US005614193A United States Patent (19) 11 Patent Number: 5,614,193 Schmaljohn et al. 45 Date of Patent: Mar 25, 1997

54). HANTAVIRUS WACCINE Schmaljohn et al. "Hantaan Virus M RNA: Coding Strategy, Nucleotide Sequence, and Gene Order", Virology 157:31-39 (75) Inventors: Connie S. Schmaljohn; David J. (1987). McClain, both of Frederick; Joel Yoo and Kang "Nucleotide sequence of the M segment of Dalrymple, deceased, late of the genomic RNA of Hantaan virus 76-118", Nuc. Acids Myersville, all of Md., by Lonnie Res. 15:6299-6300 (1987). Dalrymple, Legal Representative Schmaljohn et al. "Baculovirus Expression of the Small Genome Segment of Hantaan Virus and Potential Use of the (73) Assignee: The United States of America as Expressed Nucleocapsid Protein . . . ', J. Gen. Virol. represented by the Secretary of the 69:777-786 (1988). Army, Washington, D.C. Rossietal."Diagnostic potential of abaculovirus-expressed nucleocapsid protein for hantaviruses', Arch. Virol. (21) Appl. No.: 218,943 S1:19-28 (1990). 22 Filed: Mar. 28, 1994 Schmaljohn et al. "Antigenic Subunits of Hantaan Virus Expressed by Baculovirus and Vaccinia Virus Recombi Related U.S. Application Data nants', J. Virol. 64:3162-3170 (1990). Schmaljohn et al. "Preparation of candidate vaccinia-vec I63 Continuation-in-part of Ser. No. 799,479, Nov. 14, 1991, tored for haemorrhagic fever with renal syn Pat. No. 5,298,423. drome', 10:10-13 (1992). (51 Int. Cl...... A61K 39/285; A61K 39/12; Hsiang, C.M. 1989, Virus Genes vol. 2 pp. 367–369. C12N 7/01 Tartaglia, J. et al., 1990. Crit Rev. Immunol. vol. 10 pp. 52 U.S. C...... 424/186.1; 424/199.1; 13-30. 424/204.1; 424/232.1; 435/235.1; 435/320.1; Xu, X. et al. Am. J. Trop. Med. Hyg. vol. 47 pp. 397-404. 935/65 58) Field of Search ...... 424/186.1, 199.1, Primary Examiner Mary E. Mosher 424/204.1, 232.1; 935/65; 435/235.1, 320.1 Attorney, Agent, or Firm-John Francis Moran 56) References Cited (57) ABSTRACT PUBLICATIONS Vaccine formulations for inducing protective immune response to Hantaviruses in humans are disclosed. These Lee et al. "Isolation of the Etiologic Agent of Korean formulations include an attenuated vaccinia virus vector Hemorrhagic Fever, J. Infect. Dis. 137:298–307 (1978). containing cDNA's encoding Hantavirus nucleocapsid N Schmaljohn and Dalrymple "Analysis of Hantaan Virus protein, G1 and G2 glycoproteins. Methods for the use of RNA: Evidence for a New Genus of Bunyavirdae”, Virology these formulations also are disclosed. 131:482-491 (1983). Schmaljohn et al. "Coding Strategy of the S Genome Seg ment of Hantaan Virus”, Virology 155:633–643 (1986). 28 Claims, 8 Drawing Sheets U.S. Patent Mar 25, 1997 Sheet 1 of 8 5,614, 193

/-/9/3/ U.S. Patent Mar 25, 1997 Sheet 2 of 8 5,614, 193

2-/(9/3/

- VTV(TEISVIVV^IV&(X_L(\TEITEITIOTIO U.S. Patent Mar. 25, 1997 Sheet 3 of 8 5,614,193

XIÐATOSXT(ToHasIvaSATIvaRUHLNSVNTOXTIOOŽIV(IHL(18T(IHLL"IVAYISISnTOYTVXTOHAULSAOEDBITIyTySXTasy sxiasvnanSDHoxiavivvTvSIHnaIATOGTINIÐwassxonToXIÐwasSIHSIHsxoo?va?haIHLwasXIÐ

(THSSXTSIH?ÃVTVAoHasxonaIGTIaegsn?TO2

9]

U.S. Patent Mar 25, 1997 Sheet S of 8 5,614,193

pyp3DyL.LOL33yLOVOLVVVOOVLOVLOOOVLVOJ.VVOOOLVOVOVLOLLOEDLOJ,VOWVOOOLL oovvvvoLy?lyIvyoÐI.voÐIvoLLVvoovLOLLYDLYDVLL2voyovvooloyILovLYyOoLLOLOLS 000vypLoLLLLvvoLDLvolvoÐÐLooL?ÐVOLVLLVLOSOLLLDvovL2O3VOSLOLoÐVVDSVLovL wyoyyoyooLoooloLvoIvoVVDDODLVOVOOVOLV?VOLLDLVV90VOVLLLVVOOLVDOLYDYLÝÐ oOoLLVDLLOOVOLLVOVLVOVOJLLOOLOVLYOLLOEDVYVVOOLYWOOOOJLOVLVLOJ.LVLYOOLLY DLLyyÐLLDLOOLLVLOLLVOVOOVLOEDYLOVLLVOLXXXOOVVOVVLYLOVLVLVLVOYOYOÐLLÐO2

U.S. Patent Mar 25, 1997 Sheet 7 of 8 5,614,193

vooovÐLOÐLLLVvoLDLvov00LVOVLOLOOLSVOVVVVLVVLOVVDLLODODVOÐOLLVILLGç VLOLLVOÐLLVVLOoLovoLLvLv00ÐVOLVVDDOOYVYLLOLLLDOLLDLVVVVLV?00VVO?OO

OOOLolDJLDLLYDOVVLOVLLLLOOLIDOJ.DLLOLLLLLYLOLOLLVOLOGHzVOJLyyyyvvvLvo9VVOOVVLOED

OVL

5,614, 193 1 2 HANTAVIRUS WACCNE Schmaljohn and Dalrymple, Virology 131: 482-491 (1983). Hantaan virus particles contain three major structural pro teins: the nucleocapsid or "N" protein and the "G1" and BACKGROUND OF THE INVENTION "G2' glycoproteins. Id. In addition, virions contain an This application is a continuation-in-part of U.S. Ser. No. 5 RNA-dependent RNA polymerase function. Id. Like other 07/799,479, filed on Nov. 14, 1991, issuing as U.S. Pat. No. members of the Bunyaviridae family, the viral genome is 5,298,423, hereby incorporated by reference. comprised of three RNA segments: small (S), middle (M) Infectious febrile diseases with hemorrhagic and renal and large (L). Id. manifestations have been recognized across the Eurasian More detailed examination of the molecular structure of 10 Hantaan virus was provided following cloning and sequenc land mass for more than 50 years. Seven thousand cases of ing of cDNA's for the S and M segments. In their 1986 a "war nephritis' clinically similar to nephorpathia epi paper, Schmaljohn et al. reported that unlike other Bunyad demica (NE) were reported among British soldiers stationed viridae, the 1696 nucleotide Hantaan virus S segment did not in Flanders during World War I. Brown, Lancet i., 316–395 code for a non-structural protein (NS). Schmaljohn et al., (1916). Nevertheless, these disorders were not given much Virology 155:633-643 (1986). Rather, they found a single attention by Western physicians until the early 1950's during 15 open reading frame encoding a putative 428 amino acid the Korean conflict, when over 3000 cases were diagnosed polypeptide, presumed to be the N protein, a result consis among United Nations forces. Earle, Am. J. Med. 16: tent with earlier data from cell-free translation studies. Id. 617-709 (1954). The cDNA for the N product is disclosed and claimed in Subsequently, it was recognized that Korean hemorrhagic U.S. Pat. No. 5,298,423. fever and clinically similar diseases, collectively termed 20 In 1987, Schmaljohn et al. reported the 3616 base nucle hemorrhagic fever with renal syndrome (HFRS), pose a otide sequence for the Hantaan virus M segment. Within this significant health threatin much of Asia and parts of Europe region, a single open reading frame encoding 1135 amino and Scandinavia. Non-pathogenic of rodent popu acids was found. Schmaljohn et al., Virology 157: 31-39 lations apparently provides a reservoir for the causative (1987). Amino-terminal sequence of the G1 and G2 glyco agent. Infection of humans occurs via aerosol of the agent, 25 proteins demonstrated that this single open reading frame a Hantavirus of the family Bunyaviridae, from rodent urine, coded for both glycoproteins (5'-G1-G2-3"), probably feces and saliva. Mortality rates have decreased from the 10 expressed in the form of a polyprotein precursor that is to 15% seen during the Korean conflict to 5% or less, if cleaved post- or cotranslationally, both to separate the two improved fluid and electrolyte management and/or renal glycoproteins and to remove the amino-terminal 17 amino 30 acids of the G1 glycoprotein. Id. These data were confirmed dialysis are available. Currently, an estimated 50,000-100, later the same yearby a different group. Yoo and Kang, Nuc. 00 case occur annually in the Peoples' Republic of China, Acids Res. 15: 6299-6300 (1987). The cDNA's for the G1 with mortality rates ranging from 5 to 20%, in various and G2 products are disclosed and claimed in U.S. Pat. No. provinces. 5,298.423. A recent randomized, placebo-controlled of 35 A second Hantavirus, the "Seoul" serotype, has been intravenous ribavirin in HFRS patients presenting within the isolated and sequenced. Antic et al., Virus Res. 19: 47-58 first six days of clinical symptoms demonstrated a signifi (1991): Antic et al., Virus Res. 19: 59-66 (1991). The cant reduction in mortality. Huggins et al., J. Infect. Dis. Hantaan and Seoul serotypes show about 75% sequence 164: 1119-1127 (1991); Lee and Ahn, J. Korean Soc. Virol. homology at the RNA level. Antic et al., Virus Res. 24: 18: 143-148 (1988). Otherwise, management of all HFRS 35-46 (1992). Patients with HFRS caused by the Seoul patients is highly individualized and focused on supportive serotype appear to suffer a milder clinical form of the Ca. disease. Recent studies indicate that six other Hantaviruses A Hantavirus vaccine has been approved and is in use in exist: Puumala, Prospect Hill, Thailand, Dobrava, Thotta South Korea. Lee and Ahn (1988). This vaccine was devel palayam and Four Corners. Arikawa et al., Virology 176: oped by serial passage of the virus in brains of suckling pigs, 45 114-125 (1990); Xiao et al., Virology 198: 205-217 (1994); followed by inactivation with formalin. Initial claims Chu et al., Virology 198: 196-204 (1994); Nichol et al., reported that two doses of the vaccine, given via a subcu "Genetic Identification of a Novel Hantavirus Associated taneous route one month apart, resulted in 100% serocon with an Outbreak of Acute Respiratory Illness in the South version as measured by immunofluorescence. The produc western United States, ' Science in press (1994). Only tion of this vaccine, however, was not in compliance with 50 Hantaan, Seoul, Puumala, and Dobrava are known to cause current U.S. Food and Drug Administration guidelines on HFRS. Four Corners was described recently and linked to an "GoodManufacturing Practices," and the protective efficacy extremely deadly form of pulmonary distress in the south has not yet been determined. Furthermore, adventitious west United States. Thottapalayam, Prospect Hill and Thai agents in the product were not rigorously excluded and the land are not known to cause human disease. animal colony used for the vaccine's development was not 55 In 1988, the first use of a recombinant Hantavirus antigen pathogen-free. Other vaccines are currently undergoing test as a diagnostic agent was reported. Schmaljohn et al., J. ing in Japan, North and South Korea and China. Both Gen. Virol. 69: 777-786 (1988). cDNA's corresponding to Korean vaccine trials involved the use of formalin-inacti the S segment were used in a Baculovirus expression wated, Hantaan virus-infected mouse brain. Suh et al., Virus system, and the resulting N polypeptide showed specific Information Exchg. Newsl. 6: 131 (1989); Lee and Ahn reactivity with immune sera from rabbits, rats and humans (1988). The Chinese and Japanese trials involve inactivated, or Hantaan virus-specific monoclonal antibodies. Id. These tissue culture-derived Hantavirus preparations. Yu and Zhe, data were extended and confirmed in a second study, also Virus Information Exchg. Newsl. 6: 131 (1989). involving a recombinant, Baculovirus-expressed N protein. "Hantaan' virus, the etiologic agent of Korean hemor Rossi et al., Arch. Virol. S1: 19-28 (1990). Together, these rhagic fever, was originally isolated from the Korean striped 65 reports proved that recombinant Hantavirus proteins could field mouse. Lee et al., J. Infect. Dis. 137: 298–307 (1978). serve effectively as diagnostic reagents in screening for This serotype is the prototype for the Hantavirus genus. Hantavirus infection. 5,614, 193 3 4 Having confirmed that recombinantly-produced Hantavi tavirus. In addition, it is an object of the invention to provide rus antigens were immunologically related to those pro specific methodology for the safe and effective implemen duced by Hantavirus infection, researchers next sought to tation of such a vaccine. determine whether such antigens might be used as a vaccine In satisfying the foregoing objective, there is provided a to protect animals against infection by Hantavirus. In one 5 vaccine formulation suitable for use in a human, comprising: such study, with a vaccinia virus vectors (i) infectious vaccinia virus that comprises a polynucle expressing Hantaan virus glycoproteins not only elicited otide encoding antibody responses to Hantaan virus antigens in mice, they (a) the Hantavirus polypeptides designated N, G1 and also protected hamsters from Hantaan virus infection, as G2 (SEQID NOS 3, 1 and 2 respectively), and measured by indirect immunofluorescence of lung and kid 10 (b) all vaccinia virus polypeptides necessary for repli ney tissue. Schmaljohn et al., J. Virol. 64; 3162-3170 cation of said virus in a cell derived from said (1990). While promising, these data were deemed only to human, but not a functional thymidine kinase; and "demonstrate the feasibility of using expressed Hantaan (ii) a pharmaceutically-acceptable carrier, excipient or virus proteins to immunize animals to Hantaan virus' and diluent, wherein said vaccine formulation comprises a "should provide a basis for future exploitation of recombi 15 single dose of 1x10 to 7x10" plaque-forming units of nant-expressed Hantaan virus proteins as potential human vaccinia virus. vaccines." Id. at page 3170. In addition, there is provide a method for inducing a A later report by Schamljohn et al. reported the develop Hantavirus-protective immune response in a human, com ment of a different vaccinia-based vaccine containing both 20 prising the steps of: the M and S segments of Hantaan virus. Schmaljohn et al., (i) providing a vaccine formulation suitable for use in a Vaccine 10:10-13 (1992). In Dec. 1992, preliminary immu human comprising nization data for the M+S vaccinia virus vaccine was pre (a) infectious vaccinia virus activity and comprising a sented at a meeting in India. There, it was shown that the polynucleotide encoding M+S vaccine protected 3 out of 4 hamsters after a single immunization and 4 out of 4 hamsters given a secondary 25 (1) the Hantavirus polypeptides designated N, G1 immunization. These data were admitted to be of no statis and G2 (SEQID NOS 3, 1 and 2 respectively), and tical relevance and "repeat preclinical experiments ... with (2) all vaccinia virus polypeptides necessary for this potential human product” were reportedly in progress. replication of said virus in a cell derived from said Even though mortality rates for HFRS have dropped over human, but not a functional thymidine kinase; and 30 (b) a pharmaceutically-acceptable carrier, excipient or the past several decades, this syndrome still represents a diluent, significant threat to populations around the world, especially (ii) administering said vaccine formulation to said human, those in regions where high quality supportive care is not wherein said vaccine formulation comprises a single readily available. Therefore, it remains of utmost importance dose of 1x10 to 7x10' plaque-forming units of vac to develop a safe and effective vaccine against Hantavirus. 35 cinia virus. Currently, no vaccines have been demonstrated to meet both In a preferred embodiment, the expression of vaccine of these criteria. encoded polypeptides is under control of the vaccinia virus SUMMARY OF THE INVENTION 11 kD promoter. In another preferred emobidment, the Hantavirus It is, therefore, an object of this invention to provide a 40 polypeptides are derived from the Hantaan serotype. Pre vaccine, suitable for use in humans, that is capable of ferred sequences in this regard are the G1 sequence (SEQ10 inducing a protective immune response specific for Han NO: 1):

MET GLY LE TRPLYSTRP LEU VAL METALASER LEU VALTRPPROVAL LEU THR LEU ARG ASN VALTYR ASP METYS LE GLU CYS PRO HIS THRVAL SERPHE GLY GLUASN SERVAL LE GLYTYR VAL, GLU LEU PRO PROVAL PRO LEU ALA ASPTHRALAGLN MET WAL PROGLUSER SER CYSASN METASPASN HIS GLN SER LEU ASN THRILE THRLYSTYRTHR GLNWAL SER TRPARG GLYLYS ALAASPGLN SER GLNSER SER GLN ASN SERPHE GLUTHRVAL SER THR GLU WALASPLEULYS GLY THR CYS WALLEULYS HISLYS MET WAL GLU GLUSERTYRARG SER ARG LYSSERVAL THR CYSTYRASPLEU SER CYSASN SER THRTYRCYS YS PRO THR LEU TYR MET LEVAL PRO LE HISALA CYSASNMETLYS SERCYSLEU LEALALEU GLY PROTYRARGWAL, GLN VAL VALTYR GLU ARG SERTYRCYS MET THR GLY WALLEULE GLU GLY LYS CYS PHE WAL PRO ASP GLN SERVAL VAL SER LEILE LYS HIS GLY LEPHE ASPLEALASER WALHISILE WAL CYSPHEPHE WALALAWALLYS GLY ASN THRTYRLYSLEPHE GEU GLNWALLYSLYSSERPHE GLU SER THRCYSASNASPTHR GLUASNLYSWAL, GLNGLYTYRTYRILE CYSLE WAL, GLY ASN SERALAPRO LE TYRVAL PRO THR LEU ASPASP PHE ARG SER MET GLUALA PHETHR GLY LEPHE ARG SER PRO HIS GLY GLUASPHIS ASPLEUALA GLY GLU GLULEALASERTYRSERILE WAL, GLY PROALAASNALALYSWAL PRO HIS SER ALASER SER ASPTHR LEU SER LEU ILEALATYR SER GLY LE PROSERTYRSER SER LEU SER 5,614, 193 5 6 -continued ILE LEU THR SER SER THR GLUALALYS HIS WALPHE SER PRO GLY LEU PHE PRO LYSLEU ASN HIS THRASN CYS ASPLYS SER ALAILE PRO LEU ILE TRPTHR GLYMETILEASP LEU PRO GLYTYRTYR GLUALAVAL HIS PRO CYS THRVAL PHE CYS WALLEU SER GLY PRO GLYALASER CYS GLUALA PHE SER GLU GLY GLY LE PHEASNLE THR SER PRO MET CYSLEU WAL SERLYS GLNASNARGPHE ARG LEUTHR GLUGLNGLNWAL ASN PHE WALCYS GLN ARG WALASP METASPELE VAL VALTYRCYS ASN GLY GLN ARG LYS WALLELEU THRLYSTHRLEU VALILE GLY GLN CYSILETYR THRELE THR SER LEU PHE SER LEULEU PRO GLY WAL ALA HIS SER LEALAVAL GLU LEU CYS VAL PRO GLY PHE HIS GLY TRPALA THR ALA ALA LEU LEU VAL THRPHE CYSPHE GLY TRPVAL LEU ILE PROALALE THRPHEILE ILE LEU THRWALLEU LYSPHE ILEALA ASNILE PHE HIS THR SER ASN GLNGLUASNARG LEULYSSER WALLEU ARG LYSLELYS GLU GLU PHE GLULYSTHRLYS GLY SER MET VAL CYS ASP WALCYSLYSTYR GLU CYS GLUTHRTYRLYS GLULEULYS ALAHIS GLY WAL SERCYS PROGLN SER GLN CYS PROTYR CYSPHE THRHIS CYS GLU PRO THR GLUALA ALA PHE GLNALAHISTYRLYS WALCYS GLN WATHRHIS ARG PHEARGASPASPLEULYSLYSTHRWAL THR PRO GLNASN PHE THR PRO GLY CYSTYRARG THRLEUASNLEU PHEARGTYRLYSSER ARG CYSTYR LEPHE THRMET TRPILE PHE LEULEU WALLEU GLUSERILE LEUTRPALAALASER ALA the G2 sequence (SEQID NO: 2):

SER GLUTHR PRO LEUTHR PRO WALTRPASNASPASNALA HIS GLY WALGLY SERVAL PROMETHIS THR ASP LEU GLULEU ASPPHE SER LEU THR SER SER SERLYSTYRTHRTYRARG ARG LYSLEU THRASN PRO LEU GLU GLUALA GLN SER LEASPLEU HIS ILE GLUILE GLU GLU GLN THRILE GLY WALASP WALHIS ALA LEU GLY HISTRP PHE ASP GLY ARG LEU ASN LEULYSTHR SERPHE HIS CYSTYR GLY ALACYSTHRLYS TYR GLUTYR PRO TRPHIS THRALALYS CYS HISTYR GLUARG ASPTYR GLN TYR GLUTHR SER TRPGLY CYSASN PROSER ASPCYS PRO GLY WAL, GLY THR GLY CYSTHRALA CYS GLY LEUTRYLEUASP GLN LEU LYS PROVAL GLY SER ALA TYRLYSILE ILE THRILEARG TYR SER ARG ARGVAL CYS VAL, GLN PHE GLY GLUGLUASN LEU CYSLYSIELEASP METASNASP CYSPHE WAL SERARGHIS WALLYS WALCYSLE LE GLY THRVAL SERLYSPHE SER GLN GLY ASPTHRLEULEU PHEPHE GLY PRO LEU GLU GLY GLY GLY LEU ILE PHELYS HISTRP CYSTHR SER THRCYS GLN PHE GLY ASP PRO GLY ASPILE METSER PRO ARG ASPLYS GLYPHE LEU CYS PRO GLU PHE PRO GLY SERPHE ARG LYSLYS CYS ASN PHEALATHRTHR PROILE CYS GLUTYRASP GLY ASN MET WAL SER GLYTYRLYSLYS VAL METALATHR LEASP SERPHE GLNSERPHE ASN THR SER THRMETHIS PHETHRASP GLUARGILE GLUTRPLYS ASP PROASP GLY MET LEU ARG ASP HIS ILEASNILE LEU WALTHRLYS ASPILE ASPPHE ASPASN LEU GLY GLUASN PRO CYSLYSILE GLY LEU GLNTHRSER SERILE GLUGLYALATRP GLY SER GLY WAL, GLYPHE THRLEUTHR CYSLEU VAL SER LEU THR GLU CYS PROTHRPHE LEU THR SERILELYS ALA CYS ASPLYS ALA LE CYSTYR GLYALA GLUSER WALTHR LEU THRARG GLY GLNASN THRWALLYS WAL SER GLY LYS GLY GLY HISSERGLY SER THRPHE ARG CYSCYS HIS GLY GLUASP CYSSER GLN ILE GLY LEU HISALA. ALAALAPRO HIS LEU ASPLYS WALASN GLY ILE SER GLUILE GLU ASN SERLYS VALTYRASPASP GLY ALA PRO GLN CYS GLY LELYS CYSTRPPHE WALLYS SER GLY GLUTRPILE SER GLY LEPHE SER GLY ASN TRPLE WALLEUILE WALLEU CYS VAL PHE LEULEU PHE SER LEU WALLEU LEU SERILE LEU CYS PROVAL ARG LYS HIS LYSLYS SER 60 and the N sequence (SEQ ID NO:3):

METALATHRMET GLU GLULEU GLNARG GLUILEASNALAHIS GLUGLY GLN LEUVAL LEALAARGGLN LYS WALARG ASPALA GLULYS GLN TYR GLULYS ASP PROASP GLULEUASNLYSARG THR LEU THRASP 5,614, 193 7 8 -continued ARG GLU GLY WALALAVAL SERILE GLNALALYSILE ASP GLULEULYS ARG GLN LEU ALAASPARG LE ALATHR GLY LYSASN LEU GLY LYS GLU GLNASP PRO THR GLY WAL GLU PRO GLY ASPHISLEULYS GLU ARG SER MET LEU SERTYR GLY ASN WALLEU ASPLEUASN HIS LEU ASPLEASP GLU PRO THR GLY GLN THRALAASP TRPLEU SERILE ILE VALTYRLEUTHR SERPHE WAL VAL PRO ILE LEULEULYSALALEUTYR MET LEU THRTHRARG GLY ARG GLN THRTHRLYS ASPASNLYS GLY THRARGILE ARG PHELYS ASPASP SER SERPHE GLUASP VAL ASN GLY ILEARG LYS PRO LYS HISLEUTYRVAL SER LEU PRO ASNALA GLN SER SER METLYS ALA GLU GLUILE THR PRO GLY ARG TYRARG THRALAVAL CYS GLY LEU TYR PRO ALA GLNILE LYS ALA ARG GLN METILE SER PROVAL METSERVALILE GLYPHE LEUALA LEU ALALYS ASP TRPSER ASPARGILE GLU GLN TRPLEUILE GLU PRO CYSLYSLEU LEU PROASPTHRALAALAVAL SERLEU LEU GLY GLY PRO ALATHRASNARG ASPTYR LEU ARGGLN ARG GLN WALALALEU GLY ASN MET GLU THRLYS GLUSERLYS ALAILE ARG GLN HISALA GLUALAALAGLY CYSSER METILE GLUASPILE GLU SER PRO SER SERELETRPVAL PHEALAGLYALAPROASPARG CYS PRO PRO THRCYSLEU PHE ILEALAGLY ILEALA GLU LEU GLYALA PHEPHE SERILE LEU GLNASPMETARG ASNTHER LE METALASERLYSTHR VAL GLY THR SER GLU GLULYSLEU ARGLYSLYSSER SERPHETYR GLNSERTYR LEU ARG ARG THR GLN SER MET GLYILE GLN LEU GLY GLNARGILE ILE WALLEU PHE METWAL ALA TRP GLY LYS GLUALAWAL ASPASN PHE HIS LEU GLY ASPASPMETASP PRO GLULEU ARG THRLEU ALA GLN SER LEU ILEASP WAL LYS WALLYS GLUILE SER ASN GLN GLU PRO LEU LYS LEU, of which the first 17 amino acids of G1 are apparently cleaved to form the mature G1 protein and, therefore, are 30 optional. The preferred coding sequence for the G1, G2 and N polypeptides is (SEQ ID NO: 4):

ATGGGGATA TGGAAGTGGCTA GTGATG GCCAGTTTA GTATGGCCT GTFTTGACA CTGAGAAAT GTC TAT GACATGAAAATTGAGTGC CCC CAT ACA GTAAGTTTT GGG GAAAACAGT GTGATA GOST TAT GTA GAATTACCCCCC GTG CCATTG GCC GACACA GCACAGATG GTG CCT GAGAGTTCTTGT AACATG GAT AAT CAC CAATCGTTGAAT ACAATAACAAAA TATACC CAA GTA AGT TGG AGAGGAAAG GCT GAT CAG TCA CAGTCT AGT CAAAATTCATTT GAGAGA GTGTCC ACT GAA GTTGACTTGAAA GGAACA TGT GTT CTAAAACACAAAATG GTG GAA GAATCATAC CGTAGTAGGAAATCA GTA ACC TGT TAC GAC CTG TCT TGC AAT AGC ACTTACTGCAAGCCA ACA CTATACATGATT GTA CCAATT CAT GCATGC AATATGATG AAAAGCTGTTTGATT GCATTGGGACCATACAGR GTA CAG GTGGTTTAT GAGAGA AGT TACTGTATG ACAGGA GTC CTGATT GAAGGGAAATGCTTT GTCCCAGAT CAAAGT GTG GTCAGTATTATCAAG CAT GGG ATC TTT GATATT GCAAGTTTT CAT ATT GTATGTTTCTTT GTT GCA GTTAAA GGGAAT ACT TATAAA ATTTTT GAA CAGGTTAAGAAATCCTTT GAATCA ACATGCAAT GATACA GAGAATAAA GTG CAAGGA TAT TATATTTGTATT GTAGGGGGAAACTCT GCACCAATATAT GTT CCA ACA CTTGAT GATTTCAGA TCC ATG GAA GCATTT ACA GGAATCTTCAGATCA CCA CAT GGG GAA GAT CAT GAT CTG GCT GGA GAA GAAATT GCATCTTATTCTATA GTCGGA CCT GCCAAT GCA AAA GTTCCT CAT AGT GCTAGCTCAGAT ACATTGAGCTTGATT GCCTATTCAGGTATA CCA TCTTATTCTTCC CTTAGCATC CTAACAAGTTCA ACA GAAGCTAAG CAT GTATTCAGCCCT GGGTTGTTC CCAAAACTTAAT CACACAAATTGT GATAAAAGT GCC ATA CCA CTCATA TGG ACT GGGATGATT GATTTACCTGGATACTAC GAA GCT GTC CACCCT TGT ACA GTTTTTTGC GTATTATCAGGT CCT GGG GCATCATGT GAA GCCTTTTCT GAA GGC GGG ATTTTC AACATAACCTCTCCC ATGTGCTTAGTGTCAAAACAAAATCGATTC CGGTTA ACA GAA CAG CAA GTG AATTTT GTGTGT CAG CGA GTG GACATG GACATT GTT GTG TACTGAAACGGG CAGAGGAAA GTAATA 5,614, 193 9 10 -continued TTAACAAAAACT CTA GTTATTGGA CAGTGTATATAT ACTATA ACAAGCTTAXXXTCATTACTA CCT GGA GTA GCA CAT TCTATT GCT GTT GAATTGTGT GTA CCT GGGTTC CAT GGTTGG GCCACA GCT GCT CTG CTT GTT ACA TTC TGTTTCGGA TGG GTT CTTATA CCA GCAATTACATTTATCATA CTA ACA GTC CTA AAGTTCATT GCT AATAATTTT CACACAAGTAAT CAAGAGAAT AGG CTAAAATCA GTACTTAGAAAG ATAAAG GAA GAGTTT GAAAAAACAAAAGGCTCAATG GTATGT GAT GTCTGCAAG TAT GAGTGT GAA ACA TATAAA GAATTAAAG GCA CAC GGG GTATCATGC CCC CAATCT CAATGT CCTTACTGTTTT ACT CAT TGT GAA CCT ACA GAA GCA GCATTC CCA GTC CATTACAAG GTATGC CAA GTT ACT CACAGATTC AGG GAT GAT (CTAAAGAAAACT GTT ACT CCT CAAAATTTTACA CCA GGATGT TAC CGGACA CTAAAT TTATTT AGATACAAAAGCAGGTGCTAC-ATCTTTACAATGTGGAATTT CTT CTTGTCTTAGAATCC ATA CTGTGG GCT GCA AGT GCATCA GAG ACACCATTAACT CCT GTCTGGAATGACAAT GCC CAT GOG GTA GGTTCT GTT CCTATG CAT ACA GATTTAGAG CTTGATTTCTCTTTAACATCCAGTTCCAAG TATACATAC CGTAGGAAGTTAACAAACCCA CTTGAG GAA GCACAATCCATT GAC CTA CATATTGAAATA GAA GAA CAGACAATT GGT GTT GAT GTG CAT GCT CTAGGA CACTGGTTT GAT GOTCGT CTTAAC CTTAAAACA TCCTTT CAC TGT TAT GGT GCTTGTACAAAG TAT GAA TAC CCTTGG CAT ACT GCA AAGTGC CAT TAT GAA AGAGATTAC CAA TAT GAA ACGAGCTGGGGTTGTAAT CCATCA GATTGT CCT GGG GTG. GGCACA GGCTGT ACA GCATGTGGTTTATAC CTAGAT CAA CTGAAA CCA GTT GGTAGT GCT TATAAAATTATC ACAATAAGGTACAGC AGGAGA GTC TGT GTT CAGTTT GGG GAG GAAAAC CTTTGT AAGATAATAGAG ATGAAT GATTGTTTT GTATCT AGG CAT GTTAAG GTC TGCATAATTGGT ACA GTATCT AAATTCTCT CAG GGT GAT ACCTTATTGTTTTTT GGA CCG CTTGAAGGT GGT GGT CTA ATA TTT AAA CACTGGTGTACA TCCACATGT CAATTT GGTGAC CCAGGA GATATCATGAGT CCAAGAGACAAA GGTTTTTTATGCCCT GAGTTTCCAGGTAGTTTCAGGAAGAAATGC AACTTT GCT ACT ACC CCTATTTGT GAG TAT GAT GGA AAT ATGGTCTCAGGTTACAAGAAA GTGATG GCGACAATTGATTCCTTC CAATCTTTT AATACAAGC ACT ATG CACTTCACT GAT GAAAGGATA GAGTGGAAA GAC CCT GAT GGAATG CTA AGG GAC CATATA AACATTTTA GTAACGAAG GACATTGACTTT GATAAC CTTGGT GAAAATCCTTGCAAAATT GGCCTA CAAACA TCT TCTATTGAG GGG GCCTGGGGTTCT GGT GTG GGGTTCACATTA ACA TGT CTG GTATCA CTA ACA GAATGT CCT ACCTTTTTGACC TCAATAAAG GCTTGT GATAAG GCTATCTGT TAT GGT GCA GAGAGT GTA ACATTGACAAGAGGA CAA AAT ACA GTCAAG GTATCAGGGAAA GGT GGC CAT AGT GGT TCA ACATTTAGGTGTTGC CAT GGG GAG GACTGTTCACAAATT GGA CTC CAT GCT GCT, GCA CCT CAC CTTGACAAG GTA AAT GGGATTTCT GAGATAGAAAAT AGTAAA GTATAT GAT GAT GGG GCACCG CAA TGT GGGATAAAATGTTGGTTT GTTAAATCAGGG GAATGGATTTCAGGGATATTCAGTGGT AATTGG ATT GTA CTCATT GTC CTCTGT GTATTT CTATTGTTCTCCTTGGTTTTACTA AGCATT CTCTGT CCC GTA AGGAAG CATAAAAAATCA

and (SEQ ID NO:5)

ATG GCA ACT ATG GAG GAATTACAGAGGGAAATC AAT GCC CAT GAG GGT CAATTA GTG ATA GCC AGG CAGAAG GTG. AGG GAT GCAGAAAAA CAG TAT GAAAAG GAT CCA GAT GAGTTGAACAAGAGA ACATTA ACT GAC CGAGAGGGC GTT GCA GTATCTATC CAG GCAAAAATTGAT GAGTTAAAAAGGCAA CTG GCA GATAGGATT GCA ACT GGGAAAAAC CTTGGGAAG GAA CAAGAT CCA ACA GGG GTG GAGCCTGGA GAC CAT CTGAAA GAGAGGTCAATG CTCAGT TAT GGTAAT GTG CTG GATTTAAAC CATTTG GATATTGAT GAA CCTACA GGA CAGACA GCA GACTGGCTGAGCATCATC GTC TAT CTTACATCCTTT GTCGTCCCG 5,614, 193 11 12 -continued ATACTT CTG AAA GTC CTG TATATGTTGACA ACA AGG GGG AGG CAA ACT ACCAAG GATAATAAA GGG ACC CGGATT CGATTTAAG GAT GAT AGCTCGTTC GAG GAT GTTAACGGT ATC CGG AAA CCAAAA CAT CTT TAC GTGTCCTTG CCAAAT GCA CAG TCA AGC ATGAAG GCA GAA GAGATTACA CCT GGT AGATAT AGA ACA GCA GTCTGT GGG CTCTAC CCT GCA CAGATTAAG GCACGG CAG ATG ATC AGT CCA GTTATG AGT GTAATT GGTTTT CTA GCATTA GCA AAG GACTGGAGT GAT CGT ATC GAA CAATGGTTAATTGAA CCTTGCAAG CTT CTT CCA GATACA GCA GCA GTTAGC CTC CTCGGT GGT CCT GCA ACA AAC AGG GAC TACTTACGG CAG CGG CAA GTG GCATTAGGCAAT ATG GAG ACA AAG GAG TCA AAG GCTATA CGC CAG CAT GCA GAA GCA GCT GGCTGT AGC ATGATT GAA GATATTGAGTCA CCATCATCAATATGGGTTTTT GCT GGA GCACCA GAC CGT TGT CCACCA ACATGTTTGTTTATA GCA GGTATT GCT GAG CTTGGG GCA TTTTTTTCC ATC CTG CAGGAC ATG CGAAATACAATCATG GCATCTAAG ACR GTT GGA ACA TCT GAG GAGAAG CTA CGGAAGAAATCATCATTTTAT CAG TCC TAC CTCAGAAGGACA CAATCAATGGGGATA CAACTA GGC CAGAGAATTATT GTG CTCTTCATG GTT GCCTGGGGAAAG GAGGCT GTG GAC AACTTC CACTTAGGG GAT GATATG GAT CCT GAG CTA AGG ACA CTG GCA CAGAGCTTGATT GAT GTCAAA GTG AAG GAAATCTCCAAC CAA GAGCCTTTGAAA CTC.

In a series of preferred embodiments, the vaccine single 25 DETALED DESCRIPTION OF THE dose comprises 1x10 to 1x10' plaque-forming units, 1x10 NVENTION to 1x10 plaque-forming units, 1x10 to 5x10 plaque Vaccinia virus is a member of the Orthopox genus of the forming units or 3.4x10' plaque-forming units. Poxvirus family with little virulence for humans. Although In yet another preferred embodiment, the pharmaceuti the exact origin of vaccinia virus is obscure, it is related to cally-acceptable carrier, excipient or diluent further com 30 the cowpox virus used by Jenner and strains of vaccinia prises lactose and human serum albumin, most preferably at virus became the vaccines of choice for the prevention of 5% (w/v) and 1% (wiv) of said formulation, respectively. smallpox. Baxby, “Vaccinia Virus,” in VACCINIA VIRUSES In still another preferred embodiment, the vaccine formu AS VECTORS FOR WACCINE ANTIGENS. G. V. Quinnan, lation further comprises neomycin of no more than 25g per ed., Elsevier, New York, N.Y., pp. 3-8 (1985). The smallpox single dose of said formulation. 35 vaccines used in the eradication effort were prepared on In yet another preferred embodiment, the single dose is large scale by inoculating the shave abdomens of calves, provided in a volume of 0.1 to 1.0 ml, with increments of 0.1 sheep or water buffalo with seedstocks of vaccinia virus and ml. harvesting the infected exudative lymph from the inocula In still another preferred embodiment, the vaccine formu tion sites. Henderson and Arita, "Utilization of Vaccine in lation is in a form suitable for a route of administration 40 the Global Eradication of Smallpox,” VACCINIA VIRUSES selected from the group consisting of subcutaneous, intra AS VECTORS FOR WACCINE ANTIGENS. G. V. Quinnan, muscular and intradermal. ed., Elsevier, New York, N.Y., pp. 61-67 (1985). The Other objects, features and advantages of the present novelty of the procedure used by Jenner caused invention will become apparent from the following detailed alarm with some of his contemporaries. The ultimate eradi description. It should be understood, however, that the 45 cation of smallpox following implementation of the Inten detailed description and the specific examples, while indi sified Smallpox Eradication Program of the World Health cating preferred embodiments of the invention, are given by Organization proved that skepticism to be without founda way of illustration only, since various changes and modifi tion. cations within the spirit and scope of the invention will Vaccinia virus has several biological properties which become apparent to those skilled in the art from this detailed 50 make it an excellent candidate for use as alive vaccine. First, description. it possesses a high degree of physical and genetic stability under even severe field conditions, reducing problems and expense in transport and storage. In addition, genomic BRIEF DESCRIPTION OF THE DRAWINGS stability makes the incorporation of one or more foreign 55 genes for the antigens to be expressed more feasible than in FIG. 1 Amino acid sequence (SEQID NO: 1) of Hantaan other systems. Second, because vaccinia replicates in the virus G1 glycoprotein. Bracketed region (. . .) indicate cytoplasm of host cells and uses its own DNA and RNA potentially cleaved residues. polymerases, effects on the host cell's physiologic functions are minimized. Third, vaccinia virus has a wide host range, FIG. 2 Amino acid sequence (SEQID NO: 2) of Hantaan thus permitting use of a single vaccine in a large number of virus G2 glycoprotein. species. Fourth, both humoral and cellular immunity are FIG. 3 Amino acid sequence (SEQID NO:3) of Hantaan mediated by vaccinia virus-based vaccines. And fifth, the virus nucleocapsid N protein. duration of effectiveness of vaccinia immunization is rela FIG. 4 Nucleotide sequence (SEQID NO: 4) of Hantaan tively long. See Haber et al., Science 243:51 (1989). Much virus M segment cDNA. 65 of the early work geared towards a vaccinia virus vector was FIG. 5. Nucleotide sequence (SEQ 10 NO: 5) of Hantaan undertaken with vaccine development in mind. Weir et al., virus S segment c DNA. Proc. Nat'l Acad. Sci. USA 79: 1210-14 (1982); Mackett et 5,614, 193 13 14 al., Proc. Nat'l Acad. Sci. USA 79: 7415-19 (1982); Smith the pharmaceutical composition are adjusted according to et al., Nature 302: 490-95 (1983); Smith et al., Proc. Natl routine skills in the art. Goodman and Gilman, THE PHAR Acad. Sci. USA 80: 7155-59 (1983). MACOLOGICAL BASIS FOR THERAPEUTICS (7th ed.). As with any vaccine, safety is a major concern with the Typically, such vaccines are prepared as injectables, either use of vaccinia virus as a immunizing agent. The adverse as liquid solutions or suspensions; solid forms suitable for reaction rate of 1 in 50,000, reported during smallpox solution in, or suspension in, liquid prior to injection may , was tolerated only because the disease it also be prepared. The preparation also may be emulsified. prevented was so devastating. Baxby (1985). Generalized The active immunogenic ingredient is often mixed with an vaccinia among persons without underlying illnesses is excipient which is pharmaceutically-acceptable and compat characterized by a vesicular rash of varying extent that is O usually self-limited. In the event of the formation of skin ible with the active ingredient. Suitable excipients are, for lesions as a result of virus replication, there is a risk of example, water, saline, dextrose, glycerol, ethanol, or the bacterial superinfection. In addition, there is also a risk of like and combinations thereof. In addition, if desired, the the formation of a scar at the site of skin lesions if they occur. vaccine may contain minor amounts of auxiliary substances Several attenuated strains were developed Such as wetting or emulsifying agents, pH-buffering agents, but, due to lower potency, were not adopted for general use. 5 adjuvants or immunopotentiators which enhance the effec Recent efforts towards genetic engineering of vaccinia virus tiveness of the vaccine. have resulted in strains with decreased virulence. These The vaccines are conventionally administered parenter efforts targeted the viral thymidine kinase, growth factor, ally, by injection, for example, either subcutaneously or hemagglutinin, 13.8 kD secreted protein and ribonucleotide intramuscularly. Additional formulations which are suitable reductase genes. Bulleret al., Nature 317: 813 (1985); Buller 20 for other modes of administration an include oral formula et al., J. Virol. 62: 866 (1988); Flexner et al., Nature 330: 259 tions. Oral formulations include such typical excipients as, (1987); Shida et al., J. Virol. 62:4474 (1988); Kotwal et al., for example, pharmaceutical grades of mannitol, lactose, Virology 117: 579 (1989); Child et al., Virology 174: 626 starch, magnesium stearate, sodium saccharine, cellulose, (1990). There also is interest in using other members of the magnesium carbonate and the like. The compositions take poxvirus family, such as avipoxviruses, as limited host range 25 the form of solutions, suspensions, tablets, pills, capsules, vaccine vectors. Taylor et al., Virology 6: 497 (1988). For Sustained release formulations or powders and contain instance, U.S. Pat. No. 5,266,313, hereby incorporated by 10%-95% of active ingredient, preferably 25-70%. reference, discloses and claims a raccoon poxvirus-based The term "unit dose" refers to physically discrete units vaccine for rabies virus. Thus, discussion of vaccinia in the suitable for use in humans, each unit containing a predeter following examples is not intended to suggest limitation of 30 mined quantity of active material calculated to produce the the possible vaccine vectors. desired therapeutic effect in association with the required Recombinant vaccinia viruses have been used to express diluent, i.e., carrier or vehicle, and a particular treatment genes of nonviral pathogens such as bacteria, rickettsia and regimen. The quantity to be administered, both according to protozoa and, in some cases, have protected experimental 35 number of treatments and amount, depends on the subject to animals from infection. Fields, Science 252: 1662-67 be treated, capacity of the subject's immune system to (1991). In addition, vaccinia-based rabies and rinderpest synthesize antibodies, and degree of protection desired. vaccines have been tested. Id. The human immunodeficiency Precise amounts of active ingredient required to be admin virus type 1 (HIV-1) envelope glycoprotein (env) gene has istered depend on the judgment of the practitioner and are been cloned into a vaccinia vector and a phase trial was 40 peculiar to each individual. However, suitable dosage ranges conducted with this virus. The vaccine appeared safe, and are on the order of one to several hundred micrograms of demonstrated the development of readily detectable, persis active ingredient per individual. Suitable regimes for initial tent in vivo T-cell proliferative and serum antibody administration and boostershots also vary but are typified by responses to HIV-1 in vaccinia-naive persons. Cooney et al., an initial administration followed in one or two week Lancet 337: 567 (1991). A neutralizing antibody response 45 intervals by one or more subsequent injections or other was not seen but the expression of the env gene was low administration. compared to levels now obtainable. The following examples are given by way of illustration The pharmaceutical compositions of the present invention and should not be construed as limiting the scope of the are advantageously administered in the form of injectable invention. The examples are drawn from a Investigational compositions. A typical composition for such purpose com 50 New Drug Application, submitted to the U.S. Food and Drug prises a pharmaceutically acceptable carrier. For instance, Administration on Jun. 25, 1993, entitled "Hantaan M-S the composition may contain human serum albumin in a (Vaccinia virus-vectored) Recombinant Vaccine (TSI-GSD phosphate buffer containing NaCl. Other pharmaceutically 264), FDA IND NO. 5166, hereby incorporated by refer acceptable carriers include aqueous solutions, non-toxic C. excipients, including salts, preservatives, buffers and the like 55 (REMINGTON'S PHARMACEUTICAL SCIENCES, 15th EXAMPLE 1. Ed., Easton ed., Mack Publishing Co., pp 1405-1412 and 1461-1487 (1975) and THE NATIONAL FORMULARYXIV, Construction of a Vaccinia-Based Hantavirus 14th Ed., American Pharmaceutical Association, Washing Vaccine ton, DC (1975), both hereby incorporated by reference). Examples of non-aqueous solvents are propylene glycol, VACCINE CONSTRUCTION-Sequences pertinent to polyethylene glycol, vegetable oil and injectable organic the construction and production of the candidate vaccine are esters such as ethyloleate. Aqueous carriers include water, illustrated in FIGS. 4 and 5 (SEQID NOS 4 and 5). alcoholic/aqueous solutions, saline solutions, parenteral 1. Expression of HFRS antigenic subunits in vaccinia virus: vehicles such as sodium chloride, Ringer's dextrose, etc. 65 a. Construction of transfer vector plasmids. Intravenous vehicles include fluid and nutrient replenishers. Bgll restriction sites were engineered near the 3'- and The pH and exact concentration of the various components 5'-ends of complementary DNA (cDNA) representing the 5,614, 193 15 16 Hantaan virus Mgenome segment by site-directed mutagen tion consisting of TBST and 10% calf serum. The filters esis. Digestion with Bgll generated a restriction fragment of were rocked at room temperature for 1 h. approximately 3.5 kB which contained the entire coding The blocking solution was then replaced with polyclonal, regions of the G1 and G2 envelope glycoproteins. For hyperimmune, mouse ascitic fluid or monoclonal antibody subcloning into the SmaI site of the vaccinia virus transfer ascitic fluid to Hantaan virus proteins diluted 1:500 in TBST vector pSC11, the DNA was treated with the large (Klenow) and 10% calf serum. Normal mouse asciitic fluid was used fragment of DNA polymerase I to produce blunt ends. The for control assays. The filters were again rocked for 1 hour lac Z gene in the plasmid was deleted using the restriction at room temperature, after which they were washed three enzymes Pstl and XhoI. times for 10 minutes each with TBST. Alkaline phosphatase Because animal experimentation with recombinants indi 10 conjugated rabbit anti-mouse IgG (Promega Biotec, Madi cated that Hantaan virus nucleocapsid (N) protein, encoded son, Wis.), diluted 1:4000 in TBST was incubated with the by the S segment, conferred protection against challenge washed filters by rocking at room temperature for 10 min with Hantaan virus in concert with the glycoproteins, the S with TBST followed by incubation with a color developer segment was included in construction of the recombinant solution consisting of 330 pig NBT and 165 ug BCIP/ml vaccine candidate. 15 (Promega Biotec, Madison, Wis.) dissolved in 100 mM b. Preparation of vaccinia virus recombinants. Tris-HCl (pH 9.7) and 100 mM MgCl. Color development For vaccinia virus recombinations, confluent monolayers was stopped after 5-10 minutes by rinsing the filters with of Vero E6 cells in 25-cm’ flasks were infected at a multi sterile distilled water. plicity of infection of 0.03 plaque forming units (PFU) per To recover recombinant viruses, dark purple spots on the cell with a seed vaccinia virus diluted in liquid Eagle's 20 filters were cut out with sharp scissors and placed into sterile Minimal Essential Medium (MEM) containing Earles' salts. 10 mM Tris-HCl (pH 8.8). The filter pieces in buffer were EMEM; Gibco, Gaithersburg, Md. This virus seed carried either frozen at-70° C. until needed or were sonicated on ice the designation "Conn 3E1, Vero 1, D3 in Saline A, 7, Mar. at maximum output in a cup sonicator three times for 30 87, 4x10 PFU/ml." The Conn 3E1 seed was a 3X plaque seconds and then assayed immediately. Three successive purified derivative of the licensed Connaught smallpox 25 immunoselections were then performed. For all assays vaccine. (Conn-Master 17633). Before use in recombinant beyond the first selection, recombinant viruses were soni construction, it had been passaged once in Vero cells and cated and then filtered through 0.45 um filters in order to harvested on day 3, titering at 4x10' PFU/ml. ensure that plaques resulted from infection of a single After incubation at 37° C. for 4 h, the medium was vaccinia virus. Plaques obtained from recombinants express removed and 2 ml of fresh EMEM was added. Prior to 30 ing both the M and S segments were detectable with transfection of infected cells, 20g of the plasmid transfer monoclonal antibodies to N, G1, G2, or with anti-Hantaan vector was diluted to 0.5 ml in a buffer containing 150 mM virus polyclonal ascitic fluid. NaCl, 0.7 mM NaHPO, 5 mM KC1, 20 mM HEPES Following the three immunoplaque selections, two stan (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and dard plaque assays under agarose were then performed by 6 mM dextrose, adjusted to pH 7.05. Calcium chloride (25 35 infecting confluent monolayers of Vero E6 cells in 60 mm ul of 2.5M) was added slowly and the solution was incu dishes for 1 h and overlaying the infected cells with EMEM bated at room temperature for 0.5 to 1.0 h. The transfection containing 5% fetal bovine serum, 50 g/ml gentamicin and mixture was added dropwise to the infected cell culture 1% agarose (SeaKem, FMC Corp., Marine Colloids Div., supernatants and cells were then incubated at 37° C. for 3 h, Rockland, Me.). Random, visible plaques were picked with after which the medium was removed. A virus-neutralizing a sterile Pasteur pipette. antibody and fresh EMEM was added. The cells were The recombinant viruses were then transferred to a vac incubated at 37° C. until cytopathic effects were extensive cine production laboratory where additional plaque purifi and most of the cells had detached from the flask (4 to 5 cation in certified cells was performed. Individual plaques days). Cells were then pelleted from the cell culture super previously recovered were diluted in EMEM, sonicated and natant by centrifugation (8,000xg) and suspended in 10 mM 45 plaque-purified three additional times from agarose-covered Tris-HCl (pH 8.8). After two cycles of free-thawing (-70° monolayers of a certified lot of MRC-5 human diploid lung C/37 C.), cells were sonicated on ice four times for 30 cells (ATCC, CCL171). Assays were performed in a decon seconds each at a maximum output in a cup sonicator. taminated biological safety hood within the Vaccine Pro Because the lac Z gene had been removed from the duction Laboratory at United States Army Medical Research plasmid transfer vector, recombinants could no longer be 50 Institute of Infectious Disease (USAMRIID) in which no selected from wild-type vaccinia viruses by color. Conse other infectious agents were being investigated. At each quently, an immunoplaque assay selection procedure was passage, plaque harvests were sonicated in sterile, physi implemented. Confluent monolayers of Vero C1008 cells ologic saline and filtered through 0.45 um filters to reduce (Vero E6 cells, ATCC, CRL 1586) in 60 mm Petri dishes the possibility of vaccinia aggregation. An individual plaque were infected with 10 to 10 dilutions of recombinant 55 picked from the third assay was used to infect certified vaccinia viruses overlaid with EMEM, 5% fetal bovine MRC-5 cells and a small virus seedstock was prepared. This serum and 50 g/ml gentamicin. Two days after infection, or thrice plaque-purified virus stock, designated "HTN-REC when vaccinia virus plaques were clearly visible, medium VAX#3, 3pp MRC-5/VERO, HTN-M+HTN-S, MAR 90." was removed and monolayers rinsed once with physiologi was transferred to The Salk Institute-Government Services cal saline. Division (TSI-GSD) in Swiftwater, Pa. There, it was A 57mm dry, nitrocellulose filter (Schleicher and Schuell, expanded to produce a master seed in certified MRC-5 cells Keene, NH, BA85) was placed on the top of the monolayers (see below). and a piece of Whatman 3MM filter paper soaked in TBST c. Expression of Hantaan genes. buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% The lot #1 candidate double-recombinant vaccine Tween 20) was placed on top of the nitrocellulose. The filter 65 (expressing Hantaan virus glycoprotein and nucleocapsid and paper were left in place for about 5 min, after which the products) was examined by radiolabeling and electrophore nitrocellulose was removed and placed in a blocking solu sis of immunoprecipitated proteins. The proteins produced 5,614, 193 17 18 were indistinguishable from the authentic Hantaan virus freeze-dried as "Lot 1-1-90' of the vaccine candidate, with proteins. Comparison of previous vaccinia recombinants the product number designation of TSI-GSD-264. expressing the M and S gene demonstrated that comparable Control (uninfected) fluid was prepared at each step by levels of Hantaan envelope proteins G1 and G2 were pro shell-freezing the cells in EMEM and then blending, soni duced with each recombinant. Amounts of radiolabeled N cating, centrifuging and supplementing with HSA. Routine protein appeared considerably greater in the new recombi safety testing of final bulk, final container and control fluids nant vaccine. This might be due to improved expression of was satisfactory. These tests included bacterial sterility, the S gene under control of the vaccinia virus 11 kD including in vitro Mycobacterium culture, mycoplasma cul promoter, as opposed to the 7.5 kD promoter used in the ture in broth and on agar, tissue culture testing for adven previous recombinant. Wittek et al., J. Virol. 49:371-378 0 titious agents and general safety in mice after intracerebral (1984). . EXAMPLE 2 EXAMPLE 3

Manufacture of Seed Stocks and Vaccine Lots 15 Composition and Storage of Live Hantavirus Methods (TSI-GSD 264, Lot #1-1-90 (Lot #1)) 1. Seed Stocks: The product is a freeze-dried preparation of cell-cultured, a. Master Seed. Hantaan M+S recombinant vaccinia virus grown in certified The thrice plaque-purified virus stock, designated "HTN MRC-5 cells at 35° C. in Eagle's Minimum Essential REC VAX#3, 3 pp MRC-5/VERO, HTN-M+HTN-S, MAR 20 Medium (EMEM) with 5% fetal bovine serum. After puri 90' (see above), was transferred to TSI-GSD for manufac fication, the fetal bovine serum was reduced to an insignifi ture in certified MRC-5 cells. The production process is cant amount. The fetal bovine serum is removed during virus described briefly below. A 1:100 dilution of the seed material concentration with the Minitan tangential flow device (Mil was used to inoculate 200 tissue culture flasks (150 cm) of lipore). The 300,000 MW cut-off retains virus particles and MRC-5 cells for production of master seed virus. Infected flushes out fetal bovine serum components. Any residual cells were harvested by centrifugation. After blending and fetal bovine serum is incapable of penetrating the 30-60% sonicating the infected cells, virus was partially purified by sucrose gradient through which virus was run. Virus was centrifugation in sucrose. The resultant freeze-dried master retrieved from the 41-49% sucrose fractions. Final purified seed, designated NTOO87, was stabilized with 1 gm percent vaccine product was stabilized to a final concentration of 5% human serum albumin (HSA). 30 lactose and 1 gm percent human serum albumin (HSA), b. Production Seed. freeze-dried and sealed in an atmosphere of sterile, dry The Master Seed (NTOO87) was used for the manufac nitrogen. Each vial of dried product contains approximately ture of a production seed according to the following proto 6.76x107 PFU/ml. Due to variability in assaying for PFU, col. Two hundred tissue culture flask (150 cm) cultures of 35 some variations may occur from test to test. MRC-5 cells were infected with a 1:100 dilution of the master seed virus. Infected cells were incubated to 4+ EXAMPLE 4 cytopathic effect (CPE). "4+ CPE' indicates total or near Safety Test of Hantavirus Vaccine for Adventitious total loss of cell viability, characterized by loss of adherence Agents to the growth vessel. Cells were harvested, blended and sonicated and the virus was then partially purified by sucrose Objective centrifugation. HSA was added to 4% (v/v) as stabilizer and To remove vaccinia virus from a preparation while other then this Production Seed, designated NTOO88, was freeze viruses remain. dried understandard conditions. A Production Seed Control Background Fluid, designated NTO588, was prepared in parallel from 45 1. Type protocol for removal of vaccinia with 0.45 um uninfected cultures. filter (Nalgene model 245-0045) was provided by Dr. Smith c. Lot 1. of the Food and Drug Administration (FDA). This protocol Lot 1 of the recombinant vaccinia-vectored Hantaan vac had been abstracted from a submission accepted by the FDA cine was prepared by infecting 280 tissue culture flask (150 for a vaccinia product manufactured by an anonymous cm) cultures of MRC-5 cells with a 1:100 dilution of 50 company. Production Seed virus (NTOO88). The cultures were incu 2. Results reported in subject protocol could not be bated for 3 days at which time cytopathic effect was maxi duplicated. It is possible that the difference is in the effect of mum (4+). Infected cells were harvested and later frozen, sonication. In the original study, sonication may have failed thawed, blended, sonicated, clarified and resuspended. The to dissociate virus from membranes so that it was removed resuspended material was frozen again. To begin purification 55 by filtration. In contrast, we may have successfully disso of the bulk, the frozen material was thawed, sonicated, and ciated the virus using what is presumed to be the same pooled before concentration to 320 ml volume using a procedure and, as a consequence, could only partially 300,000 MW low-protein binding membrane. This concen remove the virus by filtration. trated bulk was sonicated and then partially purified by 3. Preliminary experimentation with a number of vac centrifugation through sucrose. Fractions were titered and cinia-specific antisera of human, rabbit and lymphocyte those with an acceptable titer (2.0x10 PFU/ml) were hybridoma origin demonstrated that vaccinia virus is not pooled, giving a volume of 380 ml. This pool was dialyzed efficiently neutralized. We found that monoclonal antibody versus phosphate buffered saline (PBS) without calcium or 7D11 was superior to all others tested, but could not neu magnesium (MR0039) to reduce the sucrose from 38% to tralize more than 10 virions. Thus, it was obvious that it 2.0% and then combined with an equal volume of stabilizer 65 would be necessary to remove any virus in excess of that (10% lactose with 2 gm percent HSA). This material was amount (vaccine typically contains 10 virions) before it then dispensed in 1 ml aliquots in sterile 6.5 ml vials and could be tested successfully in the tissue culture safety test. 5,614, 193 19 20 4. These factors necessitated additional modification of is dependent upon dermatropic properties. These properties the protocol. Therefore, the filtration process was repeated a are estimated in the rabbit scarification test. second time to reduce the pre-neutralization titer to 10s 2. Presently, vaccine induced scars are highly undesirable. virions. Therefore, TSI-GSD 264 vaccinia vectored Hantaan vaccine Materials and Methods has been produced in cell culture so that it can be admin 1. Test Articles and Reagents: istered by subcutaneous or intramuscular inoculation. Con a. Vaccinia-infected MRC-5 cells were frozen at -70° C. sequently, retention of dermatropic properties are not essen until used. The material was thawed and the cell debris tial. Nevertheless, the seed virus has been tested to ensure pelleted by centrifugation. The supernatant fluid was dis that the cell culture passage has not enhanced dermatropic carded and the pellet resuspended to its original volume. 10 . This material most closely approximated that used in vac 3. Acceptability criteria is that the vaccine seed does not cine manufacture. This suspension was sonicated twice for cause a more severe dermatropic reaction than the FDA 20 seconds. reference vaccine. b. A BHV-1 herpes virus sample was prepared using Materials standard methodology. 15 1. Test article-Hantaan (vaccinia-vectored), production c. Filter-0.45 um Nalgene filter (Model #245-0045). seed, Lot 1 (NT0088). 2. Virus Titration: 2. Control article-FDS reference vaccinia, Lot2. a. Vaccinia was titrated in triplicate using monolayers of 3. Test system-New Zealand White rabbits, 2 to 3 Kg, Vero cells in 6-well plates. After adsorption of samples for female. 2 hours, cells were overlayed with media containing 0.6% 20 Procedure agarose and incubated at 37°C. in a CO-rich environment 1. Rabbits were sedated with a Ketamine/Rompun mix for 48 h. A second overlay, the same as above except for ture. addition of neutral red, was added and the cells were 2. Hair was clipped from four, 2x5 cm areas on each side incubated an additional 24 h. Plaque titers were determined of the rabbit and scarified by vigorous rubbing with a by standard procedures. 25 "comb.' b. Herpes virus was titrated in triplicate using monolayers 3. Serial dilutions of recombinant Hantaan vaccine seed of bovine turbinate cells in 6-well plates as described above and reference vaccinia virus were prepared in two except that a single overlay was used. After 72 hours sequences: (1) logo dilutions 1:10 through 1: 10,000 and (2) incubation, the cells were fixed with 10% buffered formalin logo dilutions 1:30 through 1:30,000. and the agar overlay was removed. The cells were then 30 4. Rabbit 1 was inoculated by coating the scarified skin stained by flooding with crystal violet and plaque titers surface with 0.2 ml of dilution sequence 1 for the recom determined. binant Hantaan vaccine on the four areas on the left side of Procedures and Results rabbit 1. Corresponding dilutions of sequence 1 for the The vaccinia and herpesvirus pools were titrated before reference vaccine were placed on the right side of rabbit 1. filtration. The pools were then filtered through a Nalgene 35 5. Rabbit 2 was treated similarly with dilution sequence 2. 0.45 um filter (#245-0045), sampled, refiltered through a 6. Two additional rabbits were inoculated at two sites with second filter and then resampled. Samples were titrated as the first two dilutions of the reference virus from either summarized below. dilution sequence 1 or 2. Additionally, tissue reactions caused by the scarification were monitored in two rabbits by TABLE 1 observing two scarification sites on each rabbit that were either left uninoculated or inoculated with diluent. Virus titer (logo PFU/ml) Results Sample Vaccinia Herpes Comparison of skin reactions were purely subjective. It was concluded, however, that the recombinant Hantaan Prefiltered virus pool 8.0 7.56 45 Filtered one time 6.13 7.12 vaccine produced a much less severe skin reaction than the Filtered two times 5.19 6.44 FDA reference vaccine. The skin lesions produced by the Hantaan vaccine resolved and the scab was removed while the reference vaccine lesions retained a tightly attached scab. Conclusion Summary Vaccinia virus could be reduced to approximately 10 50 The Hantaan (Vaccinia-Vectored) seed virus was less virions by sequential passage through two Nalgene 0.45um reactive than the FDA reference virus in the rabbit scarifi filters. The residual virus could then be neutralized with monoclonal antibody. cation test. EXAMPLE 6 EXAMPLE 5 55 Testing of Hantavirus Vaccine for Pathogenicity in Assessment of Hantavirus-Induced Dermatotropic a Murine Model Reaction Objective Objective To compare the mouse lethality of Hantaan M+S (vac To compare the dermatropic properties of Hantaan (vac cinia virus-vectored) vaccine with the FDA reference vac cinia-vectored) seed virus with an FDA reference virus. cinia vaccine Lot 2. Background Materials 1. Traditionally, smallpox vaccine has been administered 1. Vaccine by multiple pressure . Successful primary vac 65 a. Hantaan M--S (vaccinia virus-vectored) vaccine, cination or a "take' induces a localized skin lesion that FB0264, Lot 1. resolves into a permanent scar. Production of this response b. FDA reference vaccinia, Lot 2. 5,614, 193 21 22 2. Animals -<24 h old suckling mice, CD-1 strain, Charles River, Raliegh, N.C. TABLE 4 Procedure 1. Comparative titration of TSI and FDA vaccines were Vaccinia Virus Strains Used for Virulence Comparisons conducted in a single test using the final bulk of the Hantaan TTER vaccine M+S (titer 2.1x10PFU/0.3 ml) and FDA reference (plaque forming vaccine (5.4x10" PFU/0.3 ml), respectively. STRAIN units/ml) 2. Logarithmic dilutions of the vaccine were prepared. Wyeth Strain from ATCC 2.3 x 10 Two litters of 5 mice each were used for each dilution. Mice Lederle Strain from ATCC 9.5 x 10 were inoculated intracerebrally with 0.03 ml of the appro 10 Wyeth Licensed Vaccine (Dryvax) 2.4 x 10 Eistree Strain (Lister Vaccine) from ATCC 1.2 x 0. priate dilutions and observed for 21 days. Connaught Licensed Vaccine 2.3 x 10 3. Mouse lethality was calculated as the dose in PFU that IHD Strain from ATCC 7.0 x 10 killed 50% of mice (e.g., PFU/0.03 ml of vaccine divided by Bureau of Biologics Standard Strain 1.1 x 10 LDso of vaccine). Western Reserve (mouse-adapted) Strain 6.5 x 10 Results TSI-GSD-24 2.0 x 10 15 TSI-GSD-264 1.2 x 10 TABLE 2 Test 1. TABLE 5 Vaccine Dose/0.03 ml LD/0.03 ml PFUILDso 20 Virulence Comparison of Vaccinia Virus Strains TS 2.1 x 106 1.2 x 10 175.0 (minimal dose in FDA 5.4 x 107 1.3 x 10 42.9 log'giving lesions) ADULTMOUSE DOSE DOSE STRANS WRULENCE ERYTHEMA EROSION TABLE 3 25 Wyeth ATCC -- 2 6 Test 2 (only data for FDA reference vaccinia shown) Lederle -- 2 5 Wyeth Vaccine -- 2 7 Vaccine Dose/0.03 ml LDo/0.03 ml PFUILDso Lister -- 2 6 30 Connaught -- 3 6 FDA 3.3 x 10 1.5 x 10° 2.2 EHD -H- 1 2 BOB Standard -- 2 5 2. Mathematical standardization of data: The data shown WR -H 1 1 TSI-GSD-24 -- 3 7 above demonstrates that when the FDA reference vaccine TSI-GSE)-264 4 7 was diluted 1:16, virulence in mice was diluted approxi mately proportionately. Using that observation, the mouse 35 Relative rating with H- most virulent and -avirulent. virulence for the FDA vaccine is calculated to be 1.4 The virus strains were examined for pock formation on PFU/LDo when given at the same titer as the M+S Hantaan chorioallantoic membranes of embryonated chicken eggs, virus vaccine (2.1x10 PFU/0.3 ml). formation of lesions after intradermal inoculation of adult Discussion rabbits, and intracerebral and intraperitoneal virulence for 1. Mouse lethality of TSI Hantaan MS (vaccinia virus adult and suckling outbred mice. Representative data are vectored) vaccine was 175 PFU/LDs. presented in Table 5 above. For purposes of comparison, 2. Mouse lethality of FDA vaccinia vaccine was calcu data are illustrated for both the vaccinia-vectored Hantaan lated to be 1.4 PFU/LDs. vaccine candidate (TSI-GSD-264) and a cell-cultured small 3. The mean time-to-death for the Hantaan was 10.76+/ pox vaccine (TSI-GSD-241, reference IND #4984) derived -4.7 days compared to 0.8+/-1.9 days for the FDA reference 45 from the same virus seed ("Conn 3E1"), which in turn was vaccinia. a thrice plaque-picked derivation from the licensed Con Conclusion naught vaccinia (Conn-Master 17633). Using these tests, The Hantaan M+S (vaccinia virus-vectored) vaccine is calculated to be 100-times less virulent than the FDA both the vaccinia-vectored Hantaan vaccine candidate (TSI 50 GSD-264) and a cell cultured smallpox vaccine (TSI-GSD reference vaccine when administered by intracerebral inocu 241) were among the least virulent vaccinia strains tested lation to suckling mice less than 24 hold. The time-to-death and were comparable to the New York Board of Health, also was increased markedly for the Hantaan vaccine which Bureau of Biologics reference strain. further indicates reduced virulence. A primary study compared the undiluted vaccinia-vec 55 tored Hantaan vaccine, TSI-GSD-264, to the licensed Wyeth vaccine, as well as to a cell-cultured smallpox vaccine EXAMPLE 7 (TSI-GSD-241, reference IND #4984). The TSI-GSD-264 booster was given to 4 monkeys in the study. An additional six monkeys received TSI-GSD 264 one year after immu Preclinical Investigation and Characterization of nization with vaccinia virus lacking any foreign gene insert Hantavirus Vaccine either the cell-cultured parent virus or Wyeth DryvaxTM. The preclinical characterization of the vaccinia-vectored Intradermal and subcutaneous administration of the vaccine Hantaan vaccine, TSI-GSD-264, included comparisons with candidate was well tolerated and all monkeys seroconverted. a collection of numerous vaccinia virus strains. The strains Cutaneous lesions were minimal and comparable among the are listed in Table 4 below and the infectious virus titers 65 three vaccines tested. noted are for virus seeds prepared by a single passage in Of the four monkeys receiving primary immunization MRC-5 cells and plaque assayed on VERO cells. with TSI-GSD 264, neutralizing antibody to Hantaan virus 5,614, 193 23 24 was detected at 42 days post-inoculation in three monkeys. The fourth monkey developed neutralizing antibody after a TABLE 6 boost. All monkeys had seroconverted, as measured by it of vaccinia boo ELISA, after primary immunization. Of the monkeys ini Group if volunteers immunity dose Ster tially immunized with a non-recombinant vaccinia and later 4. immune 3.4 x 10 PFU no immunized with TSI-GSD 264, none developed neutralizing (inpatient) antibody and two developed low ELISA titers to Hantaan. 2 4 non-immune 3.4 x 10 PFU no One monkey from the study died 6% months after receiv (inpatient) ing the vaccine candidate as a primary immunization. It had 3 4. non-immune 3.4 x 10 PFU no 10 (inpatient) not had any unusual response to immunization and its death 4 4. non-immune 3.4 x 107 PFU no was determined to be the result of gastric perforation. No (inpatient) pathologic lesions suggestive of vaccinia were noted. 5 24 (outpatient) randomized: Another monkey which was inoculated with TSI-GSD 264 A non-immune 3.4 x 107 PFU no as its booster, was euthanized 8 months later because of B non-immune 3.4 x 107 PFU yes chronic diarrhea and a generalized wasting condition attrib 15 uted to Simian Retrovirus infection (SRV). Blood specimens On day 0, Groups 3A and 3B receive the vaccine candidate obtained from the monkey four days prior to euthanasia subcutaneously. After a 30 minute observation period for were both seropositive and culture positive for SRV. In light immediate untoward reactions, subjects are followed on an of the fact that (i) most severe reactions to vaccinia virus outpatient basis. Subjects have clear instructions on what to inoculation occur within 30 days and (ii) these monkeys had 20 do if they experience an adverse reaction. On day 42, Group no lesions suggestive of vaccinia virus infection, it is 3B receives a second injection of the vaccine. Outpatient unlikely that these deaths were caused by inoculation with follow-up, periodic bleeding and further clinical examina the candidate vaccine. The pathology reports are filed with tions as provided as needed. the IND. 1. Selection of Vaccine Dose: 25 a. The cell-cultured vaccinia virus candidate vaccine has EXAMPLE 8 a titer of 6.8x107 PFU/ml. A dose of 0.5 ml of undiluted vaccine is administered subcutaneously, equaling an inocu Human Clinical Studies with Hantavirus Vaccine lum of 3.4x107 PFU. Preclinical studies in animals support the immunogenicity and safety of this undiluted vaccine General Study Design 30 dose. McClain et al., American Soc. Tropical Med. Hygiene The preliminary phase of the study is an open label Annual Meeting (1993). inoculation of four individuals with documented vaccinia 2. Clinical Procedures: immunization (Group 1), succeeded by inoculation of four a. General Considerations. vaccinia-naive individuals (Group 2) 21 days after the first All times mentioned below are referenced against the day group is inoculated. Previous vaccinia immunization are 35 of inoculation, which is customarily designated as Day 0. documented by medical records and unequivocal vaccinia Physical activity one week prior to and during the first 4 scar. This is followed by a randomized trial in 24 vaccinia weeks following inoculation is limited to each subject's naive volunteers (Group 3). is usual level of activity. This minimizes abnormalities in available on site for the treatment of serious adverse reac serum enzyme levels associated with strenuous activity. tions such as ocular, generalized or progressive vaccinia. b. Inoculation and Immediate Observation. Group 1 (smallpox-immune) volunteers are admitted as Subjects remain under the direct supervision of the Prin inpatients in groups of two to four people and receive the cipal Investigator or Associate Clinical Investigator for 30 vaccinia-vectored Hantaan vaccine candidate (open label) minutes after inoculation. Injectable epinephrine and resus by the subcutaneous route. Groups 2, 3 and 4 (vaccine 45 citative equipment are available for the treatment of acute naive) subjects are administered the vaccine candidate, in anaphylactic reaction. An aliquot of the remaining vaccine escalating doses, in like fashion. Inoculation is given in the virus is retained for determination of virus titer and the deltoid region of the arm. The four vaccinia-immune indi remainder of the vaccine is destroyed by incineration in viduals in Group 1 receive the vaccine in the arm in which accordance with state regulations for disposal of medical there is no vaccinia scar from previous immunization. Pre 50 Waste. vious vaccinia immunization is documented by medical c. Continued Inpatient Observation. records and unequivocal vaccinia scar. Members of Groups 1 and 2 are monitored for four days Group 1, 2, 3 and 4 subjects are admitted 3 days prior to after inoculation on the inpatient ward. Temperature, pulse, inoculation and remain as inpatients until 4 days after respiratory rate, and blood pressure are monitored three inoculation. Subjects are then discharged and assessed on an 55 times a day for three days prior to, and four days after, outpatient basis without periodic bleeding with clinical inoculation on the clinical ward. The interval between examinations for potential reactions or complications measurements is shortened in subjects with symptoms or through day 21. Subjects developing a vesicular or open fever (oral temperature >100.5 F). Inoculation sites are lesion suspected to contain vaccinia virus have the lesion daily examined by measurement of erythema and induration, covered with a dressing and are followed with subsequent measuring 2 perpendicular directions across the lesion. daily clinical examinations and dressing changes by an Photographs are taken as needed. Any vesicular eruption or investigator or the nursing staff until the lesion forms a scab. crusting is noted and cultured for virus. During the inpatient Group 5 volunteers, all vaccinia-naive, are enrolled phase, responses to inquiries about local or systemic com depending on availability as outpatient subjects. Group 5 plaints (pruritus, pain, malaise, fatigue, etc.) are recorded volunteers are randomly assigned to one of two subgroups: 65 daily. Attempts are made to quantify the duration and one receives a single injection and the other receives a severity of complaints. A scale of 0 to 3 is used to measure booster. The vaccinees are grouped as follows: the degree to which symptoms interfere with activities of 5,614, 193 25 26 daily living. Evaluations are detailed on clinical evaluation serum samples are maintained frozen at -40°C. in the forms. USAMRIID serum bank and available for additional studies d. Outpatient Observations. and/or repeat of selected studies as needed, unless volunteers Volunteers are followed with vital signs and physical specifically exclude use of samples for additional studies. examinations for up to 11 days following inoculation during Fluid in vesicular or weeping lesions at the site of inocula State p of that rt tion or at distant sites is be sampled and placed in viral advised that staff are available on a 24-hour basis for the isolation transport medium to attempt virus recovery. Serum duration of the study in order to screen any subject who is antibody determinations include ELISA reactivity with includesuffering fever, any side itching, effect rash of the or inoculation. any other symptoms, These side regard- effects 10 'hol y'. H altaal antigenw andd vaccinia antigenity and less of their opinion as to the relevance of the symptom to plaque reduction neutralization of Hantaan virus. the inoculation. The nursing staff evaluates the complaint and notes the complaint in the clinical protocol record. The nursing staff then reports all such complaints to the Principal EXAMPLE 9 Investigator or Associate Clinical Investigator for evaluation 15 and instructions about required follow-up care. Any subject developing g a vesvesi cular lesion at the site of inoculation has Results of Clinical Trials With M--S Hantavirus the lesion covered with a dressing until such a lesion has Vaccinia-V d) Vacci formed a scab. In addition, subjects return for scheduled (Vaccinia-Vectored) Vaccine being E. appeal bleed st A. 6 days " Clinical trials of the vaccinia-vectored M+S Hantavirus tionfter is inoculation entered into a clinicalthe clinical summary record offor protocol each volunteer. participal A vaccine are expected to show the - vaccine to be safe and copy of this summary is sent to the volunteer's official effectived inignifi humans. hVaccine al recipients are expectedd bto medical record unless a volunteer specifically indicates that produce sign cant umoral response as measured by he/she does not want this done. 25 enzyme-linked immunosorbent assays (ELISA). This 3. Laboratory Studies: response should be characterized by the production of both a. Baseline screening laboratory studies are as described neutralizing and non-neutralizing antibodies, the latter being above. Protocol participation studies include periodic measured by plaque reduction neutralization assays. In addi examinations of the following: CBC with differential and tion, lymphocyte blastogenesis assays are expected to dem platelets, urinalysis, serum chemistries, serum viremia, 30 onstrate that lymphoytes from vaccine recipients proliferate serum vaccinia antibody, serum Hantaan antibody and lym- and produce cytokines upon exposure to Hantavirus antigen phocyte immune responses to Hantaan antigen. Remaining in vitro.

SEQUENCE LISTING

( 1) GENERAL INFORMATION: ( i i i ) NUMBER OF SEQUENCES: 5

( 2) INFORMATION FOR SEQID NO:l: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 647 amino acids (B) TYPE:amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( xi ) SEQUENCE DESCRIPTION: SEQDD NO:1: Me t Gly I 1 e Trip Lys T rp Le u Val Me t Al a Ser Le u Val T rip Pro V a 5 1 O 15 Le u Th r Le u A rig As n Val Ty r A s p Me t Lys I le G 1 u Cys Pro His Thr 20 25 30 V a l Ser P he G 1 y G 1 u As n Ser V a 1 I le G l y Ty r V a 1 G i u Le u Pro Pro 35 40 45 Val Pro Le u A 1 a. A s p Thr Al a G 1 in Met W a Pro G 1 u Se r S c r Cy s As n 50 55 6 O Mc t A s p As n H is G 1 in Ser Le u As n Th r I le Thr Lys Ty r Thr G l n V a l 65 7 0. 7 5 8 0. Set Tr p A rig Gil y Lys A la A s p G 1 in Ser G 1 n Sle r S c r G 1 n. As n S c r Phe 85 90 9 5 G 1 u Thr v a 1 Ser Thr G 1 u v a 1 A s p Le u Lys Gl y T hr Cys v a 1 Le u Lys

5,614, 193 29 30 -continued

A rig Ly is I c L y s G 1 u Glu Ph C Glu Lys Th Lys Gly Sc r Mct Wa 1 Cys 530 53 5 5 40

As p Wa l Cy is Lys Ty r G 1 u Cy S Glu Thr Ty Ly is G Lys A 1 a 45 550 555

Gly Wall Ser Cys Pro G 1 in G 1 in P r O Ty r Ph c Thr H is Cys 5 65 57 O 57 5

G u P ro Thr Gil u A la A 1 a Ph C G n Hi S Wa l Cys G n Wa 580

Thr H is A rig Ph e A rig As p As p L. c u Thr Wa 1 Th I Pro Gl in 60 0 6 O 5

P he Th I Pro G 1 y Cys Ty r Thr L. c u. P he A rig 6 1 0. 6.20

Ty r I 1 c Phc Th r T r p I le Ph L. ( u Lic u Wa L clu G 1 u S. c. r 6 30 6 35 6 4.0

T r p A 1 a A la Ser A l al 6 4.5

( 2) INFORMATION FOR SEQID NO:2: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 487 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: singlc (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( xi ) SEQUENCE DESCRIPTION: SEQID NO:2:

Sc r Gil u Thr Pro Le u Thr P 0. Wa 1 As in A 1 a H is Wa 5

Gly Sle r Wa 1 Pro Me t His Th I P he Ser Thr 20 30

Ser Lys Ty r Thr Le u Thr As in Pro G 1 u 45

G I u I 1 e G 1 I le G 1 u G 1 u Gl. In Th I lic 60

A s p V a l His A 1 a Gly H is Phc As p Gly A rig Le u 7 0 7 5

T Ser Ph. H is Gly a Thr G u 95

H is Th r A la Lys H is A rig A sp Glu 10 0

Thr G 1 y Cys As in Pro Gly Wa Gly T. r Gly 1 2 5

Th I r G 1 Lys P ro Wa 1 Gly Sc r 1 30 1 4 0

A 1 a Ty r Lys I le I le Thr Wa Wa 45 1 50

P he G 1 y G 1 u Gil u As n L. eu I le As p Me t Cy s Ph c 1 65 5

Wa A rig H is Wall Lys I 1. G 1 y Th Wa Sc r Lys Ph. c. 180 1 9 O

G In y . A s p Thr Le u P a c Ph c Pro G 1 u Gly G 1 y Gly 5 20 0 2 05

P he Lys H is Trip S Thr S e r Th G 1 in P he Gly A sp P ro i 22 O

G y I e Me t S e r Pro Gl Phc P P he 5,614, 193

-continued

225 2 30 235 2 40 Pro G 1 y Ser P h e Arg Lys Lys Cy s As n Phc A 1 a T h r Thr Pro I 1 c Cys 2 4 5 25 O 25 5 G 1 u , Ty r A s p G y As n Me t Val Sc r G y Ty r Lys Lys Wal Me t A a Thr 26 O 2 6 5 2 7 0 I e A s p Ser P he G 1 a Ser Phe As n Thr Ser Thr Me t H is Phe Thr. As p 275 28 0 2 85 Glu A rig I e G 1 u T r p . Lys A s p Pro A s p G 1 y Me t L. eu A rig A s p H is I lic 29 O 2 9 5 3 O O As n I 1 c L e u V a l T h r Lys A s p I e A s p Phc As p As n L. c u G l y G u A sm 3 O 5 3 O 3 15 3 20 Pro Cy s Lys I le G 1 y Le u G l n Th r S e r S e r I le G 1 u G y Al a T r p G ly 3 25 3 30 3 35 Ser G 1 y V a 1 G l y Phe Thr Le u Thr Cys Le u V a Ser Le u . Thr G 1 u Cys 3 40 3 45 350 Pro Thr P he Le u T h r S e r I e Lys A 1 a Cys A s p Lys A 1 a I le Cys Ty r 355 3 6.0 3 65 G l y A 1 a G 1 u Se r V a l Thr Le u Thr Arg G : y G l n As n Th r V a l Lys V a l 3 O 375 3. 80 Ser G 1 y Lys G 1 y G l y H is Ser G 1 y Ser Thr Phi e A rig Cys Cy s H is Gly 385 3 9 O 395 O O Glu As p. Cy s Ser G 1 in I 1 e G l y Le u H is A a A 1 a. A l a Pro H is L. eu As p 4 05 4 O 4 15 Ly is V a l As in G y I 1 e Ser G 1 u I le G 1 u As n Ser Lys V a 1 Ty r A s p . As p 4 20 42 5 4, 30 G l y A la Pro G 1 in Cys G y I e Lys Cy s Trip Phi e V a 1 Lys Ser G 1 y G 1 u 435 4 4 O 4 45 T r p I e S c r G 1 y I 1 e Ph e Ser G 1 y As n : T r p I 1 e V a 1 Le u I le V a 1 Le u O 4 55 4 50 Cy is V a 1 P he Le u L. eu Ph c Ser Le u V a 1 Le u Le u Se r I le Le u Cys Pro 4 6 5 4 TO 475 4 80 Wa A rig Lys H is Lys Lys Ser 4 85

( 2) INFORMATION FOR SEQID NO:3: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 429 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: protein ( x i ) SEQUENCE DESCRIPTION: SEQID NO:3: Mct A la Thr Me t G i u G 1 u Le u G 1 in Arg G 1 u I 1 c As n A 1 a H is G 1 u G 1 y 5 1 0 15 G l n Le u V a 1 I e A 1 a. A rig G 1 in Lys V a 1 Arg A s p A 1 a G 1 u Lys G 1 in Tyr 20 25 30 G 1 u Lys A s p Pro. A s p G 1 u Le u As n Lys Arg Th r Le u Th r A s p A rig G 1 u 35 4 0 45 G l y V a l A 1 a V a l S e r I 1 e G 1 m A la Lys I 1 e A s p G 1 u Lc u Lys Arg G 1 n 5 O 55 5 O L. c u A 1 a. A s p A rig I l c A a Thr G y Lys As n Le u G y Lys - G 1 u G n . As p 65 70 5 80 Pro Thr G l y V a l G 1 u Pro G 1 y A s p H is Le u Lys G 1 u A rig Ser Me t Le u 85 9 O 95 5,614, 193 33 34 -continued

Sc r Ty r G 1 y As n Wa L c u As p L clu As in H is Lic u As p c Glu P O 1 00 1 05

Thr Gly G 1 in Thr A la A s p Trip L. c u Sc r . I l c I le Wa 1 Ty r Th I 1 15 1 2 0. 1 2 5

Phc Wa 1 Wa 1 Pro I e Le u Lc u Lys A 1 a Lic u Ty r Me t L. c u Thr Th 35 1 4 0

Gil y Arg G 1 in Th r Th r Lys As p Gly r A rig I e P he

A s p As p Phc G1 u A sp G A rig Pro 65 1 7

Wa Le u Pro As n A 1 a Gl I Me t A 1 a G 1 u s o 185 1 90

I c Th I Pro Gly Thr A l a Wa 1 Gly L. eu Ty r A l a 195 20 0 2 O 5

A rig Gil in Mct I c Sle r Mc t S : I Wa 1 Gly 215 2 20

Ph c L e u A la L. eu A la Lys As p A r I lic G 1 u G 1 in T r p L. c u 225 240

I c G 1 u Pro Cys L y S L e l L c l Thr A 1 a A l a Sc I Le u I c u 25 0 255

G l y G 1 y Pro A 1. a. As p T y A rig G 1 in G 1 in A 1 a 2 60 27 0

As In Me t G 1 u Thr Lys G 1 u A l a I le G l n H is A 1 a 2 75 28 O

G u A l a A la Gly I e I e. G 1 u S e r 29 0. 3 O 0

Wa 1 P he A la Gl Al a P r 0 Thr Le u 1. i 3 2 0.

Phi e I e A l a G 1 y Gly Al a P he P he I le G l n 33 0

Thir I c Mc t A l a Sc r Thr Wa Gly T. r G 1 u 3 40 3 4 5 350

G 1 u Lys Lic u Arg S e r Ph. c. G 1 in A rig 355 3 60 3 65

Th I G n S. e i Me t G 1 y I 1 c G 1 n L. eu Gly G 1 In I 1 e Wa P he 37 0 3 75

Me t Wa A la Trip Gil y Lys G 1 u A 1 a Wa H is Gly 3 85

A s p Me t Glu L. c u Arg Thr Al al I le As p 4 05 4 10 4 15

Lys V a 1 Ly is G 1 u I l e Ser As in G G u P r L c u Ly is 4 20 4, 25

( 2) INFORMATION FOR SEQID NO:4: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH; 3401 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA (genomic) ( xi ) SEQUENCE DESCRIPTION: SEQID NO:4:

A T G G G GATA T G GAA G T G G CT AG T G A T G G C C A GT T TA GTA T G G C C T G T TTT GACA CT G A GA 6 O

AA T G T CTA T G A CAT GAA AAT T G A G T G C C C C CAT ACA GT AA GTTTT GGGGA AAA CAG T G T G 2 0

5,614, 193 39 40 -continued

TT TAT CA GT C CTAC CT CAGA A G GACA CAAT CAA T G GGG AT AC AACTA. GGC CAGA GAA TTA. 1 40

TT G T G CT CTT CA T G GT T G C C T G G G GAA A GG AG G CTG T G GA . CAA CTT C CAC TTA. GGG GATG 1 20 O

ATA T G GAT C C T G A G CTA AG G ACA CT G GCAC AGA G CTTGAT T G AT GT CAAA GT GAAG GAAA 1 2 5 O

TCTCCAA CCA AGAGCCTTT G AAA CT C 28 6

We claim: 10 wherein said vaccine formulation comprises a single dose 1. A vaccine formulation suitable for use in a human, of 5x10 to 7x10' plaque forming units of vaccinia comprising: virus. (i) Reinia virus that comprises a DNA mol- 2. The vaccine formulation according to claim 1, wherein ecule(a) the encoding Hantaan virus polypeptides designated N, G1 15 thee nucleotidnucleotide sequence encodingding saidsai G1 popolypeptide ypep ae anand and G2, and said G2 polypeptide is operably linked to the vaccinia WS (b) all vaccinia virus polypeptides necessary for repli- 7.5 kD promoter, and wherein the nucleotide sequence cation of said virus in a cell derived from said encoding said N polypeptide is operably linked to the human, but not a functional thymidine kinase; and vaccinia virus 11 kD promoter. (ii) a pharmaceutically-acceptable carrier, excipient or 3. The vaccine formulation according to claim 1, wherein diluent, said G1 has the sequence (SEQ ID NO: 1):

MET GLY LETRPLYSTRP LEU VAL METALASER LEU WALTRPPROVAL LEU THR LEU ARG ASN WALTYR ASP MET LYSILE GLU CYS PRO HIS THRVAL SER PHE GLY GLUASN SERVALILE GLYTYRWAL, GLULEU PRO PRO VAL PRO LEUALA ASPTHERALAGLN MET WAL PROGLU SER SERCYS ASN METASPASN HIS GLN SER LEU ASN THR LE THRLYSTYRTHR GLNWAL SER TRPARG GLY LYS ALAASP GLN SER GLN SER SER GLNASN SERPHE GLUTHRVAL SER THR GLU WALASPLEULYS GLY THRCYS WALLEULYS HISLYSMET VAL GLU GLU SERTYRARG SER ARG LYS SERVAL THRCYSTYRASPLEU SERCYS ASN SER THRTYRCYS LYSPRO THR LEU TYR MET LEVAL PRO LEHSALACYSASN METLYSSERCYSLEU ILEALALEU GLY PROTYRARGVAL GLN VAL VALTYR GLU ARG SERTYR CYS MET THR GLY WALLEU ILE GLU GLY LYS CYS PHE WAL PROASP GLN SERVAL VAL SERILE LELYS HIS GLY LEPHE ASPILE ALASER WALHISILE WAL CYSPHEPHE WALALAVALLYS GLY ASN THRTYRLYSILE PHE GLU GLN WALLYSLYSSERPHE GLU SER THRCYS ASNASPTHR GLU ASNLYS VAL GLN GLY TYRTYRILE CYSILE VAL GLY ASN SER ALAPRO LE TYRVAL PRO THR LEU ASPASP PHE ARG SER MET GLUALA PHETHR GLY LEPHE ARG SER PRO HIS GLY GLUASPHS ASPLEUALA GLY GLU GLUILE ALASERTYRSER LE WAL, GLY PRO ALAASNALALYSWAL PRO HIS SER ALASER SER ASPTHR LEUSER LEU LEALATYR SER GLY LE PROSERTYRSER SERLEU SER ILE LEU THR SER SER THR GLU ALALYS HIS WALPHE SER PRO GLY LEU PHE PROLYSLEU ASN HIS THRASN CYS ASPLYS SER ALA LE PRO LEU ILE TRPTHR GLY MET LEASPLEU PRO GLYTYRTYR GLUALAWALHIS PRO CYSTHRVAL PHE CYS WALLEU SER GLY PROGLYALASER CYS GLUALA PHE SER GLU GLY GLY LE PHE ASNILE THR SER PRO MET CYSLEU VAL SERLYS GLNASNARG PHEARGLEUTHR GLU GLNGLN VAL ASN PHEVALCYS GLN ARG VAL ASPMETASPIE VAL VALTYR CYSASN GLY GLN ARG LYS WALLELEU THRLYSTHR LEU VALILE GLY GLN CYSLETYR THRDLE THR SER LEU PHE SER LEULEU PRO GLY WAL ALA HIS SER LEALAVAL GLULEU CYS VAL PRO GLYPHE HIS GLY TRPALATHRALAALALEULEU VAL THRPHE CYSPHE GLY TRPVAL LEU ILE PRO ALA LE THRPHE LEELE LEU THRWALLEULYSPHE ILEALA ASNILE PHE HIS THR SER ASN GLNGLUASN ARG LEULYSSERVAL LEU ARG LYSLELYS GLU GLU PHE GLULYSTHRLYS GLY SER MET WALCYS ASPVAL CYSLYSTYR GLU CYS GLUTHRTYRLYS GLULEULYS ALA HIS GLY WAL SERCYS PRO GLN SER GLN CYS PROTYRCYSPHETHRHISCYS GLU PRO THR GLUALA ALA PHE GLNALA HISTYRYS WALCYS GLN WALTHRHIS ARG PHE ARG ASPASPLEULYSLYSTHRWAL THR PRO GLN ASN PHE THR PRO GLY CYS TYRARG THR LEU ASN LEU PHEARGTYRLYS SER ARG CYSTYR 5,614, 193 41 42 -continued ILE PHE THR MET TRPELEPHE LEU LEU VAL LEU GLU SERILE LEU TRPALA ALASER ALA, said G2 has the sequence (SEQID NO; 2):

SER GLUTHR PRO LEUTHR PROVAL TRPASNASPASNALAHIS GLY WAL, GLY SERVAL PROMETHIS THR ASP LEU GLU LEU ASPPHE SER LEU THR SER SER SERLYSTYRTHRTYRARGARGLYSLEU THRASN PRO LEU GLUGLUALAGLN SERELEASPLEU HIS ILE GLUILE GLU GLU GLN THRILE GLY WALASP WALHIS ALA LEU GLY HISTRP PHEASP GLY ARG LEUASN LEU LYSTHR SERPHE HIS CYSTYR GLYALACYSTHRLYS TYR GLUTYR PRO TRP HIS THRALALYS CYS HISTYR GLU ARG ASPTYR GLN TYR GLUTHR SER TRP GLY CYSASN PRO SER ASP CYS PRO GLY VAL GLY THR GLY CYS THRALA CYS GLY LEUTRYLEU ASP GLNLEU LYS PROVAL GLY SER ALA TYRLYSILE ILE THRILE ARG TYR SER ARG ARG VAL CYS VAL GLN PHE GLY GLU GLUASN LEU CYSLYSILE LEASP METASNASP CYSPHE WAL SERARGHIS WALLYS WALCYSLEILE GLY THRVAL SERLYSPHE SER GLN GLY ASPTHREEULEU PHEPHE GLY PRO LEU GLU GLY GLY GLY LEU ILE PHELYS HISTRP CYS THR SER THR CYS GLN PHE GLY ASP PRO GLY ASPILE METSER PRO ARG ASPLYS GLYPHE LEU CYS PRO GLU PHE PROGLY SERPHE ARG LYSLYS CYS ASN PHEALATHRTHR PROILE CYS GLUTYRASP GLY ASN MET WAL SER GLYTYRLYSLYS WAL METALATHRELEASP SERPHE GLN SERPHE ASN THR SER THRMETHIS PHE THRASP GLU ARGILE GLUTRPLYS ASP PROASP GLY MET LEU ARG ASP HIS ILEASNILE LEU VALTHRLYS ASPLEASPPHE ASPASN LEU GLY GLUASN PRO CYSLYSILE GLY LEU GLN THR SER SERILE GLU GLYALATRP GLY SER GLY WAL, GLY PHE THRLEUTHR CYSLEU VAL SER LEU THR GLU CYS PRO THRPHE LEUTHR SERILELYS ALA CYS ASPLYS ALA LE CYSTYR GLYALAGLUSER VAL THRLEUTHR ARG GLY GLNASNTHRWALLYS WAL SER GLY LYS GLY GLY HIS SER GLY SER THRPHE ARG CYS CYS HIS GLY GLUASP CYSSER GLNILE GLY LEU HISALAALAALAPRO HIS LEU ASPLYS WALASN GLY LESER GLUILE GLUASN SERLYS WALTYRASPASP GLYALAPRO GLN CYS GLY LELYS CYSTRP PHE WALLYSSER GLY GLUTRPLE SER GLY LEPHE SER GLY ASN TRPLE WALLEUILE WALLEU CYS VAL PHE LEULEU PHE SER LEU WALLEU LEU SERILE LEU CYS PROVAL ARG LYS HISLYSLYS SER 40 and said N has the sequence (SEQID NO: 3):

METALATHRMET GLU GLU LEU GLNARG GLULEASNALAHIS GLU GLY GLN LEU VALILEALA ARG GLN LYS WALARGASPALA GLULYS GLN TYR GLULYS ASP PROASP GLULEUASNLYS ARG THR LEU THRASP ARG GLUGLY WALALAVAL SERILE GLNALALYSILE ASP GLULEULYSARG GLN LEU ALAASPARGILE ALATHR GLY LYSASN LEU GLY LYS GLU GLNASP PRO THR GLY WAL GLU PRO GLY ASPHIS LEU LYS GLU ARG SER MET LEU SERTYR GLY ASN WALLEU ASPLEU ASN HISLEUASPILEASP GLU PRO THR GLY GLN THRALAASPTRPLEU SERELEILE WALTYRLEUTHR SERPHE WAL VAL PRO LELEULEULYS ALALEU TYR MET LEU THRTHRARG GLY ARG GLN THRTHRLYS ASPASNLYS GLY THRARGILE ARG PHELYSASPASP SER SERPHE GLUASP WALASN GLY LEARG LYS PRO LYS HIS LEU TYR VAL SER LEU PROASNALA GLN SER SER METLYS ALA GLU GLUILE THR PRO GLY ARG TYRARG THRALAVAL CYS GLY LEUTYR PRO ALA GLNILE LYS ALAARG GLN METILE SER PROVAL METSER WALLE GLYPHE LEU ALALEUALALYSASP TRPSER ASPARGILE GLU GLN TRPLEUILE GLU PRO CYSLYSLEULEU PROASPTHRALAALAVAL SER LEU LEU GLY GLY PROALATHRASN ARG ASPTYR LEU ARG GLNARG GLN WALALALEU GLY ASN MET GLU THRLYS GLUSERLYS ALAILE ARG GLN HISALAGLUALAALAGLY CYSSER METILE GLUASPILE GLU SER PROSER SERILE TRPVAL PHEALAGLYALAPROASPARG CYS PRO PROTHRCYSLEU PHE LEALAGLY LEALA GLULEU GLYALAPHEPHE SERILE LEU GLNASPMETARGASN THRILE METALASERLYSTHR 5,614, 193 43 44 -continued WAL GLY THR SER GLU GLULYSLEU ARG LYSLYS SER SERPHETYR GLNSERTYRLEU ARG ARG THR GLN SER MET GLY ILE GLN LEU GLY GLN ARGILE ILE VAL LEU PHE MET VALALA TRP GLY LYS GLU ALA VAL ASPASN PHEHSLEU GLY ASPASPMETASP PRO GLULEU ARG THRLEU ALA GN SER LEULEASP WAL LYSWALLYS GLUILE SER ASN GLN GLU PRO LEULYSLEU.

4. The vaccine formulation according to claim3, wherein said DNA molecule comprises a wild-type vaccinia virus 10 genome with the cDNA of the M and S genomic segments of the Hantaan serotype inserted in the vaccinia virus thymidine kinase coding region. 5. The vaccine formulation according to claim 4, wherein said cDNA comprises the sequence (SEQED NO: 4):

ATG GGG ATA TGGAAGTGG CTA GTG ATG GCCAGTTTA GTATGG CCT GTTTTGACA CTGAGAAAT GTC TAT GACATGAAAATTGAGTGC CCC CATACA GTAAGTTTT GGG GAAAACAGT GTGATA GOST TAT GTA GAATTACCCCCC GTG CCATTG GCC GACACA GCA CAGATG GTG CCT GAGAGTTCTTGTAACATG GAT AAT CAC CAATCGTTGAAT ACAATAACAAAA. TATACCCAA GTA AGT TGG AGAGGAAAG GCT GAT CAG TCA CAGTCT AGT CAAAATTCATTT GAGAGA GTG TCC ACT GAA GTTGACTTGAAA GGAACATGT GTT CTAAAACACAAAATGGTG GAA GAATCA TAC CGT AGTAGGAAATCA GTA ACCTGT TAC GAC CTG TCT TGC AAT AGC ACTTACTGCAAGCCA ACA CTATAC ATGATT GTA CCAATT CAT GCATGC AATATGATG AAAAGCTGTTTGATT GCATTGGGACCATACAGR GTA CAG GTGGTTTAT GAGAGA AGT TACTGTATG ACA GGA GTC CTGATTGAAGGGAAATGCTTT GTCCCAGAT CAAAGT GTG GTCAGTATTATCAAG CAT GGG ATC TTT GATATT GCAAGTTTT CAT ATT GTA TGTTTCTTT GTT GCA GTT AAAGGGAAT ACT TATAAA ATTTTT GAA CAG GTTAAGAAATCCTTT GAATCA ACATGCAAT GATACA GAGAATAAA GTG CAAGGA TAT TATATTTGTATT GTAGGGGGAAACTCT GCACCAATATAT GTT CCA ACA CTTGAT GATTTCAGA TCC ATG GAA GCATTTACA GGAATCTTCAGATCA CCA CAT GGG GAA GAT CAT GAT CTG GCT GGA GAA GAAATT GCATCTTATTCTATA GTCGGA CCT GCC AAT GCA AAA GTTCCT CAT AGT GCT AGCTCA GAT ACATTGAGCTTGATT GCC TATTCAGGT ATA CCA TCT TAT TCT TCCCTTAGC ATC CTA ACA AGT TCA ACA GAA GCTAAG CAT GTATTCAGCCCTGGGTTGTTC CCAAAACTTAAT CACACAAATTGT GATAAAAGT GCC ATA CCA CTCATA TGG ACT GGG ATGATT GATTTA CCTGGATACTAC GAA GCT GTC CAC CCTTGT ACA GTTTTTTGC GTATTATCA. GGT CCT GGG GCATCATGT GAA GCCTTTTCT GAA GGC GGGATTTTC AAC ATA ACCTCT CCCATGTGCTTA GTG TCAAAACAAAATCGATTC CGGTTA ACA GAA CAG CAA GTG AATTTT GTGTGT CAG CGA GTG GAC ATG GACATT GTT GTG TACTGAAACGGG CAGAGGAAA GTAATA TTAACAAAAACT CTAGTTATTGGA CAGTGTATATATACTATAACAAGCTTAXXXTCATTACTA CCT GGA GTA GCA CATTCTATT GCT GTT GAATTGTGT GTA CCT GGGTTC CAT GGTTGG GCCACA GCT GCT CTG CTT GTT ACATTCTGTTTCGGATGGGTT CTTATACCAGCAATTACATTTATCATA CTA ACA GTC CTA AAGTTCATT GCT AATAATTTT CACACAAGTAAT CAAGAGAAT AGG CTAAAATCA GTA CTTAGAAAG ATA AAG GAA GAGTTT GAAAAAACAAAAGGCTCA ATG GTATGT GAT GTCTGCAAG TAT GAG TGT GAA ACA. TATAAA GAATTAAAG GCA CAC GGG GTATCATGC CCC CAATCT CAATGT CCTTACTGTTTT ACT CATTGT GAA CCTACA GAA GCA GCATTC CCA GTC CATTACAAG GTATGCCAA GTTACT CACAGATTC AGGGAT GAT CTAAAGAAAACT GTT ACT CCT CAAAATTTT ACACCAGGATGT TAC CGG ACA CTAAAT TTA TTT AGATACAAAAGCAGGTGCTACATCTTT ACAATGTGGATA TTT CTT. CTTGTCTTAGAATCC ATA CTGTGG GCT GCA AGT GCATCA GAG ACA CCATTAACT CCT GTCTGGAATGACAATGCC CAT GGG GTA GGTTCT GTTCCTATG CAT ACA GATTTAGAG. CTTGATTTCTCTTTAACATCCAGTTCCAAG TATACA. TAC

5,614, 193 47 48 -continued CAACTA GOC CAGAGAATTATT GTG CTCTTCATGGTT GCCTGG GGAAAG GAG GCT GTG GAC AACTTC CACTTAGGG GAT GATATG GAT CCT GAG CTA AGG ACA CTG GCA CAGAGCTTGATT GAT GTCAAA GTG AAG GAAATCTCCAAC CAA GAGCCTTTGAAA CTC. 6. The vaccine formulation according to claim 1, wherein 15. A method for inducing a Hantavirus-protective said single dose comprises 5x10 to 7x10" plaque-forming immune response in a human, comprising the steps of: units. 7. The vaccine formulation according to claim 1, wherein 10 (i) providing a vaccine formulation suitable for use in a said single dose comprises 5x10 to 1x10 plaque-forming human comprising units. (a) infectious vaccinia virus activity and comprising a 8. The vaccine formulation according to claim 1, wherein DNA molecule encoding said single dose comprises 5x10 to plaque-forming units. (1) the Hantaan virus polypeptides designated N, Gl 9. The vaccine formulation according to claim 1, wherein 15 and G2, and said single dose comprises 3.4x10" plaque-forming units. (2) all vaccinia virus polypeptides necessary for 10. The vaccine formulation according to claim 9, replication of said virus in a cell derived from said wherein said pharmaceutically-acceptable carrier, excipient human, but not a functional thymidine kinase, and or diluent further comprises lactose and human serum albu (b) a pharmaceutically-acceptable carrier, excipient or 20 diluent, and 11. The vaccine formulation according to claim 10, (ii) administering said vaccine formulation to said human, wherein said lactose is 5% (w/v) of said formulation and said human serum albumin is 1% (w/v) of said formulation. wherein a single dose of said vaccine formulation com 12. The vaccine formulation according to claim 11, prises 5x10 to 7x10' plaque forming units of vaccinia wherein said formulation further comprises neomycin of no 25 WS. more than 25 pig per single dose of said formulation. 16. The method according to claim 15, wherein the 13. The vaccine formulation according to claim 12, nucleotide sequence encoding said G1 polypeptide and said wherein said single dose of said formulation is in a volume G2 polypeptide is operably linked to the vaccinia virus 7.5 of 0.1 to 1.0 ml. kD promoter, and wherein the nucleotide sequence encoding 14. The vaccine formulation according to claim 1, 30 said N polypeptide is operably linked to the vaccinia virus wherein said formulation is in a form suitable for a route of 11 kD promoter. administration selected from the group consisting of subcu 17. The method according to claim 16, wherein said G1 taneous, intramuscular and intradermal. has the sequence (SEQID NO: 1):

MET GLY LE TRPLYSTRPLEUVAL METALASER LEU WALTRPPROVAL LEUTHRLEU ARG ASN WALTYR ASP METLYSILE GLU CYS PRO HIS THRVAL SERPHE GLY GLUASN SERVALILE GLYTYRVAL GLULEU PRO PROVAL PRO LEUALAASPTHRALAGLN METVAL PRO GLUSER SERCYSASN METASPASN HIS GLN SER LEU ASN THRILE THRLYSTYRTHR GLN VAL SER TRPARG GLY LYS ALAASP GLN SER GLN SER SER GLNASN SERPHE GLUTHRVAL SER THR GLU WALASPLEULYS GLY THRCYS WALLEULYS HISLYS MET VAL GLU GLUSERTYRARG SER ARG LYSSERVAL THRCYS TYRASPLEU SERCYS ASN SER THRTYR CYS LYS PRO THR LEU TYR METILE VAL PRO ILE HIS ALA CYSASN METLYS SER CYSLEU ILEALALEU GLY PROTYRARG VAL GLN VAL VALTYR GLU ARG SERTYR CYS MET THR GLY WALLEUILE GLU GLYLYS CYS PHE WAL PROASP GLNSERVAL VAL SERILE LELYS HIS GLY IEEPHE ASPILEALASER WALHIS ILE WAL CYSPHEPHE WALALAWALLYS GLY ASN THRTYRLYSLEPHE GLU GLNWALLYSLYSSERPHE GLU SER THRCYSASNASPTHR GLUASNLYS VAL GLNGLYTYRTYRILE CYSELE WAL GLYASN SERALAPRO LE TYRVAL PROTHR LEU ASPASPPHE ARG SER MET GLUALA PHE THR GLY LEPHEARGSER PRO HIS GLY GLUASPHIS ASPLEUALA GLY GLU GLU ILEALASERTYRSERILE WAL, GLY PROALAASNALALYS WAL PRO HIS SERALASER SER ASPTHRLEU SER LEU ILEALATYR SER GLYILE PROSERTYRSER SER LEU SER ILELEU THRSER SER THR GLUALALYS HIS WALPHE SER PRO GLY LEU PHE PROLYSLEU ASN HIS THRASN CYS ASPLYS SERALALE PRO LEU ILE TRPTHR GLYMET LEASP LEU PROGLYTYRTYR GLUALAWALHIS PRO CYSTHRVAL PHE CYS WALLEU SER GLY PROGLYALASERCYS GLUALA PHE SER GLU GLY GLY LE PHEASNLE THR SER PRO MET CYSLEUVAL SERLYS GLN ASN ARGPHEARGLEUTHR GLU GLNGLN WAL ASN PHEVAL CYS GLN ARG VAL ASPMET ASPILE VAL VALTYR CYS ASN GLY GLN ARG LYS VALILE LEU THRLYS THR LEU VALILE GLY GLN CYSILE TYR THRILE THR SER LEU PHE SER LEULEU PRO GLY VAL 5,614, 193 49 50 -continued ALAHIS SERILEALAWAL. GLU LEU CYS VAL PRO GLY PHE HIS GLY TRPALATHRALA ALA LEU LEU WAL THRPHE CYSPHE GLY TRP WALLEUILE PROALAILE THRPHE ILE ILELEU THRWALLEULYSPHE ILEALA ASNILE PHE HIS THR SER ASN GLNGLUASNARGLEULYSSER WALLEU ARG LYSELELYS GLU GLU PHE GLULYS THRLYS GLY SER MET WALCYS ASP WALCYSLYSTYR GLU CYS GLUTHRTYRLYS GLULEULYS ALAHIS GLY WAL SERCYS PROGLN SER GLN CYS PROTYRCYSPHE THRHIS CYS GLU PRO THR GLUALA ALA PHE GLNALAHISTYRLYS WALCYS GLN WALTHRHIS ARG PHE ARG ASPASPLEULYSLYSTHRWAL THR PRO GLNASN PHE THR PROGLY CYSTYRARG THR LEU ASN LEU PHEARGTYRLYSSER ARG CYSTYR ILE PHE THR MET TRPILE PHE LEU LEU VAL LEU GLU SERILE LEU TRP ALA ALASER ALA, 15 said G2 has the sequence (SEQID NO: 2):

SER GLUTHR PRO LEU THR PRO WALTRPASNASPASNALAHIS GLY WAL GLY SERVAL PROMETHIS THR ASP LEU GLU LEU ASPPHE SER LEU THR SER SER SERLYSTYR THRTYRARG ARG LYSLEU THRASN PRO LEU GLU GLUALA GLN SER LEASPLEU HISELE GLUILE GLU GLU GLN THRILE GLY WALASP WALHISALA LEU GLY HISTRP PHEASP GLY ARG LEU ASNLEULYSTHR SERPHE HIS CYSTYR GLYALACYSTHRLYS TYR GLUTYR PRO TRPHIS THRALALYS CYS HESTYR GLU ARG ASPTYR GLN TYR GLUTHR SER TRP GLY CYSASN PRO SER ASP CYS PRO GLY WAL, GLY THR GLY CYSTHRALA CYS GLY LEUTRYLEUASP GLN LEU LYS PROVAL GLY SER ALA TYRLYSILE ILE THR LEARGTYR SER ARG ARGVAL CYS VAL, GLN PHE GLY GLU GLUASN LEU CYSLYSELE LEASP METASNASPCYSPHE WAL SER ARG HIS WALLYS VAL CYSILE LE GLY THRVAL SERLYSPHE SER GLNGLY ASPTHR LEULEU PHEPHE GLY PRO LEU GLU GLY GLY GLY LEU LEPHELYS HISTRP CYSTHRSER THRCYS GLN PHE GLY ASP PRO GLY ASPLE METSER PRO ARG ASPLYS GLYPHE LEU CYS PROGLU PHE PRO GLY SERPHE ARG LYS LYS CYSASN PHEALATHRTHR PRO LE CYS GLUTYRASP GLY ASN MET WAL SER GLYTYRLYSLYS VAL METALATHRILE ASPSERPHE GLN SERPHE ASN THR SER THR METHIS PHE THRASP GLU ARGILE GLUTRPLYS ASP PRO ASP GLYMET LEU ARG ASP HIS ILEASNILE LEU WALTHRLYS ASPILE ASPPHE ASPASN LEU GLY GLUASN PRO CYSLYSILE GLY LEU GLNTHRSER SERILE GLU GLYALATRP GLY SER GLY VAL GLY PHETHRLEUTHR CYSLEU WAL SER LEU THR GLU CYS PRO THRPHE LEUTHR SERILELYS ALACYS ASPLYS ALAILE CYSTYR GLYALAGLUSER WALTHRLEU THRARG GLYGLNASNTHRWALLYS VAL SER GLY LYS GLY GLY HIS SER GLY SER THRPHE ARG CYS CYS HIS GLY GLUASP CYSSER GLNILE GLY LEU HIS ALA ALA ALA PRO HIS LEU ASPLYS VAL ASN GLYILE SER GLUILE GLUASN SERLYS WALTYRASPASP GLYALAPRO GLN CYS GLYILELYS CYSTRPPHE WALLYS SER GLY GLUTRPILE SER GLY LEPHE SER GLY ASN TRPILE WALLEUILE WALLEU CYS WALPHE LEULEU PHE SER LEU WALLEU LEU SERILE LEU CYS PROVAL ARG LYS HIS LYSLYSSER and said N has the sequence (SEQID NO:3):

METALATHRMET GLU GLU LEU GLNARG GLUILEASNALAHIS GLU GLY GLN LEU WALILEALAARGGLN LYS WALARG ASPALA GLULYS GLN TYR GLULYS ASP PROASP GLULEUASNLYSARG THR LEU THRASP ARG GLU GLY WALALAVAL SERILE GLNALALYSILEASP GLULEULYSARG GLNLEUALAASPARGILE ALATHR GLY LYSASN LEU GLY LYS GLU GLNASP PRO THR GLY WAL GLU PRO GLYASPHISLEULYS GLU ARG SER MET LEU SERTYR GLY ASN WALLEU ASPLEU ASN HIS LEU ASP ILEASP GLU PROTHR GLY GLN THRALAASP TRPLEU SERILE ILE WALTYRLEUTHR SER PHE WAL VAL PROILE LEULEU LYS ALALEUTYR MET LEU THRTHRARG GLY ARG GLN THRTHRLYS ASPASNLYS GLY THRARGILE ARG PHELYSASPASP SER SERPHE GLUASP WALASN GLY LEARG LYS PRO LYS HIS LEU TYRVAL SER LEU PRO ASNALA GLN 5,614, 193 51 52 -continued SER SER METLYS ALA GLU GLUILE THR PROGLY ARG TYRARG THRALAWAL. CYS GLY LEUTYR PRO ALA GLN LELYS ALAARG GLN METILE SER PRO WALMETSERVAL LEGLYPHE LEU ALALEUALALYSASP TRPSER ASPARGILE GLU GLN TRPLEU ILE GLU PRO CYSLYSLEULEU PROASPTHRALAALAVAL SERLEU LEU GLY GLY PROALATHRASNARG ASPTYR LEU ARG GLN ARG GLN WALALALEU GLY ASN MET GLU THRLYS GLU SERLYS ALAILEARG GLN HIS ALA GLUALAALAGLY CYSSERMETLE GLUASPLE GLU SER PROSER SER LE TRPVAL PHEALAGLYALAPROASPARG CYS PRO PROTHRCYSLEU PHE LEALA GLY ILE ALA GLU LEU GLY ALA PHE PHE SERILE LEU GLN ASPMETARG ASN THRILE METALASERLYS THR WAL GLY THR SER GLU GLULYSLEUARG LYSLYSSER SERPHETYR GLNSERTYRLEU ARG ARG THR GLN SER MET GLY LE GLN LEU GLY GLNARGILE LE WALLEU PHE MET WALALATRP GLY EYS GLUAE AWAL ASPASN PHE HIS LEU GLY ASPASPMETASP PRO GLU LEU ARG THR LEUALA GLN SER LEU ILEASP WAL LYS WALLYS GL LE SER ASN GLN GUPRO LEULYSLEU. 20 18.The method according to claim 17, wherein said DNA molecule comprises a wild-type vaccinia virus genome with the cDNA of the M and S genomic segments of the Hantaan serotype inserted in the vaccinia virus thymidine kinase coding region. 25 19. The method according to claim 18, wherein said cDNA comprises the sequence: (SEQID NO: 4):

ATGGGGATATGGAAGTGG CTA GTGATG GCCAGTTTA GTATGG CCT GTTTTGACA CTGAGAAAT GTC TAT GACATGAAAATTGAGTGC CCC CAT ACA GTAAGTTTT GGG GAAAACAGT GTGATAGGTTAT GTA GAATTACCCCCC GTG CCATTG GCC GACACA GCA CAGATG GTG CCT GAGAGTTCTTGTAAC ATG GAT AAT CAC CAATCGTTGAAT ACAATAACAAAA TATACC CAA GTAAGTTGGAGA GGAAAG GCT GAT CAG TCA CAG TCT AGT CAAAATTCATTT GAGAGA GTG TCC ACT GAA GTTGACTTGAAAGGAACATGT GTT CTAAAACACAAAATG GTG GAA GAATCATAC CGTAGTAGGAAATCA GTA ACC TGT TAC GAC CTG TCT TGC AAT AGC ACTTACTGCAAGCCA ACA CTATACATGATT GTA CCAATTCAT GCATGC AAT ATGATG AAAAGCTGTTTGAT GCATTGGGACCATACAGR GTA CAG GTGGTTTAT GAGAGA AGT TACTGTATG ACA GGA GTC CTGATTGAA GGG AAA TGCTTT GTCCCA GAT CAAAGT GTG GTCAGTATTATCAAG CAT GGG ATC TTT GATATT GCAAGTTTT CATATT GTATGTTTC TTT GTT GCA GTTAAA GGGAAT ACT TATAAA ATTTTT GAA CAGGTTAAGAAATCC TTT GAATCA ACATGCAAT GATACA GAGAATAAA GTG CAA. GGA TAT TATATTTGTATTGTAGGGGGAAACTCT GCACCAATATAT GTT CCA ACA CTTGAT GATTTCAGA TCC ATG GAA GCATTT ACA GGAATCTTCAGATCA CCA CAT GGG GAAGAT CAT GAT CTG GCT GGA GAA GAAATT GCATCT TAT TCTATA GTCGGA CCT GCCAAT GCA AAA GTTCCT CAT AGT GCTAGCTCAGAT ACATTGAGCTTGATT GCC TATTCAGGTATA CCA TCTTATTCT TCCCTTAGC ATC CTA ACAAGTTCA ACA GAA GCTAAG CAT GTATTCAGCCCTGGGTTGTTC CCAAAACTTAAT CACACAAATTGT GATAAA AGT GCC ATA CCA CTCATA TGG ACT GGGATGATT GATTTA CCT GGATACTAC GAAGCT GTC CAC CCTTGT ACA GTTTTTTGC GTATTATCA GGT CCT GGG GCATCATGT GAA GCCTTTTCT GAA GGC GGG ATTTTC AAC ATA ACCTCTCCC ATGTGCTTAGTGTCAAAACAAAATCGATTC CGGTTA ACA GAA CAG CAA GTG AATTTT GTGTGT CAG CGA GTG GACATG GACATT GTT GTG TACTGAAACGGG CAGAGGAAA GTAATA TTA ACAAAAACT CTA GTTATTGGA CAGTGTATATATACTATA ACAAGCTTAXXXTCATTACTACCT GGA GTA GCA CAT TCTATTGCT GTT GAATTGTGT GTA CCT GGGTTC CAT GGTTGG GCCACA GCT GCT CTG CTT GTTACATTCTGTTTCGGATGGGTT CTTATA CCA GCAATTACATTT ATCATA CTA ACA GTC CTA AAGTTCATT GCT AATAATTTT CACACAAGTAAT CAA GAGAAT AGG CTA AAATCA GTA CTTAGAAAG 5,614, 193 53 54 -continued ATAAAG GAA GAGTTT GAAAAAACAAAA GGCTCA ATG GTATGT GAT GTCTGCAAG TAT GAGTGT, GAA ACATATAAA GAATTAAAG GCA CAC GGGGTATCATGC CCC CAATCT CAATGT CCTTACTGTTTT ACT CAT TGT GAA CCTACA GAA GCA GCATTC CCA GTC CATTACAAG GTATGC CAA GTT ACT CACAGATTC AGG GAT GAT CTAAAGAAAACT GTT ACT CCT CAAAATTTT ACACCAGGATGT TAC CGG ACA CTA AAT TTATTTAGATACAAAAGCAGGTGCTACATCTTTACAATGTGG ATA TTT CTT CTT GTCTTAGAATCC ATA CTGTGG GCT GCA AGT GCATCA GAG ACA CCATTAACT CCT GTCTGGAATGACAAT GCC CAT GGG GTA GGTTCT GTTCCTATG CAT ACA GATTTAGAG. CTTGATTTCTCT TTA ACA TCCAGTTCCAAG TATACA TAC CGTAGGAAGTTAACAAAC CCA CTTGAG GAA GCACAATCC ATTGAC CTA CATATTGAAATA GAA GAA CAGACAATT GGT GTT GAT GTG CAT GCT CTAGGA CACTGGTTT GAT GGT CGT CTTAAC CTTAAAACA TCCTTT CACTGT TAT GGT GCTTGTACAAAG TAT GAATAC CCTTGG CAT ACT GCA AAGTGC CAT TAT GAA AGAGATTAC CAA TAT GAA ACGAGCTGGGGTTGT AAT CCATCA GATTGT CCT GGG GTG GGCACA GGCTGT ACA GCATGT GGTTTATAC CTA GAT CAA CTGAAA CCA GTT GGT AGT GCT TATAAAATTATC ACAATAAGGTACAGCAGGAGA GTCTGT GTT CAGTTT GGG GAG GAAAAC CTTTGTAAGATAATAGAG ATGAATGATTGTTTT GTATCT AGG CAT GTTAAG GTCTGCATAATT GGT ACA GTATCT AAATTCTCT CAG GGT GATACCTTATTGTTTTTT GGA CCG CTTGAA GGT GGT GGT CTA ATA TTT AAA CACTGGTGTACA TCCACATGT CAATTT GGT GACCCAGGA GATATCATGAGT CCAAGA GACAAA GGTTTTTTATGC CCT GAGTTTCCA GGTAGTTTCAGGAAGAAATGC AACTTT GCT ACT ACC CCTATTTGT GAG TAT GAT GGA AAT ATGGTCTCAGGTTACAAGAAA GTGATG GCGACAATTGATTCCTTC CAATCTTTTAAT ACAAGC ACTATG CACTTCACT GAT GAAAGGATAGAGTGGAAA GAC CCT GAT GGAATG CTA AGG GAC CATATA AACATTTTA GTAACGAAG GACATTGACTTT GATAAC CTTGGT GAAAATCCTTGCAAAATT GGC CTA

CAAACA TCTTCTATTGAGGGGGCCTGGGGTTCT GGT GTG GGGTTCACATTAACATGT CTG GTATCA : CTA ACA GAATGT CCT ACCTTTTTGACCTCAATAAAG GCTTGT GATAAG GCTATCTGT TAT GGT GCA GAGAGT GTA ACATTGACAAGAGGA CAAAAT ACA GTCAAG GTATCAGGGAAA GGT GGC CAT AGT GGT TCA ACATTTAGGTGTTGC CAT GOGG GAG GACTGTTCACAAATT GGA CTC CAT GCT GCT GCA CCT CAC CTTGACAAG GTA AAT GGG ATTTCT GAGATA GAA AATAGTAAA GTATAT GAT GAT GGG GCACCG CAA TGT GGG ATAAAATGT TGGTTT GTT AAATCA GGG GAATGGATTTCAGGGATATTCAGT GGTAATTGG ATT GTA CTCATT GTC CTCTGT GTATTT CTATTGTTCTCCTTGGTTTTACTA AGCATT CTCTGT CCC GTA AGGAAG CATAAAAAATCA and (SEQ ID NO. 5):

ATG GCA ACT ATG GAG GAATTACAGAGGGAAATCAAT GCC CAT GAG GGT CAATTA GTGATAGCCAGG CAGAAG GTGAGGGAT GCAGAAAAACAG TAT GAAAAG GAT CCAGAT GAGTTGAACAAGAGA ACATTA ACT GAC CGAGAGGGC GTT GCA GTATCTATC CAG GCAAAAATT GAT GAGTTAAAAAGG CAA CTG GCA GAT AGG ATT GCA ACT GGG AAA AAC CTTGGG AAG GAA CAAGAT CCA ACA GGG GTG GAG CCTGGA GAC CAT CTGAAA GAGAGGTCA ATG CTCAGTTAT GGT AAT GTG CTG GATTTAAAC CATTTG GATATTGAT GAA CCTACA GGA CAG ACA GCA GACTGG CTGAGCATCATCGTCTAT CTTACATCCTTT GTCGTCCCG ATACTT CTGAAA GTC CTG TATATGTTGACAACAAGGGGGAGG CAAACTACCAAG GATAATAAA GGG ACC CGGATT CGATTTAAG GAT GATAGCTCG TTC GAG GAT GTTAACGGTATCCGGAAA CCAAAA CAT CTT TAC GTG TCCTTG CCAAAT GCA CAGTCAAGCATGAAG GCA GAA GAGATTACA CCT GGT AGATAT AGA ACA GCA GTCTGT GGG CTCTAC CCT GCA CAGATTAAG GCA CGG CAGATGATCAGT CCA GTTATG 5,614, 193 SS 56 -continued AGT GTAATT GGTTTT CTA GCATTA GCA AAG GACTGG AGT GAT CGT ATC GAA CAATGGTTAATTGAA CCTTGCAAG CTT CTT CCA GATACA GCA GCA GTTAGC CTC CTC GGT GGT CCT GCA ACAAACAGGGAC TACTTACGG CAG CGG CAA GTG GCATTAGGCAAT ATGGAGACAAAG GAG TCA AAG GCTATA CGC CAG CAT GCA GAA GCA GCT GGCTGT AGCATGATT GAA GATATTGAG TCA CCATCATCAATATGGGTTTTT GCT GGA GCA CCA GAC CGT TGT CCACCA ACATGTTTGTTTATA GCA GGTATT GCT GAG CTTGGG GCA TTTTTTTCC ATC CTG CAG GAC ATG CGAAATACAATCATG GCATCTAAG ACR GTT GGAACATCT GAG GAGAAG CTA CGGAAGAAATCATCATTT TAT CAG TCC TAC CTCAGAAGGACA CAATCAATGGGGATA CAACTA GOC CAGAGAATTATT GTG CTCTTCATG GTT GCCTGG GGAAAG GAG GCT GTG GAC AACTTC CACTTAGGG GATGAT ATG GAT CCT GAG CTA AGG ACA CTG GCA CAGAGCTTGATT GAT GTCAAA GTG AAG GAA ATCTCCAAC CAA. GAGCCTTTGAAA CTC. 20. The method according to claim 15, wherein said single 25. The method according to claim 24, wherein said dose comprises 5x10 to 7x10' plaque-forming units. lactose is 5% (wiv) of said formulation and said human 20 serum albumin is 1% (w/v) of said formulation. 21. The method according to claim 15, wherein said single 26. The method according to claim 25, wherein said dose comprises 5x10 to 1x10' plaque-forming units. formulation further comprises neomycin of no more than 25 22. The method according to claim 15, wherein said single ug per single dose of said formulation. dose comprises 5x10 plaque-forming units. 27. The method according to claim 26, wherein said single 23. The method according to claim 15, wherein said single 25 dose of said formulation is in a volume of 0.1 to 1.0 ml. dose comprises 3.4x10" plaque-forming units. 28. The method according to claim 27, wherein said administering is by a route selected from the group consist 24. The method according to claim 23, wherein said ing of subcutaneous, intramuscular and intradermal. pharmaceutically-acceptable carrier, excipient or diluent further comprises lactose and human serum albumin. k . . g :