SEQID NO:50: i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 54 base pairs (BTYPE: nucleic acid ( C STRANDEDNESS: single 5,766.598 99 100 -continued ( D ). TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:50: A G A G T G GTA AAA TT CA G C T G CT G T GCC TT C T G CTAA CTA G T G C T G C. A 5 4

( 2 INFORMATION FOR SEQIE NO:51: i SEQUENCE CHARACTERISTICS: ( A LENGTH: 54 base pairs (B) TYPE: Eucleic acid ( C ), STRANDEDNESS: single D ) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:51:

TAAG CAA G CA CTA GT A G CA GAA. A. GGC AAC AA G CAG CGA. A TT AC CAC T CAT 5 4

2 ) INFORMATION FOR SEQID NO:52: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 29 base pairs (B) TYPE; nucleic acid C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:52: A CAT CAA, GC GAT CT A TT CAT AC 2 9

( 2) INFORMATION FOR SEQID NO:53: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs ( B. ) TYPE: nucleic acid (C) STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:53:

CA. G. C. GAA. T. TAC CA CT CA TAC GATA CA AACT TAACG 3 9

( 2 INFORMATION FOR SEQID NO:54: i SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs ( B. ) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:54:

TAAG CAA G CA CTA GT TAG

( 2 INFORMATION FOR SEQID NO:55: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: mucleic acid ( C ) STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:55:

CCG C C T C T G A C CAGAC

( 2) INFORMATION FOR SEQ ID NO:56: { i SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 39 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear 5,766,598 101 102 -continued ( x i ) SEQUENCE DESCRIPTION: SEQID NO:56: A CAT C T C T A GAATAAA AAT TA CAG GA. GGG CAAT CT G 3 9

( 2) INFORMATION FOR SEQID NO:57: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:57:

A T CAT C T C T A GAA AAAAAT TAT C T C T TA T G T C T C C C T G G 40

( 2 INFORMATION FOR SEQID NO:58: i y SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS; single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:58:

AAT AACT TT ACA G CA C C 8

( 2) INFORMATION FOR SEQID NO:59: { i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 44 base pairs B TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:59: CGA TAT C C GT TAA GT T T G T A T C G T AA T G GG AT G T CTTGG G AATC 4 4

( 2) INFORMATION FOR SEQID NO:60: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:60:

CAAG GCT TTA TT GAG G T C T C 2 0.

( 2) INFORMATION FOR SEQID No.61: ( i SEQUENCE CHARACTERISTICs: (A) LENGTH: 18 base pairs (B) TYPE: mucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQ Id No.61:

C CTG GCCTT G G CAGA TAG 1 8

( 2) INFORMATION FOR SEQID NO:62: { i ) SEQUENCE CHARACTERISTICS: A LENGTH: 58 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID No:62: 5,766.598 103 104 -continued AT CAT C GAA. T T CAAAAAA. A. CAAA GA G C G T G A G CCAA GT CCTT C C AA. T C C T C C 58

( 2) INFORMATION FOR SEQID NO:63: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 27 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:63:

C C C C C C A A G C T TTT ATC TATA CTT 2 7

( 2) INFORMATION FOR SEQID NO:64: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:64:

CAA. GGC A T G A G G T C T C O

( 2 INFORMATION FOR SEQID NO:65: i SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs B ) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D.) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:65:

CA GT G. G. A C C ACT scTATT TT ATT CAG 29

( 2 INFORMATION FOR SEQID NO:66: ( ; ) SEQUENCE CHARACTERISTICS: (A) LENGH: 61 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D.) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:66:

TAC CAATT C C T C C A G C C C G G T TAT A G CTAATTAG CAAA T G GA GT TAA TA, TA 6 O

G 6

( 2 INFORMATION FOR SEQID NO:67: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGH: 66 base pairs (B) TYPE: mucleic acid C ) STRANDEDNESS: single ( D.) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:67:

T C GC GAATT C GA. TAT CAA. G. C. A. C. GAT TTA T G A CTA GT TAAT CAAA AAAAA G CAT 6 O

ACA A G C 6 6

( 2 INFORMATION FOR SEQID NO:68: ) SEQUENCE CHARACTERISTICS: A ) LENGH: 30 base pairs (B) TYPE: mucleic acid ( C ) STRANDEDNESS: singie D.) TOPOLOGY: linear 5,766,598 105 106 -continued ( x i SEQUENCE DESCRIPTION: SEQID NO:68:

TAT C CAG C T C T G T AACA C A SAT CT AAC 3 O

( 2) INFORMATION FOR SEQID NO:69: { i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 70 base pairs BYPE: nucleic acid C) STRANDEDNESS: single (O TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQED No.69:

CGA T G A GA AAA G CAAA AATA TA CA. TATATA CA AG TACAATTA. T A G G T TA 6 O

AT CAT G. G. C. G. O

( 2) INFORMATION FOR SEQID NO:70: i SEQUENCE CHARACTERISTICS: (A) LENGTH: 65 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:70:

C C CAT GA. TA AAC CTAAA TA, ATT ACT T G T AAA. TAA T G CTATATA TT TT CAC TT TAT 6

C CAC 65

( 2 INFORMATION FOR SEQID No.:71: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: mucleic acid ( C ), STRANDEDNESS: single (D) TOPOLOGY: linear ' ( x i SEQUENCE BESCRIPTON: SEQID NO:71:

AAT CAGAGA G CAG G CT 5

( 2) INFORMATION FOR SEQID NO:72: i SEQUENCE CHARACTERISTICS: ( A LENGTH: 23 base pairs (B) TYPE: nucleic acid C STRANDEDNESS: single { D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQ ID No.72:

TT GAT C C C T A G CCA C C T C T CT 2 3

( 2) INFORMATION FOR SEQID NO:73: ( i y SEQUENCE CHARACTERISTICS: ( A LENGH: 56 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:73:

A GAC CAA CA CAC CAT CTA G C. G. G. CAAG G A G GAA ATTACT AA TTT TATT CTA GAC 5 6

2 y INFORMATION FOR SEQID NO:74: ( i ) SEQUENCE CHARACTERISTICS: A LENGTH: 58 base pairs BIYPE: nucleic acid CSTRANDEDNESS: single 5,766,598 107 108 -continued (D) TOPOLOGY: linear ( x i ). SEQUENCE DESCRIPTION: SEQID No.:74: GA T C C T C TAG AATAAAAA TT AG TAA TTT C C T C C T C GCC G CTAGA T G G T G C T G T T G GT 5 8

( 2) INFORMATION FOR SEQID No.75: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 79 base pairs (B) TYPE: nucleic acid ( Cy STRANDEDNESS: single ( Dy TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:75:

TAGA CAA AAT T GAA AA TATA TAATTACAAT ATAAAA G C C A GTA CAA CAA AT AG G T G G T A 6 o

A CTA T G T C C A C C T GCC AT T 7. 9

( 2) INFORMATION FOR SEQID NO:76: ( ; ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 80 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ). SEQUENCE DESCRIPTION: SEQID No.:76: G CT TAA T G G C A G G T G GA CAT AG T TAC CACC TAT TT G T T G T ACT G G CAT TT TATA TT G T AA 6 O

T TATATATTT T. CAATT TT GT 8

( 2) INFORMATION FOR SEQID No.77; i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 16 base pairs (B) TYPE: nucleic acid ( Cy STRANDEDNESS: single (d) TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID No.77:

TGGA T G T ACA GAC AAC 6

( 2) INFORMATION FOR SEQ ID NO:78: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( d.) TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID No.78: AAG GAT C C G A A TT CT TA CAT TAAT CTA G C C T C 3 3

( 2) INFORMATION FOR SEQID NO:79: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid ( Cy STRANDEDNESS: single ( D ) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:79: Cy is As in Th r A rig Lys Arg 1 e A rig I le G 1 in Arg G 1 y Pro G 1 y Arg A 1 a 5 1 0 15 P he w a 1 T h r G 1 y Lys 2 O 5,766,598

-continued ( 2 INFORMATION FOR SEQID NO:80: ( . ). SEQUENCE CHARACTERISTICS: (A) ENGTH: 20 amino acids ( B. ) TYPE:amino acid ( C ) SIRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:80: Cys As in Lys Arg y s Arg 1 e H is 1 e G y Pro G 1 y Arg Al a Phe Tyr 5 1 O 1 5

Th T Th T L y s As in 2

( 2 INFORMATION FOR SEQID NO:31: { i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 amino acids (B) TYPE: amino acid ( C ) STRANDEENESS: single (D) TOPOLOGY: linear x i ) SEQUENCE DESCRIPTION: SEQID NO:31: Cys As in Thr Arg lys Ser e Ty T 1 le G 1 y Pro G y Arg A 1 a Ph e H is 5 O 5 Thir Thr G 1 y Arg

( 2) INFORMATION FOR SEQID NO:82: { i SEQUENCE CHARACTERISTICS: A LENGTH: 20 base pairs B ) TYPE: nucleic acid ( C ), STRANDEDNESS: single (D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:32:

GGG TATAA T G A C T G TAG 20

( 2) INFORMATION FOR SEQID NO:33: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 30 base pairs (B) TYPE: mucleic acid C ), STRANDEDNESS: single D TOPOOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:83:

AT CA C : A G C C T G TT C C T G G G CTTAC 3

( 2 INFORMATION FOR SEQD NO:84: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 43 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:34:

ATCAT C CTA CAATAAA AA T TATA CAAA G C C C C CAA. G. C. C. 4 3

( 2) INFORMATION FOR SEQID NO:85: SEQUENCE CHARACTERISTICs: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear 5,766,598 111 112 -continued

( x i ) SEQUENCE DESCRIPTION: SEQID No:85:

AT CAC GA. G. C T C C A C GC GCT. C. 4

( 2) INFORMATION FOR SEQID NO:86: ( i ). SEQUENCE CHARACTERISTICS: ( A LENGTH:55 base pairs B TYPE: nucleic acid CSTRANDEDNESS: single (d) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:86:

AG CTT CT TA. T. CTATA CT AAAAAG GAA AATAAA TACA. A. A G G T C T T G A G G GT 55

( 2 INFORMATION FOR SEQID NO:87: ( i ) SEQUENCE CHARACTERISTCS: ( A LENGTH: 73 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQDD NO:87:

T G T G T TAAA T GAA A G CAG AAATAAT CAT AAA TTA TTT C A TTT C C GA. TAT C C G T AA 5 O

GTTT G T A C G T AC 3

2 INFORMATION FOR SEQID NO:88: ( i ) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 56 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D.) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:88:

TTAT TA GTE AT TTAATAAA GT AAA G C G CA TAG G CAA. T C AAA CATA G CA. T G A G C. 5 6

( 2) INFORMATION FOR SEQID NO:39: ( ; ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 72 base pairs ( B. TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID NO:89:

A GAA. ATA AGA AT GAA TTT CA CT TA TA GT T C C A A GAA C T C C C, AACACAAT 6 0.

AACTT CGCT CT 2

( 2) INFORMATION FOR SEQID NO:90: ( i y SEQUENCE CHARACTERISTICS: A. LENGTH: 35 base pairs B TYPE: mucleic acid CSTRANOEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:90:

A CA CAAT C T GAA T G T AAA T G TATA CTT G 35

( 2) INFORMATION FOR SEQID NO:91: ( i) SEQUENCE CHARACTERISTICS: A LENGTH: 28 base pairs B TYPE: nucleic acid 5,766,598 113 114 -continued ( C ) STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No.91:

G. G. GGG T A C C T TT G A GA GTAC CA CT CAG 28

( 2) INFORMATION FOR SEQID NO:92: ( i SEQUENCE CHARACTERISETCS: A LENGTH: 35 base pairs B TYPE: nucleic acid C STRANDEDNESS: single { D TOPOLOGY: near ( x i SEQUENCE DESCRIPTION: SEQD NO:92:

A. CAT C C T C A G GTATT CTA AA CT AG CAA AGAT G 35

( 2) INFORMATION FOR SEQID No.93: i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 35 base pairs ( B, ) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D TOPOLOGY: linear ( x 1 SEQUENCE DESCRIPTION: SEQID NO:93:

AT CAT C C GC AG GATT CTA AA CAC GAAT AGATG 3 5

2 INFORMATION FOR SEQID NO:94: ( . ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 17 base pairs (B) TYPE: nucleic acid C STRANDEDNESS: single { D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQDD NO;94:

AA TA, CGAC T C ACTA AG 7

( 2) INFORMATION FOR SEQID No.95: { i SEQUENCE CHARACTERISTICS: (A) ENGTH: 45 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D ). TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQD NO:95:

AT CA CTC TA GAATAAA AA TAT CTTTT CTCTCT GCAC CA. C. C. 45

( 2) INFORMATION FOR SEQID No.96: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs B TYPE: mucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:96:

GAAATAATAA AACAATAAC O

( 2 INFORMATION FOR SEQID No.97: i SEQUENCE CHARACTERISTICS: ( A LENGTH: 35 base pairs ( B, ) TYPE: nucleic acid (CSIRANDEDNESS: single D.) TOPOLOGY: Einear 5,766,598 115 116 -continued

x i ) SEQUENCE DESCRIPTION: SEQID NO:97:

C C C CTATT C C C A C T CA GT TT T C T C T C T G CAC 35

( 2) INFORMATION FOR SEQID NO:98: ( i SEQUENCE CHARACTERISTICS: (A) LENGH: 31 base pairs ( B. ) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQED NO:98:

GT C C T G CA. G. A G GAAAA GA. A G C C C CAA G C 3

( 2 INFORMATION FOR SEQED NO-99: { i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid C ), STRANDEDNESS: single D ). TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID NO;99;

G. G. C. G. G. A. GGCA AACTA, CCAAG G 2 1

( 2 INFORMATION FOR SEQID NO:100: ( i y SEQUENCE CHARACTERISTICS: A LENGTH: 35 base pairs B TYPE: nucleic acid ( C STRANDEDNESS: single D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:100:

A CAT C T CTA GAAAAAAA. T TACA GT CA. AAG GC 35

( 2) INFORMATION FOR SEQID No:101: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: Einear ( x i y SEQUENCE DESCRIPTION: SEQED No:101:

C GCCA G CAT G CA. GAA C A G C 20

( 2) INFORMATION FOR SEQID NO:02: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:102:

ATCA CTC A GAATAAA AA T TATA G GAA. A G C C C T C CAA GCC 4 3

( 2 INFORMATION FOR SEQID NO:103: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS; single ( D.) OPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQED NO:03: 5,766,598 117 118 -continued

G GCA GA GAA AAAA CT CAG T G G GAAT AG. A. GC 3 3

( 2) INFORMATION FOR SEQID NO:104: i SEQUENCE CHARACTERISTICS: ( A LENGTH: 47 base pairs (B) TYPE: mucleic acid ( C STRANDEDNESS: single D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:104:

ATCAT. CT CA GAA AAA AAT TA CAAA C T G C C CAT TAT C CAATTCC 4

( 2) INFORMATION FOR SEQID NO:105: ( ; ) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 47 base pairs B ) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D ) TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID NO:105:

A CAT C T CTA GAATAAAAA TA CAAA CTTG. C C CAT TEAC TAATTCC

( 2) INFORMATION FOR SEQID NO:106: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single d TOPOLOGY: incar ( x i SEQUENCE DESCRIPION: SEQID No:106:

GCC T C CT ACT ATCATA T GA ATAAT CTT T T C T C CT G 3. 8

( 2) INFORMATION FOR SEQID NO:107: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 87 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:107: TTA TCATAA. TATAT AG G. A. G. G. CT G A G GT TAAGAA. TA GTT T G C T G A C C T CT 6 O

GA GT CAATA GA GT TA G G CA. G. G. GATAA 8

( 2) INFORMATION FOR SEQID No:108: ( i SEQUENCE CHARACTERISTICS: (A) ENGTH: 87 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D ). TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:108: T TAT C C C GC CTA A C T C T A T CA CTA CA, GA, GA GT ACA GCA AAAA CT. A. T C AAA C CTAC 6 O

CAA C C C T C C T A CAT CAT TA T GAATAA 8

( 2) INFORMATION FOR SEQID NO:09: ( 1 ), SEQUENCE CHARACTERISTICS: ( A LENGTH: 43 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single (D) TOPOLOGY: linear 5,766,598 119 120 -continued

( x i SEQUENCE DESCRIPTION: SEQED NO:109:

AT CA. C. C. A GAATAAA AA TAT C C C GCC TAA C CAT CAC 4 3

2 INFORMATION FOR SEQID NO:110: ( i SEQUENCE CHARACTERISTICs: ( A LENGTH: 82 base pairs ( B. YPE: nucleic acid ( C ), STRANDEDNESS: single (D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:110:

GA CGT GACT AAT AG CAT AAAAA G CA C C G G A C C C C G A G C A GAA. T C G A C C C G G 6 0.

GTTT TAT GA CTA GT A.A. C. A 8 2

( 2) INFORMATION FOR SEQID No:111: ( i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 82 base pairs B TYPE: nucleic acid C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID No:111:

G C C G T GAT AACTA GT CA AAAAA C C C G G GAT C GATT CT AGA C T C GAG G G T A C CGG ATC 6 0

CT TT TATA G CTA AT TA GT C. A. C. 8

( 2) INFORMATION FOR SEQID NO:112: { i SEQUENCE CHARACTERISTICS: (A) LENGTH: 70 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D.) TOPOLOGY: Einear ( x i ). SEQUENCE DESCRIPTION: SEQID No:112:

GA CT TAA AA TA GT CAT CA. G. G. C. A. GGG C GA. GAA CGAGA CTAT C GC T C G T AAT TAA 6 O

TAG GT CGA CG 7 O

( 2 INFORMATION FOR SEQ ID NO:113: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 70 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No: 113:

GA C C GT CGA C CAATAAT TAA CGA G CAC ATA GT C C G C C G C C C G C C GA. GACT 6

AATAAT TAA O

( 2 INFORMATION FOR SEQID NO:114: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQDD NO:114:

G GT CGA CG GA. T C CT 1 4

( 2 INFORMATION FOR SEQID NO:115: 5,766,598 121 122 -continued

i SEQUENCE CHARACTERISTICS: ( A LENGTH: 22 base pairs B TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No: 115:

GAT CA. G. GAT C C C C GAC CTC CA

( 2) INFORMATION FOR SEQID NO:16: { i SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs ( B.) TYPE: nucleic acid CSTRANDEDNESS: single ( D TOPOLOGY: linear x i ) SEQUENCE DESCRIPTION: SEQID NO:116:

G. G. G. CAAAG AAG G G 1 6

( 2 INFORMATION FOR SEQ ID NO:17: ( ; ) SEQUENCE CHARACTERISTICs: ( A ) LENGTH: 26 base pairs (B ) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQD NO:117:

TT G GA. T C CTT A TT G T G A CGA G G G GT C 2 6

( 2) INFORMATION FOR SEQID No: 118: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 106 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single D.) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:118:

GA T C T G A GA TAAA GT CAAA. A. TATA ACA TATATA CA AA GTA CAAT A TA G G T T 5 O

AAT CA G G T G C G A GA G C G CAG TATA AG C G G G - A GAA TT AGAT O 6

2 ) INFORMATION FOR SEQID No. 119: i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 104 base pairs (B) TYPE: mucleic acid ( C STRANDEDNESS: single { D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:119.

CGA CTA A C C C C C C GC I AA TA, C G A C GC T C C GCA C C CAATAA AC CTA AATAA is 0

T G T ACT T G T AAT ATAAT A TATA A TT T. CA CTTA, C T CAA 4

( 2) INFORMATION FOR SEQID NO:120: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 68 base pairs ( B, ) TYPE: nucleic acid C STRANDEDNESS: single (D) TOPOLOGY: linear x i SEQUENCE DESCRIPTION: SEQID No:120:

C T G ACA CAGC A CACA G CAAT CAG GT CAGCC AAAATT ACTA AT TTA. T C C G A G G CGAC 6 5,766,598 123 124 -continued

A G GA C C C G 6 8

( 2 INFORMATION FOR SEQID No:121: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 72 base pairs (B) TYPE: nucleic acid ( Cy STRANDEDNESS: single ( D.) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:121:

GA T C C G G G T C C T G T C GA C C T C GA GATAAAA A T TA GT AATT TT G G C T G A C C T G AT T G CTGT 6 0

GT C C T G T G T C AG 7 2

( 2 INFORMATION FOR SEQID NO:122: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (d) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:122:

AA GAA AAT TA TAGGAC 6

2 INFORMATION FOR SEQID No:123: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 24 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID No:123:

T. G. GAT C C C T AAT C C T CAC CT GT 2 4.

( 2) INFORMATION FOR SEQID NO.124: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: mucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:124:

AAA GT CGA C C CATAT CAC CT AGAA C 25

( 2) INFORMATION FOR SEQID NO:125: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:125:

TTT G GA. T C C T TA CAAAAC T C T T G C C T TA T 29

( 2) INFORMATION FOR SEQID No:126: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 105 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID NO:126; 5,766,598

-continued T CGA CAA AA TT GAAAAAT AT AAT TA CAA TAAAAA T G C CAT AG, GCA GAA CAT C CAC 5 O

GG G CAAA T G G T A CAT CA. GGC CATAT CAC CT A GAA CT TAA A G CA O 5

( 2 INFORMATION FOR SEQ ID No:127: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 97 base pairs ( B, ) TYPE: nucleic acid CSTRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:127:

AAA T C A. G. G. GATA T G C C C T A G A C CAt G. C C C C T G GA GT C T G CA CTA A 6 O

GG CA. TAT AT T G T AATTA. TATA TT CA A TT GC 9 7

( 2 INFORMATION FOR SEQ d NO:128: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 43 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: Einear ( x i , SEQUENCE DESCRIPTION: SEQED NO:28:

G C C T C CTA. C. A. CAT A T GA ATAAA CT CAT G. G. A. GGG CA. TAC 4 3

( 2) INFORMATION FOR SEQID No:129: { i SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 9 base pairs (B) TYPE: nucleic acid ( Cy STRANDEDNESS: single D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:129:

GGC C C CAA. C.

( 2) INFORMATION FOR SEQID NO:130: ( i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 9 base pairs ( B. ) TYPE: nucleic acid ( C ) STRANDEDNESS: single D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO: 130:

T CGA GT T G C

( 2) INFORMATION FOR SEQID NO; 131: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 base pairs By TYPE: nucleic acid ( Cy STRANDEDNESS: single D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID No:131:

G G CCAAA C

( 2) INFORMATION FOR SEQID NO:132: ( ; ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 8 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear 5,766.598 127 128 -continued ( x i y SEQUENCE DESCRIPTION: SEQID No:132:

CGA GT TT

( 2) INFORMATION FOR SEQID NO:133: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 32 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQED NO:133:

C C C C C C A A G C T TA CAT CA. T G CAG T G G T TAA A C 3 2

( 2) INFORMATION FOR SEQID NO:134: { i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 base pairs ( B, ) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:134:

GATT AAA C C T AAA AA TT GT 20

( 2) INFORMATION FOR SEQID NO:135: ( i y SEQUENCE CHARACTERISTICS: ( A LENGH: 22 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID No:135:

A CAAT TATTT AG GT TAA C T G CA 22

( 2 INFORMATION FOR SEQID No:136: ( . ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:136:

GT TAAC CTA A. ATA AT TGT 8

( 2) INFORMATION FOR SEQID NO:137; ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 75 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D.) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID No:137: TAA CAT GAA. A CAAAT TATT AATA T G T G GC AA GAA GAG GA. AAA G CAT G T A C G CTTGACT 6 0

A GT TAAT CAC T C GAG 7 5

( 2) INFORMATION FOR SEQID NO: 138: ( i SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 80 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D TOPOLOGY: linear 5,766,598 129 130 -continued ( x i SEQUENCE DESCRIPTION: SEQID NO:138:

GA T C C T C G A G T GATA ACA GT CAA, GC GTA CAT AG C TT T C C T ACTT C T T G CCA CATA TT 6 0

AATAATT G TT CAT GATA 80

( 2) INFORMATION FOR SEQID NO: 139: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 78 base pairs ( B TYPE: nucleic acid CSRANDEDNESS: single { D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQ ID NO:139:

A C C C AA g G A C G T A A G CAC GAA gATAT AT T CTTT G T G G GA. T CAA. T C T A 5 O

AAA GA CTA G T TAAT CAG 8

2 INFORMATION FOR SEQID No:140: ( ; ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 82 base pairs ( B ) TYPE: mucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:140:

GAT C C CAT AACTA GT CAT TT TAAA GATT GA. T C C CA CAA A GAAATAATA CT C GT C CA 5 O

T TAC GATA CA. AACTTAACGG AT 8

( 2) INFORMATION FOR SEQID NO:141: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs ( B, ) TYPE: nucleic acid (C)STRANDEDNESS: single ( D.) TOPOLOGY: linear ( x i ). SEQUENCE DESCRIPTION: SEQID No:41:

AAT AAT A G C GCA C C C C C G G G CAAAAA AAA AAA T G 4 0

( 2 INFORMATION FOR SEQID NO;142: ( i SEQUENCE CHARACTERISTICS: ( A LENGH: 53 base pairs B TYPE: aucleic acid ( C STRANDEDNESS: single D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:142:

C C T G T ACA CT CAATTAC T CAT C CA T TAA T G T A A ATA AT C 53

( 2) INFORMATION FOR SEQID NO:43: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 107 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:143:

T CAAAAAAAT ATAAA T G AT CAC CAT C T G T AGAAAAAAA ATT A T G G AA AA TAT CA 6

T AATA TT T. G. GATT CAAA. A TTA.A.A.A.A. TATAATTACA ATA AAA O 7

( 2) INFORMATION FOR SEQID NO;144: 5,766,598 131 132 -continued ( i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 73 base pairs (B) TYPE: nucleic acid C STRANDEDNESS: single Ed TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID No:144: A T G GAT AGAG AA T GAA GT AGA CAAG GA. G. CT TATA GA G CTAT TA GA T G A CTA GT TAAT 6

CA C C GA. GGA T C C 7 3

( 2) INFORMATION FOR SEQID NO:145: { i SEQUENCE CHARACTERISTICS: ( A LENGTH: 73 base pairs B TYPE: nucleic acid ( C STRANDEDNESS: single { D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:145: GA C C C G A G GATT AA A G CAT CAA TAG CT CTA A A G C T C C T G T ACT ACT CAA 6 O

TT ACT CAT C CAT 3

( 2) INFORMATION FOR SEQID NO:146: { i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 43 base pairs ( B TYPE: nucleic acid ( C STRANDEDNESS: single { D TOPOLOGY: linear ( i SEQUENCE DESCRIPTION: SEQID No:146; A CA C G GA C C T C CAG GA TAAA C A G CAT CTA ATA G C. C. 4 3

( 2) INFORMATION FOR SEQID NO:147: i SEQUENCE CHARACTERISTICS: ( A LENGTH: 84 base pairs B ) TYPE: nucleic acid (CSTRANDEDNESS: single D TOPOLOGY: linear ( x ) SEQUENCE DESCRIPTION: SEQID NO:147: TT AA CAG GA. T C CTTAATA AT TA GTATT AGA CAAG G T G AAAAC GAA AC TA TTT G TAG C 6 0.

AATAATT AC G CA C C C G G G 8 4

( 2 INFORMATION FOR SEQ ID No:148: ( i ). SEQUENCE CHARACTERISTICS: (ALENGTH: 84 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:148: C C C C G G CTG C A G CTAATAA TTA A G CTA CA. AATA GT C G T TT CAC CT T G T C T AATAAC 6 0

AAT TAATA AG GAT C C T G A TTA. A 8 4

( 2 INFORMATION FOR SEQID NO:149: ( ; ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 34 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D.) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO: 149: 5,766,598 133 134 -continued

TTAA CAG GA. T C C T AA TA, ATTA. TATT AGAC 3 4

( 2 INFORMATION FOR SEQID No:150: ( 1 ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 42 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single D ) OPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQED NO: 150:

AT CAC G GAT C C T C G A G T G A T TAA CTA G C AT CAAT AG C T C. 4. 2

( 2 INFORMATION FOR SEQID No:151: ( i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 12 base pairs B TYPE: nucleic acid ( C STRANDEDNESS: single { D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:151:

A.A.T. G. C. GGCC GC 1, 2

( 2) INFORMATION FOR SEQID No: 152: { i SEQUENCE CHARACTERSICS: ( A LENGTH: 66 base pairs ( B TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear x i ) SEQUENCE DESCRIPTION: SEQDD NO:152:

AGAAAAAI CA GT TA G CTAAG ACT C C C G G G C C GAG G G T A C C G GA C C T G AT TA GT TAAT 6 O

TTTT GT 6 6

( 2) INFORMATION FOR SEQID No. 153: i SEQUENCE CHARACTERISTICS: ( A ) ENGTH: 70 base pairs (B) TYPE: nucleic acid ( Cy SRANDEDNESS: single (D TOPOLOGY: linear ( i ) SEQUENCE DESCRIPTION: SEQD NO:153:

GAT CACAA AA ATT. AACTA A. C.A. GGA C C G G A C C C T C G A G C C C G G G A GA C A G CAA C 5 O

T GATT T. CT 7 0

( 2) INFORMATION FOR SEQID No:154: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids ( B, ) TYPE: artino acid (C)SIRANDEDNESS: single (d) TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID NO:154: Me t Lys G 1 u G in Lys Thr w a 1 A a Met A rig Wa Lys G 1 u Lys Tyr G 1 in 5 O 5 H is L. eu Trip A 1 g Tr p G 1 y Trip A rig Tr p G y Thr Me t Le u Le u G 1 y Me t O 5 30 eu Me t 1 e Cy s Ser A 1 a Thr G 1 u Lys Le u Trip V a 1 T hr V a 1 Tyr Ty T 4 O 45

G 1 y w a P ro 5,766,598

-continued

50

( 2) INFORMATION FOR SEQID NO:155: ( i SEQUENCE CHARACTERISTICS: A ) LENGTH: 62 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: Linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:155: Pro P h e A rig L y s G in I I e I l e A s in Met T r p G 1 in G 1 u v a 1 G y Lys A 1 a 5 O 15 Me t Ty r A 1 a Pro Pro P h e A rig Lys His G 1 u A s p 1 1 e 1 1 e Ser Le u Trip 2 0. 25 3 O A s p G 1 in Ser L. eu Lys Pro Pro Phe Arg Lys A s p A rig V a I e G 1 u v a 1 3 5 4 0 45 W a 1 G 1 in G 1 y A a Ty r A rig A 1 a I l e A T g Pro Pro P h e A rig Lys 5 O 55 6 O

( 2) INFORMATION FOR SEQED NO:156: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids B ) TYPE: amino acid ( C STRANDEDNESS: single (D) TOPOLOGY: inear ( x i ). SEQUENCE DESCRIPTION: SEQID No:156: Le u Ph e l l e Me t I le V a G 1 y G 1 y Le u v a 1 G1 y Le u Arg 1 1 e V a 1 P he 0. 5 A a Wal Le u S e r Wa 1 v a 1 As in Arg Wa 1 Arg G 1 in G y 20 25

( 2 INFORMATION FOR SEQID No:157: ( . ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (d) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQDD NO:157:

A CTA CTA A G C T C T TA TTC TATA CAAA AA G T G 35

( 2) INFORMATION FOR SEQID NO:158: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid ( C ) SIRANDEDNESS: single D.) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:158:

CAT AT TAATT T G T TTT CTAA AAG GAG G T A C C C CATAAAG ACT G T G 4 6

( 2) INFORMATION FOR SEQID NO:159: ( i y SEQUENCE CHARACTERISTICS: (A) LENGTH: 94 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:159:

G C T C C T C C T T TTAGAAAACA C GAA GATA. T AT T C T G T G G GA. CAA. T C T T TAA AAC CT 6 O 5,766,598 137 138 -continued

CCTTAGAA AA GAT AGA GT AAT GAA GTA GT AC 9 4

( 2) INFORMATION FOR SEQID NO:160: ( i ) SEQUENCE CHARACTERISTICS: A LENGTH: 94 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single D TOPOLOGY: Einear ( xi ) SEQUENCE DESCRIPTION: SEQID No:160: GT ACT ACT I C AAT TAC CTA T C T TT CTAA AAG GA. GGTTT TAAA GATTGA T C C CA CAA AG 6 O

AAA AAA C C G T G T CT AAAAG GAG ACC 9 4

( 2) INFORMATION FOR SEQ ID NO:161: i SEQUENCE CHARACTERISTICS: A LENGTH: 70 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:161: AAA CAAATTA TT AAA T G T G G CAAGAA GTA G GAA AA G CTA T G T A C GC T C C T C C T TTTAGA 6 O

AAA CA, CGA AG

( 2) INFORMATION FOR SEQID No:162: ( 1 SEQUENCE CHARACTERISTICS: ( A ) LENGTH:56 base pairs B ) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D ). TOPOLOGY: linear x i ) SEQUENCE DESCRIPTION: SEQID No:162: A CAC I C A GA A CT AA TA G C CATA A G C T C C TT ACA CTT CAA TA CTC 5 5

2 INFORMATION FOR SEQID NO:163: ( ; ) SEQUENCE CHARACTERISTICS: ( A y LENGTH: 74 base pairs By TYPE: nucleic acid ( Cy STRANDEDNESS: single { D TOPOLOGY: linear ( x SEQUENCE DESCRIPTION: SEQID NO:163: A CAT CAT A GAA AAT T CAAAAG G A G CAA. T.A. G. C. CTATA A G C T C C T G T AC 6 O

TAC T CAATT ACT C 4

( 2) INFORMATION FOR SEQID NO:164: i SEQUENCE CHARACTERISTICS: ( A LENGTH: 32 base pairs (B) TYPE: nucleic acid C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:164:

AT CA, CG GAT C CA. A. G. C. A. C. A. CAT G CAG T G G 3 2

(2) INFORMATION FOR SEQID NO:165: ( i ) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 49 base pairs ( B, ) TYPE: nucleic acid 5,766.598 139 140 -continued ( C ) STRANDEDNESS: single ( D ). TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:165;

C GT T GACC AT G. CCA C C C A GATT AAA CC TAAA TAA T G T ACT TG 49

( 2) INFORMATION FOR SEQID NO:166: ( . ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs ( B ) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D ). TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQED NO:166:

A GTA CAATTA. T. T. A. G. G. T. A. A. T A T G G G T G. G CAAAT G. G. C. A. AAA CG 46

( 2 INFORMATION FOR SEQID NO;167: i y SEQUENCE CHARACTERISTICS: A LENGTH: 30 base pairs ( B TYPE: nucleic acid ( C ), STRANDEDNESS: single { D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:167:

A T C T C G GAT C C T AA CA C T C T C T C C C G G 3 O

( 2) INFORMATION FOR SEQID NO:168: ( i SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 55 base pairs B TYPE: nucleic acid ( C ), STRANDEDNESS: single { D TOPOLOGY: Einear ( x i SEQUENCE DESCRIPTION: SEQID NO:168:

AT CAT C G GAT C C T AA CA C T C C C C C G G G CA CAT C CAT C C T G G C CAT AG 55

( 2) INFORMATION FOR SEQID No:169; { i SEQUENCE CHARACTERISTICS: ( A LENGTH: 70 base pairs ( B TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:169:

AATAA C C C G GA. T C CAA. G. C. TT CTAG CAG CTAATT. A. A C G C C C C ATAATCG 50

AACTTAT TAC 7 O

( 2 INFORMATION FOR SEQ ID NO:170: ( i SEQUENCE CHARACTERISTICs: A LENGTH: 28 base pairs ( B TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: Einear i SEQUENCE DESCRIPTION: SEQID NO:170: GCTA GAAA. T C C T TA GT TT TA A G T G 28

2 INFORMATION FOR SEQID NO:171: { i SEQUENCE CHARACTERISTICS: A LENGTH: 28 base pairs B TYPE: nucleic acid 5,766.598 141 142 -continued C ), STRANDEDNESS: single D ). TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID No:171:

A CAA T G T ACA GAA. T C T G AT CAT AG 8

2 INFORMATION FOR SEQID No:172: { i SEQUENCE CHARACTERISTCS: ( A LENGTH: 67 base pairs ( B. TYPE: nucleic acid C ) STRANDEDNESS: single D ) TOPOOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO;172:

C T A G CAA A G C T G GAT C C C C G G T AAT AA AATAAA AA C C C GC C C C G T AAA GA G 30

A. AAA ATG 6

( 2) INFORMATION FOR SEQID NO:173: ( i y SEQUENCE CHARACTERISTICS: A LENGTH: 67 base pairs B TYPE: nucleic acid ( C STRANDEDNESS: single { D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:173:

CGC C CAT GAT TAAA C C T AAA AA T G T ACT T G T AA TATA AT GAATAA TTT CACT 6 O

A T C CAC 6

2 ) INFORMATION FOR SEQID NO:174: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID NO:174:

A. GG CA GT T C A T G CA 1 7

2 INFORMATION FOR SEQID No:175: { i SEQUENCE CHARACTERISTICs: ( A LENGTH: 31 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:175:

T C C C G G GAG ACT CAT C C CATT C C CA. T. 3

( 2) INFORMATION FOR SEQID NOT6: ( . ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 5 base pairs B ) TYPE: nucleic acid C). STRANDEDNESS: singie d) IOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID NO:176:

G. G. T. 5

( 2 ) INFORMATION FOR SEQID NO:177: ( i) SEQUENCE CHARACTERISTICs: 5,766,598 143 144 -continued ( A LENGTH: 11 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D TOPOLOGY: Einear ( xi ) SEQUENCE DESCRIPTION: SEQID No:177:

AATT CAA C C G C 1 1

( 2) NFORMATION FOR SEQID NO:178: i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 78 base pairs ( B, ) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:178:

C T A G CTA AG TAAG G CAG G G G TATA G G CCA G T G T T C T C T T C C C CA C C C T C T ATTT C CAG 6 0.

CA GA CT CATA C C CAA CAG 7 8

( 2) INFORMATION FOR SEQID NO:179; ( i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 77 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: Einear ( xi ) SEQUENCE DESCRIPTION: SEQED NO.179: GT C C T G T G G G T A T GA GT C T G C G GAA ATA AG. A. GGG T G G G GAA GA GAA C A C T G GCC TATA 6 O

C C C C T G CCTT. AACTTAG 7 7

( 2) INFORMATION FOR SEQ ID NO:180: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C)SIRANDEDNESS: single D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:180: AAA G CAT C C C C C G G G T TAAA AAT TIAAA G T G CAA C C 3 6

( 2) INFORMATION FOR SEQID NO;181: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID NO:181:

C GATAAA C C G C 1

( 2) INFORMATION FOR SEQID No:182: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 7 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:182:

G GT TAT 7

( 2 INFORMATION FOR SEQID NO:183: 5,766,598 145 146 -continued

( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 7 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:183:

G G CAAA G

( 2) INFORMATION FOR SEQID NO:184: ( i SEQUENCE CHARACTERISTICS: A ) LENGH: 11 base pairs B TYPE: mucleic acid C STRANDEDNESS: single (dy TOPOLOGY: linear x i SEQUENCE DESCRIPTION: SEQID NO:184:

GAT C C T TT C C C

( 2) INFORMATION FOR SEQID NO:185: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 12 base pairs ( B. ) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D TOPOLOGY: inear ( x i SEQUENCE DESCRIPTON: SEQID NO:185:

(CTAGAAAA C C GC 1 2

( 2) INFORMATION FOR SEQID NO;186: ( i SEQUENCE CHARACTERISTICS: A LENGTH: 6 base pairs B TYPE: nucleic acid ( C STRANDEDNESS: single { D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID No:186:

G G T TT

( 2 INFORMATION FOR SEQID No:187: SEQUENCE CHARACTERISTICS: (A) ENGTH: 18 base pairs (B) TYPE: Nucleic acid (C)SIRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:187:

(CAGAA GT AG. C. ATA AGT 18

( 2) INFORMATION FOR SEQID No: 188: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 45 base pairs B ) TYPE: nucleic acid ( C STRANDEDNESS: single { D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO;188:

G C C T C CTACT A CAT TAGA ATAAT C T CTT A T G T C T C C C T G GA. GC 45

( 2 INFORMATION FOR SEQID No:189:

SEQUENCE CHARACTERISTICS: 5,766.598 147 148 -continued ( A LENGTH:55 amino acids (B) TYPE: amino acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:189: Me t Lys G i u G 1 in Ly S T h r V a 1 A a Met A 1 g V a l Lys G 1 u Lys Ty r G 1 in 5 1 O 1 5 H is Le u Trip Arg Tr p G : y T r p A 1 g Tr p G l y T hr Me t Le u Le u G 1 y Me t O 25 3 O Le u Me t 1 e Cy s Ser A la Thr G 1 u Lys Le u T r p V a 1 T h r V a 1 Ty T T y T 35 4 O 4 5

G y Wa Pro Pro P be A rig L y is 5 O 55

2 INFORMATION FOR SEQID NO;190: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 89 amino acids (B) TYPE: amino acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:190: Pro P the Arg Lys G 1 y Pro Lys G 1 u Pro P h e A rig A s p Ty r Wa A s p A rig 5 O 5 Phi e Tyr Lys Pro Pro P h e Arg Lys v a 1 G 1 in I 1 e A s in Cy s Th r A rig Pro 0 25 3 O As a Ty r A s in Lys A rig Lys A rig I 1 e H is I le G 1 y Pro G y A T g A 1 a Phe 35 4 O 4 5. Ty r T b r Th r Lys As n I I e I le G 1 y T hr 1 1 e A r g G 1 in A 1 a H is Cy s A s in 5 O 55 6 O I e Ser Arg A 1 a Lys Pro Pro Ph e A rig L y is G 1 in I le 1 e A s in Me t Trip O 7 5 8 O G l n G u Wa 1 G 1 u Lys Al a Me t Ty r A 1 a

( 2) INFORMATION FOR SEQID NO:191: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 38 base pairs ( By TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: Einear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:191: A G GT C C C T C CT GAA T G A G G T A C C C CATA AT AGA CTG 3 8

( 2) INFORMATION FOR SEQED NO:192: ( i SEQUENCE CHARACTERISTICs: (A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D ) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQ ID NO:192: C CATT CAG GA AG G GAC CTA A A GAA C C T TTT AGA GATA T G T AGAT AGATT T TATAAA C CA 6

CCTTTTAGAA AA 7 2

( 2) INFORMATION FOR SEQID No:193: ( i) SEQUENCE CHARACTERISTICS: 5,766,598 149 150 -continued ( A LENGTH: 72 base pairs B ) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:193:

TT CAA AA C C T G T AT AAAA. T CTA C T A CATA A CT CAAAAG G T C T TAG CC 5 O

C T C C GAA T GG 2

( 2) INFORMATION FOR SEQID NO:194: i SEQUENCE CHARACTERISTICS: A LENGTH: 43 base pairs (B) TYPE: nucleic acid C ) STRANDEDNESS: single D ) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:19.4:

GTA CAAT TAA TTT G T ACT TT T. CEAAAA. G. C. G. AAAA A C 4 3

( 2) INFORMATION FOR SEQID NO:195: ( i ) SEQUENCE CHARACTERISTICS: (A LENGTH: 72 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO;195:

GG T G G T AT AAAATCTAT C T ACA AA T C C AAAAG GTT C T TA GT C C CATTACA. TA 5 O

CAAACTTAAC GG 7

( 2) INFORMATION FOR SEQID NO:196: ( i SEQUENCE CHARACTERISTICS: (A) LENGH: 35 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO;196: AAA C CAC CTT TT AGAAAAG A CAAATT AAT T G T AC 35

( 2 INFORMATION FOR SEQID NO:197: { i SEQUENCE CHARACTERISTICS: ( A LENGTH: 42 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID No:197:

C GCT ACG. G. AACGG T G T T T G CT CACT AA GT ACAA T G. 4

2 INFORMATION FOR SEQID No:198: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 63 base pairs B ) TYPE: nucleic acid C STRANDEDNESS: single D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:198:

C CA C C GT T C C G T A A G CAG AT AATAAA CAT G T G G CAAGAA G T A GAAAAA C A T G A G CT 6 O

AA 6 3 5,766,598 151 152 -continued

( 2 INFORMATION FOR SEQID No: 199: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 63 base pairs (B) TYPE: nucleic acid ( Cy STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:199: T TAAG CAT AC AT AG CTTTTT CTA CT C T G CCA CAT G T TT ATTA. T C T G C T TA CG GAA CG G 6 O

G. G. 6 3

( 2 INFORMATION FOR SEQID NO:200: i SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID NO:200: T CAT CAAA G C T T C T C G A GAA AAAT TAAG CA TA CATA G CTT TTTC 4 4

( 2) INFORMATION FOR SEQED NO:20: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D.) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:201:

AT CAT C G A G C T C C C G G C C GC CTAT CAA AA G T C T TAA T G A G TT 42

( 2 INFORMATION FOR SEQID NO:202: ( i) SEQUENCE CHARACTERISTICS; (A) LENGTH: 73 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:202:

GAAT T C C C G A G C T G CAG C C C GGGTTTA, TAG CTA AT TA GT CATT TTT CG TAA GTA AG 6 O

TATTTT ATT TAA 7 3

( 2) INFORMATION FOR SEQID NO:203: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 72 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (d) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID No.203: C C C G G G CT GC A G C T C G A G GA AT T C T TT A TT GATT AACT A GT CAAATGA GTATATATA A 6 0.

T GAAAAA GT AA 7 2

( 2) INFORMATION FOR SEQID NO:204: { i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 5,766.598 153 154 -continued ( x i SEQUENCE DESCRIPTION: SEQID NO:204:

GA T G A G G T A C C TT CATAAA ACA AG T G AT AAA CTTA A GT C. 45

2 INFORMATION FOR SEQID NO:205: i ) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 21 base pairs ( B ) TYPE: nucleic acid ( C ), SRANDEDNESS: singie { D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:205: T TAACGG AT A T C GC GATA A T G

What is claimed is: 10. The recombinant virus of claim 1 wherein the gene 1. A recombinant attenuated ALVAC canarypox virus product is selected from the group consisting of: gp160; comprising an exogenous DNA segment encoding a lentivi rus gene product. gp120; Gag and Pol; Gag. Pol and gp120; Gag, Pol and 2. The recombinant virus of claim 1 wherein the exog gp160; Gag. Pol and truncated Env, non-cleavable gp160; enous DNA segment encodes a human immunodeficiency gp120 anchored with transmembrane; Gag, Pol, and gp120 virus or simian immunodeficiency virus gene product. anchored with transmembrane; signal domain of Env and 3. The recombinant virus of claim 2 wherein the exog enous DNA segment encodes a human immunodeficiency 25 p24 fused to T1 and V3 loop of Envip24 fused to T1 and V3 virus gene product. loop of Envi Nef; V3 loop fused to 88 epitope; T1, T2, and 4. The recombinant virus of claim 2 wherein the exog TH4.1 epitopes; Env signal domain, T1, T2, and TH4.1 enous DNA segment encodes a simian immunodeficiency epitopes; and, Gag, Pol protease and gp120 anchored with a virus gene product. transmembrane sequence. 5. The recombinant virus of claim 1 wherein said exog 30 11. An immunogenic composition comprising a recombi enous DNA segment further encodes a human T-helper nant attenuated canarypox virus as claimed in any one of lymphocyte epitope derived from tetanus toxoid fragment C. claims 1 to 10 and an adjuvant. 6. The recombinant virus of claim 5 wherein said exog enous DNA segment encodes a human immunodeficiency 12. A method for expressing a lentiviral gene product virus or simian immunodeficiency virus gene product. comprising infecting a suitable host cell with a recombinant 7. The recombinant virus of claim 6 wherein said exog 35 attenuated canarypox virus as claimed in any one of claims enous DNA segment encodes a human immunodeficiency 1 to 10. virus gene product. 13. A method of inducing an immunological response to 8. The recombinant virus of claim 6 wherein said exog a lentivirus gene product comprising administering a recom enous DNA segment encodes a simian immunodeficiency binant attenuated canarypox virus as claimed in any one of virus gene product. claims 1 to 10. 9. A recombinant attenuated ALVAC canarypox virus 14. A method of inducing an immunological response to selected from the group consisting of vCP95, vCP112, a lentivirus gene product comprising administering an vCP60, vCP61, vCP125, vCP124, vCP126, vCP144, vCP120, vCP38, vCP117, vCP130, vCP152, vCP155. immunogenic composition as claimed in claim 11. vCP156, vCP146, vCP148, vCP154, WCP168, vCP153, and 45 vCP172.