PH26855A - Vaccines against acquired immune deficiency syndrome - Google Patents

Description

1. FIELD OF THE INVENTION

The present invention is directed to viruses that express peptides and proteins related to epitopes of Lymphadenopathy Associated Virus (LAV)/Human T-Cell

Leukemia Virus (HTLV-III), the etiological agent of

Lymphadenopathy Syndrome (LAS) and Acquired Immune .

Deficiency Syndrome (AIDS). The viruses of the present {avention may be used as immunogens in viral vaccine formulations for LAS or AIDS er im multivalent vaccine formulations. Im fact, infections viruses of the pre- seat inventiom which can multiply im a host without causing disease can be used in live viral vaccine for- i mulatioms that provide a prolonged immunogenic stimulus and cam give rise to substantial immunity.

The present imvention is alse directed te peptides and proteins related te epitepes of LAV/HTLV III that cas be used as inmunegeans im subunit vaccine fermula- ~ tioms fer LAS er AIDS, er in multivalent vaccine fermu- latiens, er as antigens in diagnestic immuneassays for

LAS er AIDS. These peptides and proteins may be pre- duced usimg recembimamt DNA techmiques in any vecter~ hest system er they may be synthesized by chemical me- theds. Accordingly, the invention is alse directed te the censtructioa ef mevel DNA sequences amd vecters in- cluding plasmid DNA, and viral DNA such as human viruses,

Ca.

animal viruses, insect viruses er bacteriephages which can be used te direct the expression eof LAV/

HTLV III related peptides and proteims im appre- priate host cells from which the peptides and pre- teins may be purified. Chemical metheds fer the synthesis ef LAV/HTLV III related peptides amd pre- teins are alse described.

In a specific embediment of the preseat imvent- jon, a recembimanat vaccinia virus was used te preduce

LAV/HTLV III related proteins. Te this end, DNA se- - quences ceding fer the gyceproteins eof LAV/HTLV IIl were imserted imtoo a vaccinia vecter which is capable of directing the expression eof the LAV/HTLV III glyce- protein geme im an appropriate hest. The LAV/HTLV III proteins preduced by the recombinant baccimia virus were found to be antigenic and immunegenic.

These recombinant vaccinia viruses themselves can be used in viral vaccine formulations. Alternatively, the LAV/HTLV III related proteims preduced by the re-~ combinant viruses can be purified or chemically synthe- gized and used as immunogens in subunit vaccime fermu- lations. Since the LAV/HTLV III related glycoprotein (8) will be recognized as "foreign" in the host snimal, a humemal and/er cell mediated immune response will be raised against this protein. In a properly prepared vaccine formulatiom, this should protect the host against subsequent LAV/HTLV III infections.

This invention also provides for the product- jon eof LAV/HTLV III antégems which are of general im- portance im human medicine. These include the use of the peptides and proteins of the present invention ag reagents in immunoassays such as ELISA tests and radieimmunoassays which are usefpl as diagnostic tools for the detection of LAV/HTLV III in blood samples,body fluids, tissues, etc. In addition, this reagent will be a valuable tool in elucidating the mechanism ef pa- thogenesis ef LAV/HTLV III. 2. BACKGROUND OF THE INVENTION 2.1. AIDS VIRUS

Acquired Immumedeficiency Syndreme (AIDS) is a disease characterized by severe immune deficiency due primarjly to impairment of the patient's cell mediated immune respomse (Gottlieb, M. et al., 1981, N. Engl. J.

Med. 305: 1425; Masur, J. et al., 1981, N. Engl. J. Med. 30531431). Two clinical presentations of the disease are recognized: (a) a prodromal phase called Lymphadene~ pathy Syndrome (LAS) characterized by chronic Lymphadene- pathy, leukopenia and a qunatitative decrease in periphe- ral blood helper cells (OKT ht cells) leading to a rever- gal of the mormal peripheral helper te superésser T= lymphocyte ratie (OKT4: OET8) which shift from 2 te 0.1 or less as the disease pregresses; and (b) an immune-deficient state characterized by a decrease in OKT4 cells and reversal ef the mormal OKTL4:OKT8 ratio, absolute lymphepemia, ard repetitive epportu- nistic imfectioms mainly by Pmeumecystiscaraii; this latter phase is ultimately asseciated with death in the majority of cases. Certain gubsets of patients have increased incidemce of lymphema and Kaposi's

Sarcoma.

Epidemiological data along with information cen- cerning the types of patieats that acquired the disease suggested that an infectious ageat tramsmitted by in- timate comtact might be the cause of the disease, Sub- sequently three greups have provided stremg evidence that the causative agent ef AIDS is a retrevirus with a trepism fer helper T-lymphecytes.

These groups are: (a) R.C. Galle and cawerkers at the Natiomal Ims- titute of Health were able to isolate a cyte- pathic retrevirus (HTLV III) frem patiemts with AIDS amd pre-AIDS (Galle, R.C. et al. 1984

Sciemce 2243500; Popovic, M. et al., 1984,

Sciemce 224:497)., They alse detected antibedies againgt HOULV II im the serum of patients with

AIDS. (») L. Montagmnier and coworkers at the Pasteur

Institute isolated a T-lymphotrepic retro- virus (LAV) from a patiemt whe presented with cervical lymphadenepathy amd was at risk fer

AIDS(Barre Simoussi, F., et al., 1983, Science 2201868). This group was alse able te demoms- trate antibedies against LAV im serum from AIDS patients (Kalyamsramam, V.S. et al., 1984,

Science 225: 321). Mereever, they were able to iselate LAV from the lymphecytes ef a pa- tient whe developed AIDS after receivimg bleed from a démer who developed AIDS (Feorine, Fa No et al. 1984, Sciemce 225:69). (¢) J.Levy amd coworkers isolated ififectious re- trevirusea (termed AIDS-associated retrevirus er ARV) from the peripheral mememuclear cells of patients with AIDS (Levy, JeA., et al., 1984

Science 225:8u40).

Although all three virusea were isolated indepen- dently, thay all probably belomg to the same retreviru- ses subgroup (Levy, J.A. et al., 1984, Sciemce 2251840) and will be collectively referred to herein as LAV/HTLV

III.

The general structure of retroviruses is that of a ribonycleoprotein core surrounded by a lipid contain- ing envelope which the virus acquires duriag the course of cell budding. Embedded within the envelope and pro- jecting outward are the viral encoded glycoproteins. 5S These determime the host range of the virus and react with specific receptors om the surface of susceptible cells. Neutralizing antibodies are thought to bind to envelope glycoproteins and block their interaction with receptors on the surface of cells (pp. 534.535 in, The

Molecular Biology of Tumer Viruses ed. Jehm Tooze, 1973, “old spring Harbor Laboratory; pp.226-227 and 236-237 ) '{a, RNA Tumer Viruses, ed. R. Weiss, Teich, N., Varmus,

H. and Coffim, J., 1982, Cold Spring Harbor Laberatery.).pps 226-227 amd 236-237 im, RNA Tumor Viruses, ed. R. Weiss,

N., Varmus, H. and Coffin, J., 1982, Celd Sprimg Harber

Laboratory). In the specific case of LAV/HTLV III,there jg evidence that the T), antigen, present on a subset of

T-lymphocytes, is the receptor or 2a component of the receptor for the virus (Dalgleish, A.G., et al., 1984

Nature 312:763; Klatzman, D., et al., 1984, Nature 312: 767).

The RNA genome of LAV/HTLV III is diagrammed im

Fig. 1. Three genes are generally recognized: the gag gene codes for the intermal structural proteins (cere proteins) of the virus and defines the viral group-

specific antigens. The pol gene codes for the viriom associated reverse tramscriptase. The env gene codes for the viral glycoproteins. Other regions marked ser and 3'=-orf denote areas of the genome containing open 6 reading frames; the functions ef these regioms is met knewn at present. 2.2. VACCINES

A number of methods are currently in use for the prevention and treatment #f viral infections. These jmcludes vaccines which elicit am active immume res- pomse, treatment with chemetherapeutic agents and in- terferon treatment.

Traditional ways of preparing vaccines include the use imactivated or attendated viruses. Inactivation of the virus renders it harmless as a biological agent

Put does mot destrey its immunegenicity. Injection eof these "killed" virus particle into a heat will them elicit an immume response capable of neutralizing a future infectiom with a live virus. Hewever, a majer concern im the use of killed vaccines (using inactivat- ed virus) is failure to jpactivate all the virus par- ticles. Even when this is accomplished, since killed viruses do not multiply in their hest, the immunity achieved is often shert lived and additional immumiza- tions are usually required. Finally, the inactivation process may alter the viral proteins remdering them less effective as immunogems.

Attenuation refers to the production eof virus strains which have essentially lest their disease producing ability, Ome way to accemplish this is to subject the virus to unusual grewth comditioms amd/ or frequent passage im cell culture, Viral mutants are thea selected which have lost virulemce but yet are capable of elicitimg an immume respomse. The at~- tenuated viruses gemerally make good immumeogens as they actually replicate im the hest cell and elicit long lasting immunity. However, several preblems are encountered with the use of live vaccines, the mest wer- risem is imsufficieat attemunatien.

An alternative te the abeve metheds ig the use of subunit vaccines. This imvelves immunizatiom enly with these proteims which centaim the relevaat immume- legical material. Fer many eaveloped viruses, the vi- rally encoded glycepreteim cemtaims these epitepes which are capable of eliciting meutralizimg antibedies; these include the glyceproteims of La Cresse Virus (Gem- zalez-Scarame, F., Shepe, R.E., Calisher, C.E. and

Nathamsen, N., 1982, Virelegy 120:42). Neematal Calf

Diarrhea Virus (Matsume, S. and Imeuye, S., 1983 Ia- fection and Immumity 39: 155), Vemezualam Equime Enmcew.

phalemyelitis Virus (Mathews, J.H. and Roehrig, J.T., 1982, J. Imm. 129:2763), Punta Tere Virus (Dalrympe,

JeMe, Peters, C.J., Smith, J.F. and Gentry M.K.,1981,

In "Replication of Negative Stramd Viruses", D.H.L.

Bishep and R.W. Compams, eds., Pp. 167. Elsevier, New York)

Murime Leukemia Virus (Steeves, R.A., Stramd, M. and Au- gust, J.Te., 1974, J. Virel. 14:187), and Meuse Mammary

Tumer Virus (Massey, R.J. amd Schochetmanm, G., 1981,

Virelegy 115:20). Ome advantage of subunit vaccines is that the irrelevamt viral material is excluded.

Vaccines are often administered im conjunction with various adjuvants. The adjuvants aid in attaining a more durable amd higher level of iimunity using small- er amounts of antigen im fewer doses than if the immume- 8 gen were administered alone. The mechaniam of adjuvant action is complex and not completely undergtopd. Hew- ever, it may invovle the stimulation ef phagecytesis and other activities of the reticuloendothelial system as well ag a delayed release and degradatiom of the antigen.

Examples of adjuvants include Freund's adjuvant (complete or incomplete), Adjuvant 65 (containing peanut oil, man- nide monoeleate and aluminum monostearate), the pluronic polyol L~121, Avridine, and mineral gels such as alumi- ~ num hydroxide, aluminum phosphate, or alum. Freund's ad- juvant is me longer used im vaccime formulations for hu-

mans because it comtaims nommetabolizable mineral eil and is a petential carcimegen. 2.2.1. RECOMBINANT DNA TECHNIQUES AND VACCINIA

VIRUS

The use of recembinant DNA techrelegy fer the production ef subunit vaccines invelves the melecular cleming amd expression im an appropriate vecter of the viral gemetic informatiem coding fer these preteins which cam elicit a neutralizimg respomse in the host amimal. All ether genetic information of the virus is excluded and enly these proteinas required te esta- »lish a meutralizimg respoase are presented to the hest animal. The host is mever expesed te the whele virus and stands po risk ef becoming infected.

Recently, & movel appreach has been described which is peotectially usefully in the preductiom of gubumit vaccimes (Mackett, M. Smith, G.L. and Mess, B., 1982, Proc. Nat'l. Acad. Sci. 79, 7415-7419; Mackett,

M., Smith, G.L. and Mess, B. 1984, J. Virel. 49 357-864

Panicili D. and Paoletti, E. 1982, Prec. Nat'l. Acad

Sei. 79 4927-4931). Thisapproach iavelves the use of vaccinia virus as a vecter te express foreigm genes in- serted into its gemome. Upom introduction inte host animals, the recombinant vaccinia virus expresses the jmgerted foreign gene and thereby elicits a host immune respense to such geme products. Since live recom- binant vaccinia virus can be used as a vaccine, such an approach combines the advantages of beth subunit and live vaccines.

Vaccinia virus contains a linear double-strand- ed DNA genome of approximately 187 kilobase pairs and replicates within the cytoplasm of infected cells.

These viruses contain a complete transcriptional em- zyme system (including capping, methylating and poly~ adenylating enzymes) within the virus core that are necessary for virus infectivity.

Vaccinia virus tramscriptomal regulatory sequences . (promoters) allow for jnitiation of transcriptiom by vaccinia DNA polymerase but not by host cell RNA pely- merase.

Expression of foreign DNA in recombinant vaccinia viruses requires the ligation of vaccinia promoters to ’ protein coding DNA sequences of the foreign gene, Plas- mid vectors, also called insertion vectors have been constructed te insert chemeric genes into vaccines vi- rus. One type of insertion vector ie composed of: (a) vaccinia virus promoter including the transcriptional jmitiation site: (W) several unique restriction endomuc- lease cloning sites located downstream from the transcrip4 jonal start site for insertion of foreigm DNA fragments}

(e¢) nonessential vaccinia virus DNA (such as the TK gene) flanking the promoter and cloning sites which direct insertion of the chemeric gene inte the homo- logous nonessetial region of the virus genome; and 8 (d) a bacterial origim of replication and antibietic resistance marker for replication and selection in E. coli. Examples of such vectors are described by Mackett (Mackett, M. , Smith, G.L. and Mess, B., 1984, J. Virel. hg, 857-864).

Recombinant vaccinia viruses are produced by trans- fection of recombinant bacterial insertion plasmids con- taining the foreigm gene into cells previously infected with vaccinia virus. Homologous recombination takes place within the infected cells and results in the insert- jon of the foreign gene into the viral genome. The infect- ed cells can be screened using immunolegical techniques,

DNA plaque hybridization, or genetic selection for recom- binat viruses which subsequently can be isolated. These vaccinia recombinants retain their essential functions and infectivity and can be constructed to accomodate ap- proximately 35 kilobases of foreign DNA.

Foreign gene expression can be detected by enzyma- tic or immunolegical assays (for axample, immuneprecipi- tation, radioimmunoassays, or immunoblotting). Naturally occurring membrane glycoproteins produced from recombi-

a nant vaccinia infected cells are glycosylated and may pe transported to the cell surface. High expression levels can be obtained by using strong promoters or by cloning multiple copies of a single gene. ° 3. SUMMARY OF THE INVENTION

Viruses which direct the expression of peptides or proteins related to epiptopes of LAV/HTLV III are described. These viruses may be formulated as viral vaccines to protect humans against LAV/HTLV III infect~ jon. In a particular embediment of the present invent- jon, live virue vaccines formulations can be prepared using infectious viruses which express LAV/HTLV III re~ lated epitopes in jnfected host but do net cause disease in the host.

The invention is also directed to peptides or pro= teins related to the epitopes of LAV/HTLV III. These may ve formulated in subunit waccines to protect humans againat LAV/HTLV III infection or they may be formulated in multivalent vaccines. The peptides or proteins of the present jpvention may be produced in and isolated from any host cell -expression vecter system; these includey fer example, animal er jnsect cell cultures infected with apprepriate recombinant virus§ micreerganisms such as vacteria transfected with recombinant plasmids, cesmids er phages; and yeast transformed with recombinant plasmids. - 1h -

The present imvention alse prevides metheds, prece- dures and DNA constructions which are used fer the expression of genetic infermation coding for the epi- topes eof LAV/HTLV III. Alternatively, the peptides and proteins of the present invention can be chemical- ly synthesized.

In a specific embodiment of the present invent- ion the gene coding for the envelope glycoproteins of

LAV/HTLV III (LAV/RHTLV III env gene) is inserted inte a plasmid so that am early vaccinia virus prometer is pesitioned 5' to the initiation methiomine sequence ( (ATG) ef the LAV/HTLV III eav gene resulting in a che- meric geme which in turm is flamked by vaccinia thymidime kinase (TK) DNA sequences. This plasmid is thea tramns- fected into cells which had been previously infected with wild type vaccinia virus thus allowing the chemiric LAV/

HTLV II1 eav gene flanked By TK sequences to be recom- bined inte the TK gene of the vaccinia virus. The cells are allowed to lyse and the reculting virus is plaqued or TK cells. Recombinant virus is selected by its abili- ty te plaue om these cells in the presence of S-brome-dee-~ xyuridime as well as by DNA-DNA hybridization to radio- labeled LAV/HTLV III-envelope probe,

Hybridization pesitive plaques are purified, expand- ed and the resulting recombinant virus is tested for its a — . ability to produce LAV/HTLV III envelope glycoproteins.

Virus stocks producing these proteins are used to im~ fect test animals. Serum obtained from these animals can be assayed for antibody against LAV/RTLV III glyeo- proteins, for its ability to prevent LAV/HTLV III im- duced viremia, and finally for its ability te confer protection against AIDS. 4, BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 represents the integrated proviral genome structure of LAV/HTLV III. Hatched areas imdicate re- gions of open reading frames. Such regions emcoding fer the groupspecific antigen, the reverse tramscriptase and envelope proteins are designated as Eag.pol and eav respectively. Overlapping open reading frames are shown by cross-hatched areas. Numbers refer to numbers of base pairs downstream from the capsite, where the viral transcript starts. Restriction sites are marked as fol- lows: Bg, BglII; Ec, EcoRI: Hm, HindIII: Kp, KpnI: Ss, sstle.

Fig. 2 represents the nucleotide sequence of the

LAV-specific region (EcoRI to Sstl) present in plasmid pRS~3 DNA; the entire LAV envelope gene (Nucleotide 5766 to 8349) is contained within the LAV insert. Restrict- jon sites used in the construction of pv-envl, pv-eav2 and pv-env5 are jadicated. The entire amino acid sequence of the envelope gene, as deduced from the nucleotide sequence data, is alse indicated.

Fig. 3 is a schematic representatiom of the cCoens- truction ef plasmids containing a portion ef the LAV envelope protein coding sequence inserted downstream from a vaccinia virus prometer., The LAV envelope ced- ing sequence is represented by the opem bar and the vaccinia promoter Wy the shaded bar. The mucleotide sequence at the junctiom ef the vaccinia prometer region and the

LAV eavelepe codimg sequence is indicated at the lewer portion of the figure. The underlined sequences indi- cate the presumed initiatimg codons and the reading frame fer the chimeric gene, Nete that the third amd feurth amine acids im the translated sequence (Pre-val) of the recombinant pv-env2 correpoad to amino acide mum- ber 43 and 4k of the LAV emvelepe codimg sequence.

Fig. 4 is a shematic representation ef the cens- . truction ef plasmids centaining the entire LAV envelope protein coding sequence imserted dowastream from a vac cimnia virus premeter. The vaccimia virus premeter is represented by the shaded bar. Plasmid pv-eav5 cemntain- ing the entire LAV eavelope coding region was cemstrcted in twe stages. The 5' and 3' pertioms ef the ceding re gion were first cloned imte pGS20 separately te form pv- 25 .enavl which contains the amine coding terminus ef the LAV envelope gene and pv-eav2 which contains the carbexyl coding terminus of the LAV envelope geme, These twe portions were rejoimed at the Stul site as shewn.

Fig. 5 represents the construction and select- ion for recombinant vaccinia virus. Solid bars rep- resent the thymidine kinase (TK) gene of vaccinia vi- rus. This TK geme is alse present in plasmid pv-env5

DNA, but in the plasmid the TK gene is imterrupted by the chimeric gene consisting ef the vaccinia 7.5K promoter (shaded bar) and the LAV envelopes coding region (open bar). “tter cells are infected with vaccinia, the re- combinant plasmid containing the TK gene interrupted by the LAV enevlepe geme is imtreduced inte the infected cells. Recombinatiomg which occur in TK sequences flamk- ing the chimeric gene introduce the LAV envelope gene sequence inte the vaccinia viral genome. The resultant recombinant virus containing the LAV envelope gene is

TK .

Fig. 6A represents a Western immuneblot analysis of the proteins produced by vaccinia-LAV eav recombinants.

Froteins from the following sources were resolved on 7- 15% gradient SDS-pelyacrylamide gel and electretransfer- red to mitrecellulese paper; LAV-viriom proteins (LAV); wild-type vaccinia virus infected cells (WTvv); mminfect- ed cells (mock) ; cells infected by recombinant viruses v-env2

(v-env2), er v-env5 (v-env5). Proteins immumereact- jve to AIDS patient serum were detected through the action ef peroxidase conjugated to anti-human IgG anti- bodies. LAV-eav geme products are indicated as gpl50, gpll0 and gp4l. Medecular weight standards are expressed in kilodaltonms.

Fig. 6B represents a Westerman Immunoblot analysis of the proteins preduced by vaccinia-LAV env recombi- nants in twe cell types. * proteins derive from either

BSC-40 or Hela cells were resolved by SDS-pelyacrylamide ’ 'gél electrophoresis (SDS-PAGE) and elctretransferred to nitrocellulose paper and reacted with pooled serum from

LAV/RETLV-111 seropositive jndividuals; lames 1 and 5 re- present mock-infected cells; lames 2 and 6 represents cells infected with wild type vaccinia virus; lames 3 and 7 re=- present cells infected with v-env5; and lanes & and 8 re- present cells infected with v-env2. Immunoreactive pro- toins were detected using proteins A labeled with 1251.

LAV-envgene products are indicated as gpl50,gpllO and gpll.

Fig. 7A represents the results of a radieimmune- precipitation analysis of the proteins synthesized in cells infected with either wild type vaccinia virus (WIvv) er recombinant vaccinia virus (v-eav2 and v=-env5)e.

Proteins were labeled with 355.methionine from 10-12 hours after infection. Labeled proteins im cell lysates were reacted with either control human serum (N) er

AIDS patient serum (1) and the immune complexes were precipitated by Staphylecoccus aureus protein

A. Immuneprecipitated proteins were resolved by elec- trophoresis om a 15% SDS~-polyacrylamide gel and detect- ed by fluorography. Molecular weights are indicated im kilodaltens, :

Fig. 7B represents the results of radioimmunopre- cipitation analysis of 34-glucosamine labelled proteins produced by the vaccinia-LAV recombinants of the imvemt- jon. Hela cells were either mock infected (lanes 1 amd 5), or infected with wild type vaccinia virus (lanes 2 and 6), v-envS(lanes 3 and 7) or v-eav2(lames U4 and 8) and labeled with 3§-glucosamine. Cell lysates were im=- muneprecipitated with either mormal humam serum (lanes 1-4) or pooled serum from LAV/HTLV III seropesitive ia- dividuals (lames 5-8) and proteim A. The immumeprecipi- tated proteins were resolved by SDS-PAGE.

Fig. 7C represents the results of a "pulse-chase radioimmunoprecipitation amalysis ef LAV emvelope pre- teims produced by vaccinia-LAV recombinants. Hela cells were infected with wild type vaccinia virus, v-emv5 er v-env2 as indicated, labeled with 355 -methionine, and washed with chase-medium. At the following time inter-

tervals, the cells were washed, lysed aad immumopre- cipitated with pooled serum from LAV/HTLV III sereo- pesitive jmdividuals and eeselved by SDS#PAGE: O heur (lames 1,7,13)3 0.5 heur (1amed 2,8, 14); 1 hour(lanmes 3, 9,15); 2 heurs (lanes %,10,16); 6 hours(lanes 5, 11, 17) and 12 hours (lames 6,12,18).

Fig. 7D represents the results of a radieimmune- precipitation analysis ef the LAV envelope~-related pro- teins foumd im cells amd ip media from ells imfected with the recombinant yaccinia-LAV viruses ef the in- vention, HeLa cells were either meck imfected (lame 1), or infected with either wild type vaccinia virus (lame 2) v-env5) (lane 3) er v-eavZ (lame 4), and labeled with 35g. methienime. The cells were separated from medium and lysed. The cell lysates (pellet) amd the media (supe) were each immumeprecipitated using pecled serum fren

LAV/HTLV III gerepssitive imdividualse The immumepre- cipitated proteins were resolved by SDS-PAGE.

Fig. 8 represents a Western immoneblet analysis : of serum samples from pice immumized with vaccinia-LAV recombinant viruses. Mice were immunized with v-envS or V-~-emv2 recombinant vaccinia vipus. After 8 weeks, 8 serum samples were reacted with LAV virion proteins which had been resolved by SDS-PAGE and electrotrans- ferred to ajitrecellulose paper. A goat anti-meuse inm-

muneglebulin conjugated te alkaline rhosphatase was used to detect those LAV proteims which were recog- aized by the meuse sera. Lares a to e represeat serum samples from 5S jndividual mice inoodlated mice jmoculated with v-env5 and lanes f to k represent serum samples from 5 jmadividual mice ineculated with v-env2e.

Pooled sera from LAV/HTLV III seropesitive individuals (AIDS) and umimmunized ¢57B163 mice (NMS) were used as pesitive and negative control, respectively. Positions of LAV envelope glycoproteins gpl50,gpll0, and gph3 are indicated.

Fig. 9 represents a Wwestera immunoblot analysis ef serum samples from macaque monkeys immunized with vacci- pia-LAV recombinant virus. Four macaque monkeys (numbers 168, 175,180, 182) were inoculated with 2 x 108 pfu eof recombinant virus v-env5 by skin scarification. One men- key was gnoculated with 2 x 107 pfu of vaccinia-herpes simplex gD recombimant (v-HSgD1). Serum samples were collected previous to imoculation (lanes p) and at 3,4, and 6 weeks pest-ineculation(lanes 3,L,and 6 respective- ly). Aliquots ef serum were diluted 50-fold and reacted . with LAV virion protein which had been resolved by SDS~

PAGE amd immebilized en pitrocellulese filters by elec- trotransfer. LAV proteins recognized by these sera were detected by goat anti-human jmmunoglebulin conjugated with alkaline phosphatese. Pooled sera from LAV/HTLV

I11 seropositive individuals (AIDS) were used as pesi- tive contrel.

Se DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to viruses that produce peptides and proteins related to epitopes ef

LAV/HTLV III. The invention is alse directed to peptides and protein related to epitopes ef LAV/HTLV III which can be preduced using recombinant DNA methods or by chemical synthésig, The viruses or peptides. and proteins of the present invention can be used as immunegens im varicus vaccine formulations, including umltivalemt vaccine fermu- lations to protect agaimst infection with LAV/HTLV I1I, the etiological agent of LAS and AIDS.

According to one embodiment of the present invention recombinant DNA techniques are used to insert nucleotide sequences encoding LAV/HTLV III epitopes into expression vectors that will direct the expression of the LAV/HTLV

II1 sequences in appropriate host cells, These express- jon vector-host cell system can be used to produce LAV/

HTLV 111 related peptides and proteins in vitroin which case, the gene products can be purified from the cells in culture and used as immunogens in subunit vaccine for- mulationse.

Alternatively, the amino acid sequence of these pep- - at tides and proteins may be deduced from the LAV/HTLV

III nucleotide sequences contained im recombinants that express immunogenke LAV/HTLV III related peptides and proteins. These peptides and proteins may then be chemically synthesized and used in synthetio subunit ~ vaccine formulations. where the expression vector is a virus, the virus itself can be formulated as a vaccine. Infectioms re- combinant viruses that de not cause disease in host can be used in live virus vaccine preparations which pre- oo vide for substantial immumity. Alternatively, inactivat- ed virus vaccines can be prepared using "killed" viruses.

In addition, multivalent vaccines containing epitopes eof

LAV/HTLV III as well as those of bther disease causing agents may be prepared.

The methed of the imventiom may be divided inte the following stages for the purpose of description: (a) isolation of a gene, or geme fragment, encoding LAV/

HLTV III viral proteins, (W) imsertion of the geme or gene fragment into expression vectors, (c) identification and growth ef the recombinant expression vecter in a hest system which is capable eof replicating and expressing the gene, (d) identificatiom and purification of the gene product, (e) determination of the immunopotency ef the preduct and (f) formulation of a vaccine. - 2h -

In & specific embodiment of the present inventioa we describe the construction of recombinant vaccinda viruses containing the envelope geme of LAV/HTLV III which direct the expression of proteins immumelogical- ly related to the emvelope proetims of LAV/HTLV ELI im tissue culture cells infected by the recombimant viruses.

However, the cempesitioms amd methods described herein are not limited to the comatructiog ef recombimant vace cinia viruses expressimg LAV/HTLV emvelepe related pre- teins and may be used te comstruct recombinants ia amy expression vecter system for the productiom of pelypep= tides related to antigeas of any etiolegical agemt of AIDS,

For clarity of discussiom, the entire methed will pe discussed in terms of the LAV/HTLV III emvelope gens.

The same technique, however, may be applied in am ama- legous fashion to construct recombinant expression vecters and to produce polypeptides related to any of the proteins of LAV/HTLV E&I aes well as these of related viruses, 5.1. ISOLATIONS OF GENES OR GENE FRAGMENTS

ENCODING LAV/HTLV III VIRAL PROTEIN lgolation of the LAV/HTLV III envelope geme imvolves first isolating DNA fragmemts which contaim the envelope gene sequences. As previously explained, LAV/HTLV I1I has an RNA geneme, therefore, the corresponding DNA which en- codes the LAV/HTLV III geme can be obtaimed either (a) by cDNA cloning of RNA isolated from purified LAV/HTLV III virioms (b) by cDNA cloning of poly/E/-containing RNA obtained from LAV/HTLV III-infected cella er (c) by clom- ing genomic DNA purified from LAV/HTLV III infected cells.

Hereinafter, PNA encodimg LAV/HTLV III genes will be re- ferred to as LAV/HTLV III DNA.

IN order to generate LAV/HTLV BII DNA fragments, the

LAV/HTLV III DNA may be cleaved at specific sites using various restriction enzymes. Alternatively ome may use

DNase in the presence of manganese to fragmemt the DNA, or the DNA can be physically sheared, as for example, by sonicatien. The linear DNA fragments can tne be separated according to size by standard techniques, including, but not limited to, agarose and polyacrylamide gel electrephe- resis and column chromategraphye.

Any restrictiom enzyme or combination of restrict- jon enzymes may be used to generate LAV/HTLV 1I1I DNA frag- ments containing the envelope sequence provided the em- zymes do met destroy the immumepotency of the envelope pro- | tein gene product. For example, the antigenic site of a protein can consist of from about 7 to about 14 amine acids.

Thus, a protein of the size of theeenvelope peptide precur- sor (approximately 97,000 daltoms) may have many discrete antigende sites, possibly thousands considering overlapp- ¥ ing sequences, secondary and tertiary structure considera-

tions, and processing events such as acetylation, gly- cosylation or phosphorylation. Therefore, many partial envelope polypeptide gene sequences could code for an i antigenic site. Consequently, many restriction enzyme combinations may be used to generate DNA fragments which, when inserted into an appropriate vector, are capable of directing the production of envelope specific amino acid sequences comprising different antigenic determinants,

Once The DNA fragments are generated, jdentifica- tion of the specific DNA fragment containing the LAV/HTLV 111 envelope gene may be accomplished in & humber of ways:

Firstly, it is possible to sequence the DNA fragments corresponding to the entire LAV/HTLV III genome and then identify the gragment containing the envelope protein gene sequence based upon a comparison of the predicted amino acid sequence to the amino acid sequence of the em- velope protein. Secondly, once the entire genomic se- quence has be determined, the large open reading frames can be ordered from 5' to 3t, As the genomic organiza- tion of all retroviruses examined te date is 5!'egag=pole env-3t, the large open reading frame closest to the 3! end will most likely code for the envelope gene. Alterna- tively, the fragment containing the envelope protein gene may be jdentified by mRNA gelection. In this procedure the LAV/HTLV III DNA fragments are used to isolate comple~ mentary mRNAs by hyBridization. Immunoprecipitation analysis of the in vitro translation products of the isolated mRNAs identifies the mRNA and, therefore,the complementary LAV/HTLV III DNA fragments that centain the envelope protein sequences. Finally, ehvelope protein-specific mRNAs may be selected by adsorption of polysomes isolated from LAV/HTLV III-infected cells to immebilized antibodies directed against envelope pro- tein. A radiolabelled envelope protein ¢DNA (complemen- tary DNA) can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template, The radie- jabeled mRRKA or cDNA may then be used as a probe te iden tify the LAV/HTLV III DNA fragments containing envelope protein gene sequences. Alternative to isolating the envelépe gene include but are not limited te, chemically synthesizing the gene sequence itself (provided the se- quence is known) er making cDNA to the mRNA which encodes the envelope gene.

Once identified and isolated, the LAV/HTLV III DNA fragment containing the sequences of interest may be first inserted inte a cloning vecter such as a plasmid cloning vecter which is used to transferm apprepriate hest cells im erder te replicate the DNA se that many copies ef the LAV/HTLV III sequences of interest are geme- rated. This can be accemplished by ligating the LAV/HTLV

III DNA fragment iite a cloning vecter which has cemplemen-

tary cohesive termini. However, if the complemen- tary restriction sites used to fragmeat the LAV/

HTLV III DNA are met present in the cloning vecter, the ends of the DNA molecules may be modified. Such modifications include producing blunt ends by digest- ing back single-stranded DNA termini or by filling the single-stranded termini so that the ends can be blunt-end ligated. Alternatively, any site desired may be preduced by ligating nucleotide sequences (linkers) onte the DNA termini these ligated linkers may comprise specific che mically synthesized oligmnucleotides encoding restrict- jon site recognition sequences. Accerding to other metheds, the cleaved vector and the LAV/HTLV III-DNA fragment may

Ye modified by homepolymeric tailimg.

Transformation of hest cells with recombinant DNA molecules that incorporate the isolated gene, cONA er synthesized DNA sequence enables generation eof multiple copies of the gene. Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.

If the ultimate goal is to insert the gene inte virus expression vectors such as vaccinia virus or ade~ nevirus, the recombinant DNA molecule that incorporates

- the LAV/HTLV II1 gene can be modified so that the gene is flanked by virus sequences that allew genetic recombinantion in cells infected with the virus se that the gene can be inserted inte the viral genome.

The entire LAV/HTLV III genome has been cloned and sequenced by Wain-Hobson etlal. (Wain-Hobson, S. ot al., 1985, Cell 40:9). One clone, referred to as lambda J19 contained a 9.2 kilobase pair DNA fragment of an LAV genomic sequence inserted inte the Hind III site of lambda L 47.1.

A particularly useful subclone of labda J19 con= taining the LAV envelope gene is pRS-3 which consists of a 3,840 base pair EcoRI to Sstl fragment of the LAV nucletide sequence jnserted into the EcoRI and Sat] site of pUC18. The LAV specific DNA contained im pRS=3 is from the EcoRI site located at nucleotide 5289 to the Sstl site lecated at nucleotide 9129 on the LAV genome (Wain-Hobson et al., 1985, Cell 40:9); see Fig. 2 which depicts the

LAV nucleotide sequence contained in pRS-3. However, due to the degeneraey of nucleotide coding sequences, other

DNA seugnces which encode substantially the same amino acid sequence as depicted in Fig. 2 may be used in the practice of the present jnvention for the cloning of the envelope gene of LAV/HTLV III. These include but are not limited to nucleotide sequences comprising all or pertions of the envelope nucleotide sequence depicted in Fig. 2 which are altered by the substitution of different codons that encode the same or a functionally equiva- lent amino acid residue within the sequence (for exam- ple, an amino acid of the same polarity) thus preducimng a silent change,

S5e2. INSERTION OF THE LAV/HTLV III PROTEIN

CODING SEQUENCES INTO EXPRESSION VECTORS

The nucleotide sequence coding for LAV/HTLV III envelope protein, or a portion thereof, is inserted in- : 10 - ‘te an appropriate expression vecter, i.e. a vector which contains the necessary elements for the transcript- ion and translation of the inserted protein-coding se- qunece. A variety of hostpvector systems may be utilized to express the protein-coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g. vaccinia virusg, adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus); micreorga- nisms such as yeast containing yeast vectors or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid

DNA. The expression elements of these vectors vary in their strength and specificities. Depending on the host- vector system utilized, any one of a number of suitable transcription and translation elements may be used, Fer instance, when cloning in mammalian cell systems, preme- ters isolated from the genome of mammalian cells.(e.g.,

—-_— mouse metallothienien premoter) of from viruses that grow in these cells, (e.g. vaccinia virus 7.5K promo- ter) may be used. promoters produced by recombinant

DNA er synthetic techniques may also be used to pro- vide for tranacription of the inserted sequences.

Specific jnitiation signals are alao required for efficient translation of inserted protein ceding sequences. These signals include the £TG initiation cedon and adjacent sequences. In cases where the en- tire LAV/HTLV III envelope gene including its own ini- tiation codon and adjacent sequences are inserted into the appropriate expression vectors, no additional trams- lation control signals may be needed. However, im cases where only & portion of the emvelepe coding sequence is inserted, exogenous translational control signals, in- cluding the ATG jnitiation codon must be provided. The initiation codon must furthermore be in phase with the reading frame of the envelope protein coding sequences to ensure translation eof the entire insert. These exe= genous translational control signals and initiatiom ce- dons can be of a variety of origins, beth matural and synthetic.

Any of the methods previously described for the insertion of DNA fragments into = vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translation- al control signals and the protein coding sequences.

These methods may include in vitro recombinant DNA and synthetic techniques and in vive recombinations (gene- tic recombination).

In the particular embodiment detailed in the exam- ples of the present invention, vaccinia virus was chosen as the expression vector. However, the invention is not limited to the use of vaccinia virus. As previously ex- plained, the expression vectors which can be used include, but are net limited to the following vectors or their de- rivatives; human er animal viruses such as, vaccinia vi- rus or adenoviruses; insect viruses such as baculoviruses; yeast vectors; bacteriophage vectors, and plasmid and cos- mid DNA vecters to name but a few.

In cases where an adenovirus is used as an express- jon vector, the LAV/HTLV III envelop gene is ligated te an adenovirus transeriptional/Translatiom control complex, e.

Key the late promoter and tripartite leader Bequences,

This chimeric gene is then inserted in the adenovirus ge- nome by in vitre or in vive recombination. Insertiom in a non-essential region of the viral genome (e.g., region

El or B3) will result in a recombinant virus that is via- ble and capable of expressing the LAV/HTLV III envelope related protein in infected hosts. Presently, there are two strains of adenovirus (types 4 and 7) approved and used as vaccinés fer military personnel, They are prime candidates for use as vecters to express LAV/HTLV

III envelop genes.

An alternative expression system which could be used te express the LAV/HTLV III glyceprotein is an in- sect system. In one such system, Autegrapha califernica nuclear pelyhedrosis virus (AcNPV) is used as a vecter to express fereign genes. The virus grews in Spodoptera frugiperda cells. The LAV/HTLV III envelope geme can be clened inte nen-essential regiens (for example the peoly- hedrin gene) eof the virus and are placed under contrel of an AcNPV prometer (for example the pelyhedrin prome- ter). Successful insertion of the LAV/HTLV III gene will result in inactivation of the pelyhedrin gene and preduct- jon of non-eccluded recombinant virus (i.e., virus lacking the proteinaceous ceat coded for by the polyherdin gene). 3 These recombinant viruses are then used to infect Spodop~ tera frugiperda cells in which the inserted gene is ex- pressed.

In addition, @ host cell strain may be chosen which modulates the expression ef the inserted sequences, or modifies and processes the chimeric gene product in the specific fashion desired. Expression from certain prome~- ters can be elevated in the presence of certain inducers,(e.g-y zinc and cadmium ions for metallothionein prometers).

Therefore, expression of the genetically engineered

LAV/HTLV I1I-env protein may be controlled. This is important if the protein product of the cloned foreign gene is lethal to host cells. Furthermore, modifica- tions (e.g., glycesylation) and processing ‘e.g. clea- vage) of protein preduce are important for the function of the protein. Different host cells have charactris- tic and specific mechanisms for the posttranslational proceasing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreigm pro- tein expressed.

In the particular embodiment detailed in the exam- ples of the present invention, we have ligated LAV- envelope coding sequences (LAV env sequences), both in its complete form and pertions thereof, to the 7.5K promoter of vaccinia virus te form chimeric genes in va- rious plasmids. The chimeric genes in these plasmids were flanked by additional vaccinia virus sequences homole- gous to the vaccinia virus TK gene. The construction of the chimeric gene involved the use of both natural and synthetic nucleotides encoding control signals for transcription and translation of the LAV env sequences.

The chimeric gene was tehn introduced into vaccinia virus

— expression vectors through in vive recombination between the homelogous TK region present on both the plasmid vector and vaccinia viral genome. These recombinant viruses containing the chimeric gene were used as expression vectors to produce LAV envelope- related proteins. 5.3. IDENTIFICATION OF RECOMBINANT EXPRESSION

VECTORS CAPABLE OF REPLICATING AND EXPRESSe

ING THE INSERTED GENE

Expression vectors containing foreign gene inserts can be identified by three general approaches: (a) DNA-

DNA hybridization, (b) presence or absence of "marker" gene functions, and (¢) expression of inserted sequences,

In the first approach, the presence of a foreign gene in- gerted in an expression vector can be detected by DNA-DNA hybridization using probes comprising sequences that are homologous to the foreign inserted gene. In the second approach, the recombinant vector/host system can be iden- tified and selected based upon the presence or absence of 20 . certain "marker" gene functions (e.ge, thymidine kinase activity, resistance to antibiotics, transfermation phene- type,ete.) caused by the insertion of foreign genes in the vector, ¥or example, if the LAV/HTLV 111 gene is insert- ed within the marker gene sequence of the vector, recom- binante containing the LAV/HTLV III insert can be identi-

fied by the absence of the marker gene function. In the third approach, recombinant expression vectors can be jdentified by assaying the foreign gene pro- duct expressed by the recombinant. Such assays can be based on the physical, immunolegical, or function- al properties of the gene product.

Once a particular recombinant DNA molecule ia identified and isolated, several methods may be used te prepogate it, depending on whether such as & re- combinant constitutes a self replicating unit (a re- plicon). A self replicating unit, e.g. plasmids, vi- ruses, cells etc., can multiply itself in the appre- priate cellulas environment and grewth conditioms. Re- combinants lacking a self-replicatimng unit will have to be integrated into a molecule having such a unit in order to be propagated. For example, certain plas- mid expression vectors upon introduction into a host cell need to be integrated into the cellular chrome- some to ensure propagation and stable expression ef the recombinant gene. Once a suitable host system and growth conditions are established, recombinant expression vecters can be propagated snd prepared in quantity.

In the particular embodiment of the invention de- tailed in the examples, the chimeric gene containing the

LAV envelope coding region was inserted into TK gene of the vaccinia virus genome, thereby converting the vi- rus into TK , i.e.,destroying the ability of the virus to make thymidine kinase. Such recombinants were se- lected by their ability to grow in media containing 5- bromo-deoxyuridine, a nucleotide analeg that is lethal to TK' cells but not TK cells. Recombinants were fur- ther identified by DNA-DNA hybridization, using LAV-env specific probes. TK~ recombinant virus was isolated by plaque-purification and stoks were prepared from infect- ed tissue culture cells.

Selte IDENTIFICATION AND PURIFICATION OF

THE EXPRESSED GENE PRODUCT

Pnce a recombinant which express the LAV/HTLV III gene is identified, the gene product should be analyzed.

This can be achieved by assays based on the physical, immunological or functional properties of the product.

Immunological abalysis is especially important because the ultimate goal is to use the gene products or recom- binant viruses that express such products in vaccine for- mulations and/or as antigens in diagnostic immunoassays.

A variety of antisera are available for analysing jmmunereactivity of the product, including but net limi- ted to serum derived from LAS or AIDS pateints and poly- valent antisera directdd against the LAV/HTLV III virus, the viral envelope protein and its analogs produced in bacterial system, or synthetic peptides containing antigenic determinents of the LAV/HTLV III envelope,

Identification of the peptides and proteins describ- ed in this invention is based on two requirements,

First, the LAV/HTLV III envelope-related protein should be produced only in recombinant virus infected cells. Second, the LAV/HTLV III envelopes related pro- tein should be immunorcactive to the serum of AIDS pa- tients er to a variety of antibodies directed against

LAV/HTLV III envelope protein or its analogs and deri- vatives.

The protein should be immunoremctive whether it results from the expression of the entire gene sequence, a portion ef the gene sequence or from two or more gene sequences which are ligated to direct the production ef fusion proteins. This reactivity may be demonstrated by stander immonulogical techniques, such as radio=-immuno~ precipitation, radioimmunelogical techniques, such as radio~immunoprecipitation, radioimmune competition, er immunoblots.

Once the LAV/HTLV envelope related protein is iden- tified, it may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, différential solubility, or by any other standard technique for the pumi-

fication of proteins.

Alternatively, once an immunereat¢tive LAV/HTLV

III related protein produced by a recombinant is iden- tified, the amino acid sequence of the immunoreactive protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant . As a result, the protein can be synthesized by standard che~ mical methods known in the art (e.g., see Hunkapiller, Mo et al., 1984, Nature 310: 105 -1I1).

In a particular embodiment of the present imvent=- jon such peptides, whether produced by recombinant DNA techniques or by chemical synthetic methods, include but are not limited to all or part of the amino acid sequence substantially as depicted in FIG. 2 including altered se- 15% quences in which functionally equivalent amino acid re- are substituted for residues within the sequence resulting in a silent change. For example, one er more amine acid : within the sequence can be substituted by another amino acid of a similar polarity which as a funct= jonal equivalent, resulting in a silent alteration. Subs@ tutes for an amine acid within the sequences may be gelect- } ed from other members of the class to which the amine acid belongs. For example, the non polar (hydrophobic) amine acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The pelar neutral - U0 = amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, The positively changed ( basic) amine acids include arginine, lysine and histidine. The negatively charged (acidic) amine acids include aspartic and glutamic acid. 5¢5. DETERMINATION OF THE IMMUNOPOTENCY

OF THE RECOMBINANT PRODUCT

Immunopotency of the LAVJHTLV III envelope rela- ted product can be determined by monitoring the immune response of test animals following immunization with the purified protein or synthetic peptide or protein.

In cases where the LAV/THLV III envelope related protein is expressed by an infectious recombinant virus, the recom- binant virus itself can be used to immunize test animals.

Test animals may include mice, rabbits, chimpanzees and eventually human subjects. Methods of introduction ef the immunogen may include intradermal, intramuscular, in- traperitoneal, intravenous, subcutaneous, fimdranasal or any other standard routes of immunization. The immune response of the test subjects can be analysed by three approaches: (4) the reactivity of the resultant immune serum to authen- tic LAV/HTLV III viral antigens, as assayed by known tech- niques, e.g., enzyme linked immunosorbent assay (ELISA), immunoblots radioimmuneoprecipitatiens, etc., (b) the abi- lity of the immune serum to neutralize LAV/HTLV III infect-

Cab ivity in vitro (Robert-Guroff, M., 1985, Nature 316: 72-74), and (¢) protection from LAV/HTLV III infect ion and/or attenuation of infectious symptoms in im- munized animals (Francis, D.P., 1984, Lancet 2:1276-1277

Gujdusek, D.C., 1985, Lancet 1:55-56).

S«6. FORMULATION OF A VACCINE

The purpose of this embodiment of the invention js to formulate a vaccine in which the immunogen is : related to an LAV/HTLV III epitope or a recombinant virus which expresses such an immunegen that protect against LAV/HTLV III virus infections for the prevent-

Co jon of LAS or AIDS. Additionally, multivalent vaccine formulations can be prepared in which the LAV/HTLV ZII gene preduct or a recombinant virus expressing the chi- meric gene product is used in combination with other immunogens for the prevention of LAS or IADS and other diseases. Examples of various formulations are discuss- ed below.

S.6.1. VIRAL VACCINE FORMULATIONS

Either a live recombinant viral vaccine or an in- activated recombinant viral vaccine can be formulated.

The choice depends upon the nature of the recombinant virus used recombinant virus is infectious to the host te be im munized but does not cause disease, a live vaccine is pre~ ferable because multiplication in the host leads to a pro- w hI?

longed stimulus of similar kind and magnitude to that occurring in natural subclinical infections and, therefore, confers substantial long-lasting immunity.

The infectious recombinant virus, upon introduction into a host animal, can express LAV/HTLV I11 envelope related proteins from its chimeric gene and thereby stimulate an immune response against LAV/HTLV 1II an- tigens. In case where such immune response is protective against subsequent LAV/HTLV IIT challenge, the live re- combinant virus by itself may be used as a preventative vaccine against AIDS virus infection. Production of such recombinant virus to be used in these formulations may in- volve both in vitro(e.g., tissue culture cells) and in vive (e.g., natural host animal -cow) systems. Yonvent- ional methods for the preparation and formulation of smallpex vaccine may be adapted for the formulation of live recombinant virus vaccine.

Multivalent live virus vaccines can be prepared . from a single or few infectious recombinant viruses that express epitopes of organisms that cause other diseases addition to the epitopes of LAV/HTLV III. For example, a vaccinia virus (which can accomodate approximately 35 kilobases of foreign DNA) can be engineered to contain cod- ing sequences for other epitopes in addition to those for

LAV/HTLV III; such as recombinant virus itself can be used as the immunogen in a multivalent vaccine. Alternative- ly, a mixture of vaccinia or other viruses, each capable of directing the expression of A different gene conding for different epitopes of LAV/HTLY III and/or other di- dease causing organisms can be formulated in a multiva- lent vaccine. whether or not the recombinant virus is infectious te the host to the immunized, an inactivated vaccine fore mulation may be prepared. Inactivated vaccines are "dead" in the sense that their jnfectivity has been destroyed,

Co : ‘usually by treatment with formaldehyde, -ldeally, the in- fectivity of the virus is destroyed without affecting the capsid or envelope proteins which carry the immunogenicity of the virus. In order to prepare jnactivated vaccines, large quantities of the recombinant virus must be grown in culture in order to provide the necessary quantity of rela- vant atigens. A mixture of inactivated viruses which ex- press different epitopes may be used for the formulation of "multivalent" vaccines. In some instances this may be pre- ferable to live vaccine formulations because of potential ’ difficulties with mutual interference of live viruses ad ministered together. In either case, the inactivated re- combinant virus or mixture of viruses should be formulated with a suitable adjuvant in order to enhance the immunele- gical response to their antigens. Suitable adjuvants ince - hh clude, but are not limited to, mineral gels, C.fejy aluminum hydroxide; surface active substances such as lysolecithin, pluronic polyols; polyanions;jpep~- tides: and oil emulsions.

Many methods may be used to intpoduce the vac- cine formulations described above; these include but are not limited to jntradermal, intramuscular, intra-~ peritoneal, intravenous, sucutaneous and intranasal routes. When a live recombinant virus vaccine formu- lation is used, it may be introduced via the natural

Cl B route of jnfection of the parent wild type virus which was used to make the recombinant virus in the vaccine formu- lation. 5.602. SUBUNIT VACCINE FORMULATIONS 13 IN an alternative to viral vaccines, the LAV/HTLV 111 envelope related protein itself may be used as an jmmunogen in subunit vaccine formulations, As previous- ly explained, subunit vaccines comprise solely the rele- vant imunegenic material necessary to immunize & host.

Accordingly, the LAV/HTLV III envelope related protein may be purified from recombinants that express the LAV/

HTLV III epitopes. Such recombinants include any of the previously described virus jnfected culture cells, bacterial transformants, er+yeast transformants that ex- press the LAV/HTLV III epitopes (see Sections 5y2y De3 and 5.4). In another embodiment of the present in- vention, the LAV/HTLV III related peptides or proteins may be chemically synthesized. To this end, the amine acid sequence of such a peptide or protein can be de- duced from the nucleotide sequence of the chimeric gene which directs its expression (see Section S.U).

Whether the immunogens are purified from recombi- nants or chemically synthesized, the final product may be adjusted to an appropriate concentration and formula- ted with any suitable vaccine adjuvant and packaged for use. Suitable adjuvants include, but are not limited to: mineral gels, e.g., aluminum hydroxide; surface active substance such as lysolecithin, pluronic polyols; poly- anions;peptides; and oil emulsions. The immunogen may also be incorporated into liposomes, or conjugated to polysaccharddes and/or other polymers for use in a vac~ } cine formulation.

In instances where the LAV/HTLV III related peptide or protein is a hapten, i.e., & molecule that is antige- pic in that it can react selectively with cognate anti- bodies, but not immunogenic in that it cannot elicit an immune response, the hapten may be covalently bound to a carrier or immunogenic molecule; for instance, & large protein such as protein serum albumin will confer immuno- genicity to the hapten coupled to it. The hapten-carrier may be formulated for use as a vaccine. 5.6.3 PASSIVE IMMUNITY AND ANTI-INDIOTYPIC

ANTIBODIES oo

Instead of actively immunizing with viral or su- bunit baccines, it is possible to confer short-term protection to a host by the administration of pre-form- ed antibody directed afainst an epitope of LAV/HTLV III.

Accordingly, the vaccine formulations described above can be used to produce antibodies for use in passive immunotherapy. Human immuneglobulin is preferred in human medicine because a heterologous immunoglobulin will provoke an immune response to its foreign immnmo- genic components. Such passive immunization could be used on an emergency basis for immediate protection of unimmunized individuals exposed to special risks, €.f., those exposed to contact with AIDS patients, for ins- tance, in hospitals and other health-care facilities.

Alternatively, these antibodies can be used in the pro- tection of antidiotypic antibedy, which in turn can be used as an antigen to stimulate an immune response against

LAV/HTLV 111 epitopes. 507 LUMUNOASSAYS

In an alternate embodiment of the present invent- jon, the LAV/HTLV III related peptides and proteins of the present jnvention may be used as antigens in immuno- eo Ih assays for the detection of antibodies to LAV/HTLV III in various patient tissues and body fluids as well as blood in blood banks and hospitals.

The antigens of the present invention may be used in any immunoassay system known in the art including but not limited radio immunoassays, ELISA assays, "sand- wich" assays, precipitim reactions, gel diffusion preci- pitin reactions, immunodiffusion assays, agglutination assays, complementfixation assays, immumpadiometrio as- says, fluorescent immunoassays, protein A immunoassays " and immunoelectrophoresis assays, to name but a few, 6. EXAMPLE

In the following examples, various plasmid vectors were constructed containing chimeric genes comrising LAV envelope coding sequences located downstream with respect to the transcriptional control sequences of vaccinia virus.

These chimeric genes containing the vaccinia promot-~ er and the LAV envelope coding sequence were inserted in- to the genome of vaccinia virus through in vive recombina- tion. Such recombinant viruses were identified and puri- fied, and viral stocks were prepared from infected tissue culture cells. Immunoreactive LAV envelope related pro- teins were shown to be produced by these recombinant vac- cinia viruses in vitro. These recombinations were tested in egperimental animals for their ability to elicit neutra- - LE =

1izing or protasctive immune responses and for their use a8 a vaccine against AIDS. A detailed descript- jon of each step in this embodiment of the invention is presented in the subsections below. ° 6.1 GENERAL PROCEDURES ' 6.1.1e CELLS AND VIRUSES

African green monkey kidney cells (strain BSC=- : 40, a continuous line of African Green Monkey Cells derived from BSC-1 cells, ATCC NO. CCL26) were ob- ' tained from R, Condit (Associate Professor, Department of Biochemistry, State University of New York, Buffale,

N.Y.) and were propagating in Dulbecco modified Eagle's medium (DMEM, Gibco, Grand Island, NY) supplemented with 108 fetal bovine serum and 100 units per ml each of penicillin and streptomycin, Human 143 TK™ cells (Phim, J.S. et ale, 1973, Intl J. Cancer 15: 23-29) were obtained from M. Botchan (* rofessor, Department of Molecular Biology, University of California, Berk- ley, Calif) and were propagated in the above medium with the addition of S-bromo-deoxyuridine (BUdR) at ug/ml. vaccinia virus (strain WR, ATCC No. VR-119) was obtained from R. Condit and was grown in BSC-40 cells in DMEM + 5% gamma globulin free calf serum + 100 unit/ 25 nl each penicillin and streptomycin. TK~ recombinants - ha were selected on 143 TK cells in the same medium containing 25 ug/ml of BUdR and plaque-purified on the same cell line in DMEM containing 1% Noble agar (DIFCO, Detroit, MICH), 5% gamma globulin free calf serum, 100 units/ml each of penicillin and strepto- mycin and 25 ug/ml BUdR. Dilutions of virus stocks were made in phosphate buffered saline (BS, per liter;

NaCl, 8 gm; KCl, 0.2 gm; NaH,PO,. 1.5 gm; K HPO, 0.2 gm) supplemented with 1 mM MgCl, and 0,01% bovine sdrum albumin (PBSAM). Co 6.1020 PREPARATION , - RESTRICTION AND

MODIFICATIONS OF DNA

Unless specified otherwise, all methods used for the following procedures were as described in the indicated pages of Maniatis et al., 1982, Molecular Cloning,

Cold Spring Harbor Laboratory; preparation of plasmid DNA (pp. 86-96), restriction digest of DNA (pp. 98-106) and purification of restriction fragments from low-melting temperature agarose gels (pp. 157-161 and p.170) ,react- jon conditions for the Klemow fragment E. coli DNA poly- nerase enzyme (pp.107-114), calf intestine alkaline phos- phatase (pp. 133-134) and ligase reaction (p.146), prepa- ration of nick translated probes (pp. 109-112) ard pro- cedures for DNA-DNA hybridization (pp.324=-325). - S50 =

6.2. CONSTRUCTION OF PLASMID VECTORS CONTAIN-

ING VACCINIA VIRUS PROMOTER LIGATED TO

THE CODING SEQUENCES OF LAV ENVELOPE GENE

The following susections describe the construct jon of various plasmid vectors containing coding sequences of the the 1,AV envelope gene preceeded by vaccinia virus transcriptional control sequences; these chimeric sequences are flanked by TK DNA. These recombinant plasmid vectors were later used to insert the 1.AV envelope coding sequences into the genome of vaccinia virus through in vivo recombina- tion.

In the subsections below, the LAV envelope coding sequence was purified from pRS-3 a subclone of lambda

J19 (Wain-Hobson, S. et.al., 1985, Cell 40:9) and in- serted into plasmid pGS20 (Mackett, M., Smith, G.L. and

Mess, B., 1984, J. Virol, hg, 857-864) downstream with respect to the vaccinia 7.5K prmoter contained inpGS20, in order to construct pv-envl, pv-env2, and pv-env5. In each of these constructions, the chimeric gene (i.e, the 7.5K vaccinia promoter ligated to the LAV specific nucleotide sequence) is flanked by vaccinia TK gene se- quences.

As previously repluimed, PRS-3 consists of a 3,840 base pair EcoRl to ssTI fragment of the LAV DNA insert contained in Lambda J19 cloned jnto the EcoRI and Sstl site of pUC18. It was obtained by cloning the Sstl restriction fragment of J19 into the Sstl site of pUC18 to create pBT-1 which was then digested with

EcoRI and re-ligated. This DNA was used to trans- form E. coli and plasmid pRS-3 was isolated. The

LAV specific DNA contained in pRS-3 is from the EcoRI site located at nucleotide 5289 to the Sstl site leo- cated at nucleotide 9129 on the LAV genome (Wain-

Hobson, S. et.al., 1985, Cell 40:9); see Figs. 2¢ 6.2.1e CONSTRUCTION OF PLASMID VECTORS

CONTAINING VACCINIA VIRUS PROMOTER

LIGATED TO THE 3' CODING SEQUENCES ' OF LAV ENV GENE

Five micrograms of pRS-3 plasmid DNA was di- gested to completion with restriction enzyme Kpa I and the resulting fragments were resoved on a 1% loe- melting temperature agarose gel. A 2.68 Kbp (Kiloba- se pair) fragment was isolated and purified. This fragment contained LAV= specific DNA from nucleotide pumber 5889 to 8572 including sequences (5889-8349) that encode the C-terminal portion of the LAV envelope protein.

One microgram of this fragment was mixed with 0.5 ug of pGS20 DNA which was previously linearized with "restriction enzyme BamHI. Ligatien of these two fragments was allowed to proceed for 2h hours at 4°C in the pre- gence of oligodeoxynucleotide linkers consisting of 0.6 got 51 GATCCACCATGGTAC-3'-OR and 0.3 ug of §1-CATGGTC~3"

-OH. These linkers served (a) to convert the Kpnl cohesive ends of the 2.68 Kbp fragment derived from pRS~3 plasmid DNA to BamHI cohesive ends which are complementary to the BamHI cohesive ends of the cleav- ed pGS20 DNA and (b) to provide a translation initia- tion sequence (ATG) in the correct reading frame with respect to the LAV envelope gene sequence asp well as nucleotide sequences required for efficient translation of the transcribed mRNA. The ligation mixture was used to transform Ek. coli strain MC100OQ. Plasmid DNA from ampicillin resistant tranformants was tested for the orientation of the insert and the regeneration of BamHI,

Ncol and Kpnl sites at the ligation junctions. The con- firmed structure of the desired plasmid, pv-env2, is shown in FIG. 3 in which the carboxyl coding portion of the LAV envelope gene, corresponding to nucleotide numbers 5889- 8572 (as shown in Fig. 2), is located downstream with res- pect to the 7.5K vaccine promoter. The LAV envelope se- quence ims positioned in the correct reading frame with respect to the initiation ATG supplied by the linker DNA. 6.2.2 CONSTRUCTION OF PLASMID VECTORS

CONTAINING VACCINIA VIRUS PROMOTER

LIGATED TC THE 5! CODING SEQUENCE

Five micrograms of pRS=3 plasmid DNs was digested to completion with Avall restriction enzyme and the re- sulting fragments were resolved on a 1% low-melting tenm=~ oo =z —

perature agarose gel. A 0.82 Kbp fragment was isolated and purified. Tjis fragment contained LAV-specifie sequences from nucleotide number 5671 to 6490 ( as shown in Fig. 2). This includes sequences (nucleotide numbers 5766-6490) encoding the N-terminal portion of the envelope protein and 95 base pairs of 5' proximal un- translated sequences of LAV. This fragment was treated with the Klenow fragment E. coli DNA polymerase in the presence of excess deoxyribonucleotide triphosphates. The resulting blunt-ended fragment was ligated to 0.5 ug of

PGS 20 DNA which was previously linearized with Smal and treated with calf-intestine alkaline phosphatase (CIAP).

Ligation was allowed to proceed for 16 hours at 12°C .The ligation mixture was used to transform E.Coli strain MC1000.

Plasmid DNA from ampieillia resistant transformants was tested for the orientation of the LAV insert with respect to the vaccinia virus transcriptional control. sequences,

The confirmed structure for the desired plasmid, pv-enl, is shown in Fig. 4 in which the amino coding portion of the LAV envelope gene containing its own initiation ATG, corresponding to nucleotide numbers 5671 to 6490 (as shown in Fig. 2), is located downstream with respect to the vac- cinia 7.5K promoter, - S54 =

6e2e3. CONSTRUCTION OF PLASMID VECTORS

CONTAINING VACCINIA VIRUS PROMOTER LIGA-

TED TC THE ENTTRS CODING SEGUENCES OF

LAV ENVELOPE GENE

Two micrograms of pv-envl plasmid DNA was digest- ed to completion by restriction enzyme Stul Pvul, and ¥phol. The resulting fragments were resolved on a 1% low- melting temperature agarose gel. A 4 kbp fragment con- taining the vaccinia virus transcriptional control ele~ ment and the 5' portion of the LAV envelope coding sequences was isolated and purified. This fragment was ligated to a 6.5 kbp fragment generated by Stul and Fvul restriction di- gests of pv-env2. The ligation mixture was used to trans- form E. coli strain MC1000. Ampicillin-resistant transform- ants were selected. Flasmid DNA from individual transfor- mants was tested for the regeneration of Stul and Pvul res- triction sites and the presence of the parenteral 4 Kbp gnd 6.5 kbp fragments. T,e desired plasmid, pv-env5, depicted in Figs 4 contains the envelope gene of LAV corresponding to nucleotide numbers 5766 to 8349 (as shown in Fig. 2),41i- gated downstream from the vaccinia virus transcriptional con- trol elements. 6e3. CONSTRUCTICN OF RECOMBINANT VACCINIA

VIRUS CONTAINING CHIMERIC LAV.ENV GENE

Insertion of the chimeric LAV-env sequences into the 5 vaccinia virus genome is achieved by in vivo recombination, made possible by the inet that the chimeric genes in plas=-

mids pv-env2 and pv-env5 are flanked by vaccinia vi- rus sequences coding for the thymidine kinase (TK) gene. Introduction of these plasmids into cells in- fected with vaccinia virus allowed recombination to occur between the TK sequences on the plasmid and the homologous sequences in the vaccinia viral genome.

Insertion of the chimeric Bene occurs as the result of double recombinations in the flanking sequences. Such recombinants will have the chimeric gene inserted in the vaccinia TK gene and, consequently, will be pheno- typically TK~. These TK recombinants can be selected for growth in medium supplemented with BUdR which is lethal . to TK cells but not to TK~ cells. The general principle of this procedure has been described (Mackett, M., Smith,

G.L. and Moss, B., 1984, J. Virol. 49, 857-864).

A 100 mm dish of 80% confluent African Green Monkey

Kidney Cells (strain BSC-40) were infected with vaccinia virus (strain WR) at the multiplicity of infection (moi) of 0.05. After 2 to 4 hours of incubation at 37°C, in- fected cells were overlaid with calcium phosphate coprecipi=~ tates of plasmid pv-env2 or pv-env5 DNA. The precipitates were prepared by adding 0.5 ml of 2XDNA-CaCl, solution drop- wise to 0.5 ml of 2XHeBS solutions (2XDNA-CaCl, solution contains 20 jug of plasmid DNA in 0.5 ml of 0.25 M CaCl,3 2XHeBS contains, per ml, 16mg of NaCl, 0.74 mg of KCl, 0.25 mg of Na HPO, 2H ,0, 2 mg of dextrose, and 10 mg of HZPES, at pH 7.08. DNAi-calcium phosphate co-precipitates were allowed to form at room temperature for 30 minutes). Four after the overlay of precipitates, cells were washed once with 1XHeBs, incubated at 37°¢ for 3 minutes in the pre- sence of 2 ml of 15% glycerol in 1XHeBs, and then washed once more with lXHeBs and incubated with 10 ml of growth medium (DMEM + 10% fetal calf serum + 100 unit{ml each penicillin and streptomycin). Two days later, infected cells were harvested and collected by centrifugation (°c, 10 minutes at 2000 x g)e. Virus stocks were prepared by resuspending these cells in 1 ml of PBSM, followed by two cycles of freezing and thawing and three 15 second somicationse.

Recombinants were selected by plating 0.1 ml of 1077 dilution of the viral stocks from above on 60 mm dishes of confluent human 143 TK~ cells, overlaid with Sml/dish of confluent human 143 TK™ cells, overlaid with 5ml/dish of 1% Novel agar (Difco, Detroit, MICH) 5% calf serum,25 yue/m BU4R in DMEM. Two days after platingg cells were stained by overlaying 2 ml/dish of agar-medium containing the same ingredients as above, plus 0.01% neutral red. ln- dividual plagues were picked one day after staining, re- suspended in 0.5 ml PBSAM, and aliquots (0.25 ml) of vi- rus suspensions were used to infect confluent 143 TK cells

— ee TT seeded in 16 mm diameter wells under selective medium (GMEM + 10% calf serum + 100 units/ml each streptomycin and penicillin + 25 ug/ml BUdR. Infected cells were collected by centrifugation and resuspended in 100 jut. of PBS containing 0.5 mg/ml of trypsin and 0.2 mg/ml of EDTA. Ce Ls were lysed by incubation at 37% for 30 minutes, followed by 3 cycles of sonication, 20 se- conds each, Cell lysates were collected on nitrocellulose filters by use of a multi-well filtering manifold (Schlei-~ cher and Schuell, Arlington, MA). The presence of LAV-env specific DNA sequences in these samples was determined by

DNA-DNA hybridization as described (Mackett, M., Smith, G.

L. and Moss, B. 1982, Froc, Nat'l, Acad, Scie 79, 7415=- 7419). 32p_1abeled Plasmid pRS~3 DNA prepared by nick- translation was used as a pybridization probe. Recombin- nants that gave positive hybridization to this probe were further plaque.purified twice on 143 TK~ cells under se- jective conditions (medium containing BUdR) and once on

BSC~-U40 cells under non-selective conditions. After confir- mation by DNA-DNA hybridization, virus stocks were prepared from the thrice purified plaques on BSC.4O cells and used for subsequent characterisation.

A schematic representation of the construction of the recombinant viruses is shown in Fig. 5 in which the

LAV envelope gene sequence (env) located downstream from the vaccinia 7.5K promoter (p ) is flanked by TK DNA se-~ quences within the vaccinia genome. Virus stocks de- rived from recombination between vaccinia virus ge- nome and plasmid pv-envS5 were designated v-envS5 and contain the entire LAV envelope gene. In the particu- lar embodiment described in the examples herein, v-env5 contains the entire envelope gene as well as 96 basepairs of the %'-proximal and 223 basepairs 6f the 3'-proximal untranslated sequences. Virus stocks derived from pv- env? were designated v-env2 ~nd contain most of the LAV envelope gene (i.e., the KpnI fragment) but lack that part of the sequence which encodes the first 42 amino acids of the LAV envelope protein. Since the presumed signal se- quence of the LAV envelope protein is located within the first 49 amino acids of the protein vwenv2 should produce 15% a protein that lacks the signal sequence; therefore, one would expect that the LAV related protein produced by this recombinant virus will not be transported to the membrane.

By contrast, V-env5, which contains the complete LAV env sequence should produce a protein that is transported to the membrane, 6.4. EXPRESSION OF LAV ENVELOPE RELATED PROTEINS

IN TISSUE CULTURE CELLS INFECTED BY RECOMBI-

The recombinant vaccinia viruses carrying the chime~ ric LAV-env genes were shown to be capable of exiressing

LAV envelope related proteins upon infection of cells in tissue culture, These proteins were glso found to be immu-

‘ noreactive with serum from AIDS Patients. 6eltels IDENTIFICATION OF LAV ENVELOPE RELATED

PROTEINS EXPRESSED IN CELLS INFECTED

WITH RECOMBINANT VACCINIA VIRUS USING

IMMUNOBLOTTING TECHNIQUES

A 100 mm dish of confluent BSCU4O cells was in- fected at a moi of 10 by wild type vaccinia virus or with its recombinant derivatives, v-env5 or v-env2. In- fection was allowed to proceed for 12 hours, at which time the cells were harvested, washed once with PBS,and collected by centrifugation. Infected cell pellets were resuspended in 1 ml of Laemmli sample buffer (Laemmli U.K., 1970, Nature 227:680) and lysed by boiling for 4 minutes.

Total cellular protein, in a 75 uk aliquot of cell lysate was resolved by electrophoresis on a 7-15% gra- dient SDS-polyacrylamide gel. A sample of purified LAV virion and an aliquot of mock infected cell lysate were included as controls. The contents of the gel were elec- trotrnasferred to a sheet of nitrocellulose filter. The filter was first incubated in 5 ml PBS + 5% non-fat dry milk for 30 minutes at room temperature and then for 2 hours at room temperatur in PBS ¥ 5% non-fat fry milk + human serum from AIDS patients (1:100 dilution of heat jpnactivated serum), The filter was then washed 5 times with PBS + 0.05% Tween 20 (polyoxyethylene sorbitan monolaurate) and once wit. PBS alone. The washed fil- ter was incubated for 2 hours at room temperature with ml of PBS containing 1% normal goat serum (heat in- activated) plus a 1:3000 dilution of goat anti-human 5 IgG~horseradish peroxides congugate. The same filter was again washed 5 times with PRS + 0.05% Tween 20, and once with TBS (0.5 M NaCl + 20 mM Tris-HCl, pH 745)

Horseradish peroxidese conjugate bound on the filter was detected by reacting chloro-naphthol coloring re- agents with the filter for 10 minutes at room temperature in the dark (chloronaphthol coloring reagent was prepared by mixing solutions A and B just prior to use. Solution

A: 20 ml cold 30% methanol + 60 mg h chloro-l-naphthol; solution B: 60 nt cold 30% hydrogen peroxide + 100 ml

TBS). Proteins bound on the filter that reacted with anti- bodies in AIDS patient serum would be detected by the co- loring reagent through its binding to the goat anti-human

IgG-horseradish peroxidase conjugates.

Results of this analysis, as shown in Fig. 6A, in~- dicated that vaccinia virus recombinant v-env5 produced a family of three proteins that were immunoreactive specifi- cally with serum from AIDS patients. These proteins had similar electrophoretic mobilities as the authentic LAV/

HTLV III glycoproteins gpl50, gpll0, and gphl, which are believed to be encoded by the env gene (Robey, E.G, et al.,

1985, Science 228; 593-595). These proteins were not produced is mock infected or wild-type vaccinia virus infected cells. Recombinant v-env2, which lacks the St proximal sequences that code for the presumed initiat- ing methionine and the first 42 amino acids of LAV en- velope proteins, produced a protein of truncated size, but still immunoreactive with AIDS patient gerum. Fre- sumably, translation of the env sequence in recombinant v-env? initiates from the AUG codon, trans€ribed from the linker sequence used in the construction of this recombi- nant (gee Section 6.2.1.)

In a second experiment, two cell line (BSC-40 and

Hel) were infected with v-envS and v-env2. The LAV speci- fic proteins in the infected cell lysates were assayed by

Western immunoblots as described velow.

Confluent monlysate of BSC-40 or HeLa cells were in. fected with recombinant viruses v-env3 or vQenv2 at a mul- tiplicity of infection (moi) of 50 plaque -forming units (pfu) per cell. Twelve hours after infection cells were washed twice in phosphate-~buffered saline, resuspended in

Laemmli sample buffer and boiled for 5 minutes. Proteins from infected cells were resolved by sodium dodecyl sul- fatepolyacrylamide gel electrophoresis (SDS- PAGE) ,elec- trotransferred to nitrocellulose membrane, and reacted with pooled serum from LAV/HTLV-III seropositive indi-=-

viduals. Imnunoreactive proteins were detected by pro- teins A, which was labeled with 125 by the chloramine

T method. In Fig. 6B, lanes 1 and 5 contain proteins from mock-infected cells; lancs 2 and 6, from cells in- fected with wild-type vaccinia virus; lanes 3 and 7, from v-env5 infected cells; and lanes 4 and 8, from v-env? infected cells. Sucrose-gradient purified LAV virion proteins were used as control (LAV) and the posi- tions of envelope proteins gpl50, gpllO and gpl wers as indicated. Molecular weight standards were expressed in kilodaltons (Kd).

Three major proteins immunoreactive with pooled serum from seropositive individuals were detected in the v-envS infected cells (Fife 6B, lane 3 and 7). The molecular weights of these proteins were estimated to be 150 Kg, 120 Kd and 41 Kd, similar to those of LAV envelope glycoproteins gpl50, gpllO and gpll.

Recombinant virus v-env2 lacked the putative signal sequence for LAV env, but was able to produce at least three immunoreactive polypentides of molecular weights 99

Kd, 68 Kd and 40 Kd (Fig. 6B, lanes 4 and 8).

6.4.2. IDENTIFICATION OF LAV ENVELOPE RELATED

PROTEINS EXPRESSED IN CELLS INFECTED WITH

RECOMBINANT VACCINIA VIRUS USING IMMUNO-

PRECIPITATION TECHNIQUES

The immunoprecipitation assay described below de- monstrated that cells infected with the recombinant vac- cinia viruses of the present invention synthesize proteins that are immunoreactive specifically with serum from AIDS patients.

A 100 mm dish of confluent BSC-40 cells was infect- ‘ 10 ed by type vaccinia virus, or its recombinants v-env5 or veenv2 at a moi of 10. At 9.5 hours post infections, growth medium was replaced by methionine-free DMEM with no serum supplements, At 10 hours post infection, the media was re- placed by 2 ml of methionine-free DMEM containing 100 juc1/ ml of />%s 7- methionine and labeling was allowed to pro- ceed for 2 hours at 32%. At the end of the labeling pe- riod, cells were washed once with PBS and collected by centrifugation. Cell pellets were resuspended in 1 ml of lysis buffer; 1% NP 40 (polyoxyoxyethylene (9) p-tert- octylphenol), 0.5% sodium deoxycholate, 0.1 M NaCl, 0.01 M

Tris-HEl, pH 7.4, lmM EDTA and the lysate was cleared by centrifugation for 1 minute in an Eppendorf microcentri- fuge.

Immunoprecipitation was carried out by adding 5 pr of heat inactivated human serum, either from a control po-

pulation or from AIDS patients, to 100 nb. of cell ly- sate. After 1 hour incubation at 4°, 60 ul. of acti- vated Staphylococcus aureus cells (Pansorbin cells,

Calbiochem Behring Corp., La Jolla, CA) was added and incubation was allowed to continue for another hour at 4°c. Immunoprecipitation complexes were collected by centrifugation for 30 seconds in an kppendorf microcen- trifuge at 4°c and washed once in 1 M NaCl + 0.1% NP 40 + 0,01 M Tris-HCl, pH 7.4 and twice in RIPA buffer (10 mM Tris-H Cl, pH 7.2, 0.15 M NaBl, 1% sodium deoxycho- late, 1% Triton-X 100 (polyoxyethylene (9-10) p-tert- octylphenol), 0.1% sodium lauryl sulfate). Washed im- munoprecipitates were resuspended in 50 ul. of Laemmli sample buffer, boiled for 1 minute and centrifuged for 1 minute in an bppendorf microcentrifuge. Immunopreci- pitated proteins present in the supernatant were analysed by electrophoresis on 15% SDS-polyacrylamide gels. After electrophoresis, the gels were stained with Coomassie blue dye, treated with sodium salicylate (30 minutes at room temperature in 1M sodium salicylate) and dried for fluo- rographye.

Results of this analysis, as shown in Fig. 7A, in- dicated that recombinant v-env5 synthesized a family of proteins immunoreactive specifically with AIDS pateints serum. These proteins had apparent molecular weights of

160,140, 120,42 and 40 kilodaltons (Kgq), correspond- ing approximately with the apparent molecular weights of the envelope related glycoproteins reported for LAV/

HTLV III (Robey, W.G., et al., 1985, Science 228: 593- 595), These proteins were not present in mock infected of wild-type vaccinia virus infected cells, nor were they : jmmunoprecipitated by control human serum. In v-env2 in- fected cells, a truncated protein of an apparent molecu- lar weight 95 Kd was produced and recognized by AIDS pa- tients serum. This truncated form of LAV-env related pro- teins is most likely initiated from the AUG codon encoded by the linker sequence jocated 5' proximal to the LAV in- gert in this recombinant. 6.4030 > H-GLUCOSAMINE LABELLING OF LAV

ENVELOPE-RELATED PROTEINS PRODUCED BY

VACCINIA-LAV RECOMBINANT VIRUSES

The radioimmunoprecipitation assay described velow indicated that the vaccinia-LAV recombinant viruses of the present jnvention produced glycosylated envelope proteins.

Hela cells were either mock infected (see Fige 7B lanes 1 and 5), or infected separately by wild-type vacci- nia virus (1anes 2 and 6), recombinants v-env5 (lane 3 and 7) or v-env2 (lanes 4 and 8), all at an moi of 50 pfu/ cells. >4-glucosamine (0.25 uc1 at 23 mCl/mg, Amersham) : was added to culture medium from 4 to 16 hours after infect-

jon. Cells were washed twice with phosphate-buffered saline and lysed in buffer containing 0.1 M NaCl, 0.01

M Tris-HCl pH 7.ly 1 mM EDTA, 1% NF-40 (polyoxyethylene (9) p-tert-octylphenol) and 0.5% sodium deoxycholate,

Aliquots of cell lysates were mixed with either normal human serum (lanes 1-4) or pooled serum from LAV/HTLV-

III seropositive individuals (lanes 5-8). Immunoreact- ive proteins were precipitated by fixed Staphylococcus aureus cells bearing protein A, resolved by SDS-PAGE and detected by fluorography.

The results of the radioisoptic labeling with glu- cosamine as shown in Fig. 7B lane 7 indicated that the v- env5 recombinant-made proteins, like the envelope proteins of LAV, were also glycosylated. Differences in the glyco- sylation patterns could account for the slight variations observed in the electrophoretic mobilities of recombinant- made proteins as compared to LAV virion glycoproteins.

As would be expected from proteins lacking signal peptides, no N-linked glycosylation with J4-glucosamine was observed in v-env? infected cells (Fig. 7B and 8).

Go lili, PULSE-CHASE IMMUNOPRECIPITATION ANALYSIS

OF LAV ENVELOPE =-RELATED PROTEINS PRODUCED

BY VACCINIA-LAV RECOMBINANT VIRUSES

It has been suggested that gpl50 of LAV is the a5 precursor from which an exterior protein gp 110 a trans-

membrane protein gphl were derived. The "pulse-chase" immunoprecipitation assay described below, indicate that the vaccinia-LAV recombinant -made 150 Kd, 120 Kd and bh

Kd proteins have a precursor-product relationship similar to that suggested for gp150, gpllO and gphl of authetic

LAV/HTLV-III. . Confluent monolayers of HeLa cells were infected with wild-type vaccinia virus (gee Fig. 7C, lanes 1-6), recombinant v-env 5 (lanes 7-12), or v-env2 (lanes 13-18), all at a moi of 50 pfu/cell., At 10.5 hours post-infect- ion, cells were labeled with 355_methionine (greater than 800 Ci/mmol, Amersham) at 100 uCi/ml for 15 minutes. At the end of the labeling period, cells were washed once 2 ml of prewarmed chase-medium (Dylbecco modified Eagle's medium + 3 mg/ml L_methionine + 5% calls serum + 100 units/ ml penicillin and 100 ug/ml streptomycin) and re-fed with 1 ml of the same medium before being returned to the in- cubator. At various times afterwards, cells were washed and lysed as previously described in gection 6.4.3, and pro- teins from cell lysates were jmmunoprecipitated with pooled serum from LAV/HTLV-III seropositive individaals. Immuno- precipitated proteins were resolved by SDS-PAGE and detect- ed by fluororarhy. The duration of each chase as follows: 0 hours (janes 1,7,13); 0.5 hours (1anes 2,8,14)3 1 hour (janes 3,9,15); 2 hours (1anes 14,10,16); 6 hours (lanes 5, 11,17) and 12 hours (1anes 6.12,18). Results shown in Figo 7C, lanes 7-12 indicated the same precursor-product rela- _ GR a tionship for the recombinant-made 150 Kd, 120 Kd and 41 proteins as the avthetic LAV/HTLV III gpl50,gpllO and pghtl. The processing of the 150 Kd protein appeared to be slow and inefficient in Hela cells, since by 6 hours after 5S the pulse-labeling less than 50% of the radioactivity in the 150 Kd protein was chased into 120 Kd and 41 Kd pro- teins. Preliminary results indicated this processing is more efficient in certain types of human peripheral blood cells infected with the same recombinant virus. These ex- periments also indicated that the env sequence in v-env? which lacked the putative signal sequence for LAV-env was expressed as an unmodified 87 Kd precursor (780 amino acids), which was processed to a 99 Kd intermediate (Fig. 7C, lanes 13-18). 6.4.5. PRESENCE OF LAV ENVELOPE-RELATED PROTEIN

IN THE GROWTH MEDIUM OF CELLS INFECTED

WITH VACCINIA-LAV RECOMBINANT VIRUSES fhe radioimmunoprecipitation assay described below demonstrated that the immunoreactive proteins produced by the recombinant vaccinia virus of the invention can be ex- pressed and processed in a pattern similar to that of the authentic LAV envelope glycprotein.

Confluent monolayers of Hela cells were either mock infected (see Fig. 7D, lane 1), or infected separately by wild -type vaccinia virus (lane 2), recombinants v-env5 (lane 3) or v-env? (lane 4}, all at an moi of 20 pfufcell,

Cells were labeled with 355 methionine (greater than 800

C1/mmole, “mersham) at 100 uCi/ml from 10 to 12 hours after infection. #t the end of the labeling period, medium was removed and clarified by centrifugation for 2 minutes at 12,000 x g before use for immunoprecipita~- tion by pooled serum from LAV/HTLV III seropositive indi- viduals. Proteins from infected cells immunoprecipita- ted by the same serum were shown in the panel labeled "pellet" and those from the medium in the panel labeled "Supe'',

As would have been expected for gp 110 of LAV, the : recombinant-made 120 Kd protein was also detected in in- fected cell medium (Fig. 7D, lane 3). These results de- monstrated that the recombinant virus v-env5 was able to express the LAV env gene and to produce immunoreactive pro- teins that were processed in a patter similar to authentic

LAV envelope glycoproteins.

As would have been expected for proteins lacking signal peptides, no LAV envelope -related polypeptide was de- tected in the v-env2 infected cell medium (Fig. 7D, lame by, 6.5 IMMUNOPOTENCY OF VACCINIA LAV ENV RECOMBINANT

VIRUS

The recombinant viruses carrying chimeric LAV-env gene were shown to be capable of eliciting antibody res- ponse against LAV in two gtrains of mice and one species of sub-human primate.

6e5e1e 1MIUNOGLNICITY CF VACCINIALLAV

NV RuCCMBINANTS IN MICE

The immonugenicity of proteins expressed by re- combinant vaccinia viruses v-onv) and v-envy was exa- mined. The experiments outlined below indicate the abi-~ 1ity of these recombinant viruses to elicit an immune response to all major glycoproteins of LAV.

Two strains of mice, one inbred (C57B16J) and one outbred (ICR) were jnoculated with recombinant viruses v- env? or v-envS. All animals were 5-7 weeks old at the time of jnoculation. Four routes of inoculation were : used: footpad, tail scarificaticn, intranasal and intra- peritoneal. Footpad inoculatinn was done py injecting 25

ML (5 x 10° pfu) of recombinant viruses into and of the rear footpads. Tail scarification was done by roughing up the skin at the base of the tail with a bifurcated needle and applying 10 jul (2 = 107 pfu) of recombinant viruses onto the scarified surface. Intranasal inoculation was done by placing 10 jul (2 x 107 pfu) of recombinant vi- ruses on the nonese of the mice and allowing the mice to breathe én the inoculum.

Intraperitoneal injecticns were done by injecting 10 ul (2 x 107 pfu) of recombinant viruses into the peritoneal cavity of the mice. A11 viral stocks and diluents were made as described in section S.1.1e Serum samples from in- dividual mice were collected at two-week intervals after inocu-

lation and analyzed by both enzyme linked immunosorbant assay (ELISA) and Western blot analysis as described be=- lowe. 6.5.1.1. SEROCONVERSION OF MICE IMMUNIZED

WITH VACCINIA-LAV ENV RECOMBINANTS

AS DEMONSTRATED BY ELISA

ELISA DATA om the 6-weeks serum samples are summa- rized in Table 1. Recombinants v-env? and v-envS sero- ’ converted 100% and 95% of the C57B16J mice, respectively.

For ICR mice, the rate of sero-conversion was B4¥% for v- . : env2 and 100% for v-env5. Total sero-conversion as well Co as the highest average ELISA titer was cb8ained in mice im- munized by tail scarification.

LIABLE I

Seroconversion of Mice Inoculated with

Recombinant Viruses Carrying Chimeric LAV-env Gene } Recombinant Route of Seroconvenrsion of Mice } Virus Inoculation Inoculation *

ICR C57B16J v-env2 Footpad 1/3 4/5

Tail Scarification 3/3 L/s

Intranasal 0/3 b/s

Itraperitoneal Not done 3/5 v-env5 Footpad L/h 5/5

Tail Scarification 3/3 5/5

Intranasal 3/3 l/s

Intraperitoneal not done 5/5

* Seroconversicn, determined by ELISA as described in the accompanying text, is expressed as the num- ber of mice sercconverted sver the total number of __ mice inoculated within each group,

The KLISA data was generated as follows: Furi- fied, inactivated 1.AV virions were diluted in carbonate buffer (50 nM sodinm carbonate, pH 9.6) and added to 96 well microtiter plates (100 ul/well containing 0.2 ug of inactivated LAV). Binding was allowed to proceed at 4°¢ overnight. Unbound protein was aspirated and washed first with 200 ul/well of PBS + 5% non-fat dry milk and then with 300 ul/well of 4% sucrose solution. After ex- cess sucrose solution was aspirated off, plates were al- lowed to dry at room temperature (hour), Then, 50ul of

PBS containing 2.5 ul of méuse serum samples was added to each well and allowed to react at 37% for 1 hour. At the end of the incubation period, wells were washed 5 times with FBS + 0.0% Tween-?0 (polyoxyethylene sorbi- tan monolaurate). Fifty ul of goat anti-mouse horseradish peroxidase conjugates (3:5000 dilution in PRS + 0.05%

Tween-20 was added to each well and allowed to react at 37°C for 45 minutes. After 5 washes with PBS + 0.05%

Tween-20, 100 ul of ¢itrate-O-phenylenediamine-peroxide substrate was added. The substrate was made s follows: 0.294 g sodium dihydrate + 0.537 g sodium phosphate dia- basic crystal were dissolved in 10 ml B,0 and adjusted to pH 5.0 with HCl; 2 ul of 30% HO, and 4 mg of O-phenyl- ene-diamine were added just before use. Plates were in- cubated 30 minutes in the dark at roomtemperature. The reaction was stopped by adding 100 ul Of 1.3N sulfuric acid to each well and the optical absorbance of the re- action mixture at 490 nM was measureds

Results in Table I are expressed as ratios of sero- positive mice to the total mice inoculated. Serum samples were collected 6 weeks after inoculation and analyzed by

ELISA. Serum samples from 5 uninoculated mice were used as controls. The mean ELISA titers for the conyrol group were 0.021 + 0.005 and 0.017 + 0.010, for ICR and C57B15J mice, respectively. Serum samples from immunized mice that gave ELISA titers more than three standard deviations high- er than the control group were scored as positive. : 6e5e1e20 SEROCONVERTED MICE PRODUCE ANTIBODIES

AGAINST AUTHENTIC LAV ENVELOPE GLYCO-

PROTEINSAS DEMONSTRATED BY WESTERN IM~

MUNOBLOT ASSAY

To demonstrate that mice inoculated with the recom- binant viruses produced antibodies against authentic LAV envelope glycoproteins, serum samples from immunized mice were analyzed by a Western immunoblot technique as follows: five to seven week old male inbred mice (c57B16J, Jackson

Laboratory) were jmmunized by tail gcarification, which consisted of applying a 10 ul inoculum containing 2 x 107 pfu of recombiant vaccinia virus to abrasions generat- ed with a bifurcated needle at the base of the tail.

Animals were bled from the retro-orbital sinus at 8 weeks post inoculation and serum maintained frozen until use. Aliquots of serum samples diluted 50-fold in phos- phate-buffered saline plus 0.2% NP-40 and 3% nonmsfat dry milk were reacted with LAV virion proteins which had been resolved by SDS-FAGE and immobilized on nitrocellulose filters by electro-transfer. LAV proteins recognized by these sera were detected by goat anti-mouse immunoglobulin conjugated with alkaline phosphatase.

Results of the 8-week serum samples C57B16J mice inoculated with the recombinant viruses by tail scarifica= tion are shown in Fig. 8. All animals immunized with the recombinant viruses produced antibodies that reacted with

LAV envelope glycoprotein gpl. Serum from some of the ani- mals immunized with v-env5 (Yig. 8, lane a) also recognized gpl50 and gpllO, indicating the ability of this recombinant virus to elicite an immune response to all major glycopro- teins of LAV/HTLV-III. 6.5020 1MMUNOGENICITY OF VACCINIA-LAV

RECOMBINANT V-ENVS5 IN SUB-HUMAN PRIMATES

Five long-tailed macaques (Macaca fascicularis), of juvenile to adult age, were used to study the immuno- genicity of vaccinia-LAV recombinant v-envS. All animals

SSS ——————— were pre-screened for the absence of antibodies to vac- cinia virus and LAV and the absence of simian T-lympho~- tropic or simian AIDS viruses or antibodies to these vi- ruses. Four monkeys were jnoculated with 2 x 108 pfu of v-env5 and one with a 107 pfu of a control recombinant vaccinia virus (a vaccinia-herpes simplex gDI recombinant v-HSgD1l), all by skin gcarifieation. Serum and whole blood samples were collected prior to inoculation and at 3,4 and 6 weeks post-inoculation. All animals showed self-healing skin lessions typical of vaccinia infections and normal phy- giological indicators throughout the course of experiment,

Humoral and cell-mediated immune responses were analyzed by a Western immunoblot assay and proliferation assays, res-= pectively. 6e5+2.1s HUMORAL IMMUNE RESPONSE IN MACAQUE MONKEYS

AS DEMONSTRATED BY WESTERN IMMUNOBLOT ASSAY

Serum samples were collected previous to inoculation and at 3, 4, and 6 weeks post-inoculation. Aliquots of se~ rum were diluted 50 fold and reacted with LAV virion protein which had been resolved by SDS-PAGE and immobilized on nitro- cellulose filters by electrotransfer. LAV proteins recognized by these sera were detected by goat anti-human jimmunog: lebulin conjugated with alkaline phosphatase. Pooled sera from LAV/

HTLV III seropositive individuals (AIDS) were used as positive control.

Besults of Western blot analysis of the macaque serum samples are shoen in ¥ige. 9. Three out of four monkeys(mon-

key numbers 168, 175 and 180) produced antibodies to

LAV envelope glycoprotein gpil starting at 3 weeks post- inoculation. Monkey 182, which did not ahow detectable label of response to LAV, also had lower serum-neutralizing titer against vaccinia virus as compared to the rest of the groupe 65.2.2. IN VIVO PRIMING OF T CBLLS IN MACAQUES VACCINATED

WITH THs VACCINIA-ENY RECOMBINANT VIRUS

Feripheral blood lymphwsyte (PBL) from macaques, 6 weeks post vaccination were jsolated by Ficoll hypaque centri- fugation and suspended at 1 x 10° PBL/ml in RPMI 1640 medium centaining 10% heat-inactivated normal human serum. Then 0.1 ml resronding PBL were cultured, in wells of 96 well plates, together with either no antigen, ultraviolet (UV)- inactivat- ed vaccinia-env recombinant, or UV-inactivated parental vaccinia (1 x 10° PFU/ml prior to UV-inactivation), seven days later T- cell proliferation was assessed by determining S4—thymidine incorporated during the last 6 hours of a 7 day culture.

Results shown in Table II indicate that peripheral blood Tcells of macaques, 6 weeks post vaccination, undergo significant proliferative responses following in vitro sti- mulation with either parental vaccinia or v-env5 recombinants virus. Furthermore, the response was similar in magnitude to thut of T-cells from macaque 173, which was vaccinated with a vaccinia-herpes simplex gD recombinant virus v-HSgDI.

These results indicated that the expression of the LAV env gene by the recombinant virus in macaque monkeys did not suppress T-cell mediated immune responses in vitro or in vivo.

TABLE II

In Vivo Priming of T Cells in Macaque Vaccinated

With the Vaccinia-env Recombinant Virus 34-thymidine Incorporation (cpm)

Macaque into T-Cells After Culturing 7 Days

Number Vaccination No Antigen ParentalVaccinia Vaccinia-env 168 Paccinia-env 520 36,755 36,942 175 Vaccinia-env 7,640 36,405 35,142 180 Vaccinia-env 3,962 32,960 30,237 vo 182 Vaccinia-env 929 11,077 12,692 173 Vaccinia-herpes 1,757 35,837 31,860 simplex gD 208 None 6,102 4,867 9,420 233 None L,100 k,755 5,530

J ——

Taken together, the results shown in Fig. 6 - 9 and

Tables 1 and II indicate that the recombinant vaccinia viruses we describe were capable of (a) expressing the inserted LAV- env gene, (b) modifying or processing such gene products to © approximate the authetic viral envelope related proteins of

LAV, (c) producing immunogenic proteins specifically immuno- reactive with IADS patient serum, (d) eliciting LAV-specific responses in mice and sub-human primates; and (e) the recom-

binant virus did not suppress Tw~cell mediated immune responses in vitro or in vivo. 2. DEPOSIT OF MICROORGANISMS

The following E. coli strains carrying the list- ed plasmids have been deposited with the Agricultural He. search Culture Collection (NRRL), Feoria, Il. and have been assigned the following accession numbers:

E.Coli Strain glasmid Accession Number -——— ria ——— | S—————————— on ——————————————TA—

K12, MC1000 pv-envl NRRL B-18003

K12, MC1000 pv-envd NRRL B-1800L

K12, MC1000 pv-env5 NRRL B-18005

The following recombinant viruses have been depo- sited with the American Type Culture Collection, Rockville,

MD, and have been assigned the listed accession numbers:

Recombinant Virus Accession Number veenva ATCC VR 211k veenvH ATCC VR 2113

The present invention is not to be limited in scope by the microorganisms and viruses deposited since the dépo- sited embodiment is intended as single illustration of one aspect of the jnvention and any microorganisms or viruses which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invent- jon in addition to those shoen and described herein will be-

come apparent to those skilled in the art from the fore- going description and accompanying drawings. Such modifi- cations are intended to fall within the scope of the ap- pended claim.

It is also to be understood that all pair sizes given for nucleotides are approximate and are used for purpose of description.

Claims (1)

1. WHAT IS CLAIMED 1s:

   l. A recombinant virus the genome of which comprises a nucleotide sequnce encoding an envelope or gag epitope of LAV/HTLV I1I, or an antigenic por- tion thereof which is under the control of a second nucleotide sequence that regulates gene expression so that a peptide or protein related to the envelope or gag epitope of LAV/HTLV III is expressed in a host infected with the virus,

   2. The virus according to claim 1 in which the nucleotide sequence encoding the epitope of LAV/HTLV III comprises that of the envelope gene of LAV/HTLV IIT or any portion thereof encoding an antigenic peptide or pro- tein. 3, The virus according to claim 2 in which the envelope nucleotide sequnce comprises the nucleotide se- quence substantially as depicted in Fig. 2 from nucleo- tide number 5767 to 8549, or any portion thereof encoding an antigenic peptide or protein. 4, The virus according to claim 1 in which the peptide or protein expressed in the infected host is re- lated to an envelope epitope of LAV/HTLV III, ot any anti- genic portion thereof,

   5. The virus according to claim 4 in which the O41 oo

   —_— eee apvalobe spitoys culprit ose oop Eide or proteins having an amino acid sequence subetantially as depicted in

   Fig. 2 from amino 8cid copidde puwber 1 to Bole OF any aptigenis portiun thereofe

   €. ‘The virus spec oding be iui 1 gouprising an savaloped viruge

   7. The virus cccording to claim 0 cunpriving a vaccinia virube

   8. vaccinia virus y-env9 £5 deponited with the 0 ¢ fue and apoigned wocassion nueber yr 211% oF a mutant, recombinant or gene tically cugincered derivative thereof. Ge Yacciuia virus younVe SB gupoaited with the pos und apsigned acCuseivh sukes Vi J11h or 8 aucunte sscouvinant ov gene ticully ongineeysd derivative thereofe 106 ihe virus accor aing Lo clauln 1, couprising o nuked viruse

   11. ihe viyws Lgeooelig po claim 10 soups 16108 au gonoviyuae

   12. hu virus cecording fo cledm 1, conpriving @ pace tf Lolyhisdrosic viruse

   13%. phe ViTU3 acco: ding ye cid 12 cowpricing & pocuioviruse . Bz - BAD ORIGINAL 9 bara .

   - 14, The virus according to claim 1, comprising a bacteriophage.

   15. A substantially pure antigenic peptide re- lated to an epitope of LAV/HTLY I11X.

   16. The peptide according to claim 15 in which the epitope comprises an envelope glycoprotein of LAV/ HTLV IIT.

   17. The peptide according to claim 15 having an amino acid sequence comprising the amino acid sequence subs- ” 10 tantially as depicted in Fig. 2 from amino acid residue number 1 to 861 or any antigenic portion thereof. 18, The peptide of claim 15, in which the peptide or protein was purified from a cultured cell containing a nucleotide sequence encoding the peptide or protein which js under the control of a second nucleotide sequence that regulates gene expression so that the peptide or protein js expressed by the cultured cell.

   19. The peptide of claim 18 in which the cultured cell comprises 8 microorganisme

   50. The peptide of claim 19 in which the micro- organism comprises a bacteria.

   21. The peptide of claim 19 in which the micro- organism comprises a yeasto

   YY ——————

   22. The peptide of claim 18 in which the cultured cell comprises an animal cell line.

   23. The peptide of claim 18 in which the cultured cell comprises an insect cell line.

   2h. The peptide of claim 15, in which the peptide was chemically synthesized.

   25. A live virus vaccine formulation compris- ing virus of claim 1, in which the virus is infectious without causing disease in a host to be vaccinated,

   26. The live virus vaccine formulation accord- ing to claim 25 in which the virus comprises an envelop- ed virus.

   27. The live virus vaccine formulation accord- ing to claim 26 in which the enveloped virus comprises a vaccinia virus,

   . 28. A live virus vaccine formulation compris- ing an infectious dose of the vaccinia virus v-env5 of claim 8. : 29. A live virus vaccine formulation compris- ing an infectious dose of the vaccinia virus v-env2 of claim 9.

   30. The live virus vaccine formulation accord- - 84 J ing to claim 25 in which the virus comprises a naked virus. 31, A live virus vaccine formulation accord= ing to claim 30 in which the naked virus comprises an adenovirus. 224 An inactivated virus vaccine formulation comprising an effective dose of the virus of claim 1, in a non-infectious state mixed with a pharmaceutical carrier. Lo , 33, An jnactivated virus vaccine formulation comprising an effective dose of the enveloped virus of claim 6 in a non-infectious state mixed with a pharma- ceutical carriers 34, An inactivated virus gyaccine formulation comprising an effective dose of the vaccinia virus of claim 7 in a non-infectious state mixed with a pharma- ceutical carriero

   35. An jnactivated virus vaccine formulation comprising an effective dose of the vaccinia virus v= envS of claim 8 in a non-infectious state mixed with a pharmaceutical carrier. 36, An inactivated virus gaccine formulation comprising an effective dose of the vaccinia virus v-env2 of claim 9 in a pon-infectious state mixed with pharma- ceutical carriere. - B85 =

   -— 37, An inactivated virus vaccine formula- tion comprising an effective dose of the naked virus of claim 10 in a non-infectious state mixed with a pharma- ceutical carriers. 38, An inactivated virus vaccine formulation comprising an effective dose of the adenovirus of claim 11 in a non-infectious state mixed with a pharmaceutical carrier. 39, An inactivated virus vaccine formulation comprising an effective dose of the nuclear polyhedrosis virus of claim 12 in a non-infectious state mixed with a pharmaceutical carrier.

   40. An inactivated virus vaccine formulation comprising an effective dose of the baculovirus of claim 13 in a non-infectious state mixed with a pharmaceutical carrier.

   41. *“n inactivated virus vaccine formulation comprising an effective dose of the bacteriophage of claim 14 in a non-infectious state mixed with a pharma- ceutical carrier.

   42. A subunit vaccine formulation in which the jmmunogen comprises an effective dose of the peptide or protein of claim 15, mixed with a pharmaceutical carriers.

   hz, A subunit vaccine formulation in which the immunogen comprises an effective dose of the pep- tide or protein of claim 18 mixed with a pharmaceuti- cal carrier. 4h, A subunit vaccine formulation in which the immunogen comprises an effective dose of the peptide or protein of claim 19 mixed with a pharmaceutical carrier. 4s, A subunit vaccine formulation in which the immunogen comprises an effective dose of the peptide or protein of claim 20 mixed with a pharmaceutical carrier.

   46. A subunit yaccine formulation in which the immunogen comprises an effective dose of the peptide or protein of claim 21 mixed with a pharmaceutical carrier.

   47. A subunit vaccine formulation in which the immunogen comprises an effective dose of the peptide or protein of claim 22 mixed with a pharmaceutical carrier,

   48. A subunit vaccine formulation in which the immunogen comprises an effective dose of the peptide or protein of claim 23 mixed with a pharmaceutical carrier. 4g, A subunit vaccine formulation in whiféh the immunogen comprises an effective dose of the peptide or protein of claim 24 mixed in with a pharmaceutical carrier. ¢ 50. A recombinant DNA vector comprising pv-envl,

   Se

   51. A unicellular organism containing the recombinant DNA vector of claim 50,

   52. A bacterium containing the recombinant DNA vector of claim 50. 53 The bacterium of claim 52 comprising Egcherichi@ coli as deposited with the NRRL and assign- ed accession number B18003, or a mutant, recombinant or genetically engineered derivative thereof. Ske A recombinant DNA vector comprising pv-env2,

   55. A unicellular organism containing the recom- binant DNA vector of claim 54,

   56. A bacterium containing the recombinant DNA vector of claim Sh,

   57. A pacterium of claim 56 comprising Egcheri- chia coli as deposited with the NRRL and assigned acces-~ sion number B18004, or a mutant, recombinant or genetical- ly engineered derivative thereof,

   58. A recombinant DNA vector comprising pv-envS.

   59. A unicellular organism containing the re- combinant DNA vector of claim 58.

   60. A bacterium containing the recombinant DNA vector of claim %8.

   61. The bacterium of claim 60 comprising Egcherichia coli deposited with the NRRL and assigned accession number B-18005, or a mutant, recombinant or genetically engineered derivative thereof.

   SH1U-LOK HU ANTHONY F.

   PURCHIO Inventors ~- Po