JPS6368075A - Vaccine relating to acquired immune deficiency syndrome and immunoassay - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (10) Field of the Invention The present invention relates to lymph node disease-associated viruses [tymphad
enopathy As5ociated Virus
l(LAV)/human subcellular leukemia group uman
Epitopes of T-Cell Leukemia Virusl (elbow LVII), i.e. lymph node disease symptoms;
Group [Lymphadenopathy Syndrome]
e] (LAS) and acquired immunodeficiency syndrome [Acqu
ired Immune oerrcrency Sy
ndrome] (AIDS). Viruses of the invention expressing LAV/elbow [v■-related peptides that elicit a protective immune response can be used as immunogens in viral vaccine formulations against LAS or AIDS or in multivalent vaccine formulations. In fact, the infectious virus of the present invention, which can reproduce in the host without causing disease, can be used in live virus vaccines to provide a prolonged immunogenic stimulus and is capable of generating substantial immunity. be.

The present invention is also useful as an immunogen in subunit vaccine formulations or in multivalent vaccine formulations against LAS or AIDS.
The present invention relates to peptides and proteins related to epitopes of LAV/former LV [1] that can be used as antigens in diagnostic immunoassays related to LV.

These peptides and proteins can be produced using recombinant DNA technology in any vector-host system, or alternatively they can be synthesized by chemical methods. Accordingly, the present invention also provides novel and NA
It relates to the formation of vectors containing sequences and plasmid DNA and viral DNA, such as, for example, human viruses, animal viruses, insect viruses or bacteriophages.

Chemical methods for the synthesis of LAV/HTLVIII-related peptides and proteins are also described.

In a particular embodiment of the invention, recombinant vaccinia virus was used to produce LAV/HTLVIII envelope or core associated proteins.

To this end, the env or QaQ DNA sequences encoding for the glycoproteins or core structural proteins of LAV/HTLVIII can be used in LAV/HTLVIII in a suitable host.
V/HTLVl [inserted into a vaccinia vector capable of directing the expression of the gene. The LAV/HTLVIII envelope-associated protein produced by a recombinant vaccinia virus is antigenic and immunogenic and can elicit humoral and cell-mediated immunity in anthropoid primates. The LAV/HTLVIII aaa-related protein is an immunoreactive protein that contains the major epitope of the authentic core protein.

Recombinant vaccinia viruses expressing LAV/HTLVIII envelope-associated proteins can be used alone or in combination with LAV/HrLVII-associated proteins (such as core structural proteins) in viral vaccine formulations. can. Alternatively, the LA produced by the recombinant virus
V/HTLVIII-related proteins can be purified or chemically synthesized and used as immunogens in subunit vaccine formulations. The LAV/H
The TLVII-related glycoprotein(s) will be recognized as "foreign" in the host animal and therefore a humoral and/or cell-mediated immune response will be generated against this protein or combination of proteins. In a suitably prepared vaccine formulation, this will protect the host against subsequent LAV/HTLVVIII infection.

In certain embodiments of the invention, recombinant -29 type baculovirus (Autographa californica nuclear polyhedrosis virus [Autographa cali
fornica nuclear polyhedro
sis virus or AcNPV) is LAV/H
was used to produce TLVIII envelope and core related proteins. To this end, they encode envelope or core structural proteins, respectively.
The env or gag DNA sequence was inserted into a baculovirus vector capable of directing the expression of the LAV/HTLVIII gene in a suitable host. The LAV/HTLVIII protein produced by the recombinant baculovirus was found to be antigenic as determined by radioimmunoprecipitation and ELISA (enzyme-linked immunosorbent assay).

The present invention also provides for the production of LAV/HTLVI antigens, which are of general importance in human medicine. These are [Aν] in blood samples, body fluids, tissues, etc.
/HTLVIII, such as enzyme-linked immunosorbent assays (ELISA), which are useful as diagnostic tools for the detection of
It includes the use of the peptides and proteins of the invention as reagents in tests and immunoassays such as radioimmunoassays. Furthermore, this reagent is LAV/
This will be a valuable tool in elucidating the mechanism of pathogenesis of HTLVIII.

(20 Background of the Invention) (2,1, AIDS Virus) Acquired immunodeficiency syndrome (AIDS, AIDS) is a disease characterized by severe immunodeficiency primarily attributable to impairment of the patient's cell-mediated immune response. (Gotrieb, M. et al., 1981, The New England Journal of Medicine 305:1425; Masher, J. et. al., 1981, The New England Journal of Medicine 305:1431 [
Gottlieb, M. et al., 1981. N.

Engl, J. Hed, 305:1425. )(asu
r, J, et al, 1981. N.

Enc+1. J, Hed, 305; 14311). Two clinical manifestations of this disease are recognized, which are (
a) Normal peripheral helper to presser T lymphocyte ratio (OKT4: fluctuating from 2 to 0.1 or less with chronic lymph node disease, leukopenia, and disease progression)
Peripheral blood helper cells (OK 8) lead to the reversal of
a prodromal phase called lymph node disease syndrome (LAS) characterized by a quantitative decrease in T4) and (b)
Decreased and normal 0KT4 in OKT4 cells: 0
KT8 ratio reversal, absolute lymphopenia, and predominantly Pneumocystis carinai
t i 5 carni i ], and this latter stage is ultimately associated with death in the majority of cases. A minority of patients have an increased incidence of lymphoma and Kaposi's sarcoma. Currently, there is no treatment or treatment for this disease. Epidemic infectious disease data along with information on the types of patients who acquired the disease
It was suggested that an infectious agent transmitted by intimate contact may be the cause of the disease. Three groups subsequently provided strong evidence that the causative agent of AIDS is a retrovirus with tropism for hypohelper lymphocytes. These groups are:

(a) National Institute of Health [
National Intitude of Hea
lthl's R, C, Gallo and their collaborators are AIDS (AI)
The cytopathic retrovirus group nvm> could be isolated from patients with DS) and pre-AIDS (Gallo, R., C. et al., 1984, Science 224:500; Bopodzuk, M. .

et al., 1984, Science 224:497 [Ga
l Io, R.

C,et,al,, 1984.5science
224:500; Popovic, H.

1984.5science 224:497]). They also detected antibodies to II and VII in the serum of AIDS patients.

(b) The Pasteur Instegote [theP
Baresinossi, F., et al., who wanted to isolate T-lymphotropic retroviruses (LAVs) from patients presenting with cervical lymph node disease and at risk for AIDS. 1983, Science 220:868 [Barre-3inOuSS
i, F,.

et al., 1983, 5science 220:8
[68]), this group was also able to demonstrate antibodies against LAV in serum from AIDS patients (Kalyan Sraman, Bui, Nis, et al. 1984, Science 225:321EKa+VanSraman, V.
S.

et al., 1984, 5science 225;3
211). Additionally, they were able to isolate LAV from the lymphocytes of patients who developed AIDS after receiving blood from a donor who developed AIDS (Fueolino, P., M. et al., 1984, Science 225: 69 [
Feorino, P. M. et al., 1984, 5
science225:69]).

(C) Jie, Levy, and collaborators were able to extract infectious retroviruses (AIDS-associated retroviruses [AIDS-associated retroviruses] from peripheral mononuclear cells of AIDS patients).
It was named irusko or ^RV. ) were isolated (Levy, J. E., et al.

1984, Science 225:840 [Lavv, J.
,^,,et,al,,1984,5science 2
25:8401).

Although all three viruses were isolated separately, they probably all belong to the same retrovirus subgroup. Levy, J.

A, et al. 1984, Science 225:840)
, and will be correctly referred to herein as LAV/HTLVI.

The general structure of retroviruses is that of a ribonucleoprotein core surrounded by lipids containing an envelope that the virus acquires during the process of cell budding. Embedded within the envelope and projecting outward are virus-encoded glycoproteins. These determine the host range of the virus and react with specific receptors on the surface of acceptable cells. Neutralizing antibodies are thought to bind to envelope glycoproteins and block their interaction with receptors on the surface of cells (The Molecular Biology of Tumor
Wills, John Toews, ed., 1973, ]-
534th in Ludo Spring Harbor Laboratory
~535 pages, RN^Te Humor Wills, R, Uniis, Teich, N.

Voormuth, H., and Cotsfin, Gray.

ed., 1982, Cold Spring Harbor Laboratory, pp. 226-227 and 236-2.
37 pages [pp, 534-535 in, dinghe
) locular Biology of Tu
mor Viruses ed, John Cho
oze, 1973. Co1d Spring ) (a
rbor Laboratory; pp226-227
and 236-237in, RNA template
Viruses, ed. R. Weiss, Dingeic.
h, N,, Vat”mus, H, and Coff1n
, J., 1982. Co1d Spring )(a'
rborLabor4toryl). LAV/HTL
There is evidence that the T4 antigen, present on a subset of T lymphocytes, is a receptor or component of a receptor for the virus in the specific case of V. VIII (Dalgleish, A., G. et al., 1984).
Nature 312 Near B3; Kratzmann, D. et al., 1984, Nature 312 Near 67 [Dal
gleish, A, G, et al,, 1984゜Na
ture 312 near 63; IatZmann, D
, et al., 1984. Nature
312 Near 67]).

The RNA genome of [^V/HTLVnl is illustrated in Figure 1. Three genes are commonly recognized: the ino genes code for the internal structural proteins (core proteins) of the virus and define virus group-specific antigens.
The injector gene encodes for the pyrion ([vir1onl, virus particle)-associated reverse transcriptase. ” The gene is
Codes for viral glycoproteins. Other areas are in the open reading frame.
Indicate the display area of gnome "π o" and 3 °f including "e]. The functions of these regions are currently unknown.

(2,2, Vaccines) Several methods are currently used for the prevention and treatment of viral infections. These include vaccines that elicit an active immune response, treatment with chemotherapeutic agents and interferon treatment.

Traditional methods of vaccine preparation involve the use of inactivated or attenuated viruses. Inactivation of a virus renders it harmless as a biological agent, but does not destroy its immunogenicity. Injection of these "killed" virus particles into the host will then elicit an immune response that can neutralize future infections with live virus. However, a major problem with the use of killed vaccines (using inactivated virus) is the failure to inactivate all virus particles.

Even if this is achieved, the immunity obtained is often short-lived and additional immunizations are usually required because killed viruses do not reproduce in their hosts. Finally, the inactivation process alters the viral proteins to make them less effective as immunogens.

Attenuation results from the production of a virus strain that has essentially lost its ability to cause disease. One way to accomplish this is to subject the virus to unusual growth conditions and/or frequent passages in cell culture. Virus variants that have lost virulence but are still capable of eliciting an immune response are then selected.

The attenuated viruses usually constitute good immunogens since they actually replicate in the host cells and give rise to long-lasting immunity. However, several problems are encountered in the use of live vaccines, the most worrisome of which is insufficient attenuation.

An alternative to the above methods is the use of subunit vaccines. This involves immunization with only those proteins containing the appropriate immunological substances.

For many enveloped viruses, the virus-encoded glycoproteins are those that contain epitopes that can elicit neutralizing antibodies. These are La Crosse Virus (
Gonzalez-Scarano, F9. Shope, Earl, E0. Carriger, C., E., and Nathanson, N.

1982, Virology-120:42 [GOnZal
eZ-3Caf”ar) 0, F, , 5hope,
R, E, , Cal 1sher, C, E, a
nd Nathanson, N.

, 1962. Virolooy 120:42]),
Neonatal bovine diarrhea virus [Neonatal Ca1
f Diarrhea Virusl (v horn.

Varnish, and Innouni, Varnish 9. 1983, Infection and Immunity 39:155 [)f
Atsuno, S., and Inouye, S., 19
83. Infection and Immunity
39:155]), Venezuela Equine Equine Ence
phalomyelitis VirUS] (Matthews, J.H. and Rohrig, J.T., 1982, Journal of Immunology 129:
2763 group atheWs, J, H, and ROehr
i(] J, ■, 1982. J, Imm, 129:2
763]), Punta Toro virus [Punta Toro virus]
ro Virusl
．． Peters, C., J. A., , Smith, J. E., F., and Gientry, M.

K. 0. 1981, “Replication of Negative Strand Wills' D., H., L., Bishop and R., W.

Edited by Combans, p. 167, Lesever, New York [Dalrymple, J.] 1. , Peters,
C, J., Sm1th, J. F.

and Gentry, H., 1981. In”
Replication of NeQat+ve 5
trandViruSeS”, D, H, L, B15h
op and R,W.

Compans, eds, p. 167, Elsevei
R, Nev Yorkl), Murine Leukemia Virus] (Steves, R, A 0. Strand, M.

and August, J.I., T. 0.1974.

Journal of Pyrorrhea 14:187 [5te
eves, R,A,, 5trand, H,an
d August, J. T., 1974. J
, Virol.

14:187]), and mouse mammary tumor virus [MOu
Se) Lammary Virusl(
Matsusei, Earl, and Jay.

and Schochetman, G., 1981, Pyrolocy 115:20[)1assey, R.J., a.
nd Schochetman, G., 1981, Vi
115:20]) contains glycoproteins (7) T-. One advantage of subunit vaccines is that irrelevant viral material is eliminated.

Vaccines are often administered with various adjuvants. Adjuvants help achieve more durable and higher levels of immunity using lower amounts of antigen in fewer doses than if the immunogen were administered alone. The mechanism of adjuvant action is complex and not completely understood. However, it will involve stimulation of phagocytosis and other activities of the reticuloendothelial system and delayed release and degeneration of antigens. Examples of adjuvants include Freund's adjuvant (complete or incomplete), adjuvant 65 (
bean oil, mannide monooleate and aluminum monostearate), synthetic polyols
L-121 [pluronic polyol L-1
[21], abridine [AVridirel], and mineral glues such as aluminum hydroxide, aluminum phosphate or alum. Freund's adjuvant is no longer used in vaccine formulations for humans because it contains non-metabolizable mineral oil and is a potential carcinogen.

2, 2, 1. Recombinant DNA Technology and Vaccinia Virus The use of recombinant DNA technology for the production of subunit vaccines involves the molecular cloning and coding for viral proteins that can elicit a neutralizing response in the host animal. It involves the expression of the viral genetic information in an appropriate vector. All other genetic information of the virus is excluded and only those proteins needed to mount a neutralizing response are provided to the host animal.

The host is completely unexposed to the virus and is at no risk of becoming infected.

Recently, new approaches have been described that are potentially useful in the production of subunit vaccines (Maquette, M. O. Smith, G., L.).

and Moss, B., 1982, Proceedings of the National Academy of Sciences 79.7415-7419. Maquette, M0. Smith, G., L., and Moss, B.

1984 Journal of Pyrorrhea 49, 8
57-864; Panikali, D., and Paoretsti.

E., 1982, Proceedings of
The National Academy of Sciences 79.4
927-4931 [) (ackett, H,, Smlt
h, G, L, and Mo5s.

B., 1982. Proc, Nat'1. 8 cad, s
ci, 79.7415-7419;) (ackett,
H,, Sm1th, G, L, and Mo5s, B,,
1984. J.

Virol, 49, 857-864;
, D., and Paoletti, E.

1982, Proc, Nat' 1. ^cad, sci
, 79.4927-49311>.

This approach involves the use of vaccinia virus as a vector to express foreign genes inserted into the genome. Upon introduction into the host animal,
Recombinant vaccinia virus expresses inserted foreign genes and thereby elicits a host immune response against such gene products. Because raw silk recombinant vaccinia virus can be used as a vaccine,
Such an approach combines the advantages of both subunit and live vaccines.

Vaccinia virus contains a linear double-stranded DNA genome of approximately 187 kilobase pairs and replicates in the cytoplasm of infected cells. These viruses contain a complete transcriptase system (including capping, methylation, and polyadenylation enzymes) required for viral infectivity. The vaccinia virus transcriptional regulatory sequence (promoter) It allows initiation of transcription by RNA polymerase, but not by host cell RNA polymerase.

Expression of foreign DNA in a recombinant vaccinia virus requires ligation of the vaccinia promoter to a protein encoding the DNA sequence of the foreign gene. Plasmid vectors, also called insertion vectors, were formed to insert chimeric genes into vaccinia virus. One type of insertion vector includes (a) a vaccinia virus promoter containing a transcription start site, (b) several unique restriction endonuclease cloning sites located downstream of the transcription start site for insertion of foreign DNA fragments; (C) a non-essential vaccinia virus DNA flanked by the promoter and cloning sites that directs the insertion of the chimeric gene into a homologous non-essential region of the viral genome (such as a single gene); and (d) replication. Bacterial sources and Escherichia coli (E
It consists of an antibiotic resistance marker for replication and selection in , coli). An example of such a vector is described by Maquette (Maquette.

M1. Smith, G., L., and Moss.

B., 1984, Journal of Pyrorrhea 49.875-864 [) (ackett,)! ,
, Sm1th, G, L, , and Mo5s.

B., 1984. J, Virol, 49.8
57-864]).

Recombinant vaccinia virus is produced by transfection of a recombinant bacterial insertion plasmid containing a foreign gene into cells previously infected with vaccinia virus. Homologous recombination occurs within infected cells and results in the insertion of foreign genes into the viral genome.

Infected cells can be screened by immunological techniques, DNA plaque hybridization, or genetic selection for recombinant viruses that can then be isolated. These cottontail recombinants retain their essential function and infectivity and can be formed to accommodate approximately 35 kilobases of foreign DNA.

Foreign gene expression can be determined by enzymatic or immunoassay methods (e.g., immunoprecipitation, radioimmunoassay or immunoblotting [III1111r10blQtti+
'4Q]). Naturally occurring membrane proteins produced from recombinant cotton-infected cells will be viscoattached and transported to the cell surface. High expression levels can be obtained by using strong promoters or by cloning multiple copies of a single gene.

(2,2,2, Recombinant DNA technology and baculoviruses) Recently, potentially useful approaches for the production of recombinant proteins have been described (Penotsuk et al., 1984;
Molecular Cell Biology 4:399, Smith et al., 1983, Journal of Pyropathy 46
+584 [Penn0Ck et al, 1984.
) tol, cell Biol, 4:399. Sm1th
et al., 1963. J, Virol, 46:5.
84] ). This approach involves using Haki10 virus vectors to express foreign genes inserted into the genome.

Upon introduction into a suitable host cell, the recombinant baculovirus expresses the foreign gene.

The prototype of the baculoviridae family is Autographa californica nuclear polyhedrosis (AcNPV). The AcNPV genome has been mapped for several restriction sites (Smith, G., E., & Summers, M.).

D. 1978, Pirogy 89:517 [Sm1
th, G, Eand Summers, H, D,...
197B, ν1roloooy 89:517]) 128
It consists of a double-stranded circular DNA species of kilobase pairs. The virus replicates in the nucleus of infected insect cells. Two forms of virus are produced as a result of wild-type AcNPV infection of susceptible cells: occluded and non-occlusive virus particles. The occluded virus particles contain the polyhedrin gene [polyhedrin gene].
It is a virus particle surrounded by a protein called edra] (Pyrology 131:5B1~
563.1983 [V i r o l , 131
: 561-563゜1983]. ).

Occluded virus particles can be easily visualized in infected cells by using light microscopy.

Expression of foreign DNA in a recombinant baculovirus requires ligation of the baculovirus promoter to the protein-encoding DNA sequence of the foreign gene. Plasmid vectors, also called insertion vectors, are AcN
Formed for inserting chimeric genes into PV. One type of insertion vector consists of (a) an AcNPV promoter containing a transcription start site, (b) several unique restriction endosphrase cloning sites located downstream of the transcription start site for insertion of foreign DNA fragments. , (C) a non-essential Ac NPV DN flanked by the promoter and cloning sites leading to insertion of the chimeric gene into the homologous non-essential region of the viral genome.
A (such as the polyhetrin gene), and (
d) consisting of a bacterial source of replication and an antibiotic resistance marker for replication and selection in Escherichia coli. An example of such a vector is described by Miyamoto et al. (1985, Molecular Cell Biology 5:2860 [1985,) lol, cell
Biol 5:2860]).

Recombinant baculovirus is produced by co-transfection of cells with a recombinant bacterial insertion plasmid containing the gene, assembled with baculovirus DNA. Homologous recombination occurs within infected cells and results in the insertion of foreign genes into the viral genome. Once the foreign gene is inserted into the polyhetrin gene, occluded virus particles are no longer produced and the resulting recombinant plaques can be visually screened for the absence of occluders. Infected cells can also be screened by immunological techniques, DNA plaque hybridization, or genetic selection for subsequent isolable recombinant virus. These baculovirus recombinants maintain their essential functions and infectivity.

Foreign gene expression can be detected by enzymatic or immunological assays (eg, immunoprecipitation, radioimmunoassay or immunoblotting).

High expression levels can be obtained by using strong promoters or by cloning multiple copies of a single gene.

30 SUMMARY OF THE INVENTION Viruses are described that direct the expression of peptides or proteins related to epitopes of LAV/HTLVIII. Viruses of the invention that express LAV/HTLVIII-related epitopes induce a protective immune response.
It can be prescribed as a viral vaccine to protect humans from TLV infection. In one particular embodiment of the invention, a live virus vaccine formulation is prepared and obtained using an infectious virus that expresses LAV/former Lv-related epitopes in an infected host but does not cause disease in the host.

The present invention also provides peptides or proteins related to epitopes of LAV/H and V[[.

These, which lead to a protective immune response, are LAV/former LVI [l
They can be formulated into subunit vaccines to protect humans from infection, or they can be formulated into multivalent vaccines. Alternatively, the peptides or proteins of the invention can be used in diagnostic assays for AIDS or LAS.The peptides or proteins of the invention can be produced in any host cell-expression vector system;
And it can be isolated from these. These include, for example, animal or insect cell cultures infected with suitable recombinant viruses; microorganisms such as bacteria transfected with recombinant plasmids, cosmids or Contains transformed yeast. The present invention also provides LAV/HTLV
Methods, procedures and DNA formation used for the expression of genetic information encoding epitopes of III are provided. Alternatively, the peptides and proteins of the invention can be chemically synthesized.

In certain embodiments of the invention detailed in the Examples, gene sequences encoding for envelope glycoproteins or core structural proteins of LAV/HTLVII (
That is, [Av/elbow tvIIIenv or QaQ gene sequence], respectively, locates the early vaccinia virus promoter 5' to the initiation methionine sequence (ATG) of the LAV/HTLVIII gene sequence, resulting in vaccinia thymidine kinase (1K) D N
It is inserted into the plasmid resulting in a chimeric gene flanked by A sequences. These plasmids are then transfected into cells previously infected with wild-type vaccinia virus, thereby allowing TK to recombine into the TK gene of vaccinia virus.
Chimeric LAV/HTLVI flanked by sequences
II env or ' subgene. The cells are
The virus is lysed and the resulting virus is plated onto TK- (which lacks thymidine kinase activity) cells. The recombinant virus is expressed by its ability to form plaques on these cells in the presence of 5-promodeoxyuridine, as well as by radiolabeled probes (LAV/HT).
tv11-Envelope Mori, < ia LAV/H
Selection is made by DNA-DNA hybridization to TLV [consisting of either I-Oag (7)].

Hybridization-positive plaques are purified, expanded, and
The obtained recombinant virus is LAV/HTLVI.
It is tested for its ability to produce II envelope glycoprotein or core structural protein. LAV/HTL expressed by the recombinant vaccinia virus
It has been demonstrated that the VIII envelope-associated vaccinia virus is antigenic and immunogenic and capable of eliciting both humoral and cell-mediated immunity in anthropoid primates. The A/HTL VIII gag-related protein expressed by the recombinant vaccinia virus was an immunoreactive protein containing the major epitope of the putative core protein.

In other specific embodiments of the invention, LAV/HTL
The env or qag sequence of VIu was derived from the vector 10 virus polyhetrin promoter followed by a polyhetrin DNA sequence at the 3′ end.
5° to the starting methionine sequence (ATG) of the I gene
inserted into the plasmid so that it is located at These plasmids were then co-transfected into cells with wild-type Haki10 viral DNA, which assembled the chimera [AV/HTLVIII gene flanked by additional AcNPV into the baculovirus polyhetrin gene]. allowed to be replaced. Cells were allowed to lyse and the resulting virus was plaqued. Recombinant virus was tested by visual observation for removal of the obstructor or by radiolabeled LAV/elbow LVI.
II envMoL/<HaLAV/HTLVIII
Selection was made by DNA-DNA hybridization to the pag probe. Recombinant plaques were purified, propagated, and the resulting recombinant viruses were screened for their ability to produce LAV/HTLV associated proteins by immunoprecipitation with analysis on SDS polyacrylamide gels. It was done. Infected cell lysates were tested by ELISA for their ability to detect LAV/HTLVIII antibodies in serum.

(49) The detailed description of the invention focuses on viruses that produce peptides and proteins related to epitopes of LAV/HTLVIII. Tokuru LAV/HT
The focus is on peptides and proteins related to epitopes of LVII. The viruses or peptides and proteins of the invention are LAV/HTLVIII
, which can be used as an immunogen in a variety of vaccine formulations (including multivalent vaccine formulations) to prevent infection with #5 LAS and AIDS (7) etiological agents. Alternatively, the peptides or proteins of the invention can be used as antigens in diagnostic immunoassays for the detection of patient antibodies specific for LAV/HTLVVIII. The peptides or proteins used in the immunoassays of the invention are antigenic (i.e., IAV/
The antibody must be reactive with antibodies against HTLVIII, but need not be immunogenic (ie, capable of eliciting an immune response). Furthermore, the antigen used in the diagnostic immunoassay can be used in vivo.
[in vivo] is not necessary to elicit a protective immune response.

According to one embodiment of the invention, recombinant DNA techniques are used to insert the nucleotide sequence encoding the LAV/HTLV* epitope into an expression vector that directs the expression of the LAV/HTLV* sequence in a suitable host cell. used for. These expression vector-host cell systems were incubated in vitro with LAV/HTLVI (VitrO).
II-related peptides and proteins, in which case the gene product can be purified from cells in culture medium. Peptides or proteins capable of eliciting a protective immune response can then be used as immunogens in subunit vaccine formulations. Alternatively, the immunogenic or merely antigenic peptides and proteins of the invention may be
It can be used as an antigen in immunoassays intended to detect patient antibodies specific for elbow [v■]. In an alternative approach for the production of the peptides and proteins of the invention, the amino acid sequences of these peptides and proteins may be included in recombinant organisms expressing antigenic and/or immunogenic [Av/HTLVIII-related peptides and proteins. LAV/HT
LVl [can be derived from the nucleotide sequence. These peptides and proteins can then be chemically synthesized,
and can be used in synthetic subunit vaccine formulations (if immunogenic) or as antigens in diagnostic immunoassays (if antigenic and/or immunogenic).

If the expression vector is a virus that directs the expression of an immunogen related to an epitope of LAV/HTLV that is capable of eliciting a protective immune response, the virus itself can be formulated as a vaccine. Infectious recombinant viruses that do not cause disease in the host can be used in live virus vaccine preparations that confer substantial immunity. Alternatively, inactivated virus vaccines can be prepared using "killed" virus. Additionally, multivalent vaccines containing two or more epitopes of AV/H and V[[ or one epitope of LAV/HTLVIII as well as epitopes of other disease-causing agents can be prepared.

The method of the invention is divided into the following steps for the purpose of nest description. (a) isolating a gene or gene fragment encoding a LAV/former LVI viral protein; (b) inserting the gene or gene fragment into an expression vector; (C) replicating and expressing the gene. (d) identification and purification of the gene product; (e) determination of the immunopotency of the product; and (f) formulation of vaccines.

In a particular embodiment of the invention, we provide LAV/H
Recombinant vaccinia virus and baculovirus containing the envelope gene of TLVIII (LAV in tissue culture cells infected with the recombinant virus)
/HTLVI, which directs the expression of proteins immunologically related to the envelope gene. ) formation. However, the compositions and methods described herein are not limited to the generation of recombinant viruses expressing LAV/HTLVIII envelope or gag-related proteins and related to the antigens of any etiological agent of AIDS. It can be used to form recombinants in any expression vector system for the production of polypeptides.

For clarity of discussion, the whole method is based on LAV/+1
The TLVI envelope and pagJ will be described by Buddha. However, similar techniques are used to form recombinant expression vectors as well as LAV/H
It can be applied in a similar manner to produce polypeptides related to any protein of TLVI and that of related viruses. Such proteins are ga
LAV/HTLVI such as g, pot and env
[l gene product, as well as SOr, tat, 3'
-orf and at least four additional genes named to date, variously named (art or trs) one gene, but are of course not limited to these.

(4, 1, LAV/H Ding LVm Wi)l Stamper':y
Isolation of the LAV/HTLVIII gene)
It involves isolating a DNA fragment containing the desired gene sequence, such as envelope or gag. As already explained kT, LAV/HTLV
III has an RNA genome and therefore LAV
The homologous DNA encoding the /HTLV■ gene is
(a) by CDNA cloning of RNA isolated from purified LAV/HTLVI pillions; (b) LA
Poly[A]-containing RNA obtained from V/HTLVIII-infected cells [poly[A]-containing R
By cDNA cloning of NAI or (C
) can be obtained by cloning genomic DNA purified from LAV/HTLVIII-infected cells. Hereinafter, the DNA encoding the LAV/HTLVIII gene, LAV/HTLV111D
It is called NA.

To generate LAV/HTLVIn DNA fragments, LAV/HTLVI[l DNA can be cleaved at specific sites using various restriction enzymes. Alternatively, DNase (deoxyribonuclease) can be used in the presence of manganese to fragment the DNA;
It can be physically cut, for example by sonication.

The linear DNA fragments can then be separated according to size by standard techniques including, but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.

Any restriction enzyme or combination of restriction enzymes may be used to protect the envelope or c+ag protein gene product, so long as the enzyme does not destroy the envelope or c+ag protein gene product immunity.
LAV/old LVI containing c+ sequences can be used to generate DNA fragment(s). For example, an antigenic portion of a protein can be comprised of about 7 to about 14 amino acids. Therefore, a protein of this size (approximately 97,000 daltons) of the envelope peptide precursor has many separate antigenic sites, overlapping sequences, secondary and tertiary structure considerations, and acetylation, glycolylation or phosphorylation. Considering processing events like , there are probably thousands of them.

Therefore, many partial envelope polypeptide gene sequences can encode for a given antigenic site. Thus, many restriction enzyme combinations can be used to generate DNA fragments that, when inserted into an appropriate vector, can lead to the production of envelope-specific amino acid sequences consisting of different antigenic determinants.

Once the DNA fragment is generated, the desired LAV/
Identification of specific DNA fragments containing the HTLVIII gene can be performed in several ways. 1st
, sequence a DNA fragment that matches the complete LAV/elbow LVm genome, and then sequence a fragment containing the envelope protein gene or QaQ protein gene sequence with a predetermined amino acid sequence of the envelope protein or Qa (J core They can be identified based on comparison with the protein's amino acid sequence.Second, once the complete genome sequence has been determined, a wide open reading frame [Iaroe open reading f'r
ame] can be fixed from 5' to 3'. Since the genome structure of all retroviruses investigated to date is 5'-gag-pol-env-3°, 3
°The wide open reading frame closest to the progeny probably encodes for envelope genes. On the other hand, the wide open reading frame closest to the 5' end would probably encode for the<gaQ gene. Third, prospective identification of specific genes can be achieved by recognition of homology to other known retroviral genes, either by nucleic acid hybridization analysis or by sequence comparison (if the sequence is known). .

Alternatively, fragments containing envelope or gag protein genes can be identified by mRNA selection. In this method, LAV/HTLVVIII
The DNA fragments are used to isolate complementary mRNA by hybridization.

Immunoprecipitation analysis of in vitro translation products of the isolated mRNA identifies the mRNA and therefore the complementary LAV/QaQ protein sequence containing the envelope or QaQ protein sequence.
Identify the old LVI [1 DNA fragment. lastly,
The specific mRNA is isolated from LAV/HTLV to an immobilized antibody directed against the envelope or gag protein.
selected by adsorption of polysomes isolated from nl-infected cells. Radiolabeled envelope protein CD
NA (complementary DNA) can be synthesized using this selected mRNA (from the adsorbed polysomes) as a template. Radiolabeled mRNA or C
The DNA can then be used as a probe to identify LAV/HTLVIII DNA fragments containing envelope or QaQ gene sequences. Alternative means for isolating the envelope or QaQ gene include chemical synthesis of the gene sequence itself (if the sequence is known) or generation of CDNA for the mRNA encoding the envelope or Q8Q gene. Including, but of course not limited to.

Once identified and isolated, the LAV/HTLVIn DNA fragment containing the sequence of interest is
first inserted into a cloning vector, such as a plasmid cloning vector, used to transform a suitable host cell to replicate the DNA;
Many copies of the LAV/HTLVIII sequence of interest are thereby produced. This is inserted into a cloning vector with complementary cohesive ends [^V/HTLVII
This is accomplished by ligating I DNA fragments. However, LAV/former LVIII DNA
If the complementary restriction sites used to fragment the DN are not present in the cloning vector, the DN
The end of the A molecule is modified.

Such modifications include - generating plant ends by back-digesting the heavy chain DNA ends or filling the heavy chain ends so that the ends can be subjected to plant end ligation reactions. It includes.

Alternatively, any desired site is created by ligating a nucleotide sequence (linker) onto the DNA terminus. These linkers are
It is composed of specific chemically synthesized oligonucleotides encoding restriction site identification sequences. According to other methods, the cleaved vector and LAV/HTLVIII DNA fragments are isolated from the hemopolymeric Tilling group emo
[polymeric tailing].

Transformation of a host cell with a recombinant DNA molecule incorporating an isolated gene, CDNA or synthetic NA sequence allows the production of multiple copies of the gene. Therefore, the gene is used to propagate the transformant, isolate the recombinant DNA molecule from the transformant, and, if necessary, recover the inserted gene from the isolated recombinant DNA. By doing so, you can obtain Oma.

If the ultimate goal is to insert the gene into a viral expression vector such as vaccinia virus or adenovirus, a recombinant DNA molecule incorporating the [^V/H and V■ genes may contain To enable insertion into the viral genome, the gene can be modified such that it is flanked by viral sequences that allow genetic recombination in cells infected with the virus.

The complete [^V/H and V■ genome has been cloned and sequenced by Uniin Hobson et al.
Wayne-Hobson, Niss, et al. 1985.

Cell 40:9 [Wein-Hobson, S. et. a.
l,, 1985. Ce1l 40:91).

One clone, called lambda J19 (λJ19), was derived from lambda L47. 'l (λL47.1) day ind
It contained a 9.2 kilobase pair DNΔ fragment of LAV genomic sequence inserted into the m site.

[^V/HTLVI[lλJ containing envelope genes
19 particularly useful subclones are EC0 of pUc18.
RI and 3st (LAV/HTL inserted into site
V [1 nucleotide sequence of 3,840 base pairs Dan CORI
→pR8-3 consists of a 5StI fragment.

pR8-3 contained LAV/HTLVI [specificity D
NA is from the CORI site located at nucleotide 5289 in the AV/H and V■ genome to the DAN1 site located at nucleotide 912968 (Wayne-Hobson, Niss, et al. 1985 Cell 40: 9).

See Figure 2, which depicts the LAV/HTLVIII NucL/,t sequence contained in pRS-3. However, the degeneracy of nucleotide coding sequences [den
Therefore, other DNA sequences encoding substantially the same amino acid sequence as depicted in FIG. 2 can be used in the practice of the present invention for cloning the LAV/HTLVIII envelope gene.

These are substitutions of different ]tons (thus altering (thus producing silent mutations) that encode the same or functionally equivalent amino acid residues (e.g., amino acids of the same polarity) within the sequence. This includes, but is of course not limited to, a nucleotide sequence consisting of all or part of the envelope nucleotide sequence depicted in .

λJ containing LAV/HTLVIII oaol Buddha
19 particularly useful subclones include DKS-5 and 1
) 385. DK'S5 plasmid is pUC18
Middle (i') 3148 base pairs (7) S St I →K
l) Consists of the n I LAV/HTLVIII fragment. LAV/HTLV contained in DK S-5
III Specificity DNA Ha, LAV/HTLVIII
Nucleotide 224 (located in the genome)
Kl)n located at nucleotide position 3.372 from the site
This is up to the I site. The DBS-5 plasmid is I) UCl
3 out of 7 (7) 5. 1 kbp ノ5stl-+Sal
ILAV/HTLVIII consists of 77 components. 1)
[Av/HTLVI[specificity D] contained in 83-5
From the 5StI site located at nucleotide 224 to the 5aI 1 site located at nucleotide 5331 (Wayne-Hobson, Niss, et al., 1985).

cell 40:9). See Figure 14 depicting the LAV/HTLVI nucleotide sequences contained in DKS-5 and p88-5. However, due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same amino acid sequence as depicted in Figure 14 include the LAV/HTLVIII(7) Qag gene (D
') can be used to implement the present invention regarding rowing. These are altered by the substitution of different codons within the sequence that encode the same or functionally equivalent amino acid residues (e.g., amino acids of the same polarity) (thus producing silent mutations), as depicted in Figure 14. These include, but are of course not limited to, nucleotide sequences consisting of all or part of the gag nucleotide sequence.

(4, 2, Coating the array LAV/HTLV I
[Insertion of I protein into expression vector] LAV/HTLV such as envelope, gag
The nucleotide sequence encoding the III protein, or a portion thereof, is inserted into a suitable expression vector, ie, a vector containing the necessary elements for transcription and translation of the inserted protein coding sequence. A variety of host-vector systems may be utilized to express the protein coding sequence. These include mammalian cell lines infected with viruses (e.g. vaccinia virus, adenovirus, etc.); insect cell lines infected with viruses (e.g. baculovirus); and yeast or bacteriophage DNA containing yeast vectors.

This includes, but is not limited to, microorganisms such as bacteria transformed with plasmid DNA or cosmid DNA. The expression elements of these vectors vary in their strength and specificity. Depending on the host-vector system utilized, any one of several preferred transcription and translation elements may be used. For example, when cloned in a mammalian cell system, promoters isolated from the genome of mammalian cells (e.g. the mouse metallothionein promoter) or promoters isolated from viruses grown in these cells (e.g. the vaccinia virus 7.5 promoters) may be used. Promoters produced by recombinant DNA or synthetic techniques are also used to effect transcription of inserted sequences.

Specific initiation signals are also required for sufficient translation of inserted protein-coding sequences.

These signals include the A-D-G initiation codon and adjacent sequences. Complete [Aν/HTLVII[envelope or Q
No additional translational control signals are required if the aQ gene is inserted into a suitable expression vector. However, if only the envelope or gag encoding sequence portion is inserted, an exogenous translational control signal must be provided, including an ATG initiation codon. Furthermore, the initiation codon is located in the open reading frame of the envelope protein coding sequence to ensure translation of the complete insert. These exogenous translational control signals and initiation codons can be of a variety of sources, natural and synthetic.

Any of the methods previously described for insertion of DNA fragments into vectors can be used to form expression vectors containing chimeric genes consisting of appropriate transcriptional/translational control signals and protein coding sequences. It can be done. These methods include in vitro recombinant DNA and synthetic techniques, as well as in vivo recombination (genetic recombination).

In specific embodiments detailed in the Examples of the invention, vaccinia virus and baculovirus were chosen as expression vectors.

However, the invention is not limited to the use of vaccinia virus or baculovirus. As previously explained, expression vectors that can be used include, but are not limited to, the following vectors and derivatives thereof:
Human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors; and plasmid and rosmid DNA vectors.

In cases where an adenovirus is used as an expression vector, the LAV/HTLVIII envelope gene is linked to the adenovirus transcription/translation control complex, such as the late promoter and tripartite leader sequence [trip
artite 1 leader 5 sequence]. This chimeric gene is then inserted into an adenovirus by in vitro or in vivo recombination. Insertions in non-essential regions of the viral genome (eg, regions E1 or [3) result in recombinant viruses that are viable and capable of expressing LAV/elbow LVII-related proteins in infected hosts. Two of the adenoviruses currently approved and used as vaccines for military personnel.
There are two strains (types 4 and 7). These are LAV
/HTLVIII gene is the prime candidate for use as a vector to express the gene.

Additionally, a host cell line is chosen that modulates the expression of the inserted sequences or otherwise modifies and processes the chimeric gene product in the particular manner desired. Expression from some promoters can be enhanced in the presence of some inducers, such as zinc and cadmium ions for the metallothionein promoter. Therefore, genetically engineered LAV/
Expression of H and Vl proteins can be regulated. This is important if the protein product of the cloned foreign gene is lethal to the host cell. Additionally, modification (eg, glycosylation) and processing (eg, cleavage) of protein products are important to the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to follow the correct modification and processing of the foreign protein expressed.

In two specific embodiments detailed in the Examples of the Invention, we used Vaccinia virus 7.5 to form chimeric genes in various plasmids.
The LAV/HTLVII envelope coding sequence (LAV/HTLVIII env-sequence), both in its complete form and a portion thereof, or the LAV/HTLVII Oag coding sequence was linked to the promoter. The chimeric genes in these plasmids were flanked by additional vaccinia virus sequences homologous to the vaccinia virus TK gene. The formation of chimeric genes involves the use of LAV/HTLVI and W-sequences or LAV/HTLVI
It involved the use of natural and synthetic nucleotide-encoded control signals for the transcription and translation of AV/HTLVIII gaa sequences. The chimeric gene was then introduced into the vaccinia virus expression vector through in vivo recombination between homologous TK regions present in both the plasmid vector and the vaccinia virus genome. These recombinant viruses with chimeric genes are LAV
/HTLVIII envelope-associated protein or L
It was used as an expression vector for producing AV/HTLVIII gapmil-related protein.

In other specific embodiments of the invention detailed in the Examples, we use either the LAV/HTLVII envelope coding sequence or the LAV/elbow LVIII gag coding sequence to form chimeric genes in various plasmids. Out graph?・Californica nuclear polyhedrosis virus [Autooraphacali
fornica nuclear polyhedro
sis virus] (AcNPV) polyhetrin [
polyhedrin] promoter. The chimeric genes in these plasmids contain additional Ac
The formation of this chimeric gene involves the use of natural nucleotides encoding control signals for transcription and translation of the LAV/HTLV muaa or env sequences. The chimeric gene is then flanked by (plasmid vector Ac
was introduced into the NPV expression vector. These recombinant viruses containing chimeric genes are LAV/HTLV■envelope-associated protein or LAV/HTLVII.
It was used as an expression vector to produce IQaQ-related proteins.

4.3. Identification of Recombinant Expression Vectors Capable of Replicating and Expressing the Inserted Genes Expression vectors containing foreign gene inserts can be prepared using three general approaches: (a) DNA-DNA hybridization; b) the presence or absence of a "marker" gene function; and (C) the expression of the inserted sequence. In the first approach,
The presence of the inserted foreign gene in the expression vector
It can be detected by DNA-DNA hybridization using a probe consisting of a sequence homologous to the foreign inserted gene.

In the second approach, a recombinant vector/host system is used to detect certain "tagged" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformed phenotype,
occlusion body formation in baculovirus) caused by insertion of a foreign gene into the vector.
Can be identified and selected based on presence or absence. For example, LAV/HTLVIII M Nobuko,
When inserted within the labeled gene sequence of a vector, LAV
Recombinants containing the /HTLVII insert can be identified by the absence of the marker gene function. In a third approach, recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays may be based on physical, immunological or functional properties of the gene product.

Once a particular recombinant DNA molecule has been identified and isolated, several methods are available for determining whether such recombinant forms a self-replicating unit (replicon) or not. Can be used for breeding. Self-replicating units, such as plasmids, viruses, cells, etc., are capable of replicating themselves in an appropriate cellular environment and growth conditions. Recombinants lacking self-replicating units must be integrated into molecules containing such units in order to reproduce.

For example, upon introduction into a host cell, some plasmid expression vectors require integration into the cellular chromosome following propagation and proper expression of the recombinant gene. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in large quantities.

In specific embodiments of the invention detailed in the Examples, LAV/HTLVII env or QaQ':
A chimeric gene containing a J-dosed region was inserted into the TK gene of the vaccinia virus genome, thereby converting the virus to TK- (ie, destroying the ability of the virus to produce thymidine kinase). Such recombinants are lethal to TK+ cells but are lethal to TK− cells.
Cells were selected for their ability to grow in media containing 5-promodeoxyuridine, a nucleoside analog that is not. The recombinant further has
AV/HTLVIII env specificity or LAV/
DNA using old LVIII 08g specificity probe
-Identified by DNA hybridization. TK-
Recombinant virus was isolated by plaque purification and strains were prepared from infected tissue cultures.

In other specific embodiments of the invention detailed in the Examples, LAV/HTLVIII env or aa
A chimeric gene containing the a-coding sequence was inserted into the polyhetrin gene of the AcNPV genome, thereby converting the virus into an unoccluded prototype. Such recombinants were visually selected by their lack of occluded virus particles. The recombinant is Zarani, [^V/HTLVVIII
env specific probe or s^V/HTLV ga
Northern blot analysis using g-specific probes [N
Identified by alternative blot analysis. Recombinant virus was isolated by plaque purification and strains were prepared from infected tissue culture cells.

4.4. Identification and Purification of the Expressed Gene Product Once a recombinant expressing the LAV/IITLV nu gene has been identified, the gene product should be analyzed. This can be assayed based on the physical, immunological or functional properties of the product. Immunological analysis is of particular importance since the end goal is the use of gene products or recombinant viruses expressing such products in vaccine formulations and/or as antibodies in diagnostic immunoassays.

Various antisera may be useful for analyzing the immunoreactivity of the products, such as those derived from LAS or AIDS patients, as well as LAV/former LV [[
Virus, viral envelope protein or Q
These include, but are not limited to, polyvalent antisera directed against the core protein encoded by the aQ gene. The immunogenic molecule may be a homologue produced in bacterial systems or LAV/HTLVII.
These include synthetic peptides that contain antigenic determinants of the I envelope or core antigen. The identification of peptides and proteins described in this invention is based on two demands. First, LAV/elbow tv11-related proteins should be able to be produced only in recombinant virus-infected cells. Second, the LAV/HTLVIn
The relevant protein should be immunoreactive with relevant protein serum or with a variety of antibodies directed against LAV/HTLVII [envelope or core protein or its homologs and derivatives.

The protein, whether from two or more gene sequences linked to direct the expression of the complete gene sequence, the expression of a portion of the gene sequence, or the production of a fusion protein, Should be reactive. This reactivity can be demonstrated by standard immunological techniques such as radioimmunoprecipitation, radioimmunocompetition or immunoplot.

Once a LAV/HTLVIII-related protein is identified, it can be analyzed by chromatography (e.g. ion exchange).

by standard methods including affinity, size-control column chromatography), centrifugation, differential solubility, or by other standard techniques for protein purification.
isolated and purified.

Alternatively, once an immunoreactive LAV/former LV-associated protein produced by a recombinant is identified, the amino acid sequence of the immunoreactive protein can be derived from the nucleotide sequence of the chimeric gene contained in the recombinant. It can be inferred. As a result, the protein can be synthesized by standard chemical methods known to those skilled in the art (e.g. Vankapillar, M. et al., 198
4th year, Nature 310:105-111 [Hun
Kapi Ier, H, et al.

See 1984, Nature Hoshi 105-1111. ).

In certain embodiments of the invention, such peptides (produced by recombinant DNA technology or by chemical synthesis methods) include substitutions of residues within the sequence with functionally equivalent amino acid residues. The entire amino acid sequence substantially as depicted in FIG. 2 or substantially as depicted in FIG. 14, including altered sequences resulting in silent mutations, or Although it includes some, it is of course not limited to these. For example, one or more amino acid residues within a sequence can be replaced by amino acids of similar polarity that act as functional equivalents, resulting in a silent modification. Substitutions for an amino acid within a sequence may be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, and tyrosine. Positively charged (basic) amino acids include arginine, lysine and histidine. Positively charged (acidic) amino acids include aspartic acid and glutamic acid.

(4, 5, Measurement of immunity of recombinant forest products) LAV/HT
The immunopotency of LVIII envelope-related products 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 LAV/HTLVIII-related proteins are expressed by an infectious recombinant virus, the recombinant virus itself can be used to immunize test animals. Test animals are mice, rabbits,
Includes chimpanzees and ultimately human subjects. Methods of immunogen introduction include oral, intramuscular, intraperitoneal,
including intravenous, subcutaneous, intranasal or any other standard route of immunization. A subject's immune response can be analyzed by three approaches. That is (a)
Known techniques such as enzyme-linked immunosorbent assay (ELIS)
A) Authentic LAV/HTL of the obtained immune serum as assayed by immunoplot, radioimmunoprecipitation etc.
VIII virus antigen reactivity; (b) ability of immune serum to neutralize LAV/HTL VIII infectivity in vitro (Roberto Garoff, M., 1985
Year, Nature 318: 72-74 [Robert-
Guroff, H., 1985゜Nature 31
6:72-74); and (C) LAV/HTLVII [protection from infection and/or
or attenuation of infectious symptoms (Francis, Dee.

P0.1984, Lancet 2: 1276-1
277; Gajusek, D.C., 1985
Year, Lancet 1:55-56 [Francis
, D., P., 1984. Lan5et 2
:1276-1277; Gujdusek, D, C
, 1985, Lan5et 1:55-56]
It is.

(4,6. Vaccine formulation) The objective of this embodiment of the invention is that the immunogen is LAV/HT.
The objective is to formulate a vaccine for the prevention of LAS or AIDS that is associated with a recombinant virus that expresses the LV epitope and an immunogen that protects against LAV/former Lv virus infection. Additionally, multivalent vaccine formulations containing one or more immunogenic LAV/HTLVIII-related determinants can be prepared.

Such a vaccine may contain LAV/HTLV I[I envelope-associated epitopes alone or, e.g.
Other LAV/HTLVn such as g-related epitopes
1 epitope, including, but not limited to, those formulated in combination with this epitope. env encoded epitope and gag
Such a multivalent vaccine containing both encoded epitopes would significantly prevent the development of AIDS. Individuals who are asymptomatic appear to produce anti-gag antibodies more frequently than disease patients, whereas patients in both groups produce antibodies directed against envelope determinants ( Science, 198B, 223:419 [
5CienCe, 198B, 223:419]) Studies have also shown that AIDS patients in the early asymptomatic stage produce antibodies against envelope and core (pag) proteins. As the disease progresses and symptoms develop, anti-QaQ antibodies are reduced, while anti-envelope antibodies are maintained (Science, 1986, 223:
282>. Therefore, the inclusion of gag-related epitopes in vaccine formulations, the method of preparation of which is mentioned as a specific embodiment of the present invention, is important in the immunoprophylaxis or immunotherapy of LAV/elbow [v]-related diseases.

Multivalent vaccines can be formulated containing recombinant viruses expressing the LAV/HTLVIU gene product and/or the chimeric gene product. They may also be used in combination with other immunogens for the prevention of LAS to AIDS and other diseases. Examples of various formulations are discussed below.

(4, 6, 1, Virus vaccine formulation) Either a live silk recombinant virus vaccine or an inactivated recombinant virus vaccine can be formulated.

The choice depends on the status of the recombinant virus used to express the LAV/HTLVI [related epitope].

If the recombinant virus is infectious but does not cause disease in the host to be immunized, replication in the host will be stimulated to a prolonged period similar in type and degree to that which occurs in natural asymptomatic infection. Live vaccines are preferred because they confer , and therefore substantially permanent immunity. Upon introduction into a host animal, the infectious recombinant virus transforms its chimeric genes into LAV/H and Vl.
can express related proteins and thereby LAV
/HTLV■ can stimulate an immune response against antigens.

Such an immune response is responsible for subsequent LAV/HTLVII
In cases where it is protective against I challenge, the live silk recombinant virus can be used alone as a prophylactic vaccine against AIDS virus infection. Production of such recombinant viruses for use in such formulations involves both in vitro (e.g. tissue culture cells) and in vivo (e.g. natural host) systems. Particularly useful. Well-known methods for the preparation and formulation of scar vaccines can be applied for the formulation of live silk recombinant virus vaccines (e.g., Factors Related to Vaccinia Virus and Vaccine Antigens, Kinan, C., Bui, ed. Elsebear). , 198
5, pp. 109-116 [Vaccinia v;r
uses and factor for Vacci
ne AntLgens, Ed, Quinnan.

G., V., E.I.V., 1985.
See pp. 109-1161. > Multivalent live virus vaccines include several LAV7HTLVI[I
/HTLV-related epitopes can be prepared from a single infectious recombinant virus. The vaccine may also contain a virus that, in addition to epitopes of LAV/HTLVIII, expresses epitopes of other disease-causing organisms. For example, vaccinia virus (which can harbor approximately 35 kilobases of foreign DNA) can be engineered to contain coding sequences for both the envelope and Qa (J-related epitopes) of LAV/HTIV. Also L
In addition to these regarding AV/HTLVIII,
Other epitopes may be encoded. Such recombinant viruses can themselves be used as immunogens in multivalent vaccines. Or again, LAV
Mixtures of vaccinia or other viruses, each capable of directing the expression of different genes encoding for different epitopes of the disease-causing organism, can be formulated in a multivalent vaccine.

Inactivated vaccine formulations can be prepared regardless of whether the recombinant virus is infectious to the host to be immunized. Inactivated vaccines are "'dead'" in that their infectivity has been destroyed, usually by formaldehyde. Ideally, the infectivity of the virus would be reduced without affecting the capsid (1), which is responsible for the immunogenicity of the virus, without affecting the envelope proteins. To prepare an inactivated vaccine, large amounts of recombinant virus must be grown in culture to provide the required amount of the relevant antigen.

Mixtures of inactivated viruses expressing different epitopes can be used to formulate "multivalent" vaccines.

In some instances, this is preferred over live vaccine formulations due to the potential difficulty in interacting with live viruses administered simultaneously. In either case, inactivated recombinant viruses or mixtures of viruses should be formulated with appropriate adjuvants to enhance the immunological response to their antigens. Suitable adjuvants include, for example, mineral glues such as aluminum hydroxide, surfactants such as lysolecithin and constituent polyols, polyanions, peptides, and oil emulsions, and B.
CG (Cametoglan rod) and Corynebacterium parvum [Corynebacterium parvum]
including, of course, potentially useful human adjuvants such as, but not limited to.

Many methods may be used to introduce the vaccine formulations described above, including, but not limited to, buccal, intramuscular, intraperitoneal, intravenous, subcutaneous and intranasal routes. do not have.

When a live silk recombinant virus vaccine formulation is used, it is based on the parent wild type that was used to produce the recombinant virus in the vaccine formulation.
e] introduced through the natural route of viral infection.

(4, 6, 2, subunit vaccine formulation) As an alternative to virus vaccines, LAV/HTLVIII
The related proteins themselves can be used as immunogens in subunit vaccine formulations (which may be multivalent). As explained above, subunit vaccines are composed only of the relevant immunogenic substances necessary to immunize the host.

Accordingly, LAV/HTLVIII related protein(s) are purified from recombinants expressing LAV/HTLVIII epitopes. Such a recombinant is LAV
/HTLVIII epitope (Section 4.2.).
See Sections 4.3 and 4.4. ). In another embodiment of the invention, the LAV/HTLVIII-related peptide or protein is chemically synthesized. To this end, the amino acid sequence of such a peptide or protein can be deduced from the nucleotide sequence of the chimeric gene directing its expression (see Section 4.4). The final product, whether purified or chemically synthesized, is adjusted to the appropriate concentration and formulated with any suitable vaccine adjuvant and packaged for use. Suitable adjuvants include, for example, mineral glues such as aluminum hydroxide; surfactants such as lysolecithin, plutonic polyols; borianions; peptides; oil emulsions; as well as BCG.
(Cametogran rod) and Corynebacterium parvum (Corynebacter;um parvum)
], including potentially useful human adjuvants such as
Of course, it is not limited to these. Immunogens can also be incorporated into liposomes or conjugated to polysaccharides and/or other polymers used in vaccine formulations.

LAV/former LVI-related peptides or proteins are haptens, i.e., molecules that are antigenic in that they react selectively with cognate antibodies, but are not immunogenic in that they are unable to elicit an immune response. In some cases, the hapten is a carrier or an immunogenic molecule,
- For example, a hapten is covalently linked to a large protein, such as the protein serum albumin, which would confer immunogenicity when bound. This hapten
The carrier is formulated for use as a vaccine.

(4,6,3, Passive immunization and anti-idiotype anti-95
(1) Instead of active immunization with viral or subunit vaccines, it is possible to confer short-term protection to the host by administering preformed antibodies directed against epitopes of LAV/H and v■. .

Accordingly, the vaccine formulations described above can be used to produce antibodies for use in passive immunotherapy. Human immunoglobulins are preferred in human medicine because heterologous immunoglobulins will stimulate the immune response against their foreign immunogenic components. Such passive immunization provides rapid protection for unimmunized individuals at particular risk, such as those exposed to contact with AIDS patients, such as in hospitals and other health care facilities. It could be used in emergency situations. Alternatively, these antibodies can be used to produce anti-idiotypic antibodies that can be used in turn as antigens to stimulate an immune response against LAV/HTLV III epitopes.

(4, 7, Immunoassay) The LAV/HTLVIII-related peptides and proteins of the present invention can be used to detect LAV/HTLVIII in various patient tissues and body fluids as well as in blood in blood banks and hospitals.
can be used as an antigen in immunoassays for the detection of antibodies against. For this purpose, the antigens of the present invention can be used in radioimmunoassays, ELISAs (to name but a few).
Enzyme-linked immunosorbent assay) ``Sandwich immunoassay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay [immuno-ra
Any method known in the art including, but not limited to, competitive and non-competitive assay systems using techniques such as diometric assays, fluorescence immunoassays, protein A immunoassays, and immunoelectrophoretic assays. Can be used in immunoassay systems.

The immunoassay of the invention can be used to test blood and patients in blood banks for exposure to LAV/HTLVIII and to monitor patients undergoing treatment for LAS or AIDS. LAV/H
Any peptide and protein related to the antigen of TLVIII can be used in the immunoassay according to the invention. Such antigens include env, QaQ,
The gene products of pol, as well as SOr, tat,
It includes, but is of course not limited to, peptides and proteins related to the four other genes named to date: 3°-orf and art (or trS). In a particular embodiment of the invention, a panel of antigens was used in an immunoassay to test patients for a profile of antibodies directed against different epitopes of LAV/HTLv■. In other embodiments of the invention, patients vaccinated with the vaccine formulations of the invention have subsequent LAV/HTLV
was monitored to detect exposure to III. In such cases, the antigen used in the immunoassay is preferably a peptide or protein of the invention that is not an immunogen of the vaccine formulation used to immunize the patient, since such immunization This is because individuals who have been tested will be seropositive for the specific epitope used in the vaccine formulation and, as a result, will test positive regardless of exposure to the virus.

For example, if a patient has been vaccinated with a LAV/HTLVIII envelope-associated epitope of the invention, the patient may be vaccinated with any other non-enveloped epitope to determine the presence of patient antibodies specific for LAV/HTLVIII. Using LAV/HTIVIII-related proteins [
Tested for Av/HTLVIII infection.

Thus, patients vaccinated with envelope-associated epitopes include (but are not limited to) the LAV/HTLVIII core antigen, or the sor and 3°-orf gene products and the like.

) can be tested for antibodies specific for other antigens of LAV/HTLVII.

In an alternative embodiment, the [Av/HT
LVIII-related peptides and proteins can be used in competitive immunoassays to detect and determine the presence of LAV/HTLVIII-encoded proteins in patient blood, serum, etc.

(5. Example: Vaccinia envelope recombinants) In the following examples, various plasmid vectors contain a chimeric gene consisting of LAV/HTLVJII envelope coding sequences located downstream to the transcriptional regulatory sequences of vaccinia virus. was formed.

These chimeric genes containing the cotton sinus promoter and the AV/H and V[l envelope]-coded sequences are
It was inserted into the vaccinia virus genome via in vivo recombination. Such recombinant viruses have been identified and purified, and virus stocks have been prepared from infected tissue culture cells. It has been shown that immunoreactive AV envelope-associated proteins can be produced by these recombinant vaccinia viruses in vitro. These recombinants were tested in laboratory animals for their ability to elicit neutralizing or protective immune responses and for use as vaccines against AIDS.

(5,1, General Procedures) (5,1,1, Cells and Viruses) African Green Monkey [AfriCan pree]
n monkey cells (BSC-1 cells, ATCCN
o, a continuous line of African green monkey cells derived from CCL2B, strain B5C-40).
Dulbecco's Modification obtained from Conduit [R, Conditl, Associate Professor, Department of Main Lines, State University of New York, Buff 70-] and supplemented with 10% fetal bovine serum and 100 units/d each of penicillin and streptomycin. Eagle's medium (D) IEM was grown in Gibcol (Grand Island, NY). Human 143TK-cells (Rim, Jie, Nis.

et al., 1975, International Journal of Cancer 15:23-29 [Rh1m, J.
S,et,al,.

1975, Intl J, Cancer 15:23-
29]) is M, Botchan [8, Botchanl
(Professor, Department of Molecular Biology, UNIVERSITY OF CALIFORNIA, Berkeley, CA), and 5-promodeoxyuridine (BtldR)
The cells were grown in the above medium supplemented with 25 μg/−.

Human multiphasic cells (IRc-5) are available from the American Type Culture Collection [American Type Culture Collection].
culture Co11ection] (
ATCC NO., CCL 171) and grown in the same medium used for BSC-40.

Vaccinia virus (strain WR, ATCCNO, VR-1
19) HAR, obtained from conduit, and DME
BSC-40 cells were grown in M with 5% gamma globulin-free bovine serum and 100 units/d each of penicillin and streptomycin. TK- recombinants were selected in 143TK- cells in the same medium containing 25 μg/d of BLJdR;
and 1% Noble agar [Noble agar (
[DJFCO], Detroit, Michigan),
Plaque purified in the same cell line in DMEM containing 5% gamma globulin-free bovine serum, 100 units/d each of penicillin and streptomycin, and 25 μl/d BIJdR. Virus strain [5ta
ck] dilutions were made in phosphate-buffered saline (PBS, supplemented with 1rngM IIcI, 2 and 0.01% bovine serum flumine per gram of NaCl JF, KCI
O, 2g, NaH2PO41,59°K HP
Contains O40.2g. ) (PBSAH).

Vaccinia Virus The New York Board of Health
f Health] strain is available from Wise Laboratories [W
The variola vaccine was purified from a commercial product of variola vaccine (Lot 321501G) sold by Marie Tac Laboratories, Pennsylvania. The variola vaccine was diluted in PBSAM (see below) and B10-4011 A strain (hereinafter referred to as v-NY) was serially plaque-purified three times in B.
10-40 and used in comparison with the recombinant virus. Recombinant virus strains derived from V-NY were propagated in MRT-5000 cells.

(5,1,2, DNA Preparation, Restriction, and Modification) Unless otherwise noted, all methods used in the following procedures were described in Maniatis et al., 1982, Molecular Cloning, Cold Spring Harbor Laboratory [ ) 1aniatis et, al.

1982, Molecular Cloning, Co.
1d Spring ) (arbor Laborato
ry] (7) Instructions: Hesi, Ta Ne #3, Preparation of Plasmid DNA (Pages 86-96), Restriction Digestion of DNA (Pages 98-106) and Purification of Restriction Fragments from Low Melting Point Agarose Gels (Pages 86-96). pages 157-161 and 170), Flenow fragment Escherichia
] Reaction conditions for DNA polymerase enzyme (No. 10)
7-114), bovine intestinal alkaline phosphatase (1st page)
33-134) and ligase reaction (page 146),
Nick translation probes (109th to 11th)
Page 2> and Procedures for DNA-DNA Hybridization (pages 324-325).

(5,2, Formation of a Plasmid Vector Containing a Vaccinia Virus Promoter Linked to the Coding Sequence of the LAV/HTLVIII Envelope Gene) This article describes the formation of various plasmid vectors containing . Note that these chimeric sequences are flanked by TK DNA. These recombinant plasmid vectors were later used to insert the LAV/old tv11 envelope coding sequence into the vaccinia virus genome via in vivo recombination.

In the following sections, the LAV/HTLVIII envelope coding sequence is shown in pR3-3 (Wayne-Hobson, Niss), one of the subclones of λJ19.

et al., 1985, Cell 40:9 [Wa 1n-Hob
son, S, et, a1. .

1985, Ce1l 40:9]) and pV-envl, pV-env2. and pv
- To form env5, plasmid pGs20 (
Maquette, M9. Smith, C., L., and Moss;
B., 1984.

Journal of Pyrology [trackett,
)1. .

Sm1th, G, L, and )1oss, B,, 19
84. J, Virol, 49.857-864])
Vaccinia 7.5 contained in ρG520 was inserted downstream of the promoter.

In each of these formations, a chimeric gene (i.e. 7,5 linked to a LAV-specific nucleotide sequence)
The vaccinia promoter) is flanked by vaccinia TKm Nobuko sequences.

As explained earlier, PBS-3
'4'1. cloned into the oRI and StI sites. λJ 191. ;: Marel-containing LAV/HTLVIII
3.8.40 base pairs of D NΔ inserts EcoRI-
Consists of +5stI fragment. It is pBT-1
5s into the 5stI site of pLIc18 to form
insert the tI restriction fragment and then pBT-1
was obtained by digesting with ECORI and religating. This DNA was used to transform Escherichia coli and plasmid pRS-3 was isolated. The LAV/HTL contained in pRS-3
VIII-specific DNA t,t,LAv genome from the COR7 site located at nucleotide 5289 to the 5st1 site located at nucleotide 9129 (Wayne-Hobson, Niss et al., 1985
year, cell 40:9). See Figure 2.

(5, 2, 1, 3 of LAV/HTLVlnenv gene
Formation of a Plasmid Vector Containing a Vaccinia Virus Promoter Linked to a Coded Sequence) 5 g of pRS-3 plasmid DNA was completely digested using the restriction enzyme K pn I, and the resulting fragment was 1%
Low melting point, resolved in agarose gel. 2.68
A Kbp (kilobase pair) fragment was isolated and purified. This fragment is LAV/H Ding LV
II [nucleotide numbers 5889 to 8572, containing the sequence (5889 to 8349) encoding the C-terminal position of the envelope protein (7) LAV/I'
It contained ITLV Ill-specific DNA.

A 1 μm portion of this fragment was preliminarily treated with the restriction enzyme BamHI.
1) GS20 DNA was linearized with 0.5 μg. The ligation reaction of these two 7-ragments is
5°-GAdingCCAcCATGGTAc-3°-DHo
, 6 μ3 and 5°-CATGG Ding G-3°-DHo,
The cells were incubated at 4° C. for 24 hours in the presence of oligodeoxynucleotides consisting of 3μ3. These linkers (a) connect the Kl)III cohesive end of the 2,68 Kbp fragment derived from pRS-3 plasmid DNA to the coined HI cohesive end that is complementary to the coined HI cohesive end of the cleaved DGS20 DNA; and (b) to provide a translation initiation sequence (ATG) in the correct reading frame for the LAV/elbow LVI [I envelope gene sequence and the nucleotide sequences required for sufficient translation of the transcribed mRNA. given to. The ligation mixture was prepared using Escherichia coli strain MC10.
was used to transform 0O. Plasmid DNA from ampyridine-resistant transformants was tested for alignment of the insert and regeneration at the junction of the BamHI, NC0I and K pn 1 sites to confirm the desired plasmid, pv-env2. The structure is shown in Figure 3, in which LAV/
The carboxyl-encoding portion of the HTLVI envelope gene is located downstream to the vaccine promoter at 7.5. The LAV/HTLVIII envelope sequence consists of the starting AT supplied by the linker-DNA.
-108 to G and is located in the correct reading frame.

(5,2,2, LAV Envelope Gene) Formation of a Plasmid Vector Containing a Vaccinia Virus Promoter Linked to a 5° Coding Sequence) 5μ3 of pRS-3 plasmid DNA is N! Complete digestion with II restriction enzyme and the resulting fragments were resolved in a 1% low melting point agarose gel. A 0.82 KbD fragment was isolated and purified. This fragment contained the LAV/HTLVIII specific sequence from nucleotide numbers 5611 to 6490 (shown in Figure 2). It contains the sequence encoding the N-terminal portion of the envelope protein (nucleotide numbers 5166-6490) and 95 base pairs of LAV(7) 5° proximal untranslated sequence. This fragment was treated with Flenow fragment Escherichia coli DNA polymerase in the presence of excess deoxyribonucleotide triphosphate. The resulting plant terminus was ligated to 0.5 μg of ρG520 DNA that had been previously linearized with a 3 ma process and treated with calf intestinal alkaline phosphatase (CIAP). The ligation reaction was allowed to wait for 16 hours at 12°C. The ligation mixture was used to transform Escherichia coli strain HClOOO. Plasmid DNA from ampyridine-resistant transformants was tested for coordination of the LAV/HTLVIII insert to vaccinia virus transcriptional control sequences. The desired plasmid pv-env
The confirmed structure for l is shown in Figure 4, where nucleotide numbers 5611 to 6490 (
It is shown in FIG. ), the amino-encoding portion of the LAV envelope gene, including its own initiating ATG, is located downstream to the vaccinia 7.5 promoter.

(5, 2, 3, LAV/HTLVIII to []-1
311 Gene (Formation of a Plasmid Vector Containing the Vaccinia Virus Promoter Linked to the D Complete Coding Sequence) 2μ9 of pv-envl plasmid DNA was completely digested with the restriction enzymes Dantub, Danbetsu■, and XhoI. The resulting fragments were resolved in 1% low melting acarose glucose. S tu I and PV
Cleavage of pv-envi at UI results in one side LAV/HT
Two 4 Kbp fragments were obtained, one containing the 5° portion of LVIII and the other containing pGs20. X
hOI cleaves the 4 KbD fragment containing DO320 into smaller fragments, and thus
Vaccinia virus transcriptional regulatory elements and LAV/HTL
The 5° subarray of the V envelope encoding sequence contains 4 bρ
allows identification of the 5tuI/PVuI noragment.

This fragment was isolated, purified, and pv-env
6 created by restriction digestion of 2 Dankan I and 2 Betsu I
, 5Kbl) fragment.

The ligation mixture was used to transform S. coli strain MC100O. Ampyridine resistant transformants were selected. Plasmid DNA from individual transformants was tested for regeneration of the foot locus 1 and vul restriction sites and the presence of the parental 4 Kbp and 6.5 Kbp fragments. The desired plasmid, pv-env5, depicted in Figure 4 is a LAV/env5 plasmid corresponding to nucleotide numbers 5166 to 8349 (shown in Figure 2) linked downstream from the vaccinia virus transcriptional control element. It contains the complete envelope gene of HTLVII.

(5, 2, 4, transmembrane [transmem
LAV/HT lacking the brainet (anchor) sequence
Formation of a plasmid vector containing the vaccinia virus promoter linked to the LVIII en+4 gene) Plasmid pv-env5 DNA (>10 μg was completely digested with 1-(ind■) and the resulting fragment was digested with 1% low melting point (LHP) ) was resolved in an agarose gel. A 3 Kbp fragment containing the promoter and the AV/former LV [l env coding sequence] promoter was purified and then digested with Flenow enzyme to fill in the cohesive ends. This fragment was then pre-digested with EC0RI and
It was then ligated into the multi-site cloning vector 926, which was sequentially treated with Flenow enzyme and calf intestinal alkaline phosphatase (CIAP). pV-env
tatHindI [1 site and plasmidl 26 in S.
The ligation reaction of CORl during filling produces a stop codon in phase with the coding sequence for env. This intermediate was designated pv-env5/26.

Plasmid pv-env5/26 was used to transform S. coli) 1c100O, expanded and purified. It was then digested with BalllHI and 1-1paI, and the resulting fragments were resolved in a 1% agarose gel. LAV
2. /HrLVIII envelope coding sequence.
A bp fragment is isolated in 1, and then previously B
Plasmid 1 digested with a1llHI and βmaI
) linked to GS20. The resulting plasmid pv-e
nv7 contains a vaccinia virus 7.5 promoter linked to a LAV/HTLVVIII specific sequence starting 96 base pairs upstream of the env start codon to the lindl11 site (nucleotide number 7698) (Figure 5). This plasmid is therefore LAV/
It lacks the putative anchor sequence of the HTLVI 8nV gene.

(5, 3, Chimeric LAV/)ITLVIII env Gene Formation and Characterization of Recombinant Vaccinia Virus Two parental strains of vaccinia virus were used for the formation of recombinant virus (i.e., standard research strain). WR and v-NY, a derivative of the New York City Board of Health stock (see Section 5.1.1).

Chimeric LAV/HTLVII into vaccinia virus
Insertion of env sequences was performed on plasmid pV-env2. p
This is accomplished by in vivo recombination, made possible by the fact that V-env5 and pv-env7 are flanked by Cuccinia virus sequences that encode for the thymidine kinase gene.

Introduction of these plasmids into vaccinia virus-infected cells allowed recombination to occur between the TK gene in the plasmids and homologous sequences in the vaccinia virus gnomome.

Insertion of the chimeric gene occurs as a result of double recombination in flanking sequences. Such a recombinant will have the chimeric gene inserted into the vaccinia TK gene and will therefore be expressively TK-.

Such a TK- recombinant is a BLId that is lethal to TK+ cells but not to TK- cells.
Selected for growth in medium supplemented with R. The general principles of this procedure have been described previously (Maquette, M. 1. Smith, G.).

L., and Moss, B. 1984, Journal of Pyrology 49.857-864 [) (acke
tt, H,, Sm1th, G, L, and M
o5s, B., 1984. J, Virol,
49.857-864] ) (blank below) (5, 3, 1, Chimera LAV/HTLVIII env
Formation of recombinant vaccinia virus containing M Nobuko) 8
0% confluent African green monkey kidney cells (strain B5C-
40) 100 m dishes were infected with vaccinia virus (strain WR) at a multiplicity of infection (moi) of 0.05. After 2-4 hours of incubation at 37°C, infected cells were infected with plasmid pv-env2 or pv
-env5 DNA was overlaid with a calcium phosphate co-precipitate. The precipitate was mixed with 0.5 m of 2X DNA-CaCl 2 solution in 0.5 d of 2X He 83 solution.
was prepared by adding dropwise. (2XD
N^-CaC12 contains 20 μfj of plasmid DNA in 0.258 CaC120.5d, 2X HeBS contains 16 mg of NaCl per 1rd, KCI O,
74mg, Na2HPO4-211200, 25mg dextrose 2m9 and HEPES 10
mg at pH 7.08. ) A DNA-calcium phosphate coprecipitate was allowed to form for 30 minutes at room temperature. Four hours after overlaying the precipitate, cells were incubated with lXH.
washed once with eB5, incubated for 3 min at 37°C in the presence of 15% glycerol 2d in 1X HeBS, and then washed once more with 1X HeB5, supplemented with growth medium (10% fetal bovine serum in DHEH and penicillin and 10 each of streptomycin
0 units/ml) was incubated at 10-. After 2 days, infected cells were harvested and centrifuged (4°
CC12000X for 10 minutes). Virus strain [5tockl these cells in PBSA)
Prepared by resuspending to 11 mR followed by two cycles of freeze-thaw and three 15 second sonications.

Recombinants were grown on 1% Nobel agar (Dirco, Detroit, MI), 5% bovine serum, 25119/1ill Bud in DMEM.
Selected by plating 0.1me of a 10-3 dilution of the virus strains from above in 60# dishes of confluent human 143TK- cells overlaid with 5I/JR per dish by R. It was done. Two days after plating, cells were treated with the same ingredients as above and 0
．． The colors were colored by overlaying 2d per dish of agar medium containing 0.01% Neutral Red. Individual plaques were removed 1 day after staining and 0.1 d
Resuspended in PBSAM and aliquots (0.25 d ) of the virus suspension were supplemented with selective medium (10% fetal bovine serum in DHEH, 100 units/d each of streptomycin and penicillin, and (25 μg confluent 1 seeded in 16 Inn diameter wells (wells) under conditions of
43TK- was used to infect cells. Infected cells were collected by centrifugation and resuspended in PBS 10-Oμ, lJ containing trypsin 0.51n9/ml and EDTAo, 2ml/me. Cells were lysed by 3 cycles of incubation at 37°C for 30 minutes followed by sonication for 20 seconds each. The cell lysate was filtered into multi-well filtration branch tubes [
multi-vell filtering mani
fold (Schleser and Schuell [5c
hleicher and 5 Chuell, ? (Arlington, S.C.) was collected on nitrocellulose phycitter. The presence of LAV/former LVIII env-specific DNA sequences in these samples was determined by NA-DNA hybridization as previously described (Maquette, M1. Smith, G.).

L., and Moss, B., 1982, Proceedings of the National Academy of Sciences.
Science 79.7415 N7419[)1acke
tt, H,.

Smith, G. L., and )loss
, B., 1982. Proc, Nat'
1. Acad.

SCi, 79.7415-74191). 32p labeled plasmid pR3-3DN prepared by nick translation
A was used as the hybridization probe. Recombinants that give positive hybridization to this probe are 143 under selective conditions (medium containing BtJdR).
B10 in TK- cells twice or under non-selective conditions
-40 cells was further plaque purified once. D
After final confirmation by NA-DNA hybridization,
Virus stocks were prepared from triple-purified plaques in B10-40 cells and used for subsequent characterization.

A schematic representation of the formation of the recombinant virus is shown in FIG. 5, in which the (P→) LAV/HTLV envelope gene sequence (env) located downstream from the promoter in Botaninia 7.5 is: It is flanked by the TK DNA sequences in the cotton sinosome. The virus strain derived from the recombination between the vaccinia virus genome and plasmid pV-env5 is V-env
5 and contains the complete LAV envelope gene. In certain embodiments described in the Examples herein, V-env5 comprises the complete envelope gene and 96 base pairs of 5' proximal untranslated sequence and 223 base pairs of 3' proximal untranslated sequence. The virus strain derived from apv-env2 was designated V-env2 and LAV/HTLVJ
Most of the II envelope genes (i.e. KpnI
fragment), but a portion of the sequence encoding the first 42 amino acids of the LAV/HTLVIII envelope protein is deleted. [Aν/HT
Since the putative signal sequence of the LVl[l envelope protein is located within the first 49 amino acids of the protein, the v
-env2 should produce proteins lacking signal sequences.

Therefore, the LA produced by this recombinant virus
One would expect that V/HTLVI[l-associated proteins would not be transported to the membrane. On the other hand, complete LA
v-env5, which contains the V/HTLVIII env sequence, should produce proteins that are transported to the membrane. pv-
The virus strain derived from env7 is designated v-env7 and contains the complete N-terminus of the LAV/HTLVrfi envelope gene, while lacking sequences downstream of the lindJ1 site. In this formation, the putative transmembrane (anchor) sequence is incorrect [mi
ssing], it can be expected that the [Av/old[v■-related protein produced by this recombinant] is secreted into the growth medium. This property is advantageous for the purification of this protein for use as a diagnostic reagent or for formulation as a subunit vaccine.

(5, 3, 2, Watashini-shi AV/H-cho LVI [I
Restriction Pattern of Envelope Recombinants) DNA from the parental vaccinia virus strains WR and V-NY, and their derivatives v-env5 and v-env5NY, respectively, was previously described (Esbosito et al., 1988).
1 year.

Journal of Pyrology Methods 2:175~
180 [Esposito et al, 1981.
J, shi1r01. HettlOds, 2:175-18
01). These are restriction enzymes)
1indlll and the resulting fragments were resolved in a 0.7% agarose gel. The DNA from the parent strain WR was previously described (Defilibs, 1982).
Journal of Pyrology 43:136-14
9[[)eFi l 1ppes, 1962. J.
Virol, 43:136-149]) shows a typical restriction pattern (Fig. 2A). v-NY
The restriction pattern for WR was also similar, but different from that for WR.

The most obvious difference was in the length of )lindl[IB and C, the terminal fragments that open up the vaccinia virus genome. Recombinant V-env5
and v-env5NY are Hindm of these parent strains.
It does not have a J fragment. Instead, size 5.
Two new fragments of 4Kbl) and 3.2Kbp were observed. This observation suggests that LAV/
Consistent with the results of a biphasic sequence event that inserted the HTLVIII envelope coding sequence. [AV/HTLV
Due to the presence of the (7) Hindm site in the III sequence, two fragments were generated by the insertion. This result also indicates that LAV/H against the vaccinia virus TK gene
Confirm the localization of the TLVIII insert.

(5, 3, 3, Wakushi X 7- LAV/H Ding LV [l
Southern plot test for envelope recombinants [5o
utern Blot Analysis'> Two novel 1s in the restriction digest of the recombinant virus genome
(indl[I fragment identity was confirmed by Southern blot assay. DNA fragments resolved on agarose gels, such as those shown in Figure 7A, were transferred to nitrocellulose filters and LAV/H and hybridized to a nick translation probe specific for the V■ envelope sequence. As shown in Figure 7B, hybridization
was detected only in bp fragments. This result confirms the genome structure of the recombinant virus as shown in FIG.

(5, 4. Expression of LAV/HTLVVIII envelope-associated proteins in tissue culture cells infected with recombinant vaccinia virus) Recombinant vaccinia virus carrying a chimeric LAV/HTLVIII env gene was used to infect LAV/HTLVVIII cells in tissue culture. It was shown that HrLVI[I envelope-related proteins can be expressed. These proteins were also found to be immunoreactive with serum from AIDS patients.

(5,4,1, Identification of LAV/)'ITLVII Envelope-Associated Proteins Expressed in Cells Infected with Recombinant Vaccinia Virus Using Immunoplot Techniques) vaccinia virus or with its recombinant derivatives V-env5 or v-env2 at an moi (multiplicity of infection) of 10. Infection is 12
A period of time was used, at which point the cells were harvested, washed once with PBS, and collected by centrifugation. Infected cell pellets were prepared in Laemmli sample buffer [La
elllli sample bufferl (Raemli, Nie, Kay 0.1970, Nature 227
:680 [Laelllllll i, υ, ni,,19
70. Nature 227:6801)
1 and dissolved by boiling for 4 minutes. All cellular proteins in 75 μj aliquots of cell lysates were resolved by electrophoresis in 7-15% gradient 5DS-polyacrylamide gels.

Samples of purified LAV pillions and aliquots of mock-infected cell lysates were included as controls. The components of the gel were electrotransferred to a sheet of nitrocellulose filter. The filters were first incubated for 30 minutes at room temperature in 5IIJR with 5% nonfat dry milk in PBS, and then incubated with 5% nonfat dry milk in PBS and human serum from AIDS patients (heat-inactivated serum). No. 1:
100 dilution) for 2 hours at room temperature. The filter is then diluted with PBS at 0.05
% Tween20 (polyoxyethylene sorbitan monolaurate) and once with PBS alone. The washed filters were incubated for 2 hours at room temperature in 5m PBS containing 1% normal goat serum plus a 1:3000 dilution of goat anti-human gG-horseradish peroxidase conjugate. The same filter was washed again 5 times with 0.05% Tween 20 in PBS and TBS (0,5HN
aCl to 20mt4 Tris-HCl [Tris-
HCII, pH 7.5). The horseradish peroxidase complex bound on the filter was detected by reacting the chloro-naphthol stain with the filter for 10 minutes at room temperature in the dark (the chloro-naphthol stain was added to solutions A and B immediately before use). Solution A was prepared by mixing 4 chloro-1 in 20 yd of chilled 30% methanol.
- 60 m3 of naphthol added, solution B cooled 30 m3
% hydrogen peroxide 60 μt plus 1B3100 d. 〉. Proteins bound on the filter that reacted with antibodies in AIDS patient serum were detected by the colorant through its binding to the goat anti-human JgG-horseradish peroxidase conjugate.

The results of this analysis demonstrated that vaccinia virus recombinant v-env5 produced a family of three proteins that were specifically immunoreactive with serum from AIDS patients, as depicted in Figure 8A. It was something to show. The three proteins contain the authentic LAV/HTLV Ill glycoprotein Op, which appears to have been modified by the env gene.
150. QD'I 10 and 111p41 (Robie, W., C., et al., 1985. Science 22
8:593-595 [Robey, W., G.,
1985. Sc 1ence228:593-595
] ) had similar electrophoretic mobility.

These proteins were not produced in mock-infected cells or in wild-type vaccinia virus-infected cells. Recombinant v-env2, which has a deletion of the 5° proximal sequence encoding for the putative initiation methionine and the first 42 amino acids of the LAV envelope protein, produces a truncated-sized protein but is not present in AIDS patient serum. It is immunoreactive. Probably recombinant V-env
Translation of the env sequence in 2 is initiated from the AUG codon transcribed from the linker sequence used in the formation of this recombinant (see section 5.2.1). In a second embodiment , two cell lines (BSC-40 and HeLa) were infected with v-env5 and v-env2.
l immunoplot.

Confluent cell monolayer of BSC-40 or HeLa cells [c
onfluent monolayer] with a multiplicity of infection (m
oi), recombinant virus v-env5 or v-e
infected with nv2. 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 sulfate-polyacrylamide gel electrophoresis (SO3-PAGE), electrotransferred to nitrocellulose membranes, and reacted with stored serum from LAV/HTLVIII-seropositive individuals. Ta. Immunoreactive proteins were detected by protein A labeled with 1251 by the chloramine-T method. In Figure 8B, the 5th rune and the 5th rune
Lanes contain proteins from mock-infected cells, lanes 2 and 6 contain proteins from wild-type vaccinia virus-infected cells, and lanes 3 and 7 show proteins from cells infected with vaccinia virus.
Lanes 4 and 8 contain proteins from v-env2 infected cells. Sucrose gradient purified LAV pillion protein was used as a control (LAV) and
Envelope protein QD150. The positions of DI) 110 and 0l) 41 are as shown. Molecular weight standards were expressed in kilodaltons (d).

Three major proteins immunoreactive with pooled serum from seropositive individuals were detected in v-env5 infected cells (Figure 8B, lanes 3 and 7). The molecular weight of these proteins is 150 lj, 120 d
and 41 Kd, LAV envelope glycoprotein 1111) 150. It was similar to that of QD120 and 111p41.

Recombinant virus v-env2 is LAV/HTLVme
Although the putative signal sequence for nv has been deleted, it has molecular weights of 99 Kd, 68 Kd, and at least 3 Kd of 40 Kd.
(Figure 8B, lanes 4 and 8)
.

(5,4,2, LAV/HTLVII expressed in m'PIA infected with recombinant vaccinia virus
Identification of I-Envelope-Associated Proteins Using Immunoprecipitation Techniques) The immunoprecipitation assay described below is a method for determining whether cells infected with the recombinant vaccinia virus of the invention are specifically immunoreactive with serum from AIDS patients. It was shown that this protein can be synthesized.

1 of 100 confluent B5C-401111 cells were infected with wild-type vaccinia virus or with its recombinant resting v-env5 or v-env2.
were infected at a multiplicity of infection (moi) of At 9.5 hours post-infection, growth medium was replaced with methionine-free DMEM without serum supplement.

10 hours after infection, the medium was [35S]-methionine 1
Methionine-free DME containing 00μCi/mff
M 2rd was substituted and labeling proceeded for 2 hours at 37°C. At the end of the labeling period, the cells are P
Washed once with BS and collected by centrifugation. Cell pellets were prepared using lysis buffer (1% NP40 (polyoxyethylene (9) p-tert-octylphenol), 0.5% sodium deoxy]-lurate, 0.IHN
aCl, 0.01)1 Tris-MCI, pl-1
7, 4, 1m HEDT) and the lysate was resuspended in Etzpendorf microcentrifuge Knta.
[Eppendorf microcentrifuge
The cells were clarified by centrifugation at 1 min for 1 min.

Immunoprecipitation was performed by adding 5 μρ of heat-inactivated human serum, either from control populations or from AIDS patients, to cell lysates of 10
This was done by adding 0 μg. 1 at 4°C
After one bastion of ink for an hour, activated Staphylococcus aureus [5 taphylococcus au
reusl cells (Bansor Pincells, Calbiochem Beering Corporation, Ujola, California) Pan5orbin cells, Cal
biochem-Behring Corp, LaJ
o! 60 μg of Ia, CAl) was added and incubation continued for an additional hour at 4°C. Immunoprecipitated complexes were collected by centrifugation for 30 seconds in an Etzpendorf microcentrifuge at 4°C;
and 0.1% NP-40 and 0 in IM NaCl.
．． 01M Tris-HCl, pH 7°4 and then RIPA buffer (10mM Tris-HCl, pH 7,2,0,15t4 NaC1,1
% Sodium deoxy]-rurate, 1% Triton X 10
0[Dingrit.

n X 1001 (polyoxyethylene (9-10)
p-tert-octylphenol), 0.1% sodium lauryl sulfate). The washed immunoprecipitates were resuspended in 50 μp of Laemmli sample buffer, boiled for 1 minute and centrifuged for 1 minute in an Elapendre 7 microcentrifuge.

The immunoprecipitated proteins present in the supernatant were 15
%5DS-polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie blue dye, treated with sodium salicylate (30 minutes at room temperature in 1H sodium salicylate) and dried for fluorography.

The results of this analysis, as shown in Figure 9A, show that the recombinant V
-env5 was shown to synthesize a family of proteins that are specifically immunoreactive with AIDS patient serum. These proteins have apparent molecular weights of 160, 140°, 120.42 and 40 kilodaltons (KO), and LA
V/HTLVIII t: Apparent molecular weight of envelope-related proteins reported (Robbie, Double, G. et al., 1985, Science 228: 593-
595 [Robey, W. G. et al., 1985
, 5science228:593-5951). These proteins were not expressed in either mock-infected or wild-type vaccinia virus-infected cells, nor were they immunoprecipitated by control human serum. In v-env2 infected cells, a truncated (incomplete) protein with an apparent molecular weight of 95 KD was produced and recognized by AIDS patient serum. -This truncated form of the LAV/elbow LVIII env-associated protein is
It appears to start with an AUG codon encoded by a linker sequence located 5° proximal to the AV/elbow tvB insert.

(5,4,3,')')Sini7- LAV/HTLVI
II recombinant virus produced [Ashi/elbowL
VIII-Glue, Cosamine Labeling of Envelope-Associated Proteins) The radioimmunoprecipitation described below demonstrated that the Botany LAV/HTLVIn recombinant virus of the present invention produced glycosylated envelope proteins.

) (ela @ cells were mock infected (see Figure 98, lanes 1 and 5) or infected with wild type vaccinia virus (lanes 2 and 6), recombinant vaccinia virus (lanes 2 and 6), respectively.
nv5 (lanes 3 and 7) or recombinant null V-en
v2 (lanes 4 and 8) separately. In all cases, the multiplicity of infection (mo) was 50 pfu/cell.
i). 3H-glucosamine (23 mCi/my
0.25μci, Amersham [Amersham]
) was added to the culture medium 4 to 16 hours after infection. Cells were washed twice with phosphate-buffered saline and 0, IM NaCl, 0, OIM Tris-HCl.
pH7,4,1111M EDTA, 1%NP-40
(polyoxyethylene (9) p-tert-octylphenol) and 0.5% sodium deoxycholate. Aliquots of cell lysate were prepared using normal human serum (lanes 1-4) or LAV/H
Stored serum from TLVI seropositive individuals (lanes 5-8)
mixed with either. Immunoreactive proteins were precipitated by immobilized Staphylococcus aureus cells bearing protein A, resolved with 5DS-PEGE and detected by fluorography.

The results of radioisotope labeling with glucosamine showed that the v-envs recombinant-produced protein was similar to the LAV/elbow [v■ envelope protein, but also glycosylated, as shown in lane 7 of Figure 96. It was shown that Differences in glycosylation patterns indicate that LAV/HT
This could explain the slight change in electrophoretic mobility of the recombinantly produced protein when compared to the LV ■ pyrion glycoprotein.

As expected from a protein lacking a signal peptide, the nitrogen bond with 3H-glucosamine [N-1inked
] No glycosylation was observed in v-env5 infected cells (Figure 96, lane 8).

(5, 4, 4, '7 Kushini 7- LAV/HTLVII
LAV/HTLV produced by I recombinant virus
Pulse-Chase Immunoprecipitation Analysis of I Envelope-Associated Proteins)
1) 110 and transmembrane protein [t
transmembrane protein(It)
It has been proposed that 41 is the derived precursor.

Papasu Chase ([pulse-ch
Asel, pulse chase)'' immunoprecipitation assay showed that vaccinia-LAV/HTLVI recombinant produced proteins 150KD, 120KD and 41KD proteins were compared to authentic LAV/HTLV nogp150.QD''I.
10 and Qr) have a precursor-product relationship similar to that proposed for 41.

A confluent cell monolayer of He1a cells was infected with wild-type vaccinia virus (see Figure 9C, lanes 1-6).

), recombinant V-env5 (lanes 7-12) or V-env2 (lanes 13-18). In all cases, the multiplicity of infection was 50 pfu/cell (
moi). At 10.5 hours postinfection, cells were
Labeling was performed with 100 μCi/rnl of 5S-methionine (>800 Ci/mmo 1, Amersham) for 15 minutes. At the end of the labeling period, cells were submerged in pre-warmed chase medium (3 m'j in Dulbecco's modified Eagle's medium).
/ml monomethionine, 5% bovine serum, and 100
It was washed with >2 d of units/d penicillin and 100 μ9/d streptomycin and re-fed with 1 rnl of the same medium before being returned to the incubator. At various times thereafter, the cells were washed and lysed as previously described in Section 5.4.3, and the proteins from the cell lysates were isolated from the LAV/elbow [v ■ pooled from seropositive individuals]. immunoprecipitated with serum. Immunoprecipitated proteins were resolved by 5DS-PAGE and detected by fluorography. The duration of each trace was 50 hours (lane 1
゜7.13); 0.5 hours (lane 2.8.14);
1 hour (lane 3.9.15); 2 hours (lane 4.10.16); 6 hours (lane 5.11.17)
) and 12 hours (lane 6, 12.18).

The results shown in lanes 7-12 of FIG.
, 1111) 110 and Qr) 41 showed a similar precursor-product relationship. Processing of the 150KD protein is slow and insufficient in He1a cells as less than 50% of the radioactivity in the 150KD protein was traced to the 120KD and 41KD proteins by 6 hours after pulse-labeling. I think that the. Preliminary results showed that this processing is more efficient in certain types of human peripheral blood cells infected with the same recombinant virus. These experiments also included LAV/HTLVIII en
The env sequence in V-env, which lacks the putative signal sequence for v, is expressed as an unmodified 85KD precursor (780 amino acids), which is processed to a 99KD intermediate and cleaved into 68KD and 40KD polypeptides. (9th C.
Figure, lanes 13-18).

(5,4,5, L in the growth medium of cells infected with vaccinia-LAV/HTLVVIII recombinant virus)
Presence of AV/HTLVIII Envelope-Associated Proteins) The radioimmunoprecipitation assay described below shows that the immunoreactive proteins produced by the recombinant vaccinia viruses of the invention are similar to those of the authentic LAV/elbow tvB envelope glycoproteins. showed that it can be expressed and processed in a pattern.

A confluent cell monolayer of 1-1ela la cells was incubated with mock infection (
(see Figure 9D lane 1) or wild-type vaccinia virus (lane 2), recombinant-13, respectively.
9 integrated v-env5 (lane 3) or v-env2 (
Lane 4) was infected separately. In all cases, the multiplicity of infection (moi) was 201) fu/cell. Cells were incubated with 358-methionine (800Ci/mm
o 1 or more, Amersham) at 100 μCi/m for 10-12 hours post-infection. At the end of the labeling period, the medium is removed and the LAV/old LVll
i clarified by centrifugation at 12000×9 for 2 min before use for immunoprecipitation with pooled sera from seropositive individuals. Proteins from infected cells immunoprecipitated by the same serum are labeled as a “pellet”.
Those from the culture medium are shown in the sections labeled "Soup."

At the same time as expected for LAV/HTLVI gI) 110, recombinantly produced 120KD protein was also detected in the infected cell culture medium (Fig. 9D, lane 3).
) These results indicate that the recombinant virus v-env5 is
It was shown that the AV/HTLVIII env gene can be expressed and produce immunoreactive proteins that are processed in the same pattern as the authentic LAV/HTLVVIII envelope glycoprotein.

As expected for a protein lacking a signal peptide, no LAV/HTLVIII envelope-associated polypeptide was detected in the v-env2 infected cell media (Figure 9, lane 4).

On the other hand, cells infected with v-env7 expressed a 130 KD truncated protein. This protein lacked the C-terminal 217 amino acids containing the anchor sequence of the [^V/HTLVI envelope protein. This truncated protein (op130) was o
pllo or its precursor gp150 was secreted into the growth medium (Figure 9F). At the same time, gp13
0 is one that is not fully processed to QDll 0 even though the cleavage site is still present on this truncated protein snow.

(5, 5, Immunity of cotton cininium LAV/former LVII env recombinant virus) A recombinant virus carrying the chimeric LAV/HTLVIlj env gene has been found to be LAV/LAV/formerly in two strains of mice and a type of flame primate. We have shown that it is possible to elicit a primary immune response against HTLVIII.

(5, 5, 1, in mouse (7) 9 Cucinia LAV/
Immunogenicity of HTLVIIIenv recombinant) Recombinant vaccinia virus v-env5 and v-env
The immunoreactivity of the protein expressed by 2 was examined. The experiments outlined below demonstrate the ability of these recombinant viruses to elicit an immune response against the major glycoprotein of [Av/elbow].

Two strains of mice, one inbred (c57B16J)
and one is a distantly related line (1 (R)).
is a recombinant virus v-env2 or v-env5
was inoculated with. All animals were 5-7 at the time of inoculation.
He was a week old. Four routes of inoculation were used.

Namely, scrotum, tail sting, intranasal and intraperitoneal.

For heel inoculation, 25μ of recombinant virus, 1! (5xlO6pro) injection. For the tail rash, the skin was roughened at the base of the tail using a bifurcated needle, and 10 μg of recombinant virus (2x
This was done by applying lO7pfu) onto the scarred surface. Intranasal inoculation was done by placing 10 μρ (2×10 7 pfu) of recombinant virus in the nose of the mouse and allowing the mouse to inhale the inoculum. lI
! Intracavitary injection of recombinant virus 101 into the peritoneal cavity of mice
1.1! (2xlO7pfu). All virus strains and dilutions are listed in Section 5.1.
Prepared as described in Section 1. Serum samples from individual mice were collected at 2-week intervals post-inoculation and subjected to enzyme-linked immunosorbent assay (ELISA) as described below.
and Western blot analysis.

Seroconversion of mice immunized with vaccinia AV/HTLV Iu env as shown by 5,1,1,1,ELtSA ELISA data on 6-week serum samples are summarized in Table 1. Recombinant dormant v-env2 and v-env5 seroconverted 95% and 100% of 057B16J mice, respectively. For ICR mice, the seroconversion rate was 44% for V-env2 and for v-env5.
The percentage was 100%. The highest mean ELISA titers as well as overall seroconversion were given in mice immunized by tail barb.

(Margins below) Serum conversion of mice inoculated with recombinant virus carrying the first person chimera LAV/HTLV II env gene Recombinant virus Route of inoculation Sero conversion of inoculated mice I CRC57B16J V-env2 Foot vi3/3 415
Tail Random Sting 3/3 415 Intranasal
0/3 415 Intraperitoneal Not performed
315V-env5 Foot kick 4/4
515 tails of random stabbing 3/3 51
5 Intranasal 3/3 415 Intraperitoneal
Not performed 515* Seroconversion measured by ELISA as described in the text is expressed as the number of seroconverted mice relative to the total number of mice inoculated in each group.

ELISA data was obtained as follows.

That is, purified, inactivated LAV/HTLVIII virus particles were dissolved in carbonate buffer (50 m), sodium carbonate, p.
H9,6) in a 96-well microtiter plate [96-well microtiterplate].
el (10μJ)/well inactivated LAV/HTL
Contains 2 μg of VII O. ) was added.

Binding was continued overnight at 4°C. Unbound protein was aspirated and initially added to 200 μl/well of PBS.
+5% skimmed milk powder and additionally 4% sucrose solution 300
μm/well was washed. After removing the excess sucrose solution by aspiration, the plates were dried at room temperature (1 hour). Next, 5 μm of PBS containing 2.5 μt of mouse serum sample was added to each well and reacted for 1 hour at 37°C. At the end of the incubation period the wells are 5
Washed twice with PBS + 0.05 Tween 20 (polyoxyethylene sorbitan monolaurate). Goat anti-mouse radish peroxidase 50 μJl (PB
S + 1:5,000 dilution in 0.05% Tween 20) was added to each well and incubated at 37°C - 14'6
- The reaction was carried out for 45 minutes. After washing 5 times with PBS + 0.05% Tween 20, citric acid-〇-phenylenediamine peroxidase [Citrate-
o-phenylend iamine-perox
ide1M quality was added. The substrate was made as follows. That is, 0°294g sodium dihydrate + 0.5
379 Sodium phosphate dibasic crystal
crysta+ was dissolved in 10 d of water and adjusted to I)H5 with hydrochloric acid. 2 μp of 30% hydrogen peroxide and 4 IQ of O-phenylenediamine were added just before use.

Plates are incubated for 30 minutes in the dark at room temperature. The reaction was stopped by adding 100 μ'' of 1,3N sulfuric acid to each well and
The absorbance of the reaction mixture at 0 nM was measured.

The results in Table 1 are expressed as the ratio of blood positive mice to the total number of mice inoculated. Serum samples were collected at 6 of inoculation.
They were collected after a week and analyzed by ELISA. Serum samples from five uninoculated mice were used as comparison controls. Control -147 = Mean ELISA titer for group is ICR and C57
0.021±0.00 respectively for B16J mice
5 and 0.017±0.010. Serum samples from immunized mice that gave ELISA titers 3 standard deviations higher than the control group were scored as positive.

(5, 5, 1, 2, Serum-converted mouse-produced antibodies against authentic LAV/HTL envelope glycoproteins shown by Western immunoplot assay) Mice inoculated with recombinant virus showed authentic LAV/HTLVII.
Serum samples from immunized mice were analyzed by Western immunoblot techniques as follows to demonstrate production of antibodies against type I envelope glycoproteins. 5-7 week old male late-onset mice (C57
B16J), Jackson Laboratory [Jackso
Animals were immunized by a tail barb consisting of administering 10 μg of inoculum containing 2×10 7 pfu of recombinant virus to an epidermal detachment created at the base of the tail using a bifurcated needle. The animals are
Blood was collected retroorbitally 8 weeks after inoculation, and serum was stored frozen until use. Phosphate buffered saline (0.2%
50 in NP-40 and 3% skim milk powder added
(Alicon of 1 diluted serum sample is SDS-
It was resolved by PAGE and reacted with LAV/HTLVII virus particle proteins immobilized on nitrocellulose filters by electrotransfer. L recognized by these sera
AV/HTLVIII protein was detected by goat anti-mouse immunoglobulin conjugated to alkaline hostase.

C57 inoculated with recombinant virus by tail barb
8. from B16J mice. The weekly serum sample results are shown in FIG. All animals immunized with recombinant virus were infected with LAV/HTLVIII envelope glycoprotein o
An antibody that reacts with p41 was produced. Sera from some animals immunized with V-env5 were also 01)1
It also recognizes LAV/HTLVII 50 and QD'I 10 of this recombinant virus.
This demonstrated the ability of I to elicit immune responses against all major glycoproteins.

(5, 5, 2, Watacinia LAV in fire primates/
Immunogenicity of the HTLVII recombinant v-env5 The immunogenicity of the vaccinia LAV/HTLVIII recombinant V-env5 was studied in two flammoid primate species.

That is, the macaque fasciculae [)facca
It was shown that v-env5, described in the following passages, is capable of eliciting humoral and cell-mediated immunity in both species.

(Humoral responses in macaque monkeys immunized with 5,5,2,1,v-env5)
ca], short-tailed monkey), macaque fasciculae [)i
acca-fasciularies] was used to study the immunogenicity of the cotton cininia LAV/HTLVIII recombinant v-env5. All animals lack antibodies to vaccinia virus and LAV/HrLVIn;
and previously screened for the absence of simian [31m1anlT-lymphocytes or simian AIDS virus or antibodies to these viruses. Four monkeys were inoculated with v-env5 2x108 pfu and four with 2X 107 pfu of the same virus. −
One animal received 2X 107 pfu control recombinant virus (vaccinia-herpes simplex gD1 recombinant V-H3).
pD1). All animals were inoculated by skin pricking. Serum and heparinized blood samples were collected prior to inoculation and at 4, 6 and 8 weeks post-inoculation. Ten weeks after the first inoculation, all animals received a second inoculation of 2×10 8 pfu of the same virus. All animals showed self-healing skin lesions and normal physiological indications of vaccinia infection throughout the course of the experiment. Fluid responses were analyzed by ELISA and Western blot assay.

For ELISA analysis, serum samples were mixed with 3% nonfat dry milk and 0.2% NP-40 (polyoxyethylene (9) p
-tert-octylphenol) in PBS containing
L diluted two-fold and immobilized on microtiter wells.
Reacted with AV/HTLVIII virus particle protein. The ELISA procedure was identical to that described in Section 5.5.1.1, except that the second antibody used was a goat anti-human antibody conjugated with horseradish peroxidase. . The results shown in Figure 11 show that only two animals seroconverted after the first inoculation, but all animals seroconverted after the second inoculation.

To determine what antibodies were produced by the immunized animals, serum samples collected 4 weeks after the secondary vaccination were analyzed by Western blot. Two fixed formats were used for optimal detection of two envelope glycoproteins, gp41 and Opl 10. Dp
For optimal detection of 41, serum was diluted 50 times in PBS plus 3% unpasteurized milk and 0.2% NP-40 and purified LAV/HTLVIII immobilized on nitrocellulose filters. It was allowed to react with virus particle proteins for 1 hour at room temperature. Filters were incubated with alkaline phosphatase-conjugated goat anti-human immunoglobulin antibody (Zyme) at a 1:2000 dilution in PBS plus 0.2% NP-40.
dl, California).

For gpllo detection, serum was diluted 50 times with 3% pasteurized milk in PBS and purified LA immobilized on a nitrocellulose filter.
reacted with V/HTLV virus particle proteins. Filters were washed with 0.05% Tween 20 in PBS and further reacted with alkaline phosphatase-conjugated goat anti-human immunoglobulin antibody (Zymed, CA) at a 1:2000 dilution in PBS buffer. I was made to do it. In each procedure, after the final wash, O, IM NaCl and 511
0.1M Tris(p)f containing 1MMQ Cl2
The second antibody reaction was performed in 9.5). The antibody bound to the antigen on the Suiltor was dissolved in 0.1M Tris (pH 9).
,5) 0.1M NaCl, 5mM MgCl
, 0°33m5/ld bromo-chloro-intris phosphate and 0.33m5/ld bromo-chloro-intris phosphate. 1711 Fl/ml nitro-
It was detected by reacting the filter with a solution containing blue-tetrazolium. After the filters were allowed to react with the chromogen, they were rinsed with water, air dried and photographed.

As shown in Figure 128, all animals that received the two vaccinations showed strong antibody responses with DI)41. In addition, four of these animals also produced antibodies that strongly reacted with Ωρ110.

The results shown in FIG. 12A also show that the recombinant V-env
5 shows that it is possible to produce LAV/LVIII-specific envelope antibodies in macaque monkeys.

(Immunization with 5,5,2,2,v-env5';5at
= cell-mediated immune response in macaques) Peripheral blood lymphocytes (PBLs) contain V-env5 or herpes simplex virus glycoprotein trade D (V-H8VQdl).
Heparinized blood of macaques obtained 4 weeks after secondary inoculation with a control recombinant cotton cinia virus expressing
Isolated by paquel centrifugation. PBL is
Additionally, 8 macaques vaccinated only once with v-env5
1 and from a macaque that was not vaccinated. PBLs were suspended in RP) I11640 medium (GIBCO, Grand Island, New York) supplemented with 10% heat-inactivated normal human serum and 1 x 105 in a final volume of o,iy medium. PBL of
were transferred into the wells of a round bottom 96-well plate. Each well contains ultraviolet (UV) light-inactivated v-env5.
(1×106 pfu/me before υV inactivation),
Alui L;! The supernatant of LAV/formerly LVII-infected CEM cells (an HLA OR-negative T-cell leukemia line) was purified by two cycles of sucrose gradient centrifugation. Non-divided LAV/H and VI[I (1#Ig/d, approx. 1
×105TCID50) was added. After 6 days of stimulation, each PBL well was injected with 1 μCi for 6 hours.
3HTDR (3H-thymidine, New England Nuclear)
,? (Boston, Sask.), cells were harvested, and the Cpm of incorporated 3H-Td R was determined as the average of four replicate wells. The stimulation index was calculated by dividing the cpm of 3H-TdR taken up into stimulated cells by the cpm taken up into unstimulated cells.

(Left below) Macaque 67 after immunization with a recombinant virus expressed in the second person LAV/HTLVVIII envelope glycoprotein V-env5 1,586 7.46
5 4.7 60.415 3B, 168 V-
env5 2,245 9,075 4.0 2B,
638 12.874 V-ellV5 1,
5B5 4,732 3.0 21,487 13.
675 V-env5 581 18,645
14.9 37,847 14.176 V-e
nv5 2,479 5.987 2.5 3B,
657 14.880 V-env5 1,0
77 13,922 12.9 24,752 23
．． 081 V-env5 965 3,965
4.1 40,572 42.082 V-
env5 2,581 8,580 3.3 46,
847 18.173 V-H3VgDI
B12 553 1.0-5B, 217 91.
926 None 2,911 1,82
2 1.0~2,240 1.0~=15
7 - As shown in Table 2, all macaques, including macaque number 81 that received a single vaccination of v-env5, proliferate in vitro in specific response to LAV/elbow Lv stimulation. Lymphocytes were produced.

In contrast, vaccinia-herpes simplex recombinant V-
11 Lymphocytes from animal 73 that received SVODI responded solely to stimulation with vaccinia virus and LAV
/HTLVII did not respond. Unimmunized control animals did not produce lymphocytes responsive to either antigen. Furthermore, v-
Lymphocyte responses from macaques immunized with env5 were comparable to those from macaque 73 vaccinated with V-H3VODI. These results showed that expression of the LAV/elbow LVIII gene by recombinant viruses in macaque monkeys did not suppress TIa vacuole-mediated immune responses in vitro or in vivo.

Any subpopulation of lymphocytes from immunized macaques
To determine whether stimulated by V/HTLVIII, we examined stimulated PBL by two-color immunofluorescence assay for the expression of IL-2 receptors present in activated TIII and 8H cells. CD4 and CD8 antigens are present in the cells, and CD2
0 (Bp35) antigen is present on B cells. Third
The results shown in the table show that almost all of the virus-stimulated PBLs that express the IL-2 receptor also express CD4 or CD8 antigens, but not CD20 (Bp35
) are co-expressed in only 1.5% to 2%. This result indicates that the stimulated lymphocytes are ■ cells.

(Margin below) 1 160 - The results in Table 3 were obtained as follows.

That is, PBL is interleukin 2 receptor (J
Algal red-conjugated monoclonal antibody 2A against L-2R)
3 (Becton-Ditzkinson Inc., Mountain View, California)
Dickinson, Inc.) fountain
VieW, CAl) and a fluorescein-conjugated, monoclonal antibody against CD20 (Genetain Inc. Systems Corporation, Seattle, WA [
Genetic Systems Corp, 5eat
tle, WAI), G10-1 for CD8 (Genetic Systems - Poration, Seattle, WA), or T4a for CD4 (Ortho Diagnostics Corporation, Rariton, New Chassis);
cs, Raritan, N.

stained at the same time. Antibody is modified FAc3I
V sorter [FAc3IV 5orterl (Becton-Ditzkinson Mountain Viny, California) -[ecton-oickinson, MOUn
The samples were analyzed by flow rate microfluorometry using a sample (in View, c, Al) and were used at predetermined saturation concentrations by titration in bulbs. quantitative 2
Color analysis was previously described in detail (Ledbetter, J. E., et al.

1984, Perspective Vectors in Immunogenetics and Histocompatibility 6, 11.
9-129 [Ledbetter, J.A., et.a.
l,, 1984. Perspectives in I
mmunogenetics and Histoco
mpatability, 6119-129]). The forward and right angle scatter gate was adjusted to include lymphoblasts and a substantial number of small lymphocytes.

Values are for total IL-2R±cells, and CD4
, CD8 or CD20(Bl)35).

Upon antigen stimulation, T cells activate natural killer cells that can promote the analysis and/or proliferation of T cells and B cells, and can lyse cells infected by various viruses, including the AIDS virus. It produces IL-2 containing lymphocytes, which can be converted into proteins. We therefore demonstrate that T cells from vaccinated macaques are LAV/HTL.
We asked whether IR-2 would be produced upon stimulation by VIII.

Two experiments were performed for this purpose. For experiment 1, PBLs were isolated from heparinized blood of macaques 4 weeks after secondary iron immunization with v-env5 or V-H3VgD1 produced by the WR strain of vaccinia virus. For experiment 2, 2X 10” p
fu v-env5, V-H3Vg old or v-envS recombinant (V-e
nv5NY) was isolated from a macaque 4 weeks after the first subcutaneous immunization. PBLs were suspended in RP)IIi640 medium supplemented with 10% heat-inactivated normal human serum, and 2x105 PBLs were transferred into the wells of a round-bottom 96-well plate. UV light inactivation v-env5
(IX 106 pfu/d before UV light inactivation
>Matahali LAV/HTLVIII (1μ9
/d) was then added to the wells. Two days after stimulation,
Supernatants were collected from replicate wells and washed with I'-2 for 24 hours prior to analytical testing.
L-2-dependent CT LL-2 cells (Dr. S. Siris, Immunox Corporation, Seattle, WA [
Kano, G11lis Immune Corp.

5eattle, W, Al〉
were tested for their ability to support the growth of. During the last 6 hours of incubation with the supernatant, cells were labeled with 3H-TdR and the amount of 3H-TdR incorporated into the cells was measured. The units of IR-2 activity present in the supernatant are determined by the standard curve obtained by testing the effect of recombinant human IL-2 (given by Dr. Gillis) on the proliferation of C-Ding LL-2 cells. It was calculated to be drawn from. The results are shown in Table 4.

(Margin below) Torture IR-2 production by PBL from isolated macaques.

68 v-env5 0 16.
0, 124.074 v-env5
0 9.0 52. '073
V-H3VIIIDI O010B, 02
6 None Q
0 0° Experiment 2203 v-env5 0 14.4
58.805 v-env5NY O1B, 8
33.349 v-env5NY O7
,126,752v-env5NY O2,4,27
,659v-hsvjot o O27,6
26 None 0 0
027 None 0
0 0 The results of Experiment 1 in Table 4 show that supernatants from V-env5 or LAV/HTLV[l stimulated PBLs were obtained from macaques immunized twice with v-env5. , IL-2-containing, as shown by their ability to induce proliferation of C-Ding LL-2' cells, an IL-2-dependent cell line. Similarly, experiment 2 in Table 4
IL-2 was produced from macaque 3 immunized once with v-env5 and from a similar recombinant (
env5NY, see section 5.3) “vaccine’”
V-e from all three macaques immunized once with strain
nv5 or LAV/IITLVIII stimulated PB
It was detected in the supernatant of L. On the other hand, vaccinia H.
Macaque 7 immunized with 3VgDI recombinant virus
PBLs from 3 and 59 produced 2(7)mi IL-2 after stimulation with V-env5te but not after stimulation with LAV/HTLVII. Unimmunized macaques 26 and 27 produced no detectable IL-2 after stimulation with either LAV/HTLVI or recombinant vaccinia virus. Because primary T1'Jll cells with helper/inducer activity produce l-2 after antigen stimulation, these results demonstrate that LAV/elbow LV in macaques immunized with recombinant virus.
In addition to their substantial role in the differentiation of B lymphocytes to produce antibodies against the 0LAV/HTLVVIII envelope antigen, which indicates the presence of helper T cells that recognize I[I], these IL-2-producing Dye cells may be involved in the differentiation and/or proliferation of effector cells that can kill virus-infected cells, such as cytotoxic T lymphocytes, natural killer cells, and the like.

(Humoral responses of chimpanzees subjected to 5,5,2,3,v-env5NYr immunization).
erlV5111Y, the ``v-env5 r vaccine'' strain. - animals are v-env5
Consisting of the same parental cotton sinus strain (V-NY) as NY, and with the same titer, the cotton sinus-herpes simplex QD recombinant strain V
-H3V (within 101 NY). All animals received a secondary vaccination of the same dose 8 weeks after the primary immunization. Serum samples were collected once every 2 weeks after the next immunization.
ELISA and Western plot [W
eStern blOt] LAV/HTLVIII
- Assayed for specific antibodies. All animals exhibited self-healing skin lesions and normal physiological indicators typical of vaccinia virus infection throughout the course of the experiment.

The ELISA method for chimpanzee serum was identical to the method used for macaque serum.

The results shown in Table 5 are consistent with both experimental animals (124 and 149
) showed seroconversion by week B after the first immunization, and antibody levels continued to rise after the second immunization. The comparison animal (134) remained seronegative.

(Left below) LAV/HTLV detected in immunized animals
■ To demonstrate that the specific antibody was directed against the envelope glycoprotein sequence, a Western plot (We
The same serum was analyzed by the tern blot method. The method used was optimized for (JD4'!) antibody detection and was identical to that used for the analysis of macaque serum. We show that the LAV/HT, LV-specific antibodies produced in the body are in fact directed against envelope glycoproteins and that these antibody levels increased after the secondary immunization.

(Cell-mediated immune response in chimpanzees immunized with 5,5,2,4,v-env5NY) Section 5.5.2
．． ．． peripheral blood isolated from the same animal described in Section 3;
Lymphocytes (PBL) were used to demonstrate cell-mediated immune responses in these animals. PBL was administered with Ficol I-hypaque 4 weeks after the first inoculation with recombinant vaccinia virus.
It was isolated from heparinized blood by centrifugation. P, B
RP supplemented with 10% heat-inactivated normal human serum and penicillin/streptomycin in plates with 96 wells at 1 x 10 cells per well.
Inoculated into M11640 medium. Non-splitting IAV/HTL
VII [(5 μg/IId or purified LAV/old [ν
LAV/HTLV envelope glycoproteins (1 μg/m!) isolated from lentil lectin chromatography were then added to the wells. After 6 days,
Thymidine incorporated into the cells was measured by liquid scintillation counting.

As shown in Table 6, lymphocytes from both animals (124 and 149) immunized with v-env5NY
LAV/old [V] proliferated in response to stimulation with either pilions or purified envelope protein (env). On the contrary, vaccinia-herpes simplex recombinant V-H3
qDINY The receiving animal 134 is LAV/HTLV
It did not produce lymphocytes that recognized I.

PBL from all Chipanzees showed high levels of 3H-thymidine incorporation (38,870-10
9,790 cpm) and also showed high levels of 3-day-thymidine uptake (42,172-12.067 cpm) after stimulation with x-irradiated pooled xenograft human PBLs. PBLs from non-immunized chimpanzees did not proliferate in response to vaccinia virus stimulation, whereas V-
PBL from env5- and V-H3VpDINY-immunized chimpanzees showed strong proliferation in response to vaccinia virus.

Furthermore, the results shown in Tables 1 to 4 and Figures 11 to 13 show that (a) recombinant vaccinia virus v-env5
and its corresponding vaccine strain V-env5NY
can elicit LAV/HTLVJI [specific humoral and cell-mediated immune responses] in two species of flammoid primates, namely macaques and chimpanzees, and (b) the recombinant virus can be used in vitro [1nvitrol or in vivo]. This indicates that the cell-mediated immune response was not suppressed in vivo.

(5,6, Reduced Neurotoxicity of Recombinants Formed with the v-NY Strain of Vaccinia Virus) Aliquots of virus containing 5X1071) fu at 25-50 ufJ were intracerebrally administered to groups of 5 mice. (ICR strain). Mortality was assessed 10 days after inoculation.

Deadly double inoculation of mice intracerebrally inoculated with the first cotton cininia recombinant Dead/number of injected v-N
Y 015v-env5
NY 015v-env2N
Y 015V-env7NY
O15 Small Digestive Vaccine (Wy
eth) 315v-env5
515V-env2
515v-env7
515 Salt solution 015M
The results shown in Table 7 show that plaque-purified tissue culture-derived vaccinia virus (strain V-NY) and its derivatives v-env5NYSv-env2NY and V-env
7NY shows reduced neurotoxicity compared to WRa and its derivatives. new york city board
Since the Op Health strain has been used as a scar vaccine, recombinants based on this strain should be more suitable for developing vaccines for use in humans.

(6, Example: Baquiulovirus gag recombinant) In the following example, various plasmid vectors are AcN
LAV/1 located downstream with respect to the transcriptional control sequence of PV
A chimeric gene containing the tTLVIIl (llao coding sequence) was formed.

Ac NPV polyhedrin promoter and LAV/
The HTLVm aac+coding sequence chimeric gene was inserted into the AcNPV genome through in vivo recombination. Such recombinant viruses have been identified, purified, and virus stocks prepared from infected tissue culture cells. It was found that immunoreactive [^V/H and v■QaQ-related proteins can be produced by recombinant AcNPV in vitro. Cells infected with recombinant virus are
It was found to exhibit positive immunofluorescence. Ultimately, lysates from cells infected with recombinant AcNPV
ELISA (E) when tested with serum from patients with AIDS
It was positive in the LISA) assay. A detailed description of each step in this practical IM aspect of the invention is provided in the sections below.

(6,1, General Procedures) (6,1,1, Cells and Viruses) Spodoptera frugiperda (5podoptera
frugiperda) cells, clone sf9 are available from the American Type Culture Collection [A-Ding CC
No. CR11711), 3.39 m/A yeast oleate (type D [Dircol), 3.3 gm/1 lactalbumin hydrolyzate (Difco), 10% fetal bovine serum, 0.06 g111 /fJ penicillin and 0.19m
Grace's Antheraea medium (Gibco, Cage Biological [GibCO, C.
a;o+ogica+sl) (Ding NM-F
H medium>. Cells were grown at 28°C. Autographa cali
f.

rnica) nuclear polyhedrosis virus, Dr. Roy Miller.

department store and of genetics and
Endomorosis, University of George 7.
Athens, Georgia 30602 (oepartmen
of Genetics and Entomol
ogy, tJuniversity of Georgi
a, Athens, GA 30602)], 1
Sf9 containing % agarose and 0.8XTNM-FH
Plaque purified on cells. Virus strain is TNM-FH
diluted in.

(6,1,2, DNA preparation, restriction and modification) Restriction enzymes were purchased from Bethesda Research Laboratories [Beth
esda Research Laboratory
Restriction digestion conditions were as directed by the manufacturer.
II DNA・1g1j merase clenounonogumene 1-1.1, bυIII M tris-MCI (
1) 87.4), 6mM MCICj2, 10mM 2
- used at a rate of 1200 units/me in mercaptoethanol for 30 minutes at 37°C.

Viral DNA for transfection was obtained from Miller, L., K. 0. Mirror D, W, and Surfer, P, [)filter, L, to.

) filter D, W, and 5afer, P,]
Unoccluded virus of wild-type virus (
NOV particles were pelleted from the supernatant by centrifugation at 90.0 OOX 9 for 1 hour at 5 °C through a cushion of 25% sucrose, 5111 M NaCp and 10 mM ED. It was done.

Remove the supernatant and collect the virus pellet at 0.5 In! ! Dissolution buffer y--(10m) 1 Tris, pH 7,6,1
0m)I EDTA, 0.25% 5OS). After the virus pellet was resuspended, proteinase K was added to a final concentration of 500μ3/ltd! and incubated overnight (approximately 16 hours) at 37°C with necessary agitation.

DNA was extracted with equal volumes of phenol:chloroform:isoamyl alcohol (25:24:1).

Transfer DNA to 1/10 volume of 3M sodium acid solution (pH 5)
and 2 volumes of cold ethanol were added to precipitate at -20'C. After pelleting, the DNA was washed with 70% ethanol, dried, and diluted with 10 ml of Tris, 1 ml of ED before use for transfection or restriction enzyme analysis.
TA, pH 7.5).

Plasmid DNA following Summers, M.

Du and Smith, G., E. [SUmlle
rS, M, D.

and Sm1th, G, E, ] Maxiprep [
maxprep] method: 1 ml of Luria broth (Luria B) supplemented with appropriate antibiotics.
roth) was inoculated with a single colony of bacterial cells from a freshly prepared plate. After incubation for 12-18 hours at 37°C, 1 d of medium was diluted with 500-1000 II
JI! 200IIJ in the flask! ! [B plus antibiotics] and incubated overnight (12-18 hours) in a stirred incubator (37°C). The 200 d culture medium was transferred to a centrifuge container and centrifuged at 2.50 Orpm for 10 minutes. After removing the supernatant, incubate each cell pellet with 30I
ID! Stead's buffer [5TET buffer (
8% sucrose, 0.5% Trimene X-100,50m
)I EDTA (pH 8,0110mM Tris-J)
, pH 8,0] at room temperature and transferred to a 125d flask. 3d"l0m1/lid! lysozyme (freshly prepared in Stead's buffer) was then added to each flask which was vortexed for mixing and incubated for approximately 5 minutes at room temperature. Each flask was then added to the cell -11 When bubbles form, place the flask in a boiling water bath for 45 minutes.
Transfer for 2 seconds, then cool the flask in an ice Eater bath for 2 minutes or more. Gradually pour the viscous mixture into a 50 d round bottom centrifuge tube and centrifuge for 50 min in a preparative centrifuge.
Centrifugation was performed using a W28 day-tor (Beckman CA) at 1B, OOOrpm for 15 minutes.

The supernatant from each test tube was carefully poured into disposable polypropylene centrifuge tubes containing 1 volume of isopropanol and 1 volume of ethanol, and the DNA was incubated at -80°C for 20 minutes.
Sedimentation for minutes (or 2 hours or more at −20° C.) followed by centrifugation at 2,5 OOXg for 15 minutes or more. After removing the alcohol, add 2.5-g of extraction buffer (per 11: 12.1 g of Trim)
, 33.6 g Na2EDTA2N20, 14.9 g KCI, pH 7.5) was added to the pellet followed by vortexing to keep the lysate off the bottom of the tube. 100
μρ 10#I5F/me ribonuclease A (0,
1X and pretreated with boiling water for 10 minutes) was added and the ink was incubated for 30 minutes at 37°C. Approximately 200μ
Add q of proteinase K to each tube and incubate for approximately 30 minutes.
Incubation was at 0-50°C at which time 250 Mg of 10% Sarkosyl was added and ink 1 basion continued for 3 hours or more. C3CΩ
To prepare the gradient, add 1 of 801 per d of DNA solution.
0#Ig/d ethidium bromide was added to each tube. Thereafter, 1.04 g of Cs C1 per portion of the DNA solution was added to each test tube, and swirling was gradually performed to dissolve it. Quick-3eal 1
H) After transfer to a test tube, the upper band consists of linear and nicked DNA and the lower band consists of covalently closed D
Two well-separated visible DNAs consisting of NA
Centrifuged at 55,000 rpm in a 70° I Ti fixed angle rotor for 16 hours to equilibration to yield a band of . Collect the lower band (covalently closed ring DNA) and add water to reduce the volume of each tube to approximately 6IiJ!
! The mixture was placed in a closed 15 Biao polypropylene centrifuge tube. Ethidium bromide was extracted from the DNA using an equal volume of isoamyl alcohol and the pink (top) phase was removed and discarded. The extraction was repeated until the top phase was colorless and the bottom (DNA') phase was clear and colorless. D of about 5d
The NA solution should be left in each tube with 2 volumes of absolute ethanol added (if not, add water to a final volume of 5 InIl). Sedimented for minutes and pelleted at 2,500Xg for 20 minutes. After removing all the alcohol, 500μm of 0.1XTE was added to each pellet and then resuspended at 65°C for 10 minutes or more. DN
A can be stored at 4°C.

Plasmid DNA was also prepared as described by Maniatis et al. (1982, Molecular Cloning, Cold Spring Harbor Laboratory (p. 8696)) [Maniatis et al.
, 1982. Molecular Cloning, C
o1d sprang ) (arbOr Labora
(1) p, 8696)]), DNA ligase was purchased from New England Biolapos (New E
purchased from England Biolabs), 5011
1M Tris-HCI (1)87.8), 10mM Mc+CI2.20mM DTT, 1mM ATP
Inside, the air was used at a rate of 10,000 units/d. Agarose gel-based analysis of DNA was as described by Maniatis et al. [Maniatis et al., 1982.
Molecular Cloning, Cold Spring
Barber Laboratory Aniatis et al.
, 1982. Molecular Cloning, Co
1d Spring ) (arbor Labo
ratory)ko.

(6,2, Formation of a Plasmid Vector Containing the AcNPV Promoter Linked to the Coding Sequence of the LAV/HTLVIII gag Gene) The next segment is preceded by the AcNPV transcriptional regulatory sequence and followed by polyhetrin DNA. (J
The formation of plasmid vectors carrying the aQ gene coding sequence is described. These recombinant plasmid vectors were then used to introduce the 1AV/HTLVI QaQ coding sequence into the baculovirus genome through in vivo recombination. The following segmental niote, LAV
/HTLVIII oap co->:
in-HObSOn, S, , et, al, ,
1985. eel 140:9]) and inserted into plasmid Ac610 downstream of the baculovirus polyhetrin promoter contained in t) Ac610 to form DAc-tJagl (see Figure 15).

In this formation, the tAv/Hrtvm specific nucleotide sequence is preceded by the polyhetrin promoter and followed by the polyhetrin gene sequence forming a chimeric gene.

As previously described, pKs-5 consists of 3 of the lambda J19-containing mulelic LAV/HTLVIII DNA insert cloned into the 3sB and KpmI sites of pUClB.
, 148 base pairs 5StI-+KI) mI fragment. It was obtained by cloning the restriction fragment of J19 into the 3sB site of f) LJC18 to form 08-1, which was then digested with K pn I and religated. This DNA was used to transform Escherichia coli and plasmid 1) KS-5 was isolated.

I) LAV/HTLVI\f different DN included in KS-5
A is [^V/H Ding LVI[I] from the 5Stl site located at nucleotide 224 in the genome to nucleotide 3
(Kl) nI site located at 372 (Wayne-Hobson).

Niss, et al., 1985, Cell 40:9; see Figure 14.
1985. Ce1l 40:9]).

10 μs DKS-5 with HinCII and 3Stl
The resulting fragments were resolved on a 1% low melting point agarose gel. The 1818 base pair fragment (approximately 0.2 μg) was excised from the gel. This fragment has nucleotide number 224 as shown in Figure 14.

It contained LAV/)ITLVi-specific sequences up to number 2042.

It contains the coding region for the complete OaO gene.

5 μ9 of DAc610 was completely digested with Tin and Tin I and fractionated on a 1% low melting point agarose gel: DN
The A band (approximately 0.4 μg) is the first sample excised from the gel.
(See Figure 5).

Both gel sections were lysed for 10 min at 65°C, and 3 μρ of the 1818 base pair AV/HTLVVIII-specific fragment (digested with HincI and 5stI) was added to 1 μg of DAc610 (S m
a I and Dandan■) were ligated.

The ink was baked for 16 hours at 23°C (room temperature). The ligation mixture was then heated at 65° C. for 10 minutes and used to transform NijiI Lychia coli strain HB101. Plasmid DNA from ampicillin-resistant transformants was tested for the presence of the LAV/HTLVI insert. The structure of this plasmid pAc-gagl is shown in FIG.

(6,3,Kime7LAV/HTLVIII QallJ
iden-jJflllA engineering type baculovirus a
al ) formation) AcNPV was plaque purified and Sf9
Cells were grown in cell layers. Wild-type Ac NPV DNA was isolated and pAc-gagl was purified as described above (see Section 6.1.2).

g) (ULAV/HTLVI [(AcNP of laQ sequence
Insertion into the V genome was achieved by in vivo recombination, enabled by the fact that the gag sequence in pAc-gagl is flanked by AcNPV coding sequences. Co-transfection of pAc-(la(]1 plasmid DNA and wild-type Ac NPV DNA resulted in recombination between polyhetrin sequences on the plasmid and homologous sequences in the AcNPV genome. Insertion of the chimeric gene , arose as a result of double recombination in the flanking sequences. Such recombinants carry a chimeric gene in the AcNPV polyhydrin gene,
After all, it will not be possible to produce an occlusion body. Recombinant plaques can be selected by visual observation for deletion of occluded virus.

1μ9 Ac NPV DNA, 5μ7 pAc-g
agl and 15 μg of calf breast JIII DNA were mixed and subjected to ethanol precipitation. The precipitate was washed with 70% ethanol, placed in a tissue culture hood and air-dried for 4 hours.
37t1. Resuspend in Q H2O. GaC1 0.2
5M added (63.u, 11 of 28 CaCL.

to 4371 DNA in H2O>. DN while bubbling air through 2XHEBS of 500/jJ)
Solution A was added dropwise. (1 drop/4 seconds). 2XI (EBS is 167+1!? Na C+ per 1d, 2m5F
D-glucose, KCI of 0.75 m'j, 0.5
5 ml of HEPES (time 7.1).The mixture was incubated at room temperature for 30 minutes and added with 10% fetal bovine serum (FBS').
and a brace of 2d (Gra
ce) containing 3X106sf9 cells in insect medium
It was added to the cultured date 1. [) NA solution was added dropwise to the cells, and the Digitator 1 was incubated at 28° C. for 4 hours. Rinse the block with 4-brace medium + FBS + antibiotics, then rinse with brace medium 10% FBS + antibiotics + 20%
Incubated with dimethyl sulfoxide for 90 seconds.

The plates were rinsed three times with complete medium and incubated with complete medium for 4 d at 28°C. After 5 days, the supernatant was collected and cleared at 1000×g for 5 minutes.

Virus strains were titrated on 5f9IIB cells and non-occluded plaques were identified visually. Non-occluded plaques were selected and replaked twice more. These plaques were grown in 5f9IB cells and virus stocks were prepared and used for subsequent characterization. The formation of recombinants is illustrated in FIG.

(LAV/HTLVIII in tissue culture cells infected with 6,4,11 recombinant baculoviruses (
1a (expression of 1-related proteins) Chimeric LAV/HTLVIII (The recombinant AcNPV carrying the 11aQ gene is LAV/1 (TLVIug
It was possible to express the aaM series RI'48 and the protein upon infection of cells in tissue culture. These proteins were also found to be immunoreactive with AIDS patient serum.

(6,4,1, Ac-aaalte-infected site aG:
Octle LAV/1 (TLVIII gag-specific RN
Identification of A) 100 dates of Sf9 cells (approximately 107 cells/date) were infected with wild-type Ac NPV or its recombinant Ac-gag1 at a multiplicity of infection (m01 moi) of 4. Cells were harvested 24 hours post-infection and 0.15M
Na C1, 0,01 M Tris-HcI, 1) H7
, 2 twice. Poly7-denylated RNAΔ was isolated according to the literature (Perthio, A., F.).

and Farid, G., C., 1979, Journal of Pyrology-29 Near B3-769 [Purchio
,A,F.

and Fareed, G. C., 1979, J.
Virol, 29 Nia 63-769]).

RNA was transferred to 1% agarose-formaldehyde glue (Rerlach, H. et al., 1977, Biochemist! J
-16:4743-4251 [Lehrach,)1
．． , et al, 1977゜Biochemistr.
y 16:4743-42511), transferred to a nylon filter (Hybond), and irradiated with ultraviolet light. filter with 0.9M Na
C1,50111M sodium phosphate (pH 7,0)
, 5mM EDTASo, 1% SDS, 4X Denhardts, 0.4m! i/d
(D tRNA, 0.25 m9/rid! of calf thymus tissue was hybridized to 32p-labeled pKs-5 DNA for 116 h at 42°C in 50% formamide. Filters were incubated with 0.1% SO8 ,0,25x
Washed 4 times for 30 minutes at 65°C in ssc and washed with Cronex 14 using a Lic+htening Plus intensifying screen.
Exposure to X-ray film. Figure 17 shows that the main band of 2,2 kilobase pairs
It is shown that it was detected in 1 infected cell and not detected in wild type infected cells.

(6,4,2, LAV/
HTLVIII (Identification of lall-related proteins)
Immunoprecipitation as described below.
On) assay involves infection with recombinant baculovirus of the present invention.

The present invention shows that these cells synthesize proteins that are specifically immunoreactive with AIDS patient serum.

5f9 cells were seeded onto 60-day dishes (5x106 cells/day) and wild-type AcN in Ac-gagi.
infected with PV. At 24.48 and 72 hours post-infection,
The medium was replaced with methionine-free medium and the cells were incubated at 28
Incubated at ℃ for 30 minutes. After this, the medium was replaced with a methionine-free medium containing 100 μCi/s [3″S 2 ]methionine, and labeling was carried out at 28° C. for 2 hours.

At the end of the labeling period, cells were treated with 0.01 M Tris-1-(
CI (1) 87.2), washed twice with 0.15M NaCl, and collected by centrifugation. Cell pellets from each 60 mm dish were washed with 1 d of lysis buffer (1% MP-4).
0 (polyoxyethylene (9) p-tert-octylphenol), 0.5% sodium deoxycholate, O, IH NaCl, 0,0 IH Tris-HCl
(t)87.4)) and the lysate was
Cleared by centrifugation for 30 minutes at OO, 0OOX g.

Immunoprecipitation was performed by adding 4 μg of heat-inactivated human serum from either control populations or AIDS patients to 500 d of cell lysate. After incubation for 1.5-1 h at 4°C, 80μ' of activated Staphylococcus aureus [5 taphylococc.
usaureus] @cells (Pan5Orbin Ce11S, California)
CC0Ib1OChe Behring Corp.
La Jolla, CAI) and heat in a colander at 14°C.
Incubation continued for 5 hours. Immunoprecipitated complexes were collected by centrifugation at 5000 rpm for 5 min at 4°C in a Sopal A384 rotor, 1M NaCl + 0.1% NP-40 + 0°01
Once at M Tris 1-ICI, pH 7.4, Lipa (R
IPA) buffer (10ml) 1 Tris-HCl, pH
7,2,0,15) 1 NaC1, 1% Sodium Deoxycholate, 1% Sodium Lauryl Sulfate, 1%
Washed three times with Triton X-100). The washed immunoprecipitate was diluted with 80 μl of Laemmli (ta).
emmli) resuspended in sample buffer, boiled for 1 minute, and centrifuged for 5 minutes at 50 oorpm in a Sawpal A3840-tar. 10 immunoprecipitated proteins in the supernatant.
% 5DS-polyacrylamide gel. After electrophoresis, the gel was coated with Kumatush (C
oomassie) blue dye and dyed with sodium salicylate (1 M sodium nalycylate at room temperature for 30 min.
minutes) and dried for fluorography.

Mature QaQ protein (24,000 daltons, p24
; 16,000 Daltons, p16; 14.000
Dalton, p14) is proposed to be processed from a larger 55,000 Dalton precursor protein. Figure 14 shows molecular weights of 67,000 daltons, 55,000 daltons, 40,000 daltons, and 3
4.000 Daltons, 32.000 Daltons and 24
．． 1,000 Dalton protein was specifically immunoprecipitated by AIDS patient serum.

To determine if any precursor/product relationships exist between any of the immunoreactive proteins described in FIG. 18, pulse-chase experiments were performed. Sf9 cells were infected as described above.

At 24 h postinfection, cells were labeled with [35S]methionine for 5 min: cells were washed with complete medium,
Incubated for 4 and 8 hours.

At these times, cells were harvested and immunoprecipitation was performed. Immunoprecipitate proteins at 10% as above.
Fractionation was performed in a DS-polyacrylamide gel. 19th
The figure shows the fluorogram of this experiment. This data is I)67. D55. D40°D34 and D32 are 5
showed uptake of the label after minutes. After 8 hours of chase, labeling decreases at D67 and p55 and increases at D34.

1) The amount of radiolabel at 4'0 is relatively constant.

(6, 4, 3, E L I S, 4kl; LAV/H
Identification of TLVIII aaa-related proteins) Each contains 107 sf9IIB cells (Il(7) 1
00trm dishes were infected with Ac-gagi.

At 24 hours post-infection, cells were harvested and treated with 0.01 M Tris-HCI (1)H7,2), 0.15 M NaCl.
The cells were washed twice and frozen and thawed three times. 2/IJ! containing 0.5% NP-40 on ice for 5 minutes. ! Split with PBS. The lysate was centrifuged at 1000×g for 10 minutes and the supernatant was removed. 4 d of carbonate buffer (50 ml of sodium carbonate, ρ89.6) was added and the mixture was washed in an Eppendorf.
Spin in a centrifuge for 20 minutes. The supernatant was removed and diluted into carbonate buffer as shown in Table 3. These lysates were used to coat 96-well microtiter plates (56 μp/well) overnight at 4°C. 300μ
(blocking reagent (5% skim milk powder).

0.01% (M-Meosal, 0.01% antifoaming agent A,
10X PBS) was added to each well and incubated for 45 minutes at room temperature. The blocking reagent was aspirated and 50 μM of 1:100 diluted human serum in blocking reagent was added to each well. Before adding to the wells, the diluted serum (AIDS patient serum or normal human serum) was incubated for 45 min at 37 °C. It was done.

Each well was allowed to react for 1 hour at 37°C. Each well was then washed three times with sodium chloride + Tween 20 (polyoxyethylene sorbitan-monolaurate).

100 μM goat anti-human horseradish peroxidase conjugate (1:100 dilution in normal goat serum containing 0.01% thimerosol) was added to each well and incubated at 37°C.
The mixture was reacted for 1 hour.

Plates were washed and 100μ, 0/well of buffered substrate and chromogenic reagents were added: buffered substrates were hydrogen peroxide, citric acid, and phosphate buffer, and chromogenic reagents were added in dimethyl sulfoxide. Tetramethylbenzidine.

Plates were incubated for 30 minutes at room temperature. 100
The reaction was stopped by adding μI/well of 3N sulfuric acid. The absorbance of the reaction mixture was based on the reference absorbance at 630 μm.
Measured at 50 μm. The results are shown in Table 8. T
R1, Y''-1, CF22C and CF9 are AIDS patient sera, and 2.16, 21 and 48 are normal human sera.

(Left below) In the end, the results shown in Figures 17 to 19 and Table 8 are as follows:
The recombinant baculovirus has (a) inserted LAV/HTI
VII fJag gene woshin, (b) Kaka6M4
xi products can be modified or processed to (C) produce antigenic proteins that are specifically immunoreactive with AIDS patient serum.

(7, Example: 99927080 recombinant) In the following example, a plasmid vector containing a chimeric gene consisting of the LAV/1lTL and CJaQ coding sequences located downstream to the transcriptional regulatory region of vaccinia virus was created. Ta. The chimeric gene containing the promoter OJl-hi LAV/elbow LVI[loag sequence in vaccinia virus 7.5 was inserted into the vaccinia virus genome by in vivo recombination. Such recombinant viruses have been identified and purified, and virus stocks have been prepared from infected tissue culture cells. Immunoreactive LAV/HTLVIII QaQ associated protein was shown to be produced by recombinant vaccinia virus in vivo. A detailed description of each step in this embodiment of the invention is provided in the sections below. The general method used in this embodiment is as described in Section 5.1.

(7,1, LAV/HTLVII QaQ gene (7)
Formation of plasmid vectors containing vaccinia virus promoters linked to +- and LAV/11T sequences of various lengths containing gag''- and ligated sequences.
Five types of plasmid vectors containing vaccinia virus 7.5 promoters linked to the LV ■ genome were formed. The source of the LAV/HTLVIII 1llaOD-coded sequence is lambda J19 (lambda Jl9) [
Wain-Hobson et al.
al) Cell (Ce11) 40:1985], pKs-5 and 1) SS-
It was 5. Plasmid pKS-5 is a pucia
inserted into the corresponding site of tL fc LAV/HTLV
It contains a 3148 base pair I→, I (nucleotide number 224 to 3,372) fragment of DNA.

Plasmid pSS-5 was inserted into the corresponding site of LAV/elbow LVI [ID DNA (7) 5,107
Salt M pair 3sLJ → 5aII (nucleotide number 224 ~
5221') fragment. pKS-5
1) G S2
0 () (Ackett et al., 1984
．． J. Virol, 49:851-864) (see Figure 20). LA inserted into ρG562
The V/HTLVII-specific sequences all begin with an EhaI (Dll) site (nucleotide 257) located 76 base pairs upstream from the start codon of the gaΩ gene. Plasmid DV-QafJ1. DV-QaQ2. DV
-1lla03.1)v-111a04 and pv-g
ag5 is connected to each CORI from this Tha1 site.
(in 4194), K Ho I (in 3372)
, containing LAV/HTLVIII-specific sequences up to the RV (at 2523), Gravity I (at 1975) or BOIII (at 1642) sites.

The formation of each plasmid described below will be clarified by referring to FIG.

Formation of pv-gagl: 5μ9 of plasmid I) SS-5 DNA was completely digested with restriction enzymes 1 and 1 and CORl.

The resulting fragments were resolved in 1% LMP agar gel. A 3.9 kbp fragment containing the LAV/HTLVII QaQ coding sequence was isolated and DGS predigested with 3mal and EC0RI.
62 DNA. The ligation mixture was used to transform S. coli -18101 and ampicillin resistant clones were selected. Plasmids from individual colonies were tested for the presence of the 3.9 kb ρ insert by restriction analysis and regeneration of CORl at the ligation point. The resulting product contains the complete coding sequence of the QaQ and protease (prt) genes, as well as the ρ01 sequence up to the CORJ site at nucleotide 4194.
It contains the main part of the reading frame.

Formation of pv-gag2: 5μ9 plasmid pKs-5 DNA was digested with restriction enzyme Tha
It was completely digested with I and 5IIlaI.

The 3ma■ site of pKs-5 is adjacent to Kl)nI, and the field■ site is the point of attachment between the LAV/HTLVIII-derived insert sequence and 1) the LIC18 plasmid multiple cloning site. The resulting fragments were resolved in a 1% LMP agar gel,
A 3°2 kbp fragment containing the T LAV/HTLVIII pag coding sequence was isolated and previously
DGS62D digested with aI and treated with CIAP
It was linked to NA. The ligation mixture was used to transform S. coli H8101 and ampicillin resistant clones were selected. Plasmids from individual colonies were tested for the presence of the 3.1 kbp 7-ragment, and the orientation of the insert was determined by restriction analysis. The resulting formation, pv-gag2, has a complete Qa
Q gene, protease gene and nucleotide 33
It contains the main part of the pol open reading frame up to the 12th K I)n I site.

Formation of pv-gag3 was performed using pv-gag3, except that a few kbl) fragment generated by ThaI and EC0RV digestion of pKs-5 was inserted into pGs62 at the 3mai site.
- It was similar to the case of gag2. The final formation is p
V-QaQ3 contains the complete Dag and protease genes and a small portion of Pol fused to the 3' portion of the 3i gene in the vaccinia virus.

Formation of pv-oap4: 5μ9 of plasmid 983-5 DNA was completely digested with 3stI and BclI. ShiAV/HTLVn
The resulting fragment containing the lqag sequence was purified. This fragment was combined with 5StI and BamH
Plasmid vector plc19R [?
- Arsh et al., 1984 Gene 32:4B1-485] to form an intermediate plasmid. l) Ba[[I] (Insertion of gag sequence into I site of I C19R
/HTLV I[I resulting in juxtaposition of the Bcl I site in the I sequence to the ECoRt site in this multi-site cloning vector.

This intermediate formation of restriction enzymes T ha and ECo
Digestion with Rt allows rapid insertion into plasmid I) GS62 at the 5I11aI and ECoRI sites.
, generated a 72 kbp fragment. The final product, pV-qag4, is derived from LAV/HTLVI Qa (]
All genes and 3 of the vaccinia virus TK genes
Contains part of the protease gene fused to one part.

Formation of plasmid pv-oaq5 was performed using pV-qa (14
was carried out using a two-step method similar to DSS-5 no 5
Digest micrograms of restriction enzymes with 3 Stl and B (It crazy). 1 containing LAV/HTLVI Qag sequences.
．． The 42 kbD fragment was isolated, purified on a 1% LMP agar gel, and inserted into plasmid DIC19R at the 5stt and Bgl[ sites. The ligation of the gag sequence at the B(+lI[ site to the corresponding site on pEC19R involves (a) the generation of a stop codon in phase with the gag open reading frame and (1)) the B(+lI[ site) to the adjacent ECoRI site on the plasmid. This intermediate then allows for the juxtaposition of Tha
Digested with I and ECORI. LAV/HTLVI
A 1.39 kbp 7-ragment containing the IIQaQ sequence was purified and ligated into plasmid pGs62 at the 5IIIaI and ECQRI sites. The resulting plasmid, pv-gag5, contains CJaQ linked to an in-frame translation stop sequence.
It contains most if not all of the coding sequence (up to the BQ11I site).

(7,2, Chimera LAV/HTLVIII (llaU
Formation of recombinant vaccinia virus containing genes) Plasmid vectors pv-oaol, pv-gaQ2
．． DV-DaQ3゜pv-gaga and pv-ga
Chimeric LAV from g5 to vaccinia virus gnom/
Insertion of the HTLνm pap gene was performed using v-env5f3
and V-env2 (see Section 5.3). In the following examples, all recombinant viruses were purified by plaque-purified New York City Board of・Health stocks (New Yo
rHaC1ty Board or Health 5t
rain) (v-NY, see section 5.1.1).

TK-recombinants were selected and subjected to three rounds of plaque purification before propagation into virus strains used for subsequent characterization. 0V-QaQl, ρV-ga02. pv-ga
g3. Recombinant viruses derived from pv-QaQl and pv-gags were v-paplNy, respectively.
, v-pap gene, V-Qa03NY, V-Q
They were designated as aQ4NY and v-oap5NY.

(7, 3, IAν/HT [vmgaΩ-related protein-related protein above chimeric LAV/) in tissue culture cells infected with recombinant vaccinia virus The recombinant vaccinia virus carrying the ITLVm pap gene can be used to infect cells in tissue culture. Some of these recombinants are able to process the precursor QaQ protein, p55, into the mature proteins p25 and p18. These proteins can be expressed in AIDS patients. It was found to be immunoreactive against serum and/or monoclonal antibodies against QaQ proteins p25 and 1)18.

Confluent B5C-401Ia cells in 60# dates
V-QaQlNY, V-QaQ2NY, V-QaQ by a different type of vaccinia virus (V-NY) or its recombinant derivative with a multiplicity of infection of 1QIllOi (multiplicity of infection)
3NY, V-QaQ4NY or v-gag5NY. Infection proceeded for 12 hours, at which point the cells were harvested, washed once with PBS, and collected by centrifugation. Infected cell pellets were collected in a 0.3 m Laemmli (t
aemli) resuspended in sample buffer and boiled for 4 minutes to dissolve.

Total cellular protein in 20 μm cell lysate was 15%
Resolved electrophoretically on an SDS polyacrylamide gel. A sample of purified LAV/elbow [shiviral particles and an aliquot of mock infected cell lysate] was included as a control.

The contents of the gel were electrotransferred to a sheet of nitrocellulose filter. Initially, the Neulter reacted either with AIDS patient serum or with a combination of monoclonal antibodies against the gag proteins ρ25 and I)18. After extensive washing, the filters are conjugated with alkaline phosphatase (AP-
Conjugate) Reacts with either a goat anti-human immunoglobulin antibody or, if a mouse monoclonal antibody is used, an AP-conjugated goat anti-mouse immunoglobulin antibody. The conditions for the first and second antibody reactions and washing are described in 5.4.
As stated in Section 1.

The final wash after the second antibody reaction was performed using 0.1M NaCl
and 0.1M Tris (pH 9) containing 5mM MQCI2
, 5>. Antibodies bound to the antigen on the filter are washed with 0.1M Tris(1)H9,
5) , 0.1 M NaCl, 5mM MgCl2,
It was detected by reacting with a solution containing bromochloroindolyl phosphate at 0.33 to 1/d and nitroblue tetrazolium at 0.17 ffig/d. After reacting the filters with the chromogen, they were washed with water, air dried, and photographed.

The results of this analysis are shown in FIG. Recombinant V-gag
lNY and v-qaa2NY contain the complete gaq and protease genes. These recombinants are L
I)5, which is a major protein of AV/HTLV Iu
5,940°1) A family of Shi^V/former LVI-related proteins that co-migrates with 25 and C18.
ly) was expressed.

These proteins are immunoreactive with both AIDS patient blood clots (Figure 218) and mouse monoclonal antibodies against 925 and 1) 18 (Figure 21A).

Recombinant v-aaa3NY contains both the qag and protease genes, but its Pol open reading frame is linked in phase with the carboxy-terminal portion of the vaccinia TK gene. v-gag3NY can also express the original QaQ gene Nobuko ρ55. However, V-11180
C25 against p55 in cell lysates infected with 3NY
and 1) p55 processing appears to be less efficient than V-QaQINY and v-gag2NY since there is less 18. Recombinant V-C) aQ4NY
contains the complete CJaQ gene, but only a portion of the protease gene. It is ρ5 in size
A LAV/HTL sym-related protein similar to 5 was synthesized. Recombinant v-gag5NY contained only part of the QaQ gene and expressed a 43 KD truncated protein (Figure 21C). V-gag4NY and v-g
Although both ag5NY appeared to process their primary fJaQ-related proteins poorly, their products are immunoreactive with monoclonal antibodies against ρ25 and ρ18.

In summary, the five recombinants, albeit with differences in their processing capacity, are immunoreactive-2 containing major epitopes of the LAV/)IrLVIII core protein.
11 = Capable of expressing protein.

(8, Example: Baquiulovirus envelope recombinant) In the following example, the plasmid vector is AcN
LAV/H located downstream with respect to the transcriptional control sequence of PV
A chimeric gene containing the TLVul envelope coding sequence is formed. A chimeric gene containing the AcNPV polyhedrin promoter and the LAV/HTLV ■ envelope coding sequence was inserted into the AcNPV genome by in vivo recombination. The recombinant virus was identified and purified, and the virus strain was
prepared from i cell supernatant. Immunoreactive LAV/old LVI
[[It has been shown that envelope-associated proteins can be produced by recombinant baculovirus in vitro.

A detailed description of each step in this embodiment of the invention is presented in the following sections. The general method used in this embodiment is as described in Section 6.1.

(8,1. Formation of a Plasmid Vector Containing the AcNPV Promoter Linked to the Coding Sequence of the LAV/HTLVIII Envelope Gene) It was purified from ρv-env5 (see Section 5.3), which was used to form vaccinia virus v-env5.

The envelope coding region in pv-envS was bordered by amH1 sites on both ends, with 96 base pairs of untranslated sequence 5° proximal and 223 base pairs of untranslated sequence 3" proximal. Partial digestion of the plasmid (including the HI site) with the restriction enzyme danam)
2.9kbp containing only AV/HTLVIII specific sequences
7 fragments were generated. This fragment (approximately 0.
5μ3) was digested with 1% LMP agar glue and purified from this and previously linearized with 3μm)-11,
0.5 μg of CIAP-treated plasmid vector 1) was ligated to Ac610.

This ligation mixture was prepared using Escherichia coli strain HCI 00
0 and ampicillin antibiotic colonies were selected. Plasmid DNA from individual transformants LAV/1-ITLVI
The presence of II envelope sequences (i.e. generation of 2, 3 kbp and 0.54 kbp fragments by 1.βamHI digestion) and orientation of the insert were tested.

Plasmids with LAV/HTLVII [envelope sequences inserted in the correct orientation were purified and pAc-
It was defined as env5. This structure is depicted in FIG.

(Formation of recombinant baculovirus containing chimeric 7 LAV/HTLVIII envelope genes)
Insertion into the V genome is accomplished by in vivo recombination as described in Section 6.3. AcNPA DNA (
IJIlg)l) Ac-env5 DNA (5mg
) and calf thymus DNA (15111g) were transfected into Sf9 cells by the method described in the same column. After 1 b. ink at 28°C in complete medium λ for 4 d, the supernatant was collected and iooox
Clarification was carried out at g for 10 minutes. This virus strain was titrated on sf9 cells, and recombinant Lyles was first detected by Summers, M., D., and Smith, G., E. (Su.
mmers, )], D, and Sm1th, G, E,
) was identified as follows by the plaque hybridization assay method.

5f91111 cells were grown in serum-free TNM-Fl- (60X1 at a density of 2.5X106 cells/plate in medium).
It was inoculated onto a 5# culture plate. After cell attachment, the medium was removed and 1 ml of diluted virus inoculum was added to each plate. At the end of the 1 hour incubation at 27°C, the entire inoculum was removed from each plate and 4 L
MP agar overlay was added gradually to the edge of each plate. After the overlay had solidified, the plates were incubated in a humid nM environment for 4-6 days or until plaques were successfully formed. The plates were then left in a non-contaminated environment overnight or longer to dry. Once completely dry, remove the agar overlay and
A dry nitrocellulose filter was placed on top of the remaining cells on the plate. A 47 m circular small Whatman (Whatman > 3
) 1 of Tris-HCl, pl-17,4,0,15H of NaCl) and placed on a nitrocellulose filter on a plate. After plotting, carefully remove the nitrocellulose filter and solution B (
Placed on a small Whattmann filter saturated with 0.5H Mail, 1.58H NaCl (on cell side)
. After 2-3 minutes, the nitrocellulose filter was air-dried on paper towels and transferred to a small Whattman filter saturated with solution C (1,000 OM Tris-HCl, 1,000 mL NaCl, DH7,4,1,5). did. After 2-3 minutes (2-3 minutes), the nitrocellulose filter was air-dried on a paper towel and the solution D(0,3) 1° NaC 1°0.
03) Washed by gently immersing in a Petri dish filled with sodium citrate (1). It was then removed and dried on paper towels and incubated in vacuum for 2 hours at 80'
C fired. Using this filter as a probe, 32p-
Labeling 1) Hybridized to RS-3. Recombinant virus was removed and titered again on sf9 cells. These were plaque purified three times more and used to prepare virus stocks for subsequent studies.

(8, 3, Tissue culture cells infected with recombinant baculovirus Ac-env5 (LAV/HTLV
Expression of I envelope-associated proteins) It was shown that recombinant Ac NPV carrying a chimeric LAV/) I TLVm envelope gene was capable of expressing [A/HrLVm envelope-associated proteins in infection of cells in tissue culture. These proteins are A
IDS patient serum and mat: LAV/HTLVVIII
envelope glycoprotein (It) 110 and 111
1) It was found that it immunoreacts with a monoclonal antibody that restricts 41.

Sf9 cells (1XI) seeded in a 100 m dish
O7) at 5 moi of wild type AcNPV or its I!
The cells were infected with a single recombinant Ac-env5. Twenty-eight hours after infection, the medium was replaced with methionine-free medium without serum supplement and incubated for 30 minutes. After this period, add 100 μC1/In! of this medium! ! of[
Methionine-free containing 35S comethionine @Jt
! 12ml and labeling was carried out for 2 hours at 28°C. At the end of labeling, cells were washed twice with PBS and
Resuspended in 1 d post-lysis buffer as described in Section 5.4.2. Immunoprecipitation was performed with AIDS patient serum and mouse monochrome against QDllo or QD41.
The procedure was performed as described in Section 5.4.2 using the bold antibody.

As shown in FIG.
This shows that the VIII envelope-related protein was exclusively synthesized. Cotton sinus recombinant V-env5
and AIDS patient serum as well as Ql)
This protein represents the glycosylated precursor envelope protein g0150, as it co-migrates with QD150, which is immunoreactive with monoclonal antibodies directed against 110 and 0p41. The processing of this molecule is
Not effective as no gollo or (II)41 was detected during the time labeling period. however,
Since the major epitopes of both QDI 10 and g+) 41 are present in the precursor, this protein is valuable as a diagnostic agent and as an immunological agent.

(9. Deposit of Microorganisms) The following Escherichia coli strains carrying the indicated plasmids were deposited in Peoria, Illinois, United States.
ia) Agricultural Research Culture
Collection [Agricultural Re5ea
rch Culture Co11ection (NR
It has been deposited with RLH and has the following deposit number.

Esieriki strain plasmid Deposit number A. coli 12. HCl00Opv-ENVI NR
12. to RL B-18003. HCl
00Opv-ENV2 NRRL B-180
04 to 12. MC100Opv-ENV5 N
12. to RRL B-18005. H.C.
l00Opv-ENV7 NRRL B-18
110 to 12. HBOI
1) V-(Ja(11NRRL B-18111)
2. ttBlol pAc-
gagl NRRL B-18105 12.
NF1829 pAc-env5 NRRL B
-18109 The following recombinant virus is located in Rockville, Maryland, USA.
American Type Culture Collection (A
merican Type Culture Co11
action) and has been assigned the following deposit number:

Recombinant virus deposit number v-env2 ATC
CVR2114v-env5 ATCCV
R2113v-env7
ATCCVR2148v-env5NY
ATCC VR2149v-gaol
NY ATCC VR215
0Ac-gaol AT
CCVR2147Ac-env5
The present invention extends beyond the deposited microorganisms, as the ATCC VR 2151 deposited embodiments are merely illustrative of one aspect of the invention, and any functionally equivalent microorganism or virus is within the scope of the invention. The range is not limited in any way by the virus. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

It should also be understood that all base pair sizes given for nucleotides are approximate and for descriptive purposes only.

[Brief explanation of the drawing]

FIG. 1 depicts the complete proviral genome structure of LAV/HTLVI. The shaded area indicates the area of the reading frame. Such regions encoding group-specific antigens, reverse transcription and envelope proteins are called gag, pot and env, respectively. Overlapping reading frames are indicated by crossed diagonal lines. Numbers indicate the apical site [CapSit] where viral transcription begins.
This relates to the number of base pairs downstream from [e]. Restriction sites are marked as below. Figure 2 shows plasmid pR8-3D.
It represents the nucleotide sequence of the LAV/HTLVIII-specific region (from Tania R1 to Tanuchi I) present in NA, and contains the complete LAV/HTLVIII envelope gene (nucleotide 576B and 834.9), LAV /HTLVI insert. pV-
envl, ρv-env2. and the restriction sites used to form pv-env5 are indicated. The complete amino acid sequence of the envelope gene, as deduced from the nucleotide sequence data, is also shown. FIG. 3 is a schematic representation of the formation of a plasmid containing a portion of the LAV/HTLVIII envelope protein coding sequence inserted downstream from the vaccinia virus promoter. The LAV/HTLVul envelope coding sequence is represented by a blank box and the vaccinia promoter is represented by a shaded bar. vaccinia promoter region and [^V/HTLVIII
The nucleotide sequence at the junction of the envelope encoding sequences is shown further down in the figure. The underlined sequences indicate the putative start codon and reading frame for the chimeric gene. Translated sequence of recombinant pV-env2 (P
Note that the third and fourth amino acids in rO-Val) match amino acid numbers 43 and 44 of the LAV/HTLVI envelope coding sequence by -222. FIG. 4 is a schematic representation of the formation of a plasmid containing the complete LAV/HTLVVIII envelope protein inserted downstream from the vaccinia virus promoter. The vaccinia virus promoter is represented by a shaded bar. Plasmid pv-env5, containing the complete A/HTLVI envelope coding region, was formed in two steps. The 5° and 3° portions of the coding region are
Initially cloned separately into DGS20, LAV/
pv-envi and LAV/HTLVIII containing the 7-mino-encoded end of the HTLVIII envelope gene
pv-env2 was formed containing the carboxyl-encoded end of the envelope gene. These two parts are
Religated at the 5tuI site as indicated. FIG. 5 is a schematic representation of the formation of a plasmid lacking the transmembrane (anchor) sequence and containing the LAV/former LVI envelope protein coding sequence inserted downstream from the vaccinia virus promoter. Plasmid pv-env7 was formed in two steps. p
An intermediate plasmid, pv, in which a 5° portion of v-env5 is inserted into D26, providing an in-frame translation end sequence (d-AA) resulting in a truncated env gene.
-env5/2B was formed. The truncated 8nV sequence ending in dA is then ρG
20 and the truncated erlV gene is flanked by the vaccinia gene sequence pv-env.
7 was formed. FIG. 6 depicts the formation and selection of recombinant vaccinia viruses. The middle black bar represents the thymidine kinase (Ding K) gene of vaccinia virus. This TK gene is also present in the plasmid pv-envsDNA, but in the plasmid, the TK gene is attached to vaccinia 7.5 with a promoter (shaded bar) and LAV
/HTL is interrupted by a chimeric gene consisting of an envelope-encoding gene (blank box). After cells are infected with vaccinia, a recombinant plasmid containing the TK gene interrupted with the LAV/HrLVI envelope gene is introduced into the infected cells. Recombination that occurs in the TK gene flanking the chimeric gene introduces the LAV/HTLVIII envelope gene into the tuxinia virus gnomome. LAV/HTL ■
The resulting recombinant virus containing the envelope gene is TK-. FIG. 7 shows that recombinant env5 and env5NY containing LAV/HTLVIII env genes and the DNA fragments produced by their respective parental plants were resolved by electrophoresis in an agarose gel.
The bands were then colored with ethidium bromide to visualize them. Figure 7B shows the transfer of degraded restriction fragments to nitrocellulose and the transfer of AV/HTLVI[l
Figure 8 shows nick translation proteins specific for envelope sequences. Proteins from vaccinia LAV/HTLVIII env recombinant sources were resolved in a 7-15% gradient 5DS-polyacrylamide gel and electrotransferred to nitrocellulose paper. Made: LAV/)ITLVVIII
e li, t protein (LAV/elbow LVII))
; Wild-type vaccinia virus-infected cells (revised VV); Uninfected cells (mock); Cells infected with recombinant virus v-env2 (v-env2) or cells infected with recombinant virus v-env5 (v-e
nv5). Proteins immunoreactive with AIDS patient serum were detected by the action of peroxidase conjugated to anti-human HIG antibodies. LAV/)ITIVenv M
The gene product is Opl 50. QDll 0 and gp
41. The numerator M reference is expressed in kilodaltons. Figure 8B shows vaccinia-[AV
Proteins derived from any of the LAV/HTLVI env recombinants were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to nitrocellulose paper. were reacted with pooled serum from seropositive individuals: lanes 1 and 5 represent mock-infected cells; lanes 2 and 6 represent cells infected with wild-type vaccinia virus; lanes 3 and 7 represent vaccinia virus-infected cells;
- represents cells infected with env5; and lane 4
and 8 represent cells infected with v-env2. Immunoreactive proteins were detected using 125I-labeled protein A. LAV/formerly LV envy Densan Shinko, DI) 150 designated as SQD110 and QD41. Figure 9A shows that cells infected with either wild-type vaccinia virus (WTvv) or recombinant vaccinia viruses (v-env2 and v-env5'') were labeled with 353-methionine for 10-12 hours. Labeled proteins in cell lysates were reacted with either control human serum (N) or AIDS patient serum (1), and the immune complexes were reacted with Staphylococcus spp.
aureus protein A. Immunoprecipitated proteins were resolved by electrophoresis in 15% 5DS-polyacrylamide gels and detected by fluorography. Molecular weights are given in kilodaltons. Figure 96 shows the vaccinia AV/HTLVI of the present invention.
3H-Glucosamine (
lanes 1 and 5) or wild-type vaccinia virus (lanes ? and 6), V-env5 (lanes 3 and 7) or v-env2 (lanes 4 and 8).
and labeled with glucosamine for 3 days. Cell lysates were prepared using normal human serum (lanes 1-4) or I
AV/elbow [vm was immunoprecipitated with pooled serum and protein A from seropositive individuals. Immunoprecipitated proteins were resolved by 5DS-PAGE. Figure 9C shows wild-type vaccinia virus, v-en, as expressed by the LAV/HTLVM envelope protein expressed by the cottontail LAV/elbow LVJII recombinant.
v5 or v-env2, labeled with 35S-methionine, and washed with chase medium. At the following time intervals, cells are washed, lysed, and LAV
/elbow tv ■ immunoprecipitated with pooled serum from seropositive individuals and resolved by 5DS-PAGE for 20 hours (lanes 1, 7.13); Q, 5 hours (lanes 2, 8.
14), 1vIfJ (L/-n 3, 9.15); 2
time m (L/- lane 4, 10.16), 1 interval (lane 5.1
1.17) and 12 hours (lane 6, 12.18). Lane M represents a marker consisting of a 14C-labeled molecular weight standard. Figure 9D shows recombinant vaccinia AV/H of the present invention.
AV/HTLVVIII envelopes (lane 1) or wild-type vaccinia virus (
lane 2), v-env5 (lane 3) or v-
env2 (lane 4) and labeled with 35s-methionine. Cells were separated from the medium and lysed. Cell lysates (pellets) and media (soup) were immunoprecipitated using pooled serum from LAV/H and V[l seropositive individuals, respectively. Immunoprecipitated proteins were resolved by 5DS-PAGE. Figure 9E shows recombinant vaccinia LAV/HT of the present invention.
LV[lIll from being infected with the virus
There is a cell inside. Recombinant v-env5 is a complete LAV/H
TLV ■ Contains an envelope encoding sequence,
On the other hand, V-env7 lacks the transmembrane (anchor) sequence and contains the AV/HTLVI [l envelope coding sequence. l-1ela cells were mock infected (lane 1) or infected with wild-type vaccinia virus (lane 2), v-env5 (lane 3) or V-env7 (lane 4), and
Labeled with 5s-methionine. Cells were separated from the medium and lysed. Cell lysates (pellets) and media (supernatants) were each immunoprecipitated using pooled serum from LAV/elbow [v■ seropositive individuals. Immunoprecipitated proteins were resolved by 5DS-PAGE. Figure 10 was immunized with vaccinia-LAV/HTLVIII recombinant vaccinia virus. After 8 weeks, serum samples were resolved by 5DS-PAGE and electrotransferred to nitrocellulose paper [A
V/HTLVIII pyrion protein. Goat anti-mouse immunoglobulin conjugated to alkaline phosphatase was used to detect these [Av/)ITing LVm proteins that were recognized by mouse serum. Lanes a-e represent serum samples from five individual mice inoculated with v-env5 and lanes f-c represent serum samples from five individual mice inoculated with v-env2. . LAV/HTLν ■ Stored serum from seropositive individuals (AI
DS) and unimmunized C57B16. J Mouse (N)
Is) were used as positive and negative controls, respectively. LAV/HTLVI x Nheop glycoprotein! I
The positions of Ip150, opllo and (JD41 are indicated. Figure 11 shows recombinant vaccinia virus V-env5.
1 is a histogram showing seroconversion of brachytailed monkeys vaccinated with . Four monkeys had 2×101) f of V-env5.
and 4 were inoculated by skin prick with 2X107 pfu. One animal received 2X of vaccinia-herpes simplex 00 recombinant (V-H3VIIIDI) as a control.
Inoculated with 107 pfu. 10 weeks after the first vaccination,
All animals except No. 81 received 2X of the same virus.
Received secondary inoculation of 108 pfu. Serum samples were collected before vaccination (blank donation); 4 weeks after the first vaccination (
(hatched bar): and collected 4 weeks after the secondary inoculation (Nakakokusho). Seroconversion uses purified L as the target antigen.
Assayed by ELISA using AV/old LVIII pillion. Figure 12 shows vaccinia-L as described in Figure 11.
Prophylaxis with AV/elbow tvm recombinant virus v-env5 were collected before the first vaccination (lane pre) and 4 weeks after the second vaccination. Aliquots of serum were diluted 50 times and digested by 5DS-PA, GE, and further modified to give optimal detection of the two envelope glycoproteins QD110 (Figure 12A) and Qρ41 (Figure 12B). LA immobilized on nitrocellulose filters by electrotransfer using format
V/HTLVI pillion protein. V/H-TV11 recognized by brachytailed monkey serum was detected by goat anti-human immunoglobulin conjugated to alkaline phosphatase. LAV/old [v■ stored serum (AIDS) from seropositive individuals (AIDS) was used as a positive control. Genuine LAV/HTLVIII Hiri, t
(AIDS) C. The comparative control L/T was operated. Figure 13 shows ") ') Sini, i'NY-LAV/H
TLVIII recombinant virus V-env5NY immunized with 5X10'' of chimpanion V-env5NY, while one animal was inoculated with vaccinia-herpes simplex gD formed from the same parental vaccinia NY strain.
were inoculated with the same dose of recombinant v-H3VgDINY. All animals received a second vaccination 8 weeks after the first vaccination. Serum samples were collected before inoculation (lane 1), 8 weeks after the first immunization (lane 2) and 2 weeks after the second immunization (lane 3). Serum aliquots are
Reaction with LAV/HTLVJII pillion proteins diluted 50 times and resolved by 5DS-PAGE and immobilized on nitrocellulose filters by electrotransfer using a fixed format that provides optimal detection of Qρ41. I was made to do it. The LAV/HT protein protein recognized by chimpanzees was detected by goat anti-human immunoglobulin conjugated to alkaline phosphatase. Stored serum (AIDS) from LAV/HTLVI seropositive individuals was used as a positive control. Figure 14 shows the LAV/elbow tvB specific region (from SStI to depression I) present in plasmid ρKS-5 DNA.
represents the nucleotide sequence of the complete LAV/
HTLVIII QaQ gene (NukuL/Ochito 340
1i color 18351t mar) 4.1:, LAV/
Contained in the HTLVIII insert. pAc-gagi
The restriction sites used in the formation of are indicated. Also shown is the complete amino acid sequence of the QaQ gene, as deduced from the nucleotide sequence data. Figure 15 is a schematic representation of the formation of plasmid pAc-gagt containing the LAV/HTLVIII (lag protein coding sequence) inserted downstream of the hecNPV promoter. The pAc610 cloning vector corresponds to the polyhetrin gene promoter. The cloning site at position 18 contains the nucleotide sequence and contains a 5' leader sequence and a 3' polyhetrine sequence interrupted by a cloning site located downstream from the transcription start site: EcoRI, 5stI,
SmaI (XmaI), BamHI, K fallen I
and 2 so I. Danchou I, A animal I and) line
lI is not non-repetitive. K pn I or DangHc
There are no related parts. FIG. 16 shows recombinant A CNPV (Ac-pa
Figure 2 schematically shows the formation and selection for al). Shaded bars indicate the polyhedrin promoter and 5° leader sequence. The black bar represents the baculovirus polyhetrin gene. Part of this gene is LAV/
The plasmid DAc-QaQI DN is interrupted by a HTLVII gap-coated region
Exists in A. Cellular LAV/HTLVIII 0a
OI gene: one of the polyhetrin genes interrupted by
A recombinant plasmid DNA containing wild-type AcNP
co-transfected in combination with V DNA. Recombination that occurs in the polyhetrin sequences flanking the chimeric gene will cause LAV to enter the AcNPV genome.
/HTLVm f) Introduce aQ gene gene array. FIG. 17 shows Spodoptera frugiperda “parallel op” infected with wild-type AcPNV and Ac-gagl.
Extracted from tera frugiperda cells
t: R Figure 18 shows recombinant baculovirus Ac
-Yes. Proteins were detected 24 hours postinfection (lanes 1 and 2).
) for 2 h at 48 h (lanes 3, 4) and 72 h (lanes 5, 6) [35S1 methionine was labeled. Labeled proteins in cell lysates were detected in control human serum (lanes 1, 3.5) or AIDS patient serum (lanes 1, 3.5).
lanes 2.4.6) and the immune complexes were precipitated with Staphylococcus aureus protein A. Immunoprecipitated proteins were resolved by electrophoresis in a 10% 5DS-polyacrylamide gel and detected by fluorography. Figure 19 shows the LAV/former LV mgap-related cells found in cells infected with the recombinant AcNPV of the present invention.
Frugiperda (sf9) cells were infected with Ac-gagl and pulse-labeled for 5 min at 24 h postinfection. Cells were then washed with complete medium and in complete medium for 0 hours (lanes 1.2), 2 hours (lanes 3, 4), 4 hours.
(lanes 5, 6) and 8 hours (lane 7.8). At these times, cells were washed, lysed, and immunoprecipitated with either stock serum from IAV/TLVIII seropositive individuals (lanes 2.4.6.8) or normal human serum (lanes 1, 3, 5. Proteins were resolved by 5DS-polyacrylamide gel electrophoresis. Figure 20 shows vaccinia virus 7.5 promoter linked to [Av/)ITLVIII genomes of various lengths containing the QaCJ coding sequence Five plasmids DV-QaC) containing 1. DV-QaQ2. DVD-D
aC)3. Figure 2 schematically represents the formation of DV-QaQ4 and 0V-QaQ5. The DGS62 cloning vectors used to construct these were pGs20 (see Figure 3) and the unique EC0R1 vector located downstream of the unique 3ma1 site of DGS20.
They are the same except for having different parts. Figure 21 shows recombinant vaccinia AV/HTLV [l
Virus (v-aaqlNY, v-c+aa2NY,
v-c+aa3NY, v-c+aq4NY and V
- in cells infected with either (la(151'lY) or the parental vaccinia virus (v-NY), and in mice. Samples of purified LAV/HTLVul pillions were fermented and the positive control Concentrated B5C-40 saturation was associated with a multiplicity of infection of 10 (
moi). Infections were allowed to mature for 12 hours, at which time cells were harvested and lysed. All lllll follicular proteins are 5DS-
Resolved by PAGE and fixed on nitrocellulose by electrotransfer. The filter can contain (1) AIDS patient serum that is then detected using goat anti-human immunoglobulin conjugated to alkaline phosphatase (Figure 21B), or 2)
Qa, which is next assayed using alkaline phosphatase IJ-conjugated goat anti-mouse immunoglobulin.
were reacted with mouse monoclonal antibodies (Figures 218 and 21C) that limit the g proteins p25 and ρ18. FIG. 22 schematically represents the formation of plasmid pAc-env5 containing the complete [^V/HTLVIII envelope coding sequence inserted downstream of the AcNPV polyhedrin promoter. The pAc610 cloning vector is described in Figure 15 above. FIG. 23 shows recombinant vector 10 virus Ac-. Mock infected cells (mock) and parent Baki 10 virus strain (^c
-NPV) is included as a control. Proteins expressed by infected sf9 cells were labeled with 35s-methionine for 2 min at 28 h postinfection. The labeled protein in the cell lysate may be combined with AIDS patient serum or Dρ110 or! 111) was reacted with a mouse monoclonal antibody limiting 41, and immune complexes were precipitated using Staphylococcus aureus protein A. The immunoprecipitated proteins were
Resolved by 5DS-PAGE and detected by fluorography. Patent applicant Oncogun agent Patent attorney 8 1) Miki M (and 1 other person) (Sushi- 7
7 senior will person moxibustion Saraba nζ Hemaka 7 voices) IELISA with one Uri Komone Power A face pre-liquid friend FIG, II F/G/7 FIG I8 1 234567g FIG, /9

Claims (1)

Claims: (1) LAV/HTLVIII under the control of a second nucleotide sequence that regulates gene expression such that a peptide or protein related to the epitope of LAV/HTLVIII is expressed in a host infected with the virus; A recombinant virus having a genome consisting of a nucleotide sequence encoding an epitope of HTLVIII or an antigenic portion thereof. (2) The nucleotide sequence encoding the LAV/HTLVIII epitope consists of the nucleotide sequence of the LAV/HTLVIII envelope gene or any part thereof encoding an antigenic peptide or protein. The recombinant virus described in section. (3) The nucleotide sequence of the envelope gene is substantially nucleotide numbers 5767 to 854 in FIG.
A recombinant virus according to claim 2, consisting of a nucleotide sequence as depicted in Figure 9 or any part thereof encoding an antigenic peptide or protein. (4) The recombinant virus according to claim 1, wherein the peptide or protein expressed in the infected host is related to the envelope epitope of LAV/HTLVIII or any antigenic portion thereof. (5) Claim 4, wherein the envelope epitope consists of a peptide or protein having an amino acid sequence substantially as delineated as amino acid residue numbers 1 to 861 in FIG. 2, or any antigenic portion thereof. The recombinant virus described in . (6) The recombinant virus according to any one of claims 1 to 5, which is an enveloped virus. (7) Claim 6 consisting of vaccinia virus
The recombinant virus described in section. (8) Recombinant vaccinia virus v-env5 deposited with ATCC and assigned deposit number VR2113
or a mutant, recombinant or genetically engineered derivative thereof. (9) Recombinant vaccinia virus v-env2 deposited with ATCC and given deposit number VR2114
or a mutant, recombinant or genetically engineered derivative thereof. (10) Deposited with ATCC and deposit number VR2148
Recombinant vaccinia virus v-env marked with
7 or a mutant, recombinant or genetically engineered derivative thereof. (11) Deposited with ATCC and deposit number VR2149
Recombinant vaccinia virus v-env marked with
8. The recombinant virus according to claim 7, which is 5NY or a mutant, recombinant or genetically engineered derivative thereof. (12) Claim 1 consisting of baculovirus
The recombinant virus according to any one of Items 5 to 5. (13) The recombinant virus according to claim 12, which comprises a polyhedrosis virus. (14) Deposited with ATCC and deposit number VR2151
Recombinant baculovirus Ac-env marked with
13. The recombinant virus according to claim 12, which is 5 or a mutant, recombinant or genetically engineered derivative thereof. (15) The recombinant virus according to any one of claims 1 to 5, which is a naked virus. (16) Claim 15 consisting of adenovirus
The recombinant virus described in section. (11) The recombinant virus according to any one of claims 1 to 5, which is a bacteriophage. (18) The nucleotide sequence encoding the epitope of LAV/HTLVIII is
2. The recombinant virus according to claim 1, which consists of the nucleotide sequence of the g gene or any part thereof encoding an antigenic peptide or protein. (19) The gag gene nucleotide sequence is substantially the first
19. A recombinant virus according to claim 18, consisting of a nucleotide sequence as delineated in Figure 4 as nucleotide numbers 340-1835 or any part thereof encoding an antigenic peptide or protein. . (20) The recombinant virus according to claim 1, wherein the peptide or protein expressed in the infected host is related to an epitope of the core protein of LAV/HTLVIII or any antigenic portion thereof. (21) The composition according to claim 20, wherein the epitope consists essentially of the amino acid sequence delineated as amino acid residue numbers 1 to 500 in FIG. 14 or any antigenic portion thereof. Replaceable virus. (22) The recombinant virus according to any one of claims 18 to 21, which is an enveloped virus. (23) The recombinant virus according to claim 22, which is composed of vaccinia virus. (24) Deposited with ATCC and deposit number VR2150
Recombinant vaccinia virus v-gag marked with
24. The recombinant virus according to claim 23, which is 1NY or a mutant, recombinant or genetically engineered derivative thereof. (25) Claim 1 consisting of baculovirus
The recombinant virus according to any one of Items 8 to 21. (26) The recombinant virus according to claim 25, which comprises a polyhedrosis virus. (27) Deposited with ATCC and deposit number VR2147
Recombinant baculovirus Ac-gag marked with
26. The recombinant virus according to claim 25, which is 1 or a mutant, recombinant or genetically engineered derivative thereof. (28) The recombinant virus according to any one of claims 18 to 21, which is a naked virus. (29) Claim 28 consisting of adenovirus
The recombinant virus described in section. (30) The recombinant virus according to any one of claims 18 to 21, which comprises a bacteriophage. (31) Substantially pure antigenic peptide or protein related to the epitope of LAV/HTLVIII. (32) The peptide or protein according to claim 31, wherein the epitope is an envelope glycoprotein epitope of LAV/HTLVIII. (33) The peptide according to claim 32, wherein the amino acid sequence consists essentially of the amino acid sequence delineated as amino acid residue numbers 1 to 861 in FIG. 2 or any antigenic portion thereof. protein. (34) from a cultured cell in which the peptide or protein comprises a nucleotide sequence encoding the peptide or protein under the control of a second nucleotide sequence that regulates gene expression such that the peptide or protein is expressed by the cultured cell; The peptide or protein according to claim 32 or 33, which is purified. (35) The peptide or protein according to claim 34, wherein the cultured cells are microorganisms. (36) The peptide or protein according to claim 35, wherein the microorganism is a bacteria. (37) The peptide or protein according to claim 35, wherein the microorganism is yeast. (38) The peptide or protein according to claim 34, wherein the cultured cells are animal cell lines. (39) Deposited with ATCC and deposit number VR2113
Recombinant vaccinia virus v-env marked with
39. The peptide or protein according to claim 38, which is produced by an animal cell infected with 5 or a mutant, recombinant or genetically engineered derivative thereof. (40) Deposited with ATCC and accession number VR2114
Recombinant vaccinia virus v-env marked with
39. The peptide or protein according to claim 38, which is produced by an animal cell infected with 2 or a mutant, recombinant or genetically engineered derivative thereof. (41) Deposited with ATCC and deposit number VR2148
Recombinant vaccinia virus v-env marked with
39. The peptide or protein according to claim 38, which is produced by an animal cell infected with 7 or a mutant, recombinant or genetically engineered derivative thereof. (42) Deposited with ATCC and accession number VR2149
Recombinant vaccinia virus v-env marked with
39. The peptide or protein according to claim 38, which is produced by an animal cell infected with 5NY or a mutant, recombinant or genetically engineered derivative thereof. (43) The peptide or protein according to claim 34, wherein the cultured cells are insect cell lines. (44) Deposited with ATCC and deposit number VR2151
Recombinant vaccinia virus Ac-en marked with
44. The peptide or protein according to claim 43, which is produced by an animal cell infected with v5 or a mutant, recombinant or genetically engineered derivative thereof. (45) The peptide or protein according to claim 31, wherein the epitope consists of an HLV/HTLVIII core protein epitope. (46) The amino acid sequence according to claim 45, wherein the amino acid sequence consists essentially of the amino acid sequence delineated as amino acid residue numbers 1 to 500 in FIG. 14 or any antigenic portion thereof. peptide or protein. (47) from a cultured cell in which the peptide or protein contains a nucleotide sequence encoding the peptide or protein under the control of a second nucleotide sequence that regulates gene expression such that the peptide or protein is expressed by the cultured cell; The peptide or protein according to claim 45 or 46, which is purified. (48) The peptide or protein according to claim 47, wherein the cultured cells are microorganisms. (49) The peptide or protein according to claim 48, wherein the microorganism is a bacteria. (50) The peptide or protein according to claim 48, wherein the microorganism is yeast. (51) The peptide or protein according to claim 47, wherein the cultured cells are animal cell lines. (52) Deposited with ATCC and deposit number VR2150
Recombinant vaccinia virus v-gag marked with
52. The peptide or protein according to claim 51, which is produced from an animal cell infected with 1NY or a mutant, recombinant or genetically engineered derivative thereof. (53) The peptide or protein according to claim 47, wherein the cultured cells are animal cell lines. (54) Deposited with ATCC and deposit number VR2147
Recombinant vaccinia virus Ac-ga marked with
54. The peptide or protein according to claim 53, which is produced from animal cells infected with g1 or a mutant, recombinant or genetically engineered derivative thereof. (55) Claims 31, 32, and 3 in which the peptide or protein is chemically synthesized.
The peptide or protein according to item 3, item 45 or item 46. (56) A live virus vaccine preparation comprising the virus according to any one of claims 1 to 5, wherein the virus is infectious without causing disease in the host to be vaccinated. (57) The live virus vaccine preparation according to claim 56, wherein the virus is an enveloped virus. (58) The live virus vaccine preparation according to claim 57, wherein the enveloped virus is vaccinia virus. (59) A live virus vaccine preparation comprising an infectious dose of vaccinia virus v-env5 according to claim 8. (60) A live virus vaccine preparation comprising an infectious dose of vaccinia virus v-env2 according to claim 9. (61) A live virus vaccine preparation comprising an infectious dose of vaccinia virus v-env7 according to claim 10. (62) A live virus vaccine preparation comprising an infectious dose of vaccinia virus v-env5NY according to claim 11. (63) The live virus vaccine preparation according to claim 56, wherein the virus is a naked virus. (64) The live virus vaccine preparation according to claim 63, wherein the naked virus is an adenovirus. (65) A recombinant virus according to any one of claims 2 to 5 and claim 1.
A multivalent live virus vaccine preparation comprising a recombinant virus according to any one of Items 8 to 21, wherein both recombinant viruses are infectious without causing disease in the host to be vaccinated. . (66) The multivalent live virus vaccine preparation according to claim 65, wherein the recombinant virus is an enveloped virus. (67) The multivalent live virus vaccine formulation according to claim 66, wherein each enveloped virus consists of vaccinia virus. (68) (a) Vaccinia virus v-env5 according to claim 8, vaccinia virus v-env2 according to claim 9, vaccinia virus according to claim 10 A multivalent vaccine formulation comprising an infectious dose of v-env7 or the vaccinia virus v-env5NY according to claim 11 and (b) vaccinia virus v-gag1NY according to claim 24. (69) An inactivated virus vaccine preparation comprising an effective amount of the virus according to any one of claims 1 to 5 in a non-infectious state mixed with a pharmaceutical carrier. (70) An inactivated virus vaccine preparation comprising an effective amount of the enveloped virus according to claim 6 in a non-infectious state mixed with a pharmaceutical carrier. (71) An inactivated virus vaccine preparation comprising an effective amount of the vaccinia virus according to claim 7 in a non-infectious state mixed with a pharmaceutical carrier. (72) An effective amount of vaccinia virus v- according to claim 8 in a non-infectious state mixed with a pharmaceutical carrier.
An inactivated virus vaccine preparation consisting of env5. (73) An effective amount of vaccinia virus v- according to claim 9 in a non-infectious state mixed with a pharmaceutical carrier.
An inactivated virus vaccine preparation consisting of env2. (74) An effective amount of vaccinia virus v according to claim 10 in a non-infectious state mixed with a pharmaceutical carrier.
- An inactivated virus vaccine formulation consisting of env7. (75) An effective amount of vaccinia virus v according to claim 11 in a non-infectious state mixed with a pharmaceutical carrier.
- An inactivated virus vaccine preparation consisting of env5NY. (76) (a) the recombinant virus according to any one of claims 2 to 5, which expresses an LAV/HTLVIII envelope-related epitope; and (b) a LAV/HTLVIII core-related epitope. each recombinant virus expressing a non-infectious virus when mixed with a pharmaceutical carrier. A polyvalent inactivated virus vaccine formulation. (77) The multivalent inactivated virus vaccine preparation according to claim 76, wherein each recombinant virus is an enveloped virus. (78) The multivalent inactivated virus vaccine preparation according to claim 77, wherein each enveloped virus consists of vaccinia virus. (79) (a) Vaccinia virus v-env5 according to claim 8, vaccinia virus v-env2 according to claim 9, vaccinia virus according to claim 10 A multivalent inactivated virus comprising an effective amount of v-env7 or vaccinia virus v-env5NY according to claim 11, and (b) vaccinia virus v-gag1NY according to claim 24. Vaccine preparation. (80) A subunit vaccine preparation, wherein the immunogen consists of an effective dose of the peptide or protein according to claim 32 or 33, mixed with a pharmaceutical carrier. (81) A subunit vaccine preparation, wherein the immunogen consists of an effective dose of the peptide or protein according to claim 34, mixed with a pharmaceutical carrier. (82) A subunit vaccine preparation, wherein the immunogen consists of an effective dose of the peptide or protein according to claim 35, mixed with a pharmaceutical carrier. (83) A subunit vaccine preparation, wherein the immunogen consists of an effective dose of the peptide or protein according to claim 36, mixed with a pharmaceutical carrier. (84) A subunit vaccine preparation, wherein the immunogen comprises an effective dose of the peptide or protein according to claim 37, mixed with a pharmaceutical carrier. (85) A subunit vaccine preparation, wherein the immunogen consists of an effective dose of the peptide or protein according to claim 38, mixed with a pharmaceutical carrier. (86) A subunit vaccine preparation, wherein the immunogen comprises an effective dose of the peptide or protein according to claim 39, mixed with a pharmaceutical carrier. (87) A subunit vaccine preparation, wherein the immunogen consists of an effective dose of the peptide or protein according to claim 40, mixed with a pharmaceutical carrier. (88) A subunit vaccine preparation, wherein the immunogen consists of an effective dose of the peptide or protein according to claim 41, mixed with a pharmaceutical carrier. (89) A subunit vaccine preparation, wherein the immunogen consists of an effective dose of the peptide or protein according to claim 42, mixed with a pharmaceutical carrier. (90) A subunit vaccine preparation, wherein the immunogen comprises an effective dose of the peptide or protein according to claim 42, mixed with a pharmaceutical carrier. (91) A subunit vaccine preparation, wherein the immunogen comprises an effective dose of the peptide or protein according to claim 43, mixed with a pharmaceutical carrier. (92) (a) the first peptide or protein according to claim 32 or 33; and (b) the second peptide or protein according to claim 45 or 46. A multivalent subunit vaccine formulation comprising an effective dose of protein mixed with a pharmaceutical carrier. (93) Effective dosage of (a) the first peptide according to claim 32 or 33, and (b) the second peptide or protein according to claim 47 A multivalent subunit vaccine formulation consisting of a mixture of the following and a pharmaceutical carrier. (94) Effective dosage of (a) the first peptide according to claim 32 or 33, and (b) the second peptide or protein according to claim 48 A multivalent subunit vaccine formulation consisting of a mixture of the following and a pharmaceutical carrier. (95) Effective dosage of (a) the first peptide according to claim 32 or 33, and (b) the second peptide or protein according to claim 49 A multivalent subunit vaccine formulation consisting of a mixture of the following and a pharmaceutical carrier. (96) Effective dosage of (a) the first peptide according to claim 32 or 33, and (b) the second peptide or protein according to claim 50 A multivalent subunit vaccine formulation consisting of a mixture of the following and a pharmaceutical carrier. (97) Effective dosage of (a) the first peptide according to claim 32 or 33, and (b) the second peptide or protein according to claim 51 A multivalent subunit vaccine formulation consisting of a mixture of the following and a pharmaceutical carrier. (98) Effective dosage of (a) the first peptide according to claim 32 or 33, and (b) the second peptide or protein according to claim 52 A multivalent subunit vaccine formulation consisting of a mixture of the following and a pharmaceutical carrier. (99) Effective dosage of (a) the first peptide according to claim 32 or 33, and (b) the second peptide or protein according to claim 53 A multivalent subunit vaccine formulation consisting of a mixture of the following and a pharmaceutical carrier. (100) (a) Claim 32 or 33
A multivalent subunit vaccine comprising an effective dose of the first peptide according to claim 54 and (b) the second peptide or protein according to claim 54 mixed with a pharmaceutical carrier. formulation. (101) Recombinant DNA vector consisting of pv-env1. (102) Recombinant D according to claim 101
A unicellular organism containing an NA vector. (103) Recombinant D according to claim 101
A bacterium containing an NA vector. (104) Deposited with NRRL and deposit number B-180
104. The bacterium of claim 103, comprising Escherichia coli designated 03 or a mutant, recombinant or genetically engineered derivative thereof. (105) A recombinant DNA vector consisting of pv-env2. (106) Recombinant D according to claim 105
A unicellular organism containing an NA vector. (107) Recombinant D according to claim 105
A bacterium containing an NA vector. (108) Deposited with NRRL and deposit number B-180
108. The bacterium of claim 107, comprising Escherichia coli designated with 04 or a mutant, recombinant or genetically engineered derivative thereof. (109) Recombinant DNA vector consisting of pv-env5. (110) Recombinant D according to claim 109
A unicellular organism containing an NA vector. (111) Recombinant D according to claim 109
A bacterium containing an NA vector. (112) Deposited with NRRL and deposit number B-180
112. The bacterium of claim 111, comprising Escherichia coli designated 05 or a mutant, recombinant or genetically engineered derivative thereof. (113) Recombinant DNA vector consisting of pv-env7. (114) Recombinant D according to claim 113
A unicellular organism containing an NA vector. (115) Recombinant D according to claim 113
A bacterium containing an NA vector. (116) Deposited with NRRL and deposit number B-181
116. The bacterium of claim 115, comprising Escherichia coli or a mutant, recombinant or genetically engineered derivative thereof. (117) Recombinant DNA vector consisting of pv-gag1. (118) Recombinant D according to claim 117
A unicellular organism containing an NA vector. (119) Recombinant D according to claim 117
A bacterium containing an NA vector. (120) Deposited with NRRL and deposit number B-181
120. The bacterium of claim 119, comprising Escherichia coli or a mutant, recombinant or genetically engineered derivative thereof, designated 11. (121) Recombinant DNA vector consisting of pAc-gag1. (122) Recombinant D according to claim 121
A unicellular organism containing an NA vector. (123) Recombinant D according to claim 121
A bacterium containing an NA vector. (124) Deposited with NRRL and deposit number B-181
124. The bacterium of claim 123, comprising Escherichia coli designated with 05 or a mutant, recombinant or genetically engineered derivative thereof. (125) Recombinant DNA vector consisting of pAc-env5. (126) Recombinant D according to claim 125
A unicellular organism containing an NA vector. (127) Recombinant D according to claim 125
A bacterium containing an NA vector. (128) Deposited with NRRL and deposit number B-181
128. The bacterium of claim 127, comprising Escherichia coli designated with 09 or a mutant, recombinant or genetically engineered derivative thereof.