United States Patent (19) 11 Patent Number: 5,338,683 Paoletti (45) Date of Patent: "Aug

USOO5338683A United States Patent (19) 11 Patent Number: 5,338,683 Paoletti (45) Date of Patent: "Aug. 16, 1994

54 VACCINIAVIRUS CONTAINING DNA SEQUENCES ENCODING HERPESVIRUS FOREIGN PATENT DOCUMENTS GLY COPROTEINS 0261940 3/1988 European Pat. Off. . 9001546 2/1990 PCT Int'l Appl. . 75) Inventor: Enzo Paoletti, Albany, N.Y. OTHER PUBLICATIONS 73) Assignee: Health Research Incorporated, Muller et al (1977) J. Gen. Virol. 38, 135-147. Albany, N.Y. Piccini et al (1987) Meth. Enzymol 153, 545-563. Taylor et al (1988) Vaccine 6, 497-507. * Notice: The portion of the term of this patent Perkus et al (1985) Science 229, 981-984. subsequent to Jul. 29, 2003 has been Allen et al. (1987) J. Virol. 61,2454-2461. disclaimed. Piccini et al., Bioessays (Jun. 1986) vol. 5, No. 6, 248-52, at 248. 21 Appl. No.: 502,834 Elliot et al., J. Gen. Virol. (1991), 72, 1762–79, at 1763. Boyle, D. B. et al., J. Gen. Virol. (1986), 67, 1591-1600. 22 Filed: Apr. 4, 1990 Guo et al., J. of Virology, vol. 64, No. 5, pp. 2399-2406 (1990). Related U.S. Application Data Guo et al., J. of Virology, vol. 63, No. 10, pp. 4189-4198 60 Continuation-in-part of Ser. No. 394,488, Aug. 16, (1989). 1989, abandoned, and Ser. No. 90,209, Aug. 27, 1987, Primary Examiner-Elizabeth C. Weimar abandoned, which is a division of Ser. No. 622,135, Assistant Examiner-Deborah Crouch Jun. 9, 1984, Pat. No. 4,722,848, which is a continua Attorney, Agent, or Firm-Curtis, Morris & Safford tion-in-part of Ser. No. 446,824, Dec. 8, 1982, Pat. No. 4,603,112, which is a continuation-in-part of Ser. No. 57 ABSTRACT 334,456, Dec. 24, 1981, Pat. No. 4,769,330, said Ser. What is described is a recombinant poxvirus, such as No. 394,488, is a continuation-in-part of Ser. No. vaccinia virus, fowlpox virus and canarypox virus, con 339,004, Apr. 17, 1989, abandoned. taining foreign DNA from herpesvirus. In one embodi ment, the foreign DNA is expressed in a host by the 51) Int. Cli...... C12N 15/00 production of a herpesvirus glycoprotein. In another 52 U.S. C...... 435/320.1; 435/172.3; embodiment, the foreign DNA is expressed in a host by 135/32 the production of at least two, particularly two or three, 58) Field of Search ...... 435/320.1, 69.1, 76.1, herpesvirus glycoproteins. What is also described is a 435/76.3 vaccine containing the recombinant poxvirus for induc ing an immunological response in a host animal inocu 56 References Cited lated with the vaccine. By the present invention, the U.S. PATENT DOCUMENTS barrier of maternal immunity in a newborn offspring 4,603,112 7/1986 Paoletti et al. . can be overcome or avoided. 4,722,848 2/1988 Paoletti et al. . 4,769,330 9/1988 Paoletti et al. . 12 Claims, 83 Drawing Sheets U.S. Patent Aug. 16, 1994 Sheet 1 of 83 5,338,683

VACCNA RIGHT TERMINUS

pUC8

Sal

LIGATE

EcoRI. Ecor. pSD49VC HindIII HindII SmaI Hind BamHISmaI r Sal - BalSal Sal (Ø Rsa 2 Eag Real \2 Eag Reg PARTA '" indi Rsa g KNASE 9 MPSYN59-62 Pst NGATE SYNTHETIC Eagi DNA

Ban VP425 - he 4- - VC-2

U.S. Patent Aug. 16, 1994 Sheet 4 of 83 5,338,683

m3mpt 9 p SD466VC Nar Ban/EcoRI. EcoRI EcoRIEHVigp3N \ r E.LIGATE Nar anime Hind LIGATE pSD467VC Pst MUTAGENESIS Smal Bg Sail SmaIMHindIII Bg|II/HindII. ISOLATED FRAGMENTs- he promoter EcoRINar É pUC3 LIGATE, EcoRV LIGATE Na r m Hind

SYPstI (V BgCO Smal Sal Nsi NKER GATE

Pst KY EcoRI Nar (NERy. Nsi Bg Sai

F. G. 3

Sal Bg EcoRV vP483 enouilliamsvp425 IN VIVO RECOMBINATION LIGATE pVHA6g 13 U.S. Patent Aug. 16, 1994 Sheet 5 of 83 5,338,683

F. G. 4

pUC8 Pio Hindi Sph I Bg Hindi

Hind BamH pMP4O9BVC

Bg EcoR LIGATE Hind (WncoR

Bi SailH Sph Pst Sphi Hind Ba3 MUTAGENESS

pMP4O9DVC

Hindi i S-2EcoRI Bg Bgll Smal

LIGATE

WP458 VP4O U.S. Patent Aug. 16, 1994 Sheet 6 of 83 5,338,683

pUC8 EcoR Barn pUC8 RNBamh AEYBam Barnh LIGATE Konp Pst EcoR V EcoR Barnh ? Barn EcoR pUCCBamHI-D UCCBaraH - Pst N Pt Banh a/EcoRI) lice BLUESCRIPT Kpni SK+ Ness pUCCBamHIMPst \ ) Kpn 2N Nsi pUC- 8 KpniP W t Nsi BLUECKpnM Bamh VAT. EcoRi Bamhi El Barnh) BinhKpn S9, EcoR PMP409DVSR: FRAGMENTpucckoi/BamHis SE LIGATE EcoR Kpn C Nsi

Bg NVMO RECOMBINATION -VP458 -o- vP577

U.S. Patent Aug. 16, 1994 Sheet 8 of 83 5,338,683

243 TGGGGTGGATGCCATGGAGGCACTACACGTCAACGTCTGTCAACTGCATCGTCGAGG W G W M P W R H Y T S T S W N C W E 1254 AGGTGGAGGCGCGGTCCGTCTACCCCTACGACTCCTTCGCCCTGTCCACCGGTGATA E W E A R S W Y P Y D S F A L S T G D 28 TTGTGTACGCGTCTCCGTTTTACGGCCTGAGGGCTGCCGCTCGCATAGAGCACAATA W Y A S P F Y G L R A A. A R E H N 1368 GCTACGCGCAGGAGCGTTTCAGGCAAGTTGAAGGGTACAGGCCCCGCGACTTAGACA S Y A Q E R F R Q W E G Y R P R D L D 39 GTAAACTACAAGCCGAAGAGCCGGTTACCAAAAATTTTATCACTACCCCGCATGTCA S K L Q A E E P W T K N F I T T P H. W. 1482 CCGTCAGCTGGAACTGGACCGAGAAGAAAGTCGAGGCGTGTACGCTGACCAAATGGA T W S W N W T E K K W E A C T L T K W 357 k AAGAGGTCGACGAACTCGTCAGGGACGAGTTCCGCGGGTCCTACAGATTTACTATTC K E W D E L W R D E. F. R. G. S Y R F T 1596 GATCCATCTCGTCTACGTTTATCAGTAACACTACTCAATTTAAGTTGGAAAGTGCCC R S I S S T F I S N T T Q F K L E S A 395 CCCTTACTGAATGTGTATCCAAAGAAGCAAAGGAAGCCATAGACTCGATATACAAAA P L T E C W S K E A K E A I D S I Y K 1710 AGCAGTACGAGTCTACGCACGTCTTTAGCGGTGATGTGGAATATTACCTGGCACGCG K Q Y E S T H W F S G D V E Y Y L. A R 433 GGGGGTTCTTAATTGCATTCAGACCTATGCTCTCCAACGAACTCGCCAGGCTGTACC G G F L A F R P M L S N E L A R L. Y 824 TGAACGAGCTTGTGAGATCTAACCGCACCTACGACCTAAAAAATCTATTGAACCCCA L N E L W R S N R T Y D L. K. N. L. L. N. P 47 ATGCAAACAATAACAATAACACCACGCGAAGACGCAGGTCTCTCCTGTCAGTACCAG N A N N N N N T T R R. R. R S L L S W P 1938 AACCTCAGCCAACCCAAGATGGTGTGCATAGAGAACAAATTCTACATCGCTTGCACA E P Q. P T Q D G W H R E Q I L H R L. H 509 AACGAGCAGTGGAGGCAACGGCAGGTACCGATTCTTCCAACGTCACCGCCAAACAGC K R A V E A T A G T D S S N W T A K Q t 2052 TGGAGCTCATCAAAACCACGTCGTCTATCGAGTTTGCCATGCTACAGTTTGCATACG L. E L I K T T S S I E F A M L Q F A Y 547 ATCACATCCAATCCCACGTCAATGAAATGCTAAGTAGAATAGCAACTGCGTGGTGTA D H I Q S H V N E M L S R I. A T A W C 266 CCCTCCAAAACAAAGAGCGGACCCTATGGAACGAAATGGTGAAGATTAACCCGAGCG T L Q N K E R T L W N E M W K I N P S F.G. 6B

U.S. Patent Aug. 16, 1994 Sheet 10 of 83 5,338,683

s s

RELATIVE HYDROPHILICITY U.S. Patent Aug. 16, 1994 Sheet 11 of 83 5,338,683

Pyu Niru IMHindIIIah -E.---- HindiiTO AEALED 35 35 EATDES HindIII is 5 & 2CE AND CE

NiruNr. KIKpn -

E. Pyu I : CD NI. Bamh MPst s 4. Bgi Pati Pst 16ATE fift TTTTTTTTT - 2)BIOGESTION b 3)RELIGATE 4)MUMGENTE WITH Pyu I OLIGONUCLEATDES HindIIIMNI FPCV AND FPVC3

HindIII sy Niru pFPEHVBA EcoRI

Pyu I HindII/KpnI KpnI 2)BLUNT-END HISS" initi/NKERS ap Nui 29. Pvu 5)SOLATE 20Obp vFP44 - - HindIII/KpnI FRAGMENT N VIVO RECOMBINATION U.S. Patent Aug. 16, 1994 Sheet 12 of 83 5,338,683

Kpn/HpaI 2)BLUNT-END

3)SOLATE FRAGMENT t2OObp U.S. Patent Aug. 16, 1994 Sheet 13 of 83 5,338,683

FIG. O Construction of plasmids containing EHV-1 gp14 odification of the 5' end of EHV-l 4. Nsi. pVM2LH6g14 -> plMP14M MPSYN240 mutagenesis Neil pVM2LH6gl4-1 -> p-P14M-34 MPSYN24l mutagenesis Nae (partial)/NSI pVM2LH6gl4-1 -> pAP14M-63 MPSYN243 mutagenesis

Removal of extraneous EHV-1 DNA Drai (partial)/Pst pMPl4M-63 -> pip14M-63P MPSYN247/MPSYN248

Movement of H6 promoter/EHV-1 14 to OEES syst NiruI (partial) /XhoI pHES-4 > pHES-MP63 Niru I (partial) /XhoI pMP14M-63P isolate 2.8 kb fragment Replacement of gp14 leader with different lengths Niru (partial) pHES-MP63 isolate 7.2 kb fragment

> pHES-MP1 Niru pMP14M isolate 2.8 kb fragment Niru (partial)

pHES-MP63 isolate 7.2 kb fragment

> pHES-MP34 NI pMP14M-34 isolate 2.7 kb fragment U.S. Patent Aug. 16, 1994 Sheet 14 of 83 5,338,683

E U.S. Patent Aug. 16, 1994 Sheet 15 of 83 5,338,683

oLoLLooLowÐLLIÐoovewooowowoowowwowowowoLBB????wOLOOÐVI???OJBOJNo.b??voT

U.S. Patent Aug. 16, 1994 Sheet 17 of 83 5,338,683

69 28 LZT OST

?MIAVÌXIWCIOJI,?Ãows?ITATATSITIVI ICISHSOWVI(I"IXI?INOWXSAC?H IIIpu?H LT VNK&J."IIJ,XN&Xd.HCIÕ gOT±? I&LVIoaWXIàM.J."IH\'SXJ,ÕVV’

U.S. Patent Aug. 16, 1994 Sheet 19 of 83 5,338,683

ZO? 6 TIE

S?iW.(?)SI,"IXIVI

IIIpu?IH

?I"IÕL:XXIA.CIàI,SHIL'I?NÕVSX SWHCI?ÀIXI+wg?SOTTIV,W,s«IFIIVÌVI

60TZ LLTZ

U.S. Patent Aug. 16, 1994 Sheet 22 of 83 5,338,683

ALZ ZAL

09.*

T,‘INQHXI5)INICI?IO"ISHAXI

IeTO 1,898 909€. €1,9€. 6088

U.S. Patent Aug. 16, 1994 Sheet 25 of 83 5,338,683

08?y 929

CISSIVÌ"IAICI(IA?ISdi A&ÕX"I?IVÌXI&X"I?ICIS »?l914

Z99 •••XI“IIS

6919 90£9 £1,09 Tyws 6099 ALL99 g?799

U.S. Patent Aug. 16, 1994 Sheet 27 of 83 5,338,683

8O 6O 24O 32O 4OO U.S. Patent Aug. 16, 1994 Sheet 28 of 83 5,338,683

8O 6O 24O 32O 4OO U.S. Patent Aug. 16, 1994 Sheet 29 of 83 5,338,683

F.G. 5

OO 2OO 3OO 4OO 5OO U.S. Patent Aug. 16, 1994 Sheet 30 of 83 5,338,683

BamhI FG 16

A Apal Aat KLENOW LIGATE

Aat pcóPCsest/SmaI (2 CSmaIMApaI) CSmaIM

IVR vp668

HindIII SmIKLENow

LIGATE

pCAOO6

26 CSmaIMHindIII) CSmaIMHindII) (SmaIMHindII) VR WP668 MRIvP668 VP822 vP773 U.S. Patent Aug. 16, 1994 Sheet 31 of 83 5,338,683

Cla VP809

pCOPCS657 VR ves68

LIGATE puCAOO9

LIGATE EcoRI.

pjCAOO6

MR v P668 1. 3. CSmaIMHindII)

EcoR FG. 7 VP8 O CSmaIVHindII) U.S. Patent Aug. 16, 1994 Sheet 32 of 83 5,338,683

FG 8 F.G. 8A F.G. 8B FG 8A Sph Apa Nhe Sph Hinc Noel Sph PRV - - - - ey BamHI+ Sphi h CAP

Hind Sphi EcoR Bann Nhe - Sph Hinc Sph SOLATE SOAT MRSYN-2 CNhe MBanh

Hind Sphi Hindi + Apal Nae le Hind CAP CAP MRSYN3-4 MRSYN9 - O

EcoR E Hindi Nhe Banh EcoRV Apal

ByA * Nhe Barn Hinc Hind EcoR MRSYN7-8 CAP CSph MBanh MRSYN7-8 LINKER) CSphi/BamH Sph LINKERosy SS Sph - Hinc C2 / Sph - Nhe SOLATE Hincl Nae U.S. Patent Aug. 16, 1994 Sheet 33 of 83 5,338,683

FG. 8B

Xmal Sph Nael

Hindi + Sph SOLATE MRSYN 9-2O CHind MEcoR LINKER)

MUNG BEAN NUCLEASE EcoRV SOLATE MUNG BEANXmall ScLEAsSN 2Hind EcoRV Xrnal CAP EcoRV Pst

amonganeumoureuses RECOMBINATION VP692s

U.S. Patent Aug. 16, 1994 Sheet 38 of 83 5,338,683

Banh Sph BamH. Nico Barn Prv--- ZZZZZiza..... gpll Bamhi

BamH Z Barn

e(s Nco

Sph Sph + BamHYNco i + BamH SOLATE SOLATE Hind EcoRI + Sph Sph CAP MRSYN2-22 CNCOMEcoR LINKER) Hind 1EcoR Sph MUTOGENESIS MRSYN5MRSYN6

SD478VC Hind 5. p s h 7-xbaO EcoS2'idi 27 "ball + Hpal Nsi Xbal U.S. Patent Aug. 16, 1994 Sheet 39 of 83 5,338,683

06 0$

vIzºÐE?a izºol-l 1S&WVIWATVyW?ywTSW

#,w?ll??n?,?l?m?n???????wuyowi,s?n?,?s? }},w?s?,s?n?r?n??n?,b?,m?s?r,buvº Wd1SdT30SN?N&10v1 w0A&S3T&0A1×x9Hw1x0HA&i JV0100W19000\/001001001000990093v0190w00000000000100000\/100000000wOw0000010000100\,00w90099V ?aeuaewonºnimus,ubi,uwag 099929000\/\/0WOW0000000000\/\/09/09090101000100000000001000000000\,0010100000000000\,0000001000000 ?aeniausiaen?s?n?, #,p?s?n?,nuwiae ?aepiºnism?n?,?s?r,p?m?n?,m?sraeg U.S. Patent Aug. 16, 1994 Sheet 40 of 83 5,338,683 £9· ?woiminism?n?,?s?n?,?s?,?}} ±?????n?,?s?,m?s????????,m?sraeg &AQ0V§43?Td\'S ±??i??i??ninu,uwiaeg #::::::::::::::::::::::::::::::::::::::::prºminisminiaeg ##?,?s?,?s?r,p?s?, ¿?,m?s?,m?laeviºu!!!!! ??,?s?,s?r?m?n?, ±n?,?s?,m???n?,M,SAsv4QM\,013&TO 0291 ??y?c???,s?v?y?s?n?m?,s?n?,m?r???m?n?,m???r; (TAA31wWITN09SA_4 U.S. Patent Aug. 16, 1994 Sheet 41 of 83 5,338,683

Barnh Net2ZZ" Barn PRV - - v Pamhd

Bant

EcoR did Stu-NdeNSNVB Stu" NUCLEASE ISOATE LIGATE

EcoR Hind QS 2 Bg &22MUTAGENESIS MRSYN2MRSYN 3

MUNG BEAN NUCLEASE PARTAL

pMPI3PP

EcoR MUNG BEAN U.S. Patent Aug. 16, 1994 Sheet 42 of 83 5,338,683

06 091 0/2 092

#|23914 1d\,AWO0V5)T1\}\,ATWWT

Å0\,d9A3d.SdA1TÒAS31ÅdAVd4 HWA3NT&0A?QSITVA90&?T&N1H 30WAI3ÅTHWO9GWINMWAHNA148× 031dWAGSd1MWd1&0&10

9111w.100w9www.010101V10\/wwlww.01101w0010100000x010101000000011w9000000010w90000000099000\/91 0100\,010V0VIw0001100v300000001100011\/www.IVOOL9000x00000V001w909.199.99999999x11001100990w195 100W0000000000100000\/00100\,000000001000WOOw00000001001000000001LW10009w5901001001w90009w55 90710100\/000000000001001100000100001000\,00V0109OWJ100WO9010019w.900wOw10000w190900000000001 09?82 0999§ 0£998999999990900001101w9w10\/000000100000\/99109101w900.00WOOw00000099009109099911101w9w595)w0000w

U.S. Patent Aug. 16, 1994 Sheet 44 of 83 5,338,683

FG. 24 Sali F. Sali , Sall Big:ER VACCNA HindIIIHind B:::ERYZZZRR:Nicol Nui Sall

Salt Hind 2 Sall U.S. Patent Aug. 16, 1994 Sheet 45 of 83 5,338,683

F. G. 25

Hind Hind VACCINIA HINDIII 1 - 7-S- pUC8 / Bamha f-HR a BamH Clal Map Barn

Msp pUCl3 Cia Smal Row la LINKERS LIGATE LIGATE

pMP49-5 EcoRMspI EcoR EcoR

FRAGMENTYLIGATE Mep SE Bant Set Cial Sall Barn Xbal U.S. Patent Aug. 16, 1994 Sheet 46 of 83 5,338,683

F. G. 26

8 enFull LIGATE TO ANNEAED OLGONUCEOTDES CE4 AND CE5

Pvu

Bg Pst Pet

)LIGATE IIILLIIIImue 2.)Pst DIGESTION 3)RELIGATE 4)MUTAGENZE WITH Pyu OLIGONUCLEOTDES FPCV AND FPCV3 HindIMPARTIAL EcoRV GATE

U.S. Patent Aug. 16, 1994 Sheet 47 of 83 5,338,683

F. G. 27 HindII f 2 f s Niru Partyrriv Pvu. (S pCE . Pvu BamHIMPst PVu HindII Hin/ Nru glPstI Pst

viz:AS as 23E8 SigESTION ricos 35REIGATE pFPCV A.a) KpnI )KpnIMHpaI 2)BLUNT-END pCPCV Pyu 3)SOLATE 200bp FRAGMENT Pvu s Pvu PVu 9 V BglII Hind LIGATE "Nr. 2BLifeRDEcoRIDEcoRI pRW764.2 SmaI HindI/Nru pSD486VC LIGATE Nico Pvu Pyu I PGPT- CO BglMSna NCOI l a. Bl CoR HindIII Nru BgimDm gTSmaI pCPPRV EcoRI 2)SOLATE 67Obp EcoRI Hind PV pSV2gpSV2gpt HindIII HindIII Bgll Dra

2-d Y2NcoIMEcoRI2)BLUNT-END LIGATE 3)Hind LINKERS Pvu CPPRV 4)ISOLATE I kbp FRAGMENT Hind p gpt - - --vCP55 U.S. Patent Aug. 16, 1994 Sheet 48 of 83 5,338,683

NCO AlwN Xho

Xho Xho Nico CPARTIAL) Nicol

Aw Nico

Aat NCO Sac

PRVL5 PRVL6

AwN CPARTAL)

PRVL3 PRVL4 Aat CPARTIAL) Sac U.S. Patent Aug. 16, 1994 Sheet 49 of 83 5,338,683

F. G. 28B

NCO

Nru Niru Banh Barn

VP4 O - A -r it U.S. Patent Aug. 16, 1994 Sheet 50 of 83 5,338,683

FG.29

FG.29A HSV2 GENOMIC DNA pUC19 Bgli Banh.

Sac B OSac Sac Sac Sac CPARTAL) Set Sst

GB3 GBL4 Sac CPARTAL) Ban U.S. Patent Aug. 16, 1994 Sheet 51 of 83 5,338,683

FG.29B

GBL GBL2 EcoRV CPARTAL) Sst

Bgll Barn U.S. Patent Aug. 16, 1994 Sheet 52 of 83 5,338,683

vP6 8 Prse-1-

FG.29C

HSV2 GENOMIC DNA

Sall Xhol U.S. Patent Aug. 16, 1994 Sheet 53 of 83 5,338,683

2e M Barn Niru Xba Sf Sall Smal Apa

Sna CPARTAL) Sall FG. 3OB

GCL3 U.S. Patent Aug. 16, 1994 Sheet 54 of 83 5,338,683

F. G. 3OC

U.S. Patent Aug. 16, 1994 Sheet 55 of 83 5,338,683

FG.3

FG.3A

HSV2 GENOMC DNA plb 25 Xba e

EcoRV Smal

Smal Dra Pst Pst

GD U.S. Patent Aug. 16, 1994 Sheet 56 of 83 5,338,683

F. G. 3B

EcoRV Ahal Nae Pet VP425 - -es 7O

GDL3 GDL4 Nael Pst

U.S. Patent Aug. 16, 1994 Sheet 57 of 83 5,338,683

Sall

Sall Sal CPARTAL) Pst Pst

BR BR2

F. G. 32A U.S. Patent Aug. 16, 1994 Sheet 58 of 83 5,338,683

Sall

Tth Tth Nicol Nicol

BRL5

BR6

F. G. 32B

BRL7 BR8 Niru Apal CPARTAL) U.S. Patent Aug. 16, 1994 Sheet 59 of 83 5,338,683

U.S. Patent Aug. 16, 1994 Sheet 60 of 83 5,338,683

MRWP293

VP73

U.S. Patent Aug. 16, 1994 Sheet 62 of 83 5,338,683

86 TzT fytyI

89I??

U.S. Patent Aug. 16, 1994 Sheet 65 of 83 5,338,683

889 TT9

A?N'IXITL?IMAHGN?"IJEOM K©I,S?AHWSNÖIXAH?SIN 899******* ?IÐLwwowowwLooooo..ºoooLwow??LLOWIoo?o?o?ooLwLÐIÐo?powwowLoooº?IwºoLolºTOTZ §€9* U.S. Patent Aug. 16, 1994 Sheet 66 of 83 5,338,683 860,- TZ8 9;†8 898 T68

WXYIWSIHVIÖW"IXI?IQIàQIA?CIS -IeuS U.S. Patent Aug. 16, 1994 Sheet 67 of 83 5,338,683

L?76 wooºoºowwooLwºwotwooooºoooLoiol.LLwowowLoovII?bovo?LLIÐboowwwwwwwLOLOLT6Z8

----A'INI,GIJ,CI?Ã U.S. Patent Aug. 16, 1994 Sheet 68 of 83 5,338,683

FG 35

4-O

OO 20O 3OO 4OO 5OO 6OO 7OO 800 900 U.S. Patent Aug. 16, 1994 Sheet 69 of 83 5,338,683

FG. 36 G.36A

FG36B G36C G36D

Cal ATG m3mp 8

ACC Cla Xmal Xmal

ACC Cla

MUTAGENESIS

mp 8gp22Oc5+4) Not EcoRV

FG.36A U.S. Patent Aug. 16, 1994 Sheet 70 of 83 5,338,683

FG. 36B

Not FEcoRV

Not Nar EcoRV

Nar Nar

Not SP3 gp22O U.S. Patent Aug. 16, 1994 Sheet 71 of 83 : 5,338,683

FG36C

Not Not SP3 gp22O

Scal Partial Scal Xho Xho

U.S. Patent Aug. 16, 1994 Sheet 72 of 83 5,338,683

F. G. 36D

SP3 gp340 OR SPI3 gp22O

Not Bgll MUNG-BEAN MUNG-BEAN

U.S. Patent Aug. 16, 1994 Sheet 73 of 83 5,338,683

FG.37

FG.37A EcoR Xmn be 25

EcoR EcoR Xnn Hind

Hinc Xmn

U.S. Patent Aug. 16, 1994 Sheet 74 of 83 5,338,683

FG.37B

Sp3

EcoR EcoR EcoRV EcoRV

U.S. Patent Aug. 16, 1994 Sheet 75 of 83 5,338,683 F.G.38 E.

FG, 38A Smal Barnh — — p3 24 ATG

U.S. Patent Aug. 16, 1994 Sheet 76 of 83 5,338,683

F.G.38B

U.S. Patent Aug. 16, 1994 Sheet 77 of 83 5,338,683

FIG. 39

VACCNA ARM

53gHgBgp340 U.S. Patent Aug. 16, 1994 Sheet 78 of 83 5,338,683

FIG.4O ES33

FG.4OA

Hind Bann plB24

Hind Hind

MUTAGENESIS 24CMVgB 24CMVgBc5+3) sHam- Hind U.S. Patent Aug. 16, 1994 Sheet 79 of 83 5,338,683

FG. 4OB

24CMVgBc5+3) Sp3|

EryBgll

SP3 igB Hindi i IFS

VACCNA LEFT ARM NSE9 Bgll Bgll Hind MUNG BEAN MUNG BEAN .

U.S. Patent Aug. 16, 1994 Sheet 80 of 83 5,338,683

FG 4. FIG. 4AFG.4B

1 ATGAATCTTATAATGCTTATTCTAGCCCTCTGGGCCCCGGTCGCGGGTAGTATGCCT M. N. L. M A W A P W A G S M P

2 CTGCCCGATGTTTCGGAGTACCGAGTAGAGTATTCCGAGGCGCGCGCGGCTCCGA P D V S E Y R V E Y S E A R C W. L. R

24 CCCCGGGTGTACTACCAGACGCTGGAGGGCTACGCGGATCGAGTGCCGACCCCGGTG P R v Y Y Q T L E G Y A D R v P T P V

36 CGCACAAAACTCGGGTTCACCCCCCGCCACAATGCCAAACTATAT R T K L V L. F Y F S P C H Q C G Y Y

481 GAACGACTATTGTTCGAAGATCGCCGTCTAATGGCGTACACGCGCTCACGATTAAG E R L. L. F E D R R L. M. A Y Y A K

601 GGTTGGCTGCACCGACATTTCCCTGGATGTTTCGGACCAGTGGGA G W L H R H F P W M F S D Q. We

FG4 A U.S. Patent Aug. 16, 1994 Sheet 81 of 83 5,338,683

GAATTATCCTTGACCTTTTCGATGAACCTCCGCCCTTGGGGAGACGGAACCGTTACCGCC E L S L T L. F D E P P P L V E T E P L P PX

TCGGGCGGTCGACTGGAGGCTCTGTGGACCCTGCGCGGGAACCTGTCCGTGCCCACGCCGACA S G G R L E A L W T L R G N L S W P T P TX

GAGGACACTCCGAAAGCCTCGTCGCAAAACGCTACTGGCCCGGGACTATCGTGTTCCCCAA E D S E S L V A K R Y W L R D Y R W P Q>

GTAGAGTGCGAACCCCGGTGCCTCGTGCCTTGGGTTCCCCTGTGGAGCTCGTTAGAGGACATC W E C E P R C L V P W V P L W S S L E D >

TCGGCGCAGATACGCGATGATGGGGCAGGATCAAGTGTGGGGGCTGTAGGAAA S A G Y T L M M W A V Q. V. F W G Y V Ko FG4B U.S. Patent Aug. 16, 1994 Sheet 82 of 83 5,338,683

FG.42

1 ATGCTACGCCGGGGAAGCCTCCGGAACCCTCTCGCGACCTGCCTGTTGTGGTGGCTG M L R R G S L R N P L A T C L W W

21 ATTCAAAATCATGTACTGAAAGGGCGGTGAAACTCTATGGACAATTCCCCTCGCC Q N H W L K G A V K L Y G G F P S P

24 ATCCTCGTGGAAGGCACCGCGACAGCTACCGAGGCGCTCTACATTCTGCTGCCCACG V E G T A T A T E A L Y P

36 CGGGATTGATGAACGCTTCGGGTCCGGTAACGATTCCGGGACGCCGATGGGG R D C Y E R F W C P V Y D S G T P M G

481 TTCGGACTGTTTGCCGGGGCTGTGTCACACCCGATCCCTCCTCCTGATAGGGT F G F C R G C V T R S C G

FG.42A U.S. Patent Aug. 16, 1994 Sheet 83 of 83 5,338,683

GGAGTGGGGCGGCAGCTACGGAGGAGACGAGAGAACCGACTACTTACGGCGGCTGTGTT G V V A A A T E E T R E P T Y F T C G C VX

AAGACTTTGCGGGCCTTGGCTGGCTACACGACGGTGAAAATCACGAAAGGCACCGGCAGCCC K T L R A A H D G E N H E R H R G P2

GAGCTATCGCCGCCGGAAGGAAACCGACCCCGAAACTATTCTGTACCCTAACACTCGCCTCC E L S P P E G N R P R N Y S W T L T L A So

CTTGATGAACTTGACGTACCTCTGGTATCTAGGCGACTACGGGGCGATACTAAAAATTTAT L. L. M. N. L. Y Y L. G D Y G A K Y>

TATATCCACCCGCGAAAA Y Y P P R Ed FG. 42B 5,338,683 2 gives a poxvirus modified by the presence, in a nones WACCNAVIRUS CONTAINING DNA sential region of its genome, of foreign DNA sequences. SEQUENCES ENCODING HERPESVIRUS GLY The term "foreign' DNA designates exogenous DNA, COPROTEINS particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome CROSS REFERENCE TO RELATED into which the exogenous DNA is placed. APPLICATIONS . Genetic recombination is in general the exchange of This application is a continuation-in-part of applica homologous sections of DNA between two strands of tion Ser. No. 394,488, filed Aug. 16, 1989, now aban DNA. In certain viruses RNA may replace DNA. Ho doned, which in turn is a continuation-in-part of appli 10 mologous sections of nucleic acid are sections of nucleic cation Ser. No. 339,004, filed Apr. 17, 1989, now aban acid (DNA or RNA) which have the same sequence of doned. This application is also a continuation-in-part of nucleotide bases. U.S. application Ser. No. 07/090,209, filed Aug. 27, Genetic recombination may take place naturally dur 1987, now abandoned, which is a division of U.S. appli ing the replication or manufacture of new viral genomes cation Ser. No. 622,135, filed Jun. 19, 1984, now U.S. 15 within the infected host cell. Thus, genetic recombina Pat. No. 4,722,848, which in turn is a continuation-in tion between vital genes may occur during the viral part of U.S. application Ser. No. 446,824, filed Dec. 8, replication cycle that takes place in a host cell which is 1982, now U.S. Pat. No. 4,603,112, which in turn is a co-infected with two or more different viruses or other continuation-in-part of U.S. application Ser. No. genetic constructs. A section of DNA from a first ge 334,456, filed Dec. 24, 1981, now U.S. Pat. No. 20 nome is used interchangeably in constructing the sec 4,769,330. tion of the genome of a second co-infecting virus in FIELD OF THE INVENTION which the DNA is homologous with that of the first The present invention relates to a modified poxvirus viral genome. and to methods of making and using the same. More in 25 However, recombination can also take place between particular, the invention relates to recombinant poxvi sections of DNA in different genomes that are not per rus, which virus expresses gene products of a herpesvi fectly homologous. If one such section is from a first rus gene, and to vaccines which provide protective genome homologous with a section of another genome immunity against herpesvirus infections. except for the presence within the first section of, for Several publications are referenced in this application 30 example, a genetic marker or a gene coding for an anti by arabic numerals within parentheses. Full citation to genic determinant inserted into a portion of the homolo these references is found at the end of the specification gous DNA, recombination can still take place and the immediately preceding the claims. These references products of that recombination are then detectable by describe the state-of-the-art to which this invention the presence of that genetic marker or gene in the re pertains. 35 combinant viral genome. Successful expression of the inserted DNA genetic BACKGROUND OF THE INVENTION sequence by the modified infectious virus requires two Vaccinia virus and more recently other poxviruses conditions. First, the insertion must be into a nonessen have been used for the insertion and expression of for tial region of the virus in order that the modified virus eign genes. The basic technique of inserting foreign remain viable. The second condition for expression of genes into live infectious poxvirus involves recombina inserted DNA is the presence of a promoter in the tion between pox DNA sequences flanking a foreign proper relationship to the inserted DNA. The promoter genetic element in a donor plasmid and homologous must be placed so that it is located upstream from the sequences present in the rescuing poxvirus (28). DNA sequence to be expressed. Specifically, the recombinant poxviruses are con 45 There are two subtypes of equine herpesvirus that, structed in two steps known in the art and analogous to although they contain cross-neutralizing epitopes, can the methods for creating synthetic recombinants of the be distinguished by their antigenic profiles, restriction vaccinia virus described in U.S. Pat. No. 4,603,112, the disclosure of which patent is incorporated herein by endonuclease fingerprints and their pathogenicity for reference. 50 horses (1). Equine herpesvirus 1 (EHV-1) is associated First, the DNA gene sequence to be inserted into the with respiratory tract disease, central nervous system virus, particularly an open reading frame from a non disorders and classic herpetic abortions whereas equine pox source, is placed into an E. coli plasmid construct herpesvirus 4 (EHV-4) is predominantly associated into which DNA homologous to a section of DNA of with respiratory tract disease (1,48). Equine herpesvi the poxvirus has been inserted. Separately, the DNA 55 ruses are members of the alphaherpesvirus subfamily gene sequence to be inserted is ligated to a promoter. and display many of the typical biological and biochem The promoter-gene linkage is positioned in the plasmid ical characteristics of human herpesviruses, such as construct so that the promoter-gene linkage is flanked genomic isomerization, regulation of gene expression, on both ends by DNA homologous to a DNA sequence establishment of latent infections, generation of defec flanking a region of pox DNA containing a nonessential tive interfering virus particles, induction of neurologi locus. The resulting plasmid construct is then amplified cal disorders, and in vitro oncogenic transformation by growth within E. coli bacteria (11) and isolated (1,4,23). Thus, EHV advantageously can be used for (1220). studying the varied biological consequences of herpes Second, the isolated plasmid containing the DNA virus infections. gene sequence to be inserted is transfected into a cell 65 Herpesvirus glycoproteins mediate essential viral culture, e.g. chick embryo fibroblasts, along with the functions such as cellular attachment and penetration, poxvirus. Recombination between homologous pox cell to cell spread of the virus and, importantly, deter DNA in the plasmid and the viral genome respectively mine the pathogenicity profile of infection. Herpesvirus 5,338,683 3 4 glycoproteins are critical components in the interaction several glycoproteins encoded from the S component in with the host immune system (36,37). the other alphaherpesviruses (66,79,80). Based on its The well characterized glycoproteins of herpes sim genomic position, it has been speculated that gp17/18 plex virus include gb, gC, gD, gB, gC, gh and g could be the HSV gF analog (2). (36.37,49-55). A number of studies have indicated the Pseudorabies virus (PRV), an alphaherpesvirus, is the importance of herpes simplex virus glycoproteins in causative agent of Aujesky's disease. The disease is eliciting immune responses. Hence, it has been reported highly infectious causing serious economic losses in the that g3 and go can elicit important immune responses swine industry. The disease is associated with high mor (6,8,13,18,2122,26,27,30,4446,47). gC can stimulate bidity and mortality among piglets and is characterized class I restricted cytotoxic lymphocytes (15.32) O by severe respiratory illness, abortions, reduced litter whereas gld can stimulate class II cytotoxic T cell re size and decreased growth rates of survivors. Fatal sponses (21,22,44,46,47). g6 was shown to be a target encephalitis is a frequent consequence of infection. La for complement-dependent antibody directed virus neu tent vital infections, a characteristic of herpes viruses, tralization (38,39). A number of glycoproteins from can be established thus allowing recovered adult swine other herpesviruses have also been shown to elicit im 15 to serve as chronic carriers of the virus. For a recent portant immune responses (5,10,36,56). extensive review see Wittmann and Rziha (81). Both subtypes of EHV express six abundant glyco The PRV genome consists of a 90x 106 dalton double proteins (1,3,43). The genomic portions of the DNA stranded DNA (82) separated by inverted repeat se sequences encoding gp2, gp10, gp13, gp14, gp17/18, quences into unique long (UL) or unique short (US) and gp21/22a have been determined using lambda ft11 20 segments (83,84). The PRV genome encodes approxi expression vectors and monoclonal antibodies (3). Gly mately 100 polypeptides whose expression is regulated coproteins gp13 and gp14 were located in the same in a cascade-like fashion similar to other herpesviruses locations within the L component of the genome to (85,86). To date, five glycoproteins gp1, gp1, gp, which the gC and g8 homologs, respectively, of herpes gp63 and gp50 have been shown to be associated with simplex virus map (3). EHV-1 appears unique among 25 the viral envelope and associated with the various mem the alphaherpesviruses whose glycoprotein genes have branous structures of PRV infected cells (80,86-91). A been mapped in that five of its six major glycoproteins sixth PRV encoded glycoprotein (gx) is released into are encoded from sequences within the genome L com the culture medium (92). The physical location of these ponent while only one (gp17/18) is mapped to the Us glycoproteins on the PRV genome and their DNA region. Analyzing these data, it has been predicted that 30 sequence are currently known (62,80,91-98). As with some of the lowabundance glycoproteins identified in the glycoproteins of other herpesviruses, the PRV gly EHV-1 virions as well as EHV-1 glycoproteins not yet coproteins mediate essential vital functions such as cel identified map to the S component of the genome (3). lular attachment and penetration into or release from The envelope glycoproteins are the principal immuno cells. The PRV glycoproteins are critical in the patho gens of herpesviruses involved in eliciting both humoral 35 genicity profile of PRV infection and are critical com and cellular host immune responses (5,8,73-75) and so ponents in the resolution of disease and the immune are of the highest interest for those attempting to design Status. vaccines. PRV gpI is non-essential for virus replication in vitro Recently, the nucleotide sequence of the Kentucky and in vivo and is absent from most attenuated PRV T431 strain of the EHV-1 transcriptional unit encoding 40 strains (99). The attenuated nature of these gp-deleted gp13 has been reported (2). An open reading frame strains also indicates a possible role for gpin virulence encodes a 468 amino acid primary translation product of (99,100). Other PRV proteins, however, appear to be 51 kDa. The protein has the characteristic features of a involved in this function since expression of gp alone is membrane-spanning protein with nine potential N not sufficient to produce high levels of virulence (100). linked glycosylation sites (Asn-X-Ser/Thr) present in 45 The role gp plays in eliciting an immune response the surface domain between the putative signal and against PRV is unclear. Monoclonal antibodies against transmembrane anchor portions of the protein (2). The gp can neutralize virus in vitro (101) and passively glycoprotein was shown to be homologous to the her protect immunized mice against a lethal PRV challenge pes simplex virus (HSV) go-1 and gC-2, to the pseudo (81). Kost et al. (98) have recently described the expres rabies virus (PRV) gpIII and the varicella-zoster virus 50 sion of PRV gpI in vaccinia virus recombinants either (VZV) gpV (2). EHV-1 gp13 is thus the structural alone or in association with gp50 and gp63. Intracranial homolog of the herpesvirus gC-like glycoproteins. inoculation of the vaccinia recombinants in mice re The nucleotide sequence of EHV-1 gp14 (71,72) has sulted in increased virulence particularly when PRV recently been reported. Analysis of the predicted amino gpI was associated with coexpression of gp50 and gp63. acid sequence of gp14 glycoprotein revealed significant 55 In swine, however, neutralizing antibodies against homology to the corresponding glycoprotein of HSV, gpI are not produced (5). In addition, a recombinant gB. vaccinia virus expressing PRV gpI-encoded polypep Monoclonal antibodies directed against some EHV-1 tides (98) does not protect mice against a lethal PRV glycoproteins have been shown to be neutralizing (76). challenge (relative to the protection afforded by the Passive immunization experiments demonstrated that wildtype vaccinia virus control). These data, taken monoclonal antibodies directed against gp13 or gp14 together, suggest that PRV gp is more appropriate as a (77) or against gp13, gp14 or gp17/18 (78) could protect diagnostic probe rather than as a component in a sub hamsters against a lethal challenge. Other g and gC unit vaccine. glycoprotein analogs are also involved in protection PRV glycoprotein gp63 is located adjacent to gp50 in against diseases caused by alphaherpesviruses (8,10,73). 65 the US region of the PRV genome (80). The coding The EHV-1 gp17/18 glycoprotein, although character sequence for PRV gp63 starts with three consecutive ized as another potential protective immunogen, had ATG codons approximately 20 nucleotides down until now no known structural counterpart among the stream from the stop codon of gp50. There is no recog 5,338,683 5 6 nizable transcriptional signal motif and translation prob primary infections (113) and to suppress recurrent epi ably occurs from the same transcript as gp50. PRV gp63 sodes (114), the control and treatment of these infec is non-essential in vitro (88). PRV gp63 as a continuous tions is far from ideal. A vaccine to prevent primary and DNA sequence with PRV gp50 has been expressed in recurrent infections is therefore needed. vaccinia virus as reported by Kost et al. (98). The con The herpes simplex virus type 1 (HSV1) genome tribution of PRV gp63 to protection in mice against encodes at least eight antigenically distinct glycopro PRV challenge is difficult to assess since those studies teins:gb, gC, gD, gE, gC, gh, gland g (115). Homo did not dissect the contributions of PRV gp50 and gp63. logues for these genes appear HRPV: 224.5. PAT 12 to PRV glycoprotein gX is a non-structural glycopro be present in HSV2 (116-119). Since these glycopro tein whose end product is secreted into the extracellular 10 teins are present in both the virion envelope and the fluid (85.92). No in vitro neutralization of PRV was infected cell plasma membrane, they can induce hu obtained with either polyclonal or monoclonal sera to moral and cell-mediated protective immune responses PRVgX (102,103) and subunit gx vaccines were non (37). protective against challenge (104). The relative importance of humoral and cellular im PRV glycoprotein gp50 is the Herpes simplex virus 15 munity in protection against herpes simplex virus infec type 1 (HSV-1) gld analog (97). The DNA open reading tions has not been completely elucidated. Mice immu frame encodes 402 amino acids (95). The mature nized with purified HSV1 ges, gC or gld are protected glycosylated form (50-60 kDa) contains O-linked car against lethal HSV1 challenge (120). Mice have also bohydrate without N-linked glycosylation (95). Swine been protected against lethal HSV1 or HSV2 challenge serum is highly reactive with PRV gp50, suggesting its 20 by passive immunization with antibodies to total HSV1 importance as an immunogen. Monoclonal antibodies to (121) or HSV2 (122) virus and with antibodies to the gp50 neutralize PRV in vitro with or without comple individual HSV2 gb, gC, gD or gF glycoproteins (123). ment (97,105,106) and passively protect mice This protection, however, appears to be dependent (102,105,106) and swine (102). Vaccinia virus recombi upon a competent T-cell response since animals in nants expressing PRV gp50 induced serum neutralizing 25 munosuppressed by irradiation, cyclophosphamide or antibodies and protected both mice and swine against anti-thymocyte serum were not protected (124). lethal PRV challenge (98,107,108). The contribution of the individual glycoproteins in The PRVgpIII gene is located in the UL region of the eliciting a protective immune response is not com genome. The 1437 bp open reading frame encodes a pletely understood. Expression of these glycoproteins protein of 479 amino acids. The 50.9 kDa deduced pri 30 in a heterologous system, such as vaccinia, has allowed mary translation product has eight potential N-linked some of these parameters to be analyzed. For example, glycosylation sites (96). PRV g|II is the HSV-1 gC vaccinia virus vectors expressing HSV1 g (125) and analog (96). Functional replacement of PRV g|II by HSV1 gC (32) have been shown to induce cytotoxic HSVgC was not observed (109). Although PRV gIII is T-cell responses. In addition, it has been shown that nonessential for replication in vitro (110,111), the ma 35 mice immunized with recombinant vaccinia virus ex ture glycosylated form (98 kDa) is an abundant constit pressing either HSV1 gb(8), HSV1 gC (126) or HSV1 uent of the PRV envelope. Anti-gpIII monoclonal anti gD (26) are protected against a lethal challenge of bodies neutralize the virus in vitro with or without HSV1. A recombinant vaccinia virus expressing HSV1 complement (86,106.110) and can passively protect gD has also been shown to be protective against HSV2 mice and swine (102). The PRV glycoprotein g|I can in a guinea pig model system (44). It is not known, protect mice and swine from lethal PRV challenge after however, whether expression of multiple HSV antigens immunization with a Cro/g|II fusion protein expressed will result in a potentiation of this protective response. in E. coli (Robbins, A., R. Watson, L. Enquist, Euro Bovine herpesvirus 1 (BHV1) is responsible for a pean Patent application 162738A1) or when expressed variety of diseases in cattle, including conjunctivitis, in a vaccinia recombinant (Panicali, D., L. Gritz, G. 45 vulvovaginitis and abortion (127). It is also one of the Mazzara, European Patent application 0261940A2). most important agents of bovine respiratory disease, One of the main constituents of the PRV envelope is acting either directly or as a predisposing factor for a disulfide linked complex of three glycoproteins (120 bacterial infection (128). kDa, 67 kDa and 58 kDa) designated as PRV gpII ac BHV1 specifies more than 30 structural polypeptides, cording to the nomenclature of Hampl (86). The DNA 50 11 of which are glycosylated (129). Four of these glyco sequence encoding PRV gpII is located in the left end proteins, g, g|I, g|II and gV, have been characterized of UL. The open reading frame of 2976 nucleotides and found to be homologous to the herpes simplex virus encodes a primary translation product of 913 amino (HSV) glycoproteins gb, gC, gD, and gF (130,131). acids or 110 kDa. PRV gpII is the HSV-1 gR homolog Subunit vaccines consisting of gl, g|I and/or glV (62). Monoclonal antibodies directed against PRV gpII 55 have been shown to protect cattle from disease (using a have been shown to neutralize the virus in Vitro (5) BHV1/Pasteurella haemolytica aerosol challenge with or without complement (81). Moreover, passive model) but not from infection (132). These results indi immunization studies demonstrated that neutralizing cate the importance of these glycoproteins in eliciting a monoclonal antibodies partially protected swine but successful immune response against BHV1. failed to protect mice from virulent virus challenge 60 gI and g|II have also been cloned into vaccinia virus (102). To date, the active immunization of swine with and cattle immunized with these recombinants are PRV gpII glycoprotein has not been reported. shown to produce neutralizing antibodies to BHV1 During the past 20 years the incidence of genital (56,133). infections caused by herpes simplex virus type 2 Feline rhinotracheitis is a common and worldwide (HSV2) has increased significantly. Recent estimates 65 disease of cats which is caused by an alphaherpesvirus indicate that in the United States, 5-20 million people designated feline herpesvirus type 1 (FHV-1). Like have genital herpes (112). Although oral treatment with other herpesviruses, FHV-1 establishes a latent infec acyclovir has been shown to reduce the severity of tion which results in periodic reactivation (134). FHV-1 5,338,683 7 8 infections in breeding colonies are characterized by a the Wyeth vaccinia strain. The Wyeth strain has been high rate of mortality in kittens. Secondary infections of widely used as a vaccine strain. the upper respiratory tract are quite debilitating in Monoclonal antibodies directed against the gp85, the adults. The control of this disease is currently attempted EBV homologue to HSV1 gh, have been described as by using modified live or inactivated vaccines which in vitro neutralizing antibodies (168,169). can suppress the development of clinical signs but do Human cytomegalovirus (HCMV) is a member of the not prevent infection that results in shedding of virus. betaherpesvirinae subfamily (family Herpesviridae). Thus, asymptomatic vaccinated cats can spread virulent HCMV can produce apersistent productive infection in virus and latent infections cannot be prevented by exist the face of substantial specific immunity. Even if ing vaccines (135) or by the safer purified subunits vac 10 HCMV possesses a low pathogenicity in general, intra cines under development (136,137). uterine infection causes brain damages or deafness in Herpesvirus glycoproteins mediate attachment of the about 0.15% of all newborns and it is the most common virion to the host cell and are extremely important in infectious complication of organ transplantation (170). vital infectivity (138,139). They also determine the sub Although the efficacy of an experimental live attenu type specificity of the virus (140). Herpesvirus glyco 15 ated (Towne strain) HCMV vaccine has been demon proteins antigens are recognized by both the humoral strated (171), concerns about live vaccine strains have and cellular immune systems and have been shown to directed efforts towards the identification of HCMV evoke protective immune responses in vaccinated hosts proteins usable as a subunit vaccine. In this prospect the (44,107,141,142). FHV-1 has been shown to contain at identification of virion glycoproteins and their evalua least 23 different proteins (143,144). Of these, at least 20 tion as protective agents is an important step. five are glycosylated (144,145) with reported molecular Three immunologically distinct families of glycopro masses ranging from 120 kDa to 60 kDa. The FHV-1 teins associated with the HCMV envelope have been glycoproteins have been shown to be immunogenic described (172): gCI (gp55 and gp93-130); gCII (143,145). (gp47-52); and gCIII (gp85-p145). Like several other alphaherpesviruses, FHV-1 ap 25 The gene coding for gCI is homologous to HSVIgB. pears to have a homolog of glycoprotein B (gb) of The gCII glycoproteins are coded by a family of five HSV-1, and partial sequence of the FHV-1 gbgene has genes (HXLF) arranged in tandem and sharing one or recently been reported (146). The HSV-1 gR is required two regions of homology. More probably gCII is coded for virus entry and for cell fusion (147-149). The HSV-1 by only two of these genes (172,173). The gene coding gB and the g8 analogs of other herpesviruses have been 30 for gCIII is homologous to HSVIgh (174). shown to elicit important circulating antibody as well as In vitro neutralizing antibodies specifically directed cell-mediated immune responses (8,10,3747,73,150). against each of these families have been described The FPIV-1 gR glycoprotein is a 134 kDa complex (174-176). which is dissociated with B-mercaptoethanol into two Suitably modified poxvirus mutants carrying exoge glycoproteins of 66 kDa and 60kDa. The FHV-1 DNA 35 nous equine herpesvirus genes which are expressed in a genome is approximately 134 Kb in size (153). host as an antigenic determinant eliciting the production Epstein Barr Virus (EBV), a human B lymphotropic by the host of antibodies to herpesvirus antigens repre herpesvirus, is a member of the genus lymphocryp sent novel vaccines which avoid the drawbacks of con tovirus which belongs to the subfamily gammaherpes ventional vaccines employing killed or attenuated live virus (115). It is the causative agent of infectious mono organisms. Thus, for instance, the production of vac nucleosis (154) and of B-cell lymphomas (156). EBV is cines from killed organisms requires the growth of large associated with two human malignancies: the endemic quantities of the organisms followed by a treatment Burkitt's lymphoma and the undifferentiated nasopha which will selectively destroy their infectivity without ryngeal carcinoma (156). affecting their antigenicity. On the other hand, vaccines Since the EBV genome was completely sequenced 45 containing attenuated live organisms always present the (207) as the genomes of VZV (66) and HSV1 (158) possibility of a reversion of the attenuated organism to numerous homologies between these different herpesvi a pathogenic state. In contrast, when a recombinant ruses have been described (159). In some cases these poxvirus suitably modified with an equine herpesvirus homologies have been used to predict the potential gene coding for an antigenic determinant of a disease functions of some open reading frame (ORFs) of EBV. 50 producing herpesvirus is used as a vaccine, the possibil The EBV genes homologous to the HSV1 genes in ity of reversion to a pathogenic organism is avoided volved in immunity are of particular interest. So the since the poxvirus contains only the gene coding for the EBV BALF4 gene has homologies with HSV1 g (68) antigenic determinant of the disease-producing organ and the EBV BXLF2 gene with HSV1 gH (161). Fi ism and not those genetic portions of the organism re nally, the EBV BBRF3 gene contains homologies with 55 sponsible for the replication of the pathogen. a CMV membrane protein (162). PRV fatally infects many mammalian species (cattle, Among the EBV proteins, the two major envelope dogs, etc.). Adult pigs, however, usually survive infec glycoproteins gp340 and gp220 are the best character tion and therefore represent an important virus reser ized potential vaccinating antigens. They are derived voir. Because PRV causes severe economic losses, vac from the same gene by splicing without a change in the cination of pigs with attenuated or killed vaccines is reading frame (163,164). Monoclonal antibodies and performed in many countries. polyclonal sera directed against gp340 neutralize EBV Attempts to control PRV infection in swine and to in vitro (165). The cottontop tamarins, the only suscep reduce economic losses have been made by active in tible animal, can be protected by an immunization with munization with modified live or inactivated vaccines. purified gp340 (166) and with a recombinant EBV 65 Attenuated vaccines which generally induce long last gp340 vaccinia virus (167). In this case, the protection ing immunity and are cost efficient present the risk of was achieved with a recombinant derived from the WR insufficient attenuation or genetic instability. Inacti vaccinia strain but not with a recombinant derived from vated vaccines are less efficient, require several immuni 5,338,683 10 zations and usually contain potent adjuvants. These successfully vaccinated in the presence of waning ma latter formulations can induce post-vaccinal allergic ternal immunity. reactions such as lack of appetite, hyperthermia or abor It can thus be appreciated that provision of a herpes tion in pregnant sows. These vaccine types also suffer virus recombinant poxvirus, and of vaccines which from certain drawbacks with respect to prevention of 5 provide protective immunity against herpesvirus infec latent infections, overcoming the effects of maternal tions, which confer on the art the advantages of live antibodies on vaccination efficacy, and eliminating the virus inoculation but which reduce or eliminate the potential use of a serological diagnostic assay to distin previously discussed problems would be a highly desir guish vaccinated animals from those previously infected able advance over the current state of technology. with PRV. 10 Alternative vaccination strategies such as the use of OBJECTS OF THE INVENTION recombinant poxviruses that express immunologically It is therefore an object of this invention to provide pertinent PRV gene products would have certain ad recombinant poxviruses, which viruses express gene vantages: (a) eliminate live attenuated PRV vaccine products of herpesvirus, and to provide a method of strains from the field; and (b) allow the distinction of 15 making such recombinant poxviruses. vaccinated versus infected or seropositive animals. The It is an additional object of this invention to provide latter could be accomplished by the use of appropriate for the cloning and expression of herpesvirus coding diagnostic reagents that would precisely distinguish sequences in a poxvirus vector, particularly a vaccinia vaccinated from naturally infected animals. This is an virus, fowlpox virus or canarypox virus vector. important consideration because of existing regulations It is another object of this invention to provide a controlling the movement of seropositive animals. Fur vaccine which is capable of eliciting herpesvirus neu ther, vaccination is more economical and preferable to tralizing antibodies and protective immunity against a testing and eliminating infected animals from the lots. lethal herpesvirus challenge. The development of such vaccines requires a knowl These and other objects and advantages of the pres edge of the contributions made by appropriate PRV 25 ent invention will become more readily apparent after antigens to the induction of protective immunity. In the consideration of the following. case of PRV, as with other members of the herpesvirus family, the glycoproteins are important candidates for STATEMENT OF THE INVENTION antigens to be present in an effective subunit recombi In one aspect, the present invention relates to a re nant vaccine. 30 combinant poxvirus containing therein a DNA se The technology of generating vaccinia virus recom quence from herpesvirus in a nonessential region of the binants has recently been extended to other members of poxvirus genome. Advantageously, the herpesvirus is a the poxvirus family which have a more restricted host member of the alphaherpesvirus, betaherpesvirus or range. In particular, avipoxviruses, which replicate in gammaherpesvirus subfamily. In particular, the DNA avian species, have been engineered to express immuno 35 sequence from herpesvirus codes for a herpesvirus gly logically pertinent gene products. Inoculation of avian coprotein. More in particular, the herpesvirus glyco (42,177) and non-avian species (41) with avipoxvirus protein is selected from the group consisting of equine recombinants elicited protective immune responses herpesvirus gp13, equine herpesvirus gp14, equine her against the corresponding pathogen. pesvirus gld, equine herpesvirus gp63, equine herpesvi Attenuated live vaccines and inactivated vaccines to rus ge, pseudorabies virus gp 50, pseudorabies virus BHV1 have been available for over 30 years and have gpi, pseudorabies virus gp1, pseudorabies virus gp, successfully reduced the incidence of BHV1 related herpes simplex virus gb, herpes simplex virus gC, her diseases. These vaccines, however, do not prevent la pes simplex virus gld, bovine herpes virus gl, feline tent infection or reinfection with wildtype virus. They herpesvirus gb, Epstein-Barr virus gp220, Epstein-Barr also complicate the differentiation between infected and 45 virus gp340, Epstein-Barr virus gb, Epstein-Barr virus vaccinated animals. gH and human cytomegalovirus gb. Both types of vaccines have other significant draw According to the present invention, the recombinant backs. Vaccination of pregnant cows with attenuated poxvirus expresses gene products of the foreign herpes live vaccines can cause fetal death and subsequent abor virus gene. In particular, the foreign DNA sequence tion (127). In addition, vaccinated animals have been 50 codes for a herpesvirus glycoprotein and the foreign shown to shed virus (178). Therefore, vaccinated ani DNA is expressed in a host by the production of the mals kept with pregnant cows can spread infectious herpesvirus glycoprotein. Advantageously, a plurality virus to the pregnant animal and cause abortion of the of herpesvirus glycoproteins are coexpressed in the host fetus. w by the recombinant poxvirus. The poxvirus is advanta Inactivated vaccines do not induce abortions or pro 55 geously a vaccinia virus or an avipox virus, such as voke viral excretion. However, they necessitate the use fowlpox virus or canarypox virus. of adjuvants and can cause fatal hypersensitivity reac In another aspect, the present invention relates to a tions (anaphylaxis) and nonfatal inflammation and fever vaccine for inducing an immunological response in a (179). host animal inoculated with the vaccine, said vaccine One of the more important issues in vaccination is including a carrier and a recombinant poxvirus contain overcoming or avoiding maternal immunity. In this ing, in a nonessential region thereof, DNA from herpes respect, if a mother is immune to a particular pathogen, virus. More in particular, the DNA codes for and ex the "immunity” in the mother will be passed on to the presses a herpesvirus glycoprotein. Advantageously, a newborn via the antibodies present in the colostrum plurality of herpesvirus glycoproteins are coexpressed and/or by additional pathways. Nevertheless, the new 65 in the host by the poxvirus. The poxvirus used in the born cannot be successfully vaccinated until the level of vaccine according to the present invention is advanta maternal immunity has waned sufficiently. Therefore, geously a vaccinia virus or an avipox virus, such as there is a narrow window where the newborn can be fowlpox virus or canarypox virus. 5,338,683 11 12 In another aspect, the present invention relates to and pHES-MP34 containing modified versions of the mechanisms to bypass the issue of maternal immunity. If EHV-1 gp14 gene; the barrier is due to the presence of antibodies to a given FIG. 11 is a map of the BamHI cleavage sites of the antigen(s) then the barrier of maternal immunity may be EHV-1 Kentucky D strain indicating the inverted re overcome or avoided by using, selectively, vectors peats of the genome by boxes, showing the location of expressing defined subsets of antigens. For example, the the six major EHV-1 glycoprotein genes and showing pregnant animal can be vaccinated with a recombinant an expansion of the region of the genome which in vaccinia virus expressing pseudorabies virus glycopro cludes the gld, gp63 and gF genes; tein gp50 and the offspring can be vaccinated at birth or FIG. 12 shows the nucleotide sequence of an EHV-1 shortly thereafter with vaccinia recombinants express 10 6402 base-pair fragment containing the gld, gp63 and ing other pseudorabies virus glycoproteins gp or gE coding sequences; gp1.I or combinations thereof. On the other hand, if the FIG. 13 is a hydropathy plot of the sequence of 402 barrier presented by maternal immunity is due to the amino acids composing EHV-1 gld; vector then one may differentially vaccinate the mother FIG. 14 is a hydropathy plot of the sequence of 413 with one vector (vaccinia or avipox) and vaccinate the 15 amino acids composing EHV-1 gp63; offspring with the other vector. This procedure, of FIG. 15 is a hydropathy plot of the sequence of 552 course, takes into consideration not only the use of amino acids composing EHV-1 gF; different vectors but also vectors expressing a different FIG. 16 schematically shows a method for the con constellation of glycoproteins. Thus, the present inven struction of donor plasmids pCA006, p.JCA007 and tion relates to a method for overcoming or avoiding 20 pJCA008 containing the EHV-1 gld gene, the EHV-1 maternal immunity which would otherwise prevent gE gene and the EHV-1 gp63 gene, respectively, and successful immunization in a newborn offspring. By the generation of recombinant vaccinia virus containing present invention, the newborn offspring is inoculated these genes; with a recombinant poxvirus containing therein DNA FIG. 17 schematically shows a method for the con from a non-pox source in a nonessential region of the 25 struction of donor plasmids pCA009 (containing the poxvirus genome, said DNA coding for a first antigen EHV-1 gD and gp63 genes) and pCA010 (containing of a pathogen of the newborn offspring, and said anti the EHV-1 gld, gp63 and g. genes), and generation of gen being different from a second antigen of the same recombinant vaccinia virus containing these genes; pathogen used to induce an immunological response to FIG. 18 schematically shows a method for the con the same pathogen in the mother of the newborn off 30 struction of donor plasmid PR18 containing the PRV spring. Also by the present invention, the newborn gpI gene, and generation of recombinant vaccinia virus offspring is inoculated with a recombinant first poxvirus expressing the PRV gpI gene; containing therein DNA from a non-pox source in a FIG. 19 shows the DNA sequence of the PRV gpII nonessential region of the first poxvirus genome, said open reading frame; DNA coding for an antigen of a pathogen of the new 35 FIG. 20 schematically shows a method for the con born offspring, and said first poxvirus being different struction of donor plasmid pPR24 containing the PRV from a recombinant second poxvirus used to induce an gpIII gene, and generation of recombinant vaccinia immunological response to the same pathogen in the virus expressing the PRV gpIII gene; mother of the newborn offspring. FIG. 21 shows the DNA sequence of the PRV gpIII open reading frame; BRIEF DESCRIPTION OF THE DRAWINGS FIG. 22 schematically shows a method for the con A better understanding of the present invention will struction of donor plasmid pPR26 containing the PRV be had by referring to the accompanying drawings, in gp50 gene, and generation of recombinant vaccinia which: virus expressing the PRV gp50 gene; FIG. 1 schematically shows a method for the con 45 FIG. 23 shows the DNA sequence of the PRV gp50 struction of the recombinant vaccinia virus vp425; open reading frame; FIG. 2 shows the DNA sequence of an EHV-1 1.88 FIG. 24 schematically shows a method for the con Kb fragment containing the gp13 coding sequences; struction of plasmid pSD478VC, and pSD479VCBG FIG. 3 schematically shows a method for the con and insertion of Beta-galactoside into vaccinia virus; struction of the recombinant vaccinia virus vp483 con 50 FIG. 25 schematically shows a method for the con taining the EHV-1 gp13 gene; struction of plasmid pMP13PP; FIG. 4 schematically shows a method for the con FIG. 26 schematically shows a method for the con struction of the recombinant vaccinia virus vp458; struction of plasmid pFPPRVII containing the PRV FIG. 5 schematically shows a method for the con gpI gene; struction of the recombinant vaccinia virus vF577 con 55 FIG. 27 schematically shows a method for the con taining the EHV-1 gp14 gene; struction of the recombinant canarypox virus vCP55 FIG. 6 shows the DNA sequence of an EHV-1 3.35 expressing the PRV gpII gene; Kb fragment containing the gp14 coding sequence; FIG. 28 schematically shows a method for the con FIG. 7 is a plot of relative hydrophilicity for the struction of the recombinant vaccinia virus vp717 ex EHV-1 gp14 coding sequences; 60 pressing the PRV g gene; FIG. 8 schematically shows a method for the con FIG. 29 schematically shows a method for the con struction of the recombinant fowlpox virus vFP44 con struction of recombinant vaccinia viruses vp569 and taining the EHV-1 gp13 gene; vP734 expressing the HSV-2 g gene; FIG. 9 schematically shows. a method for the con FIG. 30 schematically shows a method for the con struction of the recombinant canarypox virus vCP48 struction of recombinant vaccinia viruses vp579, vP748 containing the EHV-1 gp13 gene; and vP776 expressing the HSV-2 gC gene; FIG. 10 schematically shows a method for the con FIG. 31 schematically shows a method for the con struction of donor plasmids pHES-MP63, pHES-MP1 struction of recombinant vaccinia viruses vp570, 5,338,683 13 14 vP761, vP775 and vP777 expressing the HSV-2 g) Referring now to FIG. 1, the 13 Kb Sal F fragment gene; of vaccinia virus which spans the HindIII A/B frag FIG. 32 schematically shows a method for the con ment junction was ligated into Sal digested pUC8 gen struction of recombinant vaccinia viruses vp637 and erating pSD419VC. The right arm of pSD419VC cor vP724 expressing the BHV-1 g gene; responding to the HindIII B portion of the Sal F frag FIG. 33 schematically shows a method for the con ment was removed by digestion with HindIII and reli struction of donor plasmid pCA001 containing the gation generating pSD456VC. pSD456VC thus con FHV-1 g gene and for the construction of the recom tains the right end of the HindIII A fragment within binant vaccinia virus vP713 expressing the FHV-1 ge which is the complete coding region for the hemagglu gene; 10 tinin (HA) gene (35) flanked by approximately 0.4 Kb FIG.34 shows the nucleotide sequence of the 3400 bp additional vaccinia sequences on each side. segment of FHV-1 DNA encoding glycoprotein gb; To generate a plasmid vector virtually devoid of HA FIG. 35 is a hydropathy plot of the sequence of 947 coding sequences, pSD456VC was cut (partial digest) at amino acids composing FHV-1 gb; the Rsal site upstream of the HA gene and at the Eag FIG. 36 schematically shows a method for the con 15 site 80 bp from the 3' end of the HA gene. The approxi struction of donor plasmids 409gp220 containing the mate 3.5 Kb Rsa/Eag fragment was isolated from an EBV gp220 gene and 409gp340 containing the EBV agarose gel. gp340 gene; Synthetic oligonucleotides MPSYN59-62 were pre FIG. 37 schematically shows a method for the con pared to replace the region from the Rsal site through struction of vaccinia donor plasmid 409gB containing 20 position 2 upstream of the HA coding sequence, imme the EBV gR gene; diately followed by BqlI, SmaI and PstI restriction FIG. 38 schematically shows a method for the con sites and an Eagl sticky end. The sequence of struction vaccinia donor plasmid 486gH containing the MPSYN59-62, with restriction sites as indicated, is as EBV gH gene; follows:

5'-ACACGAATGATTTTCTAAAGTATTTGGAAAGTTTTATAGGTAGTTGATAGAACAA 3'-TGTGCTTACTAAAAGATTTCATAAACCTTTCAAAATATCCATCAACTATCTTGTT AATACATAATTTTGTAAAAATAAATCACTTTTTATACTAAGATCTCCCGGGCTGCAGC-3' TTATGTATTAAAACATTTTTATTTAGTGAAAAATATGATTCTAGAGGGCCCGACGTCGCCGG-5 BglI SmaI Pst Eag FIG. 39 schematically shows the structure of the vaccinia donor plasmid 513ghgBgp340 containing the EBV genes gp340, gB and gh; 35 FIG. 40 schematically shows a method for the con The annealed MPSYN59-62 mixture was ligated into struction of vaccinia donor plasmid 409CMVgB con the 35 Kb Rsal/Eagl fragment from pSD456VC, gen taining the CMV gb gene; erating pSD466VC. Thus, in pSD466VC the HA gene FIG. 41 shows the nucleotide and amino acid sequen has been replaced by a polylinker region. ces of HCMV (Towne strain) HXLF1 gene; and A 3.2 Kb Bg|II/BamHI (partial) fragment containing FIG. 42 shows the nucleotide and amino acid sequen the E. coli Beta-galactosidase gene from pMC1871 (34) ces of HCMV (Towne strain) HXLF2 gene. under the transcriptional control of the vaccinia 11 kDa promoter (7) was cloned into pSD466VC which had DETAILED DESCRIPTION OF THE been digested with BgllI. A plasmid containing the 11 INVENTION 45 kDa promoter/Beta-galactosidase gene cassette in a left A better understanding of the present invention and to right orientation relative to flanking vaccinia arms of its many advantages will be had from the following was designated pSD466VCBGA and recombined into a examples, given by way of illustration. thymidine kinase deletion mutant, vp410, of the Copen hagen strain of vaccinia virus generating the vaccinia EXAMPLE 1. 50 recombinant vP425 expressing Beta-galactosidase. Construction of Vaccinia Virus Recombinants Eighty base pairs at the carboxy terminus of the HA expressing the Equine Herpesvirus gp13 Glycoprotein gene were retained so not to disrupt a short potential Replacement of the gene of vaccinia with the E. coli open reading frame transcribed right to left relative to Beta-galactosidase gene. the vaccinia genome. 55 The recombinant vaccinia virus, vp425 (184), was The Copenhagen strain of vaccinia virus obtained identified on the basis of blue plaque formation in the from Rhone Merieux, Inc. (Athens, Ga.) was utilized in presence of the chromogenic substrate, X-gal, as de this example. The virus was propagated from a purified scribed by others (9,24). Substitution of the Beta-galac plaque isolate on either VERO (ATCC#CCL81) or tosidase gene by yet another foreign gene in subsequent MRC-5 (ATCC# CCL171) cells in Eagle's minimal 60 vaccinia recombinants could be readily scored by isolat essential medium (MEM) plus 10% fetal bovine serum ing colorless plaques instead of blue plaques. (FBS). A derivative of the wildtype virus from which To facilitate future cloning steps, the SmaI site de the entire coding sequence for the thymidine kinase rived from the pljC8 multicloning region was elimi gene was deleted by standard methods (25,28) was iso nated by digestion of pSD466VC with BamHI/EcoRI, lated and designated vp410. This thymidine kinase dele 65 bluntending with the Klenow fragment of E. coli poly tion mutant was used for further manipulations. Plas merase, and religation. Thus, the single SmaI site re mids were constructed, screened, and grown by stan maining in the resulting plasmid, pSD467VC, is in the dard procedures (20,27,28). polylinker region of the HA deletion.

5,338,683 15 16 Referring now to FIG. 3, to mutate and insert the H6 Identification of sequences encoding gp13 gene promoter into pSD467VC, oligonucleotides H6SYN The DNA sequence encoding the glycoprotein oligos A-D were synthesized. The sequence of H6SYN EHV-1 gp13 resides in the 7.3. Kb BamHI-H fragment oligos A-D, with modified base as underlined and re of EHV-1 (3). Nucleotide sequence data for both 5 striction sites as indicated, is as follows:

BglII 5'-GATCTCTTTATTCTATACTTAAAAAGTGAAAATAAATACAAAGGTTCTTGAGGGTT -AGAAATAAGATATGAATTTTTCACTTTTATTTATGTTTCCAAGAACTCCCAA GTGTTAAATTGAAAGCGAGAAATAATCATAAATTATTTCATTATCGCGATATCCGTTAA CACAATTTAACTTTCGCTCTTTATTAGTATTTAATAAAGTAATAGCGCTATAGGCAATT GTTTGTATCGTACCC-3' CAAACATAGCATGGG-5 SmaI strands was obtained from the puC (BamHI-H) region The underlined bases denote modification from the utilizing overlapping subclones using the modified T7 native H6 promoter sequence. enzyme SEQUENASE (40) (U.S. Biochemicals, Cleve The 130 bp full length, double stranded DNA formed land, Ohio). Standard dideoxy chain-termination reac by the annealing of H6SYN oligos A-D was purified by tions (33) were performed on double stranded plasmid electroelution from an agarose gel and ligated to 0.5 Kb templates that had been denatured in alkali. The M13 SmaI/HindIII and 3.1 Kb BglII/HindIII fragments forward and reverse primers were used to obtain the derived from pSD467VC. The resulting plasmid, initial sequence of each clone. Custom 16-17-met prim 25 pTP15 (184), has the ATG initiation codon modified to ers, synthesized using standard chemistries (Biosearch CCC as part of the SmaI site which is immediately 8700, San Rafael, Calif.; Applied Biosystems 38OB, followed by a Pst site. An Nsi linker, 5'-TGCATG Foster City, Calif.), were used to walk along the re CATGCA-3', (New England Biolabs, Beverly, Mass.) maining fragment. The IBI Pustell sequence analysis was inserted into the SmaI site of pTP15 to generate the program was used in all sequence data analysis (29). 30 plasmid pNSI. DNA sequence analysis revealed an open reading An EHV-1 EcoRI/NarI fragment in which the frame of 1,404 bp encoding 468 amino acids with a EcoRI site is 120 bp upstream of the ATG initiation predicted primary translation product of 50.9 kDa. Sig codon and where the Nar site is 23 bp upstream from nificantamino acid homology in the carboxy half of the the TAG termination codon of EHV-1 gp13 was cloned putative gp13 open reading frame was observed to gC 35 into phage M13mp19 generating the recombinant phage of herpes simplex viruses type 1 and type 2, g|II of M13EcoRNar. Using oligonucleotide-directed muta pseudorabies virus, and gpV of varicella-zoster virus genesis (17) an Nsil site was introduced by changing the suggesting that gp13 was a member of the gC like gly sequence TTGCCT (bases 130-135 in FIG. 2) in the coproteins of herpesviruses. Further detailed analysis of EHV-1 gp13 gene to ATGCAT. The EcoRI/NarI the EHV-1 gp13 open reading frame was presented in a fragment from mutant phage M13EcoRNar was cloned previous publication (2). To facilitate the description of into puC8 at EcoRI/Nar sites generating plasmid the cloning and expression of the EHV-1 gp13 in vac pNSIEN. cinia virus vectors, the gp13 open reading frame plus Two 42-mer oligonucleotides were synthesized hav additional 5' and 3' sequences are shown in FIG. 2. In ing the sequence, with restriction sites as indicated, as FIG. 2, a presumptive TATA box and amino acids 45 follows:

Nar gp13 3'end Nde 5'-CGCCGTACAAGAAGTCTGACTTTTAGATTTTTATCTGCAGCA-3' 3' GGCATGTTCTTCAGACTGAAAATCTAAAAATAGACGTCGTAT-5 Pst comprising putative signals and membrane anchor ele In this oligonucleotide, the termination codon (TAG) is ments are underlined. The potential cleavage site of the immediately followed by a vaccinia early transcription signal sequence is noted with an arrow following the 55 terminator (ATTTTTAT). The double stranded DNA cleavage signal ASA (open circles). Potentially, nine fragment obtained by annealing the pair of 42-mers N-linked glycosylation sites exist within the signal and contains an Nari sticky end, followed by the 3' end of anchor sequences as defined by the Asn-X-Ser/Thr the coding sequence for the EHV-1 gp13 gene, as well motif (asterisks). as a vaccinia early transcription termination signal (45), a pst site, and an Nde sticky end. This fragment was Cloning of the gp13 gene into a vaccinia virus donor inserted between the Nari/Nde sites of pNSIEN gen plasmid erating pNSIENPN (FIG. 3). An early/late vaccinia virus promoter, H6, has been The NsiI/PstI fragment from pNSIENPN was iso used for the expression of foreign genes in fowlpox lated and cloned into the Nsil/Pst sites of plasmid virus vectors (41,42). This promoter element corre 65 pNSI, generating plasmid pVHA6g13Nsil (FIG. 3). sponds to the DNA sequences immediately upstream of pVHA6g13NsiI was cut at the EcoRV site in the H6 the H6 open reading frame in vaccinia HindIII-H frag promoter and the Nsil site which had been introduced ment (31). near the beginning of the EHV-1 gp13 gene. This vec 5,338,683 17 18 tor fragment was blunt ended with Mung Bean nucle ning glycoprotein (14). In a productive EHV-1 infec ase. Two complementary 32-mer oligonucleotides were tion that gp13 glycoprotein is incorporated into the synthesized having the sequence, with restriction site as various membrane systems of the cell and is transported indicated, as follows: into the cytoplasmic membrane and detectable on the external surface of the infected cell. EHV-1 gp13 is EcoRV additionally a component of the EHV-1 virion. There SATCCGTTAAGTTTGTATCGTAATGTGGTTGCC-3 fore, immunofluorescence studies were performed to 3'-TAGGCAATTCAAACATAGCATTACACCAACGG5. determine whether EHV-1 gp13 expressed by the vac H6 promoter gp13 5' end cinia virus recombinant, vp483, was similarly presented O These oligonucleotides were annealed and ligated into on the cytoplasmic membrane of infected cells. Anti the pVHA6g13Nsil vector fragment, producing plas gp13 specific monoclonal antibody followed by fluore mid pVHA6g13, which contains a precise junction at scein-conjugated goat anti-mouse IgG revealed a strong the ATG initiation codon (underlined in the 32-met membrane immunofluorescence in vF483 infected cells sequence) of the H6 promoter and EHV-1 gp13 gene 15 but not in vaccinia virus vp410 infected cells. This sug (FIG. 3). gests that the EHV-1 gp13 expressed by the recombi pVHA6g13 was transfected into vP425 infected cells nant vaccinia virus vp483 is presented on the cytoplas to generate the vaccinia recombinant vp483 containing mic membrane as expected for authentic synthesis of a the EHV-1 gp13 gene (FIG. 3). membrane spanning glycoprotein. Construction of vaccinia virus recombinants 20 Immunoprecipitation of EHV-1 qp13 products Procedures for transfection of recombinant donor synthesized from recombinant vaccinia Virus v483 plasmids into tissue culture cells infected with a rescu infected cells ing vaccinia virus and identification of recombinants by Two million cells forming a confluent monolayer in a in situ hybridization on nitrocellulose filters were as 60 mm dish were infected at 10 pfu/cell. The inocula previously described (25,28). To construct vp425 25 where the E. coli Beta-galactosidase gene replaces the tion was performed in methionine-free medium. After vaccinia HA coding sequences, plasmid DNA (25 ug of the adsorption period, the inoculum was removed and pSD466VCBGA in HeBS (16) was electroporated 2ml of methionine-free medium containing 20 u, Ci/ml (BioRad Gene Pulser, capacitance 960, 200 volts) into of 35S-methionine added. The infection was allowed to VERO cells. Subconfluent monolayers of cells were 30 proceed for 24 hours when cells were lysed by the infected at 10 pfu/cell with vp410 one hour prior to use. addition of 1 ml of 3x Buffer A containing 3% NP-40, The infected cells were harvested with trypsin and 30 mM Tris pH 7.4, 450 mM. NaCl, 3 mM EDTA, washed with HeBS before electropotation. Cells were 0.03% sodium azide, and 0.6 mg/ml PMSF. The lysed incubated in MEM --5% fetal bovine serum at 37 C. cells and supernatant were harvested, vortexed, and for 24 hours, harvested and progeny virus plated on 35 clarified by centrifugation at 10,000g for 15 minutes. VERO monolayers. Recombinant virus expressing Protein A-Sepharose CL-4B (Pharmacia, Cat. No. Beta-galactosidase was detected as blue plaques in the 17.0780.01) was prepared as a 1:1 slurry in 1XBuffer A. presence of X-gal substrate (9,24). To generate recombi A rat anti-mouse conjugate (Boehringer Mannheim, nant vaccinia virus where the EHV-1 gp13 gene re Cat. No. 605 500) was diluted to 1:100 in the slurry and placed the Beta-galactosidase gene in vp425, a similar bound to the beads at room temperature for 4 hours protocol was followed except that the donor plasmid with rocking. The beads were then washed thoroughly was pVHA6g13 and rescuing virus was vp425. The with 6 one ml washes in Buffer A to remove unbound vaccinia recombinant vp483, containing EHV-1 gp13 conjugate. A monoclonal antibody specific to gp13 was was detected as a colorless plaque in the presence of then bound to the beads at room temperature for 4 X-gal and confirmed as a true recombinant by DNA 45 hours. Excess antibody was removed by thorough hybridization after 3 cycles of plaque purification. washing. One ml of clarified infected cell lysate was precleared by incubation with Protein A-Sepharose Expression of the EHV-1 qp13 gene on the surface of beads to which normal mouse serum had been bound. cells infected with the recombinant vaccinia virus v483 These beads were removed by centrifugation. One ml of BSC-40 cells were seeded on 22mm glass coverslips 50 the clarified precleared lysate was then mixed with in 35mm dishes at 5X10 cells/dish. At approximately 80% confluency the cells were infected at 2 pfu/cell. 100ul of the beads to which the specific monoclonal After a 1 hour adsorption period the virus inoculum was antibody had been bound. This mixture was rocked at removed and MEM plus 2% fetal bovine serum added. room temperature for 4 hours. The beads were then At 20 hours post infection the coverslips were washed 55 removed by centrifugation and washed thoroughly by with phosphate buffered saline (PBS) containing 0.2% four washes in 1X Buffer A and two washes in 10 mM BSA and 0.1% NaN3 (PBS--) and exposed to 0.1ml of Tris pH 7.4 containing 0.2M LiCl and 2M urea. The anti-gp13 monoclonal antibody, 14H7 (3) diluted one to antibody-antigen complex was then removed from the a thousand in PBS--. After 1 hour in a humidified beads and disrupted by the addition of 50ul of 2X La chamber at room temperature the cells were washed 3 emmli Disrupting Solution (60,195). The sample was times in PBS--. This procedure was repeated with fluo then boiled for 5 min before electrophoresis. rescein isothiocyanate-conjugated goat anti-mouse IgG. There are two products of approximately 44 and 47 Finally, the cells were fixed for 20 minutes in 2% para kDa detectable which are somewhat smaller than the formaldehyde in PBS. The coverslips were mounted in predicted primary translation product (51 kDa) and a 80% glycerol in PBS containing 3% in-propyl gallate 65 larger product of approximately 90 kDa which is con and fluorescence was observed with a microscope. sistent with a fully glycosylated form of the EHV-1 The protein predicted from the DNA sequence has gp13 gene product. No equivalent polypeptides were the typical features characteristic of a membrane span precipitated from control vaccinia virus infected cells. 19 5,338,683 20 One WS designated pMP409DVCBG. EXAMPLE 2 pMP409DVCBG was used as donor plasmid for recom Construction of vaccinia virus recombinants expressing bination with rescuing vaccinia virus, vp410, described the Equine Herpesvirus gp14 Glycoprotein in Example 1. The novel vaccinia recombinant, desig Replacement of the M2L gene in vaccinia virus by the nated vP458, expressing the Beta-galactosidase gene E. coli Beta-galactosidase gene inserted into the M2L deletion locus was detected using In order to insert the EHV-1 gp14 coding sequences the chromogenic X-gal substrate (9,24) and purified by into a vaccinia virus vector, a recombinant vaccinia repeated plaque cloning. virus, vF458, expressing the E. coli LacZ gene was Cloning of the EHV-1 gp14 gene constructed. Substitution of the Lacz coding sequences O in the recombinant virus, vp458, with sequences encod Referring now to FIG. 5, the EHV-1 gp14 coding ing EHV-1 gp14 allows a blue to colorless plaque sequence spans the junction between the BamHI restric screening system for identifying EHV-1 gp14 contain tion fragments a and i (3). The EHV-1 DNA fragments ing recombinant viruses (9,24) in the presence of X-gal, BamHI-a (21.3 Kb) and i (7.1 Kb) (59) were isolated a chromogenic Beta-galactosidase substrate. Further 15 from agarose gels. Plasmid pJC (BamHI-i) was con more, with the intention of constructing vaccinia virus structed by inserting the EHV-1 BamHI-ifragment into recombinants expressing both EHV-1 gp14 and EH-1 plasmid puC8 at the BamHI site. The EHV-1 BamHI-a gp13, an insertion locus for EHV-1 gp14 unique from fragment was digested with EcoRI and ligated into the hemagglutinin deleted locus used for the insertion of EcoRI/BamHI digested puC8. Plasmid puC (BamHI EHV-1 gp13 in Example 1 was prepared at the M2L 20 a/EcoRI) contains a 10 Kb EHV-1 BamHI/EcoRI locus of HindIII M. The entire coding sequence of the insert. Based on the fragment size determinations re M2L gene in the vaccinia HindIII M fragment was ported (59), DNA sequences in this insert are contigu eliminated and replaced with the E. coli Lacz gene ous with those of the BamHI-i fragment in the EHV-1 encoding Beta-galactosidase. The cloning steps for the genome. construction of vp458 are schematically presented in 25 FIG. 4. Nucleotide sequence analysis Referring now to FIG. 4, an open reading frame Nucleotide sequence analysis was obtained utilizing reading right to left relative to the vaccinia genome and different subclones from the pjC (BamHI-a/EcoRI) encoding a putative protein of 220 amino acids is lo and pljC (BamHI-ii) plasmids. Sequencing of the plas cated entirely within the HindIIIM fragment from the 30 mid puC (BamHI-a/EcoRI) was started at the BamHI Copenhagen strain of vaccinia virus to the left of the site because the EHV-1 gp14 gene spans the BamHI-a/i unique Bgll site. According to convention (31), this junction (3). The orientation of the pUC (BamHI-i) gene, which is located immediately to the right of M1L plasmid was determined by restriction enzyme diges (58), was designated M2L. Deletion studies directed to tion. Since the EHV-1 BamHI terminus closest to the the vaccinia (WR) genome extending leftward from the 35 EcoRI site in plJC (BamHI-i) was found to be the unique Bgll site in HindIII fragment M. (57) indicate BamHI site at the BamHI-a/ijunction, sequencing of that vaccinia coding sequences contained in HindIIIM to the left of the BglII site are not essential for replica the fragment was initiated from this BamHI end. tion of the virus in tissue culture. Sequence data for both strands was obtained as de To facilitate use of the M2L region as an insertion scribed in Example 1. The nucleotide sequence of the locus for foreign genes, a plasmid vector, 3,351 bp fragment containing the EHV-1 gp14 coding pMP409DVC, was created in which the entire M2L sequence is shown in FIG. 6. Numbering in the left and coding sequence was replaced by a BglII site as follows. right hand margins pertains to the amino acid and nu pSD409VC, which consists of the Copenhagen vaccinia cleic acid sequence, respectively. The putative CAT HindIII M fragment cloned into the HindIII site of 45 and TATA boxes are underlined. Amino acids in the pUCS, was digested with BamHI/Bgl and self signal and membrane spanning region are also under ligated, thus removing the right end of HindIIIM and lined with the arrow indicating a potential signal pep destroying the Bgll site. The resulting plasmid, tide cleavage site. The thirteen potential glycosylation pMP409BVC, was linearized with Sph, which cuts sites using the consensus sequence (Asn-X-Ser/Thr) are within the M2L open reading frame, and was subjected 50 indicated by an asterisk. to Bal-31 exonuclease digestion for two minutes. Muta DNA sequence analysis revealed an open reading genesis was performed on the resulting DNA (19) using frame extending from nucleotide positions 300 to 3239 a synthetic 49 mer (5'-TTTCTGTATATTT reading from left to right relative to the EHV-1 ge GCAACAATTTAGATCTTACTCAAAATATG nome, i.e. the ATG start codon was contained in the TAACAAT-3'; Bgll site underlined). In the mutage 55 BamHI-a/EcoRI fragment and the stop codon TAA nized plasmid, pMP409DVC, the M2L coding sequen was contained in the BamHI-i fragment (3,59). ces have been deleted from position --3 through the Putative transcriptional regulatory signals were end of the open reading frame. The G of the initiation found in the region 5' to the ATG initiation codon at codon ATG was changed to a C to create a unique position 300. A TATA box having the sequence Bg1II site (AGATCT) at the deletion junction. A3AATATAT (nucleotides 148 to 155) was located 70 A 3.2 Kb Bgll/BamHI partial fragment containing nucleotides downstream from a putative CAT box at 3.1 Kb of the Ecoli Beta-galactosidase gene between the positions 71 to 77 having the sequence GGTCAAT. A BamHI sites of pMC1871 (34) under the transcriptional polyadenylation signal AATAAA (nucleotides 3251 to control of the 0.1 Kb vaccinia 11 kDa late promoter (7) 3256) was located 8 nucleotides downstream from the was cloned into the unique Bgll site of pMP409DVC. 65 TAA termination codon (nucleotides 3240 to 3242). A recombinant plasmid containing the 11 kDa promo Nine out of eleven nucleotides in the sequence 5'- ter/Beta-galactosidase gene cassette in a right to left TCCTGCGCGCA-3 (nucleotides 218 to 228) are com orientation relative to flanking vaccinia arms and ge plementary to the 18S ribosomal RNA sequence 3'- 5,338,683 21. 22 AGGAAGGCGU-5' (61) and may serve as the ribo glycoproteins of other herpesviruses. Thus, the EHV-1 some binding site. gp14 is homologous to g|I of PRV (62), g of BHV-1 (63), g of varicella-zoster virus (VZV) (66), gB of Analysis of the EHV-1 gp14 structure herpes simplex virus (HSV) (67,71,72) as well as to The EHV-1 gp14, open reading frame encodes 980 glycoproteins in Epstein-Bart virus (EBV) (68) and amino acids with a calculated molecular weight of 109.8 human cytomegalovirus (HCMV) (10). kDa. Analysis of the amino acid sequence revealed a number of features common to membrane-associated Oligonucleotide-directed mutagenesis of the 5' terminus glycoproteins. A region extending from amino acids 58 of the EHV-1 gp14 coding sequence to 99 had a characteristic hydrophobicity profile and is O Referring now again to FIG. 5, plasmid Blue (KpnI/- proposed to be the signal sequence (FIG. 6). An unusual BamHI) was generated by inserting a KipnI/BamHI feature of the EHV-1 gp14 gene product is that the long fragment from puC (BamHI-a/EcoRI) into plasmid hydrophobic signal sequence is preceded by a long Bluescript SK- digested with Kpn/BamHI. Oligonu hydrophilic sequence. This characteristic has also been cleotide directed mutagenesis was performed by a mod noted for the pseudorabies virus (PRV) gI (62) and for 15 ification of the procedure of Kunkel (17) using uracil the bovine herpesvirus 1 (BHV-1) g gene (63), both of containing DNA templates from plasmid Blue (KpnI/- which are also HSV g homologs. A hydrophobic BamHI) produced in the dut ung host E. coli strain region consisting of 45 amino acids (amino acids 826 to CJ236. In the mutagenized plasmid an Nsil site was 870) is predicted to function as a transmembrane anchor created at codons 1 and 2 of the EHV-1 gp14 gene, domain. The hydrophilic cytoplasmic domain contains changing the sequence ATG/TCC (Met/Set) to ATG 110 amino acids. /CAT (Met/His). The mutated sequence was verified There are eleven Asn-X-Thr/Ser (where X can be by DNA sequence analysis. The Kpn/BamHI frag any amino acid except proline) sites for potential N ment from the mutant was transferred to Kpn/BamHI linked glycosylation (64). An unusual feature is that digested pljC18 generating the plasmid puC (KpnI/- there are also two potential glycosylation sites in the 25 DamHI). cytoplasmic domain (FIG. 6). A plasmid, pUCg14, containing the complete EHV-1 A hydrophilicity plot of the EHV-1 gp14 coding gp14 gene with the Nsil site mutation was constructed sequence is shown in FIG. 7. The hydropathic index of by inserting the EcoRI/DamHI fragment from puC EHV-1 gp14 is computed by the method of Kyte and (KpnI/BamHI) into ECoRI/BamHI digested puC Doolittle (65) with a window of seven amino acids and 30 (BamHI/pstI), a 3.9 Kb subclone of pJC (BamHI-i). no smoothing. Points below the horizontal line repre sent areas of higher hydrophobicity, therefore indicat Construction of chimeric donor plasmid pVM2LH6g 14 ing potential signal and/or membrane spanning regions. pMP409DVC was cut with BglI and ligated with The characteristics of a membrane spanning glycopro synthetic double-stranded DNA containing the modi tein including signal and anchor elements and the long 35 fied vaccinia H6 (early/late) promoter, described in hydrophilic region preceding the signal sequence are Example 1, flanked by restriction sites. Restriction sites found for the EHV-1 gp14 coding sequence. for Nsi, SacI, PSt and EcCRI were created immedi ately downstream from the endogenous initiation codon Localization of the antigenic determinant recognized by in the H6 promoter. In pMGll, the polylinker sequence the anti-EHV-1 qp14 monoclonal antibody, 3F6 downstream from the H6 promoter is ATG CAT GAG Lambda gt11 expression vectors and monoclonal CTCTGC AGA ATT CGG ATCT. The unique NsiI antibodies have been useful in identifying the EHV-1 site, containing the H6 initiation codon (underlined), is DNA sequences encoding the major EHV-1 glycopro immediately followed by unique SacI, Pst and EcoRI teins (3). A lambda gt11 recombinant, 4a1, was shown sites. to express an EHV-1 gp14 epitope recognized by the 45 The EcoRI/Nsil DNA fragment from pUCg14 con specific monoclonal antibody 3F6(3). In order to deter taining the EHV-1 DNA region upstream from the mine the identity of this epitope, the EHV-1 DNA EHV-1 gp14 initiation codon was replaced by the Eco contained within 4al was sequenced and compared with RI/Nsifragment from plasmid pMGll, thus generating the DNA sequence of the EHV-1 gp14 coding sequence plasmid pMRHg14 which contains the right arm of (FIG. 6). To sequence the DNA fragment correspond 50 vaccinia HindIII M, the H6 promoter, and the entire ing to the EHV-1 gp14 epitope in the lambda gt11 re length of the EHV-1 gp14 gene. The HpaI/pstIEHV-1 combinant 4al recognized by anti-EHV-1 gp14 mono gp14 containing fragment from plasmid pMRHgl4 was clonal 3F6(3), 4al was digested with EcoRI, the EHV-1 transferred to the vector plasmid pMGll cut with fragment isolated on agarose gels and ligated into the HpaI/pstI, creating plasmid pVM2LH6g14. EcoRI site of pljCS. DNA sequencing was performed 55 pVM2LH6g 14 contains the entire EHV-1 gp14 coding as described above with the M13 universal forward and sequence (with codon 2 changed from TCC (Set) to reverse primers. CAT (His) as indicated, and approximately 1.2 Kb of The nucleotide sequence alignment indicated that this EHV-1 DNA downstream from the EHV-1 gp14 epitope was contained within the 66 amino acid region gene) under the control of the H6 promoter, inserted in corresponding to 107 (Thr) through 172 (Val) of the 60 a right to left orientation with respect to flanking vac deduced primary translation product. The epitope is cinia sequences relative to the vaccinia genome target therefore located within the amino-terminal region of ing the insertion of the EHV-1 gp14 gene to the M2L the deduced EHV-1 gp14 surface domain. locus. Recombination was performed using vP458 as rescu Comparison of the EHV-1 gp14 amino acid sequence to 65 ing virus and pVM2LH6g14 as donor plasmid. Color other herpesvirus glycoproteins less plaques were picked and analyzed for the presence Comparison of the amino acid composition of the of EHV-1 gp14 coding sequences using a specific EHV-1 gp14 gene revealed extensive homology with EHV-1 gp14 probe labeled with 32P. After repeated 5,338,683 23 24 plaque cloning the vaccinia recombinant was desig by the specific anti-EHV-1 gp14 monoclonal 3F6 (3) nated vR577. from either uninfected VERO cells or VERO cells infected with the control hemagglutinin minus vaccinia Truncation of the EHV-1 gp14 hydrophilic leader virus, vP452 (184). EHV-1 gp13 radiolabeled products sequences were precipitated by monoclonal 14H7 from VERO Using variations of the mutagenesis and cloning ma cells infected with vp483, a vaccinia recombinant ex nipulations described above, chimeric donor plasmid pressing only the EHV-1 gp13, or the vaccinia virus pVM2LH6g14-1 was constructed. To create double recombinants expressing both EHV-1 gp13 with pVM2LH6g 14-1, which contains a deletion of codons 2 either intact gp14, vP633, or truncated gp14, vP634. through 34 of EHV-1 gp14 with the substitution of 4 10 codons, in vitro mutagenesis (17) was performed on There are two products of approximately 44 and 47kDa plasmid Blue (Kpn/BamHI), creating an Nsi site in detectable which are somewhat smaller than the pre codons 32 through 34 rather than codons 1 and 2. The dicted primary translation product (51 kDa) and a Nsi/BamHI fragment from the newly mutagenized larger product of approximately 90 kDa which is con Blue (KpDI/BamHI) plasmid was substituted for the 15 sistent with a fully glycosylated form of the EHV-1 Nsi/BamHI fragment in pVM2LH6g14. Multiple Nsi gp13 gene product. Significantly, the quality and quan linkers (New England BioLabs, Beverly, Ma.) were tity of expression of EHV-1 gp13 is unaffected by coex ligated into the NsiI site to bring the initial ATG in pression of either form of EHV-1 gp14 in the vaccinia frame with the remainder of the EHV-1 gp14 coding double recombinants, vF633 and vp634. sequence. The final plasmid, pVM2LH6g14-1, contains 20 VERO cells were infected with vF633, vp634, the sequence ATG/CAT/GCA/TCC/ATT/GCT ... vP613, and vP577, respectively, and immuno ... encoding Met/His/Ala/Cys/Ile/Ala.... where GCT precipitated with the specific anti-EHV-1 gp14 mono (Ala) is codon 35 of EHV-1 gp14. The remainder of clonal 3F6 (3). With vP633 (containing full length gp14 pVM2LH6g14-1 is identical to that in pVM2LH6g14. plus gp13) and with vP577 (containing full length The vaccinia recombinant vF613 was obtained by 25 gp14), major bands at approximately 34, 47, 60-64 and recombination with rescuing virus vF458 and donor 90kDa were observed; whereas with vp634 (containing plasmid pVM2LH6g14-1. truncated gp14 plus gp13) and with vp613 (containing truncated gp14), major bands at 34, 47, 57, 72-82 and EXAMPLE 3 116 kDa were observed. Again no significant differ Construction of Vaccinia Virus Recombinants vp633 30 ences in the synthesis of EHV-1 gp14 of either form is and vP634 expressing each of the Equine Herpesvirus observed during coexpression with EHV-1 gp13. gp13 and gp14 Glycoproteins In order to construct vaccinia recombinants express Immunofluorescence analysis of EHV-1 gp13 and gp14 ing both gp13 and gp14 EHV-1 glycoproteins, recombi products synthesized by recombinant vaccinia viruses nation was performed with either vP577 or vP613 as 35 Immunofluorescence of recombinant vaccinia virus rescuing virus and the donor plasmid pVHA6g13 (de infected VERO cells was performed as described in scribed in Example 1) which contains the EHV-1 gp13 Example 1 using either EHV-1 gp13 or gp14 specific gene under the control of the vaccinia H6 promoter monoclonal antibody. inserted at the HA deletion locus of vaccinia. Insertion EHV-1 gp13 was readily detectable on the surface of of the EHV-1 gp13 sequences into recombinant viruses VERO cells infected with vaccinia recombinants was identified by in situ DNA hybridization (25,28). vP483, vF633 and vR634 as well as internally after Recombination of pVHA6g13 with vaccinia virus re acetone fixation. No significant internal or surface im combinant vp577 (containing full length EHV-1 gp14) munoreactivity toward gp13-specific antibody was seen generated the double vaccinia virus recombinant in vP410, vP577 or vP613 infected cells. Expression of vP633; recombination with vF613 (containing trun 45 EHV-1 gp14 was readily detectable in acetone fixed cated EHV-1 gp14) generated the double vaccinia re VERO cells infected with vaccinia recombinants combinant vF634. The vaccinia virus double recombi vP577, vP613, vP633 and vP634. No significant internal nants vP633 and vP634 were plaque cloned and the immunofluorescence toward gp14-specific antibody presence of both EHV-1 gp13 and gp14 coding sequen was seen in vp410 or vP483 infected cells. Using gp14 ces confirmed by DNA hybridization analysis and by 50 specific monoclonal antibody, 3F6, a weak surface in expression assays (see below). munofluorescence was observed in cells infected with Immunoprecipitation of EHV-1 qp13 and gp14 vP613 or vP634, which express the truncated form of glycoproteins expressed in vaccinia virus recombinants EHV-1 gp14 and no significant surface response above control viruses vP410 and vP483 was obtained with In order to assess the EHV-1 gp13 and gp14 glyco 55 recombinant vaccinia viruses vp577 and vp633 which proteins expressed by vaccinia virus recombinants, VERO cells were infected with the recombinants and express the full length EHV-1 gp14 gene (see also Ex proteins were metabolically labeled with 35-S-methio ample 8). nine and immunoprecipitated as described in Example EXAMPLE 4 1. The specific monoclonal antibody to EHV-1 gp13 (14H7) or to EHV-1 gp14 (3F6) (3) were bound at a Immunization of Guinea Pigs with the Vaccinia 1:1000 dilution for 4 hours at room temperature. Sam Recombinant vP483 ples were analyzed by SDS polyacrylamide gel electro In order to determine the immunogenicity of the phoresis on a 10% polymer gel at 30mA (constant cur gp13 equine herpesvirus gene product expressed by the rent) for approximately 6 hours. Autoradiograms were 65 vaccinia recombinant vp483, guinea pigs were inocu prepared. lated with the virus and the presence of serum neutraliz No significant products were immunoprecipitated by ing antibodies against both vaccinia virus and equine the specific anti-EHV-1 gp13 monoclonal 14:H7 (3) or herpes virus was assayed. 5,338,683 25 26 Fifteen guinea pigs weighing approximately 450 the recombinant vaccinia virus, vp613, expressing a grams were divided into groups of five. One group truncated EHV-1 gp14 gene induced similar levels of received 1 ml of the vaccinia recombinant EHV-1 serum neutralizing antibodies (Table 2) as did (108TCID50/ml) on day 0 followed by a 1 ml booster on the vaccinia recombinant, vp483, expressing EHV-1 day 21 by subcutaneous inoculation. The second group 5 gp13 (Table 1). Although EHV-1 serum neutralizing received similar inoculations but with vaccinia vp452 antibodies are detectable at three weeks after the pri (108TCID50/ml). The third group remained unvac mary vaccination, a more significant level is observed cinated. All the guinea pigs were bled prior to the pri two weeks after the secondary immunization (Table 2). mary vaccination and on days 21 and 35. Sera were In all immunized animals, responses were obtained prepared and tested for the presence of neutralizing O when vaccinia antibodies were assayed by ELISA. antibodies to both vaccinia and EHV-1 (strain Ken tucky) using 50 TCID50 of virus assayed on swine testic TABLE 2 ular cells. Serum neutralizing antibodies present in guinea pigs inoculated As shown in Table 1, the EHV-1 gp13 vaccinia re with a vaccinia recombinant expressing EHV-1 gp14. combinant vp483 elicits an obvious seroconversion in 15 Serum Neutralizing guinea pigs. Serum neutralizing titers obtained with - Titer (log10) on Days - vaccinia virus are shown in parenthesis in Table 1. Both Inoculum Virus O 2 35 vaccinia and EHV-1 serum neutralizing antibodies are Recombinant Vaccinia Virus 0.4 O.T 1.3 detectable 21 days after the primary inoculation and a VP63 0.2 0.7 1.2 0.2 0.7 1.7 significant increase in the titer of serum neutralizing 20 0.2 1.1 1.6 antibodies is obtained by 2 weeks after a secondinocula 0.2 1.0 1.6 tion of virus on day 21. It should be noted that the Unvaccinated Controls 0.2 0.4 serum vaccinia neutralizing titers obtained in guinea 0.6 0.4 pigs inoculated with the recombinant virus expressing 0.7 re- 0.8 EHV-1 gp13 are significantly higher (t=7.2) than the 25 0.6 ADO 0.2 titers obtained from guinea pigs inoculated with the 0.4 WA 0.4 vaccinia vp452 virus. TABLE 1 EXAMPLE 6 Serum neutralizing antibodies present in guinea pigs inoculated with either a vaccinia recombinant expressing Protection of Vaccinated Hamsters from challenge EHV-1 gp13 or a control vaccinia virus, VP452. with EHIV-1 Inoculum. Animal Serum Neutralizing Titer (logo) on Days In order to assess the efficacy of the vaccinia recom Virus No. O 21 35 binant vp483 expressing EHV-1 gp13, hamsters were Un- 26 0.24 (0.35) 0.24 (0.70) given either a primary or primary plus booster vaccina vaccinated 27 0.24 (0.35) aaaaa- 0.56 (1.05) 35 Controls 28 0.24 (0.35) W 0.80 (0.70) tion and they, along with an uninoculated control group 29 0.24 (0.35) 0.40 (0.70) or a group inoculated twice with a control vaccinia 30 024 (0.35) 0.32 (0.35) virus, vp452, were challenged intraperitoneally with a Control 191 024 (0.35). 0.36 (0.47) 0.72 (1.75) Vaccinia 192 0.24 (0.35) 0.21 (0.93) 024 (2.30) hamster adapted Kentucky strain of EHV-1. Virus 193 0.24 (0.35) 0.48 (0.58) - ... 40 Forty syrian hamsters (forty day old weighing be vP4S2 194 0.24 (0.35) 0.24 (0.82) 024 (2.10) tween 55 and 65g) were separated into four groups. 195 0.24 (0.35) - - - - Re- 186 0.24 (0.35) 0.48 (1.28) 1.20 (2.57) Group A received a single subcutaneous (1 ml) inocula combinant 187 0.24 (0.35) 0.72 (1.63) 1.68 (2.57) tion of either 108, 106, or 106, or 10 TCID50 of the Vaccinia 188 0.24 (0.35) 024 (1.52) 1.68 (2.57) vaccinia recombinant vF483, five animals per dose. Virus 189 0.24 (0.35) 0.36 (1.40) 1.56 (2.22) Group B was vaccinated with vP483 on day 0 followed WP483 190 024 (o.35 0.48 (1.635 1.56 (3.00) 45 by a booster on day 14. The (1 ml) primary and booster doses were administered subcutaneously to groups of 5 animals using 108, 106, or 10 TCID50. Group C con EXAMPLE 5 sisted of 5 hamsters and received 2 subcutaneous injec Immunization of Guinea Pigs with the Vaccinia 50 tions (108 TCID50 per injection) on days 0 and 14 of Recombinant vp577 and vR613 vaccinia vp452. Five hamsters in group D were left as Guinea pigs were immunized to evaluate their re unvaccinated controls. All the hamsters received 200 sponse against EHV-1 gp14 expressed by vaccinia re LD50 of a hamster adapted Kentucky strain of EHV-1 combinants vp577 and vp613. Guinea pigs weighing by the intraperitoneal route 14 days after the last immu approximately 450 g received 105 TCID50 of either 55 nization. Survivors were counted 7 days after chal vP577 or vp613 vaccinia recombinant by the subcutane lenge. ous route, one ml on each of day 0 and day 21. Guinea The results are shown in Table 3. All unvaccinated pigs were bled on days 0, 21 and 35, sera prepared and and vaccinia vp452 virus vaccinated hamsters died assayed for EHV-1 antibodies. Neutralization tests were within 5 days of challenge. Significant levels of protec performed on swine testicular cells against 50 TCID50 tion against EHV-1 challenge were observed in ham of EHV-1 virus, strain Kentucky. Vaccinia antibodies sters vaccinated with the vaccinia recombinant vp483 were titrated by ELISA using an anti IgG (H&L) per expressing EHV-1 gp13. No significant differences in oxidase conjugate. protection levels were observed in hamsters immunized The results are shown in Table 2. No serum neutraliz with either primary or primary plus booster doses. The ing activity against EHV-1 was obtained in guinea pigs 65 protective dose (PD50) was similar PD50=6.32 log10 immunized with the vaccinia recombinant, VP577, con primary and 6.12 log10 primary plus booster. Neverthe taining the full length EHV-1 gp14 gene (data not less, 100% protection was only observed in the group shown). On the other hand, guinea pigs inoculated with receiving two doses of 108 TCID50 recombinant virus. 5,338,683 27 28 TABLE 3 EXAMPLE 7 Protection of hamsters vaccinated with the vaccinia re combinant, expressing EHV-1 gp13, against EHV-1 challenge. Construction of Avipoxvirus Recombinants expressing Vaccinating Virus the Equine Herpesvirus gp13 Glycoprotein Recombinant Control Referring now to FIG. 8, pVHA6913 was utilized as Vaccinia vp483 Vaccinia v P452 No the source of the EHV-1 gp13 gene. To isolate the Primary Booster Booster Virus DNA segment containing the entire EHV-1 gp13 gene, Vaccinating 8 6 4 8 6 4 8 pVHA6913 was digested with NruI and HindIII. A Dose log10 fragment of approximately 1.8 Kb containing 28 bp of TCID50 O the 3' end of the vaccinia virus H6 promoter, the entire Proportion 4 1 2 5 2 O O O EHV-1 gp13 gene, and approximately 410 bp of vac Surviving 5 5 5 5 5 5 5 5 cinia virus sequences was generated by this digestion. The 1.8 Kb Nru/HindIII fragment was isolated for In order to determine the protective efficacy of a insertion into the avipoxvirus insertion vectors vaccinia virus recombinant expressing EHV-1 gp14 15 pFPCV2 and pCPCV1. alone or in combination with EHV-1 gp13, challenge The fowlpox virus (FP) insertion vector pFPCV2 studies were performed on vaccinated hamsters. provides a vehicle for generating recombinants which Twenty one-day-old syrian hamsters weighing approxi harbor foreign genes in a non-essential region of the FP mately 60 g each were inoculated subcutaneously with 20 genome designated the f7 locus. pFPCV2 was derived 1 ml of control vaccinia virus or with recombinant from pRW731.13. The plasmid pRW731.13 contains an vaccinia viruses vF483, vF577, vP613, vP633 and FP genomic PvuI fragment of approximately 5.5 Kb vP634 expressing EHV-1 gp13 and/or gp14. Primary inserted between the two Pvu Isites of puC9. Initially, vaccination was followed by an identical vaccinating a multiple cloning sequence (MCS) was ligated into the dose (pfu/ml (log10) on day 14. All hamsters, including 25 unique HincII insertion site within this 5.5 Kb PvuIIFP non-inoculated controls, were challenged 14 days after genomic fragment. The MCS was derived by annealing the last immunization with an intraperitoneal injection oligonucleotides CE4 (5-TCGC GAGAATT of 200 LD50 of EHV-1 hamster adapted Kentucky CGAGCTCGGTACCGGGGATCCTCTGAGT strain. Survivors from groups offive were calculated 14 CGACCTGCAGGCATGCAAGCTTGTT-3") and days post-challenge at which point the experiment was 30 CE5 (5'-AACAAGCTTGCATGCCTGCAGGT. terminated. The dose of inoculum giving 50% protec CGACTCTTAGAGGATCCCCGGTACCGA tion of the hamsters is evaluated as log10 TCID50/ml GCTCGAATTCTCGCGA-3). The plasmid contain inoculant. ing the MCS was designated as pCE11. As shown in Table 4, the vaccinia virus recombinant, pFeLV1A is a derivative of vaccinia insertion vector vP577, expressing the full length EHV-1 gp14 gene 35 pTP15 (184) (FIG. 3) in which the feline leukemia virus (FeLV) env gene (192), is inserted into the PstI site failed to protect hamsters against challenge with a downstream from the H6 promoter. To transfer the 2.4 PD50 calculated 29.0 log10. On the other hand, the kb expression cassette to a FP vector, (FIG. 8) the truncated EHV-1 gp14 gene as expressed by the vac H6/FeLV env sequences were excised from pFeLV1A cinia recombinant, vF613, gave good protection on by digestion with Bgll and partial digestion with pst. challenge (Table 4). The calculated PD50 is somewhat The BglII site is at the 5' border of the H6 promoter better (5.2) than that obtained with the EHV-1 gp13 sequence. The Pst site is located 420 bp downstream expressing vaccinia recombinant, vp483 (6.1). Surpris from the translation termination signal for the FeLV ingly, the coexpression of EHV-1 gp13 and gp14, envelope glycoprotein open reading frame. whether the full length gp14 gene or the truncated gp14 45 The 2.4 Kb H6/FeLV env sequence was inserted into gene in vaccinia virus recombinants vF633 and vP634, pCE11 digested with BamHI and pst. This plasmid was respectively, gave significantly enhanced protective designated as pFeLVF1. The pFeLVF1 plasmid was efficacy compared with efficacy for the EHV-1 glyco then digested with Pst to remove the FeLV env se proteins expressed singly. Hence, the amount of virus quences. The resultant plasmid containing the vaccinia inoculum to achieve a 50% protection of the vaccinated 50 virus H6 promoter within pCE11 was designated hamsters was significantly decreased when EHV-1 pFPCV1. The sequences 5' to the promoter were mut gp13 and gp14 were coexpressed in the same vaccinia agenized (19) to remove extraneous sequences using virus recombinant. oligonucleotide FPCV1 (5-CAGTAATACACGT TABLE 4 TATTGCAGAGAGGACCATTCTTTATT Protection of hamsters vaccinated with the vaccinia 55 CTATACTTAAAAAGT-3") to produce pFPCV1. recombinants, expressing EHV-1 gp13 and/or gp14, The region 3' to the promoter (multiple cloning site) against EHV-1 challenge. was mutagenized with oligonucleotide FPCV3 (5'- Inoculum EHV-1 proteins Vaccination dose/Survivors PD50 TAGAGT CGACCTGCAGGCATC vP483 gp13 8/5 6/2 4/O 6. CAAGCTTGTTAACGAC-3) to remove the SphI None m 0/0 O 60 site, which contains an ATG. The resultant plasmid was vPS77 gp14 8/1 6/0 4/O 29.0 designated pFPCV2. None 0/0 D- w vP613 gp14 8.4/5 6.4/5 4.4/1 5.2 The 1.8 Kb NruI/HindIII EHV-1 gp13 fragment, vP633 gp13 -- gp14 8/5 6/3 4/4 4.3 defined above, was inserted into the 8.0 Kb Niru/Hin vP634 gp13 - gp14 7.6/5 5.6/5 3.6/5 s3.6 dIII fragment derived by digestion of pFPCV2. This 8.0 Vaccinia - 8/O - VD- 29.0 65 Kb Nru/HindIII fragment contained the 5' portion of None m 0/1 the vaccinia virus H6 promoter (100 bp), the FP flank vP613 and vP634 express the truncated version of EHV-1 gp14. ing sequences (4.8Kb upstream and 1.5 Kb downstream from the insertion site) and 2.4 Kb of puC (BRL, Be 5,338,683 29 30 thesda, Md). Ligation of these two fragments resulted in the formation of a 9.8 Kb plasmid designated as EXAMPLE 8 pFPEHV13A. Evaluation of additional Vaccinia Virus Recombinants The plasmid pFPEHV13A was then digested with expressing unmodified and modified versions of the KpnI and HindIII to remove an approximately 600 bp 5 Gene from Equine Herpes Virus-1 encoding fragment. This fragment contained the 3' most region of Glycoprotein gp14 the EHV-1 gp13 gene (200 bp) and the 410 bp vaccinia virus DNA segment. The 600 bp KpnI/HindIII frag Construction and evaluation of additional recombi ment was replaced by a 200 bp fragment derived from nant vaccinia virus expressing EHV-1 gp14. The pNSIENPN (FIG. 3) as follows. A PstI digestion of 10 EHV-1 gp14 containing constructs (Example 2) were pNSIENPN linearized the plasmid. The PstI termini modified in three ways: (a) varying the length of the were blunt-ended by the T4 DNA polymerase (New EHV-1 gp14 leader sequence; (b) removing excess England Biolabs, Beverly, Ma.) in the presence of EHV-1 DNA 3’ from the gene; and (c) inserting the dNTPs (0.5 mM each). HindIII linkers (BRL, Bethesda, modified versions of the EHV-1 gp14 gene into a vac Md.) were then ligated to the blunt-ended fragment. 15 cinia virus vF293 host range selection system (69) for Following digestion with HindIII the linearized plas evaluation. mid was digested with KpnI to yield a 200 bp fragment The EHV-1 gp14 gene product contains an unusually containing the 3' portion of the EHV-1 gp13 gene, the long leader sequence. A long hydrophobic sequence sequence corresponding to the termination codon extending from amino acids 58 through 99 is proposed (TAG), and the TTTTTNT sequence motif known to 20 to be the signal sequence. This region is preceded by a be a vaccinia virus early transcription termination signal long hydrophilic sequence. A similar long leader se (45). The recombinant plasmid was designated as quence has also been noted for two other gbhomologs, pFPEHV13B and was used in in vitro recombination pseudorabies virus gll (62) and bovine herpesvirus 1 g| for insertion of the H6 promoted EHV gp13 gene into (63). the f7 locus of the FP genome. The recombinant fowl 25 pox virus was designated vFP44. Modification of the 5' end of EHV-1 qp14 Referring now to FIG. 9, pFPEHV13B was also To study the effect of the length of the leader se utilized to generate a 1.4Kb Nru/HindIII fragment for quence of EHV-1 gp14 on processing, presentation and insertion into pCPCV1. The pCPCV1 plasmid contains immunological efficacy of the gp14 product expressed the vaccinia virus H6 promoter in the unique EcoRI site 30 in recombinant vaccinia virus, plasmids containing the within the 3.3 Kb Pvulcanarypox virus (CP) genomic EHV-1 gp14 gene with three different lengths of leader fragment. This insertion plasmid enables the insertion of sequence were constructed by modifying the previous foreign genes into the C3 locus of the CP genome. EHV-1 gp14 containing constructs in the following pCPCV1 was derived from prW764.2, which contains ways. a 3.3 Kb Pvul CP genomic fragment inserted into a 35 Referring now to FIG. 10, plasmid pVM2LH6g14 pUC vector. pRW764.2 was linearized by digestion (Example 2) contains the entire EHV-1 gp14 coding with EcoRI. This fragment was blunt-ended using the sequence under the control of the H6 promoter inserted Klenow fragment of the E. coli DNA polymerase (Bo into the Copenhagen vaccinia M2L deletion locus. In ehringer Mannheim Biochemicals, Indianapolis, Ind.) in pVM2LH6g14, amino acid number 2 of the EHV-1 the presence of dNTPs (0.5 mM each). Vaccinia virus gp14 gene is present as His rather than the native Set. H6 promoter sequences and a multiple cloning region To change amino acid 2 to Ser, pVM2LH6g14 was cut situated 3' to the promoter were excised from pfpCV1 with Nsil (recognition sequence ATGCAT) at codons by digestion with Kpn/HpaI. This 200 bp fragment 1-(Met/His). Mutagenesis was performed (19) using was blunt-ended with T4DNA polymerase in the pres synthetic oligonucleotide MPSYN240 (5 ATCCGT ence of dNTPs (0.5 mM each) and inserted into the 45 TAAGTTTGTATC linearized blunt-ended plasmid pRW764.2. The resul GTAATGTCCTCTGGTTGCCGTTCTGTC 3'). tant plasmid was designated pCPCV1. The plasmid The resulting plasmid, pMP14M, contains the entire pCPCV1 was digested with NruI and HindIII and the EHV-1 gp14 gene with the native codon (Ser) at posi 5.8 Kb fragment was isolated for ligation to the 1.4 Kb tion 2. EHV gp13 containing fragment described above. The 50 Plasmid pVM2LH6g14-1 (Example 2) is identical to resultant plasmid was designated pCPEHV13A. This pVM2LH6g14 except for a truncation of the leader plasmid was used in vitro recombination experiments sequence and introduction of four codons derived from for insertion of the H6 promoted EHV gp13 gene into synthetic NsiIlinkers. In pVM2LH6g14-1, the sequence the C3 locus of the CP genome. The recombinant of the 5' truncated end of the EHV-1 gp14 gene is canarypox virus was designated vCP48. 55 ATG/CAT/GCA/TGC/ATT/GCT. . . encoding Following the in vitro recombination, recombinant Met/His/Ala/Cys/Ile/Ala. . . where GCT (Ala) is avipoxvirus containing the EHV-1 gp13 gene were codon 35 of EHV-1 gp14. pVM2LH6g14-1 was modi identified by a standard plaque hybridization assay. fied by mutagenesis (19) in two ways. To produce a Positive plaques were purified by 3 cycles of plaque version of the gp14 gene truncated to approximately the isolation followed by hybridization analyses. Recombi same degree as pVM2LH6g14-1 but more closely ap nants were designated as vFP44 and vCP48 for FP and proximating the native gp14 sequence, pVM2LH6g14-1 CP recombinants, respectively. Both recombinants was cut with Nsi at codons 1-2. Mutagenesis was per were analyzed using a Protein A-B-galactosidase im formed using synthetic oligonucleotide MPSYN241 (5' munoscreen assay with a monoclonal antiserum to ATCCGTTAAGTTTGTATCGTAATGAGTGTC EHV-1 gp13. The results demonstrated that CEF and 65 CCAGCAGCTGGCTCCTGGATC3"). In the result VERO cell monolayers infected with either vFP44 or ing plasmid, pMP14M-34, the EHV-1 gp14 coding se vCP48 express the EHV-1 gp13 on the surface of virus quence begins with ATG/AGT/GTC/CCA. . .Met/- infected cells. Ser/Val/Pro...where CCA (Pro) is amino acid 36 of

5,338,683 31 32 EHV-1 gp14. The EHV-1 gp14 gene contains an Nael containing the shortest version of the leader sequence. site (GCCGGC) at codons 61-63 (Lys/Pro/Ala). To The 2.8 Kb H6 promoter/EHV-1 gp14-containing produce a more severely truncated version of the fragment derived from pMP14-63P was ligated with the EHV-1 gp14 gene, pVM2LH6g14-1 was linearized NruI(partial)/XhoI vector fragment derived from with Nael, followed by digestion with Nsi and isola 5 pHES-4. The resulting plasmid, pHES-MP63, contains tion of vector fragment from an agarose gel. Mutagene the H6 promoter/EHV-1 gp14 gene cassette with no sis was performed using synthetic oligonucleotide extraneous EHV-1 DNA. To transfer the H6 promoter MPSYN243 (5' ATCCGTTAAGTTTGTATC /EHV-1 gp145' ends containing full length or moder GTAATGGCATCATCGAGGGTGG ately truncated leader sequences, plasmids pMP14M GCACAATAGTT 3 ) . In the resulting plasmid, 10 and pMP14M-34 were cut with NruI and the 2.8Kb and pMP14M-63, the EHV-1 gp14 coding sequence begins 2.7 Kb bands, respectively, isolated from agarose gels. with ATG/GCA. . .Met/Ala...where GCA (Ala) is pHES-MP63 was subjected to partial NruI digestion amino acid 63 of the native EHV-1 gp14. and a 7.2 Kb fragmentisolated from an agarose gel. The Removal of extraneous EHV-1 DNA 7.2 Kb vector fragment corresponds to pHES-MP63 15 from which the 2.6 Kb NruI fragment containing the In all EHV-1 gp14 containing plasmids discussed H6 promoter/EHV-1 gp145' end has been removed. above, the EHV-gp14 coding sequences are followed The 7.2 Kb NruI (partial) vector fragment derived from by approximately 1200 bp of EHV-1 DNA. The termi pHES-MP63 was ligated with the 2.8 Kb NruI frag nation codon (TAA) for the gp14 gene occurs within a ment from pMP14M, generating pHES-MP1. The 7.2 Drasite (TTTAAA). To remove excess EHV-1 DNA, 20 Kb Niru (partial) vector fragment derived from pHES pMP14M-63 was subjected to partial Dral digestion MP63 was also ligated with the 2.7 Kb Nrul fragment followed by isolation of linear DNA from an agarose from pMP14M-34, generating pHES-MP34. The clon gel, and digestion with Pst which cuts at the junction ing steps leading to the generation of plasmids pHES of EHV-1 DNA and the downstream vaccinia flanking MP63, pHES-MP1 and pHES-MP34 are presented arm. A 6.5 Kb Dra/PstIDNA band was isolated from 25 schematically in FIG. 10. an agarose gel. Synthetic oligonucleotides MPSYN247 Plasmids pHES-MP1, pHES-MP34 and pHES-MP63 (5' AAATTTTTGTTAACTCGAGCTGCA 3) and were used as donor plasmids for recombination with MPSYN248 (5 GCTCGAGTTAACAAAAATTT3) vP293 (69), generating recombinant vaccinia viruses were annealed and ligated with the 6.5 Kb fragment. In vP753, vP765 and vP721, respectively. Recombinant the resulting plasmid, pMP14M-63P, the EHV-1 gp14 30 progeny were selected on human MRC-5 cells. coding sequences are followed immediately by a se quence specifying termination of early vaccinia tran Evaluation of VP293-based vaccinia virus recombinants scription (45) followed by a polylinker region (contain expressing the EHV-1 gp14 gene ing HpaI, XhoI, Pst restriction sites) and the left vac To determine whether the three forms of the EHV-1 cinia flanking arm derived from HindIII M. 35 gp14 gene product expressed in recombinant vaccinia virus vP753, vP765 and vP721 were present on the Insertion of the H6 promoter/EHV-1 gp14 gene into a surface of infected cells, VERO cell monolayers were pHES/vP293 selection system infected with the three EHV-1 gp14-containing recom In all EHV-1 gp14 containing plasmids discussed binant vaccinia viruses. Infected cell monolayers were above, the EHV-1 gp14 gene is under the control of the 40 analyzed for surface immunofluorescence using the vaccinia H6 promoter inserted into the M2L deletion EHV-1 gp14-specific monoclonal antibody 3F6. Sur locus of Copenhagen strain vaccinia virus. Since the face immunofluorescence was positive for cells infected M2L insertion locus is located within a larger region of with all three vaccinia vital recombinants, vp753, the genome that can be deleted (69), the relocation of vP765 and vP721. This indicates that proper trafficking the H6 promoter/EHV-1 gp14 expression cassette to a 45 of the EHV-1 gp14 gene product in vaccinia infected potentially more stable insertion site was investigated. cells is not affected by varying the length of the leader As a preliminary step, EHV-1 gp14 gene constructs sequence. containing different lengths of the leader sequence were To compare the EHV-1 gp14 gene products ex moved to the WR pHES/vP293-based host range selec pressed by the three EHV-1 gp14-containing vaccinia tion system (69) to allow rapid generation of vaccinia 50 virus recombinants, MRC-5 cells were infected by recombinants for comparative evaluation. vP753, vF765 and vP721 and proteins were metaboli Plasmid pHES-4 contains the vaccinia H6 promoter, cally labeled with 35S-methionine. Immunoprecipita followed by a polylinker region and the K1L human tions were performed with the radiolabeled cell lysates host range gene (70), all inserted between WR vaccinia using EHV-1 gp14-specific monoclonal antibody 3F6. arms flanking a 21.7 Kb deletion (69). pHES-4 contains 55 Immunoprecipitated proteins from cells infected with two NruI sites, one within the H6 promoter and one vP753, vP765 and vP721 are indistinguishable from within flanking vaccinia sequences. pHES-4 was linear each other, and are equivalent to the proteins immuno ized by partial digestion with NruI and the band con precipitated from vP613, the EHV-1 gp14-containing taining full length linear DNA was isolated from an vaccinia recombinant produced from plasmid agarose gel. This linear DNA was cut at the XhoI site in 60 pVM2LH6g14-1. These results indicate that the varia the polylinker region. pMP14M-63P contains two NruI tions in length of the EHV-1 gp14 leader sequence sites, one within the H6 promoter and the other within tested in these recombinants neither enhance nor inter EHV-1 gp14 coding sequences, 0.2 Kb from the 3' end fere with proper processing of the gene product. of the gene. pMP14M-63P was linearized with Niru, To evaluate the protective efficacy of recombinant followed by digestion with XhoI. A 2.8Kb NruI (par 65 vaccinia virus expressing the different forms of EHV-1 tial)/XhoI fragment was isolated from an agarose gel. gp14, hamsters were inoculated with varying doses of This fragment contains part of the H6 promoter, fol vP753, vP765 and vP721 and challenged with EHV-1 lowed by the form of the modified EHV-1 gp14 gene hamster adapted Kentucky strain. All three EPV-1