HU205780B - Process for producing recombinant viruses, proteins and vaccine against aids - Google Patents

Description

The present invention relates to the production of viruses which are the pathogen of the acquired immune deficiency syndrome (Aquired Immune Deficiency Syndrome, hereinafter "AIDS") and the lymphadenopathy syndrome (LAS), Lymphadenophthy associated-virus (LAV). / Human T-cell leukemia virus (HTLV-Hl) produces epitopes of peptides and proteins called alternate names. The viruses of the present invention produce peptides of the LAV / HTLVGI type which elicit a protective immune response and are therefore useful as immunogenic agents in AIDS vaccines against LAS or in multivalent vaccines. Infectious viruses of the present invention that multiply in the host without causing disease may also be administered in a live virus vaccine; they induce lasting immunity and provide a significant increase in Immunity.

It is a further object of the present invention to provide peptides and proteins of the LAV / HTLV β-epitope type which can be used as immunogenic agents for AIDS and LAS anti-subunit vaccines or as antigens in immunoassays for the diagnosis of LAS or AIDS. These peptides and proteins can be prepared or chemically synthesized in any vector host system by recombinant DNA technology. Accordingly, the construction of novel DNA sequences and vectors such as plasmid DNA and viral DNA (e.g., human viral, animal viral, insect viral DNA, or bacteriophage) is within the scope of the present invention; these DNA sequences and vectors induce the production of LAVZHTLVIH-type peptides and proteins in a suitable host, which can then be purified from apeptides and proteins after purification. ALAV / HTLVHI virus-related peptide and protein priming mode will be described below.

In one embodiment of the invention, recombinant vaccinia fragments are used to produce the outer (envélope or env) and inner (co-40 re or gag) proteins of LAV / HTLV D1. For this purpose, env or gag-type DNA sequences were encoded into the vaccine vector that encode the LAV / HTLV ID outer or core skeletal proteins 45, which vectors in the host cell promote LAV / HTLV gazd- expression of genes. The LAV / HTLV Dt outer proteins produced by the recombinant vaccinia viruses were antigenic and immunogenic and elicited a humoral and cellular immune response in non-human primates. The LAV / HTLV D1 gag proteins produced by the recombinant vaccinia viruses are immunoreceptive and contain major eplopes of the corresponding core proteins.

The recombinant vaccinia viruses producing LAV / HTLV D1 shell type proteins may be used alone or in combination with other LAV / HTLV ΠΙ type protein (such as core skeletal protein) recombinants in a viral vaccine composition. LAV / HTLV 60 produced by recombinant viruses

The ΠΙ-type proteins can be purified, if desired, and thus used as immunogenic material in a subunit vaccine composition. As the host animal will recognize the LAV / HTLV ΠΙ protein as a "foreign" substance, it will respond to the protein or protein combination in question with a humoral and / or cellular immune response. Thus, a properly prepared vaccine will protect the host against further LAV / HTLV D1 infection.

In another embodiment of the invention, re- combinatorial baculoviruses (Autographa califomica nuclear polyhedrosis virus, or AcNPV for short) are administered.

It is designed for the recovery ofLAV / GTLVID * external and internal proteins. For this purpose, an env or gag type ^s

DNA sequences were incorporated into a baculovirus vector encoding LAV / HTLV D1 outer or envelope backbone proteins, which promote the expression of LAV / HTLV D1 genes in the host. The LAV / HTLVDI proteins produced by the recombinant baculoviruses have been shown to be antigenic by ELISA and radioimmunoassay.

The invention also relates to the production of LAV / HTLV D1 antigens which are essential for human medicine. The peptides and proteins of the present invention can be used as reagents in various immunoassays, such as ELISA and radioimmunoassays, and can be used in diagnostic assays to detect antibodies specific for LAV / HTLV ΠΙ, blood samples, body fluids, tissues, and the like. When testing. These reagents can also be a useful tool for elucidating the mechanism of action of LAV / HTLV D1.

2. State of the art

2.1 is the AIDS virus

Acquired Immune Deficiency Disease (AIDS) is known to result in severe immune deficiency, primarily due to the impairment of the patient's cellular immune response [Gottlieb, M. et al., N. Engl. J. Med.

305.1425. (1981) and J. Masur et al., N. Engl.

301, 1431 (1981)]. Clinically, the disease was observed to occur in two ways, namely: (a) an introductory phase of lymphadenapathy (LAS), in which chronic lymphadenopathy, leukopenia and peripheral blood helper cells (OKT4 cells) are expressed in a normal manner; T-lymphocyte ratio -J (OKT.OKT8) from 2 to 0.1 or less in patient | progression; and a deficiency of OKT4 cells in which the normal OKT4: OKT8 ratio is reversed; absolute lymphopenia and recurrent so-called "oppurtunist infections" (which are mainly infections caused by Pneumocystis camli) are still characteristic of the disease. This latter stage usually results in the death of the patient. Some patients have an increased incidence of lymphoma and Kaposi's sarcoma. There is currently no way to treat or cure the disease.

HU 205780 Β ί

fe

Based on the type of patient affected by the infection and the route of transmission of the disease, it appears that the infection is spreading through intimate contact. Three different research groups have provided convincing evidence to this effect, suggesting that AIDS is caused by a retrovirus that shows tropism to helper T lymphocytes. The 3 groups are as follows:

(a) R. C. Gallo et al., National Institute of

Health researchers out of 10 patients with AIDS

A cell retrovirus called HTLV ΙΠ -alali [R. C, Gallo et al., Science 224: 500 (1984); M. Popovic et al., Science 224: 497 (1984)]. Antibodies to HTLV IH have also been detected in the serum of AIDS patients.

(b) L. Montagnier and his team at the Pasteur Institute isolated T-lymphotropic retrovirus' (LAV / HTLV ΙΠ) from a suspected AIDS patient with cervical lymphadenopathy [F. Barre-Sinoussi et al., Science, 220, 868 (1983)]. They also detected antibodies to LAV / HTLV IH in the serum of AIDS patients (Kalyansraman, S.S. et al., Science, 225, 321 (1984)). In addition, LAV / HTLV ΠΙ virus has been isolated from lymphocytes of 25 patients who have contracted AIDS after receiving blood from a donor who was later found to have AIDS [P. M. Feorino et al., Science,

225, (1984)], 69th. 30 (c) J. Levy et al. Isolated an infectious retrovirus known as AIDS-associated retrovirus from peripheral single-cell cells of AIDD patients [J. A. Levy et al., Science 225: 840 (1984)].

Although the three viruses have been isolated independently, it is likely that the same retroviral subgroup [J. A. Levy et al., Science, 225, 840.

(1984)] and hereinafter referred to as LAV / HTLV HI.

The structure of retroviruses is usually composed of a ribnucleo protein core and a surrounding lipid envelope formed by the virus during cellular growth. The glycoproteins encoded by the virus are present as an extension of this envelope. These determine which host cells the virus can fuse with and respond to specific receptors on the surface of the cells in question. Presumably, the role of viral neutralizing antibodies is to bind to envelope glycoproteins and thereby prevent them from interacting with receptors on the surface of cells (J. Tooze, J. Mol. Biology of Tumor Viruses, 534-535). 1973 Cold Spring Harbor Laboratory; RNA Tumor Viruses, 236-237, published by R. Weiss, N. Teich, H. Vannus and J. Coffin, 1982, Cold Spring Harbor Laboratory). In the case of LAV / HTLV virus Hl evidence 55 to the virus-binding receptor is a component of the _T4 antigen of T-lymphocytes in a group is present [Dalgleish AG et al, Nature, 312,

763 (1984) and D. Klatzmann et al., Natura, 312, 767 (1984)],

The genome of the LAV / HTLV IH RNA is shown in Figure 1. Generally, 3 genes are recognized: the gag gene encodes for internal skeletal proteins (core proteins) and defines virus group-specific antigens; the nurse gene encodes a virion-linked reverse transcriptase; and the hormone gene carries genetic information on viral glycoproteins. The regions asor and 3'orph represent the open reading frames of the genome and their role is not yet known.

2.2Vakcinák

Several methods are known for the prevention and treatment of viral infections. These include vaccines that elicit an active immune response, and chemotherapeutic agents and interferon are known to treat viral infections.

Conventional vaccine preparation methods use attenuated or inactivated virus. The virus becomes biologically harmless by inactivation, but does not lose its ability to induce immunity. Inoculation of these "killed" portions of the virus into the body elicits an immune response that can neutralize the effect of a future infection with a live virus. However, the main problem with using killed virus vaccines (inactivated virus) is that not all virus particles may have been inactivated. Even if this trigger has been avoided, another problem is that since the killed virus does not replicate in the body, the acquired immunity is short-lived and usually requires repeated immunization. Finally, there may be concerns that viral proteins may be altered during the inactivation process and thus become less effective immunogenic agents.

By attenuation, strains of virus that have essentially lost their disease ability can be obtained. One way of doing this is by culturing the virus under unusual conditions and / or exposing it to frequent passage in cell culture. The virus mutants are then isolated by selection which have lost their viral lenses but are still capable of eliciting an immune response. Attenuated viruses will generally become good immunogenic agents because they are indeed replicated in the host, thereby providing long-lasting immunity. However, when using live virus, there are risks, the most worrying of which is the weakening of the virus.

A variant of the above methods is the use of a subunit vaccine. The aim of this method is to immunize only with proteins containing the relevant immunological material. In many enveloped viruses, glycoproteins carrying the genetic code for the virus contain epitopes that produce antibodies that block the effect. The glycoproteins of the following viruses are La Crosse Virus [F. GonzalezScarano, R.E. Shope, C.E. Calisher, and N. Nathanson, Virology 120: 42 (1982)]; diarrhae virus in newborn calves [S. Matsuno and S. Inouye, Infection and Immunity, 39, 155 (1983)]; the Venezuelan horse

HU 205780 Β enchephalomyelitis virus [J. H. Mathews and J. T. Roehrig, J. Imm. 129, 2763 (1982)]; PuntaToro virus [J.M. Dalrymple, C: J. P'eters, J.F. Smith and Μ. K. Gentry, Rep. Of the Negative Strand Viruses, edited by D.H.L. Bishop and R. W. Compans, page 167, Elsevier, New York (1981)]; murine leukemia virus (R A Steeves, M. Strand and J. T. Augug J. Viriro, 14, 187 (1974)); mouse mammary tumor virus | R. J. Massey and G. Schoohetman, Virology, 115, 20 (1981)]. The main advantage of subunit vaccines is that they do not contain non-relevant viral material.

Vaccines are often administered to a patient in combination with various adjuvants. The adjuvant allows for longer or stronger immunity and facilitates the administration of the antigen in smaller amounts and less frequently than when the immunogenic agent is used alone. The mechanism of action of the adjuvant is complex and not yet fully understood; it may stimulate phagocytosis and other functions of the reticuloendothelial system and play a role in delayed antigen release and degradation. Known adjuvants include Freund's adjuvant (total or partial), the so-called adjuvant 65, which contains peanut oil, mannitol monoleate and aluminum stearate, L-121 polyoxalene, avridine and inorganic gels. aluminum hydroxide, aluminum phosphate and potassium aluminum sulfate. Freund's adjuvant is no longer used in human vaccine formulations because it contains non-metabolizable mineral oil and is a potential carcinogen.

2.2 Recombinant DNA Technology and Vaccine Virus

Molecular cloning, as well as the expression of viral genetic information encoding viral genetic information in the host that protects against the virus and elicits an immune response, is accomplished by recombinant DNA technology to obtain a subunit vaccine. In this way, all other viral genetic information is excluded and only the desired proteins that provide a neutralizing response to the virus are introduced into the host. The host animal is never exposed to the entire virus, so there is no risk of infection.

Recently, a novel approach to the problem has been described and may also be suitable for the preparation of a subunit vaccine [M. Mackett, G.L. Smith and B.. Moss, Proc. Natl. Acad. Sci., 79, 7415-7419. (1982); M Mackett, G. L. Smith and B. Moss, J. Virol., 49, 857-864. (1984); and D. Panicali and E. Paoletti, Proc. Natl. Acad. Sci.

79, 4927-4931. (1982)]. The essence of this method is to use a recombinant vaccinia virus as a vector expressing a foreign gene. When a recombinant vaccinia virus is introduced into a host, the integrated foreign gene is expressed therein and thus elicits an immune response in the host against such gene products. Because a live recombinant vaccine virus can be used as a vaccine, the solution combines the benefits of a subunit vaccine and a live virus vaccine.

The vaccine virus contains a linear double-stranded DNA genome of approximately 187,000 base pairs in length and replicates in the cytoplasm of infected cells. These viruses have the complete transcriptional enzyme system within the virus nucleus (including initiation, methylation and polyadenylation enzymes) required for viral infection. Vaccine virus transcriptional regulatory sequences (promoters) allow transcription only initiated by vaccinia RNA polymerase, not initiated by host cell RNA polymerase.

In order for foreign DNA to be expressed in the recombinant vaccinia virus, it is necessary that the vaccine promoter is ligated with the DNA sequence encoding the foreign proteins (ligation). Plasmid vectors, also known as insertion vectors, were constructed to incorporate the resulting chimeric genes into the vaccine virus. One type of insert vector is comprised of: (a) a vaccinia virus promoter including a transcription start site; (b) a number of sites downstream of the transcription start site downstream of the anuldeotide chain, (c) non-essential vaccine DNA (e.g., the TK gene) that flank the promoter and the cloning sites to inserting a chimeric gene into a homologous, non-essential portion of the viral genome; and (d) a bacterial and antibiotic resistance-encoding gene (marker) for replication that enables replication and selection. Examples of such vectors are M. Mackett, G. L. Smith and B. Moss, S. Virol., 49, 857-864. (1984)].

Introducing recombinant bacterial plasmid vectors containing the foreign gene into cells infected with vaccine vectors yields recombinant vaccine vectors. The infected cells undergo homologous recombination, resulting in the integration of the foreign gene into the viral genome. Screening of infected cells can be accomplished by immunoassays, DNA plaque hybridization, or genetic selection of recombinant viruses and subsequent isolation. These vaccine recombinants retain their original functions and infectivity and can contain up to 35,000 nucleotide bases of foreign DNA

Expression of the foreign gene may be detected by enzymatic or immunological methods (e.g., immuno-recombination, radioimmunoassay, immunoblot analysis). Naturally occurring membrane glycoproteins produced by cells infected with recombinant vaccinia virus can be delivered to the cell surface in glycosylated form. High levels of expression can be achieved by cloning a large number of copies of a single gene or using strong promoters.

2.2.2. Recombinant DNA technique and baculovirus

Pennock et al., Mol. Cell. Biol., 4, 399 (1984)] and Smith et al., J. Biol. Virol., 46, 584 (1983)] described a method that could potentially be used for the production of recombinant proteins, the essence of which is to use a baculovirus vector in a foreign gene incorporated into the viral genome.

HU 205780 Β gene. The recombinant baculovirus expresses the foreign gene when introduced into a suitable host cell.

The prototype of the Baculoviridae family is Autographa califomica nuclear polyhedrosis virus (AcNPV). The genome of AcNPV contains double-stranded circular DNA of 128,000 bp. A gene map of this is provided by G, E, Smith and MD Summers (Virology, 89, 517 (1978)) with a number of restriction sites. The virus replicates in the nucleus of infected insects. Upon infection of susceptible cells with wild-type Ac-NPV *, two types of viral particles are formed (namely, closed and non-closed) _. Viral particles are surrounded by a protein called polyhedra encoding the polyhedron gene [Virol. 561-565. (1983)]. Closed virus particles in the infected cell can be observed by light microscopy.

Expression of foreign DNA in recombinant baculovirus requires ligation of DNA sequences encoding the foreign protein and promoters of the baculovirus. Plasmid vectors, also known as carrier vectors, were constructed to incorporate chimeric genes into AcNPV. One type of plasmid vector consists of: (a) the AcNPV promoter containing the start site of the transcription system; (b) a number of single-cleavable restriction endonuclease cloning sites downstream of the start site for insertion of foreign DNA fragments; (c) a non-essential AcNPV DNA (e.g., a polyhedron gene) flanked by the promoter and cloning sites for direct delivery of the chimeric gene to the homologous, non-essential region of the viral genome; and (d) a bacterial and antibiotic resistance coding gene for replication that allows replication and selection in E. coli. Such vectors include, for example, Miyamota et al. Cell. Biol., 5, 2860 (1985)].

Recombinant baculoviruses are prepared by co-infecting cells with the recombinant carrier plasmid containing the gene and the baculovirus DNA. In infected cells, homologous recombination occurs, whereby the foreign gene is incorporated into the viral genome. Once the foreign gene has been incorporated into the polyhedron gene, closed virus particles are no longer produced and the resulting recombinant plateaus can be visually distinguished by the absence of such closed formations in the screen. Immunological methods such as plaque hybridization or DNA $ of the recombinant viruses in infected cells _Wed: lekciójával and subsequent isolation can be screened * needles. They preserve the essential functions and infectivity of the recombinant virus.

Expression of the foreign gene can be detected by enzymatic or immunological methods (e.g., immunoprecipitation, radioimmunoassay, or immunoblot analysis). High levels of expression can be achieved by cloning a large number of copies of a single gene or by using strong promoters.

3. BRIEF SUMMARY OF THE INVENTION

The present invention relates to viruses that direct expression of peptides or proteins related to LAV / HTLVΙΠ epitopes. The recombinant viruses of the invention, which express epitopes of LAV / HTLV-ΙΠ that elicit a protective immune response, are produced using infectious viruses that express LAV / HTLV-it epitopes in humans but which do not affect the host.

The invention also relates to peptides and proteins related to LAV / HTLV ΙΠ epitopes. These protective immune responses, in the form of subunit vaccines and multivalent vaccines, can be used to prevent LAV / HTLV D1 infections. The peptides and proteins of the invention may also be used in the diagnosis of AIDS or LAS. The peptides and proteins of the invention can be prepared and isolated from any host cell expression vector system. Examples of such systems include: culturing an animal or insect cell infected with a suitable recombinant virus; A microorganism (e.g., a bacterium) exposed to transfection of a recombinant plasmid, cosmid or phage. DNA constructs and methods for expressing genetic information encoding LAV / HTLV ΠΙ epitopes are also within the scope of the present invention. It also encompasses the chemical synthesis of peptides and proteins of the present invention.

In one exemplary embodiment of the present invention, the gene sequences encoding the outer glycoproteins of LAV / HTLV ID or the core skeletal protein (i.e., the env and / or gag gene sequences of LAV / HTLV ID) are inserted into plasmids such that the early vaccine viral promoter is linked to the 5'-position of the initial methionine portion (ATG) of the LAV / HTLV ΙΠ gene sequence and the resulting chimeric gene is flanked by vaccinia thymidine kinase (TK) DNA sequences. Plasmids are introduced into cells pre-infected with wild-type vaccinia virus (transfection), where the chimeric LAV / HTLV D1 env or gag gene is inserted into the vaccinia virus TK gene by its flanking sequence. Following cell lysis, viruses are obtained which form plaques in TK cells. Recombinant viruses are then selected either to form plaques on the cells of interest in the presence of 5-bromooxyduridine or to be DNA-DNA-labeled with a sample containing .45 LAV / HTLV Dl env or LAV / HTLV ΙΠ gag sequences. hybridization. After purification, the hybridizable plaques are further cultured and the resulting recombinant viruses are assayed for the production of LAV / HTLV III external glycoproteins or internal skeletal proteins. External proteins of the LAV / HTLV ΠΙ type expressed by the recombinant vaccine virus have been shown to be antigenic and immunogenic and have been shown to be capable of inducing both humoral and cellular immunity in lower human primates. The LAV / HTLV ΙΠ type gag proteins expressed by recombinant vaccine viruses were immunoreactive and contained the major epitopes of authentic core proteins.

According to another embodiment of the invention,

LAV / HTLV ΙΠ env and gag sequences are thus translated

HU 205780 Β was introduced into the plasmid to attach the 5 'position of the abacaculovirus polyhedron gene promoter to the 3' end position of the LAV / HtlV IH gene sequence (ATG). These plasmids and wild-type baculovirus DNA are used to co-infect cells, A4. wherein the chimeric LAV / HTLV IH gene flanked by additional AcNPV sequences is integrated into the baculovirus polyhedron gene by recombination. The selection of the recombinant virus can be done either by visual observation (absence of occlusion formations) or by the use of radiolabeled LAV / HTLV ΠΙ-env or LAV / HTLV DNS-gag samples by DNA-DNA hybridization. The recombinant plaques are purified after purification and the resulting recombinant viruses are assayed for their ability to produce LAV / HTLV IH-related proteins by immunoprecipitation analysis on an SDS-polyacrylamide gel. The solution obtained by cell lysis of the infected cells is assayed by ELISA for the detection of LAV / HTLV ΠΙ antibodies in serum.

4. Description of the Figures

Figure 1 shows the structure of the LAV / HTLV H1-injected provirus genome. Hatched areas indicate regions of coding structure. In addition, the coding regions for the group-specific antigen, the reverse transcriptase and the outer proteins are designated gag, pol, and env, respectively. The overlapping coding regions 30 are indicated as transversely hatched regions. The numbers represent the order of the base pairs along the strand from the origin of viral transcription to the origin. The restriction points are designated as Bg, Bg / H; Ec,

Eco; Hn, 77 / ndIIÍ; Kp, ^ »I; ss, Ssil

Figure 2 shows the nucleotide sequence of the LAV / HILVIH specific region (EcoRI to SsiI) present in plasmid pRS-3 DNA. The 40 LAV / HTLV IH extension contains the complete LAV / HTLV H1 envelope gene (nucleotide sequence 57668349). The restriction sites used for the production of p-env1, p-env2 and p-env5, as well as the amino acid sequence deduced from the nucleotide sequence 45, are also indicated in the figure.

Figure 3 is a schematic representation of the construction of plasmids in which a portion of the LAV / HTLV IH envelope protein coding sequence is inserted downstream of the 50 vaccinia vfrus promoters. The coding sequence for the ALV / HTLV HI envelope protein is illustrated by a light bar, and the vaccine promoter by a shaded bar. The nucleotide sequence at the junction of the vaccinia promoter region with the sequence encoding the LAV / HTLVIH envelope proteins is indicated at the bottom of the figure. Structural elements encoding the chimeric gene and putative initiation codons are underlined. 60

The third and fourth amino acids (Pro-Val) in the recombinant pv-env2 coding sequence correspond to amino acids 43 and 44 in the LAV / HTLVIH envelope protein coding sequence.

Fig. 6A shows a scheme for the construction of plasmids in which the entire LAV / HTLV ΙΠ envelope protein coding sequence is downstream of the vaccinia promoter. The vaccinia virus promoter is designated by the shaded bar. THE *

Plasmid pn-env5 containing the complete sequence encoding LAV / HTLV ΠΙ envelope proteins was constructed in two steps. The 5 'and 3' portions of the coding region were first inserted into pGS20 separately to obtain a pv-envlet encoding the terminal amino group of the LAV / HTLV ΠΙ envelope gene and the terminal carboxyl encoding of the LAV / HTLV IH envelope gene pv-env2, These two details are reconnected at the Stul point shown in the figure.

f. Figure 1B shows a plasmid production scheme in which the coding sequence for LAV / HTLV H1 envelope proteins is missing downstream of the vaccinia virus promoter and lacks the cell membrane anchor sequence. Plasmid pvenv7 was constructed in two steps.

The 5 'portion of plasmid pv-env5 was inserted into p26, whereby the intermediate plasmid py-env5 / 26 was formed with a translation termination sequence (TAA), i.e. the transformation resulted in a "tailed" env gene. . The TAA-terminated carved gene was then transferred to pG20, and the resulting pvenv7 plasmid was surrounded by vaccinated TK gene sequences. FIG. 1 illustrates the arrest and selection of an arecombinant vaccine virus. The thymidine kinase (TK) gene of the vaccinia virus is shown in black in the figure. This thymidine kinase gene is also present in the pv-env5 plasmid DNA, but the TK gene in the plasmid is derived from a vaccine 7.5K promoter (shaded lane) and the LAV / HTLV H1 outer protein coding sequence (lane lane). ) interrupts a standing chimeric gene. Vaccine susceptible cells are infected and the recombinant plasmid containing the TK gene interrupted by the LAV / HTLVIH env gene is introduced into the infected cells. Through recombination in the TK sequence flanking the chimeric gene, the LAV / HTLVIH envelope gene is incorporated into the vaccinia virus genome. The received,

The recombinant virus containing the LAV / HTLV HI envelope gene is TK '.

genomic sequence of recombinant vaccine viruses containing the LAV / HTLV IH envelope gene6

HU 205780 mutatja shows hands. Figure 7A shows restriction enzyme analysis of v-env5 and venv5NY vaccine recombinants as well as the corresponding vaccine mother (elod) strains (WR and V-NY). The DNA fragments generated by digestion with the HituFSI restriction enzyme are separated by agarose gel electrophoresis and the bands are visualized by ethidium bromide staining. After separation, the stain is transferred to nitrocellulose paper and Figure 7B shows the result of a soutber hybridization-translation test specific for the ALV / HTLV H1 env sequences.

Figure 8A shows an analysis of proteins expressed by vaccine-IAV / HTLV-env recombinants by Western technique. After separation by Ί15% gradient SDS-polyacrylamide gel electrophoresis followed by transfer to a nitrocellulose filter, the following proteins were assayed: 1AV / HTLVΙΠ virion proteins (LAV / HTLVΙΠ); proteins of cells infected with wild-type vaccinia virus (WTw); uninfected cells (so-called imitation sample); proteins of cells infected with v-env2 and v-env5 recombinant viruses (v-env2 and v-env5, respectively). Proteins that confer immune response to the serum of AIDS patients were detected by their reaction with peroxidase bound to anti-human IgG antibodies. ALAV / HTLV ΙΠ env gene products are designated gp150, gp10 and gp41, and molecular weights are expressed in kilo.d.

Figure 8B shows a Western blot analysis of proteins expressed by vaccine-LAV / HTLV H1 env recombinant ⁴ cells in two different cell types. Proteins from both BSC-40 and HeLa cells were separated by SDS-polyacrylamide gel electrophoresis, passed through a nitrocellulose filter, and reacted with pooled sera from LAV / HTLV D1 serum-positive patients. Lanes 1 and 5 · with the imitation pattern

- protein from treated cells, lanes 2 and 6 from cells infected with wild-type vaccine virus, lanes 3 and 7 from cells infected with v-env5 and lanes 4 and 8 from v-env -2. from infected cells. Immune response proteins were detected with ¹²⁵ I-labeled protein antigen. The products of the LAV / HTLV Π env gene are designated gp150, gp10 and gp41.

9A. Figures 3A and 4B show radioimmunoprecipitation analysis of proteins expressed in cells infected with wild-type vaccine virus (WTw) and recombinant vaccinia virus v-env2 and v-env5. 10-12 hours after infection, the proteins were labeled with ³⁵ S-methion. The labeled proteins were reacted with control human serum (N) or serum from AIDS patients in cell lysates and the resulting immune complexes were precipitated with the Staphylococcus aureus protein antigen. After immunoprecipitation, the proteins were separated by 15% gradient SDS-polyacrylamide gel electrophoresis10 and detected by fluorography. Molecular weights are expressed in kilodaltons.

Figure A-9B shows the results of analysis of the ³ H-glucosamine-labeled forms of the proteins produced by the LAV / HTLV ΙΠ recombinants of the present invention by the radioimmunoprecipitation method. Lanes 1 and 5 from mock-treated cells, lanes 2 and 6 from cells infected with wild-type vaccinia virus, lanes 3 and 7 from cells infected with v-env5, and Lanes 8 and 8 represent H ³ -glucosamine-derived proteins from cells infected with v-env2. Cell lysed fun with normal human serum (Figures 1-4).

or LAV / HTLV HI serum-positive patients (lanes 5-8) and precipitated with protein antigen. Protein immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis.

The 9.C. Figure 2B is a pulse chase of LAV / HTLV H1 envelope proteins made by v-chimeric LAV / HTLV ΠΙ recombinants. shows the results of radioimmunoprecipitation analysis using a late blast radioactive signaling technique (lo35 late blasting followed by spinning with unlabeled media). HeLa cells were infected with wild-type vaccinia virus, v-env5 or v-env2 ( ³⁵ S-methionine-labeled samples). The infected samples were washed with chase media at various times, and following cell lysis, the proteins were precipitated with pooled serum from LAV / HTLV-serum positive patients and separated by SDS-polyacrylamide gel electrophoresis. In the figure, lanes 1, 7, and 13 are the 0 o'clock time, 2.8 and

Lanes 14 for the 0.5 hour time, 3.9 and

Lanes 15, 1 hour, 4,10,16. lanes 2h, lanes 5.11 and 17 correspond to 12h and 6h, lanes 6, 12 and 18 respectively. Lane M corresponds to markers containing ¹⁴ C-labeled molecular weight standards.

Figure A-9D shows θθ LAV / HTLV H1 envelope proteins in the shape of cells infected with recombinant vaccinia-LAV / HTLV ΠΙ viruses of the present invention and in the fluidized cell part

HU 205780 mutatja shows the results of radioimmunoprecipitation analysis. HeLa cells were infected with ³⁵ S-methionine-labeled wild-type vaccinia virus (lane 2), v-env5 (lane 3) or venv2 (lane 4) or subjected to infection imitation. (Lane 1). Cells were separated from the medium and, following cell lysis, both shaped cells (pellets) and supernatant cells (supe) were precipitated with pooled serum of LAV / HTLV Ht serum-positive patients. The protein components of the immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis.

In 9.E. FIG. 6B shows the results of radioimmunoprecipitation analysis of LAV / HTLV-envelope proteins in the shape and fluid sections of cells infected with recombinant vaccinia-ALV / HTLV H viruses of the present invention. The 20 v-env5 recombinant contains the entire LAV / HTLV IH envelope coding sequence, and the v-env7 recombinant contains the LAV / HTLV H1 envelope coding sequence which does not contain a cell membrane transfer region. HeLa cells were infected with ³⁵ S-methionine-labeled wild-type vaccinia virus (lane 2), v-env5 (lane 3), or v-env7 (lane 4), exceedingly exposed to infection imitation ( Lane 1). Cells were separated from the medium and, following cell lysis, both the shape elements (pellets) and fluid (supernatant) were precipitated with pooled serum from LAV / HTLV IH serum-positive patients. Following immunoprecipitation, the proteins were resolved by SDS-polyacrylamide gel electrophoresis.

A10. Figure 10A shows the result of a Western immunoblot analysis of serum minus 40 mice immunized with vaccinia-LAV / HTLVHI recombinant viruses. Mice were immunized with v-env5 and v-env2 recombinant vaccinia virus, respectively. After 8 weeks, serum samples were reacted with ALV / HTLV m viral proteins. (The proteins of interest were subjected to SDS-polyacrylamide gel electrophoresis separation on a nitrocellulose filter.) Detection of mouse serum reactive proteins was performed using goat anti-mouse immunoglobulin bound to alkaline phosphatase. Lanes a) - e) show results from 5 different v-env5-vaccinated mouse serum samples, lanes f) - k) show results from 5 different v-env2-vaccinated mouse serum samples. Pooled serum from LAV / HTLV H1 serum positive (AH) S patients as positive control, non-immunized as negative control

Serum from C57N16J mice (NMS) was used. The glycoproteins of the LAV / HTLV IH envelope gp150, gp10, and gp41 are shown in the figure,

Figure A1 shows a change in the serum of macaque monkey inoculated with v-env5 recombinant vaccine virus. The yam received the vaccine (v-env5) by scratching it with 4 monkeys 2 x 10 ⁸ pfu, (plggm sorming as) 4 monkeys 2 x 10 ⁷ pfu and 1 control monkey 2 x 10 ⁷ pfu in vaccinia herpes simplex recombinant (v-HSVgD1). Ten weeks after the first vaccination, the 8 g. s. except for experimental animals, all animals were inoculated with the same virus (2 x 10 ⁸ pfu). Serum samples taken 4 weeks prior to the first vaccination are marked with hatched bars and data obtained 4 weeks after the second vaccination are marked with dark bars. Change in serum (using purified LAV / HTLV IH virions as target antigen) was determined by ELISA.

A12. Figure a

As shown in Figure 11, Western immunoblot analysis results obtained from serum samples of macaque monkeys immunized with the recombinant vaccine v-env5 LAV / HTLV IH. Serum taken 4 weeks before the first vaccination ("pre" lanes) and 4 weeks after the second vaccination. Aliquots of sera were diluted 50-fold and reacted with LAV / HTLV IH virion protein. The proteins of interest were separated by SDS-polyacrylamide gel electrophoresis and immobilized on nitrocellulose filters by electrotransfer, ensuring optimal detection of the gp10 (Figure 12A) and gp41 envelope glycoproteins (Figure 12B). LAV / HTLV ΠΙ proteins recognized by macaques' serum were detected by goat anti-human immunoglobulin bound to alkaline phosphatase. Authentic LAV / HTLV HI virions (AIDS) were used as controls, and pooled sera from LAV / HTLV IH serum positive patients (AIDS) were used as positive controls.

A13. Figure 10B shows western immunoblot analysis of serum samples of chimpanzees immunized with vL-env5NY recombinant vaccineNYLAV / HTLVnavir. Two chimpanzees intradermally received 5 x 10 ⁸ pfu v-env5NY vaccines, one dose of the pediatric vaccine, and a serum sample taken from the same vaccine-NY strain prior to recombination with HAVgDINY vaccine-herpes simplex gD.

HU 205780 Β Lane 1, 8 weeks after first immunization, Lane 2, and 2 weeks after second immunization, Lane 3. Aliquots of serum were diluted 50-fold and reacted with 5 A17, LAV / HTLV IH virion protein. The proteins of interest were separated by SDS-polyacrylamide gel electrophoresis and immobilized on nitrocellulose filters by electrotransfer, ensuring that the detection of gp41 glycoproteins is optimal. Detection of LAV / HTLV E proteins recognized by chimpanzee serum was performed with goat anti-human immunoglobulins bound to alkaline phosphatase. Pooled sera from LAV / HTLV E serum positive (AIDS) patients were used as a positive control.

A14. Fig. 4A is a component of plasmid pKS-5,

Shows the nucleotide sequence of the LAV / HTLV E-specific gene region 20 (Ssu to KpnI); the LAV / HTLV E extension contains the entire LAV / HTLV E gag gene (nucleotide base sequence 340-1871). The cleavage sites that play a role in the production of pAc-gagl as well as the complete amino acid sequence within the gag gene, which can be deduced from the nulldeotide base sequence, are shown in the figure.

A15. Figure 5A shows a scheme for the construction of a plasmid pAc-gagl containing the entire LAV / HTLV III gag protein coding sequence downstream of the AcNPV promoter. The cloning vector pAcolO contains 35 nucleotide-base sequences corresponding to the polyhedron gene promoter, in which the initiation region is in the 5'-position and the 3'-polyhedron is interrupted by cloning sites downstream of the transcription start site. The 40th

The 8-position cloning sites are .EcoRi, SstI, Smal (Χηαΐ), BamHI, Xbal and PstI. Not only are SalI, AccI, and Hin cH restriction sites found. KpnA or Bgl H cloning 45 points do not occur.

A16. Fig. 3A shows the production and selection scheme of recombinant AcNPV (Ac-gagl). The shaded bar represents the polyhedron gene promoter and the 5 'initial sequence, the black antibody-bold line represents the baculovirus polyhedron gene. Details of the latter gene in the pAcgagl plasmid DNA are interrupted by the LAV / HTLV E gag coding region (light bar). Cells are co-transfected with wild-type AcNPV DNA and recombinant plasmid DNA pAc-gagl interrupted with the LAV / HTLV E gag gene, which contains portions of the polyhedron gene. Through recombination in the polyhedron gene fragment flanking the chimeric gene, the LAV / HTLV III gag gene sequence is introduced into the AcNPV genome.

Figure 3B shows the results of northem blot analysis of RNA extracted from Spodoptera frugipede cells infected with AcNPV and Ac-gagl virus using specimens specific for LAV / HTLV E gag.

Fig. 4A shows the results of radioimmunoprecipitation analysis of proteins expressed in cells infected with Ac-gagl recombinant baculovirus. Proteins were labeled with ³⁵ S-methionine 2 hours after infection 24 hours (lanes 1 and 2), 48 hours (lanes 3 and 4) and 72 hours (lanes 5 and 6). Following cell lysis, labeled proteins were either control human serum (lanes 1, 3, and 5) or AIDS patients serum (2,

Lanes 4 and 6) and the immune complexes were precipitated with the Staphylococcus aureus protein antigen. Proteins in the immunoprecipitate were separated by 10% SDS-polyacrylamide gel electrophoresis and detected by fluorography. Molecular weights are given in kilodaltons,

A19. Figure 1B shows a recombinant according to the invention

It shows the results of radioimmunoprecipitation analysis of LAV / HTLV E gag proteins in cells infected with AcNPV. In the experiment, Spodoptera frugipede (Sf9) cells were infected with recombinant Acgagl and the samples were radiolabeled 24 hours 5 minutes after infection. The cells were then washed with so-called whole medium and then in the same medium for 0 hours (lanes 1 and 2), 2 hours (lanes 3 and 4), 4 hours (lanes 5 and 6), and (lanes 7 and 8). After incubation, cells were washed and after cell lysis with pooled sera from LAV / HTLV E serum-positive patients (lanes 2, 4, 6 and 8) and normal human serum (lanes 3, 5 and 7). ) immunoprecipitates were prepared. The protein components of the precipitates were separated by SDS-polyacrylamide gel electrophoresis.

Figure 20 shows a construction scheme for 5 different plasmids, pv-gagl, pv-gag2, pv-gag3, pb-gag4, and pv-gag5. The plasmids contain gag coding sequences of different lengths for the LAV / HTLV E genome (ligase). contain a vaccinia virus 7.5K promoter. The cloning vector pGS62 used in the method is approximately identical to the vector pGS20 shown in Figure 3; the only difference

EN 205780 Β that the vector pGS20 also has an EcoRI restriction site downstream of the sole Snwzl cloning site in the vector pGS62.

Figure A21.Analyzes of Western Expressions in Cells Infected with Recombinant Vaccine-LAV / HTLVUI Viruses (v-gaglNY, v-gag2NY, vgag3NY, v-gag4NY, and v-gag5NY), Predovirus (v-NY), and Immunoprecipitated (MOCK) shows the results of an immunoblot analysis. Purified LAV / HTLV HI viral samples (LAV / HTLVIH) were used as positive controls. Contiguous BSC-40 cells were infected (multiplicity of infection = 10). After a 12-hour infection period, cells were harvested and, following cell lysis, total cellular protein was separated into its components by SDS-polyacrylamide gel electrophoresis and immobilized on a nitrocellulose filter by electrotransfer. Filters were treated in two ways: (1) by reacting with serum from AIDS patients (Fig. 21JB) and reacting proteins were detected with goat anti-human immunoglobulin bound to alkaline phosphatase, or (2) by reacting with mouse monoclonal antibodies specific for p25 and p18 gag proteins (21). and Figure 21C) and the reactive proteins were detected with goat anti-mouse immunoglobulin bound to alkaline phosphatase.

Figure A22 shows the construction scheme of plasmid pAc-anv5. The entire plasmid encodes the LAV / JTLV IH envelope coding sequence downstream of the AeNPV polyhedron gene promoter. The cloning vector pAcolO was already described in Figure 15.

Figure 23 shows the results of a radioimmunoassay of proteins expressed in cells infected with Ac-env5 recombinant baculovirus. Cells infected with the parental strain (AeNPV) and the replicate specimen (Mock) were used as controls. Proteins expressed in cells infected with strain Sf9 were labeled with ³⁵ S-methionine for 2 hours at 28 hours post-infection. The labeled proteins were reacted in cell lysate with either serum from AIDS patients or with murine monoclonal antibodies specific for gp10 or gp41, and the immune complexes were precipitated with the Staphylococcus aureus protein antigen. Protein components from the immunoprecipitate were separated by SDS-polyacrylamide gel electrophoresis and detected by fluorography.

5. DETAILED DESCRIPTION OF THE INVENTION The invention thus relates to viruses that produce peptides and proteins associated with LAV / HTLVHI epitopes. The invention also relates to peptides and proteins associated with LAV / HTLV IH epitopes, which may be produced by recombinant DNA technology or by chemical synthesis. The viruses, peptides and proteins of the invention, which are useful in inducing an aprotective immune response, can be used as immunogenic agents in vaccine formulations, optionally as immunogenic agents for multivalent vaccines, to prevent infections caused by LAS and AIDS, LAV / HTLV. The peptides and proteins of the invention may also be used as antigens in diagnostic immunoassays for the detection of LAV / HTLV-specific antibodies in patients. When used for immunoassays, the peptides and proteins of the invention must be antigenic (i.e., reactive to the patient's ALV / HTLV IH antibodies) but not immunogenic (i.e., immunogenic). Antigens used for diagnostic immunological tests are not even required to elicit a protective immune response in vivo.

In one embodiment of the invention, nucleotide sequences encoding LAV / HTLV H1 epitopes are inserted into recombinant DNA technology into expression vectors that promote the expression of LAV / HTLV H1 sequences in susceptible host cells. These expression vector host cell systems can be used in vitro to produce LAV / HTLV-related peptides and proteins, and the resulting gene products can be recovered after purification from cell culture. Peptides or protein subunit vaccine compositions suitable for eliciting a protective immune response may be used as immunogenic agents. On the other hand, the immunogenic or antigenic peptides and proteins of the present invention can be used as antigens in immunoassays for the detection of LAV / HTLV specific antibodies in patients. It is further possible from the production of the peptides and proteins of the invention that the amino acid sequences of the peptides and proteins contained therein can be deduced from recombinant LAV / HTLV H1 nucleotide sequences expressing antigenic and / or immunogenic properties. they can be synthetically produced. Synthetically produced materials can be used in a subunit vaccine (if they are immunogenic) or used as an antigen in diagnostic immunoassays (if they are antigenic and / or immunogenic).

If the expression vector is a virus which promotes the expression of an immunogenic substance that is associated with an LAV / HTLV IH epitope capable of eliciting a protective immune response, a vaccine may be prepared from the virus itself. A live virus vaccine is introduced by an infectious recombinant virus10

HU 205780 Β, which is not a disease of the host. Such compositions may be formulated to provide a sustained immunized virus vaccine. A multivalent vaccine may be prepared using two or more IAV / HTLV ΙΠ epitopes or one LAV / HTLV ΠΙ epitope and another pathogen.

Generally, the vaccine preparation steps are as follows:

(a) isolating a gene or gene fragment encoding a LAV / HILV ΙΠ viral protein;

(b) inserting the gene or gene fragment into an expression vector;

(c) identifying and culturing the recombinant expression vector in a host in which the gene is expressed and replicated;

(d) identification and purification of the gene product;

(e) determining the immuno-potency of the gene product; and (f) preparing a vaccine composition.

One embodiment of the present invention is the production of recombinant vaccinia viruses and baculovines containing the lAV / HILV H1 envelope gene which promotes expression of proteins that are immunologically linked to LAV / HILV D1 envelope proteins in cells of tissue culture infected with recombinant viruses. Another embodiment of the invention is the production of recombinant vaccinia viruses and baculoviruses containing a LAV / HTLVDIgag gene that promotes expression of proteins immunologically linked to the structural proteins of LAV / HTLVDI Core in a tissue culture cell infected with recombinant viruses. However, the present invention is not directed to viruses expressing LAV / HILV ID envelope or gag-like proteins, their preparation and use; the invention also encompasses the production of polypeptides associated with the antigen of any AIDS agent using recombinants engineered in any expression vector system.

For purposes of simplification, LAV / HILV D1 envelope and gag genes are referred to throughout this specification, but similar methods can be used to construct other recombinant expression vectors for the production of polypeptides associated with LAV / HTLVokon or related viral proteins. Examples of dyen proteins are the products of the LAV / HILV ID gene, such as gag, pol, and env, and at least four other genes that are used today for tat, 3'-orf, and fourth for art or alternative trs. [Fischer et al., Science,

233, 665-659. (1986)].

5.1. Isolation of genes and gene fragments encoding LAV / HILV D1 viral proteins

The first step in isolating the LAV / HTLV ΙΠ genes is to generate DNA fragments containing the desired gene sequence, such as env or gag. As noted above, the LAV / HTLVDI genome is of RNA type, from which the corresponding DNA encoding the LAV / HTLV ΠΙ gene can be produced in three ways: (a) cloning RNA isolated from purified LAV / HILV ΠΙ virions with cDNA; (b) cDNA cloning of RNA containing Poli A obtained from cells infected with LAV / HTLV és; and (c) cloning of genomic DNA obtained and purified from cells infected with LAV / HILV ΠΙ. In the following, NDS encoding the LAV / HTLV ΠΙ genes is referred to as LAV / HTLV ΙΠ DNA.

LAV / HTLV ΠΙ-DNA fragments can be prepared by: opening DNA at specific cleavage sites using restriction enzymes; 10 fragmenting the DNA with DNA polymerase in the presence of manganese; or by physical means, such as high frequency sonic vibration. Linear DNA fragments are known in the art, such as agarose gel or polyacrylamide gel electrophoresis, column chromatography, and the like. we can separate it.

Any restriction enzyme or enzyme combination may be used to produce the LAV / HTLV ΠΙ DNA fragment (s) containing the Envelope or gag sequences; however, it is a requirement that the antigenic character of the protein gene product is not impaired. For example, an antigenic region of a particular protein is comprised of 7-14 amino acids. Thus, an evelope-type peptide precursor-sized protein (having a molecular weight of about 96,000 song tons) has a large number of discrete, antigenic regions; the number of these may be taken into account, taking into account overlapping sequences, secondary and tertiary structural elements, and possible transformations such as acetylation, glycolysis or phosphorylation. It follows that an antigenic region can be encoded by many partial envelope polypeptide gene regions. Thus, a variety of restriction enzyme combinations can be used to produce DNA fragments which, when incorporated into a suitable vector, are capable of directing the production of envelope-specific amino acid sequences containing various antigenic determinants.

Specific DNA fragments 40 containing the desired LAV / HTLV ΠΙ gene should then be identified; There are many ways to authenticate. First, the entire LAV / HTLV ΠΙ genome is matched with the corresponding DNA fragments, and the fragment containing the envelope protein gene or gag gene sequence is amplified on the basis of comparing the expected amino acid sequence of the fragment with that of the envelope protein or gag core protein. Once the entire genome sequence has been determined, the long coding gene segments can be arranged between 5 'and 3'. 50 Since the retroviral genes examined so far corresponded to a 5'-gag-pol-env-3 'arrangement, the region closest to the 3' position of the large coding region is likely to be the envelope gene and the region closest to the 5 position likely encodes the gag gene. Identification of a particular gene is also possible on the basis that nucleic acid hybridization analysis or comparison of a given sequence with other known sequences can be verified with respect to known retroviral genes.

HU 205780 Β

Alternatively, the fragment containing the envelop or gag gene can be identified by mRNA selection. In this case, complementary mRNAs are isolated by hybridization using LAV / HTLV ΠΙ-DNA fragments. Immunoprecipitation analysis of the in vitro translation products of the isolated mRNA can identify the mRNA and thus the LAV7HTLV ΠΙDNA fragments containing the envelope or gag protein sequences. Finally, selection of specific mRNAs can also be accomplished by adsorbing polysomes isolated from cells infected with LAV / HILVIH with immobilized antibodies directed against envelope or gag proteins. Using mRNA selected from adsorbed polysomes as template, complementary DNA (cDNA) can be prepared. The isotope labeled mRNA or cDNA can then be used to identify LAV / HTLVHI DNA fragments containing envelope or gag sequences. Other alternatives to constructing the envelope or gag gene are, for example, chemical production of the gene itself (provided that it is a known sequence) or DNA complementary to the mRNA encoding the envelope or gag gene.

Once the LAV / HTLV ΠΙ DNA fragment containing the sequence of interest has been isolated and identified, it is inserted into a cloning vector, such as a plasmid cloning vector, which is introduced into susceptible cells where the DNA replicates and generates the desired LAV / HTLVIU sequences in high copy numbers. This can be done, for example, by ligating the LAV / HTLV DNS-DNA fragment with a ligase enzyme to a cloning vector with a complementary strand. However, if the complementary restriction sites required for cleavage of LAV / HTLV ΠΙ DNA are not present in the cloning vector, the DNA chain ends must be modified. One way of modification is to shorten the ends of the single-stranded DNA by enzymatic digestion, or to fill the ends of the single-stranded DNA with a substituent to be linked in an abbreviated form. An alternative solution is to construct the desired site by linking appropriate nucleotide sequences (called linkers) to the DNA terminus; linkers thus attached to the end of the chain are sequences containing chemically synthesized oligonucleotides encoding the restriction enzyme recognizable cleavage sites. Alternatively, the opened vector and the LAV / HTLV ΙΠ-DNA fragment can be modified by the formation of homopolymer tail structures.

In the host cells transformed by a recombinant DNA molecule containing the isolated gene, cDNA or synthesized DNA sequence, the gene is produced in high copy number. Thus, a large amount of gene can be recovered by culturing the transformed cells, then isolating the recombinant DNA molecules from the transformed cells and, if necessary, recovering the gene solution from the isolated DNA.

If the gene is to be inserted into the virus itself, such as a vaccinia virus or adenovirus as an expression vector, the recombinant DNA molecule containing the LAV / HTLV IH gene is kept in a milieu surrounded by viral sequences cells allow for genetic recombination, i.e. it is possible for the gene to be incorporated into the viral genome.

Cloning of the entire LAV / HTLV HI genome is described by S. WainHobson et al., Cell40: 9 (1985). One of the described clones is lambda J19, which is the lambda L

Inserted into the HituTBL cloning site 47.1 contained a LAV / HTLV ΠΙ genomic sequence, namely a 9200 nucleotide base pair DNA fragment.

A particularly important subclone of lambda J19 containing the LAV / HTLV IH envelope gene is pRS-3, which, inserted into pUC18, contains a 3840 bp LAV / HTLV H1 nucleotide sequence inserted between the EcoRI and SstI sites. The LAV / HTLV ΠΙ-specific DNA in pRS-3 is located between nucleotide bases 5289-9129 in the aLAV / HTLVIH genome (see Figure 2, which shows the LAV / HTLV in pRS-3). ΠΙ represents the nucleotide sequence). However, due to the degeneracy of the nucleotide coding sequences, other DNA sequences encoding substantially the same sequences as the amino acid sequence shown in Figure 2 may be used in the present invention to clone the LAV / HTLV ΠΙ envelope gene. For example, nucleotide sequences containing all or parts of the envelope nucleotide sequence shown in Figure 2, which have been altered by substitution with different codons. These replacement codons encode the same or functionally equivalent amino acid residues in the sequence (e.g., other amino acids with the same polarity as the original), i.e., so-called "silent" exchanges, changes in the amino acid sequence.

Other major subclones of lambda J19 containing the LAV / HILVHI gag gene are pKS-5 and pSS5. Plasmid pKS-5 contains a 3148 bp LAV / HTLV IH fragment inserted into pUC18, inserted between the SstI and ΙφηΙ sites. The LAV / HTLV? -Specific DNA in the pKS-5 between the SstI and KpttI sites is located between the nucleotides 2224 and 3372 in the LAV / HTLV H1 genome. Plasmid pSS-5 contains a 5,100 bp LAV / HTLV HI fragment inserted between pS18 and SalI binding sites. In pSS-5, the LAV / HTLVΙΠ-specific DNA between the SstI and SalI sites is located between nucleotides 224 and 5331 in the LAV / HILVHI genome. Wain-Hobson et al., Cell. 40, 9 (1985)]. Figure 14 shows the LAV / HILVHI nucleotide sequence in pKS-5 and pSS-5. However, due to the degeneracy of the nucleotide coding sequences, other DNA sequences encoding substantially the same sequence as the amino acid sequence shown in Figure 14 may be used in the present invention.

HU 205780 Β for the cloning of the LAV / HTLV IH gag gene. For example, nucleotide sequences containing all or parts of the gag nucleotide sequence shown in Figure 14 which have been altered by substitution with different codons. These replacement codons encode amino acid residues (e.g., another amino acid of the same polarity) that are identical or functionally equivalent to those in the sequence, i.e., produce "silent" changes.

5.2. Insertion of LAV / HTLVΠ3 protein coding sequences into expression vectors

A nucleotide sequence encoding a LAV / HTLV ΠΙ protein as isolated above, such as an envelope or gag protein, or a portion thereof, is incorporated into a suitable expression vector. The vector must contain the elements necessary for transcription and translation of the integrated protein coding sequence. A variety of host-vector systems can be used to express the protein coding sequence. These include, for example, mammalian cells infected with the virus (e.g., vaccinia virus or adenovirus); insect cells infected with a virus (e.g., baculovirus); microorganisms (e.g., yeast) containing a yeast vector; or bacteria. The specificity and strength of the elements of the vector vary. Depending on the host cell vector system, any suitable transcriptional and translational elements may be used. For example, when cloning in mammalian cell systems, promoters isolated from the genome of mammalian cells (e.g., the murine metallothion promoter) or promoters isolated from viruses cultured in such cells (e.g., the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA technology or synthetically can also be used for transcription of inserted sequences.

For proper translation of the inserted protein coding sequences, specific start signals are also required. In the "start" markers, the ATG initiation codon and the linked sequences are included. If the entire LAV / HTLV D1 envelope or gag gene is inserted into a suitable expression vector, together with its own initiation codon and related sequences, no further translational control signal is required. However, if only part of the envelope or gag coding sequence is incorporated, an exogenous translational control signal, including the ATG initiation codon, is required. An additional requirement for the initiation codon is that it must be in the same phase as the coding sequence of the protein coding sequences in order for the entire extension to be translated. These exogenous translational codons are of various origins, that is, either naturally or of synthetic origin.

Any of the methods described above for inserting DNA fragment doctrines into a vector can be used to generate expression vectors containing appropriate chromosome gene translation and translation control signals and protein coding sequences. The methods may be in vitro solutions using synthetic or recombinant DNA techniques or in vivo (genetic) recombinations.

In the exemplary embodiments of the invention, the vaccine virus or baculovirus is used as the expression vector. However, these are exemplary only and are not intended to limit the need for vaccine virus or baculovirus. Expression vectors include, for example, the following vectors and derivatives thereof: human and animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculoviruses, yeast vectors; bacteriophage vectors; and plasmid and cosmid DNA vectors, to name a few.

When an adenovirus is used as an expression vector, the LAV / HTLV ΙΠ gene is ligated to the adenovirus transcription / translation control complex; the latter is, for example, a promoter and a three-part starter sequence. The resulting chimeric gene is then incorporated into the adenoviral genome in vitro or by in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., Ed or E3) results in a viable recombinant virus capable of expressing LAV / HTLV-related proteins in the infected host. Currently two adenovirus strains (a

Type 4 and Type 7) are approved and used to vaccinate military personnel. These are, by the way, the number one candidates for use as a vector for expression of the LAV / HTLV D1 gene. It is also possible to select a gas cell or cell line that modulates the expression of the inserted gene sequence, or that modifies or repositions the chimeric gene product in a desired specific direction. In the presence of certain inducing agents (e.g., the presence of a metallothionin promoter in the presence of zinc and cadmium ions), expression levels from a given promoter are increased; that is, the expression of the LAV / HTLV ΠΙ-type protein produced by genetic engineering can be regulated. This is particularly important if the protein product of the cloned foreign gene is toxic to host cells. Modification (e.g., glycosylation) and conversion (eg ring opening) of protein products is also important for protein function. Each host cell type has a specific and specific mechanism for post-translational modification and conversion of proteins. With properly selected cell lines or host cell systems, the protein expressed in the cell can be modified and converted in the proper direction.

In another exemplary embodiment of the present invention, on the one hand, the LAV / HTLV D1 gag coding sequences are ligated to the vaccinia virus 7.5K promoter to obtain chimeric genes incorporated into various plasmids. These chimeric genes in the plasmid are taken from additional vaccine virus sequences homologous to the vaccinia virus TK gene. For the production of chimeric genes, either synthetic or natural nucleotides that are derived from the LAV / HTLV D1 gag sequence13 can be used.

EN 205780 Β are used for coding of the signaling system required for transcription and translation. These chimeric genes are incorporated into vaccine virus expression vectors, the vaccine virus geuomha. and by combining in vivo homologous TK sequences present in both plasmid vectors. The resulting recombinant viruses containing the Idmere gene can be used as expression vectors for the production of LAV / HTLVΙΠ envelope and LAV / HTLV gag-type proteins.

In another exemplary embodiment of the present invention, the LAV / HTLV ΠΙ envelope coding or LAV / HTLVIUgag coding sequences are ligated with a plasmid containing various chimeric genes containing the ligand enzyme Autographa califomica nuclear polyhedrosxs virus (AcNPV). to produce mids. The chimeric genes in the plasmid are surrounded by additional AcNPV sequences. Chimeric genes can be generated using either natural or synthetic nucleotides that encode the signals required for transcription and translation of the LAV / HTLV Hl envelope or LAV / HTLV ΙΠ gag 20 sequences. These chimeric genes are then incorporated into AcNPV expression vectors by in vivo recombination between homologous DNA sequences present in both the AcNPV genome and the plasmid vector. The resulting recombinant viruses containing chimeric genes can be used as expression vectors for the production of LAV / HTLV D1 envelope and LAVZHTLVHI gag proteins.

5.3. Identification of recombinant expression vectors capable of expressing and replicating the gene solution

Expression vectors containing the foreign gene as an tag can be identified by three major methods: (a) DNA-DNA hybridization, (b) the presence or absence of features characteristic of the "marker" gene, and (c) expression of the tagged sequences. In the first method, the presence of the foreign gene solution in the expression vector can be detected by performing DNA-DNA hybridization using samples containing sequences homologous to the inserted foreign gene. In the second embodiment 40, the recombinant vector / host cell system can be identified and selected based on the presence or absence of function specific to a particular "marker" gene (e.g., thymidine kinase activity, antibiotic resistance, phenotypic change, baculovirus formation), which changes in the vector. foreign genes are formed in the fossil. For example, by incorporating the LAV / HTLV D1 gene into the sequence of a vector marker gene, recombinants 50 containing the LAV / HTLV ΠΙ extension can be identified by the lack of functions of the marker gene. In the third embodiment, the recombinant expression vectors are analysis of expressed (produced) gene products. Analyzes may be based on the detection of physical, immunological or functional properties of the gene products.

A variety of methods can be used to propagate isolated and identified recombinant DNA molecules, and the method is primarily selected based on whether or not the 60 recombinants have a self-replicating moiety (replicon). A unit capable of replicating itself (plasmid, virus, cell) amplifies itself in the presence of appropriate cells and under appropriate culture conditions. Recombinants without a unit capable of self-replication must be integrated into a molecule containing such a unit in order to be propagated. For example, certain plasmid expression vectors after integration into a host cell need to be integrated into the cell chromosome to be recombinant ^; nant gene expression and reproduction. After providing the appropriate host cell system and culture conditions, recombinant expression vectors can be propagated and produced in large quantities.

In the exemplary embodiment of the present invention, the chimeric genes comprising LAV / HTLV IH env and gag coding sequences are incorporated into the TK gene of the vaccinia virus genome. As a result, the virus becomes TK-like, which means that the virus loses its ability to synthesize thymidine kinase. The recombinants thus obtained can be selected by culturing them on a medium containing 5-bromodeoxyuridine which is a nucleoside analogue that kills TK * cells but not TK cells. The recombinants are then subjected to DNA-DNA hybridization,

LAV / HTLV IH is identified by its env-specific or LAV / HTLV H1 gag-specific template. ATK recombinant viruses were isolated by plaque purification and a stock solution of infected tissue culture cells was prepared.

In another exemplary embodiment of the present invention, chimeric genes containing LAV / HTLV D1 envelope or gag coding sequences have been incorporated into the polyhedron gene of the AcNPV genome, thereby transforming the virus into a non-occlusive particle phenotype. and was identified by Northern blot analysis using LAV / HTLV IH env-specific and LAV / HTLV IH gag-specific samples. Recombinant viruses were isolated by plaque purification and a stock solution was prepared from cells of infected tissue culture.

5.4. Purification and identification of the expressed (produced) gene product

After recombinant identification of the LAV / HTLV H1 gene has been made, the gene product must be analyzed. This can be done by an analytical method based on the physical, immunological or functional properties of the product. Immunoassays are particularly important if the gene products or recombinant viruses expressing them are to be used in vaccine formulations and / or as antigens in diagnostic immunoassays.

The product is immuno-responsive by means of various antisera, such as sera from AIDS or LAS patients, or

HU 205 780 Β

It can be analyzed with LAV / HTLV envel antiviral envelope virus protein or polyvalent antiserum against gag encoded core proteins. Immunogenic molecules: They may also be analogs produced by bacterial systems or synthetic peptides containing the antigenic determinants of 5 LAV / HTLV ΙΠ envelope or core antigens. In order to identify the peptides and proteins of the invention, two conditions must be met, namely: (1) 10 LAV / HTLV M-type proteins produced in cells infected with recombinant virus only, in contrast to sera from AIDS patients or LAV / HTLVin envelope or vore proteins; should be given an immune response against various antibodies against its analogs and derivatives. 15

Whether the whole or a portion of a gene expresses a protein, or two or more ligands linked to a ligase direct the production of a fusion protein, the protein must always be immunoreactive. Reactivity can be tested by standard immunoassays, such as radioimmunoprecipitation, radioimmunoassay, or immunoblot analysis.

The identified LAV / HTLV ΙΠ-type protein is then isolated and purified, for example, by chromatography (ion exchange or affinity chromatography, column chromatography), centrifugation, solubility differences, or other standard methods applicable to proteins.

Based on the nucleotide sequence of the chimeric gene in the recombinant, the amino acid sequence of the LAV / HTLV ΙΠ-type immunoreactive protein produced and identified by the recombinant can be reconstructed and synthesized using standard methods known per se {for example: M. Hunkapiller et al., Natura, 310, 105-111. (1984)].

Thus, it is contemplated that all peptides, either synthetically or by recombinant DNA technology, which are altered sequences containing the entire amino acid sequence shown in Figures 2, 40 or 14, so called "silent alteration", are contemplated by the present invention. For example, one or more of the amino acid residues in the chair may be replaced with another amino acid of similar polarity which is functionally equivalent, i.e., the change is undetected. Within a sequence, one amino acid may be replaced by another 50 amino acids of the same class as the amino acid. For example, non-polar (hydrophobic) amino acids are alanine and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) are arginine, lysine and histidine; negatively charged (acid 55) are asparagine and glutamine.

5.5. Determination of the immuno-potency of the recombinant product

The purified natural protein or synthetically produced protein or peptide was then inoculated with experimental animals and the immune response of the LAV / HTLV M-type product can be determined. When the LAV / HTLV M-type protein is expressed in an infectious recombinant virus, the recombinant virus itself can be used to immunize the animals. Mice, rabbits and chimpanzees can be used as experimental animals. The subject may be a human subject. The immunogenic agent may be administered orally, intramedially. intramuscularly, intravenously subcutaneously, intranasally or by any standard immunization method. The subject's immune response can be evaluated from three approaches; (a) reacting the resulting serum with authentic ALV / HTLV ΠΙ viral antigens, which can be monitored by known means, such as enzyme linked immunosorbent assay (ELISA), immunoblot analysis, or radioimmunoprecipitation; (b) ability of immune serum to neutralize LAV / HTLV ΠΙ virus infection in vitro, [M. Robert-Guroff's Method for Detecting Anti-envelope Antibodies, Natur, 316, 72-74. (1985)] and protection against LAV / HTLV infection and / or attenuation of infection symptoms in immunized animals [D. P. Francis, Chain, 2 "1276-77 (1984) and D.C. Gajdusek, Chain,

7: 55-56 (1985)].

5.6. Preparation of vaccine

According to the invention, a vaccine may be prepared using an epitope or recombinant virus expressing an LAV / HTLV ffl; the immunogenic agent elicits a protective immune response against LAV / HTLV ΠΙ viral infections and can be used to prevent LAS and AIDS. Additionally, multivalent vaccines containing more than one immunogenic LAV / HTLV AV-related determinant may be prepared. Such vaccine compositions may contain, for example, an ALV / HTLV ffl envelope epitope, optionally with other epitopes, such as LAV / HTLV ΠΙ gag. Multivalent vaccines containing both envelope and gag-encoded epitopes can play a major role in preventing the development of AIDS. Asymptomatic patients are much more likely to have anti-gag antibodies than those with the symptoms; antibodies directed against envelope determinants can be detected (Science, 233: 419 (1986)). Relevant studies have also shown that AIDS patients develop antibodies to both envelope and gag proteins in the early, asymptomatic stages of the disease, and as the disease progresses, the amount of anti-gag antibodies decreases as the symptoms develop. and the amount of envelope antibodies remains [Science,

233, 282 (1986)]. It follows that gag-type epitopes produced in one embodiment of the invention may be of great importance in the prevention and immunotherapy of LAV / HTLV ώ diseases. Multivalent vaccines include recombinant viruses expressing LAV / HTLV M gene products and / or chimeric gene products

EN 205780 Β and may be used in combination with other immunogenic agents to prevent LAS or AIDS. Various formulations are described below.

5.6.1. Virus vaccine preparations

Vaccines containing live recombinant virus or inactivated recombinant virus may be prepared. Which solution we choose depends on the characteristics of the virus expressing the LAV / HTLV ΠΓ type epitope. If the recombinant virus is infectious to the subject to be immunized but not infected, the use of a live virus vaccine is advantageous because, when reproduced in the body, it produces an effect of the same magnitude and duration as natural subclinical infection, i.e., strong and lasting immunity. An infectious recombinant virus introduced into the host, expresses the LAV / HTLVHI type proteins corresponding to its chimeric genes, thereby inducing an immune response to LAV / HTLV ΙΠ antigens. In all cases where the immune response is such as to protect against further LAV / HTLV A vaccine containing the live virus itself is used to prevent infection with the AIDS virus The recombinant virus required for the preparation can be produced either in vitro (e.g., in tissue culture) or in vivo (in a host organism), and vaccine virus recombinants are particularly suitable. Methods for the preparation of a preparation known for the preparation of Vaccinia Viruses and Factors for Vaccine Antigens, edited by GV Qui nnan.Elsevier, pp. 109-116 (1985)].

Live virus-containing multivalent vaccines may be prepared using one or more infectious recombinant viruses expressing some LAV / HTLV ΙΠ type epitopes. The vaccine may also contain viruses that express not only the LAV / HTLV H1 epitopes but also the epitopes of pathogens of other diseases, such as the vaccine virus, into which 35,000 bp of foreign DNA can be inserted, can be engineered to modify the LAV / HTLV. It contains sequences encoding HTLV IH envelope and gag epitopes. The virus may contain sequences encoding epitopes other than the LAV / HTLV H1 epitope sequences, and the recombinant virus may be used as an immunogen in a multivalent vaccine. Alternatively, mixtures of vaccinia virus and other viruses that encode different epitopes of LAV / HTLVHI and / or other pathogenic organisms can be used to make a multivalent vaccine capable of directing the expression of various genes.

In any event, a recombinant virus, whether infectious to the host organism to be immunized or not, can be used to produce an inactivated virus vaccine. Inactivated viruses are considered to be dead virus in the sense that they have lost their infectivity, usually due to formaldehyde treatment. Ideally, no capsid or envelope proteins carrying the immunogenicity of the virus will be altered when the virus becomes infectious. For the preparation of a vaccine containing an inactivated virus, a large number of viruses must be cultured in tissue culture to produce the relevant antigen in the required amount. By using a mixture of inactivated viruses expressing different epitopes, "multivalent" vaccines can be prepared. This is advantageous in some ways like live viruses ké- spawned ^five multivalent vaccines, administered at the same time because the interaction of live viruses hidden potential dangers. Both the inactivated recombinant virus and a mixture of these viruses are combined with a vaccine composition together with an adjuvant that enhances the immune response to their antigen. Examples of such adjuvants include inorganic gels such as aluminum hydrocide, surfactants such as polyoxyethylene and polyoxypropylene polyols, lysolecithin, polyanions, peptides, oily emulsions, and potentially useful human adjuvants such as BCG. Bacillus Calmette-Guerín) and corynebacterium.

The vaccine compositions may be administered in a variety of ways, including oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous and nasal administration. For example, when using a vaccine containing a live recombinant virus, the vaccine may be administered to a patient as a result of the natural infection of the calf-type precursor virus used to make the recombinant virus.

5.71mmunological studies

The LAV / HTLV D1 type peptides and proteins of the invention can be used as antigens in various immunoassays for the detection of antibodies raised by LAV / HTLV H1 in patients' tissues, body fluids and blood products in blood banks. The antigens of the present invention may be used in any known immunoassay, including any of the following: radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), sandwich immuno-diffusion assay, or agglutination method, immuno-radiometric assay, fluorescent immunoassay, protein antigen immunoassay, and immunoelectrophoresis. - competitive or non-competitive immunoassays.

The above assays utilizing the antigens of the present invention are useful for screening blood products in the blood bank, screening for persons exposed to LAV / HTLV IH infection, and monitoring the course of LAS and AIDS patients. Any peptide or protein associated with the LAV / HTLV H1 antigen can be used in the immunological assays of the invention. These include, but are not limited to, the antigens of interest, env, gag, and pol gene products, and four

EN 205780 Β other peptides and proteins currently associated with a gene product known as the line, tat, 3'-orf and art (or trs) genes. In one embodiment of the diagnostic method of the present invention, a sector of antigens is used to screen patients by immunoassay for the detection of a particular type of antibody directed against various epitopes of LAV / HTLV HI. A further application of the diagnostic method of the present invention is that a subsequent LAV / HTLV ΙΠ effect can be monitored in patients vaccinated with the vaccine composition of the invention. In this case, the peptide or protein used as an antigen in the immunoassay should not be the same as that contained in the vaccine used to immunize the patient as an immunogenic substance; in fact, vaccinated individuals will certainly be positive for the particular epitope used in the vaccine, so the test will give a positive result regardless of whether they have been exposed to the virus after vaccine administration. For example, if a patient received a LAV / HTLV H1 envelope epitope in the vaccine, we would like to detect LAV / HTLV IH specific antibodies in another non-envelope strain to detect LAV / HTLV ΙΠ infection in the same patient. screening of a vaccinated patient for the detection of antibodies specific to LAV / HTLV ΙΠ type antigens, or for the detection of antibodies specific to other LAV / HTLV ΙΠ antigens (such as row and 3'-orgene products).

The LAV / HTLV kapcsolatos-related peptides and proteins of the invention may also be used in competitive immunoassays for LAV / HTLV III encoded proteins in the patient's blood, serum, etc. for quantitative measurement.

6. Examples for the preparation of envelope-type vaccine recombinants

The following examples describe the construction of various plasmid vectors containing the chimeric gene in which the LAV / HTLV ΠΙ envelope coding sequences are downstream of the vaccinia virus transcriptional sequences.

These chimeric genes containing the vaccinia promoter and LAV / HTLV IH _t velope coding sequences are inserted into the vaccinia virus genome by in vivo recombination. The resulting recombinant viruses are identified and purified and viral stock solutions are prepared from cells of infected tissue culture. Immunoreactive LAV / HTLV envel-envelope proteins are produced in vitro by these recombinant vaccine viruses. Recombinants are tested in experimental animals to elicit a neutralizing or protective immune response, that is, to be used in a vaccine against AIDS. Each step is described in detail in the following subsections.

6.1. Description of main methods

6.1.1. Cells and viruses

Cell line BSC-40 from the kidney of the African green monkey (J. Virol. 54: 569-7575 (1985)) (deposited at ATCC under CCL26,

1963, and a continuous cell line from African green monkeys derived from BSC-1 cells) R.

It was provided by Associate Professor Condit (Department of Biochemistry, State University of New York, Buffalo, NY). For Dulbecco's modified Eagle's medium (hereafter; DNEM; manufactured by Gibco, Grand Island,

N. Y.) 10% fetal bovine embryo serum and 100 to 100 units of penicillin and streptomycin per ml are added and BSC-40 cells are cultured in the thus prepared medium. The human 143 TK? cells (ATCC CRL 8303 deposited 1983) [J. S. Rhim et al., Inti. J. Cancer,

15, 23-29. (1975) Wair et al., Proc. Natl.

Acad. Sci. USA 79: 1210-1214 (1982)]. It was obtained from Professor M. Botchan (Department of Molecular Biology, University of Califomia, Berkeley, Calif.). They were cultured in the above medium after addition of 25 ng / ml 5-bromo-deoxyuridine (hereinafter: BUdR).

Human diploid cells MRC-5 (ATCC accession number CCL171) were cultured in the same medium as BSC-40 cells.

The vaccine virus strain WR (ATCC Accession No. VR-119) was obtained from Associate Professor R. Condit. The virus was grown in 25 BSC-40 cells on DNEM medium (GIBCO) containing 5% γ-globulin-free bovine serum and 100-100 units / ml penicillin and streptomycin. TK recombinants were cultured in 143 30 TK 25 of the same medium containing BUdR-ng / ml ^- cells were selected, and 1% Noble agar (DIFCO, Detroit, MICH), 5% y globulihmentes calf serum, 100 to 100 ml per unit of penicillin and streptomycin and 25 ng / ml BUdR in the same cell line cultured on DNEM medium by 35 plaque assays. The resulting virus stock solutions were diluted with phosphate-buffered saline containing 1 mmol / l magnesium chloride and 0.01% bovine serum albumin (PBSAM) (hereinafter: PBS, 8 g / l sodium chloride, 0.2 g / l potassium). chloride, 1.5 g of sodium dihydrogen phosphate, and

Containing 0.2 g of dipotassium hydrogen phosphate).

The New York City Public Health Office vaccine virus strain was prepared from a commercially available hhn45 shoot vaccine (Dryvax ^R , 32150G; available from Wyeth Laboratories, Marietta, PA) as follows: the pox vaccine was diluted with PBSAM and cultured three times in BSC-40 cells. In a plaque purification step, a strain culture (50, hereinafter referred to as "v-NY") was used to construct recombinant viruses. The strain of Av-NY-derived recombinant virus was grown in MRT cell culture. Neff, Ϊ.Μ. 1985, Vaccinia Vinueses as Vactors fór Vaccina Antigans, NY 66-74.] 55 6.1.2. Production, Cleavage, and Modification of DNA

Methods used in the following procedures are described by Maniatis et al., With the possible exceptions, in Molecular Cloning, Cold Spring Harbor Laboratory (1982) and in the preparation of plasmid DNA17.

Hü 205,780 Β sa (pages 86-96), restriction enzyme digestion of DNA (pages 98-106), purification of restriction fragments by low melting point agar gel (pages 157-161 and 170), E. coli DNA. reaction conditions of the Klenow fragment of the polymerase enzyme 5 (pages 107-114), calf intestinal alkaline phosphatase (133134) and ligase reactions (page 146), preparation of the translated samples (pages 109-112) and DNA-DNA hybridization methods 324-325. page 10)

62. Construction of plasmid vectors containing the LAV / HTLV ΠΙ env gene coding sequence for a ligase enzyme vaccinia virus promoter

The following subchapters describe the construction of plasmid vectors in which the LAV / HTLV 15 H1 envelope gene coding sequences are preceded by sequences directing the transcription of the vaccinia virus; these chimeric sequences are surrounded by TK DNA. Using these recombinant plasmid vectors, the LAV / HTLV H1 envelope coding sequences are inserted into the vaccine virus genome by in vivo recombination.

A LAV / HTLV H1 envelope coding sequence was obtained by purification of a lambda J19 subclone designated pRS-3 (S.Wain-Hobson et al., Cell, 40, 9 (1985)). Mackett, G.L. Smith and B.Moss J, Virol. 49: 857-864 (1984)], such that the tag is located downstream of the 7.5K promoter of the vaccine within the pGS20. In the pv-env-1, pv-env-2, 30 pv-env5 and pv-env7 plasmids so constructed, the chimeric gene (i.e., the LAV / HTLV HI specific nucleoid sequence linked to the 7.5K vaccine promoter by the ligase enzyme) was vaccinated with TK. gene sequences.

As mentioned above, pRS consists of a 3840 bp LAV / HTLV IH-specific DNA sequence located between the EcoRI and SstI cloning sites between the EcoRI and SRI sites of pUC18 in lambda J19 cloning. This was prepared by introducing the J19 Ssri restriction fragment 40 into pUC18 by cloning at the Ssff site, digesting the resulting pBT-1 formation with Eco RI and then ligating it again. The resulting DNA was transformed into E. coli and plasmid pRS-3 was isolated from the transformed cells. In the LAV / HTLV 45 IH genome, the LAV / HILV IH-specific DNA in pRS-3 is located between the EcoRI and SstI sites and nucleotides 5289 and 9129, as shown in Figure 2 [S. Wain-Hobson et al., Cell,

40: 9 (1985)]. 50

62.1. Construction of Plasmid Vectors Containing the LAV / HTLV β-Env Gene 3 'Coding Sequence Vaccine Virus promoter ligated with Ligase enzyme ng plasmid DNA pRS-3 was completely digested with KpnI and the resulting fragments were separated on 55 1% low melting agarose gel. The A2680 base pair fragment was isolated and purified. In this fragment, it is known that the nucleotide sequence 5889-8572 contains the LAV / HTLV ΠΙ specific DNA, which contains the sequence encoding the C-terminal portion of the LAV / HTLV IH envelope protein (5889-8349).

One ng of this fragment was mixed with 0.5 ng of pGS20DNA, previously linearized with the Eu / hHI restriction enzyme. Two fragments were ligated together with a ligase enzyme. Alignment is carried out at 4 'C for 24 hours in the presence of oligodeoxynucleotide linkers (0.6 ng 5'-GATCCACCATGGTAC-3'-OH and 0.3 ng 5'-CATGGTG3'-OH). The role of these linkers is to (a) convert end fragments of the 2680 base pair fragment of the plasmid pRS-3 DNA into a védα / ηΗΙ protecting group capable of linearizing the pGS20 -DNA complementary to EamHI-capable end groups, and to (b) provide an appropriate start sequence (ATG) for the correct encoding of the LAV / HTLV IH envelope gene sequence and the nucleotide sequences required for proper translation of the transcribed mRNA. This ligation mixture was used to transform MC1000 E. coli strain (Stratogene). Plasmid DNA containing the transformed bacterial beacepil, an ampicillin resistance-containing gene region, is tested for regeneration of the EamHI, NcoI and KpnI restriction sites at the site of the tag in question, as well as at the site of ligation. The thus confirmed structure of the desired plasmid pn-env2 is shown in Figure 3, including the carboxyl coding portion of the LAV / HTLV ΙΠ envelope gene, corresponding to the 5889-8572 nucleotide sequences and from the 7.5K vaccine protector along the DNA strands. 2, illustrated in Figure 2. The LAV / HTLV HI envelope sequence is located in the encoded region as expected with respect to the starter signal (ATG) from the linker DNA.

622. Construction of Plasmid Vectors for the LAV / HTLV ΠΙ Env Gene 5'-Coding Sequence Containing Vaccine Virus Promoter with Ligase Enzyme Plasmid ngpRS-3 DNA was completely digested with Ara II and the resulting fragments were separated on a 1% low melting agarose gel. The 820 bp fragment was isolated and purified. nucleotide sequence, which is a sequence specific for LAV / HTLV ((see Figure 2). It contains, on the one hand, a sequence encoding the N-terminal portion of the envelope protein (nucleotides 5766-6490) and, on the other hand, a 95 base pair non-coding LAV / HTLV IH sequence adjacent to the 5'-position. This fragment was treated with Klenow fragment of E: coli DNA polymerase in the presence of excess deo-ribonucleotide triphosphates. ) to treated pGS20DNA. Ligation is performed for 16 hours at 12 ° C. The ligation mixture is used to transform MC 1000 E. coli strain Plasmid DNA incorporated into ampicillin-resistant transformed bacteria is tested where the LAV / HTLV IH tag is.

HU 205 780 Β relative to the vaccinia transcriptional control sequence. The confirmed structure of the pv-envl plasmid to be produced is shown in Figure 4; according to this, the amino acid coding for the LAV / HTLVIII envelope gene is shown in SEQ ID NOs: 5671-6490. nucleotides (see Figure 2), the vaccine is downstream of the 7.5K promoter.

6.2.3. Plasmid vectors containing the full coding sequence ligase enzyme linked LAV / HTLVDI envgén vakeiniavírus promoter production ^s TOWING jig envl-pv plasmid DNA Stu I, Pvu ésXhól digested to completion with restriction enzymes. The resulting fragments were separated on a 1% low melting agarose gel. Cleavage of pv-envl with StuI and PvwI results in two 4000 base pair fragments, one containing the HI5 'portion of LAV / HTLV and the other containing pGS20; the Xhol enzyme cleaves the 4000 base pair fragment containing pGS20 into further fragments, thereby enabling the identification of the 5 'portion of the LAV / HTLVIII envelope coding sequence and the transcriptional regulatory element of the Vaccine virus in the 4000 base pair Siul / Pvul fragment. This fragment was isolated and purified and ligated to a 6500 base pair fragment obtained by digestion of pv-env2 with StuI and Pvul. The ligation mixture was used to transform MC 1000 E. coli. The ampicillin-resistant transformed bacteria are selected, and the plasmid DNA incorporated therein is tested for regeneration of the Stul and Pvul restriction sites, and for the presence of 4000 and 6500 base pairs of precursor fragments. The desired plasmid pvenv5 (see Figure 5) is set forth in U.S. Patent Nos. 5766-8349. nulldeotidsomes contain the appropriate full LAV / HTLV IH envelope gene (see Figure 2) and are downstream of the transcriptional regulatory element of the Vaccine virus.

6.2.4. Construction of Plasmid Vectors Containing a Vaccine Virus Promoter Not Containing a LAV / HTLV ΙΠ env Gene Without a Cell Membrane Anchor Sequence ng pv-env5 plasmid DNA was completely digested with HzWIIi and the resulting fragments were isolated at 1% low melting point agar. Containing the 7.5K vaccine promoter and the 5'-promoter of the LAV / HTLV en env coding sequences

The 3,000 bp fragment was purified and treated with Klenow to bind the end groups that could be attached. This fragment was ligated with the ligated enzyme to the pretreated multiple binding site p26 cloning vector (Bethesda Research Laboratories) (the vector was pretreated by digestion with <RTIgt; E1C1 </RTI> and Klenow and finally calf intestinal alkaline phosphatase [CIAP]). . Upon ligation of the pv-env5 plasmid with the binding HindIII sites and the p26 plasmid with the EcoRI binding sites, a stop codon for the envelope coding sequence was created. This intermediate formation is called pv-env5 / 26.

Plasmid DNA pv-env5 / 26 was transformed into strain M1000 E. coli, and the transformed culture was amplified and purified and digested with restriction enzymes BauwIII and Hpal. The resulting fragments were separated on a 1% agarase gel. The 2100 bp fragment containing the LAV / HTLV ΠΙ envelope coding sequence was isolated and ligated to the pGS20 plasmid, previously digested with BamHI and SmaI. In the resulting plasmid pv-env7, the LAV / HTLV? nucleotides (HindIII) (see Figure 5). Thus, the plasmid lacks the putative anchor sequence of the LAV / HTLV ΠΙ env gene.

6.3. Preparation and Characterization of a Recombinant Vaccine Virus Containing Chimeric LAV / HTLV ΠΙ Env

For the production of recombinant strains, two vaccine virus parent strains were used, namely the standard research strain WR and the strain v-NY, which is a subspecies of the New York City Board of Health (see section 6.1.1).

The chimeric LAV / HTLV ΙΠ env sequences were introduced into the vaccine virus genome by in vivo recombination, under conditions such that the pv-env2, pv-env5 and pv-env7 lumeral genes were encoded by the vaccinia virus sequences encoding the thymidine kinase (TK) gene. around. When these plasmids are introduced into vaccinia virus infected cells, recombination occurs between the TK sequences on the plasmid and the homologous sequences of the vaccinia virus genome. Incorporation of the chimeric gene results from double recombination in the surrounding sequences. In these recombinants, the chimeric gene is present in the vaccine's TK gene and consequently has a TK 'character in its phenotype. Selection of TK 'recombinants can be done by culturing them in BUDr-containing medium; this additive kills TK ⁺ cells, but TK cells survive. GL Smith and Β. Moss described in [J. Virol, 41, 857-864. (1984)],

6.3.1. Production of a recombinant vaccinia virus containing the chimeric LAV / HTLV IH env gene

An 80% confluent kidney cell culture (strain BSC-40, ATCC CCL26, deposited in 1983) from African horseshoe monkeys was infected with vaccine virus (strain WR) in a 100 mm diameter dish (multiplicity of infection = 0.05). The preparations are incubated for 2 to 4 hours at 37 ° C and then added to the infected cells to produce calcium phosphate coprecipitate pv-enc2 or pv-env5. The additive is prepared by adding 0.5 ml of 2XHeBS solution (containing 16 mg of sodium chloride, 0.74 mg of potassium chloride per ml,

HU 205780 Β

0.25 mg disodium hydrogen phosphate dihydrate, 2 mg dextrose and 10 mg HEPES at pH 7.08; (HEPES 'N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid); 0.5 ml of 2XDNSCaCl ₂ solution (containing 20 ng of plasmid DNA in 0.5 ml of 0.25 M calcium chloride solution) is added dropwise. After the addition, the culture was allowed to stand for 30 minutes at room temperature, and after 4 hours of formation of the overlay coprecipitate, the cells were first washed with 1XHeBs and incubated for 3 minutes at 37 ° C in 2 ml of 10% 15% glycerol solution. , then again washed with 1XHeBs and incubated with 10 ml medium (DNEM + 10% fetal calf serum + 100-100 units / ml penicillin and streptomycin). Two days later, the infected cells were harvested by centrifugation (4 ° C, 10 minutes, 2000 X g). The resulting cells were resuspended in 1 ml of PBSAM, frozen, thawed, then re-frozen and thawed and finally subjected to three 15-second sonic vibrations, and the resulting 20 virus stock cultures were used.

incubated in 25 ml of 25 DNEM containing 1% Growth Agar (Difco, Detroit, Mich.), 5% calf serum and 25 ng / ml BUDR. and further cultured on this apreparátumon 10 ^-3 dilution of a solution of 0.1 ml of the above virus stocks were screened for the recombinant selection. The culture was continued for 2 days, and agar medium containing the above additives + 0.01% neutral red fiber was plated on the dishes for 2 ml / dish staining. One day after staining, the stained plaques were harvested, resuspended in 0.5 ml PBSAM (PBS + 1mM MgCl ₂ , 0.91% BSA) and aliquots of virus suspension 35 in 0.25 ml aliquots. contiguous 143 TK cell cultures cultured in selective medium (DNEM + 10% calf embryo serum + 100-100 U / ml streptomycin and penicillin plus 25 µg / ml BUDR) were inoculated. The infected cells were harvested by centrifugation and resuspended in 100 µl of PBS. Following cell lysis (incubation for 30 minutes at 37 ° C), the lysate was subjected to three 20-second sonic vibrations. The cell lysate was collected on a nitrocellulose filter using a multi-well separating 45-tube filter (Schleicher and Schuell, Arlington, MA). The presence of LAV / HTLV D1 env-specific DNA sequences in the samples was determined by the DNA DNA method of M. Mackett, GL Smith and B. Moss, Proc. Natl. Acad. Sci., 79., 50 7415-19. (1982)]. ³² P-labeled pRS-3 plasmid DNA prepared by cleavage and translation was used as a hybridization sample. Recombinants that gave a positive hybridization reaction were further purified with this sample (two to five times plaque formation on 143 TK cells, BUdR-containing selective medium, once on BSC-40 cells under non-selective conditions). After final confirmation of DNA-DNA hybridization, the virus stock was isolated after three to 60-fold plaque purification on BSC-40 cells and used for further characterization.

The production scheme of recombinant viruses is illustrated in Figure 6. From here, it can be seen that the LAVZHILV ΠΙ envelope gene sequence (env) is downstream of the vaccine 7.5K promoter (p - * ·) in the vaccinia virus genome and is surrounded by IK-DNA sequences. The virus stock resulting from the recombination between the vaccinia virus genome and the pv-env5 plasmid is called v-env5 and contains the entire LAV / HTLV ΠΙ envelope gene. In addition to the full envelope gene, v-env5 produced in the embodiment of the present examples also contains a 96 bp sequence linked to the 5'-position and a 223 bp sequence linked to the 3'-position. The strain of virus derived from pv-env2 is called v-env-2 and contains a large portion of the LAV / HTLV M envelope gene (i.e., the £ pn1 fragment), but lacks the sequence portion of the LAV / HTLV ΠΙ envelope encodes the first 42 amino acids. Given that the putative signal sequence of the LAV / HTLV D1 envelope protein is located within the first 49 amino acids of the protein, v-env2 is likely to produce a protein lacking this signaling sequence; therefore, the LAVZHTLVni protein thus produced by the recombinant virus is unlikely to reach the cell membrane. In contrast, v-env5 containing the complete LAVZHTLVDI env sequence is likely to produce a protein that reaches the membrane. The strain of virus derived from pv-env7 is called v-env7 and contains the entire N-terminal portion of the LAV / HTLVDI envelope gene, but lacks a sequence downstream of the H / hdII cleavage site. Given the absence of the putative membrane transfer sequence (anchor) from this construct, it is expected that the LAV / HTLV HT-type protein produced by the recombinant is thereby secreted into the medium by the recombinant. This property is advantageous for protein purification and is suitable for use as a diagnostic reagent or for the production of a subunit vaccine.

6.3.2. Vaccine - Behavior of LAV / HTLVni envelope recombinants by restriction enzymes

DNA from the parental strains of the WR and v-NY vaccine virus, as well as their derivatives of v-env5 and v-env5NY, were isolated as described previously [Esposito et al., J. Virol. Methods, 2, 175-180. (1981)]. These were digested with restriction enzyme Hindin and the resulting fragments were separated on 0.7% agarose. DNA from the parent WR strain showed a typical restriction behavior (Fig. 2A) as described previously [DeFilippes, J. Virol.

43, 136-149. (1982)]. The restriction behavior of v-NY was similar but distinct from that of WR. The most striking difference is the terminal fragments of the Vaccine Virus genome,

It was found with respect to the length of Π-Β and -C fragments. The v-env5 and v-env5NY recombinants are free

HU 205780 Β glaze the Hindin J fragment characteristic of their parent strains; instead, two new fragments of 5,400 and 3,200 bp were observed. This observation is consistent with the result of the double recombination in which the LAV / HTLV ΠΙ envelope coding sequence is incorporated into the vaccine TK gene, which is Hindin. It is located in fragment J. Upon insertion, two fragments are generated by the HWIH restriction site present in the LAV / HTLV D1 sequence. This circumstance also confirms that the LAV / HTLV sit extension is located in the vaccinia virus TK gene.

633. Analysis of the Recombinant Southern Blot of Vaccinia -LAV / HTLV envel-Envelope Two new # / £ £? Resulting from restriction enzyme digestion of the recombinant viral genome. Southern blot analysis was performed to identify the ΙΠ-fragment. The DNA fragments separated on the agarose gel are shown in Figure 7A. As shown in FIG. 6A, they were transferred to a nitrocellulose filter and hybridized with a labeled translation sample specific for LAV / HTLV ΙΠ envelope sequences. As in Figure 7B. As shown in Fig. 3A, hybridization was only detectable for fragments of 5400 and 3200 base pairs. This result also confirms the genomic structure of the recombinant virus shown in Figure 6.

6.4. Expression of LAV / HTLV H1 envelope proteins in cells of tissue culture infected with recombinant vaccinia viruses

Recombinant vaccinia viruses carrying chimeric LAV / H1LV H1 env genes have been shown to be capable of expressing LAV / HTLV H1 envelope proteins in cells of tissue culture infected with them. The proteins produced were immuno-reacted with sera from AIDD patients.

6.4,1. Identification of LAV / HTLV ΠΙ envelope type proteins expressed in recombinant vaccinia virus infected cells by immunoblot analysis

Container in a 100 mm diameter cup

BSC-40 cell culture is infected with wild-type vaccinia virus or a recombinant derivative (v-env5 or v-env2) (multiplicity of infection = 10). 12 hour infection! over time, cellular cells were harvested, washed with PBS, and centrifuged. The pellets formed from the infected cells by centrifugation were resuspended in 1 ml of Laemmli buffer [U. K, Laemmli, Naturre, 227, 680 (1970)] and cell lysis at reflux for 4 minutes. The total cellular protein content of 75 nl of the cell lysate was separated into its components by electrophoresis on a 7-15% gradient SDS-polyacrylamide gel. Purified LAV / HTLV HI virion on the one hand and lysates of cells infected with the replicate sample were used as controls, on the other hand. Fractions on the gel were electronically transferred to a nitrocellulose filter pad. The filter was first mixed with 5 ml of PBS solution + 5% skimmed milk for 30 minutes at room temperature and then PBS solution + 5% skimmed milk + human serum (1: 100 diluted with heat-inactivated serum) for 2 hours at room temperature. The filter was then washed five times with PBS + 0.05% Tween 20 (polyoxyethylene sorbitan monolaurate) and once with PBS. The washed filter was then incubated with 5 ml of PBS containing 1% normal goat serum (heat inactivated) and 1: 3000 diluted goat anti-human IgG-horseradish peroxidase conjugate for 2 hours at room temperature. The filter was rinsed again, first washed five times with PBS + 0.05% Tween 20, then once with TBS (0.5 M sodium chloride and 20 mM tis hydrochloride, pH 7.5). The filter-bound horseradish peroxidase conjugate was detected by adding to the filter in the dark at room temperature for 10 min. it consists of solutions A and B mixed just before use (solution A: a mixture of 20 ml of 30% cold methanol and 60 mg of 4-chloro-1-naphthol; solution B: 60 ml of 30% cold hydrogen peroxide and 100 ml IBS). Protein-bound proteins that react with antibodies in the serum of AIDD patients are detected by the color reagent by binding to goat anti-human IgG horseradish peroxidase conjugates.

The analytical results are shown in Figure 8A. Figure 3B shows, in particular, how v-env5 vaccinia virus produces three recombinant proteins that give specific immune responses to the serum of AIDS patients. The electrophoretic migration rates of these proteins are similar to authentic LAV / HTLV IH glycoproteins (gp150, gp10 and gp41), which are likely to be encoded by the env gene [W. G. Robey et al., Science, 228, 593-595. (1985)] - In cells infected with the wild-type vaccine virus and the mock-up sample, these proteins were not produced. The v-env2 recombinant lacking the 5'-position sequence, which is thought to encode the initial 42 amino acids of the LAV / HTLV envel-envelope proteins, produces a tailed protein that does not immunize with the serum of AIDS patients. . The translation of the env sequence in the v-env2 recombinant may be initiated from the AUG codon transcribed from its linker sequence used to construct the recombinant (see section 6.2.1).

In another experiment, two cell lines (BSC-40 and HeLa) were infected with v-env5 and v-env2. LAV / HTLV ΠΙ-specific proteins in the lysate of infected cells were assayed by the Wesfem immunoassay described below.

Single-cell monolayers of BSC-40 and HeLa cells were infected with v-env5 or v-env2 recombinant virus (multiplication of 50 pfu / cell pfu plague forming unit). Twelve hours after infection, the cells were washed twice with PBS, resuspended in Laemmli buffer and boiled for 5 minutes. Proteins produced in infected cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, applied to a nitrocellulose membrane, and reacted with serum collected from LAV / HTLV HI serum-positive patients. The immunoreactive proteins were ¹²⁵ I-labeled protein antigen se21

HU 205780 Β 2 was detected by the chloramine T method according to the reagent manufacturer's instructions. 8B. lanes 1 and 5, proteins from cells infected with the mock-up sample, lanes 2 and 6 with the wild-type vaccine virus, lanes 3 and 7 with v-env5, and 4.5 and 8, respectively. lane 3 shows proteins from cells infected with v-env2. Sucrose gradient purified LAV / HTLVIH virion proteins (LAV / HTLV ΠΙ) were used as controls and the positions of gp150, gp101 and gp41 envelope proteins were marked. Standard molecular weights are expressed in kilodaltons (kD). In cells infected with V-env5 (Fig. 8B, 3 and 4)

Lane 7) produced three major proteins which were immuno-responsive to the serum collected from serum-positive patients. These proteins are estimated to have molecular weights of 150 kD, 120 kD and 41 kD, which are very similar to the glycoproteins of LAV / HTLV ΠΙ envelope (gp 150, gp 110 and gp 41).

The recombinant virus v-env2 did not show the expected LAV / HTLV ΠΙ-env sequence, but produced at least three immunoreactive polypeptides of 99 kD, 68 kD and 40 kD (Fig. 8B, 4 and Lane 8).

6:42. Identification of LAV / HTLV IH envelope-like proteins expressed in cells infected with recombinant vaccinia virus by immunoprecipitation method

The following describes an immunoprecipitation method that has been used to demonstrate that the recombinant vaccine viruses of the present invention synthesize a protein that elicits a specific immune response in the serum of AIDS patients.

A contiguous BSC40 cell culture in a 100 mm diameter dish is infected with wild-type vaccinia virus or its v-env5 and recombinant env2 35 (multiplicity of infection = 10). 9.5 hours after infection, the medium is replaced with DNEM medium containing no serum additive or methionine.

one hour after infection, the medium is replaced with 2 ml of DNEM 40 containing ³⁵ n-S ³⁵ -methionine, which does not contain normal (unlabelled) methionine. The mixture is reacted with the radiolabeled component for 2 hours at 37 ° C and the cells are washed once with PBS and centrifuged. The resulting cell pellets were resuspended in 1 ml of lysis buffer. Composition of lysis buffer: 1% NP-40 (polyoxyethylene-9- (p-tert-octylphenol)), 0.5% sodium deoxycholate,

0.1 molar sodium chloride, 0.01 molar sodium chloride, 0.01 molar tris hydrochloride (pH 7.4) and 1 mM EDTA The resulting cell lysate was centrifuged for 1 min in an Eppendorf microcentrifuge.

To 100-100 µl of cell lysate is added 5-5 ml of heat-activated human serum, either from AIDS patients or from a healthy control group, and incubated for 1 hour at 4 ° C. Subsequently, 60 µl of activated Staphylococcus aureus cells (Pansorbin cells, Calbiochem-Behring Corp., LaJolla, Ca) are added to the preparations and incubation is continued for 4 hours at 4 ° C. Immunoprecipitation complexes were separated and washed with Eppendorf microcentrifuge for 30 seconds at 4 ° C, once with 1 M sodium chloride (+ 0.1% NP-40 and 0.01 M Tris-Hydrochloride, pH 7.4) and twice with RIPA buffer [10 mM Tris hydrochloride, pH 7.1; 0.15 M sodium chloride, 1% sodium deoxycholate, 1% TritonX 100 (polyoxyethylene (9-10) -tertoctylphenyl) and 0.1% sodium lauryl sulfate]. The washed immunoprecipitates were resuspended in 50 µL of Laemmli buffer, boiled for 1 minute and centrifuged for 1 minute in an Eppendorf microcentrifuge. Immunoprecipitation proteins present in the supernatant were analyzed by electrophoresis on a 15% SDS-polyacrylamide gel. After electrophoresis, the gel was stained with anazolene sodium, treated with 1 M sodium salicylate for 30 minutes at room temperature and dried for fluorographic detection.

The result of the analysis is shown in Figure 9A. From here, it can be seen that the v-env5 recombinant synthesizes a series of proteins that elicit a specific immune response in the serum of AIDS patients. These apparently have molecular weights of 160,140, 120, 42, and 40 kilodaltons (kD), which approximate the LAV / HTLV HI envelope type. glycoproteins [W. G. Robey et al., Science, 228, 593-595. (1985) J. These cells were not present in cells infected with the wild-type vaccine virus or the mock-up sample, nor did they form immunoprecipitates with control human serum. V-env2-infected cells produced a tailed protein with an apparent molecular weight of 95 kD; it reacted with serum from AIDS patients. This tailed-form LAV / HTLVIH env-type protein most likely originates from the AUG codon encoded by the linker sequence at the 5 'position of the LAV / HTLV IH extension in the recombinant.

6.4.3. Labeling of LAV / HTLV H1 envelope proteins produced by vaccinia-LAV / HTLV IH recombinant viruses with ³ H-glucosamine

In the following, a radioimmunoprecipitation method is described which demonstrates that the vaccine-LAV / HTLV H1 recombinant viruses of the invention produce glycosylated envelope proteins.

HeLa cells infected with an imitation sample (lanes 1 and 5 in Figure 9B), wild-type vaccine virus (lanes 2 and 6), or v-env5 recombinant (lanes 3 and 7) and v- Experiments were performed on HeLa cells infected with env2 recombinant (lanes 4 and 8) (multiplicity of infection> 50 pfu / cell). At 4-16 hours post-infection, ³ H-glucosamine (activity: 0.25 µCi; specific activity: 22 mCi / g; prepared by Amersham) was added to the culture medium. . The buffer contained 0.1 M sodium chloride, 0.01 M tris hydrochloride, pH 7.4, 1 mM EDTA, 1% ΝΡ-40 [polyoxyethylene-9- (pterc). octylphenol)] and 0.5% sodium deoxycate. Aliquots of cell lysate for normal human serum (lanes 1-4) or pooled serum for LAV / HTLVIH serum positive patients (lanes 5-8).

HU 205780 Β beat. Immune-responsive proteins are precipitated with fixed protein antigen-bearing Staphylococcus aureus cells, separated by SDS-polyacrylamide gel electrophoresis and detected by fluorography.

The results of the radioisotope labeling with glucosamine (Figure 9B, lane 7) show that they have been glycosylated to proteins produced by the recombinant v-env5. Differences in glycosylation behavior may be due to differences in the electrophoretic migration rates of recombinant-produced proteins and the LAV / HTLVBIvirion glycoproteins.

In the case of a protein lacking the signaling peptide sequence, ^3- H-glucosamine does not produce an N-glycolysed derivative in v-env2-infected cells (Fig. 9B, lane 8).

6.4.4. Blood culture - Analysis of LAV / HTLV IH envelope-type proteins produced by LAV / HTLV ΙΠ recombinant viruses by pulse duration immunoprecipitation method

We hypothesized that the gpl50 glycoprotein of LAV / HTLV H1 is the precursor from which the gp 110 outer protein and the gp41 membrane transfer protein are derived. The following is a pulsed-chase immunoprecipitated labeled isotope labeled , which showed that the 150 kD, 120 kD and 41 kD proteins produced by the recombinant vaccinia LAV / HTLV HI are in a precursor product relationship similar to that of the authentic LAV / HTLVHI gp50, gp10 and gp41 proteins.

Single-cell monolayer HeLa cultures were infected with wild-type vaccine virus (lanes 1-6 in Figure 9C), v-env5 (lanes 7-12), or recombinant v-env2 (lanes 13-18). Multiple infections in each case were 50 pfu / cell. At 10.5 hours after infection, cells were labeled with ³⁵ S-methionine (activity: above 800 Ci / mmole, specific activity: 100 nCi / ml, produced by Amersham) for 15 minutes. At the end of the radiolabeling period, the cells are washed with 2 ml preheated chase medium (Dulbecco's modified Eagle's solution + 3 mg / ml L-methionine + 5% calf serum + 100 U / ml penicillin and 100 ng / ml streptomycin) and before incubation. again, add another 1 ml of the same solution. The cells are then washed at various times and allowed to lyse as described in 6.4.3. Subsequently, the cell lysates are immuno-reacted with pooled sera from LAV / HTLVIH serum-positive patients. The protein components of the immunoprecipitate were separated by SDS-polyacrylamide gel electrophoresis and detected by fluorography. Each start was made after the following time intervals: 0 hours (lanes 1, 7 and 13); 0.5 hour (lanes 2, 8 and 14), 1 hour (lanes 3, 9 and 15); 2 hours (lanes 4, 10 and 16); 6 hours (lanes 5, 11 and 17) and 12 hours (lanes 6-12 and 18), 9C illustrating the results. 7-12. bands refer to the same precursor-to-product ratio of 150 kD produced by the recombinant,

120 kD and 41 kD proteins such as the authentic LAV / HTLVHI gp150, gp10 and gp41 fractions. In HeLa cells, the conversion of the 150 kD protein fragment was slow and inadequate, as less than 50% of the radioactivity in the 150 kD protein was transferred to the 120 kD and 41 kD proteins 6 hours after pulse signaling. Preliminary experiments have shown that this process is much more efficient in some human peripheral blood cells infected with the same recombinant virus. The same experiments also showed that the env sequence present in v-env2, lacking the putative signaling sequence of LAV / HTLV H1 env, was produced as an unchanged 87 kD precursor (780 amino acids); this was processed into a 99 kD intermediate and cleaved to a 68 kD and 40 kD polypeptide (lanes 13-18 in Figure 9C).

6.4.5. LAV / HTLV H1 envelope protein in culture medium of cells infected with vaccinia - LAV / HTLV HI recombinant viruses

The following describes a radiolabeled immunoprecipitation assay that demonstrates that the immunoreactive proteins produced by the recombinant vaccine virus of the invention are produced and transformed in a manner similar to the authentic LAV / HTLV Ht envelope glycoprotein.

Single-cell monolayer HeLa cell culture was infected with an imitation sample (Figure 9D lane 1) or with 30 wild-type vaccine viruses (lane 2), v-env5 recombinant (lane 3), or v-env2 recombinant (lane 4). ). Multiple infections in each case were 20 pfu / cell. At 1012 hours after infection, cells were labeled with ³⁵ S-methionine (activity: above 800 Ci / mmole; specific activity: 100 nCi / mL, manufactured by Amersham). At the end of the radiolabeling period, the reconstituted portion is removed, centrifuged at 12,000 x g for 2 minutes, and subjected to an immunoprecipitation reaction with pooled serum from LAV / HTLVHI serum positive patients. Proteins obtained by immunoprecipitation from infected cells with the same serum and proteins obtained from solution media are shown in a separate field. ' ^:, ' ^J

As expected for LAV / HTLV ΙΠ gpl 10, the 120 kD protein produced by the recombinant could be detected in the soluble part of the infected cell (Figure 9D, lane 3). This result confirms that the v-env5 recombinant virus is capable of expressing the LAV / HTLV H1 env gene and producing immunoreactive proteins, which exhibit a transformation behavior similar to authentic LAV / HTLV ΠΙ envelope glycoproteins.

As expected, the LAV / HTLV ΙΠ envelope-type polypeptide was not detectable in the soluble portion of the v55 env2-infected protein (Figure 9D).

lane 4).

At the same time, cells infected with the v-env7 recombinant produced a 130 kD carcass protein. 60 These proteins are missing for the C-terminal23

HU 205780 Β

Table L

Serum titer change in mice vaccinated with recombinant viruses carrying the chimeric LAV / HTLV H1 env gene, containing the anchor sequence of the alAV / HTLVIII envelope protein. This tailed protein (gp130) is excreted into the media in much greater amounts than either gp110 or its gp150 precursor (Fig. 9E). However, gp130 is not efficiently converted to gp10, although the required cleavage sites are present on the tailed molecule.

6.5. Vaccine - LAV / HTLV H1 env recombinant virus immune strength

Recombinant viruses harboring the chimeric LAVZHTLVHI env gene have been shown to produce antibodies against LAV / HTLV ΠΙ in two mouse strains and in a non-human primate.

6.5.1. Vaccination - Immunogenicity of LAV / HTLV H1 env recombinants in mice

The immunogenicity of proteins expressed by v-env5 and v-env2 recombinant vaccinia viruses was examined. The experiments detailed below have shown that these recombinant viruses elicit an immune response against all major LAV / HTLVHI glycoproteins.

Two mice strains, cultured ICR and inbred C57B16J mice (produced by Natur 320: 557-560), were given the recombinant viruses v-env5 and v-env2. The mice were 5-7 weeks old at the time of vaccination. Four modes of administration were tested: arecombinants administered to the toe, scratching the tail, orb and intraperitoneally. When injected into the fingertip, 25 µl (5 x 10 ⁶ pfu) of recombinant virus were injected into both dorsal toe. Scratching of the tail was done by scraping the skin at the tail with a double-pointed needle and applying 10 µl (2 x 10 ⁷ pfu) of recombinant to the scratch surface. For nasal administration, 10 µl (2 x ^{10 7} pfu) of recombinant virus were applied to the nose of the mouse and the animal was allowed to inhale the vaccine. Upon intraperitoneal administration, 10 µl (2x10 ⁷ pfu) of recombinant virus were injected into the abdominal cavity of the mice. Strains of vfr and strains made from them are diluted as described in 6.1.1. were prepared as described in Subchapter II. At 2-week intervals after vaccination, the animals were sampled and analyzed by both ELISA and western blot analysis as described below.

6.5.1. Changes in serum titer of mice immunized with vaccinia -LAV / HTLV H1 env recombinants as measured by ELISA.

The data measured in serum samples by ELISA during the 6-week study period are summarized in Table I.

recombinant virus Method of vaccination Abeoltott mice sera -literének * changes 10 ICR C57B16J v-ENV2 footpad 3/3 4/5 tail scratching 3/3 4/5 intranasal 0/3 4/5

intraperitoneal not performed 3/5

v-ENV5 toe tip 4/4 tail scratching 3/3 intranasal 3/3 5/5 5/5 4/5 20 intraperitoneal gender done 5/5

^X Changes in serum titer as determined by the ELISA method detailed in the text are expressed as the ratio of mice showing a change in serum titer to the total number of inoculated mice.

In C57B16J mice, 100% and 95% change in serum titre was observed, respectively, by v-env2 and v-env5. ICR mice had a 44% change in v-env2, and a 100% change in serum titer for v-env5 recombinant. Complete change in serum titer and highest mean ELISA titers were observed in tail-scratched mice.

The EUSA method was applied as follows:

Purified and inactivated LAV / HTLV IH virions were diluted in carbonate buffer (50 nM sodium carbonate, pH 9.6) and added to 96-well microtiter culture dishes (100 µl / well, 0.2 ng inactivated LAV / HTLV HI). . The formulations were allowed to stand overnight at 4 ° C, then the unbound protein was aspirated and washed first with 200 µl PBS in 5% skimmed milk per dish and then in 300 ml 4% sucrose solution. Then, for each dish

50 η 1 PBS solution containing 2.5 η 1 mouse serum was added and allowed to react for 1 hour at 37 ° C. At the end of the incubation period, the preparations were washed five times per well with PBS solution containing 0.05% Tween 20 (polyoxyethylene sorbitan monolaurate). 50 µl of goat anti-mouse horseradish peroxidase conjugate (1: 5000 dilution in PBS; 0.05% Tween-20) was then added to the dishes and allowed to react at 37 ° C for 45 minutes. The cultures were washed 5 times with 0.05% Tween-20 in PBS and 100 µl of citrate-o-phenylenedianunperoxide substrate was added. The substrate was prepared as follows: Dissolve 0.294 g of sodium dihydrate and 0.537 g of crystalline disodium hydrogen phosphate in 10 ml of water and adjust the pH of the solution to 5.0 with hydrochloric acid; the solution thus prepared is used directly

Before the addition of 205780 µl, 2 µl of 130% hydrogen peroxide and 4 mg of o-phenylene diamine were added. The culture vessels were incubated for 30 minutes in the dark at room temperature. The reaction was then quenched by the addition of 100 µL of 3N sulfuric acid per vessel and the optical absorbance of the reaction mixture was measured at 490 nm.

The results in Table I are expressed as the ratio of serum-positive mice to all inoculated mice. Serum samples are collected 6 weeks after vaccination. needles and analyzed by ELISA. Five non-vaccinated mice formed the control group. The mean ELISA titers of serum samples from the control group were 0.021 ± 0.005 and 0.017 ± 0.010, respectively, for ICR and * C57B16J mice. Serum samples from immunized mice were considered serum positive with an ELISA titre greater than three standard deviations greater than that of the control group.

6.5.1.2. Detection of antibody production against authentic LAV / HTLV ΠΙ envelope glycoproteins in altered serum titers by Western blot analysis

To demonstrate that mice inoculated with recombinant viruses produce antibodies against authentic LAV / HTLV ΙΠ envelope glycoproteins, serum samples from immunized mice were examined by Western blot analysis as follows: 5 7-week-old male inbred mice (strain C57B16J): Jackson lab. immunized by applying 10 µl of a 2 x 10 ⁷ pfu recombinant vaccinia virus vaccine to the bruised surface of the tailbone with a double-pointed needle, 8 weeks after vaccination, the animals were bled through the retro-orbital sinus and frozen in serum until use. Serum samples · aliquots were diluted 50-fold with 0.2% NP-40 and 3% skimmed-milk phosphate buffered saline (PBS) and pre-separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filter and <RTIgt; lv </RTI> immunized. white jocks. LAV / HTLV D1 proteins reacting with these sera were detected by a goat anti-mouse immunoglobulin conjugate with alkaline phosphatase.

Figure A10 shows the results of 8 weeks serum samples of C56B16J mice immunized with recombinant viruses by tail scratching.

All animals immunized with recombinant viruses produced antibodies that reacted with the gp41 LAV / HTLV Hl ⁹¹ envelope glycoprotein. Some of the animals immunized with the v-env5 recombinant also reacted with the gp150 and gp1010 glycoproteins, i.e., this recombinant virus can elicit an immune response against all major LAV / HTLV III glycoproteins.

6.5.2. V-env5 vaccine - Recombinant immunogenicity of LAV / HTLV ΙΠ in inferior primates in humans

The recombinant immunogenicity of v-env5 vaccine - LAV / HTLV ΙΠ was tested in two sub-human strains, namely Macaco fascicularis and chimpanzees. The results presented in the following subchapters demonstrate that v5 env5 induces humoral and cellular immunity in both species.

6.5.2.1. Immune Immune Response in Macaque Monkeys Immunized with V-env5 Immunogenicity of the V-env5 vaccine - LAV / HTLV ΙΠ - was investigated in long-tailed macaques (Macaco fasciularis), a mixed son and old animal. Only animals in which no pre-screening antibody or anti-LAV / HTLV, antibody, monkey T-lymphotropic virus, monkey AIDS virus, or antibodies raised against these were found. Four monkeys received 2 x 10 ⁸ pfu, another 4 monkeys received 2 x 10 ⁷ pfu of recombinant v-env5. One animal received 2 x 10 ⁷ pfu control recombinant (vaccinia herpes simplexgD1 recombinant). Árekombi20 nancy was applied to the animals by scratching the skin. Serum and heparin-treated blood samples were collected at 4.6 and 8 weeks prior to and after immunization. 10 weeks after the first dose of a newer, second dose given to the animals (with the same virus, 2 x 10 ⁸ pfu). All animals exhibited a characteristic skin lesion that was self-healing for the vaccine infection; this phenomenon was considered a normal physiological indicator during the experiment. Immoral immune response was detected by ELISA and western blot analysis.

For ELISA, serum samples were diluted 50-fold in PBS containing 3% skimmed milk, 0.2% ΝΡ-40 [polyoxyethylene-9- (p-tercoctylphenyl)] and fixed in microtiter dishes.

Reaction with LAV / HTLV ΠΙ virion proteins. The ELISA assay itself is described in 6.4.1.1. except that a goat anti-human antibody conjugated to horseradish peroxidase was used as the second antibody.

A11. It can be seen from Figure 5A that only two animals showed a change in serum titer after the first vaccination, whereas serum conversion occurred in all animals after the second immunization.

one week after the second immunization, a new serum sample was taken and the antibodies produced in the immunized animals were determined by Western blot. The two envelope glycoproteins, gp41 and gpl10 were optimally determined differently. For optimal determination of gp41, serum was diluted 50-fold in PBS containing 3% unpasteurized milk and 0.2% ΝΡ-40 and reacted with purified LAV / HTLV ΙΠ virion protein fixed on a nitrocellulose filter for 1 hour at room temperature. The filter was then reacted with goat anti-human immunoglobulin antibody (Zymed, CA) diluted 1: 2000 in PBS solution containing 0.2% l-40. For optimal determination of gpl 10, serum contains 3% unpasteurized milk

Diluted 50 fold with PBS containing 205780 HU HU and reacted with LAV / HTLV Hl virion protein (purified) fixed on a nitrocellulose filter for 1 hour at room temperature. The filter was washed with PBS solution containing 0.05% Tween-20 and reacted with goat anti-human immunoglobulin antibody (Zymed, CA) diluted 1: 2000 in PBS solution. After reaction with the second antibody, both glycoproteins For example, the preparation was washed with a solution of 0.1 M Tris pH 9.5, 0.1 M sodium chloride and 5 mM magnesium chloride. The antigen-bound antibody on the filter was 0.1 M Tris pH 9.5, 0.1 M sodium chloride, 5 mM magnesium Worid, 0.33 mg / ml bromochlorodoline phosphate and 0.17 mg / ml nitrotetrazolium blue chromogen solution, washed with water, air-dried and photographed.

A12. It can be seen from Figure 5A that all double-inoculated animals give a strong antibody response to the agp41 glycoprotein, four of which also express antibodies specifically to the gp110 glycoprotein. Taken together, Figure 12A shows that LAV / HTLV IH envelope specific antibodies are produced by recombinant venv5 in macaque monkeys.

6.5.22. Predatory immune response in macaque monkeys immunized with V-env5 recombinant

The experiments were performed in macaque monkeys vaccinated with v-env5 recombinant and control vaccine recombinant virus (v-HSVgdl) expressing the herpes simplex virus glycoprotein Dt. Four weeks after the second intradermal immunization, the macaques were bled, heparinized, and peripheral lymphocytes (PBL) isolated by Ficoll-hypaque centrifuge. In addition, PBL was isolated from the blood of macaques immunized only once with v-env5. PBL was suspended in RPMI medium containing 10% heat inactivated normal human serum (Gibco, Grand Lsland.NY), and a final volume of 0.1 ml of medium containing 1 × 10 ⁵ PBL was added to each well of a 96 round bottom dish. in some dishes. Subsequently, v-env5 (1 χ 10 ⁶ pfu / ml before inactivation) or total LAV / HTLV ΠΙ (1 mg / ml, approximately 1 χ 10 ⁵ TCID ₅₀ ) was inactivated with ultraviolet light in 0.1 ml medium. TCID ₅₀ = ₅₀ % viral dose of Abeolytic tissue cultures), obtained from each supernatant of LAV / HTLV-infected CEM cells (a HLA DR negative T-cell leukemia cell line) by double sucrose centrifugation. Six days after PLB stimulation, each vessel (PLB preparation) was treated with 1 nCi ³ H-TdR pH-thymidine; New England Nuclear, Boston, MA) for 6 hours, cells were harvested and the incorporation of ³ H-TdR was determined from the average of 4 culture wells. The stimulation index was calculated as the quotient of the ³ H-TdR counts incorporated into the stimulated cells and the counts of the non-stimulated cells. The results are shown in Π. Table.

Π. spreadsheet

LAV / HTLVin envelope glycoprotein vaccine proliferative response of PBL isolated from recombinant virus immunized macaques to LAV / HTLV ΙΠ

Virus used to stimulate APBL

Amacaca Immunization unstimulated sample LAV / HTLV Hl v-env5

number virus counts * counts * SP * counts * SP * 67 v-ENV5 1,586 7.645 4.7 60.415 38.1 68 v-ENV5 2.245 9.075 4.0 28.638 12.8 74 v-ENV5 1,585 4.732 3.0 21.487 13.6 75 v-ENV5 581 8,645 · 14.9 37.847 14.1 76 v-ENV5 2.479 5.987 2.5 36.657 14.8 80 v-ENV5 1,077 13.922 12.9 24.752 23.0 81 v-ENV5 965 3.985 4.1 40.572 42.0 82 v-ENV5 2.581 8.580 3.3 46.847 18.1 73 v-HSVgDl 612 553 1.0- 56.217 91.9 26 2.911 1,822 L0- 2,240 L0- 27 1,228 1,072 1.0- 2,532 2.0

* Incidence of ³ H-TdR incorporated ** Stimulation index

The Π. Table 6 shows that all macaques, including V-env5, were immunized only once. monkeys - responded specifically to stimulation with LAV / HTLV,, namely, produced lymphocytes that proliferated in vitro. 60

However, in the case of Art. A monkey that received a v-HSVgD1 vaccine for herpes simplex recombinant treatment produced lymphocytes that responded to the cv vaccinia virus stimulation but not to the LAV / HILVHI stimulation. Lymphocytes from non-immunized control animals

HU 205780 Β did not react to any of the antigens. The magnitude of the response of lymphocytes from macaques immunized with v-env5 was similar to that of v. HSVgD1 inoculated with v-HSVgD1. makákóéihoz. These results indicate that the LAV / HTLV ΕΠ env gene expressed by recombinant virus does not suppress T cell-mediated immune responses in macaques in vitro or in vivo.

To determine what kind of lymphocytes isolated from immunized macaques were stimulated by LAV / HTLV ΙΠ, two-color immunofluorescence assays were performed to determine the expression of IL-2 (interleukin-2) receptors on activated T cells and B cells. CD4 and CD8 antigens are present on T cells and CD20 (Bp35) antigen is present on B cells, A ΠΙ. Table 11 shows that viral-stimulated PBLs expressing JL-2 almost all express CD4 and CD8 antigens as well; however, only 1.5-2% express CD20 (Bp35) antigen. These results confirm that stimulated lymphocytes are T-cells.

Table E.

IL-2 receptors and CD4, CD8 or CD20 (Bp35) antigens on PBL from vaccinated macaques stimulated with LAV / HTLV E or recombinant vaccine virus producing LAV / HTLV E envelope glycoproteins

Amakákó number PLBstimulálásra used virus JL-2R + + cells comp. % IL-2R + CD4% expressed by cells CD9% CD20 (Bp35)% 74 v-ENV5 27.7 48.6 52.2 2 80 v-ENV5 25.6 62.4 40.0 not tested 74 LAVZHTLVE 14.7 50.0 59.0 1.9 80 LAV / H1LVE 12.0 58.0 38.0 1.5 80 - 0.3 0.3 0.3 0.3

The data in Table AE were obtained under the following experimental conditions: PBLs were stained concomitantly with phycoerythrin-conjugated monoclonal antibody 2A3 (Becton-Dickinson, Inc., Mountain View CA) - a monoclonal specific for interleukin-2 receptor (JL2R) and fluorescein. antibodies, and is E5 (Genetic Systems Corp., Seattle, WA), which is specific for CD20, G10-l-digested (Genetic Systems Corp., Seattle, WA) to CD8, or with _the T4 (Ortho Diagnistie, Raritan, NJ), which is specific for CD4. Antibodies were used at saturation concentration obtained by titration of lymphocytes previously analyzed with a modified FACSIV cell classifier (Becton-Dickinson, Mountain View, CA) using flow microfluorimetry. Quantitative analysis of two colors by the method of JA Ledbetter et al., Prespectives in Immunogenetics and Histocompatibility, 6, 119-129. (1984)]. The anterior and perpendicular scattering gates are adjusted so that lymphatic obstructs and a sufficiently large number of small lymphocytes are observed. The table shows the% of EL-2R + cells on the one hand and the CD4, CD8 or CD20 (Bp35)% expressed by IL-2R + cells on the other hand.

Antigen-stimulated T cells are known to produce lymphokines, such as IL-2, which promote T and B cell differentiation and / or proliferation, and to activate natural killer cells that are capable of binding to a virus, e.g. to cells infected with the AIDS virus. Thus, the question was raised whether T cells from vaccinated macaques produce IL-2 when stimulated with LAV / HTLV E.

To clarify this, 2 experiments were performed. The preparation of Experiment 1 was as follows: 4 weeks after the second immunization, macaques were double-inoculated with v-env5 or v-HSVgD1 constructed using the WR vaccine virus and PBLs were isolated from the heparinized blood. Preparation of Experiment 2: Injected with v-envNY (recombinant v-env5 constructed using a virus strain of the New York City Board of Helath), v-env5 or v-HSVgD1 at 2 x 10 ⁵ pfu macaques were bled 4 weeks after the first immunization and PBLs were isolated. PBLs were resuspended in RPMI 1640 medium containing 10% heat inactivated normal human serum and placed in 2 x 10 ⁵ PBL media in each well of a 96-well bottom plate. Ultraviolet light-inactivated v-env5 (1 x 10 ⁶ pfu / ml before IV inactivation) or purified LAV / HTLV E (1 ng / ml) was then added to the dishes. After 2 days of stimulation, the supernatant from the culture vessel was harvested and tested for proliferation-enhancing activity on IL-2-dependent OTLL-2 cells (prepared by Dr, S. Gillis, Immunex Corp., Seattle, WA). The CTLL-2 cells used were washed for 24 hours prior to the assay for IL2. The supernatant was incubated with CTLL-2 cells, the cells were labeled with ³ H-TdR for the last 6 hours of incubation and the amount of ³ HTdR isotopes incorporated into the cells was determined. IL-2 activity (units / ml) was determined using a calibration curve; curve recombinant human IL-2 (produced by Dr. Gillis) prol proliferates CTLL-2 cells

HU 205780 Β. The results are shown in Table IV. Table.

ARC. spreadsheet

IL-2 production of PBLs from vaccinated macaques stimulated with LAV / HTLVHI or AIDS virus envelope glycoprotein producing vaccinated macaques

l.Kísériet: Amakákó number Virus used for vaccination no virus IL-2 (units / ml) Virus used to stimulate the supernatant of PBL LAV / HTLVIH v-ENV5 67 v-ENV5 0 28.0 96.0 68 v-ENV5 0 16.0 124.0 74 v-ENV5 0 9.0 52.0 73 v-HSVgDI 0 0 108.0 26 - 0 0 0 Experiment 2: 03 v-ENV5 0 14.4 55.8 05 v-env5NY 0 16.8 33.3 49 v-env5NY 0 7.1 26.7 52 v-env5NY 0 2.4 27.6 59 v-HSVgDl 0 0 27.6 26 - 0 00 0 27 - 0 0 0

AIV. In Table 1, the data from Experiment 1 record the following: Supernatants from PBLs immunized twice with v-env5 and stimulated with v-env5 or LAV / HTLV ak contain IL-2; this is due to their ability to induce proliferation of IL-dependent CILL cells. Similarly, in the second experiment, V3 env5 was immunized once. In macrophages and in the supernatants of PBLs immunized with all three macaques once immunized with the same recombinant 35 "vaccine" strains (y-env5NY; see section 6.3), IL-1 was also detected in supernatants of -2-t. On the other hand, vaccines HSVgD1 recombinant virus were immunized with 40 73 and 59. macaque PBLs produced IL-2 only after stimulation with venv5, not after stimulation with LAV / HTLVHI. A26. and

No. 27 unimmunized macaques were not stimulated by either LAV / HTLV or recombinant vaccinia virus. produced a detectable amount of IL-2. Since T cells with helper activity predominantly produce IL-2 after antigen stimulation, the results suggest the presence of helper T cells that recognize LAV / HTLV ΠΙ in macaques immunized with recombinant viruses. In addition to being likely to play a role in B cell differentiation, which results in the production of antibodies specific to LAV / HTLV H1 envelope antigens, T1-producing T cells are effector cells, such as cytotoxic T-lymphocytes, or viral. they may also play a role in the differentiation and / or proliferation of killer cells that kill infected cells. 6.5.2.3. Humoral immune response in chimps immunized with v-env5NY

Two young chimpanzees were injected intradermally with 5 x 10 ⁸ pfu of v-eny5 "vaccine" strain, i.e., v-env5NY. Another animal was given intradermally the same titre of the vaccinia herpes simplex gD recombinant, i.e. v-HSVgDINY (vNY was a two-recombinant "ancestor" strain). 8 weeks after the first vaccination, each animal was immunized a second time with the same dose. After immunization, animals were serum sampled every two weeks and analyzed by ELISA and Western blot for the presence of antibodies specific for LAV / HTLV ΙΠ. In all animals, self-healing skin lesions characteristic of vaccinia virus infection were observed as normal physiological reactions.

Chimpanzee sera were analyzed by ELISA as described for macaque sera. The results of Table V show that the serum titer of the experimental animals (animals 124 and 149) changed (became serum positive) 8 weeks after the first immunization and further increased after the second immunization. The control animal (No. 134) remained serum negative.

HU 205780 Β

Table V.

ELISA data from serum samples of chimpanzees immunized with recombinant vaccine viruses

Sampling Time No. 134 chimpanzee v-HSVgDINY ELISA thaw 124 for Serum Antibodies to LAV / HTLV E. chimpanzee v-env5NY No. 149 chimpanzee v-env5NY Immunization before 0.084 ± 0.015 0.100 ± 0.005 0.123 ± 0.05 8 weeks for that 1. after immunization 0.085 ± 0.010 0.185 ± 0.020 0.403 ± 0.27 2 weeks a 2. Immunization After 0.075 ± 0.012 0.237 ± 0.017 0.523 ± 0.073

Western blot analysis of antibodies specific for LAV / HTLV ΠΙ in immunized animals revealed that they are directed against envelope-type glycoproteins. The procedure was optimized for detection of gp41 antibody; the method was the same as that used in the macaque serum. Figure 13, which shows the results, shows that in both vaccinated animals the LAV / HTLV E-specific antibodies produced are indeed envelope glycoproteins and that the level of these antibodies is increased after the 2nd immunization.

6.5.2.4.

cellular immune response in chimpanzees immunized with v-env5NY

A 6.5.2.3. Peripheral lymphocytes (PBL) isolated from the same animals as those described in Subdiv. PBL isolation was performed by Ficoll-hypaque centrifugation of the animals' heparin-treated blood 4 weeks after the first intradermal inoculation with recombinant vaccinia viruses. 1x10 ⁵ cells / well PBL were inoculated into 96-well culture plate dishes with 10% heat inactivated normal human serum and RPMI 1640 medium containing penicillin / streptomycin.

Subsequently, 5 ng / ml LAV / HTLV E total or 1 ng / ml LAV / HTLV E envelope glycoproteins isolated from purified LAV / HTLV E by lens-extin chromatography were added to the culture vessels. Six days later, the amount of ³ H-thymidine incorporated in the cells was determined by liquid scintillation counting.

AVI. Table 1A shows that the lymphocytes of both animals immunized with v-env5NY (Nos. 124 and 149) proliferated under the stimulation of both LAV / HTLV E virion and purified envelope proteins (env). However, Article 134 an animal which received recombinant treatment with v-HSgDINY vaccine-herpes simplex did not produce lymphocytes recognizing LAV / HTLV E virus.

VI. spreadsheet

Cellular immune response in chimpanzees vaccinated with recombinant vaccinia

The germ PBL incorporates ³ H-thymidine if Panza immunization used to stimulate PBL number used virus antigen: - South Africa / HTLV E env

124th v-env5NY 2482 37.132 26.370 149th v-env5NY 375 20.292 27.115 134th v-HSVgDINY 367 1.987 367 64th - 452 567 222 72nd - 737 2.417 905

Following stimulation with phytohemagglutinin (PHA, 2 ng / ml), all chimpanzees incorporated large amounts (38,870-109,790 bpm) of ³ H-thymidine; high levels of ³ H-thymidine incorporation were also observed by X-ray treated xenogeneic human PBL (42,172 12,067 beats / min). Chimpanzees derived from chimps immunized with V-env5 or vHSVgDlNY reacted strongly to the vaccine virus, while PBLs from non-immunized chimpanzees did not proliferate when vaccinated with the vaccine virus.

I. A-IV. and Tables 11-13. Summarizing the results of Figures 1 to 4, we can state the following:

(a) v-env5 recombinant vaccinia virus and venv5NY, a copy of the former into a vaccine strain, are capable of eliciting a humoral and cellular immune response specific to LAV / HTLV E in two non-human primates (macaques and chimpanzees);

(b) the recombinant viruses did not repress the T cell mediated immune response either in vitro or in vivo.

6.6. Recombinants constructed using the vaccinia virus strain v-NY are diminished neuroto60

HU 205780 Β xicity

Groups of five ICR mice were injected with 5x10 ⁷ pfu / 25-50 µl virus stock solution by intracerebral injection. The mortality rate was evaluated 10 days after drug administration. The results are as follows VH. táblázatmutatja.

VH. spreadsheet

Mortality in mice vaccinated with vaccine recombinants

Vaccine Number of animals killed / inoculated / number of animals v-NY 0/5 v-env5NY 0/5 v-env2NY 0/5 v-env7NY 0/5 Smallpox vaccine (Wyeth) 3/5 v-ENV5 5/5 v-ENV2 5/5 v-env7 5/5 Physiological saline 0/5

The VH. The results of Table 1A show that the plaque purified tissue culture vaccinia virus (v-NY strain) and its derivatives (v-env5NY, v-env2NY and v-env7NY) show reduced neurotoxicity compared to the WR strain and its derivatives. Since the strain of the New York City Board of Health is used as a smallpox vaccine, the recombinants constructed from it are likely to be more suitable for use in a human vaccine.

7. Examples for the preparation of gag-type baculovirus recombinants

The following examples describe the construction of plasmid vectors containing a chimeric gene in which the LAV / HTLV g-gag coding sequences are downstream of the AcNPV transcriptional control sequences.

Chimeric genes containing the AcNPV polyhedron gene promoter and the LAV / HTLV ΠΙ-gag coding sequence are inserted into the AcNPV genome by in vivo recombination. The resulting recombinant viruses were identified, purified and virus stock cultures were prepared from cells of infected tissue culture. Immunoreactive LAV / HTLV M-gag proteins are produced in vitro by recombinant AcNPV. The lysates of the recombinant AeNPV-infected cells were then reacted with the serum of AIDS patients and found positive by ELISA. The individual steps that are briefly described are described in detail in the following sections.

7.1. Afó'bbmódszerekleírása

7.1.1. Cells and viruses

The Sf9 clone of Sopoptera frugiperda cells was obtained from American Type Culture Collectxon (ATCC; Accession No. CRL 1711). Idon Grace contains 3.3 g / l yeastolate (Difco), 3.3 g / l lactalbumin hydrolyzate (Difco), 10% calf manmum serum, 0.06 g / l penicillin and 0.1 g / l streptomycin. Cultured on Antheraea medium (Gibco, KC Biologicals) at 28 ° C. The medium containing the additives is hereinafter referred to as TNM-EH medium. Autographa califomica nuclear polyhedrosis virus [Mol. Cell. Biol. 3. 2156-2165 (1983)] from Dr. Lois Miller (Department of Genetics and Entomology, University of Georgia, Athens, GA 30602) (deposited 1978, ATCC VR1344) and 1% agarose and 0.8 fold Sf9 cells containing TNM-EH medium were purified by plaque. The virus stock culture was diluted with TNM-EH medium as needed.

7.1.2. Production, Cleavage, and Modification of DNA

Restriction enzymes were obtained from BetesdaResearchLaboratories, Inc., and the manufacturer also provided recommended digestion conditions. The Klenow fragment of E. coli DNA polymerase was in 200 units / ml in a solution containing 50 mM Tris hydrochloride, pH 7.4, 6 mM magnesium chloride and 10 mM 12-mercaptoethanol. Apply at 37 'C for 30 minutes.

The viral DNA from the non-confluent viral parts of the wild-type virus stock (nonoccluded virus, hereinafter "NOV") is L. K. Miller, D. W. Miller and P. Separated according to Safer's method, the NOV particles were centrifuged at 90 ° C for 1 hour at 5 ° C with 25% sucrose, 5 mM sodium chloride and 10 mM EDTA, and the supernatant was removed. the pellet was resuspended in 0.5 ml of lysis buffer (10 mM Tris, pH 7.6; 10 mM EDTA and 0.25% sodium dodecyl sulfate). Proteinase K enzyme was added to the suspension at a final concentration of 500 ng / ml. Incubate for about 16 hours at 37 ° C with occasional mixing. The DNA is extracted with an equal volume of phenol / chloroformisoamyl alcohol (25: 24: 1). The ADNS is then precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5) and 2 volumes of cold ethanol to -20 'Con. After pelletization, the DNA is washed with 70% ethanol, dried and resuspended in so-called TE solution (10 mM Tris, 1 mM EDTA) prior to use for restriction enzyme analysis or cellular application.

Plasmid DNA was prepared by the so-called "maxiprep" method of M.D. Summers and G.F. Smith as follows: To 1 ml of Luria media (LB), an appropriate antibiotic was added and induced with a single colony from a freshly prepared bacterial culture. The preparation was incubated at 37 ° C for 12-16 hours, then 1 ml of the culture was transferred to a 500-1000 ml flask containing 200 ml of LB + antibiotic mixture and shaken at 37 ° C for 12-18 hours. incubated. The 200 ml cultures were then centrifuged at 2500 rpm for 10 minutes. After removing the supernatant, the cell pellet was resuspended in 30 ml of STEF buffer (8% sucrose; 0.5% Triton X-100; 50 mM EDTA pH 8.0; and 10 mM tris hydrochloride, pH 8.0) at room temperature and the suspension was placed in a 125 ml flask. Then .a

Add 3 ml of a 10 mg / ml solution of lysozyme (freshly prepared in STET buffer) to 205780 Β flasks, mix and mix well and incubate for 5 minutes at room temperature. Each flask was then held over a flame with gentle shaking (about 1 shake every 5 seconds) until the cells conjugated and bleached. When bubbling begins, the flasks are placed in a hot water bath for 45 seconds and then cooled in ice water for 2 minutes or more. The viscous mixture was then centrifuged into round-bottomed centrifuge tubes (50 mL) and centrifuged at 1600 rpm in a preparative centrifuge equipped with a SW 28 rotor (Beckman, CA). Carefully transfer the supernatant from each tube into 50 mL disposable polypropylene centrifuge tubes, add 1 volume isopropanol and 2 volumes ethanol, and DNA at -80 ° C for 20 minutes (or -20 ° C for 2 hours, or slightly longer) and centrifuged at 2500 x g for 1 minute or longer. After the alcohol was removed, 2.5 ml of extraction buffer (12.1 g iris, 33.6 g Na ₂ EDTA 2 H ₂ O and 14.9 potassium chloride, pH 7.5) was added to the pellet and shaking vigorously to loosen the cell lysate from the bottom of the tube. Subsequently, 100 µl of 10 mg / ml ribonuclease A (dissolved in 0.1x TE and pre-boiled for 10 minutes) were added and incubated for 30 minutes at 37 ° C. Approximately 200 ng of proteinase K was then added to each tube and the cultures were incubated for 30 minutes at 4050 ° C, followed by the addition of 250 µl of 10% sarcose and incubation continued for at least 3 hours. Then 80 µl of 10 mg / ml ethidium bromide per ml of DNA solution was added to each tube, followed by 1.04 g of cesium chloride per Imi DNA solution, and the tubes were gently shaken until dissolved. The preparations were transferred to Quick-Seam tubes and centrifuged at 55,000 rpm (16 hours centrifugation with a special rotor set at a fixed angle) until equilibrium was reached. Two apparently well separated DNA bands are obtained, the upper band being linear and cut DNA and the lower band being covalently bound circular DNA. The lower band (covalently bound circular DNA) is collected in 15 ml polypropylene centrifuge tubes and the liquid is made up to 6 ml with water. Ethidium bromide is extracted from the DNA with an equal volume of isoamyl alcohol and the upper pink band is separated and discarded. The extraction is repeated until the upper phase is colorless and the lower phase containing DNA is clear and colorless. At the end, approximately 5 ml of DNA solution remain in each tube (if not, make up to 5 ml with water) to which are added two volumes of anhydrous ethanol. The DNA was precipitated at -80 ° C for 10 minutes or at -20 ° C overnight and centrifuged at 2500 x g for 20 minutes. After removal of the total alcohol, pellets obtained by centrifugation (65 ° C, 10 minutes or more) are resuspended in 500 µl of TE solution diluted 110 times. ADNS can be stored at 4 ° C.

Plasmid DNA was also prepared by the method of Maniatis et al. (Molecular Cloning, Cold Spring Harbor Laboratory, 8686 (1982)). DNA ligase was taken from New England Biolabs and used at a concentration of 10,000 units / ml (Solvent; 50 mM). Tris-M hydrochloride, pH 7.8; 10 mM magnesium chloride, 20 mM DTT and 1 mM adenosine trisphosphate). DNA analysis was performed on an agarose gel by the method of Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory (1982).

7.2. Construction of plasmid vectors containing the AcNPV promoter linked to the LAV / HTLV g-gag gene coding sequence by ligase enzyme (pAcgagl)

The following subchapters describe the construction of plasmid vectors in which the LAV / HTLV ΙΠ gag gene coding sequences are preceded by sequences directing AcNPV transcription and followed by a polyhedron DNA sequence. These recombinant plasmid vectors will subsequently be used to insert LAV / HTLV ΙΠ-gag coding sequences into the baculovirus genome by in vivo recombination.

In the methods described in the following subchapters, the LAV / HTLV -ί-gag coding sequence used (Figure 14) was used to construct a subclone of lambda J19, pKS-5 [S. Wain-Hobson et al., Cell, 40, 9 (1985)] and inserted into the Aco101 plasmid containing the baculovirus polyhedron gene promoter by inserting the LAV in the resulting pAc-gagl (Figure 15). / HTLV g-gag coding sequence should be downstream of the polyhedron gene promoter. In this construct, the LAV / HTLV? -Specific nucleotide sequence is preceded by a polyhedron promoter (which formerly forms a chimeric gene) and followed by a polyhedron gene sequence.

As mentioned above, pKS-5 contains a 3148 bp segment of the LAV / HTLV ΙΠDNA located between the restriction sites SstI and KpnI (the restriction site derived from J19 is cloned into pUC18 between the SstI and KpnI sites). ).

PKS-5 was prepared by cloning the SstI restriction fragment of J19 into pUC18 at the Ssd site, digesting the resulting plasmid pBT-1 with KpnI and reconstituting with the ligase enzyme. The resulting DNA-Sel. E. coli was transformed and plasmid pKS-5 was isolated from the transformed bacterium. The LAV / HTLV-specific DNA in pKS-5 is described in SEQ ID NO: 224. from the SsII cloning site at nucleotide no. at the nucleotide LpM to the LAV / HTLV HI genome [S. Wain-Hobson et al; Cell, 40, 9 (1985); see also Figure 14].

Plasmid pKS-5 was completely digested with HincH and S. and the resulting fragments were separated on a 1% low melting agarose gel. The 1818 bp fragment (about 20 ng) was excised from the gel. This fragment contains 31

EN 205780 Β glaze according to the provisions of No. 224-2042. LAV / HTLVIH specific sequences (see Figure 14) and contains the entire gag gene coding region.

jig pAc610 (mol. Cell. Biol. 5: 2860-65 (1985)) was completely digested with SmaI and Sisfl and fractionated on a 1% agarose gel; the DNA band (about 0.4 jog) was excised from the gel (Figure 15).

Both gel slices were maintained at 65 ° C for 10 minutes and 3 µl of the 1818 bp LAV / HTLV HI-specific fragment (produced by HincTL and SstI digestion) was ligated to 1 µl of pAc610 (produced by Smal and Stol). switching in a total volume of 40 µl. The coupling was performed by incubation at 23 ° C (room temperature) for 16 hours. The ligation mixture was heated to 65 'C for 10 minutes and used to transform HB10I E. coli strain (Stratagene). Plasmid DNA from ampicillin resistant transformed cells was tested for the presence of the LAV / HTLVIH extension. The structure of the resulting plasmid pAc-gagl is shown in Figure 15.

7.3. Recombinant baculovirus construction of the chimeric LAV / HTLVHI gag gene (Ac-gagl)

AcNPV was grown on Sf9 cells and purified by plaque assay. Wild-type AcNPV DNA was isolated and pAc-gagl was purified as described in 7.1.2. .

The chimeric LAV / HTLV H1 gag sequences are inserted into the AcNPV genome by in vivo recombination; this is made possible by the gag sequences in the pAc-gagl being surrounded by AcNPV coding sequences. The combined use of pAc-gagl plasmid DNA and wild-type AcNPV DNA may result in recombination between the polyhedron sequences on the plasmid and the homologous sequences in the AcNPV genome. Incorporation of a chimeric gene occurs when double recombination occurs between adjacent sequences. In such recombinants, the chimeric gene is incorporated into the polyhedron gene of AcNPV, thereby rendering the recombinant incapable of producing occlusive formations. Recombinant plaques can be selected by visual observation (absence of cyclization virus particle).

µg AcNPV DNA, 5 µg pAc-gagl and 15 µg calf thymus DNA were mixed and precipitated with ethanol. The precipitate was washed with 70% ethanol and air dried under a tissue culture hood and then suspended in 437 µl of water. To 437 µl of aqueous DNA was added 63 µl of 2M calcium chloride (thus containing 0.25M calcium chloride). To 500 µl of 2XHEBS solution containing 16 mg of sodium chloride, 2 mg of D-glucose, 0.75 mg potassium chloride, 0.5 mg disodium hydrogen phosphate 12 H 2 O and 9.5 mg N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (pH 7.1) with 1 drop of air bubbling through The DNA solution was added dropwise at 4 seconds, incubated for 30 minutes at room temperature and then in 2 ml of Grace's medium for insect cell culture containing 10% fetal calf serum (PBS) and antibiotic in 60 mm diameter tissue culture dish. Add 3x10 ^{6 to} Sf9 cells, this time medium does not contain yeast hydrolyzate and lactalbumin hydrolyzate After the addition of the DNA solution, the culture is incubated for 4 hours at 28 'C with 4 ml of Grace's medium containing FBS and antibiotic additive. flushed and incubated for 90 seconds with Grace's medium containing EBS, antibiotic and 20% dimethylsulfoxide.

Atenyészetet átöblít- three times in complete medium _and then, purified incubated at 28 ° C in 4 ml of complete medium. After 5 days, the supernatant was recovered and centrifuged at 1000xg for 5 minutes. The titre of the resulting virus stock solution was determined by Sf9 cells and plaques containing no trapped particles were visualized, harvested, and after two additional plaque formation cycles on Sf9 cells, the virus stock culture prepared from the culture was used to determine strain characteristics. Production of the recombinant by genetic manipulation is schematically illustrated in Figure 16.

7.4. Production of LAV / HTLV IH gag-type proteins in cell culture infected with recombinant baculoviruses

It was further demonstrated experimentally that recombinant AcNPV carrying the chimeric LAV / HTLVHIgag gene is capable of producing LAV / HTLV g-gag RNA and proteins in tissue culture containing cells infected with the virus. We have also shown that these proteins give an immune response to the serum of AIDS patients

7.4.1. Identification of LAV / HTLV H1-gag-specific RNA in cells infected with Ac-gagl recombinant

Wild type AcNPV or its recombinant (Ac-gagl) (multiplicity of infection: 4) is added to 10 culture vessels (about 10 ⁷ cells / well) containing Sf 9 cells. Twenty-four hours after the infection, the cells were harvested and washed twice with 0.15 mol / l sodium chloride and 0.01 mol / l trishydrochloride (pH 7.2). Apolydenylated RNA was obtained by the method of AF Purchio and GC Fareed [J. Virol, 29, 763-769. (1979)]. RNA was obtained on a 1% agarose formaldehyde gel fraction JH. Lehrach et al., Biochemistry, 16, 4743-4751. (1977)], applied to a nylon filter (Hybond) and irradiated with ultraviolet light.

The filter material was hybridized with ³² p-labeled pKS-5 DNA for 16 hours at 42 ° C in the following mixture: 0.9 M sodium chloride, 50 mM sodium dodecyl sulfate, 4XDenhardts, 0.4 mg / ml tRNA,

0.25 mg / ml calf thymus DNA and 50% formamide. The filters were then washed four times with a mixture of 0.1% sodium dodecyl sulfate and 0.25XSSC for 30 minutes at 65 ° C and applied to a Cronex 4 X-ray film using illuminating and amplifying screens.

A17. As shown in FIG. 5A, the main band of the 2200 bp fragment is detectable in cells infected with recombinant AcNPV, but not in cells infected with wild-type virus.

HU 205780 Β

7.4.2. Identification of LAV / HTLV ΙΠ gag proteins produced in cells infected with recombinant baculovirus by immunoprecipitation

The following describes an immunoprecipitation assay that demonstrates that cells infected with the recombinant baculovirus of the present invention produce proteins that express a specific immune response in the serum of AIDS patients.

Sf9 cells (5 x 10 &apos; cells / well) were inoculated into cell cultures and the cell cultures were infected with wild-type AcNPV-derived Ac-gagl. At 24.48 and 72 hours after infection, the medium was changed to methionine-free medium and the cultures were incubated for 30 minutes at 28 ° C. The medium was then replaced with 100 nCi / ml of ³⁵ S-labeled methionine (and unlabeled methionine-free) and allowed to stand at 28 ° C for 2 hours to incorporate the labeled component. The cells were then washed twice with 0.01 M Tris hydrochloride (pH 7.2) and 0.15 M sodium chloride and centrifuged. Cell pellets from each 60 mm culture dish were resuspended in 1 ml lysis buffer containing 1% NP-40 [polyoxyethylene-9- (p-tert-octylphenyl)]. 0.5% sodium deoxycholate, 0.01 M Tris hydrochloride, pH 7.4, 0.1 M sodium chloride, and 1 mM EDTA, followed by 100,000 g of cell lysate for 30 minutes. centrifuge.

To 500 ml of cell lysate was added 4 µl of heat inactivated serum (either from patients with ADS or from a healthy control) and after incubation for 1 to 1/2 hour at 4 ° C, 80 µl of activated Staphylococcus aureus cells (Pansorbin cells, Calbiochem-Behring Corp., La Jolla, CA) was added and incubation was continued for 1 - 1/2 hours at 4 ° C. Immunoprecipitation complexes were separated by centrifugation for 5 minutes at 4 ° C (Sorvall A384 rotor, 5000 rpm) and once with 1 mol of sodium chloride, 0.1% ΝΡ-40 and 0.01 mol of tris hydrochloride, pH 7.4) and three times with RIPA buffer (10 mmol Tris hydrochloride, pH 7.2; 0.15 mol sodium chloride; 1% sodium deoxycholate; 1% sodium lauryl sulfate and 1% Triton-X-100). ) wash. The washed immunoprecipitates were resuspended in 80 µl Laemmli buffer, boiled for 1 minute, and centrifuged for 5 minutes in a Sorvak A384 rotor at 5000 rpm.

Proteins isolated by immunoprecipitation are contained in the supernatant and analyzed by electrophoresis on 10% SDS-Pollacrylamide gel, stained with analosene sodium, treated with 1 M sodium salicylate at room temperature for 30 minutes and subjected to fluorographic detection.

It is likely that the mature gag-f proteins (p24 with a molecular weight of 24,000 Daltons, a plow of a molecular weight of less than 1,000 Daltons and p14 of a molecular weight of 14,000 Daltons) are derived from the larger precursor protein of 55,000 Daltons. Figure 18 shows that 67,000 daltons,

000 Dalton, 40,000 Dalton, 34,000 Dalton,

Proteins with a molecular weight of 000 Daltons and 24 000 Daltons give a specific immunoprecipitation reaction with the serum of AIDS patients.

To demonstrate that some of the immunoreactive proteins shown in Figure 18 are linked to each other by precursor products, we performed immunoprecipitation assays using the so-called pulsechase method. Infection of Sf9 cells was performed as described above. 24 hours after infection, cells were reacted with ³⁵ S-labeled methionine for 5 minutes and incubated with complete medium for 2, 4 and 8 hours. At the indicated times, the cells were harvested and subjected to an immunoprecipitation reaction. Proteins precipitated by immunoprecipitation were separated by electrophoresis on 10% SDS-polyacrylamide gel as previously described. The fluorogram for the experiment is shown in Figure 19. From this, the protein fractions p67, p55, p40, p34 and p32 were labeled within 5 minutes. In the samples started after 8 hours, the radioactivity content of p67 and p55 fractions is decreased and that of p34 is increased. The radioactivity content of p40 fraction remains relatively constant.

7.4,3. Identification of LAV / HTLV ΙΠ gag-type Proteins by ELISA The cultures of 10 ⁷ Sf9 cells in 100-well culture dishes were infected with Ac-gagl recombinant. Twenty-four hours after infection, the cells were harvested and washed twice with a solution containing 0.01 M Tris hydrochloride, pH 7.2, and 0.15 M sodium chloride, and then frozen three times and thawed. Cell lysis was achieved by addition of 2 ml PBS containing 0.5% ΝΡ-40 (5 minutes on ice). The cell lysate was centrifuged for 10 min at 100 x g and the supernatant was removed. After adding 4 ml of carbonate buffer (50 mM sodium carbonate, pH 9.6), the mixture was spun for 20 minutes in an Eppendorf centrifuge; the supernatants were removed and diluted with carbonate buffer as described in Table I. With these cell lysates, the wells of the 96-well microtiter culture plate were coated (50 µl / well) and allowed to stand overnight at 4 ° C. Then 300-300 µL of blocking reagent (5% skimmed milk, 0.01% thimerosol and 0.01% A '' - antifoam in 10XPBS solution) was added to the dishes and the cultures were incubated for 45 minutes at room temperature. The blocking reagent is aspirated and to each well is added human serum (50 µL diluted serum / dish) diluted 1: 100 with blocking reagent; however, before the diluted serum (either AIDS patient or healthy control) is added to the dishes, it is incubated for 45 minutes at 37 ° C.

The cultures in the vessel were then allowed to react at 37 ° C for 1 hour and then washed three times with a mixture of sodium chloride and Tween-20 (polyoxyethylene sorbitan monolaurate). Subsequently, 100 µl of goat antl-human horseradish peroxidase conjugate (1: 100 diluted normal goat serum containing 0.01% thimerosal) was added to the dishes and

Allow 205780 Β winds to react for 1 hour at 37 'C. The cultures were washed and 100 µl / well of buffered sucrate + homogeneous reagent mixture was added.

The buffered sucrate contains hydrogen peroxide, citric acid and phosphate buffer, a solution of homogeneous reagent in 5 tetramethylbenzidine dimethylsulfoxide. The cultures were incubated for 30 minutes at room temperature and then quenched with 3N sulfuric acid (100 N / well). The optical absorption of the reaction mixture was measured at 450 nm; reference absorption at 630 nm. The results can be found in the VIH. Table. In this, sera TRI, Y-1, GF22CeCF9 are derived from AIDS patients; 2, 16, 21, and 48; sera were normal human serum samples.

VIH. spreadsheet

Absorption values for ELISAs

Dilutions of lysates of cells infected with Ac-gagl recombinant

Atesztelt

antlszérum 50 ¹ ioo- ¹ 200 " ^l 400 ¹ 800 ¹ 1000 ' ¹ AIDS patients TRI 2.619 2,471 2,235 2,076 1,786 1,499 2.504 2.479 2,216 2,119 1.877 1,500 Y-I 2.502 2,354 2.046 1,564 1,298 1,014 2,305 2.306 2.006 1,595 1,282 1.078 - CF22C 2,486 2,317 1,924 1,393 1,009 0.811 2,319 2.241 1,838 1,331 1,068 0.848 CF9 2,303 2,110 1,339 0,855 0.636 0.512 2.209 2,012 1,305 0,850 .659 0.455

With normal serum

2 0.211 0.229 0,204 0,187 0.312 0.176 .199 .227 0.228 0.208 0.222 0.210 16 0.176 0,175 0.217 0.131 0.144 0,129 0.165 0,135 0.139 0.210 0,175 0,145 21 0.193 0,190 0.216 0.189 0.237 0.165 0,205 0.176 0,170 .199 0.197 0.224 48 0,200 0.185 0.217 0.177 0,190 0.134 0.249 0.169 0.163 0,175 0.173 0.143

Summarizing Figures 17-19. and Fig. VIIL, it can be concluded that the recombinant baculoviruses described by us are capable of

a) to express the built-inAVA / HTLVHI gag gene,

(b) modification and modification of free gene products; and

c) for the production of antigen-specific proteins which confer specific immune responses to the sera of AIDS patients.

Example 8:

Vaccine Gag Recombinants In the following example, the construction of plasmid vectors having a chimeric gene containing the LAV / HTLV ΠΙgag coding sequence and located downstream of the LAV / HTLV coding gene for the transcription of the vaccinia virus in the chimeric gene is described. The chimeric gene containing the 7.5K promoter and the LAV / HTLV g-gag sequence was inserted into the avaccia virus genome by in vivo recombination. These recombinant viruses were identified and purified, and virus stock cultures were prepared from cells of infected tissue culture. Recombinant vaccinia viruses have been shown to be immunoreactive in vitro 60

They produce proteins of the LAV / HTLV ag-gag type. Each step is described in detail in the following subsections. In this embodiment of the present invention, the same major methods as those described in Section 6.1 have been used. has already been described.

8.1. Construction of plasmid vectors containing a vaccinia virus promoter linked to the LAV / HTLV AV-gag gene coding sequence Five different plasmid vectors were constructed; they contain a vaccinia virus promoter linked to LAV / HTLV ΠΙ genomes of varying lengths containing gag coding sequences. The LAV / HTLV IH-gag coding sequences were obtained from two similar subclones of lambda J19, pKS-5 and pSS5 (Wain-Hobson et al., Cell 40: 9 (1985)). Plasmid pKS5 contains a 3148 bp LAV / HTLV ΠΙ DNA fragment from SsII (i.e., between 224-3372 nucleotides) inserted into its corresponding cloning sites in pUC18. Plasmid pSS-5 contains a 5107 base pair LAV / HTLV ΠΙ DNA fragment from SstI to SuI (i.e., between 224-5221 nulldeotides) inserted into pUC18 between the corresponding cloning sites34.

HU 205 780 Β tea. Gag coding sequences of various lengths derived from pKS-5 or pSS-5 were inserted into pGS62; this plasmid is identical to PGS20 with the difference [Mackett et al., J. Virol. 49, 857-864. (1984)] that it has 5 single EcoRI cleavage sites downstream of the unique Smal site (see Figure 20). The LAV / HTLV? -Specific sequences incorporated into PGS62 all begin at the Thal (FnuDII) cleavage site (nucleotide 257), located 76 bp upstream of the gag gene initiation codon. The LAV / HTLV ΠΙ specific sequences of pv-gagl, pv-gag2, pv-gag3, pvgag4, and pv-gag5 from this Thal site to EcoRI (nucleotide 4194) or kpnlig (nucleotide 3372), or EcoRV (15 nucleotides 2523) »or Fc / I (1975) or Fg / H (nucleotide 1642). The construction of each plasmid can be carried out as shown in Figure 20 and described below.

Construction of plasmid pv-gagl: 5 ng of plasmid pSS-5 DNA were completely digested with restriction enzymes Thal and EcoRI. The resulting fragments were separated on a 1% low melting agarose gel. A 3900 base pair fragment containing the LAV / HTLV g-gag coding sequences was isolated and ligated to pGS62 DNA previously digested with SmaI and EcoRI using 25 ligases. The ligation mixture is used to transform E. coli HB101 cells and select ampicillin-resistant clones. Plasmids isolated from each colony were examined by restriction analysis for the presence of a 3900 bp extension and examined for ligation junction EcoRI restriction site regeneration. The resulting genetic construct contains the complete gag coding sequence as well as the protease (prt) -35 gene, and contains most of the non-coding region of the pol gene up to nucleotide 4194 (to the Eco RI site).

Construction of plasmid pv-gag2: 5 ng of plasmid pKS-5 were completely digested with restriction enzymes Thal and SmaI. The SmaI restriction site of plasmid pKS-5 is located adjacent to the Kpn1 cloning site; the latter is an interface between the inserted LAV / HTLV ΙΠ-derived sequence and the multiple cloning sites of plasmid pUC18. The resulting fragments were separated on a 1% low melting agarose gel. A 3200 base pair fragment containing the LAV / HTLV g-gag sequence was isolated and ligated to pGS62 DNA pre-digested with SmaI and treated with 50 alkaline phosphatase from the calf using a ligase. The ligation mixture is used to transform E. coli HB101 cells and select ampicillin-resistant clones. Plasmids from each colony were tested for the presence of a 3100 base pair extension and the location of the extension was determined by restriction analysis. The resulting construct contains the entire gag gene and the prt gene, and also contains a portion of the non-coding region of the pol gene up to the Kpn1 site (nucleotide 3372). 60

Construction of plasmid pv-gag3: This is similar to pv-gag2 except that the 2300 base pair fragment obtained by digestion of Thal and EcoRV from plasmid pKS-5 was inserted into pGS62 at the Smal cloning site. The pv-gag3 obtained at the end of the procedure contains the entire gag gene and a small portion of the pol gene; the latter related to the 3'-part of the vaccine virus TK gene,

Construction of plasmid pv-gag4: 5 ng of plasmid pSS-5 DNA was digested with SstI and Bell. The resulting fragment containing the LAV / HTLV g-gag sequence was purified. The resulting fragment was bound to pIC19R by a binding enzyme previously digested with SstlationBamff1 and converted to an intermediate plasmid. plasmid (March et al., Gene, 32, 481485 (1984)). When the gag sequence is inserted at the BamHI cloning site of plasmid pIC19R, a Bell cloning site is located adjacent to the EcoRI cloning site in the LAV / HTLV ΙΠ sequence in the multiple cloning vector. The resulting intermediate formation is then digested with 7% and EcoRI restriction enzymes to give a 1720 base pair fragment which can be easily inserted into the plasmid pGS62 at the SmaI and EcoRI site cloning sites. The final formation, pvgag4, contains the entire LAV / HTLV ΙΠ gag gene and part of the prtgene linked to the 3 'position of the vaccinia virus TK gene.

Pv-gag5 was produced using the same two-step strategy as pv-gag4. 5 ng of plasmid pSS-5 DNA was digested with restriction enzymes SstI and BglII. The 1420 bp fragment containing the LAV / HTLV g-gag sequence was isolated, purified on a 1% low melting point agarose gel and inserted into the SICII and BglII cloning sites in pIC19R. The gag sequence at the Bgl megfelelő cloning site was ligated with the appropriate site in plasmid pIC19R. As a result of this manipulation: (a) a stop codon is formed at the non-coded portion of the gag, and (b) it is flanked by & Bgl kerül in your plasmid with multiple cloning sites. and the EcoRi cloning site. The resulting intermediate formation is then digested with Thal and EcoRI. The resulting 1390 bp fragment containing the LAV / HTLV g-gag sequence was purified and ligated to the Sma I and Eco RI cloning sites of plasmid pGS62. The resulting plasmid pv-gag5 contains almost the entire gag sequence (up to the BglII site) linked to an intracellular translation stop sequence.

.2. Preparation of recombinant vaccine viruses containing chimeric LAV / HTLV ΠΙ-gag genes

By means of pv-gagl, pv-gag2, pv-gag3, pv-gag4 and pv-gagS, the chimeric LAV / HTLV III gag genes were introduced according to the method described in v-env5 and v-env2 (Chapter 6.3). into the vaccine virus genome. In the following examples, all recombinant viruses used were plaque-purified by the New York City Board

EN 205780 Β of Health (v-NY, see section 6.1.1). TK recombinants were selected, plaque purified three times, and virus stock cultures were prepared to determine strain characteristics. Recombinant viruses produced by pv-gagl, pv-gag2, pv-gag3, pv-gag4 and pv-gag5 are referred to as v-gag1NY, vgag2NY, v-gag3NY, v-gag4NY and v-gag5NY, respectively.

8.3. Production of LAV / HTLV ΠΙ-gag Proteins in Tissue Culture Cells Infected with Recombinant Vaccines

The recombinant vaccinia viruses described above containing the Lomer / LAV / HTLV g-gag gene produce LAV / HILV ΠΙ-gag proteins in infected cells of tissue cultures. Some of these recombinants convert the early gag protein (p55) to mature p25 and p18 proteins. These proteins have been shown to be immune to sera from AIDS patients and / or gag proteins responsive to p25 and / or 8l8.

contiguous BSC-40 cell culture in culture vessels of mm diameter are infected with the precursor vaccinia virus (v-NY) or its recombinant derivative v-gaglNY, v-gag2NY, vgag3NY, v-gag4NY, or v-gag5NY: . After 12 hours of infection, the cells were harvested, washed once with PBS and centrifuged. The centrifugal infected cell pellet was resuspended in 0.3 ml of Laemmli buffer and the cells were tossed by boiling for 4 minutes. The total cellular protein content of a 20-well aliquot of the cell lysate was obtained by fractionation on a 15% gradient SDS-polyacrylamide gel. Purified LAV / HTLV IH virion on the one hand and an aliquot of the lysate of the cells infected with the replicate sample were used as controls.

Fractions on the agel were transferred electronically to a nitrocellulose filter plate. The screen was reacted with serum from AIDS patients or with a mixture of p25 and p18 gag protein-specific antibodies. After thorough washing, the filter is reacted with alkaline phosphatase-bound goat anti-human immunoglobulin antibodies (dialysis serum has been used previously) or reacted with alkaline phosphatase-bound goat anti-mouse immunoglobulin antibodies (if previously monoclonal antibodies are used). The first and second antibody reactions and the washes a

6.4.1. . The final wash after the second antibody reaction was performed with a solution of 0.1 M iris (pH 9.5), 0.1 M sodium chloride, and 5 mM magnesium chloride. To detect antibody bound to the antigen on the filter, the filter was reacted with a solution of 0.1 molar iris (pH 9.5), 0.1 molar sodium chloride, 5 mmol magnesium chloride, 0.33 mg / ml bromochlorine indolyl phosphate and 0.17 mg / ml nitrotetrazolium blue. After completion of the chromogenic reaction, the filter is washed with water, air dried and photographed.

The analysis results are shown in Figure 21. The v-gag1NY and v-gag2NY recombinants contain the entire gag gene and the prt gene. These recombinants produce a family of LAV / HTLV IH-type proteins that migrate together with the major gag proteins of LAV / HTLVIH (p55, p40, p25 and p18). These proteins, as well as serum from AIDS patients (Figure 21J3), and both p25 and p18 protein-specific murine monoclonal antibodies (Figure 21A), confer an immune reactor. The v-gag3NY recombinant contains both the gag and pol genes, but the unencoded pol construct is located (ligated) to the carboxyl-terminal portion of the vaccine TK gene. V-gag3NY is also capable of producing the primary gag gene product, p55. However, in this case, the conversion of p55 to p25 and p18 is much less extensive than that of p55 produced by v-gagINY or v-gag2NY; in fact, the lysates of cells infected with v-gag3NY contained much less p25 and p18 compared to p55. The v-gag4NY recombinant contains the entire gag gene but only a portion of the prt gene. It synthesizes a LAV / HTLV IH-like protein similar in size to p55. The vgag5NY recombinant contains only a portion of the gag gene and produces a 43,000 dalton molecular weight carved protein (Fig. 21C). Although neither v-gag4NY nor v-gag5NY efficiently convert the primary gag-type protein, their products are immune to p25 and p18 specific monoclonal antibodies. In conclusion, regardless of differences in their ability to convert, all five recombinants are capable of producing immunoreactive proteins containing the major epitope of LAV / HTLV IH core proteins.

Example 9

Preparation of Baculovirus Envelope Recombinants The LAV / HTLV H1-env coding sequence was obtained from pv-env5 (subsection 6.3) by purification; recombinant vaccinia virus ven.v5 was produced and er40 was shown to produce env-type proteins. In pv-env5, there is a 96 bp region attached to the 5'-position, an integral env coding region and a 223 bp non-transcribed region at the 3'-position at each end having a Bam45 HI cleavage site. Digestion of this plasmid (which also has an internal BamHI site) with the BamHI restriction enzyme results in a 290 o pair of fragments containing only the LAV / HTLV IH specific sequences. This fragment (about 0.5 ng) was separated and purified on a 1% low melting agarose gel and ligated with a ligase enzyme pre-linearized with BamHI and treated with calf intestinal alkaline phosphatase vector pAc010 (0.5 ng). The ligation ke55 beats transformed MC1000 E. coli and selected ampicillin resistant colonies. Plasmid DNA from each transformed cell was present in the presence of LAV / HTLV en-env sequences (i.e., 2300 and 540 bp fragments generated by BamHI digestion) and

HU 205780 Β extension. Plasma containing the LAV / HTLV ΠΙ env sequence inserted into the appropriate site is purified and termed pAc-env5. Its structure is shown in Figure 22.

9.2. Production of recombinant baculovirus containing the chimeric LAV / HTLV d-env gene

The lomerase LAV / HTLV en-env gene by in vivo recombination is shown in Figure 7.3. of the AcNPV genome. See also section 7.3. Subsequent to the preparation described in Subchapter I, add a mixture of mgAcNPV DNA, 5mlpAc-env5 DNA and 15mg calf thymus BNS to Sf9 cell culture. The culture was then incubated for 5 days at 28 ° C with 4 ml of complete medium and the supernatant was clarified by centrifugation at 1000 X g. The titer of the resulting virus stock was determined by Sf9 cells and the recombinant virus was identified by M. D. Summers and G. E. Smith by plaque hybridization as follows:

Sf9 cells (2.5 x 10 ⁶ cells / well) in serum-free TNMFH medium were inoculated into x 15 mm culture dishes. After adherence of the cells, the medium is removed and 1 ml of diluted virus inoculum is added to each well. The cultures were incubated for 1 hour at 27 ° C, then the inoculum was removed from each well and 4 ml of low melting agar topcoat was carefully added, starting at the corners of each well. After the top layer has solidified, the dishes are incubated in a humid environment for 4-6 days until plaques develop. The cultures in the pots are then allowed to dry in a dry place overnight or longer. After proper drying, the agarose topcoat is removed and a dry nitrocellulose filter is placed on top of the cells in the culture vessels. The nitrocellulose filter on the vessel was then placed on a Whatman 3 MM filter paper, 47 mm round, saturated with solution A. Solution A contains 0.05 moles of Tris hydrochloride, pH 7.4, and 0.15 moles of sodium chloride. After soaking in the filter paper, the filter is carefully removed and placed on a cellular side with Whatman filter paper saturated with B solution. (Solution B: 0.5 N sodium hydroxide and 1.5 mol sodium chloride). After 2-3 minutes, the nitrocellulose filter was air-dried on a paper towel and contacted with Whatman filter paper saturated with solution C (solution C: 1 M Tris hydrochloride, pH 7.4; 1.5 M sodium chloride). After 2-3 minutes, the filter is air-dried on a paper towel and carefully dipped in a solution filled with solution D (solution D: 0.3 M sodium chloride, 0.03 M sodium citrate). The filter was then removed, dried on a paper towel, stored in vacuum at 80 ° C for 2 hours and finally hybridized with ³² P-labeled plasmid pRS-3. The recombinant viruses were harvested and their liter confirmed by Sf9 cells again. Plaques containing no viral particles were identified by visual observation, and after three further plaque purifications, virus stock cultures were prepared for the following assays.

9.3. Production of LAV / HILV d-env type proteins in cells of tissue culture infected with Ac-env5 recombinant baculovirus

Recombinant AcNPV carrying the chimeric LAV / HTLV d-env gene has been shown to be capable of producing LAV / HILV d-env proteins in infected cells of tissue cultures. Proteins produced with both AIDS sera and gpl10 and. gp41 immunoreact with monoclonal antibodies that recognize LAV / HTLV ΙΠ envelope-type proteins.

Sf9 cells (1 x 10 ⁷ cells / well) were inoculated into a 100 mm diameter culture vessel and infected with either wild-type AcNPV or its Ac-env5 recombinant (multiple infection: 5).

28 hours after infection, the medium is changed to medium containing no serum additive and methionine and the cultures are incubated for 30 minutes. The medium is then changed to 2 ml of ³⁵ S-labeled methionine (and no unlabelled methionine) at 100 µCi / ml and allowed to stand for 2 hours at 28 ° C. At the end of the insertion time, the cells are washed twice with PBS and the cells are washed as described in 6.4.2. is suspended in 1 ml of the lysis buffer described in subsection II. The immunoprecipitation assay is described in 6.4.2. monoclonal recognition of sera from AIDS patients or of gpl10 or gp41 proteins as described in

Figure 23, which shows the results of the analysis, shows that the Ac-env5 recombinant virus predominantly produces a 150 kDM LAV / HTLV ΙΠ envelope protein. Because this protein migrates together with the gp150 protein synthesized by the recombinant vaccine of the v-env5 vaccine, and because it gives an immune response to both the AIDS patient serum and the monoclonal antibodies recognizing the gp101 and gp41 glycoproteins, to be. Given that this precursor contains the major epitopes of both gp10 and gp41, this protein can be potentially valuable as either a diagnostic reagent or an immunogenic agent.

Deposit of microorganisms

The following E. coli strains carrying the plasmids listed below were deposited with the Agricultural Research Culture Collection (NRRL) (Peoria.IL) with the following deposit numbers for each biological object:

E. coli Tribe plasmid Deposit number K12, MC1000 pv-envl NRRL B-18003 K12, MC1000 pv-ENV2 NRRL B-18004 K12, MC1000 pv-ENV5 NRRL B-18005 K12, MC1000 pv-env'7 NRRL B-18110 K12, HB101 pv Gagik NRRL B-18111 K12, HB101 Pac-gagl NRRL B-I8105 K12, NF1829 Pac-envf NRRL B-18109

The following recombinant viruses were deposited with American Type Culture Collectnn (Rockville, MD).

HU 205780 Β

Recombinant virus Accession number v-env2 ATCCVR2114 v-env5 ATCCVR2113 v-en7 ATCCVR2148 v-env5NY ATCCVR2149 v-gaglNY ATCCVR2150

Ac-gagI ATCCVR2147

Ac-env5 ATCCVR2151

The invention encompasses not only the deposited microorganisms and viruses, which are intended only to illustrate the invention, but also to all functionally equivalent microorganisms and viruses. In addition to the representations shown, various modifications of the invention which are described above and which are apparent to one of ordinary skill in the art are also within the scope of the claims.

It is also noted that the length of the nucleotides expressed in base pairs is approximate.

Claims (35)

CLAIMS 1. A method of producing a recombinant vaccinia or baculovirus which direct the production of peptides or proteins carrying an epitope of LAV / HTLV m in susceptible cells infected with the recombinant virus, characterized in that the vaccine or baculovirus has a nucleotide sequence / L a nucleotide sequence encoding the antigen-specific portion thereof. 2. The method of claim 1, wherein the entire sequence of the LAV / H1'LV H1 envelope gene, or a portion thereof encoding antigenic peptides or proteins, is inserted into the virus. The process according to claim 2, characterized in that in FIG. nucleotide sequence or a portion thereof encoding an antigenic peptide or protein is inserted into the virus. The method of claim 1, wherein the appropriate nucleotide sequence is inserted into a vaccine virus. The method of claim 1 at ATCC, VR 2113 for the production of v-env5 recombinant virus, characterized in that the appropriate nucleotide sequence is inserted into the virus. The method of claim 1 at ATCC VR 2114 for the production of the recombinant viral env2 recombinant virus, characterized in that the appropriate nucleotide sequence is inserted into an avira virus. 7. The method of claim 1, wherein said ATCC is deposited with VR-2148 for the production of recombinant virus v-env7, wherein said nucleotide sequence is inserted into said virus. 8. The method of claim 1 for producing a recombinant virus v-env5NY deposited with the ATCC under VR 2149, which comprises inserting the appropriate nucleotide sequence into the virus. 9. The method of claim 1, wherein the appropriate nucleotide sequence is inserted into a baculovirus. 10. A9. The method of claim 1, wherein the nucleotide sequence is inserted into a nuclear polyhedrosis virus. 11. The method of claim 1 for producing an ATC env5 recombinant virus deposited with the ATCC under VR 2151, which comprises inserting the appropriate nucleotide sequence into the rite. 12. The method of claim 1, wherein the LAV / HTLV IH gag gene sequence, or a portion thereof, encoding peptides or proteins with antigenic properties is deleted into a virus. 13. A12. The method of claim 14, wherein the method of claim 340-1871 of FIG. a gag gene sequence or a portion thereof encoding an antigenic peptide or protein is inserted into a virus. 14.12. or a method according to claim 13, characterized in that the appropriate nucleotide sequence is inserted into a vaccine virus. 15. The method of claim 1 for the production of a v-gaglNY recombinant virus deposited at ATCC with VR 2150, which comprises inserting the appropriate nucleotide sequence into the virus. 16. The method of claim 12 or 13, wherein the appropriate nucleotide sequence is inserted into a baculovirus. 17. The method of claim 16, wherein the nucleotide sequence is inserted into a nuclear polyhedrosis virus. 18. The method of claim 1 for producing an Ac-gal recombinant virus deposited at ATCC under the number VR 2147, characterized in that the appropriate nucleotide sequence is inserted into the virus. 19. A method of producing a peptide or protein carrying an epitope of LAV / HTLV IH, comprising infecting animal cells with a recombinant virus according to claim 1, culturing, recovering the peptide or protein from a cell culture and purifying it if desired. 20. A19. The method of claim 1 for the preparation of a glycoprotein carrying the epitope of the LAVZHTLVIII envelope, characterized in that it is infected with a virus carrying the protein of the subject. 21. A20. The process of claim 1 a2. 1-861 in FIG. a peptide or protein comprising an amino acid sequence consisting of amino acid residues, or an antigen-specific portion thereof, characterized by culturing animal cells infected with a virus encoding a peptide or protein. 22. The method of claim 21, wherein the ATCC is deposited with venv5 recombinant vaccine virus infected with CR 2113 and infected with animal cells. 23. The method of claim 21, wherein the v38 deposited with the ATCC is VR 2114. Animal cells infected with HU 205780 rek env2 recombinant vaccinia virus were cultured. 24. The method of claim 21, wherein the ATCC is deposited at VR 2148 with animal cells infected with the recombinant vaccinia virus venv7. 25. The method of claim 21, wherein the ATCC is deposited at VR 2149 with animal cells infected with the venv5NY recombinant vaccinia virus. 26. A19. A method according to claim 1 or 20, characterized in that the cells of an insect cell line are cultured. i 27. The method of claim 26, wherein the insect cells infected with the Acenv5 recombinant baculovirus deposited with the ATCC under VR 2151 are cultured. 28. A19. A method according to claims 1 to 500 of FIG. for the production of a peptide or protein comprising an amino acid residue consisting of an amino acid residue or an antigen-specific portion thereof, characterized in that the animal cells infected with the virus encoding the peptide or protein of the subject are cultured. 29. The method of claim 28, wherein the ATCC is deposited with vgaglNY recombinant vaccinia virus deposited at VR 2150. 30. The method of claim 28, wherein the cells of an insect cell line are cultured. 5 31. The method of claim 30, wherein the insect cells infected with the Acgagl recombinant baculovirus deposited at ATCC with VR 2147 are cultured. 32. A process for the preparation of a vaccine comprising a live virus, wherein the vaccine composition of any one of claims 4-8. The recombinant virus produced according to any one of claims 1 to 6 is converted into a standard vaccine form by methods known per se. 33. The method of claim 32, wherein the recombinant virus is a vaccine virus or mutant of v-env5 deposited at ATCC with VR2113. 34. The method of claim 32, wherein said ATCC is VR 2144 as a recombinant virus. The v-env2 vaccine virus or mutant deposited with 20 numbers is used. 35. The method of claim 32, wherein said recombinant virus is a vaccinia virus or mutant of v-en7 deposited at ATCC with VR 2148.